hPSC maintenance and expansion
Materials Required
Principle
This protocol maintains and expands human pluripotent stem cells under feeder-free, chemically defined conditions using E8 medium and vitronectin-coated culture surfaces; the readout is sustained adherent colony growth with undifferentiated morphology and retained pluripotency-marker expression during serial passaging[1][2][3][4]. E8-based hPSC culture relies on defined soluble factors and matrix-dependent adhesion rather than feeder cells; vitronectin supports hPSC attachment through integrin-mediated interactions, and EDTA passaging dissociates colonies as small aggregates without enzymatic digestion, centrifugation, or routine ROCK-inhibitor treatment[1][2][5].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Essential 8 medium is used as the chemically defined maintenance medium for hESCs and hiPSCs[1][2].
• Recombinant human vitronectin is used as a defined coating substrate for adherent hPSC attachment and expansion[1][5].
• EDTA dissociation solution is used for enzyme-free passaging of hPSCs cultured in E8-based conditions[1][2].
• Y-27632 may be used only when single-cell dissociation or low-survival recovery is required, because ROCK inhibition reduces dissociation-induced apoptosis in hESCs[6].
• Pluripotency assessment may use antibodies against OCT4 and SSEA4, because these markers were used to evaluate maintenance of undifferentiated hPSC/iPSC cultures in defined E8-based systems[1].
• Use a biosafety cabinet, humidified CO2 incubator, tissue-culture-treated plates, phase-contrast microscope, pipettes, and flow cytometer when morphology monitoring and OCT4/SSEA4 analysis are performed[1][2].
• Recombinant human vitronectin is used as a defined coating substrate for adherent hPSC attachment and expansion[1][5].
• EDTA dissociation solution is used for enzyme-free passaging of hPSCs cultured in E8-based conditions[1][2].
• Y-27632 may be used only when single-cell dissociation or low-survival recovery is required, because ROCK inhibition reduces dissociation-induced apoptosis in hESCs[6].
• Pluripotency assessment may use antibodies against OCT4 and SSEA4, because these markers were used to evaluate maintenance of undifferentiated hPSC/iPSC cultures in defined E8-based systems[1].
• Use a biosafety cabinet, humidified CO2 incubator, tissue-culture-treated plates, phase-contrast microscope, pipettes, and flow cytometer when morphology monitoring and OCT4/SSEA4 analysis are performed[1][2].
Experimental Procedure
• Coat tissue-culture-treated vessels with vitronectin before seeding hPSCs, because E8 culture on vitronectin-coated surfaces supported hESC and hiPSC maintenance, attachment, and expansion in defined feeder-free conditions[1][5].
• Prepare complete E8 medium and EDTA dissociation solution according to the published E8/EDTA culture workflow; the literature supports routine expansion using E8 medium with EDTA-based passaging rather than enzymatic dissociation[1][2].
• Maintain hPSCs as adherent colonies in E8 medium on vitronectin-coated vessels and monitor colony morphology by phase-contrast microscopy before passaging[1][2][5].
• For routine passaging, remove spent medium, apply EDTA dissociation solution to loosen colonies, remove EDTA, and detach cells as small aggregates into fresh E8 medium for reseeding; the published EDTA protocol reports passaging one six-well plate or 10-cm plate in about 6-7 min and avoids enzyme neutralization, centrifugation, and drug treatment[2].
• Use ROCK inhibitor only for workflows involving dissociated single cells or poor post-dissociation survival, because Y-27632 reduced apoptosis after hESC dissociation and increased cloning efficiency in the original study[6].
• Assess successful maintenance by the combination of compact undifferentiated colony morphology, expansion after serial passaging, and pluripotency-marker analysis such as OCT4 and SSEA4 flow cytometry or immunostaining[1][2][5].
• Use negative staining controls for antibody-based flow cytometry or immunostaining, and compare cultures across passages or conditions when evaluating medium, matrix, or passaging changes[1][5].
• Prepare complete E8 medium and EDTA dissociation solution according to the published E8/EDTA culture workflow; the literature supports routine expansion using E8 medium with EDTA-based passaging rather than enzymatic dissociation[1][2].
• Maintain hPSCs as adherent colonies in E8 medium on vitronectin-coated vessels and monitor colony morphology by phase-contrast microscopy before passaging[1][2][5].
• For routine passaging, remove spent medium, apply EDTA dissociation solution to loosen colonies, remove EDTA, and detach cells as small aggregates into fresh E8 medium for reseeding; the published EDTA protocol reports passaging one six-well plate or 10-cm plate in about 6-7 min and avoids enzyme neutralization, centrifugation, and drug treatment[2].
• Use ROCK inhibitor only for workflows involving dissociated single cells or poor post-dissociation survival, because Y-27632 reduced apoptosis after hESC dissociation and increased cloning efficiency in the original study[6].
• Assess successful maintenance by the combination of compact undifferentiated colony morphology, expansion after serial passaging, and pluripotency-marker analysis such as OCT4 and SSEA4 flow cytometry or immunostaining[1][2][5].
• Use negative staining controls for antibody-based flow cytometry or immunostaining, and compare cultures across passages or conditions when evaluating medium, matrix, or passaging changes[1][5].
Troubleshooting
Problem: Low survival after dissociation.
• Possible cause: Excessive single-cell stress or dissociation-induced apoptosis.• Literature-supported solution: Prefer EDTA aggregate passaging for routine maintenance, and use Y-27632 when single-cell handling is required[2][6].
Problem: Variable growth or loss of consistent culture performance.
• Possible cause: Undefined or variable medium components.• Literature-supported solution: Use chemically defined E8 conditions, which were designed to remove albumin-associated variability in hESC/hiPSC culture[1].
Problem: Poor attachment after transfer to feeder-free culture.
• Possible cause: Inadequate matrix support.• Literature-supported solution: Use vitronectin-coated surfaces, which supported hPSC attachment and expansion in E8-based defined culture systems[1][5].
References:
- [1]. Chen G, Gulbranson DR, Hou Z, Bolin JM, Ruotti V, Probasco MD, et al. Chemically defined conditions for human iPSC derivation and culture. Nat Methods. 2011;8(5):424-429. [Content Brief]
- [2]. Beers J, Gulbranson DR, George N, Siniscalchi LI, Jones J, Thomson JA, et al. Passaging and colony expansion of human pluripotent stem cells by enzyme-free dissociation in chemically defined culture conditions. Nat Protoc. 2012;7(11):2029-2040. [Content Brief]
- [3]. Ludwig TE, et al. Feeder-independent culture of human embryonic stem cells. Nat Methods. 2006;3(8):637-646. [Content Brief]
- [4]. Akopian V, Andrews PW, Beil S, Benvenisty N, Brehm J, Christie M, et al. Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In Vitro Cell Dev Biol Anim. 2010;46(3-4):247-258. [Content Brief]
- [5]. Badenes SM, Fernandes TG, Cordeiro CS, Boucher S, Kuninger D, Vemuri MC, et al. Defined Essential 8 Medium and Vitronectin Efficiently Support Scalable Xeno-Free Expansion of Human Induced Pluripotent Stem Cells in Stirred Microcarrier Culture Systems. PLoS One. 2016;11(3):e0151264. [Content Brief]
- [6]. Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol. 2007;25(6):681-686. [Content Brief]
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