i

Skeletal Muscle Myogenesis in C2C12 Self-Assembled 3D Microtissues

By

Gianna Prata Ahnrud

B.S. & B.A., University of Rhode Island, 2013

Thesis

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

of Science in the Department of Molecular Pharmacology, Physiology and

Biotechnology, and the Center of Biomedical Engineering at Brown

University

PROVIDENCE, RHODE ISLAND

MAY 2019

ii

AUTHORIZATION TO LEND AND REPRODUCE THIS THESIS

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

institutions or individuals for the purpose of scholarly research.

Date: _______________ Signature: ____________________________________

Gianna Prata Ahnrud, 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: _______________ Signature: ____________________________________

Gianna Prata Ahnrud, Author

iii

This thesis by Gianna Prata Ahnrud is accepted in its present form by the Department of

Molecular Pharmacology, Physiology, and Biotechnology, and the Center of Biomedical

Engineering as satisfying the thesis requirements for the degree of Master of Science

Date: _______________ Signature: ____________________________________

Dr. Jeffrey Morgan, Adviser

Date: _______________ Signature: ____________________________________

Dr. Jacquelyn Schell, Reader

Date: _______________ Signature: ____________________________________

Dr. Kim Boekelheide, Reader

Approved by the Graduate Council

Date: _______________ Signature: ____________________________________

Dr. Andrew G. Campbell, Dean of the Graduate School

iv

Vita

Gianna Prata Ahnrud graduated from The University of Rhode Island in 2013 after

completing her Bachelor of Arts in Biology and Bachelor of Science in Secondary

Education. She has since been employed as a Biological Sciences Technician at the

Combat Capabilities Development Command Soldier Center. There, she applied for and

received the DoD SMART Scholarship for Service Award to attend Brown University in

pursuit of a ScM. Biotechnology where she researched skeletal muscle myogenesis in 3D

microtissues in Dr. Jeffrey Morgan’s Lab. After graduation she will return to the Combat

Capabilities Development Command Soldier Center and be promoted to the role of

Research Biologist.

v

Acknowledgements

Thank you to my husband Eric for always being my partner in life and supporting my

dream to complete a Master of Science degree. Thank you to my grandfather Carl for

always supporting my educational goals and fostering a work ethic and desire to pursue

learning from the time I was very young. Thank you to my sisters Ashleigh, Marissa,

Rebecca and Teresa for always being there for me no matter what.

Next, I would like to thank all the members of the Morgan Lab at Brown University, past

and present, for all their guidance and support and providing a truly collaborative lab

environment to work in. Thank you to Dr. Jeffrey Morgan, Dr, Jacqueline Schell, and Dr.

Kim Boekelheide for your guidance in my thesis work and challenging me to develop the

skills necessary to be a successful research scientist.

Thank you to the DoD SMART Scholarship for Service, Ken Racicot, and Joshua

Magnone for funding my research efforts in this Master of Science degree. Finally, thank

you to Dr. Kevin O’Fallon and Patrick Marek for your continuous mentorship over the

years.

vi

Table of Contents

Chapter 1: Background ................................................................................................................................. 2

1.1 The Skeletal Muscle System: Overview & Medical Relevance ....................................................... 2-7

1.2 Current Research & Development Tools for Studying DMD, ALS, & Exercise Induced Damage 7-9

1.3 Comparison: 2D Skeletal Muscle Research Tools vs. 3D Technological Advancements ............. 9-13

1.4 Myogenesis in 2D Using the C2C12 Skeletal Muscle Cell Line ................................................. 14-18

Chapter 2: Materials & Methods ................................................................................................................. 20

2.1 Cell Culture ....................................................................................................................................... 20

2.2 Micromold Specifications & Formation of Non-Adherent Agarose Molds................................. 20-21

2.3 Microtissue Formation ...................................................................................................................... 22

2.4 Microtissue Formation Validation ............................................................................................... 22-23

2.5 Immunofluorescent Staining: Myogenesis 2D ............................................................................. 23-24

2.6 Immunofluorescent Staining: Myogenesis in Spheroids and Toroids (3D) ................................ 24-26

2.7 Confocal Imaging Parameters ................................................................................................... 26-27

2.8 Confocal Image Analysis ................................................................................................................ 27

2.9 H&E Staining .............................................................................................................................. 27-28

Chapter 3: Microtissue Formation & Investigation of Myogenesis in 3D Spheroids ................................. 29

3.1 C2C12 Myogenesis in 2D ............................................................................................................ 29-32

3.2 C2C12 Microtissue Formation (Spheroids) ................................................................................. 33-36

3.3 C2C12 Investigation of Myogenesis in 3D Microtissues (Spheroids) ........................................ 37-39

Chapter 4: Microtissue Formation & Investigation of Myogenesis in 3D Toroids .................................. 40

4.1 C2C12 Microtissue Formation (Toroids) ................................................................................... 40-49

4.2 C2C12 Investigation of Myogenesis in 3D Microtissues (Toroids) .......................................... 49-59

4.3 H&E Staining Demonstrates Morphology in C2C12 3D Toroid Microtissues ......................... 60-62

Chapter 5: Conclusions & Future Directions ............................................................................................ 62

5.1 Conclusions .................................................................................................................................. 62-63

5.2 Future Directions: Biological ...................................................................................................... 63-65

5.3 Future Directions: Technology .................................................................................................. 65-66

References ............................................................................................................................................ 67-71

Appendix 1&2: Harmony Analysis, H&E and Paraffin Embedding Protocols ................................... 72-76

vii

Figure 1: Schematic skeletal muscle tissue. ................................................................................. 4

Figure 2: Schematic overview of skeletal muscle cell ................................................................. 4

Figure 3: Musculoskeletal injuries dominate injuries in military personnel ................................ 7

Figure 4: Technologies for 3D skeletal muscle cell culture ....................................................... 11

Figure 5: Morgan Lab 3D microtissue platforms ....................................................................... 12

Figure 6: Myogenesis overview .........................................................................................14

Figure 7: 2D C2C12 cell culture time line. ................................................................................ 24

Figure 8: 2D MHC positive cells-graph ..................................................................................... 31

Figure 9: C2C12 Myogenesis 2D ............................................................................................ 32

Figure 10: C2C12 spheroid microtissue formation by media formulation. ............................... 35

Figure 11: C2C12 spheroid microtissue formation by seeding density ..................................... 36

Figure 12: C2C12 Myogenesis 3D-Spheroids .......................................................................... 38

Figure 13: C2C12 toroid microtissue formation (GM). ............................................................. 41

Figure 14: C2C12 toroid microtissue formation (DM). ............................................................. 42

Figure 15: C2C12 toroid microtissue stability (4 medias) ......................................................... 44

Figure 16: C2C12 toroid microtissue survival curve (4 medias) .............................................. 46

Figure 17: C2C12 toroid microtissues (SFMA) ......................................................................... 47

Figure 18: C2C12 toroid microtissue survival curve (14 days) ................................................ 51

Figure 19: Harmony analysis schematic (MHC volume/nuclei). ............................................... 52

Figure 20: MHC volume/nuclei over time-graph ....................................................................... 53

Figure 21: MHC production over time ..................................................................................... 54

Figure 22: Harmony analysis schematic (multinucleation) ...................................................... 55

Figure 23: Cellular multinucleation over time .......................................................................... 56

Figure 24: Nuclei quantities and distribution through microtissues .......................................... 59

Figure 25: H&E staining demonstrates microtissue morphology ............................................. 61

viii

ix

Table 1: Comparison of skeletal, cardiac and smooth muscle characteristics. ............................ 3

Table 2: Overview of 3D cell culture methods .......................................................................... 10

Table 3: Summary of in vitro and in vivo models for DMD, ALS, EIMD, and traumatic skeletal

muscle injury ...................................................................................................................... 17-18

Table 4: Immunofluorescent staining protocol for C2C12 microtissues ................................... 24

Table 5 Confocal imaging acquisition parameters: .................................................................... 27

Table 6: Media composition for C2C12 2D and 3D cell culture ............................................30

1

Abstract of Skeletal Muscle Myogenesis in C2C12 Self-Assembled 3D Microtissues, by

Gianna Prata Ahnrud, ScM. Brown University, May 2019.

Skeletal muscle in vitro models have traditionally utilized mammalian cell lines isolated from rat

or mouse skeletal muscles. These models have been extensively validated in traditional 2D cell

culture to demonstrate the process of normal skeletal muscle growth and development called

myogenesis. This process consists of single nucleated myoblast cells proliferating, lining up and

fusing to form multinucleated myotubes, the cells that make up adult skeletal muscle fibers. In

vitro models have begun transitioning from 2D platforms to 3D microenvironments that better

recapitulate the in vivo environment due to their ability to promote cell-cell contact, enhanced

ECM production, and limited interaction between the cells and a stiff culturing substrate. The

process of skeletal muscle myogenesis in a 3D platform is less understood and requires

investigation. Developing and understanding of myogenesis in a 3D self-assembled platform will

help inform future investigations related to skeletal muscle regeneration after injury and skeletal

muscle disease and associated interventions.

Ability of the C2C12 cell line to form self-assembled microtissues in the non-adhesive

micromolds was assessed by brightfield microscopy. Investigation of myogenesis in the self-

assembled microtissues was demonstrated by fluorescent antibody labeling of myosin heavy

chain protein and quantitated using 3D confocal microscopy analysis. The C2C12 cell line

formed stable self-assembled 3D toroids when maintained in a serum free advanced media

formulation (SFMA) and demonstrated signs of myogenesis at days 10 and 14 of growth.

2

Chapter 1

Background

1.1 The Skeletal Muscle System: Overview & Medical Relevance

The human skeletal system is comprised of the bones, muscles, tendons, and ligaments

that, when all working together, provide the body with support, joint stabilization and ability to

move. Skeletal muscle tissue makes up 40% of the entire body mass and is the muscle that is

most responsible for generating heat in the body[1]. This muscle type covering the skeleton is

crucial to providing movement and contains specific characteristics that are unique compared to

smooth muscle and cardiac tissue One of the main differences of skeletal muscle compared to

smooth and cardiac muscle is the fact that skeletal muscle is voluntary and subject to conscious

control[1]. Skeletal muscle tissue is striated in which individual muscle fiber cells are long and

have the ability to shorten and contract or extend and stretch when in a relaxed state[1].

Skeletal muscle tissue is comprised of mainly skeletal muscle cells (fibers), but also

contains blood vessels, nerve fibers, and connective tissue that anchor the muscle tissue to bone

Within each multinucleated muscle fiber or cell are thousands of myofibrils which are rod

shaped organelles that live under a surface lining called the sarcolemma and play an important

role for contraction to occur. The myofibrils are composed of two types of myofilaments, a thick

and thin filament that when stimulated by an action potential from the nervous system along the

sarcolemma surface, slide past each other to create muscle shortening or contraction[1]. The

thick filaments contain myosin molecules or bundled myosin heads which attach to the fibrous f

actin sites on the thin filaments, forming a cross bridge between the two which generates tension

and allows the muscle to contract[1] (Fig 2).

3

Table 1. Comparison of skeletal, cardiac and smooth muscle characteristics. Adapted from E.

Marieb Human Anatomy & Physiology, 6th Edition.

Characteristic Skeletal Cardiac Smooth

Body location Attached to bone or

(some facial muscles)

to skin

Walls of heart Single-unit muscle in

walls of hollow visceral

organs (other than

heart); multiunit muscle

in intrinsic eye muscles

Cell shape &

appearance

Single, very long,

cylindrical,

multinucleated cells

with obvious striations

Branching chains of

cells; uni-or binucleate;

striations

Single, fusiform,

uninucleate; no

striations

Connective tissue

components

Epimysium,

perimysium and

endomysium

Endomysium attached

to fibrous skeleton of

the heart

Endomysium

Presence of gap

junctions

No Yes; at intercalated

discs

Yes; in single-unit

muscle

Regulation of

contraction

Voluntary via axonal

endings of the somatic

nervous system

Involuntary; intrinsic

system regulation; also,

autonomic nervous

system controls;

hormones; stretch

Involuntary; autonomic

nerves, hormones, local

chemicals; stretch

Speed of contraction Slow to fast Slow Very slow

Rhythmic contraction No Yes Yes, in single-unit

muscle

Response to stretch Contractile stretch

increases with degree

of stretch (to a point)

Contractile strength

increases with degree

of stretch

Stress-Relaxation

response

Respiration Aerobic and anaerobic Aerobic Mainly aerobic

4

Figure 1. (a) Schematic of skeletal muscle tissue. (b) Histological cross section of skeletal

muscle tissue (90x). Adapted from E. Marieb Human Anatomy & Physiology, 6th

Edition.[1].

