3D Culture of HepG2 Liver Cancer Cells in Transglutaminase ...

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3D Culture of HepG2 Liver Cancer Cells in Transglutaminase-Crosslinked Gelatin

Scaffolds

By

MARY FLORDELYS DIZON AVILA

B.S., Northeastern University, 2009

Thesis

Submitted in partial fulfillment of the requirements for the Degree of Master of Science

in the Graduate Program of Biotechnology at Brown University

PROVIDENCE, RHODE ISLAND

October 2021

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AUTHORIZATION TO LEND AND REPRODUCE THE THESIS

As the sole author of this thesis, I authorize Brown University to lend it to other

institutions for the purpose of scholarly research.

Date

Mary Flordelys Dizon Avila, Author

I further authorize Brown University to reproduce this thesis by photocopying or

other means, in total or in part, at the request of other institutions or individuals for

the purpose of scholarly research.

Date

Mary Flordelys Dizon Avila, Author

10 September 2021

10 September 2021

iv

Acknowledgements

Dr. Ian Wong – Brown University

Dr. Natalia Sushkova – Boston Scientific

Dr. Michael Bachelor – Boston Scientific

Dr. Moises Rivera-Bermudez – Boston Scientific

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Table of Contents

AUTHORIZATION TO LEND AND REPRODUCE THE THESIS ....................................................... ii

Acknowledgements....................................................................................................................... iv

Table of Tables .....................................................................................................................xii

Chapter 1: Introduction ................................................................................................................ 14

Chapter 2: Response of HepG2 Cells in a Gelatin Scaffold to Drug and Device Treatments .............. 23

Appendix A: Supplementary Section – Preliminary Data from HPAF-II and Human Dermal Fibroblasts in Coculture ................................................................................................................ 58

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Table of Figures

Figure 1: Representative diagram of the cryoablation freezing zone. .......................................... 18 Figure 2: TG-gelatin casting schematic ........................................................................................ 27 Figure 3: Average HepG2 total cell, necrotic and apoptotic aggregate areas at 1, 7, 10, 14, and 17

days. Average aggregate area approximately tripled in size between days 1 to 7, and days 7 to 10.

Average aggregate area doubled in size between day 14 and 17. The large standard deviation at

day 17 was expected, based on the observation of a range of aggregate sizes from small HepG2

aggregates to larger aggregates that appeared to be comprised of multiple individual aggregates

that had merged together to form a larger conglomerate. Average apoptotic area and necrotic

areas remained relatively stable between days 1-14, with an increase at day 17. Areas were

measured from three representative field of views at 10x in one scaffold per timepoint. ............ 36

Figure 4: Average HepG2 total cell, necrotic and apoptotic aggregate diameters at 1, 7, 10, 14,

and 17 days. Average aggregate diameter increased between day 1 and 17. The large standard

deviation at day 17 was expected, based on the observation of a range of aggregate sizes from

small HepG2 aggregates to larger aggregates that appeared to be comprised of multiple

individual aggregates that had merged together to form a larger conglomerate. Average apoptotic

and necrotic diameters remained relatively stable up to day 14, and had a gradual increase at day

17. Diameters were measured from three representative field of views at 10x in one scaffold per

timepoint. ...................................................................................................................................... 37 Figure 5: Cumulative HepG2 total cell, necrotic, and apoptotic aggregate count at 1, 7, 10, 14,

and 17 days. The total number of aggregates were counted from three representative field of

views at 10x in one scaffold per timepoint. Total HepG2 aggregate count fluctuated between

approximately 600-700 aggregates over 17 days, while the number of apoptotic and necrotic

aggregates increased over time. The number of necrotic aggregates were higher than the number

of apoptotic aggregates at all timepoints. ..................................................................................... 38 Figure 6: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin

scaffold 1 day post seeding. Single cell and small aggregates with low levels of apoptosis and

necrosis. A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7).

D) Necrotic events (Ethidium Homodimer-1). 10x magnification. .............................................. 39

Figure 7: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin

scaffold at 7 days post seeding. HepG2 aggregates ranging from small to large aggregates with

low levels of apoptosis and necrosis were observed across the scaffold. A) Merged image. B)

HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium

Homodimer-1). A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events

(Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification. ...................... 40


scaffold at 10 days post seeding. Larger HepG2 aggregates were observed at a higher frequency

across the scaffold compared to previous timepoints. Aggregates were generally circular with

irregular edges in appearance. Observations of apoptotic and necrotic events were still infrequent

across the scaffold. A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events

(Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification. ...................... 41 Figure 9: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin

scaffold at 14 days post seeding. HepG2 aggregates ranging from small to large were observed

across the scaffold. Larger aggregates were more spherical in appearance. Observations of

apoptotic and necrotic events were still infrequent across the scaffold. A) Merged image. B)

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HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium

Homodimer-1). 10x magnification. .............................................................................................. 42


scaffold at 17 days post seeding. Large HepG2 aggregates were observed across the scaffold.

Several aggregates appeared to comprised of smaller individual aggregates, suggesting that those

in close proximity had merged together. More frequent and larger apoptotic and necrotic events

were observed across the scaffold, primarily located in the largest aggregates. A) Merged image.

B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events

(Ethidium Homodimer-1). 10x magnification. ............................................................................. 43 Figure 11: Percent surface area of regions with live cells, apoptotic cells, and necrotic cells in

paclitaxel-treated and untreated scaffolds from 9 samples (3 replicates, 3 scaffolds per replicate).

The percent area of regions with live cells were lower in paclitaxel-treated HepG2 cells in

comparison to untreated HepG2 cells in a gelatin scaffold (p-value 0.0009, Student’s t-test). The

areas of apoptotic and necrotic regions were higher in paclitaxel-treated HepG2 cells compared

to untreated HepG2 cells in a gelatin scaffold (p-values 0.0004 and 0.0177 respectively,

Student’s t-test). ............................................................................................................................ 45

Figure 12: Representative images of untreated HepG2 cells in TG-gelatin scaffolds at 14 days.

A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D)

Necrotic events (Ethidium Homodimer-1). 10x magnification. ................................................... 48

Figure 13: Representative images of paclitaxel-treated HepG2 gels at 14 days. Apoptotic and

necrotic events were observed to a higher degree and frequency in paclitaxel-treated

transglutaminase-crosslinked gelatin scaffolds compared to untreated scaffolds. A) Merged

image. B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events

(Ethidium Homodimer-1). 10x magnification. ............................................................................. 49

Figure 14: Proposed experimental design to evaluate HepG2 proliferation and viability in a

gelatin scaffold qt days 1, 7, 10, 14, and 17. Each arm will have 3 biological replicates and 3

technical replicates. ....................................................................................................................... 52 Figure 15: Proposed experimental design to evaluate the effect of paclitaxel on HepG2

proliferation and viability in a gelatin scaffold. Each arm will have 3 biological replicates and 3

technical replicates. ....................................................................................................................... 54

Figure 16: Representative images of cocultured HPAF-II (CMFDA green labeled) and EMEM-

HDF (CMTPX labeled) cells in EMEM (image A), EMEM-HDF monoculture in EMEM (image

B), and control HPAF-II monoculture in EMEM (image C) at Day 2 (10x magnification). ....... 68

Figure 17: Representative images of cocultured HPAF-II and EMEM-HDF cells in EMEM

(image A), EMEM-HDF monoculture in EMEM (image B), and control HPAF-II monoculture in

EMEM (image C) at Day 4 (10x magnification). ......................................................................... 69

Figure 18: Representative images of cocultured HPAF-II (CMTPX red labeled) and EMEM-

HDF (CMFDA green labeled) cells in EMEM (images A and B), EMEM-HDF monoculture in

EMEM (image C), control HPAF-II monoculture in EMEM (image D), and control HDF

(CMRA orange labeled) monoculture in FGM (image E) at Day 1 (10x magnification). Note the

difference in appearance of control HDF to EMEM-HDF (image C vs image E). ...................... 71 Figure 19: Representative images of cocultured HPAF-II and EMEM-HDF cells in EMEM

(images A and B), EMEM-HDF monoculture in EMEM (image C), control HPAF-II

monoculture in EMEM (image D), and control HDF monoculture in FGM (image E) at Day 2

(10x magnification). Note the difference in appearance of control HDF to EMEM-HDF (image C

vs image E). .................................................................................................................................. 72

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Figure 20: Representative images of cocultured control HPAF-II (CMTPX red labeled) and

control (CMRA orange labeled) HDF cells in FGM (images A and B), cocultured control HPAF-

II and control HDF cells in EMEM (images C and D), control HDF monoculture in EMEM

(image E), and control HPAF-II monoculture in FGM (image F) at Day 1. ................................ 74 Figure 21: Representative images of cocultured control HPAF-II and control HDF cells in FGM

(images A and B), cocultured control HPAF-II and control HDF cells in EMEM (images C and

D), control HDF monoculture in EMEM (image E), and control HPAF-II monoculture in FGM

(image F) at Day 2 (10x magnification). ...................................................................................... 75 Figure 22: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and

HPAF-II (CMTPX red labeled); 60:40 ratio, 10X magnification (images A and B). Both cell

lines were adapted to a 1:1 FGM:EMEM media ratio. Representative images from HPAF-II cells

adapted to a 1:1 FGM:EMEM ratio (CMTPX red labeled, image C), control HPAF-II cells

(CMTPX red labeled, image D), HDF cells adapted to a 1:1 FGM:EMEM media ratio (CMFDA

green labeled, image E), and control HDF (CMTPX red labeled, image F). ............................... 78 Figure 23: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and





green labeled, image E), and control HDF (CMTPX red labeled, image F). ............................... 79 Figure 24: Day 4 – representative images of cocultured HDF (CMFDA green labeled) and





green labeled, image E), and control HDF cells (CMTPX red labeled, image F). ....................... 82 Figure 25: Day 5 – representative images of cocultured HDF (CMFDA green labeled) and





green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that

brightness and contrast was adjusted for better visualization; original can be provided upon

request). ......................................................................................................................................... 83 Figure 26: Day 6 – representative images of cocultured HDF (CMFDA green labeled) and











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green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that image

was taken with FITC/TRITC/EOSIN filters applied in an attempt to visualize cells. Brightness

and contrast was adjusted for better visualization; original can be provided upon request). ....... 85 Figure 28: Day 4 – representative images of cocultured HDF (CMFDA green labeled) and





green labeled, image E), and control HDF cells (CMTPX red labeled, image F). ....................... 86 Figure 29: Day 5 – representative images of cocultured HDF (CMFDA green labeled) and



















green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that image

was taken with FITC/TRITC/EOSIN filters applied in an attempt to visualize cells. Brightness

and contrast was adjusted for better visualization; original can be provided upon request). ....... 89 Figure 32: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and

adapted HPAF-II (CMTPX red labeled) in FGM; 1:9 adapted HPAF-II to HDF cell ratio, 10X

magnification (images A and B). Representative images from adapted HPAF-II cells in FGM

(CMTPX red labeled, image C), control HPAF-II cells in EMEM (CMTPX red labeled, image

D), and control HDF cells in FGM (CMFDA green labeled, image E). ...................................... 92 Figure 33: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and

adapted HPAF-II (CMTPX red labeled) in FGM; 2:3 ratio, 10X magnification (images A and B).

Representative images from adapted HPAF-II cells in FGM (CMTPX red labeled, image C),

control HPAF-II cells in EMEM (CMTPX red labeled, image D), control HPAF-II cells in FGM

(CMTPX red labeled image E), and control HDF cells in FGM (CMFDA green labeled, image

E). .................................................................................................................................................. 93

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Figure 34: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and




D), control HPAF-II cells in FGM (CMTPX red labeled image E), and control HDF cells in

FGM (CMFDA green labeled, image E). ..................................................................................... 94 Figure 35: Day 2 – representative images of cocultured HDF (CMFDA green labeled) and





FGM (CMFDA green labeled, image E). ..................................................................................... 96






FGM (CMFDA green labeled, image E). ..................................................................................... 97 Figure 37: Day 2 – representative images of cocultured HDF (CMFDA green labeled) and

adapted HPAF-II (CMTPX red labeled) in FGM; 1:1 ratio, 10X magnification (images A and B).

Representative images from adapted HPAF-II cells in FGM (CMTPX red labeled, image C),

control HPAF-II cells in EMEM (CMTPX red labeled, image D), control HPAF-II cells in FGM

(CMTPX red labeled image E), and control HDF cells in FGM (CMFDA green labeled, image

E). .................................................................................................................................................. 98






FGM (CMFDA green labeled, image E). ..................................................................................... 99






FGM (CMFDA green labeled, image E). ................................................................................... 100






FGM (CMFDA green labeled, image E). ................................................................................... 101 Figure 41: Day 1 – representative images of cocultured control HDF (CMFDA green labeled)

and control HPAF-II (CMTPX red labeled) in FGM; 1:1 normal HPAF-II to HDF cell ratio, 10X

magnification (images A and B). Fibroblasts appear to surround cluster(s) of HPAF-II cells

(Image A, arrows). Representative images from normal HPAF-II cells in FGM (CMTPX red

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labeled, image C), control HPAF-II cells in EMEM (CMTPX red labeled, image D), control

HPAF-II cells in FGM (CMTPX red labeled image E), and control HDF cells in FGM (CMFDA

green labeled, image E). Clusters of HPAF-II cells in FGM appear to be loosely connected, as if

the cells were detaching from neighboring cells (Image C, arrow). Clusters of HPAF-II cells in

EMEM appear to be more tightly connected (Image D arrow). ................................................. 103 Figure 42: Day 2 – representative images of cocultured control HDF (CMFDA green labeled)

and control HPAF-II (CMTPX red labeled) in FGM; 1:1 normal HPAF-II to HDF cell ratio, 10X

magnification (images A and B). Representative images from normal HPAF-II cells in FGM



FGM (CMFDA green labeled, image E). ................................................................................... 105 Figure 43: Day 3 – representative images of cocultured HDF (CMFDA green labeled) and

control HPAF-II (CMTPX red labeled) in FGM; 1:1 normal HPAF-II to HDF cell ratio, 10X

magnification (images A and B). Fibroblast aggregation appeared to be higher around areas

containing a higher density of HPAF-II cells (Figure A, arrow). Representative images from

normal HPAF-II cells in FGM (CMTPX red labeled, image C), control HPAF-II cells in EMEM

(CMTPX red labeled, image D), control HPAF-II cells in FGM (CMTPX red labeled image E),

and control HDF cells in FGM (CMFDA green labeled, image E). HPAF-II cells with different

morphologies were also observed throughout the plate (Figure C, arrows). .............................. 107

Figure 44: Day 2 – representative images of cocultured control HDF (CMFDA green labeled)

and control HPAF-II (CMTPX red labeled) in FGM; 1:9 HPAF-II to HDF cell ratio, 10X

magnification (images A and B). Representative images from control HPAF-II cells in EMEM

(CMTPX red labeled, image C), control HPAF-II cells in FGM (CMTPX red labeled image D),

and control HDF cells in FGM (CMFDA green labeled, image E). ........................................... 110

Figure 45: Day 2 – representative images of cocultured control HDF (CMFDA green labeled)




and control HDF cells in FGM (CMFDA green labeled, image E). ........................................... 111 Figure 46: Day 3 – representative images of cocultured control HDF (CMFDA green labeled)




and control HDF cells in FGM (CMFDA green labeled, image E). ........................................... 112 Figure 47: Day 3 – representative images of cocultured control HDF (CMFDA green labeled)




and control HDF cells in FGM (CMFDA green labeled, image E). ........................................... 113

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Table of Tables

Table 1: Fibrinogen, Calcium Chloride, Transglutaminase and Gelatin Component ......... 25 Table 2: Thrombin and HepG2 Component ............................................................................ 25 Table 3: Summary of Experiments and Conclusions .............................................................. 32 Table 4: Area and Diameter Data for HepG2 Growth in TG-Gelatin Scaffolds at 1, 7, 10,

14, and 17 Days ............................................................................................................................ 35

Table 5: Percent Surface of Live, Apoptotic, and Necrotic Regions from Paclitaxel-treated

and Untreated HepG2 Cells in a Gelatin Scaffold ................................................................... 44 Table 6: Descriptive Statistics of HepG2 Total Cell, Apoptotic, and Necrotic Aggregate

Areas and Diameters from Paclitaxel-treated and Untreated HepG2 Cells in a Gelatin

Scaffold......................................................................................................................................... 47

Table 7: Proposed Experimental Groups ................................................................................. 55 Table 8: Pilot Coculture 1 – Experimental Group Overview ................................................. 62 Table 9: Pilot Coculture 2 – Experimental Group Overview ................................................. 63

Table 10: Pilot Coculture 3 – Experimental Group Overview ............................................... 64

Table 11: Pilot Coculture 4 – Experimental Group Overview ............................................... 65 Table 12: Pilot Coculture 5 – Experimental Group Overview ............................................... 66

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Abstract of 3D Culture of HepG2 Liver Cancer Cells in Transglutaminase-Crosslinked

Gelatin Scaffolds, by Mary Flordelys Dizon Avila, ScM, Brown University, September, 2021

(2021).

