How to get better cells

   Working with iPS cells is a bit like caring for a delicate garden—healthy, well-maintained cultures are the foundation for everything that follows. The quality of your starting cells has a direct impact on how well they differentiate into neurons, so it’s worth investing the time to keep them happy and consistent. In this post, I’ll share some approaches and small details that have helped me maintain robust iPS cell cultures and guide them more reliably toward neuronal differentiation. From daily maintenance habits to optimizing induction conditions, these are the kinds of practical tips that can make the difference between a struggling culture and one that thrives.

Here is a list of things I take care ordered by importance:

1. Make sure iPS cell quality is good

High-quality iPS cells are the foundation for successful experiments. If the cells are stressed, partly differentiated or over confluent, it can lead to poor survival, inconsistent differentiation, and unreliable results. Maintaining healthy, well-characterized iPS cells ensures that when you induce them to become neurons, the process is more efficient and the resulting cultures are robust and reproducible.

2. Mix culture media well

Proper mixing of culture media is essential to ensure that all components—nutrients, growth factors, and supplements—are evenly distributed. This applies at every step: when preparing fresh media, when aliquoting small amounts for use, and right before adding media to your cultures. Uneven mixing can lead to pockets of higher or lower concentration and cells not receiveing ideal concentrations of nutrients and growth factors which are essential for efficient differentiation. Thorough mixing helps maintain a stable, supportive and desired environment for your iPS cells and derived neurons.

3. Use freshly prepared culture media

Using freshly prepared media helps ensure that all nutrients, growth factors, and supplements are at their intended concentrations and remain biologically active. Over time, some biological components in the culture media can degrade, precipitate or reduce the effectiveness of certain additives. Fresh media supports cell health, improves survival, and increases the consistency and reliability of differentiation, especially when working with sensitive iPS cells and developing neurons. In my case I try to not use prepared media longer than 2-3 weeks and only prepare the amount for this time period.

4. Prewarm culture medium well

Prewarming culture medium to the appropriate temperature before adding it to your cells helps prevent thermal shock, which can stress sensitive iPS cells and developing neurons. Sudden temperature changes can affect cell metabolism, slow growth, or trigger unwanted stress responses. By ensuring the medium is properly warmed, you create a stable and supportive environment that promotes healthy cell survival and consistent differentiation. Additionally, always aliquot only the amount of culture media you need to warm, rather than warming your entire stock solution. This protects your stock media from prolonged exposure to higher temperatures, which can accelerate nutrient degradation, and allows the smaller aliquot to reach the desired temperature more quickly.

5. Be gentle with media changes

Neurons and iPS cell-derived cultures are delicate and can be easily damaged by rough handling. When changing media, add and remove liquids slowly and carefully to avoid disturbing the cells. Similarly, be gentle when moving plates or flasks from the incubator to the cell culture hood, or between workspaces, to prevent unnecessary stress or detachment. 

6. Minimize time of cells being outside of the incubator

iPS cells and neurons are sensitive to changes in temperature, CO₂ levels, and humidity. Prolonged exposure outside the incubator can stress cells, reduce viability, and negatively affect differentiation. Plan your work so that media changes, observations, or transfers are done efficiently, keeping the cells in a stable environment as much as possible. 

7. Keep cells in the back of the incubator

The back of the incubator is generally the most stable area in terms of temperature, humidity, and CO₂ levels. Placing your plates or flasks there reduces exposure to fluctuations caused by opening the incubator door or uneven airflow. 

8. Conduct media change at approximately same times

Maintaining a consistent schedule for media changes ensures a stable environment for your cells and proper exposure to signaling molecules in the culture media. This is especially important during differentiation, where protocols often call for daily media changes. Deviating from the schedule—such as changing media at night and again the next morning—can alter the timing of exposure, potentially disrupting the morphogens and growth factors that guide differentiation. Following a regular schedule helps cells receive the intended signals consistently, supporting healthy growth and reliable differentiation outcomes.

   Working with iPS cells and neurons can be challenging, but paying attention to these small details can make a big difference in the health, consistency, and differentiation of your cultures. I hope these tips and insights help streamline your experiments and reduce common frustrations. Healthy cells are the foundation of reliable results—and with careful practice, it’s possible to achieve both.