Fig 1a: (a) Schematic skeletal muscle tissue. (b) Histological cross section of skeletal

muscle tissue (90x) [1].

Figure 2. Schematic overview of skeletal muscle cell. Adapted from E. Marieb

Human Anatomy & Physiology, 6th Edition [1].

5

Research related to the skeletal muscle system are very important specifically to the

medical community in order to further investigate treatments for musculoskeletal disorders

including Duchenne’s muscular dystrophy (DMD) and amyotrophic lateral sclerosis (ALS). The

skeletal muscle system also plays a crucial role in military driven research to investigate skeletal

muscle damage and repair and associated nutritional supplementation mitigation strategies as a

result of exercise induced damage or traumatic injury in the soldier population.

It is estimated that approximately 1 in every 3,500 to 6,000 male babies are born with

DMD per year, effecting people of all races and ethnicities[2]. The disease is characterized by

the absence of, or improper functionality of the protein dystrophin which plays an important role

in muscle contraction when working properly[2] [3]. Symptoms of the disease include muscle

fatigue, weakness and muscle wasting and is more commonly found in young boys than girls[2].

DMD progresses with a patient’s age and includes long term muscle weakness and degradation

while also affecting other systems of the body including respiratory functionality[3]. DMD is

usually diagnosed when children are in the toddler years and begins with signs of a “waddling”

gait while walking or an overuse of the hands to compensate for lack of strength in the lower half

of the body[2] [3]. Blood and urine tests that measure creatine kinase levels leaking from

damaged tissues, immunostaining of muscle biopsies for lack of dystrophin, and in recent years,

genetic testing have been utilized for diagnosis of DMD[2] [3]. Treatment for DMD has

traditionally included physical, occupational and speech therapy, assisted ventilation, corrective

surgery, and corticosteroid drug therapy to delay muscle degeneration[2].

Amyotrophic lateral sclerosis commonly known as ALS and formerly called “motor

neuron disease” is a neurodegenerative progressive disorder with an incidence of approximately

20,000 people per year in the United States[4]. Progressive degeneration and death of the motor

6

neurons in the brain and spinal cord leads to muscle wasting or muscle atrophy because the brain

can no longer initiate the voluntary movement required for muscle to function properly[4]. The

onset of ALS typically occurs between the ages of 40-70, occurs in greater percentages in men

than women and is characterized by a gradual onset of painless muscle weakness usually in the

hands, arms, feet and legs[4]. While the symptoms range from patient to patient, progressive

muscle weakness and paralysis ultimately occur with an average survival time of 3-5 years from

the time of diagnosis[4]. ALS is diagnosed by blood and urine analysis, muscle or nerve biopsy,

spinal tap, X rays and MRI, and electrodiagnostic test[4]. Currently the only approved drug

treatment for ALS is Riluzole, a drug that aims to modulate glutatmatergic transmission in the

nervous system to prevent damage to motor neurons and demonstrates limited efficacy in

patients[5]. In depth reviews of current clinical trials and their associated treatment have been

conducted and demonstrated that there is a need for further drug investigations at the pre-clinical

level[5].

In addition to research for musculoskeletal disease, exercise induced damage and

traumatic injury to skeletal muscle is a frequent and commonplace issue within active military

personnel[6]. It has been estimated that injuries are the leading cause of medical treatment and

hospitalization stays which ultimately contribute to lost duty days[7] (Fig 3). One of the primary

goals of the Natick Soldier Research Development & Engineering Center’s Combat Feeding

Directorate is to provide the soldier population with targeted, nutritious food that assists in

muscle recovery after exercise and promotes wound healing. Injury prevention and rapid wound

healing are therefore of great emphasis in military applications and research[6].

7

Figure 3. Injuries dominate as the top burden of disease category across military personnel.

Adapted from Jones et. al. 2010[7].

1.2 Current Research and Development Tools for Studying DMD, ALS & Exercise Induced

Muscle Damage and Traumatic Injury

Modeling disease at the in vitro and in vivo levels are the earliest steps toward drug and

other treatment discoveries for many diseases. DMD is associated primarily with the loss of

dystrophin which leads to a cascade of other events such as calcium homeostasis upset, oxidative

stress, inflammation and fibrosis at the intracellular level[8]. In vitro models used to study the

relationships among these events vary, but all maintain the same goal of investigating modes of

treatment at the earliest possible level before recommending a treatment to clinical trials. Some

in vitro models that have historically been used to study DMD are the C2C12 mouse muscle

immortalized cell line, dystrophic induced pluripotent stem cells (iPSCs), primary

cardiomyocytes, and primary muscle fibers from mice[8]. In vivo models primarily include the

mdx mouse model and the golden retriever muscular dystrophy model (GRMD). Both animal

8

models contain a mutation in the dystrophin gene that abolishes dystrophin from the animal

allowing researchers to study DMD at the in vivo level[8].

Similar in vitro and in vivo models to DMD have guided research efforts for ALS. A

mutation in the gene SOD1 was identified as a contributor to development of ALS, leading to the

development of a mouse model with a mutation of SOD1 to be used for treatment

investigation[9]. This mouse model developed disease pathology of ALS in a similar fashion to

humans that included paralysis and motor neuron deficiency[9]. Many modifications have since

been made to the SOD1 mouse to account for differences in phenotype and age at onset of

disease and many more animal models with a mutation of SOD1 have been developed including

canines, zebrafish, Drosophila, nematodes and pigs[9]. TDP-43 mutations are rare among ALS

patients but many in vivo models including mice, rats, Drosophila, zebrafish and nematodes,

with modifications to TDP-43 have been developed for studying ALS mechanisms and

treatments as well[9].

Somewhat different from disease specific in vitro and in vivo modeling is the concept of

replicating exercise induced muscle damage (EIMD) or traumatic lacerative skeletal muscle

injury in a laboratory setting. Mechanical loading, tension and contraction of muscle is a very

important part of skeletal muscle functioning, and when muscle is overworked or damaged it is

important to have a method of analysis for this phenomenon in vitro. In this case, researchers

have utilized a system called the Flex Cell System in which skeletal muscle cells can grow,

proliferate, differentiate, and then be mechanically stretched and strained to mirror the

contraction and tension associated with exercise[10]–[12]. Specific strain protocols can be

optimized to model muscle stretch and exercise and cellular responses related to proliferation

and immune system response can be investigated[10], [11]. A traditional method for studying

9

traumatic or lacerative injury to skeletal muscle is called the scratch assay. Skeletal muscle cells

are grown in culture in a two dimensional dish and when confluent are scratched with a razor to

make a wound to the culture[13], [14]. Cell proliferation and muscle fiber regeneration at the site

of damage can then be investigated and analyzed[13], [14].

1.3 Comparison: 2D Skeletal Muscle Research Tools vs 3D Technological Advancements

As described in Table 3 there are many pros and cons to traditional in vivo and in vitro

models for disease mitigation related to skeletal muscle functioning and EIMD. Recent efforts in

in vitro modeling have transitioned from the traditional 2D cell culture platform to a variety of

3D applications all with the intention of providing research tools that are more physiologically

relevant to the human in vivo state. Over the years, shortcomings related to the physiological

relevance of 2D cell culture have been discussed citing differences in cell morphology, polarity

and extracellular matrix (ECM) interaction compared to the native in vivo state[15]. It has been

hypothesized that a 3D culturing system for drug delivery and disease treatment research is more

physiologically relevant because the 3D environment enhances cell-cell interaction, cell

signaling and is phenotypically more similar to a human in vivo tissue microenvironment[15]. A

variety of 3D culturing methods have been established and have been reviewed for their relative

advantages and disadvantages in Table 2[15].

10

Table 2. Overview of 3D cell culture methods and the pros and cons of each method in research.

Adapted from Breslin and O’Driscoll 2013 Three-dimensional cell culture: the missing link in

drug discovery.

Method

Type

Advantages Disadvantages

Forced-

floating • Relatively simple

• Inexpensive

• Suitable for high-throughput

testing

• Spheroids produced are

easily accessible

• Variability in cell size and shape

if not as fixed cell no./well

• DIY plate-coating is relatively

labor intensive

Hanging

drop • Inexpensive if using

standard 96-well plate

• Homogenous spheroids

suitable for high-throughput

testing

• Spheroids produced are

easily accessible

• More expensive if using

specialized plates

• Labor intensive if preparing

plates in-house

• Small culture volume makes

medium exchange, without

disturbing cells, difficult

(proposed easier handling with

commercially available formats)

Agitation-

based

approaches

• Simple to culture cells

• Large-scale production

relatively easily achievable

• Motion of culture assists

nutrient transport

• Spheroids produced are

easily accessible

• Specialized equipment required

• No control over cell no./size of

spheroid (can be overcome by

additional culture step; see

‘Forced-floating methods’)

• Time consuming for HTS due to

extra step required for

homogenous spheroids

• Cells possibly exposed to shear

force in spinner flasks (may be

problematic for sensitive cells)

Matrices

and

scaffolds

• Provide 3D support that

mimics in vivo

• Some incorporate growth

factors

• Can be expensive for large-scale

production

• Can have difficulty in retrieving

cells following 3D culture

formation

Microfluidic

cell culture

platforms

• Described as suitable for

high-throughput testing

• specialized equipment required

adding expense

• Further analysis of 3D cultures

produced may be difficult

11

It has been documented that long term studies are difficult when dealing with skeletal

muscle cells either from an immortalized cell line such as mouse C2C12, the rat IL6 cell line or

primary cells isolated from human tissue in a 2D monolayer[16]. As skeletal muscle myoblasts

proliferate and ultimately differentiate into myofibers they lift off the plastic or glass substrate

they are being cultured on making it difficult to utilize for long term drug screening assays[16].

Early successful muscle fiber growth and differentiation was demonstrated in 3D when cells

were grown between two PDMS posts in a collagen/MATRIGEL scaffold and showed long term

study potential when they remained functional out to two weeks post casting (FIG 4) [16], [17].

Further 3D culturing and tissue engineering endeavors related to skeletal muscle include

scaffolding matrices made from collagen, gelatin or decellularized skeletal muscle[18]. Synthetic

sources for scaffolding consist of polymers such as poly(vinyl alcohol)(PVA, poly(lactic acid)

Figure 4. Examples of technologies utilized for cell culture and 3D tissue engineering. 1)

FlexCell System for exercising and straining skeletal muscle. FlexCell International. 2)Skeletal

muscle cells attached between PDMS posts in a collagen/Matrigel scaffold. Vandenburgh et al

2002. 3) Gelatin micromold design to direct skeletal muscle alignment. Bettadapur et al 2016. 4)

Schematic of myofiber cell sheets directed for cellular differentiation. Takahashi et al 2018.

12

(PLA) and poly(captolactone) (PCL)[18]. Topographical micro or nano patterning into grooves

of different materials have allowed for direction of cellular alignment and formation of cell

sheets driving differentiation of myoblasts into myofibers[18]–[20], (FIG 4). Polymers that were

electrospun in specific patterns drove cellular alignment and increased cellular differentiation in

C2C12 cells indicating the potential physiologically relevant benefits of culturing cells in

3D[18].

