The world leader in serving science

Stem Cells and Cell Culture

Developments Within

ThermoFisher Scientific

Katrina Kidd

European Application Scientist

Cellomics

ThermoFisher Scientific

2

Cell Culture

Gene Modulation

Nucleic Acid Detection &

Analysis

Protein Sample Prep Detection &

Analysis

High Content Analysis

Thermo Scientific has a powerful tool set for cutting-edge life science research

Growing Cells for

an experiment.

• HyClone Serum

• HyClone Media

•Stem Cell Lines

•AdvanceSTEM

•Hydrogels

Instructions for a

protein – turning

genes on or off to

understand their

function in a cell.

Turn the gene down::

• Dharmacon RNAi

Technologies

• Open Biosystems

RNAi Technologies

Turn the gene up:

• cDNA

• ORF Expression

Vectors

How much of a

specific DNA or

RNA is in the

sample?

• ABgene PCR &

QPCR Reagents

Plastics for PCR

and sample

storage

• ABgene PCR &

QPCR Plastics

• Finnzymes

• Fermentas

Is the protein of

interest in my

sample. If so,

how much and

what is its

function?

• Pierce Protein

Research

• Affinity

BioReagents

• Open

Biosystems

Custom

Antibodies

Using imaging &

fluorescent

reagents to

monitor changes

in whole cells.

• Cellomics

Reagents,

Instruments, and

Software

• BioImage

Redistribution

Assays

A Typical Life Science Research Workflow

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Cell Culture

Gene Modulation

Nucleic Acid Detection &

Analysis

Protein Sample Prep Detection &

Analysis

Analysis & Characterization

•Growth

•Expansion

•Differentiation

•Cell Banking

Detection (labeling)

Assays

Quantification

Decisions

LSR-Cellomics focuses on 2 parts of the Life Science workflow

Cellomics within the Life Science Workflow

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Thermo Scientific Portfolio for Cellomics

GROW

Classical media

Serum

Specialty Serum & Media

ECM

Cells

Stem cell reprogramming (iPSC)

DETECT

GFP reporter cell lines

Immunofluorescence antibodies /kits

ANALYZE

High content imaging

Image analysis

DISCOVER

Bioinformatics

Services

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Complex, sequential, parts iterative, long timelines, regulatory, ethical

environment

Workflow: Stem Cells

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Reliable, consistent

source

Characterized

Easy access

Relevant species (Mouse,

human etc)

Meet regulatory

guidelines

Disease specific

Collection / Preparation - Cells

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SV30109.01 PRX-B6N

derived C57BL/6N blastocyst-stage

embryo

SV30098.01 PRX-129/X1

Derived 129/X1/SvJ blastocyst-stage

embryo.

Characterized

Karyotypically normal male: 40 XY

IMPACT* tested

Germline competent.

ü Reliable, consistent source

ü Characterized

ü Easy access

ü Relevant species (Mouse)

ü Meet regulatory guidelines

mESC platform provided by Primogenix Inc.

Murine ESC (mESC) Stem Cells

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• Transgenic and Knockout

Mouse production

• Researchers studying stem

cell self-renewal and

differentiation

• Drug discovery

Impact of mESC Platform

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Variety of non embryonic stem cell

lines

Can be differentiated into a number

of different cell types

Extensively characterized

Stem cell markers

Purified by Flow Cytometry

HIV, HepA, HepC certified free

NOT FOR THERAPEUTIC USE

ü Reliable, consistent source

ü Characterized

ü Easy access

ü Relevant species (Human)

ü Meet regulatory guidelines

Human non EC platform provided by CET Inc.

Human (Non Embryonic)

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Source Application Customer

Wharton’s Jelly

Adipose Derived

Amniotic derived

Bone Marrow

Most easily isolated, grow well, multi

lineage differentiation potential to to

neurons, adipocytes and osteocytes,

hepatocytes

Stem cell core

facilities

Drug discovery

labs

Amniotic Epithelial

Stem Cells

Retain embryonic stem cell

characteristics, can be differentiated into

pancreatic islet cells, liver

hepatocytes, cardiomyocytes and

neurons.

