The possible causes of cells dying in cell culture

Why are my cells dying? 

If you’ve spent any amount of time working with cultured cells, chances are you’ve asked this question. Cells grown in culture can be temperamental, and there are endless wide range of factors that can impact cell health and viability. Here is a list of common sources of cell culture death to help pinpoint problems so your cultures can flourish. 

Incubation

Incubators are essential for controlling temperature, gas exchange, and humidity of cell cultures, thereby directly impacting osmolality and pH of the cell culture medium1. Even slight fluctuations in temperature and evaporation rate can have profound effects on cell health. 

The biggest impact on temperature is frequent opening and closing of the incubator door, which can result in significant drops in temperature1. To address this, consider using additional incubators for different experiments to reduce traffic. We typically have an incubator for experiments that require frequent in and out of the incubator (like short term assays that will last 1-3 hours or passaging of the cells) and a separate incubator for long term culture or culture with very valuable or fragile cell cultures. You can also store your plates towards the back of the incubator, where temperature fluctuations will be less drastic. 

Growing in a warm environment, cell cultures face a constant rate of media evaporation1. To counteract this, ensure the water reservoir in your incubator is full, and make sure you are conducting media changes or passaging with enough frequency to counteract the effects of evaporation on the osmolality and pH of the medium. 

Media

Cell culture medium provides the necessary nutrients for cells to grow in a dissociated state2,3. Many cell lines will also require additive such as growth factor supplements, such as serum, L-glutamine, or antibiotics. The needs of different cell types vary, so make sure to research which media and additives are appropriate for your cell linetype. If you notice cell death after introducing a reagent from a new batch, inspect and replace, as the formulation may have changed. When exposed to fluorescent light, many common ingredients in cell media, such as tryptophan, riboflavin, and tyrosine react to create toxic compounds4, so m. Make sure cells and media are stored in the dark, protected from fluorescent light. 

Over-passaging and overcrowding

The number of times a cell line has been passaged has significant impacts on cellular function and genetic expression5,6. Passaging too many times can impact the health and viability of your cultures and introduce significant experimental variability. Be sure to research the recommended maximum passage number for your cell line. 

CActively dividing cells need room to grow. Overcrowding at 100% confluency can arrest cell division and cause cell death7. Cells should be passaged around 80% confluency while they still show an exponential rate of growth to ensure healthy cultures. But most cells also hate growing too sparsely…

Detachment

Although some cell types can adhere directly to plastic or glass, many require a growth substrate to adhere to the plate surface. If your cells being to pile together into clumps or detach from the plate over time, this is a sign that your coating substrate is being degraded8. The most commonly used coating substrates are poly-D-lysine (PLL and PDL) and poly-L-lysineornithine (PLLO) which are homopolymeric chains of the positively charged amino acid lysine9–12. PDL is formed from the D-lysine enantiomer and is more resistant to enzymatic degradation by proteases such as trypsin13,14 compared to the L enantiomer. If you encounter substrate degradation issues with PLL, try replacing it with PDL. If issues with substrate degradation persist with PDL, consider switching to  a non-peptide polymer such as dPGA, which mimics the structure of poly-lysine, but lacks peptide bonds making it highly resistant to degradation15. 

Cryopreservation

Cryopreservation of cell lines allows for maintenance of a stock to continue deriving future cultures from the same line without over-passaging2. However, this process places stress on the cells, and can cause cell death if not done carefully. If you notice a significant reduction in cell viability when seeding cells from a frozen stock, the issue may be with the cryopreservation process.

Cells should be frozen from early passages at around 80% confluency while at an exponential rate of growth. Slowing the cooling rateU using a cryoprotective agent such as DMSO is important to prevent the formation of damaging ice crystals7. Slowing the cooling rate U using insulated containers, such as a Mr. Frosty (Invitrogenttps://www.thermofisher.com/order/catalog/product/5100-0001), can also help towill  ensure a gradual and controlled rate of freezing2.  

When thawing, warm rapidly to 37°C and dilute gently with pre-warmed medium2. Plate cells at a higher density than usual, to account for some cell death that is normal during the freezing process7. After 24 hours, cells should be adhered to the plate. 

References

1. Ryan, J. A. Corning Guide for Identifying and Correcting Common Cell Growth Problems. (2008).
2. Masters, J. R. & Stacey, G. N. Changing medium and passaging cell lines. Nat Protoc 2, 2276–2284 (2007).
3. Philippeos, C., Hughes, R. D., Dhawan, A. & Mitry, R. R. Introduction to Cell Culture. in Human Cell Culture Protocols (eds. Mitry, R. R. & Hughes, R. D.) 1–13 (Humana Press, Totowa, NJ, 2012). doi:10.1007/978-1-61779-367-7_1.
4. Wang, R. J. Effect of room fluorescent light on the deterioration of tissue culture medium. In Vitro Cell.Dev.Biol.-Plant 12, 19–22 (1976).
5. Briske-Anderson, M. J., Finley, J. W. & Newman, S. M. The Influence of Culture Time and Passage Number on the Morphological and Physiological Development of Caco-2 Cells. Experimental Biology and Medicine 214, 248–257 (1997).
6. Hughes, P., Marshall, D., Reid, Y., Parkes, H. & Gelber, C. The costs of using unauthenticated, over-passaged cell lines: how much more data do we need? BioTechniques 43, 575–586 (2007).
7. Phelan, K. & May, K. M. Basic Techniques in Mammalian Cell Tissue Culture. Current Protocols in Cell Biology 66, 1.1.1-1.1.22 (2015).
8. Thiry, L., Clément, J.-P., Haag, R., Kennedy, T. E. & Stifani, S. Optimization of Long-Term Human iPSC-Derived Spinal Motor Neuron Culture Using a Dendritic Polyglycerol Amine-Based Substrate. ASN Neuro 14, 17590914211073381 (2022).
9. Sahu, M. P., Nikkilä, O., Lågas, S., Kolehmainen, S. & Castrén, E. Culturing primary neurons from rat hippocampus and cortex. Neuronal Signal 3, NS20180207 (2019).
10. Roppongi, R. T., Champagne-Jorgensen, K. P. & Siddiqui, T. J. Low-Density Primary Hippocampal Neuron Culture. J Vis Exp 55000 (2017) doi:10.3791/55000.
11. Brewer, G. J. & Torricelli, J. R. Isolation and culture of adult neurons and neurospheres. Nat Protoc 2, 1490–1498 (2007).
12. McKeehan, W. & Ham, R. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol 71, 727–734 (1976).
13. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. Journal of Biological Chemistry 222, 479–485 (1956).
14. Li, J. & Yeung, E. Real-Time Single-Molecule Kinetics of Trypsin Proteolysis. https://pubs.acs.org/doi/epdf/10.1021/ac801365c (2008) doi:10.1021/ac801365c.
15. Clément, J.-P. et al. Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro 14, 17590914211073276 (2022).