Growing cells in a 3D microenvironment matrix for early drug screening and treatment

delivery

assays that

utilize either a

collagen or

polymeric

based

scaffolding

system for

support has

been an

effective method for 3D cell culture and tissue engineering[15], [16], [18]. Another innovative

approach to 3D cell culture and tissue engineering is the use of a technology called the 3D Petri

Dish[21]. This petri dish, made from an agarose hydrogel, provides a platform for cells to self-

assemble into micro sized tissues without adhering to the agarose itself (FIG 5). This type of 3D

cell culturing format promotes a more physiologically relevant in vivo like platform than other

2D and 3D matrices because the cells only adhere to one another rather than adhering to a plastic

Figure 5 Non-adherent agarose gel mold platforms for creating microtissues

by cell self-assembly. 1) Toroid shaped micromold. Wilks et al 2018. 2)

Spheroid shaped micromold. Microtissues.com

13

culturing plate or another adherent engineered scaffold. The 3D Petri Dish has been utilized

across many cell lines including ovarian granulosa cells, fibroblasts and endothelial cells, and

has demonstrated applicability in drug uptake and diffusion investigation, cellular alignment of

the extracellular matrix and collagen production and the ability to translate from microtissue to

microtissue applications[22]–[25]. Based on the successful applications of the 3D Petri Dish to a

variety of cell lines from different human systems, it is of interest to utilize this self-assembled

microtissue building platform as a model for the skeletal muscle system. This platform could

ultimately have application as a treatment investigation platform to advance the 2D cell culture

in vitro and animal in vivo models that have been widely used to study DMD, ALS and EIMD. It

is important to recognize that with any new technology while the application to study the disease

of interest is the end goal, it is crucial to effectively characterize the in vitro model system’s

ability to perform under normal baseline conditions to ensure physiological relevance for the

system at hand. The following thesis work will investigate utilizing the 3D Petri Dish

microtissue self-assembly platform to effectively model baseline or normal physiological

characteristics of skeletal muscle proliferation and differentiation called myogenesis.

Confirmation and analysis of myogenesis occurrence in skeletal muscle microtissues will be

performed using brightfield microscopy, fluorescent antibody labeling and quantitative confocal

microscopy.

14

1.4 Skeletal Muscle Myogenesis Overview

Myogenesis is the process of skeletal muscle growth and development from individual satellite

cell activation to terminally differentiated myofibers and occurs during embryonic development

or as a response to injury or disease[13]. Embryonic myogenesis and skeletal muscle

regeneration are similar in that they have similar regulatory pathways, genes and transcription

factors that drive the process[13]. Myogenesis begins when a niche of committed embryonic

stem cells form a pool of skeletal muscle satellite cells with the upregulation of the transcription

Figure 6. Different transcription factors are upregulated and downregulated

during the different phases of myogenesis (proliferation, early differentiation and

late differentiation). Adapted from Bentzinger et al 2012 Building Muscle:

Molecular Regulation of Myogenesis.

15

factor Pax7[26]. Part of this pool of satellite cells remain in a quiescent dormant state throughout

adult life and are activated only when muscle is damaged and needs repair[13]. Remaining

satellite cells proliferate and form myoblasts, single nucleated cells that eventually fuse together

during the differentiation phase to form multinucleated myotubes or myofibers[13], [26]. Each of

these phases is regulated by a family of muscle specific transcription factors and proteins and is

visually demonstrated in FIG 6. Myoblasts that fuse together to form myotubes will upregulate

myogenin which drives the expression of the muscle specific protein myosin heavy chain[26],

[27].

1.4 Myogenesis in 2D using the C2C12 skeletal muscle cell line

Skeletal muscle cells like the C2C12 cell line have a very specific timeline for

proliferation and differentiation in 2D that is directly influenced by the culture media being used

to feed the cells (Fig 7). Skeletal muscle cells have been shown to differentiate in 2D cell culture

when standard growth medium containing fetal bovine serum (FBS) is switched over to

differentiation medium containing horse serum instead of FBS[28], (Table 6). Skeletal muscle

cellular differentiation is also driven by cell-cell contact and will spontaneously happen when

culturing flasks become more than 80% confluent[29]. Immunofluorescent staining for myosin

heavy chain can be used to determine if cellular differentiation and myogenesis is occurring in

the cell culture system. In standard 2D cell culture it is typical to see no cellular differentiation

within the first two days while cells are being fed with growth medium (GM) containing FBS.

Early signs of differentiation can be seen on day 3 when cell media is transitioned over to

differentiation medium (DM) and continues to increase, producing multinucleated myofibers

typical of muscle in vivo (Fig 8) (Fig 9).

16

There are limitations with 2D cell culture systems to provide an accurate representation

of biological processes that occur in vivo. The C2C12 cell line like many others has been

optimized for use under specific conditions that require a 2D plastic adherent surface for growth

and maintenance. Myogenesis has been shown to occur during a specific timeline, with specific

media formulations, however this biological process may appear differently and require different

nutritional maintenance when cells are cultivated in a 3D nonadherent environment. Therefore,

it is important to characterize initial cell seeding densities and growth patterns of cells in 3D

prior to immunofluorescence investigation of myogenesis in 3D.

17

Table 3. Summary of in vitro and in vivo models for DMD, ALS, EIMD, and traumatic skeletal muscle injury.

Disease

Relevance

In vitro or In vivo

model

Advantages Disadvantages Reference

Duchenne’s

Muscular

Dystrophy

(DMD)

Mdx mouse • Mutation of exon 23 removes dystrophin

expression

• Commercially available and well characterized

• Inexpensive

• Isolated muscles allow for muscle function

analysis ex vivo

• Long term studies due to normal lifespan

• Muscle function

loss timeline is

different from

human disease

• Skeletal muscle

is non-human

Blat & Blat

2015

DMD Golden retriever

muscular dystrophy

(GRMD)

• Large mammal, vertebrate

• Spontaneous mutation in dystrophin gene

demonstrates extensive muscle degeneration

• Expensive

• High inter-

animal

variability

Blat & Blat

2015

DMD C2C12 cell line • Can be modified for targeted screening efforts

of specific molecules

• Inexpensive for high throughput screening

• Not inherently

dystrophic

• Non-human

Blat & Blat

2015

DMD Zebrafish • Two strains identified with dystrophin

mutations

• Inexpensive

• Vertebrate

• Non-human Blat & Blat

2015

Amyotrophic

Lateral

Sclerosis (ALS)

SOD1 mouse • Reproduce clinical findings associated with

ALS

• Develop motor deficits, muscle degeneration,

and motor neuron degeneration similar to ALS

• Variations available for different phenotypes,

age of disease onset, and survival

• Poor correlation

between age of

disease onset and

disease

progression to

humans

• Non-human

Picher-Martel

et al 2016

ALS Dogs • Can display naturally occurring, progressive

neurodegenerative disorders like humans

• Expensive

• Non-human

Picher-Martel

et al 2016

18

ALS Zebrafish • Simplified vertebrate system allows access to

motor neurons

• Useful for rapid screening of early

investigative treatments or therapeutic methods

• Vertebrate

system has

limited

similarities to

humans

• Non-human

Picher-Martel

et al 2016

ALS Drosophila • Useful tool for genetic research

• Inexpensive compared to mammals and

rodents

• Short lifespan allows for quick initial studies

• Mammalian

nervous system

is much more

complex

• Non-human

Picher-Martel

et al 2016

ALS C. elegans • Transparent for fluorescent protein detection

• Short lifespan permits rapid drug screening

• Nervous system

dissimilar to

humans

• Non-human

Picher-Martel

et al 2016

Exercise

Induced Muscle

Damage

FlexCell System

(can be used with

rodent or human

cell lines

• Controlled experimental strain of cell cultures

• Plates allow for cell growth from seeding all

the way though strain

• Can be used with skeletal muscle cells isolated

from humans

• Rodent cells:

non- human

• Expensive

Tsivitse et al

2004

Traumatic

Skeletal Muscle

Injury

Scratch assay (can

be performed on

rodent or human

cell lines)

• Wound regeneration can be viewed and

analyzed in real time

• In vivo like release of cytokines from “wound”

region

• Microscopic analysis of cell morphology and

protein expression

• Inexpensive

• Low throughput

• Limited to use

with 2D cell

monolayer

• Rodent cell

line=non-human

Goetsch &

Niesler 2010

19

Intentionally left blank

20

Chapter 2

Materials and Methods

2.1 Cell Culture

C2C12 cells (ATCC CRL #1772) were purchased from ATCC, expanded to passage 8

then frozen and stored in liquid nitrogen. Cells were thawed and expanded in T75cm2 flasks

(Corning #430641U) for experimental use at passage 10. Cells were maintained at 37˚C, 10%

CO2 in Dulbecco’s Modified Eagle Media (DMEM) (Gibco #11995-065) supplemented with

10% fetal bovine serum (FBS) (Gibco #10347-028) and 10% Penicillin Streptomycin (PS) (MP

Biomedicals #1670249) herein after referred to as growth media (GM). Cell media was

exchanged every two days until the cells reached 65% confluency for passaging. At passage

time, cells were washed once with 1X phosphate buffered saline (PBS) and trypsinized with

0.05% trypsin (Hyclone #SH30042.01) in 1X PBS at 37˚C, 10% CO2 for 5 minutes. The cell

flask was gently agitated to remove any remaining adherent cells and equal parts GM was added

to the flask to deactivate the trypsin. Cells were centrifuged at 250g for 3 minutes and

resuspended in fresh GM for cell counting with a hemocytometer.

2.2 Micromold Specifications & Formation of Non-Adherent Agarose Molds

Spheroids

C2C12 spheroid microtissues were made using the non-adhesive micromolds

(Microtissues Inc. Providence, RI). The 24-96 Small Spheroid mold with dimensions of 400µm

by 800µm per recess and a working volume of 75µl was used as a platform for making agarose

molds. Agarose (Fisher Sci #BP160-500) was autoclaved, added to sterile water and heated by

microwave for a final 2% solution. Approximately 1ml of molten agarose was pipetted into the

Small Spheroid 3D Petri Dish and evenly distributed and flattened with a spatula. Once gelled,

21

agarose molds were removed from the 3D Petri Dish and added to a standard 24 well plate (Fig

5). 250ul of molten agarose was added along the edge of each well to adhere the mold to the

plate bottom. Serum free GM was added to the molds for hydration. To remove bubbles between

the agarose recesses, the 24 well plate was covered and placed in a vacuum chamber at 25-30psi

until bubbles rose to the media surface and popped. Hydrated agarose micromolds were

equilibrated and stored at 37˚C, 10% CO2 until ready for cell seeding.

Toroids

C2C12 toroid microtissues were made using a toroid shaped micromold. Toroid molds

were designed in SolidWorks (SolidWorks Corporation, Concord, MA) and printed using the

Form 1+ stereolithography printer (Form Labs, Somerville, MA) according to the design

parameters and methods of Wilks et al 2018 (Fig 5), [24]. OMOO 30® silicone rubber (Smooth-

On, East Texas, PA) was pipetted into the 3D printed negatives, placed under 25-30psi vacuum

to remove bubbles, and allowed to dry overnight at room temperature. Once molds were dried,

they were removed from the negative cast and autoclaved. To make the final micromolds,

agarose was autoclaved and added to sterile PBS for a final 2% solution. Approximately 1ml of

molten agarose was pipetted into the silicone molds and evenly distributed with blunt tweezer to

remove any bubbles. A glass microscope slide was added on top of the molten agarose so

agarose could gel evenly. Once gelled, agarose molds were carefully removed from the silicone

mold and placed into a 24 well plate. Approximately 300ul of molten agarose was added to the

edge of each well to hold the toroid mold in place in the dish.1ml of serum free GM was added

to each well and molds equilibrated in 37˚C, 10% CO2 until cell seeding.

22

2.3 Microtissue Formation

Spheroids

C2C12 cells were trypsinized and counted by hemocytometer after reaching 65-75%

confluency during standard passaging. Cells were centrifuged down in appropriate cell culture

media (GM, DM, serum free plus (SFM+) or serum free advanced (SFMA)) 200g for 2 minutes

at desired density (500-2000 cells/spheroid) for spheroid formation in agarose gels. Media was

aspirated from the gels and C2C12 cells were resuspended and seeded into agarose gels in a total

volume to allow for seeding each gel with 75 µl cell suspension. Cells in gel settled in the

incubator for 30 minutes prior to adding 1ml media to each well of the 24 well plate.

Toroids

C2C12 cells were trypsinized and counted by hemocytometer after reaching 65-75%

confluency during standard passaging. Cells were centrifuged down in appropriate cell culture

media (GM, DM, SFM+ or SFMA) 200g for 2 minutes at desired density (50,000-400,000

cells/toroid) for toroid formation in agarose gels. Media was aspirated from the gels and C2C12

cells were resuspended and seeded into agarose gels in a total volume to allow for seeding each

gel with 30 µl cell suspension. Cells in gel settled in the incubator for 30 minutes prior to adding

1.5ml media to each well of the 24 well plate.

