Four essential tips for passaging single cell hPSCs

I think it is fair to say that culturing human pluripotent stem cells is an art form. An art form perfected over time. Here are a few tips I have learned over the years that address the challenges around passaging cells in our labs.

Passaging human pluripotent stem cells (hPSCs) as single cells can lead to more consistent experimental results, as uniform cell densities can be determined and used with each experiment. Additionally, hPSCs passaged as single cells can be more efficiently scaled in culture for high-throughput automation or to generate cell numbers needed for therapeutic applications. These four essential tips address only a few of the challenges you may have seen with passaging your cells.

1. Use a compatible culture environment

• hPSCs require a culture system, both media and substrate, that promotes cell survival through single-cell passaging through fast attachment of cells and appropriate migration (for non-clonal propagation) so that cell aggregates can form small monolayer colonies that expand across the culture surface.
• Purified laminin protein has been widely published and used as a suitable basement membrane matrix for human pluripotent stem cell culture.  Laminin-521 is notable for its use in effective single-cell culture applications. 
• The use of a ROCK inhibitor (such as Y27632, Thiazovivin, Chroman 1, or the CET Cocktail) in the plating media can have a significant effect on cell survival, attachment efficiencies, and overall culture health through comprehensive cytoprotection, as is the case with CET Cocktail supplementation.

2. Use gentle cell dissociation solutions

• To avoid losing cells during harvesting and passaging, be sure not to over-incubate the cultures with the dissociation solution. Proper single-cell dissociation will allow for the cells to be primed for detachment, and able to be rinsed with culture media without detaching completely from the surface.  Harvest the rinsed cultures in complete culture media and distribute to the new wells without the need of a centrifugation step.
• Accutase® is an example of a widely used gentle cell dissociation solution made of proteolytic and collagenolytic enzymes. Accutase® is used in many cell and tissue-culture workflows, and is specifically highlighted for effective dissociation of hPSCs as single cells for routine expansion and differentiation protocols.
• EDTA is a gentle non-enzymatic cell dissociation reagent that is suitable for routine hPSC passaging. EDTA is a chelating agent that sequesters calcium from the culture environment.  It reduces cell attachment by disrupting the function of cell adhesion proteins dependent on calcium, such as cadherins.  EDTA is generally used as a 0.2% solution, which is sometimes called “Versene”.

3. Maintain proper culture density

• The plating density is a critical factor for increased cell survival with routine passaging as single cells.  Allowing single cells to aggregate on the culture surface after seeding can significantly improve culture survival and growth rates. 
• Unless working on clonal propagation, plating hPSCs at densities of 10,000 to 15,000 cells/cm² can be an optimal range for routine passaging a 1:25 or higher split ratio under proper conditions.  Higher plating densities (30,000 to 40,000 cells/cm²) can also be effective for rapid culture expansion and proliferation. 
• When culturing hPSC as single cells, do not allow the cells to grow to a full monolayer across the culture surface before passaging. Ensure that cultures are passaged by the time they reach 75% confluency to avoid differentiation caused by an overpopulated environment.

4. Check your karyotype

• Without the proper culture environment, long-term serial passaging as single cells can place selective pressure on the culture populations that survive and propagate.  This leads to genetic abnormalities across the culture, which can be detected by
• Always check a cell population’s karyotype prior to beginning any critical or long-term experiment to avoid using cells that are genetically abnormal and biased toward survival in stressful culture conditions.
• For routine culture management, seek a reputable, expert karyotyping service with experience characterizing hPSCs, such as WiCell.

In summary, implementing these four essential tips for passaging your cultures can help set you up on the path to perfecting your art form of culturing and passaging single hPSCs in the lab.