Objective: The objective of this thesis was to establish an experimental 3D culture system based

on liver cancer (HepG2) cells in a gelatin scaffold and investigate the functional phenotypic

response to drug and device treatment.

Methods: HepG2 cells were cultured in a transglutaminase-crosslinked gelatin scaffold, and

proliferation and viability were measured with paclitaxel treatment relative to untreated control.

Proliferation and viability were assessed using stains for live cells (Hoechst), apoptotic cells

(Caspase 3/7) and necrosis (Ethidium Homodimer-1).

Results: Preliminary experiments focused on the early feasibility of generating three-

dimensional HepG2 cell aggregates in a gelatin scaffold, and the use of paclitaxel as a positive

control for apoptosis. HepG2 cells were able to form 3D aggregates in the gelatin scaffold

model. HepG2 cells exhibit reduced viability and increased apoptosis and necrosis in paclitaxel

treated HegG2 cells relative to untreated HepG2 cells in the gelatin scaffold.

Conclusions and Future Directions: Early pilot studies suggest that the gelatin scaffold model

developed in this thesis could be used to evaluate the effect of drug and ablation treatments on

HepG2 proliferation and viability. Additional experiments are proposed to determine the

robustness of the preliminary observations in this thesis.

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Chapter 1: Introduction

Liver cancers are one of the leading causes of cancer-related death in the world.1,2 It is estimated

that more than 1 million patients will die from liver cancer in 2030, based on annual projections

by the World Health Organization.1 The 5 year survival rate for liver cancer is 18%, making it

the second-most lethal tumor.1 Of all primary liver cancers, hepatocellular carcinoma is the most

frequently diagnosed liver cancer, and is one of the leading causes of death in patients with

cirrhosis.1,2 Liver cancer will likely become one of the leading causes of death in developed

regions, due to the rise of nonalcoholic fatty liver disease, metabolic syndrome and obesity.1,2

Patients are often diagnosed with hepatocellular carcinoma at the point where they present with

deteriorating hepatic function where survival time is only a matter of months and treatment

options are limited to palliative care, or at a stage where tumor resection and liver transplant are

not options.1,3 Percutaneous ablation is the standard of care for patients with unresectable

hepatocellular carcinoma, and offers survival outcomes similar to resection and liver

transplation.1-6

The Barcelona Clinic Liver Cancer algorithm is a validated system used to classify patients in

one of five stages, with recommended treatments at each stage.1,2 Ablation is recommended

treatments for patients who have retained liver function and have 2-3 nodules that are less than 3

cm in diameter. From a technology perspective, future directions for ablation devices to treat

hepatocellular carcinoma include controlling the area of ablation, minimizing damage to healthy

patient tissue, and improving long-term success by improving the odds of complete tumor

treatment and patient survival.7 The ability to design an ablation device that can injure or

destroy a 3 cm liver tumor relies on appropriate preclinical models that can accurately reflect the

amount and location of the damage caused by the device. Based on the review of the literature

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and the way the probe of an ablation device needs to interact with the liver tumor, models that

can aid in early prototype device development would ideally have an extracellular matrix of at

least 3.5 cm in diameter, sufficient height to support the growth of human liver cancer cells in

three dimensions (3D), allow for the exchange of nutrients and waste, allow for the insertion of

the device into the center of the scaffold to allow for the application of ablation energy, allow for

spatial analysis of the tumor cross-section to determine tumor cell viability in relation to the

applied energy, be handled using aseptic technique in order to facilitate temporal evaluation of

cell viability at subsequent timepoints, and can be reliably reproduced in large quantities for

iterative testing. This thesis presents an early exploratory survey of a method and conditions that

could be used to generate a 3D liver cancer model that can assess how liver cancer cells respond

to an ablation device.

Physiology of the Liver

The liver is responsible for metabolizing nutrients such as carbohydrates, proteins, fats, and

hormones, as well as foreign substances such as drugs. The liver is also responsible for filtering

and storing blood, vitamins, glucose in the form of glycogen, and iron. Bile production, which

aids in digestion, and production of blood coagulation factors also takes place in the liver.9-11 In

a healthy liver, cells of the liver are organized into lobules, which contain rows of hepatocytes in

a hexagonal shape that extend from the portal and hepatic veins to the central vein. 9-11

Hepatocytes make up approximately 80% of all the cells in the liver and are exposed to a mixture

of deoxygenated blood from the gastrointestinal system through the portal vein and oxygenated

blood from the hepatic artery. Approximately 75% of the blood entering the liver comes from

the portal vein to allow for nutrient absorption from the intestines; the remaining 25% comes

from the hepatic artery. Blood from the portal vein and the hepatic artery mixes in the sinusoid

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of the lobule, before flowing over the hepatocytes. Blood processed by these cells then empties

into the central vein, which returns it to the circulation by means of the hepatic vein.9-11

Pathology of Liver Cancer

Hepatocellular carcinoma most often occurs in patients with chronic liver disease, primarily as a

result of alcohol abuse, and hepatitis B or hepatitis C viral infection.1 The pathogenesis of

hepatocellular carcinoma is a result of a complex multistep process involving sustained

inflammatory damage, fibrosis, and abnormal hepatocyte necrosis and regeneration.1,2 Cirrhosis

and chronic liver disease increases the risk of developing hepatocellular carcinoma.1,2 The most

frequent genetic alteration observed in hepatocellular carcinoma cells are mutations in the TERT

promoter, which accounts for approximately 60% of patient cases.1 Somatic DNA alterations

such as mutations and chromosomal abnormalities are also accumulated in hepatocellular

carcinoma cells.1 Other mutations in hepatocellular carcinoma cells affect genes in the cell

cycle, chromatin remodeling, and WNT signaling.1 Despite our current understanding of the

genetic alterations involved in hepatocellular carcinoma, there are very little somatic mutations

that can be targeted with molecular therapies.1

Molecular subtypes of patients with hepatocellular carcinoma falls either in the proliferation

class or the nonproliferation class.1 The proliferation class is characterized by high serum levels

of alpha-fetoprotein, chromosomal instability, poor cell differentiations, and TP53 mutations.1

Activation of oncogenic pathways is also seen in this group of tumors, including RAS-mitogen-

activated protein kinase and AKT-mammalian target of rapamycin.1 The majority of genetic

signatures associated with poor clinical outcome is enriched in this class.1 In the

nonproliferation class, tumors have more beta-catenin mutations, and have more resemblance to

normal hepatocytes in their gene expression pattern.1 Alpha-fetoprotein is a widely-used

17

serological marker of hepatocellular carcinoma, which may be valuable for determining tumor

size, but limited in its ability to determine tumor differentiation, metastasis, and predict patient

outcome.2,4 Other clinical biomarkers of hepatocellular carcinoma such as glypican-3,

cytokeratin 19, and midkine are under consideration, but require additional clinical validation

before they can be used clinically.8 Since there is a lack of reliable biomarkers that can be used

to detect the presence of liver tumors, imaging methods such as ultrasound, magnetic resonance

imaging, and computed tomography is used to identify the presence of lesions. However, the

ability to identify a tumor usually requires the detection of a vascular shift from the portal vein to

the hepatic artery that occurs when the liver tumor shifts from a benign lesion to a malignant

lesion.1-3, 9-11 Depending on how the tumor is classified using the Barcelona Clinic Liver Cancer

Algorithm, patients may be eligible for different types of treatment, including ablation.1-3

Ablation Technologies to Treat Liver Cancer

Ablation is the recommended and leading treatment for patients with early stage unresectable

hepatocellular carcinoma.1-3,12 Several ablation technologies are currently employed in clinical

practice, including radiofrequency ablation, microwave ablation, and cryoablation.1-3,12,13 The

majority of ablation systems are comprised of a generator and a needle or probe-like device that

delivers energy directly to the tumor.13,15 In cryoablation, the mechanism of tumor cell

destruction is by delivering intervals of controlled, sub-zero freezing temperatures to the tumor

mass, with periods of either passive or active warming in between freezing cycles.12-15 The

process of freezing and thawing causes physical damage as result of ice formation both inside

and outside the tumor cells. This causes a disruption to all cellular processes, initiates diverse

cell death processes, and prevents the cells from developing defensive mutations. The limitation

of this therapy is that the physics of the freezing process does not uniformly expose the entire

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tumor to the lethal temperature zone of -20º to -40º (Figure 1). The temperature range of -0º to -

20º at the outer margin of the ice ball causes incomplete destruction of tumor tissue; cells

exposed to this temperature range may undergo apoptosis within the first 24 hours after

treatment, and transition to secondary necrosis as the environment becomes increasingly

hypoxic. Some cancer cells survive in this temperature range. The lethal temperature zone can

be extended towards the edges of the tumor during the procedure by creating a positive freezing

margin; unfortunately, this technique damages healthy tissue adjacent to the tumor.14,15 One

potential solution to develop a device that can extend the lethal temperature zone to the edges of

the freezing zone.15 This improvement may have the potential to improve cryoablation as a

technique for treating liver tumors in patients.

Figure 1: Representative diagram of the cryoablation freezing zone.

Baust, John G et al. “Cryoablation: physical and molecular basis with putative immunological consequences.”

International Journal of Hyperthermia, 36:sup1, 2019, 10-16, DOI:10.1080/02656736.2019.1647355.

19

The development of medical devices occurs at a rapid pace when compared to the drug industry.

Drug development can take decades; in comparison, medical devices can be made available to

users and patients within 18 to 24 months. The speed at which devices are designed is largely

attributed to the fact that it is technology-based, which allows engineers to rapidly create device

iterations for testing. Medical devices are optimized for treating human-sized organs which

necessitates the evaluation and testing of these devices in models that reflect the target patient

population.16

Prior Work with Gelatin-based Hydrogels and HepG2 Cells

Hydrogels provide a 3D scaffold of water-swollen cross-linked networks that addresses the

limitation of 2D culture. Cells are known to behave more natively in a 3D environment in

biomaterial hydrogels.17 Three-dimensional cell culture can simulate the physical and biological

functions of tissues in the human body.18 Additionally, these hydrogels can mimic native

extracellular matrices and have mechanics similar to tissues.19 Gelatin-based hydrogels are

derived from hydrolysis of collagen and can mimics the microenvironments of natural

tissues.20,21 Gelatin-based hydrogels incorporating the use of the transglutaminase crosslinking

reaction to produce a viable scaffold that has been used to support culture of cells in 3D.22

The HepG2 cell line is a well-characterized cell line derived directly from human hepatocellular

carcinoma and is often used as an in vitro model for human hepatocytes.23,24,25 HepG2 cells have

been cultivated in both 2D and 3D, and in 3D systems such as collagen and Matrigel.24 These

cells have been used to evaluate liver-specific toxicity, metabolism of xenobiotics, and drug

discovery. 23,24,25 Currently there is no in vitro cell culture model that incorporates the use of

HepG2 cells in a transglutaminase crosslinked gelatin scaffold that can be used to test the

delivery of ablation energy by a device to a tumor. In addition, there is no model that can

20

facilitate the rapid evaluation of early ablation prototypes, with a simple visual readout to

determine if the ablation energy applied to the cells causes cell death. The experiments

conducted in this thesis presents a preliminary assessment of a set of methods and conditions to

generate an in vitro HepG2 gelatin scaffold model to evaluate the effect of drug and ablation

energy.

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References

1. Villanueva, Augusto. “Hepatocellular Carcinoma.” New England Journal of Medicine,

Vol.380 (15), 2019, p.1450-1462, DOI: 10.1056/NEJMra1713263.

2. Forner, Alejandro, et al. “Hepatocellular Carcinoma.” The Lancet, Volume 391, Issue

10127, 31 March–6 April 2018, Pages 1301-1314, https://doi.org/10.1016/S0140-

6736(18)30010-2.

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23

Chapter 2: Response of HepG2 Cells in a Gelatin Scaffold to Drug and Device Treatments

Methods

HepG2 Culture and Maintenance

Human liver hepatocellular carcinoma cells (HepG2) from the American Type Culture

Collection (ATCC, USA; Catalog Number ATCC HB-8065) were used in these experiments. All

experiments were conducted using cells at the 9th passage.

Prior to seeding in gelatin scaffolds, cells were cultured in flasks (VWR, Falcon® Tissue Culture

Flasks, Sterile, Corning®, vented) at 37˚C in an 80% humidified incubator with 5% CO2. Cells

were passaged at approximately 85% confluence. Media was removed and discarded from the

flasks, rinsed with DPBS without calcium and magnesium, and dissociated from flasks using

0.25% trypsin-EDTA (ThermoFisher, 25200-114). Cells were placed in the incubator for about

2-5 minutes while waiting for complete dissociation. Cells were collected in Eagle’s Minimum

Essential Medium (EMEM) (ATCC, USA; Catalog Number 30-2003) once completely

dissociated from the flask and centrifuged at 125g for 7 minutes. Cells were suspended in

EMEM for manual cell counting using trypan blue and a hemocytometer. Cells were seeded in

new flasks at a 1:6 ratio. HepG2 cells were maintained in EMEM supplemented with 10% fetal

bovine serum (FBS, ThermoFisher, 10437-080) and 1% penicillin streptomycin (pen strep)

(Gibco, 15140-122). Media was replaced every 2-3 days.

Gelatin Scaffold Solution Preparation

The gelatin scaffold solution based on Kolesky’s method to generate an extracellular matrix to

support vascularized tissues was prepared before casting the gel.1 Briefly, stock solutions of

12.5% W/V gelatin (Gelatin 300 bloom, type A (porcine skin), Sigma, G-1890), 50 mg/mL

fibrinogen (Bovine Blood plasma protein (fibrinogen), Sigma, 341573-1g), 250 mM calcium

24

chloride (Sigma, 21115-1L), 500 units/mL thrombin (Sigma, T7513-500units), and 10%

transglutaminase (Moo Glue, Modernist Pantry, 1202-50) solution were prepared for the

fibrinogen, calcium chloride, and gelatin component of the scaffold solution. Gelatin,

fibrinogen, and thrombin solutions were prepared and aliquoted prior to gel casting.

Transglutaminase was prepared the day of gel casting.

The 12.5% gelatin solution was prepared by dissolving gelatin in DPBS without calcium and

magnesium (ThermoFisher, 14190144) while stirring at 70ºC overnight. The pH of the solution

was adjusted to 7.5 using 1 M NaOH (Gibco, A4782901) at 40ºC. The gelatin solution was filter

sterilized (Thermofisher, 151-4020) before aliquoting and storage at 4ºC.

