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(A)For Cell Lines Refractory to Transition, rhLaminin-521 Provides Optimum Cell Survival Post-Transition from KSR-Based Medium System to CTS™ Essential 8™
(B)PSCs Can Subsequently Be Transferred to CTS™ rhVTN-Nfor Remainder of Culture
RESULTS
Figure 4. PSCs Cultured in CTS™ Essential 8™/ CTS™ rhVTN-N System Maintain Pluripotency
ABSTRACT
Pluripotent stem cell (PSC) culture using the xeno-free Essential 8™ Medium/truncated recombinanthuman Vitronectin system has been shown to supportnormal PSC properties and provide a large pool ofcells for disease modeling and drug development. Asresearch moves from translational to clinical research,general regulatory guidance from the US Food andDrug Administration (FDA) indicates that, cGMPmanufactured, or clinical grade reagents should beused whenever available as ancillary reagents tominimize downstream risk to patients. Thus, wesought to identify regulatory compliant, animal-origin-free alternatives for growth factors contained withinthe Essential 8™ Medium, producing a qualifiedancillary system for PSC expansion. Here we presentdata to support a seamless transition from the xeno-free Essential 8™ Medium system to the Cell TherapySystems (CTS™) animal-origin free system.Compatibility is shown with existing cGMP-manufactured passaging reagents: Versene Solutionfor clumped cell passaging and CTS™ TrypLE™Select combined with RevitaCell™ Supplement forsingle cell passaging. Upon expansion, PSCs areshown to maintain normal PSC properties, includingmorphology, pluripotency, karyotype, and trilineagedifferentiation potential. Together this system providesa consistent, feeder-free PSC culture medium fortranslational and clinical research.
INTRODUCTION CONCLUSIONS• The CTS™ Essential 8™/CTS™ rhVTN-N system provides
a seamless transition from bench to clinic, providing long-term maintenance of normal PSC properties
• This CTS™ system provides reliable ancillary reagents forPSC culture upstream of manufacturing of cell, gene, ortissue-based products.
• The CTS™ system is compatible with existing reagentsincluding RevitaCell™ Supplement for post-thaw recoveryand single cell passaging applications, as well ascompatible with Gibco™ differentiation kits.
• Please contact [email protected] [email protected] for additionalinformation, including additional scale-up protocols using thismedium system.
TRADEMARKS/LICENSING© 2018 Thermo Fisher Scientific Inc. All rights reserved. All trademarks arethe property of Thermo Fisher Scientific and its subsidiaries unlessotherwise specified. Essential 8 is a trademark of Cellular DynamicsInternational, Inc. TaqMan is a trademark of Roche Molecular Systems.For Research Use or Manufacturing of Cell, Gene, or Tissue-BasedProducts. CAUTION: Not intended for direct administration into humans oranimals.
Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
Mohan Vemuri, Ph.D., Lauren E. Sangenario, M.S., Rhonda A. Newman, Ph.D., and David T. Kuninger, Ph.D.,Thermo Fisher Scientific, 7311 Governor’s Way, Frederick, MD 21704
Development of Feeder-Free PSC Culture SystemEnabling Translational & Clinical Research
Figure 1. RUO to CTS™ Essential 8™ Media Conversion
To provided a seamless transition from research to translationalneeds, the CTS™ Essential 8™ Medium is formulated with animalorigin free growth factors and undergoes increased quality standardsas highlighted in the table above.
Figure 2. Simple Transition options to transfer from RUO to CTS™ PSC Culture Systems
Cultures previously cultured in the RUO PSC culture system caneasily be transitioned to the CTS™ Essential 8™/ CTS™ rhVTN-Nsystem using the above transition schemes.
OCT4 SSEA4 DAPI Merge
TRA1-60 SOX2 DAPI Merge
H9 ESCs cultured in CTS™ Essential 8™ Medium on CTS™ rhVTN-Nwere propagated for >30 passages using Versene Solution forpassaging. PSCs were shown to maintain normal pluripotency asassessed using the Pluripotent Stem Cell 4-MarkerImmunocytochemistry Kit (Cat. No. A24881).
Figure 5. PSCs Cultured in CTS™ Essential 8™/ CTS™ rhVTN-N System Maintain Normal Karyotype
Figure 9. rhLaminin-521 Can Be Used to Support Transition from Feeder-Dependent KSR-Based System to CTS™ Essential 8™/ CTS™ rhVTN-N System
(A) Feeder-Dependent iPSCs were collagenase passaged according toEssential 8™ Adaptation Kit protocol and seeded on various extracellularmatrices for recovery in CTS™ Essential 8 Medium. rhLaminin-521 wasshown to support optimum transition of challenging PSCs. (B) Cells aresubsequently Versene passaged onto CTS™ rhVTN-N for the remainderof culture.