2.4 Microtissue Formation Validation

Brightfield Imaging

C2C12 spheroid and toroid microtissue formation was assessed using a Zeiss inverted

brightfield microscope and Zen Blue 2.3 edition. Spheroid snapshots were taken with a 5x EC

Plan-Neufluar objective at 2 fields of view per sample allowing for 30 spheroids per media

condition and seeding density to be imaged at varying time points depending on the experimental

23

trial. Initial toroid formation for different media formulations and cell seeding densities was

assessed by time lapse imaging with a 2.5x EC Plan-Neofluar objective from 0-24hrs and

individual snapshots post 24hrs depending on the experimental trial.

2.5 Immunofluorescent Staining: Myogenesis (2D)

C2C12 cells were seeded at a density of 50,000 cells per slide flask (ThermoFisher Scientific

#170920) (Fig 7). C2C12 cells were seeded at a density of 50,000 cells per slide flask

(ThermoFisher Scientific #170920) in duplicate and fed with GM and DM according to a

standard 2D cell culture timeline to induce myogenesis (Fig 7). Media was changed every two

days by carefully rotating the slide flask upside down, aspirating old media, and replacing with

3ml of fresh media. Cells were fixed with 10% neutral buffered formalin at room temperature for

2 minutes at each experimental time point by rotating the slide flask, carefully aspirating 1ml of

media and adding 2ml of formalin to the remaining media for a working volume of 4ml. After

aspirating the formalin, cells were permeabilized with 0.5% TritonX100 (ThermoFisher #85112)

for 15 minutes. TritonX100 was removed and cells were fixed again in 5% neutral buffered

formalin in PBS and washed with 1ml PBS (3 x 5min). Cells were blocked in 3% bovine serum

albumin (BSA) in PBS (Sigma Aldrich #05470-5G) for 1 hour, then washed in1ml PBS (3 x

5min). After the PBS was aspirated, the slide flask chamber was carefully snapped off from the

slide using the snapping tool provided by the manufacturer. Slides were placed in an empty

pipette tip box with a moist paper towel underneath creating a humidified chamber to prevent

evaporation of antibody staining solutions. Slides were stained with 4µg/ml myosin heavy chain

(ThermoFisher Scientific #50-6503-82) in PBS (500µl/slide working volume) for 1hr then

washed with 1ml PBS (3 x 5min). Next, 2 drops/ml actin (ThermoFisher Scientific #R37112) in

24

PBS (500µl/slide working volume) was added to each slide for 30 minutes. Slides were washed

again with 1ml PBS (3 x 5min). Lastly, 2 drops/ml Dapi (ThermoFisher Scientific #R37605) in

PBS (500µl/slide working volume) was added to each slide for 20 minutes. Slides were washed

for the last time with 1ml PBS (2 x 5min), mounted with 120µl bio-gold mounting medium

(ThermoFisher Scientific #P36930) coverslipped, and sealed with nail polish to dry overnight

prior to confocal imaging.

2.6 Immunofluorescent Staining: Myogenesis in Spheroids and Toroids (3D)

Spheroids

C2C12 spheroids were seeded at 1000 cells/spheroid and fed with GM and DM following

the standard 2D cell culture timeline (Fig 7). Cell culture media was aspirated from the outside

of the gel and spheroids were fixed at each time point with 1ml 10% neutral buffered formalin

for 1hour. The formalin was removed and replaced with 1ml of formalin for 2 hours. The

formalin was aspirated, and spheroids were washed with 1ml PBS (3 x 10 minutes/wash). Fixed

spheroids were stored in PBS, the plate was wrapped in parafilm and finally stored at 4˚C until

all time points had been collected. Spheroids were then immunofluorescently stained inside the

agarose gel according to the procedure in Table 4.

Figure 7. Cell culture timeline and media formulation to induce skeletal muscle differentiation in

2D.

25

Table 4 Immunofluorescent staining protocol for C2C12 microtissues. Modified from

Boekelheide Lab original protocol.

1) Permeabilize in 0.25% Triton X100

in PBS at room temperature on

rocker

Spheroids (in gel) Toroids (outside gel)

Volume/ well 1ml 400µl

Time 30 minutes 1 hour

2) Wash in PBS at room temperature

on rocker

Spheroids Toroids

Volume/ well 1ml 500µl

Time 2 x 5 mins/wash 2x 10 mins/wash

3) Block in 3% BSA at 4˚C on rocker

Spheroids Toroids

Volume/ well 1ml 600µl

Time Overnight Overnight

4) Wash in PBS at room temperature

on rocker

Spheroids Toroids

Volume/ well 1ml 500µl

Time 2 x 15 mins/wash 2x 20 mins/wash

5) Incubate conjugated primary

antibody cocktail in 3% BSA

solution at 4˚C on rocker (dark)

Spheroids Toroids

Volume/ well &

concentration

1ml 4µg/ml 250µl 4µg/ml

Time Overnight Overnight

6) Wash in 0.25% TritonX100 in PBS

at room temperature on rocker

(dark)

Spheroids Toroids

Volume/ well 1ml 250µl

Time 1 x 10 mins 1 x 20 mins

7) Wash with PBS at room temperature

on rocker

Spheroids Toroids

Volume/ well 1ml 500µl

Time 2 x 10 mins 2 x 20 mins

8) Store samples in PBS at 4˚C until

imaging

26

Toroids

C2C12 spheroids were seeded at 300,000 cells/toroid and fed with either SFM+ or SFMA

media for multiple time points. Cell culture media was aspirated from the outside of the gel and

toroids were fixed at each time point with 1ml 10% neutral buffered formalin for 1hour. The

formalin was removed and replaced with 1ml of formalin for 2 hours. The formalin was aspirated

and toroids were washed with 1ml PBS (3 x 10 minutes/wash). Fixed toroids were stored in PBS,

the plate was wrapped in parafilm and finally stored at 4˚C until all time points had been

collected. Prior to immunofluorescent staining toroids were carefully removed from the agarose

gels using fine, blunt tweezers and deposited into a fresh 24 well plate for subsequent staining

protocol found in Table 4.

2.7 Confocal Imaging Parameters

Confocal Imaging of 2D Slides

Confocal images of C2C12 cells on slides were obtained with a Zeiss LSM 710 upright

system (Table 5). Each experimental condition contained 2 slide replicates and 10 fields of view

per slide were imaged for a total of 20 fields of view per time point.

Confocal Imaging of 3D Microtissues

Confocal images of 3D microtissues in spheroid and toroid forms were taken using the

Opera Phenix High Content Screening System (Table 5). Image acquisition for each time point

consisted of 30 z plane images per tissue at a 5µm interval per slice.

27

Table 5 Confocal imaging acquisition parameters utilized for determining myogenesis in 2D and

3D platforms.

Myogenesis 2D Myogenesis 3D

Confocal Imaging System Zeiss LSM 710 upright Opera Phenix High Content

Screening System

Objective Plan apochromat 20x Plan apochromat 20x Water

Lasers 405 561 633 405 561 633

Power 2.0% 2.0% 4.0% 50% 35% 80%

Gains/exposure times 625 360 670 40ms 20ms 35%

Labeled cell structure Nuclei Actin MHC Nuclei Actin MHC

2.8 Confocal Image Analysis

Zen Blue Analysis for 2D Immunofluorescent Staining: 2D Myogenesis

Zen Blue software was used to assess the occurrence of myogenesis by counting myosin

heavy chain positive cells in each field of view taken at each time point. Each channel was set to

best fit and an intensity of 5000 on the 633 laser was subtracted to account for any possible non-

specific staining or background noise interference prior to counting cells.

3D Harmony Analysis for 3D Immunofluorescent Staining: 3D Myogenesis

3D Harmony High Content Imaging and Analysis Software was used to assess the

presence of myosin heavy chain in C2C12 spheroid and toroid microtissues. Background

intensities were detected by staining with a Mouse IgG2b K Isotype Control (ThermoFisher #50-

5503080) and subsequently subtracted from all experimental samples. In depth step-by-step

analysis for spheroid and toroid microtissues can be found in Appendix 1.

28

2.9 H&E Staining

Microtissue sample preparation

C2C12 toroid microtissues sampled from different days were fixed in 10% formalin for 1

hour, exchanged for fresh formalin for 2 hours, washed in PBS (3 x 10 mins) and stored at 4˚C

until all time points were ready for prep. At this time PBS was gently aspirated from the outside

of the agarose well containing the tissue. 2% agarose was heated until dissolved and allowed to

cool until warm to the touch. At this point PBS within the microtissue well was gently wicked

away by capillary action using a paper towel. Approximately 200µl of the warm agarose was

pipetted into the well containing the tissue and allowed to dry. The sealed microtissue samples

were placed in large embedding capsules and stored in PBS at 4˚C until paraffin embedding.

H&E Staining and Paraffin Embedding

H&E staining and paraffin embedding protocols can be found in Appendix 2.

29

Chapter 3: Microtissue Formation & Investigation of Myogenesis in 3D Spheroids

3.1 C2C12 Myogenesis in 2D

Introduction:

To most closely compare the process of myogenesis in 3D to traditional 2D methods, GM

and DM media formulations were used in accordance with the traditional feeding timeline (Fig

7). Cell densities were chosen for seeding cells at 30% confluency and accommodating a media

volume of 4ml.

Experimental Design:

C2C12 cells were seeded at a density of 50,000 cells per slide flask in duplicate and fed

with GM and DM according to a standard 2D cell culture timeline to induce myogenesis (Fig 7).

Media was changed every two days by carefully rotating the slide flask upside down, aspirating

old media, and replacing with 3ml of fresh media. Cells were fixed and immunofluorescently

stained and imaged by confocal microscopy according to the protocols described in Chapter 2.5

and 2.7.

Results: Myogenesis was assessed by the positive detection of myosin heavy chain

(MHC) formation according to the methods described in 2.8. The early stage myoblasts shown in

days 1 and 2 demonstrated little to no MHC as the cells were still in a proliferative state and

confluency had not been met (Fig 9). By day 3 when the media was switched to DM the single

nucleated myoblasts began to fuse into multinucleated cells and show early signs of MHC

presence. Day 4 demonstrated am increase in MHC positive cells and began to level out at Day 5

(Fig 9).

Discussion: These results demonstrate the traditional progression of myogenesis in a 2D

cell culture system as shown by others in the field of muscle biology and were primarily used

here as an overall positive control for this thesis[26], [30].

30

Table 6. Media composition for C2C12 2D and 3D cell culture.

Abbreviation Media

Name

Media Components C2C12 2D cell

culture

C2C12 3D

microtissue

formation

GM Growth

Medium • 500ml DMEM

(Gibco #11995)

• 10% FBS (Gibco

#10347-028)

• 1% Pen Strep

(MP Biomedicals

#1670249)

Myoblast

proliferation

Spheroid Toroid

+ -

DM Differentiation

Medium • 500ml DMEM

(Gibco #11995)

• 2% HyClone

Donor Equine

Serum (GE

HealthCare Life

Sciences

#SH3007402)

• 1% Pen Strep

(MP Biomedicals

#1670249)

Myoblast cell

fusion and

differentiation

into

multinucleated

myotubes

Spheroid Toroid

+ -

SFM+ Serum Free

Media Plus • 500ml DMEM

(Gibco #11995)

• 1% Pen Strep

(MP Biomedicals

#1670249)

• 50 ug/mL L-

Proline (Sigma

Aldrich # P0380-

10MG)

• 1mM L-

Ascorbic Acid 2-

Phosphate

Trisodium Salt

(Sigma Aldrich

#49752-10G)

N/A Spheroid Toroid

- +

SFMA Serum Free

Media

Advanced

• 500ml Advanced

DMEM (Gibco

#12491015)

• 10ml Glutamax

(Gibco #35050-

061)

N/A Spheroid Toroid

+ +

31

Figure 8. MHC positive cells increase with time and change of media formulation. MHC positive

cells displayed as mean ± SD over 20 fields of view per time point. (α= not significant p > 0.05.

All other pairings demonstrate significant difference in MHC presence over time).

32

Figure 9. C2C12 myogenesis occurs with transition to DM in 2D cell culture as demonstrated by MF20 antibody staining for MHC

positive (red) multinucleated myofibers. Cytoskeletal actin is stained with Actin555 ReadyProbes (green) and nuclei are stained with

Dapi (blue). Images displayed as best fit. 5000 background intensity subtracted from all time points. Scale bar= 20µm.