The 50 mg/mL fibrinogen stock solution was prepared by dissolving lyophilized fibrinogen from

bovine plasma in 37ºC sterile DPBS without calcium and magnesium for 45 minutes to allow

complete dissolution. Fibrinogen was aliquoted and stored at -20ºC.

The calcium chloride solution was prepared by diluting the 1M calcium chloride solution in

water and filter sterilized. The stock solution was kept at room temperature.

The thrombin solution was prepared ahead of time by dissolving lyophilized thrombin from

porcine plasma (Sigma-Aldrich, T7513) to a stock concentration of 500 units/mL in DPBS

without calcium and magnesium and stored in aliquots at -20ºC.

Cellular Gel Casting Method

The day the scaffolds were casted, gelatin aliquots were warmed using a block heater set at an

initial temperature of 38ºC until the gelatin had thawed to a viscous solution, and then was

maintained at 37 ºC. Thrombin and fibrinogen aliquots were thawed at room temperature.

HepG2 cells were harvested the day of scaffold casting. HepG2 cells were dissociated from

25

flasks using 0.25% trypsin-EDTA (ThermoFisher, 25200-114), collected in EMEM and spun

down at 125g for 7 minutes. Cells were suspended in 1 mL of EMEM for manual cell counting

using a trypan blue and a hemocytometer. Additional EMEM was added to achieve a final

concentration of 2x106 million cells per 429 µL. The 10% tranglutaminase solution was prepared

by dissolving lyophilized powder (Moo Glue, Modernist Pantry, 1202-50) in DPBS without

calcium and magnesium and gently mixing the solution on a rocker set at a speed of 1.5 at room

temperature for one hour. The solution was filter-sterilized before use.

Moving forward, all casting steps were conducted in a tissue culture hood using aseptic

technique. The reagents of the gel were separated into two components: a fibrinogen-calcium

chloride-transglutaminase-gelatin solution, and a cell-suspension and thrombin solution. Each

solution was prepared by mixing the reagents in separate 50 mL tubes according to Table 1 and

Table 2.

Table 1: Fibrinogen, Calcium Chloride, Transglutaminase and Gelatin Solution

Reagent Stock Concentration

Final Concentration (when mixed with

solution 2) Volume per gel

Fibrinogen 50 mg/mL 10 mg/mL 0.6 mL

Calcium Chloride 250 mM 2.5 mM 30 µL

Transglutaminase 10g/100mL (10%) 0.20% 60 µL

Gelatin 12.5% 7.5% 1.875 mL

Total volume needed to cast 1 scaffold 2.565 mL

Table 2: Thrombin and HepG2 Solution

Reagent Stock Concentration

Final Concentration (when mixed

with solution 1) Volume per gel

Thrombin 500 units/mL 1 unit/mL 6 µL

HepG2 Cell Suspension

(2 million cells)

N/A N/A 429 µL

Total volume needed to cast 1 scaffold 435 µL

Once each solution was prepared, the fibrinogen-calcium chloride-transglutaminase and gelatin

solution was transferred to the tube containing the thrombin and HepG2 solution and was mixed

26

by pipetting up and down. The combined solution was mixed gently to avoid introducing air

bubbles into the solution, but quickly enough to avoid having the solution set prematurely.

The mold that was used to cast the cellular disc was comprised of sterile 35 mm x 10 mm petri

dishes (VWR Scientific, 242001 or 734-0005), and 2 mm high, 31 mm internal diameter (ID)

high-temperature silicone O-rings (McMaster Carr, 5233T157) and 100 mm x 20 mm sterile

petri dishes (Celltreat, 229620) (Figure 2). The 100 mm x 20 mm sterile petri dish was used to

hold the 35 mm petri dishes so that they never placed on a non-sterile surface. Using tweezers,

the lid of a 35 mm petri dish was placed into the bottom of the 100 mm x 20 mm sterile petri dish

with the interior surface of the 35 mm petri dish facing upward. The bottom of the 35 mm petri

dish was also placed into the larger sterile petri dish with the bottom facing upwards. An O-ring

was placed onto the inside surface of the 35 mm petri dish lid, and the prepared cellular TG-

gelatin solution was pipetted immediately after mixing into the space in the center of the O-ring.

The outside surface of the 35 mm petri dish bottom was then pressed onto the top of the O-ring

to create a flat surface for optimal imaging (Figure 2). The casted gel assembly was then

transferred into a clean 100 mm x 20 mm petri dish and placed in an incubator for 1.5 hours to

allow the gels to set. After 1.5 hours, gels were carefully detached from the petri dish and O-ring

using sterile forceps and a scalpel (if needed) and were transferred into a 100 mm x 20 mm petri

dish with 30 mL of scaffold media. Gels were incubated on an undulating 3D orbital rocker set at

a speed to prevent gel adherence to the dish without spilling media (speed setting of 1.5).

27

Figure 2: TG-gelatin casting schematic

Petri dishes and silicone rings were handled with sterile instruments to minimize contamination.

O-rings were autoclaved prior to use. The working surface of the tissue culture hood was draped

with sterilized surgical drapes cut to the size of the working area. Large sterile petri dishes were

used to maintain sterility during the casting process, and to hold the casted gels when they were

transferred into the incubator to set. Petri dishes used for the gel casting process were from

sleeves that were never opened outside the tissue culture hood. Petri dishes that were removed

from the tissue culture hood were never used for gel casting. Instruments were dipped into a 70%

isopropyl alcohol solution then DPBS without calcium and magnesium prior to handling any

surface or object that would contact the cell suspension or casted gel. Instruments used to handle

the gel such as forceps and scalpels were autoclaved prior to use.

Maintenance of HepG2 Cells in a Gelatin Scaffold: After suspension in the gelatin scaffold,

cellular gels were maintained in 100 cm petri dishes (Celltreat, 229620) on an orbital rocker set

at a speed of 1.5 at 37˚C in an 80% humidified incubator with 5% CO2. HepG2 cells grown in

the gelatin scaffold were maintained in 30 mL EMEM supplemented with 10% FBS, 1%

antibiotic-antimyotic 100X (AA) (ThermoFisher, 15240-112), and 4% 1 M HEPES (N-2-

28

hydroxyethylpiperazine-N-2-ethane sulfonic acid, ThermoFisher, 15630-080). Media was

replaced every 2-3 days.

Proliferation of HepG2 Cells in a Gelatin Scaffold

For an early screen of HepG2 aggregate growth and formation in the gelatin scaffold, HepG2 cell

scaffolds were prepared and evaluated for aggregate formation, apoptosis, and necrosis at 1, 7,

10, 14, and 17 days post-seeding. Areas and diameters of HepG2 aggregates, apoptotic events,

and necrotic events were assessed by sacrificing scaffolds at 1, 7, 10, 14, and 17 days.

Viability of HepG2 Cells in Response to Paclitaxel

HepG2 cell scaffolds were prepared and matured for 11 days. At 11 days, HepG2 scaffolds in

the treatment arm were treated with 200 nM paclitaxel, and again at 36 hours post initial dose

administration. The untreated control arm was not treated with paclitaxel. The 200 nM

paclitaxel solution was prepared by diluting a stock concentration of 1 mM paclitaxel

(Invitrogen, P3456) in 100% DMSO (Fisher Scientific, BP231-100) in EMEM. Scaffolds from

the paclitaxel-treated arm and the untreated arm were imaged at 72 hours post paclitaxel

treatment to evaluate the viability of HepG2 cells in response to paclitaxel.

Cell Labeling

HepG2 cell scaffolds were stained with NucBlue Live Reagent (Hoechst® 33342 dye,

Thermofisher Scientific, R37605) to identify live cells, CellEvent™ Caspase-3/7 Green

ReadyProbes (Thermofisher Scientific, R37111) to identify apoptotic cells, and Ethidium

Homodimer-1 (EthD-1, Thermofisher, E1169) to identify necrotic cells. A 4 mL staining

solution was prepared for each gel by adding 8 drops of Hoechst 33342, 12 drops of Caspase 3/7,

and 8 µL EthD-1 to EMEM. Gelatin scaffolds with HepG2 cells were incubated for 1.5 hours

29

before fixing in 35 mL of 10% formalin. Cellular gels were fixed overnight in order to allow for

clearance of the dyes from the gel and were imaged the next day.

Imaging

Cellular gels were imaged using a Leica M205 FCA fluorescence stereo microscope with a Leica

DFC3000 G camera. Gels were placed in a 35 mm petri dish and covered with a #1.5H 22 mm x

22 mm high precision microscope cover slip and weighed down by 3D printed 35 mm diameter

weighted O-rings in to create a flat surface for imaging. Images were acquired at 3 discrete

points in each gel using the same exposure and gain settings for all experiments.

Image Analysis

Image analysis was performed using the Leica LASX 2D analysis software. Briefly, the

thresholding feature was used to determine the surface area of live cells, apoptotic cells, and

necrotic cells in each field of view. The Subtract Background (set at 200) and Grow White

Regions (set at 1) image processing pre-filters were utilized to minimize background

fluorescence. For Hoechst 33342 positive cells, the intensity threshold was set at greater than 42,

Split Touching (setting 3) and Fill Binary Processing Pre-Filters were applied to detect

individual spheroids. For Caspase 3/7 and EthD-1 positive cells, threshold levels were

determined set by using the auto adjust feature (max entropy) in the software. One limitation of

this method was that the threshold settings for some images required manual adjustment due to

autofluorescence.

Aggregate Area, Diameter, and Count: Values for aggregate (total, apoptotic, necrotic) area,

diameter, and count from each field of view were exported from the Leica LASX analysis

software into an Excel spreadsheet for statistical analysis.

30

Proliferation: Percent area coverage values of live cells (Hoechst33342-positive cells) per field

of view were exported into an Excel sheet for statistical analysis. The percent area of live cells

was determined by subtracting the percent area coverage of apoptotic cells from the percent area

coverage of Hoechst-positive cells for each field of view.

Viability: Percent surface area coverage per field of view for apoptosis (Caspase 3/7-positive

cells) and necrosis (Ethidium Homodimer-1-positive cells) were exported into an Excel sheet for

statistical analysis.

Statistical Analysis

Descriptive statistics was used to assess average area, diameter, and count for total HepG2

aggregates (Hoechst positive), apoptotic aggregates (Caspase 3/7 positive), and necrotic

aggregates (EthD-1 positive). A Student’s t-test was performed to assess if there was a

difference between the percent surface area of regions with live cells, apoptotic cells, and

necrotic cells between paclitaxel-treated and untreated HepG2 cells in a gelatin scaffold.

31

Results

Summary Overview: In this early exploratory stage of model development, two pilot experiments

were conducted to screen for a 3D scaffold that could support HepG2 cell growth in 3D, and to

screen for a potential apoptosis control that could be used in future experiments to evaluate

ablation technologies in this model (Table 3).

The first experiment was an exploratory pilot study to explore the potential to grow HepG2 cells

in a gelatin scaffold. The gelatin scaffold was based on the Kolesky method to create an

extracellular matrix for 3D printing of thick vascularized tissues.1 The hypothesis was that the

gelatin scaffold composition used to generate the extracellular matrix of Kolesky’s model could

support the growth and formation of HepG2 aggregates in 3D.

The second experiment was conducted as a screening study to assess the feasibility of using

paclitaxel as an agent for a positive apoptosis control for future ablation experiments. Published

literature shows that paclitaxel causes apoptosis in HepG2 cells.2 The hypothesis of that

experiment was that HepG2 cells would exhibit decreased cell numbers or aggregates and

increased apoptosis relative to the untreated control.

32

Table 3: Summary of Experiments and Conclusions

Question and Goals Hypothesis and Approach Experimental Details Observations/Conclusions

Question:

How does a gelatin

scaffold affect HepG2

growth and

aggregation?

Goal:

Obtain initial proof of

concept of HepG2

aggregate formation in a

gelatin model for future

experiments

Hypothesis:

HepG2 cells will form 3D

aggregates in the gelatin scaffold

that was based off a published

method to generate bioprinted

vascular tissue that could

accommodate cell growth in 3D

Approach:

• Assess if the scaffold could

support proliferation of HepG2

cells over time • Assess if HepG2 cells could

form aggregates over time • Assess viability of HepG2 cells

in gelatin over time

Imaging Timepoints: 1, 7, 10, 14,

17 Days

Stains:

• Hoechst (total cell)

• Caspase 3/7 (apoptosis)

• Ethidium Homodimer-1

(necrosis)

• HepG2 cells could form aggregates in the

gelatin scaffold • HepG2 aggregates increased in size over 17

days • Apoptosis and necrosis were observed in all

timepoints • A timepoint within this timeframe may be

feasible for future ablation studies but replicate

studies with higher sample sizes is necessary to

confirm observations and assess HepG2

proliferation and viability in this gelatin model

over time

Question:

How does the

proliferation and

viability of HepG2 cells

in scaffold differ when

treated with paclitaxel

relative to untreated

control?

Goal:

Obtain initial proof of

concept for paclitaxel as

an agent to induce

apoptosis in the model

Hypothesis:

HepG2 cells will exhibit fewer cell

and/or aggregate numbers and

increased apoptosis relative to the

untreated control.

Approach:

• Expose HepG2 cells to

paclitaxel and evaluate viability

after 72 hours • Differentiate live and dead cells

microscopically

• Paclitaxel concentration: 200

nM

• HepG2 scaffold age at

timepoint: 11 days • Readout after 72 hours • Stains

• Hoechst (total cell)

• Caspase 3/7 (apoptosis)

• Ethidium Homodimer-1

(necrosis)

• Viability was decreased in paclitaxel-treated

HepG2 cells in a gelatin scaffold

• Apoptosis was elevated in paclitaxel-treated

HepG2 cells in a gelatin scaffold • Necrosis was elevated in paclitaxel-treated

HepG2 cells in a gelatin scaffold • Paclitaxel may be feasible as a positive control

for apoptosis

33

Proliferation of HepG2 Cells in a Gelatin Scaffold

The first experiment was an exploratory pilot study to explore the potential to grow HepG2 cells

in a gelatin scaffold. The gelatin scaffold was based on the Kolesky method to create an

extracellular matrix for 3D printing of thick vascularized tissues.1 The goal was to obtain an

initial proof of concept that HepG2 cells could form aggregates in a transglutaminase cross-

linked gelatin scaffold. The hypothesis was that the gelatin scaffold composition used to

generate the extracellular matrix of Kolesky’s model could support the growth and formation of

HepG2 aggregates in 3D. One scaffold at 1, 7, 10, 14, and 17 days was used as a preliminary

screen for timepoints to assess HepG2 aggregate formation for future experiments. The scaffold

was assessed for its ability to support HepG2 proliferation and aggregate formation. A

preliminary assessment of HepG2 cell viability was also performed using stains for total cell

(Hoechst), apoptosis (Caspase 3/7), and necrosis (Ethidium Homodimer-1).

Preliminary data in Table 4 suggests that the average area for HepG2 aggregates increases

between 1 and 17 days. At day 1, the average HepG2 aggregate size was 152 ± 30 µm2 and

increased to 3864 ± 3414 µm2 by day 17 (Figure 3). The size of these aggregates ranged from a

minimum observed area of 14 µm2 at day 1 to a maximum observed area of 98,650 µm2 at day

17. The average area of apoptotic regions ranged between 18 µm2 and 22 µm2 between days 1

and 10 and increased further at days 14 (29 ± 9 µm2) and 17 (52 ± 40 µm2). Necrotic area

averages ranged between 22 µm2 and 39 µm2 between days 1 to 14, then increased to 58 ± 31

µm2 at day 17.

