Protocols: Maintaining happy iPSC colonies

Episode 1: Let’s start at the beginning, maintaining your iPSC colonies

Keeping healthy iPSC colonies, better for clean cultures than single cell cultures, is the foundation of any reliable and reproducible experiment. When culturing colonies, you have choices for different media, air, temperature, growth surface (extracellular matrix) and density.

  • Extracellular matrix – keep it chilled!
    • Most common option: Matrigel from BD (now Corning)
    • Suitable alternative: Geltrex® (Life technologies)

Both options contain a cocktail of extracellular matrix proteins, primarily collagen I and laminin, and solidify at room temperature. More consistent colonies with Matrigel over Geltrex®, but the higher price for Matrigel and batch to batch concentration variations makes Geltrex® a worthwhile alternative. Handling of both substances is very similar, since both quickly form clumps once defrosted and coating is generally done at 1:100 dilution. (For a comparison of different coatings see 1)

To make aliquots: defrost MG or GT on ice (or fridge overnight), place sterile sample tubes (best use 1.5 or 2 ml tubes) on ice and chill 1000 ul pipette tips in the fridge. Once MG or GT are defrosted (but still on ice), move all equipment into the biosafety cabinet and quickly aliquot MG or GT into the sample tubes. You really want to avoid additional freeze-thaw cycles, so consider your aliquot size for one-time use (200 ul in my case worked well).

To coat vessels: Dilute ECM at 1:100 with DMEM/F12. Ensure that DMEM/F12, pipettes and falcon tubes are ice-chilled. Very slowly defrost (best on ice, but +4C works too) MG or GT and keep on ice until final use. Once defrosted, gently add small volumes of DMEM to the aliquot and transfer entire tube content to the chilled DMEM solution. Mix by inverting and brief vortexing. Coat your culture vessels (ca 50 ul/cm2) right away and keep either at room temperature for 1h or in the fridge for longer storage (use within 2 weeks).

  • Media – really every day?

Before we get into the different media options, one thing first: don’t use antibiotics. Use excellent cell culture and sterile techniques, to avoid the need for antibiotics. In fact, antibiotics influence mitochondria function and importantly, once used it is near impossible to stop using them later. You also lose one tool against infections, so if you do use for example Pen/Strep and still get a bacterial infection, it will be much harder to combat.

mTeSR1 has been the default media for iPSC cultures for many years. Due to its high price, less expensive alternatives have arrived on the marked, with TeSR-E8 from Stem Cell Technologies and E8 media from Life Technologies, commonly used. All three media are used in the same way: 100% media change every day. You can double-feed one day out of 6 and skip the next day, for a Sunday off. This is best done 48 h after passaging, when the cultures are still small. Several companies have introduced alternative media to allow researchers 2-day feeding cycles, but in the experience of myself, our team and other teams I have spoken too, cultures are less consistent with lower purity and I can therefore not recommend them.

When handling complete iPSC media, avoid repeated heating cycles which damage the growth factors. Best to make aliquots, take only the volume needed for the day out of the fridge and let it warm to room temperature on the bench before briefly heating the media to 37C.

  • Incubator condition (gas & temperature) – drop the O2

Stem cells are special as you know and have to be handled with delicate care. This goes beyond the surface they grow on and the media they feed on, it extents to the gas they breath and the warms of their environment. If you have the opportunity, grow your stem cell cultures in hypoxic conditions (3% O2). Oxygen levels in our body drop with further distance from the lungs and stem cells are less likely to spontaneous differentiate with low O2 than atmospheric O2 2 . Additionally, culturing stem cell colonies at 35C further reduces spontaneous differentiation 3, but also slows down proliferation, so culturing takes longer.

  • Density & culture vessel – keep them accessible

If you want to ensure high purity in your stem cell cultures, in my experience you will need to be able to (manually) remove spontaneous differentiating cells (more on that later). This means you’ll need to physically access your cultures, so a wider surface is better than a narrow well. Cell culture appropriate 6 well plates and 60 mm dishes work very well, both to allow physical access to your cultures, up-scaling stem cell production and stacking in incubators. 100 mm dishes can work too, but due to the larger volume are potentially more likely to cause spills.

Seeding density varies between lines as it depends on the proliferation rate, but as a rough indication: 40,000 cells per cm2 works pretty well for most of our lines, with a split ratio of at least 1:5 from confluent vessels (more to passaging iPSCs later).

That’s it for now on how to keep your iPSCs happy. Get in touch if you have questions, comments or suggestions for improvement.

Good luck!

p.s. back to the iPSC miniseries index

Lam MT, Longaker MT. Comparison of several attachment methods for human iPS, embryonic and adipose-derived stem cells for tissue engineering. J Tissue Eng Regen Med. 2012;6(S3):s80-s86. doi:10.1002/term.1499
Correia C, Serra M, Espinha N, et al. Combining Hypoxia and Bioreactor Hydrodynamics Boosts Induced Pluripotent Stem Cell Differentiation Towards Cardiomyocytes. Stem Cell Rev and Rep. 2014;10(6):786-801. doi:10.1007/s12015-014-9533-0
Belinsky GS, Antic SD. Mild Hypothermia Inhibits Differentiation of Human Embryonic and Induced Pluripotent Stem Cells. BioTechniques. 2013;55(2). doi:10.2144/000114065
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