Five Fundamental Tips for Cell Harvesting

The following article originally appeared in Technology Networks on January 24, 2024.

Cell culture has a wide array of applications across various fields of research and industries. Once the cells in the culture reach an optimal density or developmental stage for the intended use, they need to be detached from the culture vessel, separated from the growth medium and isolated during a process known as “cell harvesting”. This is a critical step as it can directly impact the quality and yield of the harvested cells.

In this guide, Hilary Sherman, senior scientist at Corning Life Sciences, presents five general tips for cell harvesting that can help you boost your cell culture research.

1. When is the right time to harvest your cells?

This depends on the type of cells and the desired application. For example, are you trying to get as many cells as you can for your particular application? Or do you want to make sure that those cells are in their optimal stage? It is important to know that some cells are “contact inhibited”, which means they start to become unhealthy when they get too dense in their vessel. In this case, it is important to harvest them at a lower confluence than you might do with other cells that are not as sensitive.

2. How do you choose the most appropriate harvesting reagents?

This also depends on the type of cell and application. Trypsin is a good option for robust cells, as it is economical and works well. However, it is a nonspecific protein cleaver, meaning it degrades any protein and not only the extracellular matrix (ECM). It can then destroy membrane receptors and also enter the cell, affecting cell viability. Accutase, on the other hand, is a more specific protein cleaver that degrades the ECM and is less likely to enter the cells. Finally, depending on the cell type, EDTA (Ethylenediaminetetraacetic acid) can also work well. As this is a non-enzymatic agent, it is not suitable for cultures that have a lot of ECM, because the cells are going to form clusters. However, this can be a desirable feature if you’re culturing iPS cells where you don’t want single cells.

3. Is centrifugation always necessary after harvesting?

Centrifugation is normally performed to remove any harvesting reagents from the isolated cells. However, it is a harsh process and can damage the cells. Thus, I prefer to avoid centrifugation after harvesting. For many applications, the small amount of enzymatic or harvesting reagent that remains after diluting and reconstituting the cells in a fresh buffer is not a problem. However, for some other applications, removing the harvesting reagent is essential, and thus centrifugation is necessary. For example, if accutase is used for harvesting iPS cells for single-cell applications, centrifugation is required to remove the reagent as it will have an impact on the next passage.

4. How do you avoid cell clumping?

It is important to avoid overdigesting the cells, which causes the intracellular material to escape and makes the cells “sticky”. Thus, when using trypsin, it is important to optimize the incubation time. Reagents such as accutase break down the ECM without the risk of exposing the cells to the sticky inside of damaged cells. You can also add reagents such as benzonase to degrade RNA and DNA coming from overdigested cells. Another option is to use a cell strainer to get rid of the cell clumps. This could be particularly important for applications such as flow cytometry where you require a single-cell suspension.

5. Tips to harvest cells that are difficult to detach

Some cell lines grow very fast and form super tight junctions with a lot of ECM, making it very difficult to obtain a single-cell suspension. One trick is to harvest them frequently (every 2–3 days) and keep them under 50% confluence. Another trick is to pre-soak the culture with PBS without calcium and magnesium (these ions can help the cells to attach) for 10–15 minutes before applying trypsin. Additionally, you could use a higher concentration of the reagent. Trypsin and other enzymes, such TrypLE, can be purchased at a 10x concentration, which you could then dilute down until you find the optimal concentration for your culture.

Learn more about Corning cell culture solutions

Hilary Sherman is a Senior Scientist in the Corning Life Sciences Applications Lab situated in Kennebunk, ME. She has been part of the Corning team since 2005, specializing in a diverse range of cell types, including mammalian, insect, primary, stem cells and organoids across various applications. Her primary responsibilities encompass the creation of technical documents along with offering technical support and training for both the Corning sales force and customers.