34

The average diameter of HepG2 aggregates appeared to increase between 1 and 17 days (17 ± 2

µm to 59 ± 24 µm, Figure 4). The diameters of apoptotic regions ranged between 4 and 7 µm

between days 1 and 14 and increased to 10 µm at day 17. Diameters of necrotic regions

fluctuated between 6 to 9 µm between days 1 and 14 and increased to 10 at day 17.

There were large standard deviations associated with average HepG2 aggregate areas and

diameters, which was likely due to a wide range of aggregate sizes observed throughout the

scaffold. The overall number of total cell aggregates within 3 field of views fluctuated between

500-700 over 17 days (Figure 5). The number of apoptotic and necrotic aggregates increased over

17 days, with more necrotic than apoptotic aggregates observed at each timepoint.

Representative images of HepG2 spheroids at each timepoint are located in Figures 6 to 10.

Preliminary observations suggest that HepG2 cells could form aggregates in the gelatin scaffold.

The size of HepG2 aggregates increased over 17 days. Apoptosis and necrosis were observed at

all timepoints. Average area and diameters of HepG2 aggregates had large standard deviations,

suggesting that a more controlled method to grow more consistently sized aggregates is needed.

A timepoint within this timeframe may be feasible for future ablation studies, but more replicates

with a higher sample size are needed to identify accurate trends to determine how HepG2 cells

aggregate and proliferate over time, and how their viability is affected as their diameters

increase.

35

Table 4: Area and Diameter Data for HepG2 Growth in TG-Gelatin Scaffolds at 1, 7, 10, 14, and 17 Days

Timepoint

Day 1 Day 7 Day 10 Day 14 Day 17

HepG2 Cell Aggregates

Total Number of Aggregates 583 707 725 585 655

Average Count (3 FOV) 194.33 235.67 241.67 195.00 218.33

SD 19.66 31.07 15.95 85.42 113.52

Average Area (µm2) 151.62 422.58 1451.86 1830.63 3864.19

SD 29.86 116.34 639.22 313.72 3413.90

Max Area (µm2) 1819.08 9552.61 34367.97 43203.91 98649.53

Min Area (µm2) 13.80 13.73 13.73 13.55 13.67

Average Diameter (µm) 16.69 25.46 42.10 45.73 58.66

SD 1.68 3.78 9.75 10.85 24.05

Max Diameter (µm2) 67.28 143.63 330.21 292.04 440.95

Min Diameter (µm2) 5.87 5.24 5.86 5.24 5.24

Apoptotic Events

Total Number of Clusters 3 8 13 68 73


SD 1.73 1.15 4.93 12.06 23.01

Average Area (µm2) 21.62 20.71 18.45 28.71 51.62

SD 37.45 11.34 12.27 8.70 40.24

Max Area (µm2) 96.61 35.71 96.13 223.59 877.53

Min Area (µm2) 0.00 4.12 1.37 1.37 8.20


SD 6.20 1.88 1.81 1.30 4.57

Max Diameter (µm2) 12.71 9.45 14.82 27.12 52.88

Min Diameter (µm2) 0.00 3.71 1.66 1.66 4.22

Necrotic Events

Total Number of Clusters 14 28 49 94 174


SD 4.04 5.03 6.11 20.53 35.59

Average Area (µm2) 31.58 21.59 38.72 36.04 58.17

SD 9.51 19.61 9.48 24.86 30.75

Max Area (µm2) 115.94 107.12 144.20 323.95 891.27

Min Area (µm2) 1.38 1.37 6.87 1.38 6.84


SD 0.78 3.00 0.74 2.47 3.09

Max Diameter (µm2) 14.53 15.72 18.93 25.53 62.20

Min Diameter (µm2) 1.66 1.66 4.23 1.66 4.22

Abbreviations:

FOV: Field of View SD: Standard Deviation

36

Figure 3: Average HepG2 total cell, necrotic and apoptotic aggregate areas at 1, 7, 10, 14, and 17 days. Average

aggregate area approximately tripled in size between days 1 to 7, and days 7 to 10. Average aggregate area doubled

in size between day 14 and 17. The large standard deviation at day 17 was expected, based on the observation of a

range of aggregate sizes from small HepG2 aggregates to larger aggregates that appeared to be comprised of

multiple individual aggregates that had merged together to form a larger conglomerate. Average apoptotic area and

necrotic areas remained relatively stable between days 1-14, with an increase at day 17. Areas were measured from

three representative field of views at 10x in one scaffold per timepoint.

-20.0

980.0

1980.0

2980.0

3980.0

4980.0

5980.0

6980.0

7980.0


Ave

rage

Agg

rega

te A

rea

(µm

2 )Average HepG2 Total Cell, Necrotic, and Apoptotic Aggregate

Areas Timecourse Results

Average Apoptotic Aggregate Area Average Necrotic Aggregate Area HepG2 Average Aggregate Area

37

Figure 4: Average HepG2 total cell, necrotic and apoptotic aggregate diameters at 1, 7, 10, 14, and 17 days.

Average aggregate diameter increased between day 1 and 17. The large standard deviation at day 17 was expected,

based on the observation of a range of aggregate sizes from small HepG2 aggregates to larger aggregates that

appeared to be comprised of multiple individual aggregates that had merged together to form a larger

conglomerate. Average apoptotic and necrotic diameters remained relatively stable up to day 14, and had a gradual

increase at day 17. Diameters were measured from three representative field of views at 10x in one scaffold per

timepoint.

-10.00

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00


Ave

rage

Agg

rega

teD

iam

eter

(µ

m)

Average HepG2 Total Cell, Necrotic, and Apoptotic Aggregate Diameters Timecourse Results

Average Apoptotic Aggregate Diameter Average Necrotic Aggregate Diameter

HepG2 Average Aggregate Diameter

38

Figure 5: Cumulative HepG2 total cell, necrotic, and apoptotic aggregate count at 1, 7, 10, 14, and 17 days. The

total number of aggregates were counted from three representative field of views at 10x in one scaffold per

timepoint. Total HepG2 aggregate count fluctuated between approximately 600-700 aggregates over 17 days, while

the number of apoptotic and necrotic aggregates increased over time. The number of necrotic aggregates were

higher than the number of apoptotic aggregates at all timepoints.

0

100

200

300

400

500

600

700

800


Nu

mb

er o

f A

ggre

gate

sHepG2 Total Cell, Apoptotic, and Necrotic Aggregate Count

Timecourse Results (3 FOV)

HepG2 Aggregates Apoptotic Aggregates Necrotic Aggregates

39

Figure 6: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin scaffold 1 day post

seeding. Single cell and small aggregates with low levels of apoptosis and necrosis. A) Merged image. B) HepG2

aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x

magnification.

A

C D

B

40

Figure 7: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin scaffold at 7 days

post seeding. HepG2 aggregates ranging from small to large aggregates with low levels of apoptosis and necrosis

were observed across the scaffold. A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events

(Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). A) Merged image. B) HepG2 aggregates (Hoechst). C)

Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification.

A B

C D

41


post seeding. Larger HepG2 aggregates were observed at a higher frequency across the scaffold compared to

previous timepoints. Aggregates were generally circular with irregular edges in appearance. Observations of

apoptotic and necrotic events were still infrequent across the scaffold. A) Merged image. B) HepG2 aggregates

(Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification.

A B

C D

42


post seeding. HepG2 aggregates ranging from small to large were observed across the scaffold. Larger aggregates

were more spherical in appearance. Observations of apoptotic and necrotic events were still infrequent across the

scaffold. A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events

(Ethidium Homodimer-1). 10x magnification.

A B

C D

43

Figure 10: Representative images of HepG2 aggregates in a transglutaminase-crosslinked gelatin scaffold at 17 days post

seeding. Large HepG2 aggregates were observed across the scaffold. Several aggregates appeared to comprised of smaller

individual aggregates, suggesting that those in close proximity had merged together. More frequent and larger apoptotic and

necrotic events were observed across the scaffold, primarily located in the largest aggregates. A) Merged image. B) HepG2

aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification.

A B

C D

44

Viability of HepG2 Cells in a Gelatin Scaffold in Response to Paclitaxel

The second experiment was conducted as a screening study to assess the feasibility of using

paclitaxel as an agent for a positive apoptosis control for future ablation experiments. Published

literature shows that paclitaxel causes apoptosis in HepG2 cells.2 The hypothesis of this

experiment was that HepG2 cells would exhibit decreased cell numbers or aggregates and

increased apoptosis relative to the untreated control. HepG2 cells in 11 day old scaffolds were

exposed to paclitaxel and evaluated for viability at 72 hours. Live and dead cells were

differentially stained using stains for total cell (Hoechst), apoptosis (Caspase 3/7), and necrosis

(Ethidium Homodimer-1) and analyzed microscopically. Three scaffolds from three replicates

were analyzed for percent surface area of regions with live cells, apoptotic, and necrotic cells for

a total of n = 9 samples.

At 72 hours post paclitaxel treatment, the average percent surface area of regions with live

HepG2 cells was lower in scaffolds that were treated with paclitaxel when compared to untreated

scaffolds (Table 5, Figure 11). The average percent surface area of apoptotic regions and

necrotic regions was higher in paclitaxel-treated HepG2 scaffolds. Representative images for

untreated and paclitaxel-treated HepG2 aggregates are located in Figure 12 and Figure 13.

Table 5: Percent Surface of Live, Apoptotic, and Necrotic Regions from Paclitaxel-treated and Untreated

HepG2 Cells in a Gelatin Scaffold

Group

Average Percent

Surface Area with

Live Cells

Average Percent Surface

Area with Apoptotic Cells

Average Percent Surface

Area with Necrotic Cells

Treated 9.7% ± 4.5% 5.9% ± 3.0% 2.4% ± 2.2%

Untreated 21.5% ± 7.0% 0.1% ± 0.1% 0.3% ± 0.3%

45

Figure 11: Percent surface area of regions with live cells, apoptotic cells, and necrotic cells in paclitaxel-treated

and untreated scaffolds from 9 samples (3 replicates, 3 scaffolds per replicate). The percent area of regions with

live cells were lower in paclitaxel-treated HepG2 cells in comparison to untreated HepG2 cells in a gelatin scaffold

(p-value 0.0009, Student’s t-test). The areas of apoptotic and necrotic regions were higher in paclitaxel-treated

HepG2 cells compared to untreated HepG2 cells in a gelatin scaffold (p-values 0.0004 and 0.0177 respectively,

Student’s t-test).

A table of average areas and diameters of HepG2 aggregates in treated and untreated scaffolds is

presented in Table 6. The average HepG2 aggregate area and diameter for paclitaxel-treated

scaffolds was 877 ± 2223 µm2 and 31 ± 29 µm respectively. The average area and diameter of

apoptotic regions was 705 ± 2377 µm2 and 24 ± 34 µm respectively. Average area and diameter

of necrotic regions was 262 ± 1334 µm2 and 15 ± 20 µm respectively.

The average HepG2 aggregate area and diameter for untreated scaffolds was 1555 ± 4682 µm2

and 39 ± 39 µm respectively. The average area and diameter of apoptotic regions was 530 ±

0%

5%

10%

15%

20%

25%

30%

% Area with Live Cells % Area with Apoptotic Cells % Area with Necrotic Cells

Percent Surface Area of Regions with Live, Apoptotic, and Necrotic Cells in Paclitaxel-treated and Untreated HepG2 Cell Scaffolds

Paclitaxel Treated Scaffolds Untreated Scaffolds

p = 0.0009

p = .0.0004

p = 0.0177

46

1455 µm2 and 23 ± 25 µm respectively. Average area and diameter of necrotic regions was 144

± 737 µm2 and 12 ± 13 µm respectively. A wide standard deviation for all parameters was

observed in this experiment.

With the exception of necrosis, the results are consistent with the expected outcomes in this

experiment. HepG2 viability was decreased in paclitaxel-treated scaffolds when compared to the

untreated control. Apoptosis was elevated in paclitaxel-treated scaffolds when compared to the

control. Necrosis was unexpectedly increased in paclitaxel-treated scaffolds in response to the

untreated control. Since DMSO was used as the vehicle for paclitaxel, it may be the cause of

elevated necrosis in paclitaxel-treated scaffolds. Subsequent experiments should include DMSO

as a vehicle control. These results suggest that paclitaxel may be feasible as a positive control

for apoptosis in future ablation experiments.

47

Table 6: Descriptive Statistics of HepG2 Total Cell, Apoptotic, and Necrotic Aggregate Areas and Diameters

in Paclitaxel-treated and Untreated HepG2 Cells in a Gelatin Scaffold

Total Cell

Aggregates

Apoptotic

Aggregates/

Events

Necrotic

Aggregates/

Events

Paclitaxel-

treated HepG2

Cells

Average Area (µm2) 876.51 705.12 262.16

SD 2229.86 2377.31 1334.03 Min Area (µm2) 13.538 1.354 1.354

Max Area (µm2) 64284.73 45766.91 30226.03

Average Diameter (µm) 30.88 24.37 14.70

SD 29.30 33.85 20.41

Min Diameter (µm) 13.538 1.354 1.354

Max Diameter (µm) 394.73 357.178 258.8 Number of Scaffolds/Replicate 3 3 3

Number of Replicates 3 3 3

Total number of samples 9 9 9

Untreated

HepG2 Cells

Average Area (µm2) 1555.03 530.02 144.29

SD 4683.22 1455.31 737.33

Min Area (µm2) 13.51 1.35 1.35 Max Area (µm2) 101345.54 15906.62 17204.62

Average Diameter (µm) 39.34 22.82 11.90

SD 39.25 25.50 13.48

Min Diameter (µm) 5.21 1.64 1.64

Max Diameter (µm) 546.98 182.61 177.34

Number of Scaffolds/Replicate 3 3 3

Number of Replicates 3 3 3

Total number of samples 9 9 9

48

Figure 12: Representative images of untreated HepG2 cells in TG-gelatin scaffolds at 14 days. A) Merged image. B)

HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase 3/7). D) Necrotic events (Ethidium Homodimer-1). 10x

magnification.

A B

C D

49

Figure 13: Representative images of paclitaxel-treated HepG2 gels at 14 days. Apoptotic and necrotic events were

observed to a higher degree and frequency in paclitaxel-treated transglutaminase-crosslinked gelatin scaffolds

compared to untreated scaffolds. A) Merged image. B) HepG2 aggregates (Hoechst). C) Apoptotic events (Caspase

3/7). D) Necrotic events (Ethidium Homodimer-1). 10x magnification.

A B

C D

50

Discussion

This thesis presented early exploratory experiments that surveyed methods and conditions that

could be used to generate a 3D liver cancer model to assess how HepG2 cells responds to a drug

or an ablation device. The ongoing COVID pandemic presented many challenges that limited

the amount of time that could be spent in lab to conduct additional experiments that would

generate data to support the initial observations in these exploratory pilot experiments.

However, the limitations of these experiments could be addressed by incorporating the following

proposed modifications for future experiments.

Common Limitations to Both Studies

Preliminary data from both experiments showed that there was a wide variation of aggregate

sizes of HepG2 cells at timepoints greater than day 10. The wide variation in aggregate sizes

shows that one or more aspects of the method or model is causing variability in the growth and

aggregation between different HepG2 aggregates within the same scaffold, and that the size of

the aggregates cannot be controlled over time using the methods used in the experiment.

The lab did not have equipment or a setup to evaluate the scaffolds using live cell imaging

techniques for temporal evaluation of the same scaffold. Because of this, scaffolds had to be

sacrificed to evaluate cell proliferation and viability, requiring a high number of scaffolds to be

generated for each experiment, and preventing temporal evaluation of cell proliferation and

viability within the same scaffold. This limitation could be overcome if the lab could set up an

imaging station for live cell analysis, utilizing non-cytotoxic dyes and timelapse confocal

microscopy to evaluate the proliferation and viability of HepG2 aggregates over time in response

to a drug or ablation. The use of a confocal microscope could also overcome the limitation of

using a low power magnification stereomicroscope to allow for identification and quantification

51

of the cells within an aggregate, and accurately identify apoptotic and necrotic cells. The data

presented in this thesis reflected live aggregates, apoptosis, and necrosis as areas within a field of

view that were stained positive by their respective stains as the stereomicroscope did not have

the objectives to appropriately visualize individual cells at the appropriate magnification.

