Induced pluripotent stem cells (iPSCs) have revolutionised the field of regenerative medicine, offering promising avenues for disease modelling, drug discovery, and potential therapeutic applications. However, the culture and maintenance of iPSCs can be challenging due to their sensitivity and specific requirements. Here are some essential tips for a successful iPSC culture: 

1. Starting with High-Quality Cells 

The foundation of a successful iPSC culture lies in the quality of the starting cells. Ensure that you are starting with well-characterised and authenticated iPSCs. These cells should exhibit high pluripotency potential with the ability to differentiate into all three germ layers (endoderm, mesoderm, and ectoderm), and low differentiation markers to minimise contamination with unwanted cell types. Using well-characterised iPSC lines with established properties allows for more predictable and reproducible experiments. This is especially important for collaborative research or large-scale studies. It is important to regularly monitor your iPSCs for any signs of spontaneous differentiation or contamination.  

Table 1: Lineage-specific markers.  

*Check out Atlantis Bioscience’s Live-or-Dye Fixable Dead Cell Dye 

*Checkout Atlantis Bioscience’s Annexin V Dyes & Viability assay kits 

2. Choosing the Right Culture Medium 

The choice of culture medium is critical for maintaining iPSC pluripotency and health, providing them with the essential nutrients and signals they need to thrive.  Several commercially available media formulations are tailored specifically for iPSC culture, each with its unique composition. These media can be broadly categorised by:  

• Defined vs. Undefined: Defined media contain all components in known quantities, ensuring consistency and reproducibility. Undefined media, like those containing feotal bovine serum (FBS), have unknown components that can introduce variability. 

• Xeno-free vs. Xeno-containing: Xeno-free media exclude animal-derived products (but may contain human-derived products), minimising the risk of animal-borne contaminants and ethical concerns. Xeno-containing media often utilise FBS, which can be a cost-effective option but introduces variability. 

• Feeder-based vs. Feeder-free: Feeder-based media utilise a layer of irradiated mouse embryonic fibroblasts (MEFs) to support iPSC growth. Feeder-free media eliminate the use of MEFs, simplifying culture protocols and reducing the risk of contamination from animal products. 

Apart from commercially available options, there are also in-house media where DMEM/F12 is the standard basal media used in iPSC culture. Regardless, most commercially available and in-house media are supplemented with recombinant growth factor proteins, such as FGF2, TGFβ, Activin A, and Nodal, at varying concentrations, to maintain pluripotency.  

*Checkout Atlantis Bioscience’s Growth Factors 

*Checkout Atlantis Bioscience’s Cell Culture Media 

*Checkout Atlantis Bioscience’s Embryonic Stem Cell Tested FBS 

3. Choosing the Right Coating Substrates: 

Coating substrates play a critical role in providing a suitable surface for iPSC attachment and growth. Coating substrates are often composed of extracellular matrix (ECM) components that play a critical role in providing a hospitable surface for iPSCs to adhere to and spread, mimicking their natural environment. The ECM components also interact with receptors on the iPSC surface, sending signals that influence cell behaviour, proliferation, and differentiation potential. 

Many different ECM components can be used to coat substrates for iPSC culture. Some of the common ECM include Matrigel, Geltrex, Laminin etc. Matrigel and Geltrex are basement membrane extracts derived from Engelbreth-Holm Swarm (EHS) sarcoma cells. They offer a complex mixture of ECM proteins and growth factors, supporting iPSC attachment, survival, and pluripotency. However, their source (animal tumour) raises concerns about batch-to-batch variability and potential contamination with animal products.  

Therefore, animal-free ECMs such as the recombinant human laminin – iMatrix-511 have received much attention as they provide a defined and animal-free alternative to Matrigel and Geltrex. This ensures greater consistency and reduces the risk of animal-derived contaminants. 

By carefully selecting an appropriate coating substrate based on your specific needs, you can provide a foundation that optimises the attachment, growth, and differentiation potential of your iPSCs. 

*Checkout Atlantis Bioscience’s Recombinant Laminin – iMatrix-511 

iMatrix-511 silk

(0)

$578.00

Request a Quote

4. Use ROCK Inhibitor During Passaging 

Successful passaging is crucial for maintaining the health and pluripotency of your iPSCs. One technique that can significantly improve your passaging success rate is the use of a ROCK inhibitor.  

ROCK inhibitors target a protein called Rho-associated protein kinase (ROCK). ROCK plays a role in cell adhesion, contraction, and cytoskeletal organisation. When iPSCs are detached from the culture dish during passaging, they experience stress that can lead to cell death. ROCK inhibitors mitigate cell stress, improving cell survival rates during detachment, reseeding, and attachment. 

The optimal concentration of ROCK inhibitor depends on the specific inhibitor and cell line. Typically, 1 μL/mL (10 μM) of ROCK inhibitor, Y-27632, is added to the culture medium, improving survival and adherence of the iPSCs when passaged. 