33

3.2 C2C12 Microtissue Formation (Spheroids)

Introduction

Recent advances in tissue engineering have led to a variety of methods for forming and

maintaining microtissues (Table 2). The Morgan Lab at Brown University has demonstrated

success using micromold designs to form microtissues of different sizes, shapes and for varying

biological applications [22]–[25]. It is characteristic when first investigating a new cell line to

determine whether the cells traditionally cultured in a 2D environment will self -assemble into

microtissue using one of the designated micromold designs. This section discusses the

experimental design and analysis used to identify whether the C2C12 cell line would form

spheroid microtissues in media formulations utilized for traditional 2D growth and maintenance.

Experimental Design

C2C12 skeletal muscle cells were assessed for microtissue formation using the non-

adhesive micromold platform (Microtissues Inc. Providence, RI). 96 circular recess 8x12 array

containing a 400µm diameter and 800µm depth per recess[21]. This micromold accommodates

cell seeding densities of 500-2000 cells per spheroid. Based on these metrics, C2C12 sells were

grown, maintained and seeded at cell densities of 500 and 1500 cells/spheroid according to the

methods described in 2.1-2.3. Initial characterization experiments were intended to determine if

C2C12 cells would form spheroids when fed and maintained with GM and DM cell culture

medias (Table 6). C2C12 spheroid formation was assessed using brightfield microscopy methods

described in Chapter2.4. 2 fields of view per micromold across 3 replicates per seeding density

and media formulation were imaged on day 1,2,4, and 6. Fifteen spheroids within each field of

view were captured and their diameter was measured at each time point using the circular

34

measurement tool in the Zen Lite Blue Edition allowing for a total of 90 spheroids to be

measured over time.

Results

C2C12 cells demonstrated the ability to form spheroids when seeded into the micromolds

and were maintained in either GM or DM (Fig 10). Statistical analysis by two-way ANOVA and

Tukey’s recommended post hoc test showed significant differences in spheroid diameter as a

function of time in both 500 and 1500 cell/spheroid seeding densities when they were maintained

in GM and DM (Fig 10). Results showed the most significant changes in spheroid diameter

between the first day and the remaining 3 time points. As time progressed, there were less

significant changes in diameter and no changes were seen between days 4 and 6 in all spheroids

fed with both GM and DM (Fig 10). Differences in mean diameter as a function of cell seeding

density was also seen in both GM and DM fed spheroids (Fig 10).

Two-way ANOVA was performed on the same data set rearranged to analyze spheroid

diameter change over time as well as investigate whether the media formulation influenced

spheroid diameter. As previously mentioned, spheroid size decreased slightly over time in both

the 500 c/s and 1500 c/s microtissues (Fig 11). The C2C12 microtissues seeded with 500 c/s and

fed with DM demonstrated a larger diameter across all time points than the 500 c/s microtissues

fed with GM (Fig 11). In the larger seeding density where 1500 c/s of C2C12s were used to

make the microtissues, the analysis displayed larger spheroid diameter in the DM fed

microtissues for only the first 2 out of 6 days (Fig 11).

35

Figure 10. Brightfield microscopy images of C2C12 cells show formation of spheroid

microtissues at varying cell densities maintained in different media formulations. Spheroid

diameter (µm) displayed as mean ± SD increases as cell seeding density (cells/spheroid ‘c/s’)

increases in both GM and DM (A&B, δ= Significant at p<0.0001). Spheroid diameter decreases

slightly as a function of time in both GM and DM (A&B, ****Significant at p<0.0001).

36

Figure 11. Diameter (µm) of C2C12 microtissues displayed as mean ± SD changes over time in

both 500 and 1500 c/s seeding densities (****Significant at p < 0.0001). Spheroids fed with DM

show significant difference in spheroid size than spheroids fed with GM when both cell seeding

densities are used (δ= significant at p < 0.05).

Discussion

The significant differences seen in spheroid diameter as a result of utilizing two different

cell seeding densities was to be expected and validated that the seeding methods of varying cell

amounts into the chambers worked. The results demonstrating spheroid diameter decrease over

time could in fact be due to the individual cells compacting from a looser spheroid on day 1 to a

tighter spheroid by day 4. Not seeing any significant change in diameter from day 4 to 6 in both

GM and DM fed spheroids could indicate that the spheroids have reached maximum cell- cell

contact within the seeding chamber. The change in diameter as a result of media formulation led

to the hypothesis that there could possibly be cellular differentiation occurring in the spheroids

that were fed with DM. As a result, follow on experiments to investigate cellular differentiation

within the spheroids were performed utilizing the presence or absence of myosin heavy chain as

a marker for differentiation. These data are discussed in the next section.

37

3.3 C2C12 Investigation of Myogenesis in 3D Microtissues (Spheroids)

Introduction

After the initial experiments demonstrated that C2C12 cells formed spheroid shaped

microtissues in both GM and DM, follow on experiments were run to investigate whether the

biological process of myogenesis occurred in the microtissues. In order to compare myogenesis

in the 3D spheroid model as closely as possible to the 2D adherent C2C12’s on slide flasks, the

media formulation and cell feeding timeline was the same for 3D microtissues as it was for the

2D cells.

Experimental Design

C2C12 cells were grown, passaged and seeded at 1000 cells/spheroid into micro molds

according to the protocols outlined in Chapter 2.2-2.3. The microtissues were fed every 2 days

with either GM or DM to induce myogenesis according to the standard 2D cell culture media

formulations and feeding timeline (Fig 7). C2C12 spheroids were fixed at each time point and

underwent the immunofluorescent staining protocol described in (Table 4). Spheroids were

specifically stained inside the agarose gel for actin, Dapi and MHC. The force of pipetting a

small volume of PBS into the gel allowed for transfer of whole spheroids into a clean 96 well

glass bottom plate for imaging (Grenier Bio-One #655892). Whole spheroids were imaged by z

stack image acquisition using the Opera Phenix High Content Imaging System under a 20x water

objective using laser power and exposure times referenced in Chapter 2.7.

Results

As previously demonstrated, C2C12 cells will form spheroids in both GM and DM (Fig

11). Immunofluorescent staining showed no MHC production during the first 2 days when

spheroids were fed with GM (Fig 12). A positive detection of MHC was seen when spheroid

38

media was switched from GM to DM on the third day and was displayed on Day 4 and Day 5 as

well (Fig 12).

Figure 12. Composite Z stack images of whole C2C12 spheroid microtissues demonstrate low

levels of MHC production (red) when switched from GM to DM. Scale bar = 100µm.

Discussion:

It was a promising result to see that C2C12 cells undergo early signs of myogenesis when they

were treated in the same fashion as the standard culturing method to induce cellular

differentiation in cells plated in 2D. While Fig 12 shows the progression of this timeline it is

important to note that these images are a composite Z stack of an entire microtissue. This means

that within each single plane of view within the stack, the MHC signal is even lower than can be

seen in Fig 12. There are a few reasons that could account for the low level of MHC production

in the spheroid microtissues as compared to the standard 2D MHC levels shown in (Fig 9).

Firstly, each microtissue is seeded with 1000 cells/spheroid as was determined an average

seeding density amount following successful initial microtissue formation characterization

shown in Fig 10 & 11, but those amounts vary greatly from the 50,000 cells seeded per slide

39

flask in the 2D experiments. There may not be enough cell signaling interactions occurring in

order to drive the production of MHC in the spheroids. Another reason for seeing low levels of

MHC could have to do with the shape of the model itself. As shown in Chapter 1, skeletal

muscle is attached to bones and ligaments and this attachment requires certain levels of tension

and forces that occur at the cellular and tissue levels. The nature of the spheroid model is that not

only is it non-adherent unlike the typical culturing conditions of 2D substrates, it is also free

floating with no forces or tension acting upon the cells other than gravity. Another fundamental

difference between the spheroid microtissue model and skeletal muscle in vivo is the opportunity

for skeletal muscle myoblasts to align and fuse forming multinucleated myofibers. The inability

of cells to align within a microtissue mold shaped like a sphere could also influence the cells’

ability to fuse and produce MHC.

It has been demonstrated in the field of tissue engineering that microtissue mold design

can directly influence cellular alignment and associated forces that act upon cells to drive that

alignment[23]. Since skeletal muscle tissue in vivo is characterized by bundles of long striated

multinucleated myofibers, it was logical to transition to a mold design that would be more

relevant in driving the C2C12 cells to align and fuse into long multinucleated myofibers.

40

Chapter 4 Microtissue Formation & Investigation of Myogenesis in 3D Toroids

4.1 C2C12 Microtissue Formation (Toroids)

Introduction

As mentioned in Chapter 3 many iterations of mold designs to drive cells to form self-

assembled microtissues have been developed and validated to investigate various biological

questions [23], [24], [31]. Each of these designs have demonstrated microtissue formation

utilizing varying cell lines, seeding densities and media formulations. The toroid micromold

design seen in Fig 5 has demonstrated effectiveness in driving cellular alignment and can

accommodate higher amounts of cell seeding compared to the spheroid model making it a more

physiologically relevant design to investigate myogenesis in C2C12 skeletal muscle cells. As

with the spheroid model, prior to investigating the biological question of MHC production, it is

important to characterize initial toroid formation by optimizing cell seeding densities and media

formulations necessary to maintain a stable microtissue.

Experimental Design

C2C12 cells were passaged and seeded at varying seeding densities (100k, 200k, 300k,

400k cells/toroid) into toroid micro molds according to the methods described in Chapter 2.3.

Toroid shaped microtissues were given 24hrs to assemble prior to formation assessment by

brightfield imaging under the conditions described in Chapter 2.4. Toroids were fed every 2 days

with either GM, DM, SFM+ or SFMA (Table 6). Snapshot images were taken each day that

C2C12 toroid microtissues remained intact and tissue stability in each media formulation was

assessed as a percent survival over time.

Results

C2C12 cells demonstrated the ability to form toroid shaped microtissues in the 2.5mm

micro molds in both GM and DM in less than 24hrs, but the microtissue stability varied among

41

seeding densities used. The toroids seeded with 100k and 200k cells/toroid and fed with GM

formed in less than a day, but broke after the first 24hrs. Toroids seeded with 300k and 400k

cells/toroid initially looked more stable, but by day 2 showed increased blebbing in at least half

the tissue and broke by the day 3 time point (Fig 13).

Figure 13. C2C12 cells form toroid microtissues in GM but break over time. A) Higher seeding

densities of 300k and 400k show initial formation on day 1 and increased blebbing and

nonuniformity on day 2. B) Toroids seeded with lower amounts of C2C12 cells at 100k and 200k

break by 24 hrs. Higher seeding densities of 300k and 400k remain stable until day 2 but break

by day 3. Scale bar = 500µm.

C2C12 toroids seeded at the same seeding densities but fed with DM showed even more

variability when analyzed by brightfield microscopy. All microtissue formed in less than 24hrs,

but by the end of day 1, only the 200k and 400k seeded toroids were still intact (Fig 14). The

200k toroids showed morphological variability within the tissue where part of the toroid showed

a large bleb and the other part appeared evenly stretched around the central agarose peg on day 1.

By day 2, the bleb increased in size and the evenly stretched part of the tissue became even

thinner and was broken by day 3 (Fig 14). The highest seeding density of 400k per toroid showed

initial uniformity within the microtissue by the end of day one but similarly to the 200k

42

microtissue, blebbing was seen in a large portion of the microtissue along with a thinner taut

section by day 2 and the tissue completely broke by day 3 (Fig 14).

Figure 14. C2C12 cells form toroid microtissues in DM but break over time. A) Seeding

densities of 200k and 400k show initial formation on day 1 but vary in their uniformity.

Nonuniformity and increased blebbing is seen on day 2 in both seeding densities. B) Toroids

seeded with 100k and 300k cells/toroid break by 24 hrs. Seeding densities of 200k and 400k

form and remain stable until day 2 but also break by day 3. Scale bar = 500µm.

The variability and instability seen within the C2C12 toroid microtissues when fed with

GM and DM prompted further investigation into using new media formulations hoping to

promote microtissue stability and eventually induce differentiation and myogenesis. Since 300k

and 400k seeding densities provided at least an initial stable tissue at the time of cell seeding, it

was determined going forward to use 300k cells for seeding in further experiments. Next,

C2C12s were seeded into toroid shaped agarose gels at 300k cells/toroid following the same

passaging and seeding protocols outlined in Chapter 2.1-2.3. C2C12 microtissues were fed with

GM, DM, SFM+ and SFMA (Table 6) and tissue formation was assessed by brightfield

microscopy as described in Chapter 2.4. Kaplan Meier survival curves were used to assess

microtissue stability over time.