The staining, imaging, and image analysis methods used in both studies also used Hoechst 33342

and Caspase 3/7 kits contained the dyes in a dropper, which was an imprecise way to measure

the amount of dye added to the staining solution. Additionally, the image analysis method used

for this project requires additional optimization to remove the need to manually adjust threshold

settings during the analysis process.

HepG2 Cell Proliferation in a Gelatin Scaffold Experimental Limitations

Although the preliminary results suggest that the gelatin scaffold model is a feasible scaffold to

grow HepG2 cells in 3D, the robustness of the model was not sufficiently tested. In addition to

the limitations that were common to both studies, this pilot screening study was limited by

insufficient sample size and number of replicates performed to draw significant conclusions on

proliferation and viability of HepG2 cells in the gelatin scaffold over time. This limits the ability

to determine if the size of the aggregates and the corresponding amount of apoptosis and necrosis

was an outlier or representative of their behavior in the gelatin model at 1, 7, 10, 14, and 17 days.

Future studies should increase the sample size and number of replicates performed to be able to

compare the number of live, apoptotic, and necrotic cells between time points, and characterize

the viability of the HepG2 cells over time. An ANOVA and t-test could be used to measure

significant changes in viability between timepoints.

This study revealed that HepG2 cells could form aggregates during culture. Future experiments

should have a control arm that compares the growth and aggregate formation of HepG2 cells in

52

the 3D matrix of the gelatin scaffold to the growth of HepG2 cells grown on top of a thin layer of

the gelatin matrix solution to compare how the growth, aggregation, proliferation and viability of

HepG2 cells in the gelatin scaffold compares to the growth, aggregation, proliferation and

viability of HepG2 cells grown An assay to assess HepG2 function, such as albumin, should also

be included to understand how HepG2 cell function is affected in the gelatin scaffold over time.3

A proposed experimental design to address the limitations from previous experiment to the

evaluate aggregate formation, proliferation, and viability of HepG2 cells in 2D and 3D is

presented in Figure 14. Each arm will have 3 biological replicates and 3 technical replicates.

Figure 14: Proposed experimental design to evaluate HepG2 proliferation and viability in a gelatin scaffold qt days

1, 7, 10, 14, and 17. Each arm will have 3 biological replicates and 3 technical replicates.

Effect of Paclitaxel on HepG2 Cells in a Gelatin Scaffold Experimental Limitations

Proliferation and viability of HepG2 cells were assessed at a single timepoint after treatment with

paclitaxel. The data collected indicated that the application of paclitaxel in 11 day old scaffolds

and evaluation of the effect of paclitaxel at 72 hours post administration was reasonable.

However, to ensure robustness of the design, future experiments comparing the effect of

paclitaxel on HepG2 cells on proliferation and cell viability in the gelatin scaffold should be

repeated with the inclusion of additional timepoints such as 0, 4, 8, 12, 24, 48, 72, and 96 hours

after dosing with appropriate replications.

53

During the experiment, it was observed that exposure of HepG2 to paclitaxel showed an increase

in apoptosis and necrosis. Paclitaxel is known to induce apoptosis in HepG2 cells.2 Increased

necrosis is not an expected result of paclitaxel. A potential explanation from a publication on the

effects of paclitaxel on breast cancer cells lines suggests that concentrations of paclitaxel greater

than 120 nM could cause necrosis.4 The effect of DMSO as a vehicle to deliver paclitaxel was

not assessed. A vehicle control arm to understand the effect of DMSO on proliferation and

viability should also be incorporated in future experiments. Similarly, a positive necrosis control

should be incorporated into future studies.

A control arm evaluating the effect of HepG2 cells grown on a thin layer of the gelatin scaffold

matrix should also be incorporated into future experiments to understand how 2D versus 3D

tissue culture conditions affects the proliferation and viability of HepG2 when treated with

paclitaxel.

If a live cell imaging setup can be established, time-lapse confocal microscopy and non-cytotoxic

dyes could be used in future experiments to acquire images for an image-analysis based method

to quantify proliferation and viability in response to paclitaxel by determining if the Hoechst-

stained cells counterstained by Caspase 3/7 were undergoing apoptosis. An ATP or MTT assay

for quantifying proliferation in 3D cell culture could also be used to compliment microscopy

analysis.

A proposed experimental design to address the limitations of the previous experiment and

evaluate the effect of paclitaxel on HepG2 cell proliferation and viability in a gelatin scaffold in

comparison to an untreated control is presented in Figure 15. The experimental design also

evaluates the effect of 2D and 3D, and the effect of DMSO as a vehicle for paclitaxel in both 2D

and 3D. Each arm will have 3 biological replicates and 3 technical replicates.

54

Figure 15: Proposed experimental design to evaluate the effect of paclitaxel on HepG2 proliferation and viability in a gelatin scaffold. Each arm will have 3 biological replicates and 3 technical replicates.

Proposed Future Experiment – Effect of Ablation on the Proliferation and Viability of

HepG2 Cells in a Gelatin Scaffold

Assuming that the experiments in this thesis are repeated with the proposed modifications to

address the limitations identified in the previous experiments, the following experiment is

proposed to study the effect of an ablation device on proliferation and viability of HepG2 cells in

a gelatin scaffold.

The aim of the proposed study is to determine how proliferation and viability of ablated HepG2

cells differs relative to an untreated control. The proposed study also aims to further optimize

the model so that spatial distribution of viable, apoptotic, and necrotic cells can be mapped

relative to the point of the probe (Figure 1). The hypothesis is that HepG2 cells will exhibit

increased cell death near the device relative to the untreated control.

In this proposed experiment (Table 7), HepG2 cell scaffolds will be prepared and matured for 14

days. A plastic tissue culture control arm with HepG2 cells grown in 2D on a petri dish will also

be included and maintained for 14 days and will be exposed to the same conditions as the

treatment arm. Five HepG2 cell scaffolds from 3 replicate runs will be ablated in aseptic

55

conditions with a clinically used ablation device and clinical protocol and evaluated at 8, 24, 48,

and 72 hours post treatment.

Table 7: Proposed Experimental Groups

Treatment Arm Sham Arm Apoptosis Control Necrosis Control

Plastic Tissue

Culture Control

HepG2 cells in

gelatin scaffold

ablated using a

clinical protocol of

known ablation

device

Inactive ablation

device inserted into

the HepG2-gelatin

scaffold for the

same duration as the

treatment arm

HepG2 cell

scaffolds treated

with paclitaxel

HepG2 cell

scaffolds treated

with a clinically-

used ablation device

that is known to

cause necrosis

HepG2 cells grown

on a layer of the

gelatin solution in

2D exposed to

similar ablation

conditions

Cell proliferation and viability relative to the position of the probe will be evaluated at 8, 24, 48,

and 72 hours post treatment. Cells will be visualized using Hoechst 33342 (live cells), Caspase

3/7 (apoptosis), and Ethidium Homodimer-1 (necrosis) stains. Cells will be counted using

optimized methods, preferably by confocal microscopy. The spatial distribution of live,

apoptotic, and necrotic cells will be mapped relative to the position of the probe.

Potential Directions for Future Studies

Additional studies that should investigate how different stiffness and porosities of the gelatin

scaffold affects HepG2 aggregate formation, proliferation, and viability. An investigation on

how changes in stiffness and porosity affect how HepG2 cells respond to drug and ablation

treatments. Experiments should also be performed to understand the diffusion kinetics for drug

and dye permeabilization at different gelatin concentrations or crosslinking protein

concentrations to optimize drug and dye penetration and concentration. In addition, additional

studies should investigate the ability to culture additional cell lines in single or coculture in the

gelatin scaffold.

56

Conclusions

This thesis describes pilot efforts to develop an in vitro HepG2 in a gelatin scaffold for

hepatocellular carcinoma. This model may be used to evaluate devices that can deliver energy to

destroy tumor cells at the edges of the tumor. The design of a follow-up experiment was

proposed to test an ablation device using the model presented in this thesis.

Early pilot studies suggest that the gelatin scaffold model used in this thesis could be used to

evaluate the effect of ablation treatments on HepG2 proliferation and viability. Preliminary

experiments focused on the early feasibility of generating three-dimensional HepG2 cell

aggregates in a gelatin scaffold. HepG2 cells could form 3D aggregates in the gelatin scaffold

model. Apoptosis was elevated in HepG2 cells in gelatin scaffolds treated with 200 nM

paclitaxel after 72 hours when compared to untreated scaffolds, suggesting that paclitaxel may be

an appropriate positive control for apoptosis. This also suggests that the effect of therapies

inducing apoptosis as a primary mechanism of action may be detected using this model.

Once limitations with this model are addressed, future applications using this model could be to

understand the mechanisms of cell death caused by different ablation methods or different

clinical protocols. This model may be able to predict the outcome of potential ablation therapies

if the injury response observed in this model can be correlated to clinical outcomes. This in vitro

model may to reduce the need for animal studies in the preclinical development stage. The

gelatin model could also be used to culture other tumor cells or support the coculture of two or

more cell types, such as pancreatic tumor cells and pancreatic stellate cells.

57

References

1. Kolesky, David, et al. “Three-dimensional bioprinting of thick vascularized tissues.”

Proceedings of the National Academy of Sciences, Mar 2016, 113 (12) 3179-3184, DOI:

10.1073/pnas.1521342113

2. Brenes, Oscar, et al. “Characterization of cell death events induced by anti-neoplastic

drugs cisplatin, paclitaxel and 5-fluorouracil on human hepatoma cell lines: Possible

mechanisms of cell resistance.” Biomedicine & Pharmacotherapy, Volume 61, Issue 6,

July 2007, Pages 347-355, https://doi.org/10.1016/j.biopha.2007.02.007.

3. Chang, Tammy T, and Millie Hughes-Fulford. “Monolayer and spheroid culture of

human liver hepatocellular carcinoma cell line cells demonstrate distinct global gene

expression patterns and functional phenotypes.” Tissue engineering. Part A, vol. 15,3

(2009): 559-67. doi:10.1089/ten.tea.2007.0434

4. Yeung, Tai K, et al. “The Mode of Action of Taxol: Apoptosis at low concentration and

necrosis at high concentration.” Biomedical and Biophysical Research Communications,

Volume 263, Issue 2, 24 September 1999, Pages 398-404,

https://doi.org/10.1006/bbrc.1999.1375.

58

Appendix A: Supplementary Section – Preliminary Data from HPAF-II and Human

Dermal Fibroblasts in Coculture

Pancreatic ductal adenocarcinomas remain one of the deadliest cancers both in the United

States.1 Treatment for this disease remains largely palliative, primarily due to the fact that it is

diagnosed at an advanced stage; even with curative surgery, cancer recurrence is often an

outcome for patients.2 The ability to diagnose the disease at an earlier stage or the ability to

develop more effective therapies would improve the long-term survival rate of patients. The

ability to gauge the effectiveness of medical devices targeting these malignancies often relies on

a clinically representative preclinical model to evaluate device performance and identify viable

prototype designs. Ideally, the devices tested should be the size and configuration intended for

clinical use; evaluating smaller-scale devices in smaller models is not always feasible and may

not perform similar to devices in the desired size matrix. Because of this, tumor models should

ideally present clinically representative tumors that are representative of the size the device

intends to target. In vitro tumor models have the potential to be more cost-effective than in vivo

models and may offer an efficient method for device evaluations. However, the ability to

successfully produce large (greater than 2 mm), viable, high-throughput tumors in a controlled

manner for iterative device testing has not yet been achieved. A potential direction for model

development involves coculturing tumor cells with stromal cells, as the role of the stroma in

pancreatic ductal adenocarcinomas has been implicated in its aggressive nature and

chemoresistance.3 The human pancreatic adenocarcinoma (HPAF-II) cell line and an adult

human dermal fibroblast (HDF) cell line were selected as the initial pair of cell lines to assess for

their feasibility to be cocultured in 2D, and potentially in 3D. The HPAF-II cell line is a well-

characterized ductal adenocarcinoma cell line.4 Adult human dermal fibroblasts are relatively

59

easier to obtain and maintain compared to pancreatic stellate cells for initial method development

and proof of concept. This supplementary section presents initial progress in coculturing these

two cell lines in 2D.

Materials and Methods

Cell Lines

Human pancreatic adenocarcinoma cells (HPAF-II) obtained from the American Type Culture

Collection (ATCC) (ATCC, USA; Catalog Number ATCC CRL-1997) were cultured using the

supplier recommended protocol. Human pancreatic adenocarcinoma cells were maintained in

Eagle’s Minimum Essential Medium (EMEM) (ATCC, USA; Catalog Number 30-2003)

supplemented with 10% fetal bovine serum (FBS) (ATCC, USA), and 1% penicillin

streptomycin (pen strep). Cells were cultured at 37˚C in an 80% humidified incubator with 5%

CO2.

Adult human dermal fibroblasts (HDF) were obtained from Cell Applications (CA) (CA, USA;

Catalog Number 106-05a, Lot 1503) and were cultured using the supplier recommended

protocol. Human dermal fibroblasts were initially maintained in Human Dermal Fibroblast

Growth Medium (FGM) (CA, USA; Catalog Number 116-500) with 1% pen strep. Cells were

cultured at 37˚C in an 80% humidified incubator with 5% CO2.

Media Adaptation

Human Dermal Fibroblasts: Human dermal fibroblasts were initially adapted to the media

conditions of HPAF-II cells in preparation for coculture experiments. Initially, cells were

gradually transitioned to EMEM with 10% FBS and 1% pen strep in 25% increments, increasing

60

the amount of EMEM once the fibroblasts appeared to recover from the previous medium ratio

increase. Cells were cultured at 37˚C in an 80% humidified incubator with 5% CO2. Human

dermal fibroblast cells were qualitatively observed for changes in morphology and cell viability

during the conditioning phase and compared to the morphology of HDF cells in FGM maintained

as a control line. Results from the initial subset of cells transitioned to EMEM with 10% FBS

and 1% pen strep are presented in this supplementary section.

Due to morphological differences observed between the EMEM-transitioned fibroblasts and the

control fibroblasts, adaptation of a subset of fibroblasts from the control HDF cell line to EMEM

supplemented with 10% FBS, 1% pen strep, and 100 µM ascorbic acid (AA) was initiated. The

ratio of EMEM with 100 µM AA was increased by 25% increments once fibroblasts appear to

recover from the previous medium ratio increase while maintaining the morphology observed in

the control fibroblasts.

HPAF-II Cells: A subset of human pancreatic adenocarcinoma cells were gradually adapted to

FGM in 25% increments. Cell behavior was observed as cells transitioned from EMEM to FGM.

Cell Labeling and Imaging

Cells were labeled with Cell Tracker Dyes to identify HDF and HPAF-II cell populations in

preliminary coculture experiments. Cell Tracker Red CMTPX (ThermoFisher C34552), Cell

Tracker Green CMFDA (ThermoFisher C7025), and Cell Tracker Orange CMRA (ThermoFisher

C34551) were used to label cell lines. Stock solutions of cell tracker dyes were prepared

according to manufacturer instructions. Dyes were dissolved in dimethylsulfoxide (DMSO)

(Fisher Scientific, BP231-100) to a stock concentration of 10 mM. The stock concentration was

diluted in serum-free media at working concentrations of 2.5 µM for short term experiments (less

than 4 days), and 7.5 µM for long term experiments (7 days). Cells were imaged using an

61

Olympus C61WI microscope with an X-Cite 120 fluorescence illumination system. Images were

acquired using an Olympus DP80 camera.

