Lifting the Curtain: a Beginners Guide to iPS Cell Culture

This is a post by Christine Miller, Research Assistant at Harvard University, Amy Wagers Lab:

Are you curious about the intricacies of using 24 well cell culture plates? Get in touch with us today for expert advice!

In many people's view, there's a bit of "black magic" involved in making certain cell culture protocols work, especially with iPS/hES cell cultures. From my experience, I've noticed particular challenges in this area. Through this blog series, I aim to demystify these challenges and share what methods have been successful for me while encouraging others to share their insights as well.

Despite being a relatively new technology, iPS cell culture already has numerous protocols. Each variation involves different reagents, methods of passaging, and freezing techniques. The variables to test when optimizing your culture strategy are numerous. Additionally, iPS lines exhibit differences even among those reprogrammed from normal patients. For example, in pluripotency tests, one line may form teratomas in six weeks, while another might take 10 to 12 weeks. Such differences might not seem alarming on paper but can be puzzling during experiments when some lines form teratomas quickly and others lag. Similarly, differentiating iPS cells into specific lineages like hematopoietic cells can vary significantly between lines, with differences in growth rates and passaging requirements. The key takeaway is to be aware of these differences and remain open-minded if one line's behavior doesn't align perfectly with another's.

iMEFs vs. Matrigel

Two primary methods exist for culturing iPS cells: using a feeder layer with irradiated mouse embryonic fibroblasts (iMEFs) or culturing feeder-free. Both methods have specific advantages depending on your goals.

Here’s what I found using iMEFs:

iMEFs are great for thawing a line for the first time. They offer reliable culture support for around 10 days, enabling the formation of small colonies. iPS colonies on iMEFs tend to be uniformly shaped, making it easier to identify colonies and observe differentiation. However, using iMEFs requires high-quality sources to provide the appropriate support. Poor-quality iMEFs can result in failed or improper seeding, leading to differentiation. iMEFs should be positioned snugly between colonies to keep them contained and undifferentiated. I've found that commercial iMEFs, such as those from global stem (cat # CF-1 MEF), are cost-effective. I plate them at 200k/well on a six-well plate, with even spreading important for successful seeding. On the downside, using iMEFs is time-consuming. Preparing plates involves gelatin-coating, setting, plating iMEFs, and allowing them to sit overnight, requiring one or two days of preparation before plating iPS cells.

Feeder-free culture systems like Matrigel, CELLstart, and Vitronectin XF require less preparation time, usually one to two hours. These systems simplify the process, with standardized media kits that often include passaging and freezing reagents. When choosing feeder-free systems, ensure you use hES-grade materials. Low-grade materials can degrade quickly, causing colonies to lift off the plate.

Passaging

Passaging hES and iPS cells can be done enzymatically or manually. Less than 20 colonies per 6 well cell culture plates well are best passaged manually to maintain better control. Over 20 colonies are easier to passage enzymatically, though it might pick up some iMEFs, affecting colony quality. Enzymes like collagenase IV and Dispase help keep cells in clumps, crucial for optimal seeding and growth. Preparation and storage guidelines should be followed to ensure enzyme effectiveness.

When removing cells post enzyme treatment, use the "car wash" method with a 5ml glass pipette. This gentle scraping method keeps cells in clumps, facilitating successful passaging.

Antibiotic Use

While opinions may differ, I strongly advocate for antibiotic-free culturing. It provides better control over cell status, allowing immediate detection and elimination of contamination without affecting other cultures. It also helps avoid masking mycoplasma infections, a significant concern demonstrated by a costly infection outbreak in our facility. Antibiotic-free culturing reinforces good sterile techniques and maintains optimal culture conditions.

Last Notes

Patience is paramount in iPS cell culture, especially when thawing cells for the first time. Persist even if no immediate colonies are visible – eventual success is likely with continued feeding. Helpful resources and online protocols from HSCI iPS core and WiCell are valuable for guiding your efforts.

In conclusion, for those new to hES/iPS culture, I hope this has provided some clarity on culture techniques, helping to navigate a few variables as you get started. Feel free to share your tips as well! By collaborating, we can make significant scientific advancements.

Christine Miller is a Research Assistant at Harvard University, Joslin Diabetes Center, Amy Wagers Lab.

Note: StemCultures facilitates posting on this blog, and the views expressed are those of the author(s) or interviewee(s) and not necessarily of StemCultures.