43

C2C12 cells formed microtissues within 24hrs when fed with all media formulations

however the microtissue stability varied among the different medias over time with GM and DM

fed microtissues breaking at the 24hr time point in this experimental trial (Fig 15). SFM+ and

SFMA fed toroids showed uniform shape within each tissue and remained stable at the 24hr time

point. (Fig 15). Kaplan Meier survival curves demonstrated the change over time in microtissue

stability among the different media formulations in which GM, DM, and even SFM+ all had

complete broken microtissues (n=5 per media condition) by day 3 (Fig 16). The C2C12

microtissues fed with SFMA were the only tissues that remained stable out to day 3 with a loss

of 2 out of 5 tissues on the day 3 time point (Fig 16).

44

Figure 15. C2C12 microtissues demonstrate stability as a result of media formulation. C2C12 toroid microtissues break by 24hrs when

fed with GM and DM but remain stable at 24hrs when fed with SFM+ and SFMA. Scale bar = 500µm.

45

46

Figure 16. C2C12 toroid microtissues fed with SFMA show the greatest stability over time

compared to microtissues fed with GM, DM or SFM+.

Based on experimental results demonstrating that C2C12 cells formed the most stable

toroid microtissues in SFMA as opposed to the other media formulations, the stability of C2C12

toroid microtissues over a longer period in SFMA was assessed by brightfield imaging. C2C12

toroid microtissues formed within 24hrs and were imaged by brightfield microscopy according to

the protocols described in Chapter 2.4. Images were taken on day 1, 2, 3 and 6. The toroids

showed consistent shape and uniformity on day 1 with slight visibility of blebbing on day 2 and

an increase in blebbing area compared to taught tissue area on day 3 and day 6 (Fig 17).

47

Figure 17. Brightfield images display C2C12 microtissue stability over time. Microtissues

display change in morphology but remain unbroken over time when fed with SFMA. Scale bar =

500µm.

Discussion

Prior to investigating the biological process of myogenesis in self assembled C2C12

toroid microtissues, it is critical to establish that the cells will not only form into toroid

microtissues but will also remain stable over a period that it may take for myogenesis to occur. In

order to most closely compare C2C12 microtissues in 3D to the standard 2D format, it was

critical to start by feeding C2C12 toroid microtissues with GM and DM. Initial experiments

showed that some tissues with certain seeding densities would remain unbroken for 2 days, but

follow on experiments using a higher “n” within the experimental design showed frequent

breakage of the toroid microtissues fed with GM and DM (Fig 13), (Fig 14), (Fig 15). Since GM

and DM fed toroids did not remain stable past 24hrs it is therefore impossible to investigate the

question of whether myogenesis occurs in the microtissue. Both GM and DM have serum, FBS

in the GM and HBS in the DM. It has been demonstrated in human dermal fibroblasts that serum

48

causes microtissue breakage and these experiments with C2C12 skeletal muscle cells follow that

trend [24]. In order to continue to investigate myogenesis in C2C12 toroid microtissues it was

critical to find a media formulation that would provide stability to the microtissues. Wilks et al

2018 also demonstrated that by removing serum, but adding supplements such as ascorbic acid

and l-proline, tissues remain more stable over time (SFM+) [24]. While C2C12 microtissues

showed somewhat inconsistent survivability when fed with the SFM+, a few tissues remained

stable at the 24hr time point, showing that the initial step of removing serum and adding stability

factors like l-proline and ascorbic acid provided directionality for altering media composition

(Fig 16). It has also been shown that while C2C12 cells will differentiate in 2D when fed with

DM, they will also differentiate under low serum or serum free conditions, and demonstrate an

increase in cellular differentiation making the choice of using SFM+ formulation a valid one

[32], [33].

There are a few possible explanations as to why C2C12 cells form the most stable of the

toroid shaped microtissues when fed and maintained in SFMA. C2C12 cells will differentiate

and form multinucleated myotubes when fed with serum free media formulations in traditional

2D cell culture and SFMA contains no form of serum [32]. The 3D environment provided by the

agarose gel for cells to self-assemble into microtissues is inherently different than a 2D cell

culture platform due to cell-to-cell cues and relationship between cells and the extracellular

matrix as well as eliminating the interaction effect of cells to a plastic stiff substrate [15], [34].

Progress has been made with utilizing C2C12 skeletal muscle cells in other 3D platforms. It has

been shown that when C2C12 cells are grown into 3D engineered tissues in a Matrigel platform

their differentiation is increased in 3D with the addition if insulin like growth factor (IGF-1)

[35]. The SFMA formulation used in these studies contains IGFs in the original Advanced

49

DMEM formulation, l-proline and ascorbic acid for stability and Glutamax for overall tissue

health making it the best possible option at this time for maintaining C2C12 cells in a self-

assembled toroid microtissue.

Determining the conditions in which C2C12 cells successfully form toroid microtissues

and remain stable over time was a critical step prior to investigation of biological activity within

the microtissue. The SFMA formulation was used in all subsequent experiments in which

myogenesis is investigated by immunofluorescence and confocal microscopy in C2C12 skeletal

muscle toroid microtissues.

4.2 C2C12 Investigation of Myogenesis in 3D Microtissues (Toroids)

Introduction

Once it was determined that maintaining C2C12 toroid microtissues was possible using

the SFMA formulation, extended tissue survival analysis and investigation of myogenesis was

conducted. Survival analysis was evaluated by Kaplan Meier survival curves. Myogenesis was

investigated by immunohistochemistry and quantitative confocal microscopy to determine if

there was an increase in MHC production over time. Another key aspect of the process of

myogenesis is the fusion events of single nucleated myoblasts into multinucleated myotubes or

myofibers. This phenomenon of multinucleation was also quantified in C2C12 toroids for the

later time points as an indicator of myogenesis.

Experimental Design

C2C12 cells were grown in GM and passaged according to passage and maintenance

protocols outlined in Chapter 2. C2C12 cells were then resuspended in SFMA and seeded into

agarose gels at 300,000 cells per toroid in accordance with the protocols outlined in Chapter 2.1-

50

2.2. A total of three 24 well plates was made each containing 21 toroids per plate. All

microtissues were fed with SFMA and the media was exchanged every 2 days. Each separate

plate was designated for fixation at either Day 1, Day 3, or Day 6. Upon fixation in 10%

formalin at the designated time point and washed in PBS, the plates were then stored in PBS at

4˚C until all time points were collected and ready for immunofluorescent staining. Microtissues

were immunofluorescently labeled for MHC and Dapi according to the optimized 3D staining

protocol for toroid microtissues outlined in Chapter 2.6. 3D z stack confocal images of the

C2C12 toroids were obtained using the Opera Phenix High Content Imaging System using the

imaging parameters outlined in Chapter 2.7. Quantitative analysis for MHC production and

nuclei counts were collected using 3D Harmony analysis software. All 3D analysis software

functions can be found in (Appendix 1).

A duplicate set of C2C12 microtissues following the same passage, growth and feeding

protocols was made and allowed to incubate for an extended time period. One plate containing

21 toroids was incubated for 10 days and an additional plate also containing 21 toroids was

incubated for 14 days. At the time of media change all toroids were assessed for sustained

formation (survival) or breakage (death) and recorded. Each plate was fixed in 10% formalin and

washed and stored in PBS at 4˚C until both time points were collected for immunostaining. Both

the Day 10 and Day 14 C2C12 microtissues underwent the same immunostaining and confocal

imaging protocols as the Day 1-6 microtissues.

A C2C12 toroid microtissue from each time point was designated for staining with Dapi

and a mouse IgG2b kappa Isotype Control eFluor660 (Thermo Fisher #50-4732-82) to be used as

a negative control for MHC to collect any non-specific background signal. During quantitative

analysis for MHC the background signal identified by the isotype control was subtracted prior to

51

quantification of MHC. These steps are specified in (Appendix 1). Immunostaining sample sizes

were n=8 for Days 1-6 and n=3 for Days 10 and 14.

Results

The C2C12 microtissues that were incubated for the longest time period out to Day 14

were assessed for toroid survival. Kaplan Meier survival analysis showed approximately 80%

survival of C2C12 toroids at the mid time point of Day 6 with a drop to 50% survival by Day 12

(Fig 18).

Figure 18. Kaplan Meier survival curve demonstrating toroid stability over an extended time

point of 14 days.

A 3D analysis protocol was designed in Harmony to assess the presence of MHC in the

remaining C2C12 microtissues at each time point. Software input instructions of this process can

be found in (Appendix 1). First, true MHC signal was separated from the background signal then

nuclei were identified along with the MHC region, and finally the MHC region associated

52

around each nucleus was calculated and measured in volume (µm3) and applied to each Z slice

allowing for analysis of the entire tissue (Fig 19).

Figure 19. Harmony 3D analysis software calculation steps for determining MHC volume in 3D

C2C12 toroid microtissues.

The quantitative analysis protocol for the presence of MHC in the C2C12 toroid

microtissues was run across the samples from each time point (Days 1, 3, 6, 10, and 14) and

displayed as the mean volume (µm3)/nuclei across the entire tissue. This total mean value was

determined from 10 fields of view per tissue at each time point. Statistical analyses were

performed using GraphPad Prism. Sample groups were assessed at each time point for Gaussian

(normal) distribution using the Wilks Shapiro test. The groups of Day 1-6 did not pass the Wilks

Shapiro test for normality, but the Day 10 and 14 measurements were determined to have been

sampled from a normal distribution. Since the majority of sample groups that were analyzed did

not pass the normality test, the entire dataset was analyzed conservatively using a non-parametric

53

Kruskal-Wallis test, accounting for non-normal distribution instead of traditional one-way

ANOVA. The Dunn’s multiple comparison post hoc test determined significance at p < 0.05 and

demonstrated that there were significantly different sample means of MHC volume/nuclei

between Day 1 and Day 14 as well as Day 3 and Day 14 (Fig 20). While not determined to be

statistically significantly different by these analyses, it can still be noted that there is at least a

visible increase in the amount of MHC volume/nuclei after Day 6. Results showed very low

signs of MHC in the earlier time point of Day 1-6 (Fig 20), (Fig 21). Significant levels of MHC

were detected at the Day 14 time point indicating that the C2C12 toroid microtissues were

differentiating and undergoing myogenesis.

Figure 20. Quantitative confocal microscopy analysis shows C2C12 toroids have an increase in

MHC volume/nuclei at Day 10 and Day 14 indicating that the tissue is undergoing myogenesis.

*= Significant at p<0.05 by the Dunn’s multiple comparison post hoc test.

54

Figure 21. C2C12 toroid microtissue segments demonstrate an increase in MHC (Red) over time.

Dapi stained nuclei (Blue) show increased multinucleation in late timepoints of Day 10 and Day

14. Scale bar =100µm.

An additional image analysis to investigate fusion events of single nucleated myoblast

cells into multinucleated myofibers was established using Harmony and software input

instructions can be found in detail in (Appendix 1). Background signal was removed for the

55

MHC channel and the true MHC red signal was used to identify the cell while the Dapi signal

was used to isolate the nuclei within the cells (Fig 22).

Figure 22. Harmony Analysis software shows calculation steps for identification of nuclei

quantities within cells.

Due to Harmony software limitations, this analysis was unable to be performed in a 3D

capacity through multiple Z stacks and is only available for 2D analysis. Because of this software

limitation, a representative single Z slice approximately 20-35µm into the microtissue across 10

fields of view per time point was selected for multinucleation analysis. The Wilk Shapiro

statistical analysis test for normality was used to determine that the sample measurements did

indeed come from normal distribution. As a result, one-way ANOVA was conducted, and

Tukey’s multiple comparison post hoc test was applied to determine significant differences

56

between sample means at each time point. Results are displayed showing the distribution of

maximum nuclei per cell at the Day 6, 10 and 14 time point (Fig 23).

Figure 23. C2C12 toroid microtissues show an increase in multinucleation over time.

***Significant at p< 0.05 by Tukey’s multiple comparison post hoc test.

The Tukey’s post hoc test detected statistically significant differences (p<0.05) in mean

maximum nuclei/cell counts between Day 6 and 14. This increase in the average amount of

nuclei present in a single cell over time clearly demonstrates that single nucleated myoblasts

have fused into multinucleated myofibers (Fig 23).