Coculture Experiments – Experimental Designs

Pilot experiments were performed to develop the method and media conditions to coculture

HPAF-II cells and HDF cells in 2D. Initial experiments focused on evaluating HPAF-II cells and

HDF cell coculture growth and arrangement in either EMEM or FGM.

Detailed Experimental Designs

Control HPAF-II cells and EMEM-transitioned HDF cells were cocultured at a 1:1 EMEM-

HDF/HPAF-II ratio. The number of cells seeded followed the recommended seeding density for

fast growth for fibroblasts (6,000 cells/cm2, Cell Applications). Prior to coculture, cells were

incubated in serum-free media with Cell Tracker dyes for 30 minutes, at working concentrations

of 2.5 µM. Human dermal fibroblasts adapted to EMEM were labeled with Cell Tracker Green

CMFDA. Human pancreatic adenocarcinoma cells were labeled with Cell Tracker Red CMTPX.

Cells were seeded onto 60 mm petri dishes and maintained at 37˚C in an 80% humidified

incubator with 5% CO2. Cells that were cocultured together were seeded at the same time. Cells

were imaged at 2 and 4 days. Prior to imaging, the media in the plates was replaced with fresh

media. The entire plate was assessed for cell appearance, organization, and fluorescence at each

time point. Representative images from at least 3 discrete locations within the plate were

obtained.

62

Coculture 1:

Table 8: Pilot Coculture 1 – Experimental Group Overview

Group

ID

Description Media Conditions Number of cells

seeded at Day 0

Timepoints

Assessed

A EMEM-transitioned human

dermal fibroblasts (HDF-

EMEM) cocultured with

control HPAF-II cells

EMEM with 10% FBS

and 1% pen strep

129,000 HDF-

EMEM, 129,000

HPAF-II

2 and 4 days

B EMEM-transitioned human

dermal fibroblasts only

EMEM with 10% FBS

and 1% pen strep

129,000 HDF-

EMEM

2 and 4 days

C Control HPAF-II EMEM with 10% FBS

and 1% pen strep

129,000 HPAF-II 2 and 4 days

Human dermal fibroblasts adapted to EMEM were cocultured together with control HPAF-II

cells at a 1:1 ratio similar to the first pilot experiment. Control HDF and control HPAF-II were

also cultured together in either EMEM (control HPAF-II media conditions) or FGM (control

HDF media conditions) at a 1:1 ratio. This experiment was conducted partly to confirm

observations of morphological differences of the EMEM-adapted human dermal fibroblasts

when compared to the control fibroblasts, and to assess potential experimental conditions that

may be avenues for future coculture experiments.

63

Coculture 2:


Group

ID

Description Media Conditions Number of cells

seeded at Day 0

Timepoints

Assessed

A EMEM-transitioned human

dermal fibroblasts (HDF-

EMEM) cocultured with

Control HPAF-II cells

EMEM with 10% FBS

and 1% pen strep

129,000 HDF-

EMEM; 129,000

HPAF-II

1 and 2 days

B EMEM-transitioned human

dermal fibroblasts only

EMEM with 10% FBS

and 1% pen strep

129,000 HDF-

EMEM

1 and 2 days

C Control HPAF-II EMEM with 10% FBS

and 1% pen strep

129,000 HPAF-II 1 and 2 days

D Control HDF FGM with 1% pen strep 129,000 HDF 1 and 2 days

E Control HDF cocultured with


FGM with 1% pen strep 129,000 HDF;

129,000 HPAF-II

1 and 2 days

F Control HDF cocultured with


EMEM with 10% FBS

and 1% pen strep

129,000 HDF;

129,000 HPAF-II

1 and 2 days

G Control HDF EMEM with 10% FBS

and 1% pen strep

129,000 HDF 1 and 2 days

H Control HPAF-II FGM with 1% pen strep 129,000 HPAF-II 1 and 2 days

Fibroblasts and HPAF-II cells were cultured at a 1:1 ratio. The number of cells seeded followed

the recommended seeding density for fast growth for fibroblasts (6,000 cells/cm2, Cell

Applications). Prior to coculture, cells were incubated in serum-free media with Cell Tracker

dyes for 30 minutes, at working concentrations of 2.5 µM. Human dermal fibroblasts adapted to

EMEM were labeled with Cell Tracker Green CMFDA. Human pancreatic adenocarcinoma cells

were labeled with Cell Tracker Red CMTPX. Control human dermal fibroblasts were labeled

with Cell Tracker Orange CMRA. Cells were seeded onto 60 mm petri dishes and maintained at

37˚C in an 80% humidified incubator with 5% CO2. Cells that were cocultured together were

seeded at the same time. Cells were imaged at 1 and 2 days. Prior to imaging, the media in the

plates was replaced with fresh media. Plates were assessed for cell appearance, organization, and

fluorescence at each time point. Representative images from at least 3 discrete locations within

the plate were obtained.

64

Coculture 3:


Group

ID

Description Cell Lines Used Media Conditions Number of cells

seeded at Day 0

Timepoints

Assessed

A Coculture

Ratio 60/40

Adapted HDF and

HPAF-II (see

paragraph below for

details)

50% EMEM supplemented

with 10% FBS, 1% pen

strep, and 100 µM AA; 50%

FGM with 1% pen strep

129,000 adapted

HDF; 86,000

adapted HPAF-

II

1, 4, 5, 6,

and 7 days

B Coculture

Ratio 80/20

Adapted HDF and

HPAF-II (see

paragraph below for

details)





172,000 adapted

HDF; 43,000

adapted HPAF-

II

1, 4, 5, 6,

and 7 days

C HPAF-II (1:1

FGM/EMEM)

Adapted HPAF-II

(see paragraph

below for details)



strep; 50% FGM with 1%

pen strep

129,000 adapted

HPAF-II

1, 4, 5, 6,

and 7 days

D HPAF-II

Control

Control HPAF-II EMEM with 10% FBS and

1% pen strep

129,000 control

HPAF-II

1, 4, 5, 6,

and 7 days

E HDF (1:1

FGM/EMEM

AA)

Adapted HDF (see

paragraph below for

details)





129,000 adapted

HDF

1, 4, 5, 6,

and 7 days

F HDF Control Control HDF FGM with 1% pen strep 129,000 control

HDF

1, 4, 5, 6,

and 7 days

Pilot coculture 3 was conducted to assess the behavior and arrangement of fibroblasts and

HPAF-II cells when cocultured at two different cell number ratios in a 1:1 FGM/EMEM media

ratio. In this experiment, adapted HDF cells and adapted HPAF-II cells were cocultured together

at two different ratios, based on literature describing the tumor-stroma ratio in pancreatic ductal

adenocarcinoma.3 Adapted fibroblasts and HPAF-II cells were seeded at a 60:40 HDF to HPAF-

II cell ratio, and at a 80:20 HDF to HPAF-II ratio.

Prior to coculture, cells were incubated in serum-free media with Cell Tracker dyes for 30

minutes, at working concentrations of 7.5 µM. Adapted human dermal fibroblasts were labeled

with Cell Tracker Green CMFDA. Adapted HPAF-II cells were labeled with Cell Tracker Red

CMTPX. Control HDF and HPAF-II cells in monoculture were both labeled with Cell Tracker

CMTPX. Cells were seeded onto 60 mm petri dishes and maintained at 37˚C in an 80%

humidified incubator with 5% CO2. Cells that were cocultured together were seeded at the same

65

time. Cells were imaged at 1, 4, 5, 6, and 7 days to assess for cellular appearance and

arrangement.

Coculture 4:


Group

ID

Description Cell Lines Used Media Number of cells seeded

at Day 0

Timepoints

Assessed

A Adapted HPAF-II

monoculture in FGM

Adapted HPAF-II FGM 129,000 adapted HPAF-

II

1, 2, and 3

days

B Control HPAF-II

monoculture in EMEM

Normal HPAF-II EMEM 129,000 normal HPAF-II 1, 2, and 3

days

C Control HPAF-II

monoculture in FGM

Normal HPAF-II FGM 129,000 normal HPAF-II 1, 2, and 3

days

D Control HDF

monoculture in FGM

Normal HDF FGM 129,000 normal HDF 1, 2, and 3

days

E 1:1 coculture ratio Adapted HPAF-II,

normal HDF

FGM 129,000 adapted HPAF-

II, 129,000 normal

HPAF-II

1, 2, and 3

days

F 1:9 cocuture ratio Adapted HPAF-II,

normal HDF

FGM 21,500 adapted HPAF-II,

193,500 normal HDF

1, 2, and 3

days

G 2:3 coculture ratio Adapted HPAF-II,

normal HDF

FGM 86,000 adapted HPAF-II,

129,000 normal HDF

1, 2, and 3

days

H 1:1 coculture, normal

cell lines in FGM

Normal HPAF-II,

normal HDF

FGM 129,000 normal HPAF-II,

129,000 normal HPAF-II

1, 2, and 3

days

Pilot coculture 4 was conducted to assess the behavior and arrangement of control fibroblasts and

adapted HPAF-II cells when cocultured at three different cell number ratios. In this experiment,

FGM-adapted HPAF-II cells were cocultured with control (normal) HDF cells together at three

different ratios, based on literature describing the tumor-stroma ratio in pancreatic ductal

adenocarcinoma.1 Cells were seeded at a 1:1 adapted HPAF-II to HDF cell ratio, 40:60 adapted

HPAF-II to HDF cell ratio, and at a 10:90 adapted HPAF-II to HDF cell ratio.


minutes, at working concentrations of 5 µM. Adapted human dermal fibroblasts were labeled

with Cell Tracker Green CMFDA. Adapted HPAF-II cells were labeled with Cell Tracker Red

CMTPX. Control HPAF-II cells in monoculture were labeled with Cell Tracker CMTPX. Cells

66

were seeded onto 60 mm petri dishes and maintained at 37˚C in an 80% humidified incubator

with 5% CO2. Cells that were cocultured together were seeded at the same time. Cells were

imaged at 1, 2, and 3 days to assess for cellular appearance and arrangement.

Coculture 5:


Group

ID

Description Cell Lines Used Media

Conditions

Number of cells

seeded at Day 0

Timepoints

Assessed

A Control HPAF-II

monoculture in

EMEM

Normal HPAF-II EMEM 129,000 control HPAF-

II

2 and 3 days

B Control HPAF-II

monoculture in FGM

Normal HPAF-II FGM 129,000 control HPAF-

II

2 and 3 days

C Control HDF

monoculture in FGM

Normal HDF FGM 129,000 control HDF 2 and 3 days

D 1:9 cocuture ratio Normal HPAF-II,

normal HDF

FGM 21,500 control HPAF-

II, 193,500 control

HDF

2 and 3 days

E 3:7 coculture ratio Normal HPAF-II,

normal HDF

FGM 86,000 control HPAF-

II, 129,000 control

HDF

2 and 3 days

Pilot coculture 5 was conducted to assess the behavior and arrangement of normal fibroblasts and

normal HPAF-II cells when cocultured in FGM at two different cell number ratios. In this

experiment, normal HPAF-II cells were cocultured with control HDF cells at two different ratios,

based on literature describing the tumor-stroma ratio in pancreatic ductal adenocarcinoma.1

Cells were seeded at a 3:7 HPAF-II to HDF cell ratio, and at a 1:9 HPAF-II to HDF cell ratio.


minutes, at working concentrations of 5 µM. Human dermal fibroblasts were labeled with Cell

Tracker Green CMFDA. HPAF-II cells were labeled with Cell Tracker Red CMTPX. Cells were

seeded onto 60 mm petri dishes and maintained at 37˚C in an 80% humidified incubator with 5%

CO2. Cells that were cocultured together were seeded at the same time. Cells were imaged at 2

and 3 days to assess for cellular appearance and arrangement.

67

Results

Pilot Coculture 1: Initial Coculture of HPAF-II and EMEM-Transitioned HDF in EMEM

In general, the size and morphology of the EMEM-transitioned human dermal fibroblasts

appeared to be different from the control fibroblasts (not included as a control group in this

experiment; see Pilot Coculture 2 for representative images and additional information). The

abnormal appearance of fibroblasts suggests that the media formulation may be causing the

fibroblasts to activate or differentiate; another potential cause may be that the media may be

lacking a component required for fibroblasts to retain their spindle-like phenotype. In coculture,

HPAF-II cells appear able to form small multicellular aggregates in the presence of fibroblasts,

similar to the appearance and aggregation of HPAF-II cells in the control dish (Figure 16 and

Figure 17). The arrangement of fibroblasts and HPAF-II cells in coculture suggest that one or

both cell lines may interact with the other, although a larger density of cells or a longer duration

experiment would be required to assess if any trends in cellular interaction and organization

could be observed. The proliferative capacity of the HPAF-II cells does not appear to be

significantly hindered by the presence of fibroblasts, as suggested by the approximate surface

area occupied by the HPAF-II cells at day 1 versus day 2. However, the presence of large

cellular aggregates (greater than approximately 500 µm in diameter at its shortest axis, Figure

17) does not appear to be seen in coculture at 2 and 4 days.

68

Figure 16: Representative images of cocultured HPAF-II (CMFDA green labeled) and EMEM-HDF (CMTPX

labeled) cells in EMEM (image A), EMEM-HDF monoculture in EMEM (image B), and control HPAF-II

monoculture in EMEM (image C) at Day 2 (10x magnification).

A

C B

100 µm

100 µm 100 µm

69

Figure 17: Representative images of cocultured HPAF-II and EMEM-HDF cells in EMEM (image A), EMEM-HDF

monoculture in EMEM (image B), and control HPAF-II monoculture in EMEM (image C) at Day 4 (10x

magnification).

Pilot Coculture 2: EMEM-HDF and HPAF-II Coculture in EMEM

HDF-EMEM cocultured with HPAF-II cells generally appeared to be evenly distributed

throughout the plate. HDF-EMEM cells in close proximity to HPAF-II cells tended to surround

or appeared to reach out towards the tumor cells, but generally did not appear to overlap the

HPAF-II cells in 2D when in direct contact (Figure 18). The fibroblasts in contact with HPAF-II

cells appeared to exhibit the flattened polygonal phenotype, especially when there was a high

concentration of cells in the area. In lower density areas, fibroblasts occasionally exhibited a

more spindle-like phenotype, but were generally an admixture of the elongated polygonal

A

C B

100 µm

100 µm 100 µm

70

phenotype or the more pronounced flattened polygonal phenotype. HPAF-II cells cocultured

with HDF-EMEM formed aggregates similar to the control HPAF-II plate. At day 2, EMEM-

HDF and HPAF-II cells cocultured in EMEM differed in organization compared to day 1 (Figure

18 and Figure 19). Aggregation of EMEM-HDF cells close to HPAF-II cells was more

pronounced. The arrangement of EMEM-HDF cells around HPAF-II cells suggested that the

positioning of the fibroblasts was likely not random when compared to monocultured EMEM-

HDF. The organization observed at Day 2 suggests that EMEM-HDF cells were potentially

migrating towards HPAF-II cells. Because the EMEM-transitioned fibroblasts underwent a

morphological change during media adaptation, further changes in phenotype from the control

fibroblasts could not be detected.

Morphology of HDF-EMEM in monoculture at 1 and 2 days was markedly different when

compared to the morphology of control human dermal fibroblasts (Figure 18 and Figure 19).

Human dermal fibroblasts adapted to EMEM appeared to consist mostly of two phenotypes –

one that had a pronounced flattened polygonal shape (similar to myofibroblasts), and one that

appeared to be in a transition phase with elongated, polygonal bodies. Occasional spindle-shaped

fibroblasts were observed in areas where cell density was low. HDF-EMEM in monoculture

appeared to be evenly distributed across the surface of the plate at both timepoints and did not

appear to exhibit the same organization of overlapping spindle fibers and cell bodies displayed

by a cluster of normal fibroblasts in close proximity to each other.