Stem cell core

facilities

Drug discovery

labs

Multipotent Cord

Blood Unrestricted

Somatic Stem Cells

Different to mesenchymal –

unrestricted differentiation potential,

neurons, osteocytes, hepatocytes

Stem cell core

facilities

Basic stem cell

research

Cord Blood

Hematopoietic Stem

Cells

(CD34+/Cd133+)

Mainly used in animal models to study

immune system, cardiovascular or

circulatory defects, and cancer stem

cell research. Differentiated to

progenitor cells

Stem cell core

facilities

Basic stem cell

research

Cancer research

Impact – Human Stem Cell Lines

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Reliable expansion without

differentiation

Controlled differentiation to

various lineages

Animal free / serum free

Reliably Inducing pluripotency

Eliminate feeder layers

Cell harvesting

Characterizing cells

Stem Cell Expansion / Differentiation

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Product Application Customer / Impact

Low osmolality basal media Growth of mESC – osmolality

media approximates murine

tissue

Any stem cell lab, esp. murine

Reliable growth / expansion

without differentiation

Serum replacement Growth of all ESC Any stem cell lab, esp. murine,

labs researching potential

therapeutic uses

Removes need for serum, works

with Low osmo media

ES Screened Serum Growth of all ESC Any stem cell lab

Optimized for ESC growth, highly

defined

Mesenchymal ExpansionKits

& Amniotic Epithelial

Expansion Kits

Differentiate Mesenchymal Stem

cells, Amniotic Epthelial Cells

Any stem cell lab

Expansion prior to differentiation

– improve yields of cells

Cell retain embryonic

characteristics

Expansion - Thermo Scientific AdvanceSTEM Platform

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Product Application Customer / Impact

Mesenchymal differentiation

kits

Differentiation of

mesenchymal stem cells

Neuronal

Adipocytes

Osteocytes

Chondrogenic

Any stem cell lab

Regenerative research

Clinical / restorative surgery

Geriatric / Aging

Improved differentiation –

enriched cultures

Differentiation - Thermo Scientific AdvanceSTEM Platform

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Pluripotent cells : a source for regenerative medicine

Reubinoff et al., 2000

Blastocyst Inner cell mass

Feeder cell layer

hES Colony

55

fibroblastsBiopsy

3weeks

Reprogramming factors

Oct4 Klf4 Sox2

c-Myc

Unreprogrammedfibroblasts

iPS colony

Yamanaka., 2007Wild type or mutant cells

Embryonic Stem cells (ES)-Mouse mESc

-Human hESc: Regulatory restrictions

Induce pluripotent stem cells (iPs)

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iPS technology relatively new (2006)

Genetic reprogramming of adult cells with “Yamanaka

factors”

Yields of iPS colonies low

Concerns over reprogramming

Avoids ethical concerns

Potential to create disease specific lines

Are they really pluripotent?

Will they differentiate to all tissue types

Most experience with ES cells – will it translate to iPSC

Reliably generating induced pluripotent stem cells (iPSC)

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High Efficiency of iPSC colonies formed (6 pluripotency factors)

Resemble ES cells based on morphology, stem cell markers

Differentiate into other cell types, all 3 germ layers

vIPS protocol very robust – cells every time

Self-inactivating virus

Thermo Scientific vIPS Kit

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Creation of iPS cells -> chimeric

pigs18 living pigs

üClear validation of vIPS technology

üRobust, reproducible approach

üAccelerate Research into diseases

üRegenerative medicine (diabetes)

üAnimal husbandry

Impact

Impact of vIPS technology

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ECM can’t be animal

derived

Need a customised ECM for

difficult to grow cell lines

Looking at 3D stem cell

culture or looking at effects

of physical environment on

stem cell growth

Reliable, consistent ECM for

reliable expansion

Expansion – Extracellular Matrices (ECM)