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0.5 ug/cm2 CTS™ rhVTN-N, From 0.5 ug/cm2 rhLaminin-521
(A) Post-Thaw Transition
Cryopreserved PSCs previously cultured in
RUO Essential 8™
Recover in CTS™ Essential 8™
+/- RevitaCell™ Supplement
Thaw
(B) Post-Passage Transition
PSCs cultured in RUO Essential 8™
Recover in CTS™
Essential 8™
VersenePassage
Figure 3. PSCs Cultured in CTS™ Essential 8™/ CTS™ rhVTN-N System Maintain Morphology Comparable to RUO System
Essential 8™ Medium
CTS™ Essential 8™ Medium
Gibco™ Human Episomal iPSCs cultured in CTS™ Essential 8™Medium on CTS™ rhVTN-N were propagated for 10 passages usingVersene Solution for passaging. PSCs were shown to maintain normalmorphology as assessed by phase contrast imaging.
(A) Qualitative ICC Analysis
(B) Quantitative ICC Analysis
Red = pluripotent reference set Blue = non-pluripotent reference set
(C) Pluritest™ Pluripotency PlotGibco™ Human Episomal iPSCs culturedin CTS™ Essential 8™ Medium on CTS™rhVTN-N were propagated for 3 passagesusing Versene Solution for passaging.PSCs were shown to maintain normalpluripotency as assessed by (B)Quantitative ICC using the Cellomics™CellInsight™ and (C) Pluritest™, an open-access bioinformatic assay forpluripotency of human cells based upontheir gene expression profiles.
Essential 8™ Medium
CTS™ Essential 8™ Medium
(A) G-Band Karyotype
(B) KaryoStat™ Assay
Gibco™ Human Episomal iPSCs culturedin CTS™ Essential 8™ Medium on CTS™rhVTN-N were propagated using VerseneSolution for passaging. PSCs were shownto maintain normal karyotype as assessed(A) at P10 via G-Band KaryotypeAssessment and (B) at P5 via Karyostat™Assay .
Figure 6. PSCs Cultured in CTS™ Essential 8™/ CTS™ rhVTN-N System Maintain Trilineage Differentiation Potential
(A) Trilineage Differentiation Potential Maintained As Assessed by TaqMan™ hPSC Scorecard™ Panel
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(B) Trilineage Differentiation Potential Maintained As Assessed by Directed Differentiation Using Thermo FisherScientific Kits
CXCR4 TNNT2NKX2.5
NESTINSOX2
OTX2FOXA2
Gibco™ PSC DefinitiveEndoderm Induction Kit
Gibco™ PSC CardiomyocyteDifferentiation Kit
Gibco™ Neural Induction Medium
Gibco™ DopaminergicNeuron Differentiation Kit
Following expansion of PSCs in CTS™ Essential 8™ Medium onCTS™ rhVTN-N using Versene Solution for passaging, PSCs wereevaluated for maintenance of trilineage differentiation potential via twomethods. (A) Random differentiation of PSCs with subsequentevaluation using the TaqMan™ hPSC Scorecard™ Panel. (B)Directed differentiation using kits available from Thermo FisherScientific: differentiation to the endodermal lineage using the Gibco™PSC Definitive Endoderm Induction Kit (Cat. No. A3062601),differentiation to mesodermal lineage using the Gibco™ PSCCardiomyocyte Differentiation Kit (Cat. No. A2921201), and finally tothe ectodermal lineage using the Gibco™ Neural Induction Medium(Cat. No. A1647801) and the Gibco™ Dopaminergic NeuronDifferentiation Kit (Cat. No. A3147701).
Fibroblast culture Reprogramming Growth Passaging Banking/
Recovery
CTS KnockOut Serum
Replacement XF
CTS CytoTune 2.1 Reprogramming Kit
VTN-N matrix Versene CTS Synth-a-freeze
rhLaminin-521CTS TrypLE Select
with RevitaCell Supplement
during recovery
PSC Cryopreservation
Kit
RevitaCell Supplement
CharacterizationPSC Live Staining Kits
PSC Immunocytochemistry Kits3-Germ Layer Immunocytochemistry Kit
TaqMan hPSC Scorecard Panel—for assessing tri-lineage differentiation potentialPrimeView Gene Expression Assay (PluriTest compatible)—for testing pluripotency
KaryoStat Assays—for checking genomic stability
Cellular
Molecular
Figure 7. Products Compatible with CTS™ Essential 8™ Medium
Gibco™ Human Episomal iPSC Linecultured in CTS™ Essential 8™Medium on CTS™ rhVTN-N waspassaged using TrypLE™ Select andrecovered at 25K viable cells/cm2 inCTS™ Essential 8™ on CTS™rhVTN-N (A) including (greentriangles) or excluding (blue circles)RevitaCell™ Supplement (Cat. No.A26445-01) for the first 24 hours post-passage. Media was exchanged 24hours post-passage with CTS™Essential 8™ Medium alone.
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Figure 8. RevitaCell™ Supplement Provides Support When Single Cell Passaging is Required
CTS™ Essential 8™ Medium + RevitaCell™Supplement for 1st
24 Hours
CTS™ Essential 8™ Medium Alone
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