*Checkout Atlantis Bioscience’s Y-27632 (Dihydrochloride) 

Rock Inhibitor Y-27632 dihydrochloride

(0)

Request a Quote

5. Preventing Contamination 

Contamination by bacteria, fungi, or mycoplasma can compromise cell viability, disrupt differentiation potential, and ultimately derail your experiments. To fortify your defenses and keep your iPSC cultures safe, strict aseptic techniques are essential. This includes regularly sterilising work surfaces and equipment. However, it’s important to use antibiotics sparingly to avoid masking underlying issues with your culture technique.  

Mycoplasma, in particular, presents a unique challenge. These tiny bacteria lack a cell wall, making them remarkably small and able to evade detection by conventional methods like light microscopy. Additionally, they grow slower than many other bacteria, leading to delayed identification. This is why routine testing for mycoplasma using specialised methods is crucial. Early detection allows for potential intervention with antibiotics or elimination of contaminated cultures before widespread infection occurs. 

*Check out Atlantis Bioscience’s Mycoplasma Solutions 

*Check out this YouTube Video on Mycoplasma 

6. Regular Monitoring and Quality Control 

Constant monitoring of the health and pluripotency of your iPSCs is crucial for ensuring the reproducibility and reliability of your results. Observe iPSC colonies under a microscope to confirm they exhibit typical pluripotent morphology, such as compact colony formation and a high nucleus-to-cytoplasm ratio. 

Regularly evaluate your iPSCs for key pluripotency markers like Oct4, Sox2, and Nanog. Techniques such as immunocytochemistry, flow cytometry, or qPCR are effective for this purpose. The reprogramming process and hiPSC maintenance can cause cell stress, leading to genomic instability, which may result in unwanted genomic lesions and chromosomal abnormalities. Therefore, routine karyotyping should be performed in the early passages (passages 7-10) and during propagation (every 10-15 passages) to ensure the genetic integrity of the hiPSCs. 

By incorporating regular monitoring practices, you can proactively maintain the quality of your iPSC culture, leading to more reliable and reproducible research outcomes. 

*Check out Atlantis Bioscience’s qPCR solutions 

*Check out Atlantis Bioscience’s Superior CF-Conjugated Secondary Antibodies 

7. Optimising Differentiation 

Once you’ve established healthy and pluripotent iPSCs, the crucial step of differentiation allows them to mature into specific cell types crucial for your research goals. Different cell types arise from iPSCs through specific signalling pathways. To guide your iPSCs towards their desired fate, tailoring the differentiation process is crucial. Explore various differentiation protocols that utilise factors or culture conditions tailored to your desired cell lineage. This might involve growth factors, small molecules, or specific media formulations.  

Many iPSC differentiation protocols begin with the formation of embryoid bodies (EBs). EBs are 3D structures that resemble early-stage embryos, where iPSCs self-organise and differentiate into various cell types found in the developing body. Once differentiation is achieved, employ appropriate markers and functional assays to confirm the identity and functionality of the differentiated cells. 

By carefully tailoring the differentiation process and employing appropriate verification methods, you can transform your iPSCs into the specialised cell types needed for your research endeavours. 

*Checkout Atlantis Bioscience solutions for reproducible production of uniform EB – Spherical Plate5D 

Conclusion 

Culturing iPSCs requires meticulous attention to detail and a thorough understanding of their unique needs. By following these tips, you can improve the success rate and quality of your iPSC cultures, paving the way for groundbreaking research and therapeutic applications. Remember, consistency and vigilance are key to maintaining healthy and pluripotent iPSCs. 

References 

Castro-Viñuelas R, Sanjurjo-Rodríguez C, Piñeiro-Ramil M, Rodríguez-Fernández S, López-Baltar I, Fuentes-Boquete I, Blanco FJ, Díaz-Prado S. Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells. Mol Ther Methods Clin Dev. 2021 Nov 3;23:569-581. doi: 10.1016/j.omtm.2021.10.013. 

Cheng YS, Xu M, Chen G, et al. A Protocol for Culture and Characterization of Human Induced Pluripotent Stem Cells After Induction. Curr Protoc. 2023;3(8):e866. doi:10.1002/cpz1.866 

Dakhore S, Nayer B, Hasegawa K. Human Pluripotent Stem Cell Culture: Current Status, Challenges, and Advancement. Stem Cells Int. 2018 Nov 22;2018:7396905. doi: 10.1155/2018/7396905.  

Li X, Krawetz R, Liu S, Meng G, Rancourt DE. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum Reprod. 2009;24(3):580-589. doi:10.1093/humrep/den404 

Pakzad M, Totonchi M, Taei A, Seifinejad A, Hassani SN, Baharvand H. Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev Rep. 2010;6(1):96-107. doi:10.1007/s12015-009-9103-z