Introduction:
Induced pluripotent stem cells (iPSCs) have revolutionized the field of regenerative medicine and disease modeling due to their ability to differentiate into various cell types. However, maintaining iPSCs in culture requires careful attention to detail and adherence to best practices. This guide aims to provide tips for a successful start into the world of iPSC cell culture. It includes information on culture media, coatings, and best practices for maintaining cell health and pluripotency.
1. Selecting Cell Culture Media:
Choosing the right culture medium is crucial for maintaining iPSCs’ pluripotency and promoting robust growth. Several commercially available media formulations are tailored specifically for iPSC culture, each with its unique composition:
mTeSR™ and mTeSR™ Plus: Based on DMEM/F12, these defined, serum-free media contain essential components such as FGF2, TGF-β, and insulin to support iPSC growth and pluripotency.
StemFlex™: Also based on DMEM/F12, StemFlex™ is a xeno-free medium optimized for iPSC expansion. It contains basic FGF, TGF-β receptor inhibitor, and IGF-1 to promote robust growth and maintenance of pluripotency.
E8 Medium: A simplified formulation based on DMEM/F12, E8 medium contains only eight essential components, including FGF2, TGF-β, and insulin. It provides a defined and cost-effective environment for iPSC culture.
Essential 8™ Flex and TeSR™-E8™: Both based on DMEM/F12, these media offer a balance of defined components to support iPSC growth and pluripotency.
Explore our recent survey about the use of different culture media for iPSCs.
2. Coating Substrates:
Coating substrates play a critical role in providing a suitable surface for iPSC attachment and growth. Commonly used coatings include:
Matrigel: Derived from mouse sarcoma cells, Matrigel contains various extracellular matrix (ECM) components necessary for iPSC attachment and pluripotency.
Laminin-521: A recombinant human protein, Laminin-521 provides a defined and reproducible surface for iPSC culture. It promotes cell attachment, survival, and pluripotency.
Geltrex: Derived from murine Engelbreth-Holm-Swarm (EHS) sarcoma cells (as Matrigel). Geltrex offers a two- and three-dimensional matrix for iPSC culture.
iMatrix and Vitronectin-N: These recombinant human proteins offer defined and xeno-free substrates for iPSC culture, supporting robust growth and maintenance of pluripotency.
Learn more about the use of coatings for iPSCs in our recent survey report.
3. Best Practices for iPSC Culture:
Maintaining iPSC culture requires adherence to best practices to ensure cell health and pluripotency:
Maintain a sterile environment: Work in a laminar flow hood and use sterile techniques to prevent contamination.
Regularly check cell morphology: Monitor iPSC colonies under a microscope to ensure they exhibit typical pluripotent morphology, including compact colony formation and high nucleus-to-cytoplasm ratio.
Avoid overconfluence: Passage iPSCs when they reach approximately 70-80% confluency to prevent overcrowding and maintain pluripotency.
Regularly change medium: Replace old medium with fresh, pre-warmed medium every 24 to 48 hours to provide essential nutrients and growth factors.
Handle cells gently: Minimize agitation and mechanical stress during cell passaging to prevent cell damage and maintain cell integrity.
Finally, perform regular quality control. Test media, reagents, and coatings for consistency and performance to ensure reproducibility and reliability of results.
Conclusion:
Successful iPSC cell culture requires careful selection of culture media and coating substrates. Along with adherence to best practices for maintaining cell health and pluripotency, researchers can establish robust iPSC culture systems suitable for various applications in regenerative medicine, disease modeling, and drug discovery.
If you would like to learn more, please watch our introductory video about iPSCs, discover gentle technologies ideally suited for single-cell cloning iPS-cells, or explore our free resources and insights to advance your cell-engineering projects.
Please note that iotaSciences is neither endorsing any of the mentioned culture reagents, nor is affiliated with any supplier or manufacturer of mentioned reagents.
Citations and further reading:
Stem Cells, 23 (2005), pp. 315-323
Biol. Reprod., 70 (2004), pp. 837-845
Nat. Methods, 3 (2006), pp. 637-646
Nat. Biotechnol., 24 (2006), pp. 185-187
Curr. Protoc. Stem Cell Biol. (2007), 10.1002/9780470151808.sc01c02s2
Nat. Protoc., 7 (2012), pp. 2029-2040
Nat. Methods, 8 (2011), pp. 424-429
Nature Communications, 3 (2012), 1236
Nat. Biotechnol., 24 (2006), pp. 185-187
Stem Cell Reports, 2020, pp. 256-270