Discussion

Once it was determined that the SFMA formulation was the optimal media for

maintaining C2C12 cells as toroid microtissues, long term experimental studies to investigate the

stability of the microtissues was conducted. From the time of cell seeding at 300,000 cells per

57

toroid to the final fixation time point of 14 days it was observed that by Day 12, 50% of the

microtissues remained unbroken and whole around the agarose peg. Skeletal muscle cells are

naturally contractile and under in vivo conditions can fuse and attach to tendons for support.

While the mold design of the toroid does provide tension as the cells are differentiating, it is not

the same as the forces and tension found in vivo when skeletal muscle is attached to a bone via a

tendon and this lack of stability could result in breakage of the tissue. The C2C12 mouse cell

line which has been spontaneously immortalized, has shifted greatly from the original parent cell

line and has been optimized for growth using stiff plastic substrates, is potentially in a

suboptimal growth environment when the only attachment opportunity is between cells.

The high seeding density of 300,000 cells, while optimal for tissue formation, might be a

limiting factor in tissue survival due to the lack of critical nutrients from the SFMA being able to

reach such a high number of cells in a microtissue. As stands, the agarose gel used for cell

seeding and toroid formation fits into a classic 24 well plate which can only hold between 1 and

2ml of media. A combination of an agarose mold that is optimized to be held in a larger plate

which holds a higher volume of media and a lower seeding density of C2C12 cells is a potential

solution to improving the microtissue survival rates over a two-week time period.

C2C12 cells maintained as 3D toroid microtissues do indeed undergo the process of

myogenesis as was demonstrated with an increase in MHC volume/ nuclei over time (Fig 20).

Differences in this process have been seen firstly by the media formulation used to maintain and

feed the microtissues (SFMA as opposed to traditional DM) and then secondly in the timeline

that it took for myogenesis to occur. It was not until the Day 10 mark that large increases in

MHC volume were seen in the C2C12 toroid microtissues and an even higher amount by Day 14.

Due to the low microtissue survivability at the latest Day 14 time point prior to fixation, some

58

tissue instability and fragileness upon handling when removing from the gel, and limitations on

MHC conjugated antibody quantity, only an N=3 was obtained for the Day 10 and 14 time

points. This unequal sample size compared to the earlier time points led to less powerful

statistical analyses to detect differences in MHC volume at the different time points. If repeated,

a higher N of at least 8 would be used to equally match the measurement quantities obtained

from the earlier time points. Even though sample sized for the Day 10 and Day 14 MHC volume

measurements were small, there was a noticeable increase in MHC at these time points. This

differs greatly from the timeline of C2C12 behavior in 2D where the presence of MHC can be

detected on Days 3-5 when cells are fed with specific differentiation medium containing horse

serum. Other examples of skeletal muscle differentiation seen throughout the literature in mice

show that the time it takes for a satellite cell to activate, become a myoblast and then fuse into a

myotube and expresses MHC can take approximately 7 days [36]. It makes sense therefore that

in traditional 2D cell culture with a mouse skeletal muscle cell line that this timeline is similar.

However, in 3D it has been shown in other skeletal muscle models that the differentiation time

can take longer when attempting to validate and maintain a functional muscle tissue or when

utilizing a textured substrate in attempt to direct cellular alignment[16], [20], [37].

A variety of methods to define and calculate fusion of myoblasts into multinucleated

myofibers have been established and specific metrics of the analysis usually depend on the

sample size and investigative question [33], [37]. To investigate multinucleation over time, the

Harmony Analysis software used the positive MHC signal to define the borders of the cell region

and then quantified the number of nuclei within that cell region. The MHC signal only just began

to increase enough to identify these areas at the Day 6 time point which is why only Days 6-14

were included in the multinucleation analysis. Utilizing an additional fluorescently labeled

59

antibody for a marker such as actin would be an improvement on this method and would capture

the individual cells present in the earlier time points because actin is found throughout all

skeletal muscle cells at varying stages of proliferation and differentiation. Even though early

time points of Days 1-3 could not be analyzed, an increase in fusion was seen from Day 6-14

(Fig 23). Significant differences were observed in multinucleation between each time point

analyzed, providing additional evidence of myogenesis occurring in the microtissues (Fig 23).

Total nuclei quantities decreased over time in the C2C12 microtissues (Fig 24). During

myogenesis, myoblasts have the choice to commit to the myotube formation process, revert back

to a satellite cell precursor, or undergo apoptosis[38], [39]. It is hypothesized that in these 3D

microtissues, many myoblasts underwent apoptosis prior to committing to the differentiation

process and were washed away during media exchanges, thus lowering the total cell counts by

the latest time point. The microtissue continued to develop a non-uniform bleb area containing a

high concentration of cells compared to the thinner tissue areas throughout the study timeline

(Fig 24). Allowing the microtissue to grow out to 21 days and further analyzing this area is

needed to provide additional insight into the role this morphology plays in the microtissue.

Figure 24. Total nuclei decrease over time in C2C12 toroid microtissues (A). Nuclei increase in

the thin and bleb regions of C2C12 microtissues (B). Nuclei displayed as mean ± SD.

60

4.3 H&E Staining Demonstrates Morphology in C2C12 3D Toroid Microtissues

Introduction

Hematoxylin and eosin (H&E) staining has been a useful technology for many years that

is widely utilized to analyze tissue health and morphology. During this staining process, the

hematoxylin stains nucleic acids within the tissue a dark blue and the eosin stains proteins of the

cytoplasm and extracellular matrix in pink[40]. This visualization of cellular structure provides

vital information about the overall health of the tissue of interest. C2C12 toroid microtissues

were investigated by H&E staining for tissue health and signs of cellular fusion events from

single nucleated myoblasts to multinucleated myotubes.

Experimental Design

C2C12 toroid microtissues were allotted for H&E staining from the group of microtissues

that were grown and maintained in SFMA discussed in Chapter 4.2. Duplicate microtissues from

each time point (Day 1, 3, 6, 10, & 14) were prepared for and processed by paraffin embedding,

H&E staining, microtome sectioning and slide mounting according to the methods outlined in

Chapter 2.9. Slides were entered in the Leica ScanScope and images of the tissue slices were

documented.

Results

C2C12 toroid microtissues were assessed for tissue health and overall morphology at

each time point. The earliest time point of Day 1 showed the most uniform tissue morphology

whereas by Day 6 there was a large abnormal bleb area of present in part of the tissue (Fig 25).

The thinnest part of the microtissue on Day 6 showed evidence that single nucleated cells shown

in earlier time points had fused into a multinucleated myotube. Multinucleation continued to

increase by the latest time point of Day 14 as well as an increased bleb area (Fig 25).

61

Figure 25. H&E staining shows overall morphology changes and tissue health in C2C12 toroid microtissues over time. Day 1

demonstrates uniform morphology while Day 6 and Day 14 show morphology changes and increased multinucleation of myotube

formation. Scale bar = 100µm.

62

Discussion

The use of H&E staining to investigate additional signs of myogenesis proved to be very

helpful. The staining showed overall tissue morphology and gave insight into the cellular activity

occurring in the bleb area as well as the thinner more aligned part of the microtissue. Within the

bleb space there are some signs of apoptosis as well as multinucleation. In order to decrease

apoptosis, further optimization of cell seeding density to include less cells and an increase in

media volume to the tissue is recommended. The multinucleation visibly demonstrated by H&E

beginning in the Day 6 time point correlates with the quantitative output from Harmony Analysis

showing multinucleation at Day 6 and further increasing out to Day 14. These results provide

evidence that the microtissues were undergoing differentiation and myogenesis, following the in

vivo process needed for formation of adult skeletal muscle.

Chapter 5: Conclusions & Future Directions

5.1 Conclusions

Optimization of media formulations played a critical role in the ability to investigate the

biological process of myogenesis in C2C12 self-assembled microtissues and it was successfully

determined that the current SFMA formulation is effective for tissue formation and stability. At

the time of this writing, only one other investigation into C2C12 myogenesis using s 3D non

adherent tissue formation platform to form toroid shaped microtissues is known[41]. These

investigators also used a similar seeding density of 350,000 cells/ toroid and determined that a

low serum feeding regiment was necessary to maintain microtissue in 3D, however they did not

investigate myogenesis past 7 days[41]. The instability seen in the microtissues when analyzed

out to a long time point of 14 days is possibly caused by too high of a cell seeding density, a

63

limitation in nutrient availability from media due to the small media volumes that the wells can

currently hold and the fact that skeletal muscle cells are naturally a very dynamic and contractile

cell type, rather than the type of media formulation used to maintain the tissues. Most all other

skeletal muscle investigations in 3D utilize a scaffold or adherent platform for the cells to attach

on to and while it is known that skeletal muscle cells do need attachment in vivo it is interesting

to investigate their behavior in a model strictly focused on self-assembly. The data reported in

this thesis that when allowed to self-assemble with no adherent platform and with only one cell

type (C2C12) the process of myogenesis was not significantly increased until 10 days post tissue

formation (Fig 20), (Fig 21).

Modeling the baseline process of myogenesis which occurs in normal skeletal muscle cell

development and after an exercise induced injury, is relevant in in vitro model development

because it can act as a control when studying muscle related disease and exercise induced injury

using in vitro models in the lab. It is important to know that if this were used as a 3D control

model that this critical biological process would not be detectable until 10-14 days post seeding.

5.2 Future Directions: Biological

It has been demonstrated in the literature that supportive fibroblast cell types can be

grown and maintained in self assembled toroid microtissues, align during this process and

produce ECM characteristics in this system[23], [24]. Fibroblasts are shown to make up part of

the cell population surrounding skeletal muscle and co-culture systems of skeletal muscle cells

and fibroblasts in vitro have been reported in other 3D models to be successful at inducing

myogenesis [20], [39], [40]. Therefore, it may be of interest to investigate a co-coculture model

of C2C12 cells with fibroblast cells. The production of ECM proteins such as collagen could

64

assist in stability of the microtissue and potentially induce myogenesis to occur earlier than the

10-day mark seen in these data here.

In addition to using a smaller seeding density of C2C12 cells and changing the agarose

mold design to accommodate more media, another design modification could include a larger

peg diameter. The larger peg might help eliminate the bleb area of the tissue by providing the

cells with a larger circumferential area to form around during the seeding process. This increase

in space may also help reduce signs of apoptosis since so many cells would no longer be

concentrated in the bleb area of the microtissue at the later time points.

In addition to analyzing MHC as a marker for late stage cellular differentiation,

investigation into other resident cell types and characteristics within the microtissues would

provide insight into the behavior of the C2C12 microtissue platform and its usefulness as a

control model for normal skeletal muscle behavior. Ki-67, a known marker of cellular

proliferation could be used to stain and detect resident cells in the microtissue population that

have not undergone differentiation into adult myofibers[41], [42]. It is known throughout skeletal

muscle biology that a resident group of satellite cells is always present among the adult skeletal

muscle population waiting to activate the differentiation process if the need arises after an

exercise or injury[26], [27] It would also be interesting to determine and quantify the resident

satellite cell population present in C2C12 skeletal muscle toroid microtissues. This analysis

could be accomplished also using immunohistochemistry and quantitative confocal microscopy

by staining for a satellite cell specific adhesion protein called M-cadherin [36], [41], [43].

It was relevant to utilize the C2C12 skeletal muscle cell line for investigation of

myogenesis in 3D because it was and still is a widely utilized cell line for in vitro research

related to skeletal muscle. However, as more and more in vitro models are steering towards

65

utilizing human cell lines, it would be appropriate to investigate myogenesis in self assembled

3D toroid microtissues seeded with human skeletal muscle cell lines such as the HSMM, SkC-L

or SkMC-P cell lines, all of which have recently been characterized by their ability to undergo

myogenesis in 2D[30].

5.3 Future Directions: Technology

Advancements in 3D confocal imaging have allowed for high throughput analysis

opportunities critical to drug discovery and disease intervention allowing scientists to collect

large quantities of data very quickly. There are however some limitations that were discovered

after performing the data acquisition for this thesis. The Harmony 3D Analysis software has the

amazing capability of being able to image through multiple Z planes of a microtissue however, it

is limited in that it cannot image as well through the agarose gel because the thickness of the gel

is longer than the working distance of the objectives used to capture images on the Opera Phenix.