Control fibroblasts generally exhibited a more spindle-like morphology, which was expected

from the cell line. Control fibroblasts were also distributed evenly across the plate and had not

yet organized into the expected arrangement of overlapping spindle fibers and cell bodies in

contact to each other, which was expected at the confluence observed at 1 and 2 days. The

71

HPAF-II controls also formed the expected cellular aggregates and were distributed across the

plate (Figure 18 and Figure 19).

Figure 18: Representative images of cocultured HPAF-II (CMTPX red labeled) and EMEM-HDF (CMFDA green

labeled) cells in EMEM (images A and B), EMEM-HDF monoculture in EMEM (image C), control HPAF-II

monoculture in EMEM (image D), and control HDF (CMRA orange labeled) monoculture in FGM (image E) at

Day 1 (10x magnification). Note the difference in appearance of control HDF to EMEM-HDF (image C vs image

E).

A B

C

C D

C

E

C

100 µm 100 µm

100 µm 100 µm

100 µm

72

Figure 19: Representative images of cocultured HPAF-II and EMEM-HDF cells in EMEM (images A and B),

EMEM-HDF monoculture in EMEM (image C), control HPAF-II monoculture in EMEM (image D), and control

HDF monoculture in FGM (image E) at Day 2 (10x magnification). Note the difference in appearance of control

HDF to EMEM-HDF (image C vs image E).

A B

C D

E

100 µm 100 µm

100 µm

100 µm

100 µm

73

Control HDF and Control HPAF-II Coculture in FGM and EMEM

Control HDF cells cocultured with control HPAF-II cells in FGM appeared to be evenly

distributed across the plate at day 1 and 2. Control HDF cocultured with HPAF-II cells in FGM

generally displayed similar morphology to control HDF monocultured in FGM, although there

were some fibroblasts that started to display the pronounced, flattened polygonal shape that was

observed in the HDF-EMEM fibroblasts. Heterogeneity is not unexpected in a fibroblast

population so the presence of these flattened polygonal fibroblasts may not be a result of

interactions with HPAF-II cells.5 HPAF-II cells did not tend to form tight multicellular clusters

observed in control HPAF-II cells, and instead appeared to be dispersed even when in close

proximity to each other. This was also observed with monocultured HPAF-II cells cultured in

FGM, suggesting that the media formulation may be lacking one or more components required

for HPAF-II aggregation. Control HDF cells cocultured with control HPAF-II cells in EMEM

were generally distributed across the plate. Control HDF cells generally displayed the

pronounced, flattened polygonal shape and elongated polygonal bodies displayed by HDF-

EMEM cells, although there were cells that displayed the spindle-like morphology of control

HDF in FGM. The morphology of cocultured HDF cells in this plate was similar to the

morphology of HDF cells monocultured in EMEM. HPAF-II cells also formed aggregates

similar to those seen in the control HPAF-II plate, although they also exhibited a similar

dispersion pattern that was seen with monocultured HPAF-II cells in FGM, and the cocultured

HPAF-II cells in FGM. Representative images are presented in Figure 20 and Figure 21.

74

Figure 20: Representative images of cocultured control HPAF-II (CMTPX red labeled) and control (CMRA orange

labeled) HDF cells in FGM (images A and B), cocultured control HPAF-II and control HDF cells in EMEM

(images C and D), control HDF monoculture in EMEM (image E), and control HPAF-II monoculture in FGM

(image F) at Day 1.

A B

C D

E F

100 µm 100 µm

100 µm

100 µm

100 µm

100 µm

75

Figure 21: Representative images of cocultured control HPAF-II and control HDF cells in FGM (images A and B),

cocultured control HPAF-II and control HDF cells in EMEM (images C and D), control HDF monoculture in

EMEM (image E), and control HPAF-II monoculture in FGM (image F) at Day 2 (10x magnification).

100 µm 100 µm

100 µm

100 µm 100 µm

100 µm

76

Pilot Coculture 3: Cocultured HDF and HPAF-II Cells; 50:50 FGM/EMEM Media Ratio

Representative images from Day 1 are located in Figure 22 and Figure 23. For both seeding

densities, adapted HDF and adapted HPAF-II cells cocultured in 50:50 FGM/EMEM ratio were

generally dispersed evenly throughout the plate, and were observed to be in the early stages of

migration towards other nearby cells at day 1. Adapted fibroblasts in close proximity to other

fibroblasts were occasionally observed to have thin cellular protrusions (similar to the

appearance of filpodia) extending towards nearby cells. Adapted fibroblasts were also observed

to display what appeared to be the leading edge of a more flattened protrusion of the cell body

(similar to the appearance of lamellipodia) in the direction of nearby cells. The observation of

these cellular protrusions were likely associated with fibroblast migration. Adapted fibroblasts

also appeared to surround smaller HPAF-II cells with single or multiple thin protrusions, or what

appeared to be the leading edge of migrating fibroblasts. Small aggregates (less than 10 cells) of

adapted fibroblasts were occasionally observed; this was also observed in the petri dish

containing monocultured adapted HDF cells conditioned to a 1:1 FGM/EMEM ratio.

Qualitatively, the ratio of adapted HDF cells to adapted HPAF-II cells per field of view was

generally similar to the initial seeding density (e.g. 80:20 adapted HDF cells to adapted HPAF-II

cells). There were no other notable differences in cellular arrangement or morphology observed

between the two coculture ratios at day 1.

Adapted fibroblasts in coculture generally displayed normal morphology and arrangement

appropriate to their confluence, which was also similar to the morphology observed in the

control HDF plates. Monocultured adapted HDF cells were also observed to have similar

morphology and arrangement as the control HDF plates. Compared to the HDF control plate at

day 1, the number of adapted fibroblasts in coculture appeared to be greater than the number of

77

control fibroblasts in monoculture. When compared to adapted fibroblasts monocultured in a 1:1

FGM/EMEM media ratio, fibroblasts in coculture appeared to be similar in number. These

observations were not unexpected given that the overall amount of FBS present in a 1:1

FGM/EMEM media ratio is greater than the amount of FBS in FGM alone.

Adapted HPAF-II cells in coculture at day 1 did not form the expected arrangement of tightly-

joined monolayer of cells. Control HPAF-II cells were observed to form tightly-joined clusters of

cells. Adapted HPAF-II cells were dispersed in mostly single cell arrangements generally

observed in the HPAF-II control dish. This observation was similar to the arrangement of cells

observed in the petri dish containing monocultured HPAF-II cells conditioned to a 1:1

FGM/EMEM media ratio. The dispersion of adapted HPAF-II cells may be caused by a

component in FGM that might affect the formation of cellular junctions. Mucus secretion also

appears absent in adapted HPAF-II cells; it is possible that a component in FGM might also be

inhibiting mucus production. Additional experiments would be required to determine if a

component in FGM may be affecting the production of mucins, or the ability of HPAF-II cells to

form a cluster of tightly joined cells. The lack of mucus production did allow for better

visualization of individual HPAF-II cells and their morphology, which is usually difficult to

visualize when HPAF-II cells are in their typical cellular arrangement. Control HPAF-II cells

also tend to be more difficult to visualize using current methods due to the secreted mucus layer.

HPAF-II migration could not be assessed using the methods in this experiment. There were no

other notable differences in cellular arrangement or morphology observed between the two

coculture ratios at day 1.

78

Figure 22: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and HPAF-II (CMTPX red

labeled); 60:40 ratio, 10X magnification (images A and B). Both cell lines were adapted to a 1:1 FGM:EMEM

media ratio. Representative images from HPAF-II cells adapted to a 1:1 FGM:EMEM ratio (CMTPX red labeled,

image C), control HPAF-II cells (CMTPX red labeled, image D), HDF cells adapted to a 1:1 FGM:EMEM media

ratio (CMFDA green labeled, image E), and control HDF (CMTPX red labeled, image F).

A B

=

=

A

C

=

A

D

E F

100 µm

100 µm

100 µm

100 µm

100 µm 100 µm

79

Figure 23: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and HPAF-II (CMTPX red labeled); 80:20

ratio, 10X magnification (images A and B). Both cell lines were adapted to a 1:1 FGM:EMEM media ratio. Representative

images from HPAF-II cells adapted to a 1:1 FGM:EMEM ratio (CMTPX red labeled, image C), control HPAF-II cells (CMTPX

red labeled, image D), HDF cells adapted to a 1:1 FGM:EMEM media ratio (CMFDA green labeled, image E), and control HDF

(CMTPX red labeled, image F).

Representative images from days 4-7 are located in Figure 24, Figure 25, Figure 26, Figure 27,

Figure 28, Figure 29, Figure 30, Figure 31. At 4-7 days, cocultured adapted fibroblasts and

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

80

adapted HPAF-II cells were similar in morphology, arrangement, and aggregation when

compared to day 1, with a higher density of cells per field of view. The arrangement of some of

the adapted HDF and adapted HPAF-II cells suggested that there was some overlapping of cells

despite the lack of an initial extracellular matrix. It is possible that collagen secretion by

fibroblasts allowed for localized areas of 3D arrangement for both adapted HDF cells and HPAF-

II cells. Qualitatively, there appeared to be more adapted fibroblasts with a rounded shape

compared to the elongated shape typically observed in monocultured control fibroblasts. Dye

intensity across all adapted fibroblasts in the plate was variable, which is likely due to dye

dilution with each division of the initially dyed cells. Similar to day 1, the number of adapted

fibroblasts in coculture and monoculture both appeared to be greater compared to control

fibroblasts. These observations were not unexpected given that the overall amount of FBS

present in a 1:1 FGM/EMEM media ratio is greater than the amount of FBS in FGM alone. The

morphology of cocultured and monocultured adapted fibroblasts were similar to control

fibroblasts, although there appeared to be more fibroblasts with a more rounded body in

coculture compared to monocultured control fibroblasts.

Cocultured adapted HPAF-II cells continued to display the same dispersed, mostly single-cell

arrangements at 4-7 days. An occasional cluster of 2-3 HPAF-II cells encapsulated by a mucus

layer was seen but was a very rare observation. Adapted HPAF-II cells typically did not form the

same extent of tightly joined cell clusters seen in control HPAF-II cells at any timepoint.

Monocultured adapted HPAF-II cells also continued to display the same dispersed, mostly

single-cell arrangements. At low to medium densities, the arrangement of adapted HPAF-II cells

appeared to follow a circular pattern, with individual cells surrounding round empty spaces.

Monocultured control HPAF-II cells at days 4-7 continued to proliferate and form the expected

81

arrangements of tightly-joined cell clusters. HPAF-II migration could not be assessed using the

methods in this experiment.

The CMTPX red dye was used to label control fibroblasts in this experiment. In comparison to

the CMFDA green dye, the CMTPX red dye was more difficult to visualize at all time points

using the identified imaging system. At day 7, the dye was very faint and difficult to image using

the TRITC filter. The CMFDA green dye was also difficult to image at day 7, but still provided

adequate visualization of the fibroblasts. Subsequent experiments used the CMFDA green dye to

stain control and adapted fibroblasts. Additional optimization is required to determine the

concentration and incubation time needed to achieve adequate visualization of fibroblasts for

experiments with timepoints greater than 6 days.

82





ratio (CMFDA green labeled, image E), and control HDF cells (CMTPX red labeled, image F).

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

83





ratio (CMFDA green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that brightness

and contrast was adjusted for better visualization; original can be provided upon request).

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

84







A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

85





ratio (CMFDA green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that image was

taken with FITC/TRITC/EOSIN filters applied in an attempt to visualize cells. Brightness and contrast was adjusted

for better visualization; original can be provided upon request).

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm

100 µm

100 µm

100 µm

86





ratio (CMFDA green labeled, image E), and control HDF cells (CMTPX red labeled, image F).

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

87







A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm

100 µm 100 µm

100 µm

88







A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

89





ratio (CMFDA green labeled, image E), and control HDF cells (CMTPX red labeled, image F. Note that image was

taken with FITC/TRITC/EOSIN filters applied in an attempt to visualize cells. Brightness and contrast was adjusted

for better visualization; original can be provided upon request).

A B

=

=

A

C

=

A

D

E F

100 µm 100 µm

100 µm 100 µm

100 µm 100 µm

90

Pilot Coculture 4: Cocultured HDF and Adapted HPAF-II Cells in FGM

Similar to previous experiments, fibroblasts and adapted HPAF-II cells cocultured in FGM were

generally dispersed evenly throughout the plate at Day 1; fibroblasts were observed to be in the

early stages of migration towards other nearby cells. Fibroblasts initially seeded at a 1:9 adapted

HPAF-II to HDF cell ratio were observed to have more instances of large aggregates (greater

than 10 cells) similar to that observed with higher densities of monocultured normal fibroblasts,

likely a result of a higher number of fibroblasts seeded at day 0. Fibroblasts seeded at a 2:3

adapted HPAF-II to HDF cell ratio and at a 1:1 adapted HPAF-II to HDF cell ratio were

generally observed to be in the early stages of migration; large aggregates of fibroblasts were

infrequently observed in the plate. Similar to Coculture 3, fibroblasts in close proximity to other

fibroblasts were occasionally observed to have thin cellular protrusions (similar to the

appearance of filipodia) extending towards nearby cells. Fibroblasts were also observed to

display what appeared to be the leading edge of a more flattened protrusion of the cell body

(similar to the appearance of lamellipodia) in the direction of nearby cells. The observation of

these cellular protrusions were likely associated with fibroblast migration. Fibroblasts also

appeared to surround smaller HPAF-II cells with single or multiple thin protrusions, or what

appeared to be the leading edge of migrating fibroblasts.

Aggregates of fibroblasts were also observed in the control plate containing monocultured HDF.

Qualitatively, the ratio of HDF cells to adapted HPAF-II cells per field of view was generally

similar to the initial seeding density. There were no other notable differences in cellular

arrangement or morphology observed among the three coculture ratios at day 1. Fibroblasts in

coculture generally displayed normal morphology and arrangement, which was also similar to

the morphology observed in the control HDF plates. Compared to the HDF control plate at day 1,

91

the number of fibroblasts in coculture appeared to be similar to the control fibroblasts in

monoculture when the initial number of cells were similar.

Adapted HPAF-II cells in coculture at day 1 did not form the expected arrangement of tightly-

joined cell clusters usually observed in control HPAF-II cells, similar to the observations made

in the previous experiment. Adapted HPAF-II cells were dispersed in mostly single cell

arrangements instead of forming the multicellular, mucus-secreting clusters generally observed

in the HPAF-II control dish. This observation was similar to the arrangement of cells observed in

the petri dish containing monocultured HPAF-II cells adapted to FGM. This behavior observed

in HPAF-II cells may be caused by a component in FGM that might affect the formation of

cellular junctions. Mucus secretion also appears absent in adapted HPAF-II cells; it is possible

that a component in FGM might also be inhibiting mucus production. Additional experiments

would be required to determine if a component in FGM may be affecting the production of

mucins, or the ability of HPAF-II cells to form cell junctions. HPAF-II migration could not be

assessed using the methods in this experiment. There were no other notable differences in

cellular arrangement or morphology observed between the two coculture ratios at day 1.

Representative images are found in Figure 32, Figure 33, Figure 34.

92

Figure 32: Day 1 – representative images of cocultured HDF (CMFDA green labeled) and adapted HPAF-II

(CMTPX red labeled) in FGM; 1:9 adapted HPAF-II to HDF cell ratio, 10X magnification (images A and B).

Representative images from adapted HPAF-II cells in FGM (CMTPX red labeled, image C), control HPAF-II cells

in EMEM (CMTPX red labeled, image D), and control HDF cells in FGM (CMFDA green labeled, image E).