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Build your own ECM

Add growth factors /

attachment factors

Animal free

Consistency (synthetic

product)

2D and 3D cell culture

Highly customizable

Thermo Scientific HyStem Hydrogels

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Expansion without Differentiation

Reliably expand cells to high numbers

No loss of pluripotency

No differentiation until ready

Animal component free

Feeder layer free

Animal origin free

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Nunclon Vita

Animal component-free surface

for growth of stem cells

No coating or feeder cells

necessary

Supports attachment and colony

formation of human ESC and iPS

cells

Supports growth of pluripotent

human ESC for at least 10

passages

Non-enzymatic release and

dissociation with removal or

ROCK inhibitor

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Thermo Scientific Nunc UpCell Surfaces

What is the UpCell surface? What are the benefits?

• UpCell is a thermoresponsive surface - releases adherent cells by a simple

reduction in temperature

• Enables harvesting of cells with high viability and intact surface proteins for:

• Culture passaging

• Single cell analyses

• Cell transplantation research

• Cell sheet & tissue engineering

• No trypsin need – preserve cell surface proteins

• No scraping – get high cell viability

• 36 month shelf life

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Application – Cell Sheets & 3D Tissue Models

UpCell enables harvested cell sheets to be stacked in

order to form 3D tissue models

• No scaffold – make 3D tissue models without

exogenous material

• Endless possibilities for mixing cell types and

creating 3D co – cultures

Examples of applications using cell sheet engineering

(3D models)

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Application - Single Cells

UpCell Surface versus

trypsinization and scraping in cell

harvest

• Cells that are difficult to detach

by enzymatic or mechanical

methods can be harvested from

UpCell simply by reducing the

temperature of the cell culture

Recovery rate of mouse

macrophages

A-549 Lung cancer cell line

Normal culture

at 37oC

30 min post-

cooling at 20oC

Removal after

slight agitation

• This data compares the recovery of

mouse peritoneal macrophages

harvested from UpCell using

temperature reduction with those

harvested using trypsinization or

scraping

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What is the HydroCell surface?

• Superior Low Cell Binding surface for minimal cell adhesion and protein

adsorption

Thermo Scientific Nunc HydroCell Surfaces

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Application - Single Cells in Suspension

Enables cultivation of cells that are sensitive to activation and

differentiation signals arising from cell adhesion

Macrophages from mouse peritoneum in medium with serum were incubated for 3 days.

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Application – Cell Clusters in Suspension

Certain cell types form cell clusters or spheroids - frequently used in

stem cell biology and cancer biology

• Neural stem cells (neurospheres) are clusters containing stem cells and

differentiated neural cells

• Embryonic stem cells can be differentiated in cluster (embryoid bodies) -

stem cells differentiate into cell types of the three germ layers

Problem with traditional cultureware is cells in the periphery of the cluster

can adhere - results in unwanted differentiation and lower yield

HydroCell Non-treated polystyrene Petri dish

Embryoid body formation of hES cells

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Cell Growth & Analysis - Nunc Edge Microplate

The next generation in microplate

technology

Perimeter reservoir reduces edge

effect evaporation

Increases assay performance and

efficiency; decreases costs

Superior plate flatness boosts

optical clarity and better results

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Characterization & Analysis

Are these cells

pluripotent

Are these cells the

same as last week

What markers should

I analyze for

“stemness”

What antibodies/dyes

can I use

How can I understand

/ analyze

differentiation

Investigate diseases /

toxicity in relevant cell

lines

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Pain Relief – Cell Analysis / High Content

Platform for cell analysis

• Stem cell biologies

CellInsight – personal cell imager

• Ideal for routine stem cell

characterization

• Screening with ES or iPS derived

disease lines

Arrayscan VTI HCS Reader

• Ideal for research into stem cell

biology

Range of Antibodies, Kits and Dyes

for stem cell markers

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Available Material

• Public:

• Product

Brochures

• Product Inserts

• Protocols

• COAs

Thermo Scientific AdvanceSTEM Literature

All literature can

be found on CSI