The world leader in serving science Stem Cells and Cell Culture Developments Within ThermoFisher Scientific Katrina Kidd European Application Scientist Cellomics ThermoFisher Scientific
A Typical Life Science Research Workflow Thermo Scientific has a powerful tool set for cutting-edge life science research Cell Culture Gene Modulation Nucleic Acid Detection & Analysis Protein Sample Prep Detection & Analysis High Content Analysis 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 2
Cellomics within the Life Science Workflow 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 3
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 4
Workflow: Stem Cells Complex, sequential, parts iterative, long timelines, regulatory, ethical environment 5
Collection / Preparation - Cells Reliable, consistent source Characterized Easy access Relevant species (Mouse, human etc) Meet regulatory guidelines Disease specific 6
Murine ESC (mesc) Stem Cells 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. 7
Impact of mesc Platform Transgenic and Knockout Mouse production Researchers studying stem cell self-renewal and differentiation Drug discovery 8
Human (Non Embryonic) 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. 9
Impact Human Stem Cell Lines Source Application Customer Wharton s Jelly Adipose Derived Amniotic derived Bone Marrow Amniotic Epithelial Stem Cells Multipotent Cord Blood Unrestricted Somatic Stem Cells Most easily isolated, grow well, multi lineage differentiation potential to to neurons, adipocytes and osteocytes, hepatocytes Retain embryonic stem cell characteristics, can be differentiated into pancreatic islet cells, liver hepatocytes, cardiomyocytes and neurons. Different to mesenchymal unrestricted differentiation potential, neurons, osteocytes, hepatocytes Stem cell core facilities Drug discovery labs Stem cell core facilities Drug discovery labs 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 10
Stem Cell Expansion / Differentiation Reliable expansion without differentiation Controlled differentiation to various lineages Animal free / serum free Reliably Inducing pluripotency Eliminate feeder layers Cell harvesting Characterizing cells 11
Expansion - Thermo Scientific AdvanceSTEM Platform 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 12
Differentiation - Thermo Scientific AdvanceSTEM Platform 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 13
Pluripotent cells : a source for regenerative medicine Embryonic Stem cells (ES) -Mouse mesc -Human hesc: Regulatory restrictions Induce pluripotent stem cells (ips) Reprogramming factors Oct4 Klf4 Sox2 c-myc fibroblasts Biopsy 5 Blastocyst Inner cell mass 3weeks Feeder cell layer Unreprogrammed fibroblasts hes Colony ips colony Reubinoff et al., 2000 Wild type or mutant cells Yamanaka., 2007 14
Reliably generating induced pluripotent stem cells (ipsc) 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 15
Thermo Scientific vips Kit 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 16
Impact of vips technology Creation of ips cells -> chimeric pigs 18 living pigs Impact üclear validation of vips technology ürobust, reproducible approach üaccelerate Research into diseases üregenerative medicine (diabetes) üanimal husbandry 17
Expansion Extracellular Matrices (ECM) 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 18
Thermo Scientific HyStem Hydrogels Build your own ECM Add growth factors / attachment factors Animal free Consistency (synthetic product) 2D and 3D cell culture Highly customizable 19
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 20
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 21
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 22
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) 23
Application - Single Cells UpCell Surface versus trypsinization and scraping in cell harvest A-549 Lung cancer cell line 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 Normal culture at 37 o C 30 min postcooling at 20 o C 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 Recovery rate of mouse macrophages 24
Thermo Scientific Nunc HydroCell Surfaces What is the HydroCell surface? Superior Low Cell Binding surface for minimal cell adhesion and protein adsorption 25
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. 26
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 Embryoid body formation of hes cells HydroCell Non-treated polystyrene Petri dish 27
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 28
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 29
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 30
Thermo Scientific AdvanceSTEM Literature Available Material Public: Product Brochures Product Inserts Protocols COAs All literature can be found on CSI 31