As a result, toroid microtissues had to be removed from the gels and placed in 96 well glass

bottom Grenier plates containing PBS for imaging. As the experimental timeline progressed and

the microtissues became less uniform, the sample weight was no longer consistent. This

unevenness in sample morphology caused the microtissue to tip unevenly in the imaging well.

The Opera Phenix was still able to image through the entire tissue, but this unevenness resulted

in non-level Z planes, making it very difficult to do any type of quantitative analysis in 3D

Harmony that investigated MHC production as a specific function of Z depth in the tissue. In

addition to optimizing the biological components of the tissue to obtain a more uniform tissue, it

would be useful to utilize some type of weight, adhesive, or even mounting medium that would

ensure the microtissue lies flat in the 96 well plate when it’s time for confocal imaging.

66

One of the most interesting features about the process of myogenesis is the fact that

single nucleated cells can fuse into multinucleated cells. The multinucleation protocol in

Harmony was able to detect this biological phenomenon and produce quantitative data from the

confocal images however due to current limitations in the software, this application is only

available for 2D samples (Appendix 1). In order to generate this data, individual Z planes were

isolated from Z stacks and processed through the protocol as a 2D image. An optimization in the

software platform that can process an entire Z stack at one time and thus be applied to entire 3D

microtissues would be very beneficial to the data analysis pipeline and ensure that researchers

are able to capture the biological response of an entire sample set.

67

References

[1] E. N. Marieb, Human Anatomy & Physiology, Sixth. 2006.

[2] N. I. of N. D. and Stroke, “Muscular Dystrophy: Hope Through Research.” [Online].

Available: https://www.ninds.nih.gov/Disorders/Patient-Caregiver-Education/Hope-

Through-Research/Muscular-Dystrophy-Hope-Through-Research.

[3] P. B. Shieh, “Emerging Strategies in the Treatment of Duchenne Muscular Dystrophy,”

Neurotherapeutics, pp. 840–848, 2018.

[4] A. Association, “No Title.” [Online]. Available: http://www.alsa.org/about-als/what-is-

als.html.

[5] D. Petrov, C. Mansfield, A. Moussy, and O. Hermine, “ALS clinical trials review: 20

years of failure. Are we any closer to registering a new treatment?,” Front. Aging

Neurosci., vol. 9, no. MAR, pp. 1–11, 2017.

[6] K. O’Fallon, “Technical Plan-Nutritional Factors that Promote Immune Function and

Muscle Recovery,” 2018.

[7] B. H. Jones, M. Canham-Chervak, S. Canada, T. A. Mitchener, and L. S. Moore, “Medical

surveillance of injuries in the U.S. Military: Descriptive epidemiology and

recommendations for improvement,” Am. J. Prev. Med., vol. 38, no. 1 SUPPL., pp. S42–

S60, 2010.

[8] Y. Blat and S. Blat, “Drug discovery of therapies for duchenne muscular dystrophy,” J.

Biomol. Screen., vol. 20, no. 10, pp. 1189–1203, 2015.

[9] V. Picher-Martel, P. N. Valdmanis, P. V Gould, J.-P. Julien, and N. Dupre, “From animal

models to human disease: a genetic approach for personalized medicine in ALS,” Acta

Neuropathol. Commun., vol. 4, no. 70, pp. 29–34, 2016.

68

[10] R. Tatsumi, S. M. Sheehan, H. Iwasaki, A. Hattori, and R. E. Allen, “Mechanical stretch

induces activation of skeletal muscle satellite cells in vitro,” Exp. Cell Res., vol. 267, no.

1, pp. 107–114, 2001.

[11] S. K. Tsivitse, “Mechanical loading and injury induce human myotubes to release

neutrophil chemoattractants,” AJP Cell Physiol., vol. 288, no. 3, pp. C721–C729, 2004.

[12] “FlexCell International Corporation.” [Online]. Available:

http://www.flexcellint.com/index.html.

[13] J. G. Tidball and S. A. Villalta, “Regulatory interactions between muscle and the immune

system during muscle regeneration,” Am J Physiol Regul Integr Comp Physiol, vol. 298,

pp. 1173–1187, 2010.

[14] K. P. Goetsch and C. U. Niesler, “Optimization of the scratch assay for in vitro skeletal

muscle wound healing analysis,” Anal. Biochem., vol. 411, no. 158–160, p. 249, 2010.

[15] S. Breslin and L. O’Driscoll, “Three-dimensional cell culture: The missing link in drug

discovery,” Drug Discov. Today, vol. 18, no. 5–6, pp. 240–249, 2013.

[16] H. Vandenburgh et al., “Drug-screening platform based on the contractility of tissue-

engineered muscle,” Muscle and Nerve, vol. 37, no. 4, pp. 438–447, 2008.

[17] C. A. Powell, B. L. Smiley, J. Mills, and H. H. Vandenburgh, “Mechanical stimulation

improves tissue-engineered human skeletal muscle,” AJP Cell Physiol., vol. 283, no. 5,

pp. C1557–C1565, 2002.

[18] M. Beldjilali-Labro et al., “Biomaterials in tendon and skeletal muscle tissue engineering:

Current trends and challenges,” Materials (Basel)., vol. 11, no. 7, 2018.

[19] A. Bettadapur et al., “Prolonged Culture of Aligned Skeletal Myotubes on Micromolded

Gelatin Hydrogels,” Sci. Rep., vol. 6, pp. 1–14, 2016.

69

[20] H. Takahashi, T. Shimizu, and T. Okano, “Engineered Human Contractile Myofiber

Sheets as a Platform for Studies of Skeletal Muscle Physiology,” Sci. Rep., 2018.

[21] J. R. Morgan, “Microtissues.com.” [Online]. Available: https://www.microtissues.com/.

[22] T. M. Achilli, S. McCalla, J. Meyer, A. Tripathi, and J. R. Morgan, “Multilayer spheroids

to quantify drug uptake and diffusion in 3D,” Mol. Pharm., vol. 11, no. 7, pp. 2071–2081,

2014.

[23] J. Y. Schell et al., “Harnessing cellular-derived forces in self-assembled microtissues to

control the synthesis and alignment of ECM,” Biomaterials, vol. 77, pp. 120–129, 2016.

[24] B. T. Wilks, E. B. Evans, M. N. Nakhla, and J. R. Morgan, “Directing fibroblast self-

assembly to fabricate highly-aligned, collagenrich matrices,” Acta Biomater., 2018.

[25] K. L. Manning, A. H. Thomson, and J. R. Morgan, “Funnel-Guided Positioning of

Multicellular Microtissues to Build Macrotissues,” Tissue Eng. Part C Methods, vol. 24,

no. 10, pp. 557–565, 2018.

[26] C. F. Bentzinger, Y. X. Wang, and M. A. Rudnicki, “Building Muscle : Molecular

Regulation of Myogenesis 2 . MORPHOGEN GRADIENTS AND MYOGENESIS,” pp.

1–19, 2014.

[27] A. Musarò, “The Basis of Muscle Regeneration,” Adv. Biol., vol. 2014, no. Table 1, pp. 1–

16, 2014.

[28] I. Grabowska, A. Szeliga, J. Moraczewski, I. Czaplicka, and E. Brzóska, “Comparison of

satellite cell-derived myoblasts and C2C12 differentiation in two- and three-dimensional

cultures: changes in adhesion protein expression,” Cell Biol. Int., vol. 35, no. 2, pp. 125–

133, 2011.

[29] “ATCC C2C12 (ATCC® CRL-1772TM).” [Online]. Available:

70

https://www.atcc.org/Products/All/crl-1772.aspx#culturemethod.

[30] J. Owens, K. Moreira, and G. Bain, “Characterization of primary human skeletal muscle

cells from multiple commercial sources,” Vitr. Cell. Dev. Biol. - Anim., vol. 49, no. 9, pp.

695–705, 2013.

[31] T. Achilli, J. Meyer, and J. R. Morgan, “Cellular Spheroids,” Expert Opin Biol Ther, vol.

12, no. 10, pp. 1347–1360, 2012.

[32] H. Fujita, A. Endo, K. Shimizu, and E. Nagamori, “Evaluation of serum-free

differentiation conditions for C2C12 myoblast cells assessed as to active tension

generation capability,” Biotechnol. Bioeng., vol. 107, no. 5, pp. 894–901, 2010.

[33] A. Shima, J. Pham, E. Blanco, E. R. Barton, H. L. Sweeney, and R. Matsuda, “IGF-I and

vitamin C promote myogenic differentiation of mouse and human skeletal muscle cells at

low temperatures,” Exp. Cell Res., vol. 317, no. 3, pp. 356–366, 2011.

[34] F. Pampaloni, E. Reynaud, and E. Stelzer, “The third dimension bridges the gap between

cell culture and live tissue,” Nat. Rev. Mol. Cell Biol., vol. 8, no. 10, pp. 839–845, 2007.

[35] D. Gawlitta, K. J. M. Boonen, C. W. J. Oomens, F. P. T. Baaijens, and C. V. C. Bouten,

“The Influence of Serum-Free Culture Conditions on Skeletal Muscle Differentiation in a

Tissue-Engineered Model,” Tissue Eng. Part A, vol. 14, no. 1, pp. 161–171, 2008.

[36] P. S. Zammit et al., “Kinetics of myoblast proliferation show that resident satellite cells

are competent to fully regenerate skeletal muscle fibers,” Exp. Cell Res., vol. 281, no. 1,

pp. 39–49, 2002.

[37] P. Y. Wang, H. Te Yu, and W. B. Tsai, “Modulation of alignment and differentiation of

skeletal myoblasts by submicron ridges/grooves surface structure,” Biotechnol. Bioeng.,

vol. 106, no. 2, pp. 285–294, 2010.

71

[38] L. M. Schwartz, “Skeletal Muscles Do Not Undergo Apoptosis During Either Atrophy or

Programmed Cell Death-Revisiting the Myonuclear Domain Hypothesis,” Front. Physiol.,

vol. 9, no. January, pp. 1–8, 2019.

[39] L. M. Schwartz, “Atrophy and programmed cell death of skeletal muscle,” Cell Death

Differ., vol. 15, no. 7, pp. 1163–1169, 2008.

[40] R. D. Cardiff, C. H. Miller, and R. J. Munn, “Manual hematoxylin and eosin staining of

mouse tissue sections,” Cold Spring Harb. Protoc., vol. 2014, no. 6, pp. 655–658, 2014.

[41] J. Krieger, B.-W. Park, C. R. Lambert, and C. Malcuit, “3D skeletal muscle fascicle

engineering is improved with TGF-β1 treatment of myogenic cells and their co-culture

with myofibroblasts,” PeerJ, vol. 6, p. e4939, 2018.

72

Appendix 1: Harmony Analysis Protocols

73

74

Appendix 1 cont’d

75

Appendix 2: H&E Staining & Paraffin Embedding Protocols

H&E Staining Schedule

Xylene 5 MIN

Xylene 5 MIN

100% Ethanol approximately 10 dips

95% Ethanol “ “

70% Ethanol “ “

H20 dip until water sheets off (a few seconds)

1Hematoxylin 2 2 min. (specific to the hematoxylin we use)

Tap water rinse under running water, until clear, about 1 min.

*Acid alcohol 2 or 3 QUICK dips (too much will fade stain)

Tap water rinse under running water, until clear, about 1 min

**Ammonium hydroxide about 10 dips, or until desired blue color is reached

Tap water rinse under running water, until clear, about 1 min

95% Ethanol approximately 10 dips

2Eosin Y 4 seconds

95% Ethanol About 10 dips

100% Ethanol “

100% Ethanol “

All Xylenes (3x) 5 min. each

Slides can remain in last xylene while you are cover slipping.

*0.5% HCL in 70% ETOH

**0.2% aqueous solution

1Hematoxylin 2 – Richard Allan brand, Fisher #22-050-113

2Eosin Y – Richard Allan brand, Fisher #22-050-110

76

Appendix 2 cont’d

Paraffin Embedding

70% Ethanol 1 Hour

95% Ethanol 45 Minutes

95% Ethanol 1 Hour

100% Ethanol 45 Minutes

100% Ethanol 1 Hour

100% Ethanol 1 Hour

Xylene 45 Minutes

Xylene 45 Minutes

Xylene 1 Hour

Paraplast Paraffin (@ 60°) 1 Hour

Paraplast Paraffin (@ 60°) 1 Hour

Paraplast Paraffin (@ 60°) 1 Hour 15 Minutes