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(CMTPX red labeled) in FGM; 2:3 ratio, 10X magnification (images A and B). Representative images from adapted

HPAF-II cells in FGM (CMTPX red labeled, image C), control HPAF-II cells in EMEM (CMTPX red labeled,

image D), control HPAF-II cells in FGM (CMTPX red labeled image E), and control HDF cells in FGM (CMFDA

green labeled, image E).

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in EMEM (CMTPX red labeled, image D), control HPAF-II cells in FGM (CMTPX red labeled image E), and

control HDF cells in FGM (CMFDA green labeled, image E).

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At days 2 and 3, fibroblasts showed more pronounced migration towards other nearby cells or

clusters of nearby cells. Fibroblasts initially seeded at a 1:9 adapted HPAF-II to HDF cell ratio

formed more aggregates across the plate compared to the other plates with different cell ratios;

this observation is similar to normal fibroblasts in monoculture approaching confluence. The

higher number of fibroblasts at days 2 and 3 is likely due to a higher number of fibroblasts

initially seeded at day 0. Fibroblasts seeded at a 2:3 adapted HPAF-II to HDF cell ratio also

displayed areas of typical fibroblast arrangement to a lesser degree when compared to the plates

seeded at a 1:9 adapted HPAF-II to HDF ratio. Fibroblasts seeded at a 1:1 adapted HPAF-II to

HDF cell ratio were generally observed to be in the early stages of migration (similar to day 1),

with very few large aggregates of fibroblasts. Aggregates of fibroblasts were also observed in the

control plate containing monocultured HDF. Fibroblasts in coculture generally displayed normal

morphology and arrangement appropriate to their confluence, which similar to the control HDF

plates. Compared to the HDF control plates at their respective timepoints, the number of

fibroblasts appeared to be similar to the control fibroblasts in monoculture when the initial

number of cells were similar.

Cocultured adapted HPAF-II cells continued to display the same dispersed, mostly single-cell

arrangements at 2 days. An occasional cluster of 2-3 HPAF-II cells encapsulated by a mucus

layer was seen but was a very rare observation. Monocultured adapted HPAF-II cells also

continued to display the same dispersed, mostly single-cell arrangements. At low to medium

densities, the arrangement of adapted HPAF-II cells continued to follow a circular pattern, with

individual cells surrounding round empty spaces. This was also observed in Coculture 3.

Monocultured control HPAF-II cells at days 2 continued to proliferate and form the expected

arrangements of tightly-joined cluster of cells encapsulated by a mucus layer. HPAF-II migration

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could not be assessed using the methods in this experiment. Representative images are found in

Figure 35, Figure 36, Figure 37, Figure 38, Figure 39 and Figure 40.






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(CMTPX red labeled) in FGM; 1:1 ratio, 10X magnification (images A and B). Representative images from adapted



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Cocultured HDF and non-adapted HPAF-II Cells in FGM

At day 1, fibroblasts and normal HPAF-II cells cocultured in FGM were generally dispersed

evenly throughout the plate. Fibroblasts were observed to be in the early stages of migration

towards other nearby cells. Fibroblasts in close proximity to a cluster of HPAF-II cells were

occasionally observed to have thin cellular protrusions (similar to the appearance of filpodia)

extending towards the cluster, or what appeared to be the leading edge of a more flattened

protrusion of the cell body (similar to the appearance of lamellipodia) in the direction of nearby

cells. Fibroblasts also appeared to stretch around HPAF-II cell clusters. Small aggregates (less

than 10 cells) of adapted fibroblasts were observed occasionally throughout the plate; this was

also observed in the control petri dish containing monocultured HDF cells. Fibroblasts in

coculture generally displayed normal morphology and arrangement, which was also similar to

the morphology observed in the control HDF plates.

Normal HPAF-II cells in coculture at day 1 were observed to have a combination of single cells

and small clusters of cells surrounded by a mucus layer. HPAF-II cell bodies generally appeared

opacified, likely due to mucus secretions. Large clusters of HPAF-II cells were not observed in

coculture, which is expected at day 1. Normal HPAF-II cells monocultured in FGM consisted of

single and small clusters of cells surrounded by mucus, similar to HPAF-II cells cocultured in

FGM; it was noted that clusters of HPAF-II cells appeared loosely connected when compared to

clusters of cells in EMEM. Control HPAF-II cells cultured in EMEM consisted of single cells

and clusters of cells surrounded by mucus. Clusters of control HPAF-II cells in EMEM appear

tightly joined when compared to HPAF-II cells in FGM. Representative images are found in

Figure 41.

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Figure 41: Day 1 – representative images of cocultured control HDF (CMFDA green labeled) and control HPAF-II

(CMTPX red labeled) in FGM; 1:1 normal HPAF-II to HDF cell ratio, 10X magnification (images A and B).

Fibroblasts appear to surround cluster(s) of HPAF-II cells (Image A, arrows). Representative images from normal



green labeled, image E). Clusters of HPAF-II cells in FGM appear to be loosely connected, as if the cells were

detaching from neighboring cells (Image C, arrow). Clusters of HPAF-II cells in EMEM appear to be more tightly

connected (Image D arrow).

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At day 2, fibroblasts displayed more pronounced aggregates with nearby cells. Larger aggregates

were observed more frequently throughout the plate; this was also observed in the control petri

dish containing monocultured control HDF cells. Fibroblasts in coculture generally displayed

normal morphology and arrangement, which was also similar to the morphology observed in the

control HDF plates. Normal HPAF-II cells in coculture at day 2 were observed to consist mostly

of single cells and few small clusters of cells surrounded by a mucus layer. The opacity of single

cells depended on the amount of mucus surrounding individual cells. Clusters of HPAF-II cell

bodies generally appeared opacified. Large aggregates of HPAF-II cells were not observed in

coculture.

Normal proliferating HPAF-II cells in EMEM form a continuous monolayer surrounded by

mucus, which was observed in the control HPAF-II plate at day 2. Normal HPAF-II cells

monocultured in FGM consisted of single and small clusters of cells surrounded by mucus,

similar to HPAF-II cells cocultured in FGM. At day 2, the dispersion of HPAF-II cell clusters

was more pronounced. Representative images are found in Figure 42.

105



Representative images from normal HPAF-II cells in FGM (CMTPX red labeled, image C), control HPAF-II cells in

EMEM (CMTPX red labeled, image D), control HPAF-II cells in FGM (CMTPX red labeled image E), and control

HDF cells in FGM (CMFDA green labeled, image E).

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At day 3, fibroblast aggregation was more pronounced, which is typical at higher densities.

Larger aggregates were throughout the plate, similar to the aggregates observed in the control

petri dish containing monocultured control HDF cells. Fibroblasts in coculture generally

displayed normal morphology and arrangement, which was also similar to the morphology

observed in the control HDF plates. Fibroblast aggregation appeared to be higher around areas

containing a higher density of HPAF-II cells. Normal HPAF-II cells in coculture at day 3 were

observed to consist primarily of single cells. Small clusters of cells surrounded by a mucus layer

was rarely observed. Large clusters of HPAF-II cells were not observed in coculture.

Control HPAF-II cells in EMEM continued to proliferate and form a continuous monolayer

surrounded by mucus, which was observed in the control HPAF-II plate at day 3. Normal HPAF-

II cells monocultured in FGM consisted primarily of single cells, and infrequently small clusters

of 2-3 cells, similar to HPAF-II cells cocultured in FGM. HPAF-II cells with different

morphologies were observed more frequently throughout the plate. Representative images are

found in Figure 43.

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Figure 43: Day 3 – representative images of cocultured HDF (CMFDA green labeled) and control HPAF-II


Fibroblast aggregation appeared to be higher around areas containing a higher density of HPAF-II cells (Figure A,

arrow). Representative images from normal HPAF-II cells in FGM (CMTPX red labeled, image C), control HPAF-

II cells in EMEM (CMTPX red labeled, image D), control HPAF-II cells in FGM (CMTPX red labeled image E),

and control HDF cells in FGM (CMFDA green labeled, image E). HPAF-II cells with different morphologies were

also observed throughout the plate (Figure C, arrows).

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Pilot Coculture 5: Cocultured HDF and HPAF-II Cells in FGM

At days 2 and 3, fibroblasts and HPAF-II cells cocultured in FGM were generally dispersed

evenly throughout the plate. Fibroblasts were observed to be in the early stages of migration

towards other nearby cells. Fibroblasts in close proximity to other fibroblasts were occasionally

observed to have thin cellular protrusions (similar to the appearance of filpodia) extending

towards nearby cells. Fibroblasts were also observed to display what appeared to be the leading

edge of a more flattened protrusion of the cell body (similar to the appearance of lamellipodia) in

the direction of nearby cells. The observation of these cellular protrusions were likely associated

with fibroblast migration. Fibroblasts also appeared to surround smaller HPAF-II cells with

single or multiple thin protrusions, or what appeared to be the leading edge of migrating

fibroblasts. Aggregates of fibroblasts were observed; this was also observed in the control petri

dish containing monocultured HDF cells. Qualitatively, the number of fibroblasts throughout the

coculture plates and the control HDF plate seemed lower at day 2 compared to the previous

experiment. Fibroblasts in coculture also appeared to be generally larger than the control

fibroblasts.

Fibroblasts in coculture generally displayed normal morphology and arrangement, which was

also similar to the morphology observed in the control HDF plates (Figures 23E and 24E).

Compared to the HDF control plate at day 1, the number of fibroblasts in coculture appeared to

be greater than the number of control fibroblasts in monoculture. This observation is somewhat

unexpected given that there did not appear to be a difference between monocultured HDF in

FGM and cocultured normal HDF in FGM from pilot experiment 4. The control plates also

appeared to have a lower density of fibroblasts compared to control plates from previous

experiments. Fibroblasts in the control plate also appeared to be larger in comparison to

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fibroblasts from previous experiments when compared at the same timepoint. The fibroblasts

used in this experiment were from passage 8, which might be associated with a decreased growth

rate compared to the growth rate observed in previous passages.

At day 2, normal HPAF-II cells cocultured with HDF cells in FGM consisted primarily of small,

loosely connected clusters of HPAF-II cells and single cells. At day 3, HPAF-II cells consisted

primarily of single cells, with occasional loose clusters of HPAF-II cells. Both observations were

similar to what was observed in plates with cocultured normal HPAF-II cells in the previous

experiment. This was also observed in plates with monocultured HPAF-II cells in FGM at days 2

and 3. Control HPAF-II cells in EMEM formed the expected clusters of tightly-connected cells

surrounded by a mucus layer at days 2 and 3. Representative images are found in Figure 44,

Figure 45, Figure 46, and Figure 47.

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(CMTPX red labeled) in FGM; 1:9 HPAF-II to HDF cell ratio, 10X magnification (images A and B). Representative

images from control HPAF-II cells in EMEM (CMTPX red labeled, image C), control HPAF-II cells in FGM

(CMTPX red labeled image D), and control HDF cells in FGM (CMFDA green labeled, image E).

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Discussion

Results from these early pilot coculture experiments suggest that human dermal fibroblasts could

be cocultured with HPAF-II cells in different media conditions. Ascorbic acid is a required

supplement for normal fibroblast function and should be present at adequate levels for fibroblasts

to maintain normal morphology and collagen production.6 Early experiments with fibroblasts

cultured in EMEM resulted in fibroblasts with altered morphology, likely due to ascorbic acid

deficiency. Fibroblasts cultured in a 1:1 EMEM (supplemented with 100 µM ascorbic acid) to

FGM ratio appeared to retain normal morphology and increased proliferation when compared to

control fibroblasts cultured in FGM at the same timepoint. Increased proliferation could be

caused by the increased amount of FBS present in the combined media. The media used to

support fibroblast growth contained 3% FBS, and EMEM used to support HPAF-II growth was

supplemented with 10% FBS. Fibroblasts also potentially produced collagen based on

observations suggesting overlapped cells at days 4-7.

In comparison, HPAF-II cell aggregation and morphology were altered when cultured in

different ratios of FGM. Normal HPAF-II cells form a monolayer of tightly attached cells. After

exposure to FGM, HPAF-II cells appeared to detach from each other, and disperse across the

plate. This was observed during the adaptation process at a 1:3 FGM to EMEM ratio and

continued to be observed in experiments with adapted HPAF-II cells, or HPAF-II cells

introduced to FGM without adaptation. This alteration in morphology and arrangement may

potentially be due to components in FGM that might affect the ability of the HPAF-II cells to

form appropriate cellular junctions. Despite the behavior and morphology of HPAF-II cells in

FGM, future experiments will likely focus on using a 1:1 EMEM supplemented with 100 µM

ascorbic acid to FGM ratio. This combination would contain necessary components required to

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maintain fibroblast function and morphology and allow HPAF-II cells to survive and proliferate

despite their altered morphology and behavior.

One observation that requires additional assessment was that fibroblast doubling time seemed to

increase after passage 6, which was observed during experiments and during general cell

maintenance. In Coculture 5, fibroblasts used in the experiment were at passage 8. In this

experiment, the number of control fibroblasts observed across the plate at days 2 and 3 appeared

to be lower compared to previous experiments. The same number of fibroblasts were seeded onto

a control plate in both experiments. Control fibroblasts in Coculture 5 also appeared to have a

lower number of fibroblasts than expected at days 2 and 3 when the entire plate was assessed.

Discussions with a Cell Applications representative suggested that fibroblast proliferation would

decrease as the passage number approached 10, which seems to be supported by the observations

made in recent experiments and during general cell maintenance.

Future Directions

Currently, planned future work would focus on quantifying the proliferation rate of fibroblasts

and HPAF-II cells both in monoculture and coculture. Replicate experiments should be

conducted once the method to quantify cells in coculture and monoculture has been optimized to

determine the reproducibility of the results. A few methods that may be feasible for counting

cocultured cells in 2D include flow assisted cell sorting and image analysis. Dye optimization for

timepoints longer than 4 days would also be required to adequately visualize cells in coculture.

An additional factor to consider is the molecular weight of the dyes used to label cells in

coculture. Assessment of the molecular weights of CMFDA green and CMTPX red and their

effect on migration and proliferation rates of both cell lines might be something to consider in

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future experiments. Once successful methods have been developed and consistent results are

achieved, experiments evaluating the impact of various cancer treatment modalities can be

conducted to determine their effect on HPAF-II cells in monoculture and coculture. Use of

pancreatic stellate cells instead of human dermal fibroblasts would also be another avenue to

pursue in future experiments.

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References

1. American cancer society

(https://cancerstatisticscenter.cancer.org/?_ga=2.263305860.905660924.1619448287-

877799850.1619448287#!/) Access 26 April 2021

2. Gupta, Rohan et al. “Current and future therapies for advanced pancreatic cancer.”

Journal of surgical oncology vol. 116,1 (2017): 25-34. doi:10.1002/jso.24623

3. Leppänen, Joni et al. “Tenascin C, Fibronectin, and Tumor-Stroma Ratio in Pancreatic

Ductal Adenocarcinoma.” Pancreas vol. 48,1 (2019): 43-48.

doi:10.1097/MPA.0000000000001195

4. Rajasekaran et al. “HPAF-II, a Cell Culture Model to Study Pancreatic Epithelial Cell

Structure and Function.” Pancreas, Volume 29, Issue 3, pages 77-83. October 2004.

5. Lynch and Watt. “Fibroblast heterogeneity: implications for human disease.” Journal of

Clinical Investigation, Volume 128(1): pages 26-35. January 2, 2018.

6. Schafer, I A et al. “Ascorbic acid deficiency in cultured human fibroblasts.” The Journal

of cell biology vol. 34,1 (1967): 83-95. doi:10.1083/jcb.34.1.83

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