stem cells and cell culture developments within thermofisher...
TRANSCRIPT
The world leader in serving science
Stem Cells and Cell Culture
Developments Within
ThermoFisher Scientific
Katrina Kidd
European Application Scientist
Cellomics
ThermoFisher Scientific
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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
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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