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The possible causes of cells dying in cell culture

The possible causes of cells dying in cell culture

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 a 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 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 evaporation, resulting in increased osmolality and altered pH1,2. 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 diminish the effects of evaporation.

Media

Cell culture medium provides the necessary nutrients for cells to grow in a dissociated state3,4. Many cell lines will also require additives such as growth factor supplements, 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 type. 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 compounds5, so make sure cells and media are stored in the dark, protected from fluorescent light. It is also important to make sure your media and additives are fresh. Most commercially available media contains the pH indicator phenol red, which changes from yellow at a low pH to red and then pink as pH increases6. If your media changes colour while in storage, it is time to replace it with a new bottle, even if it hasn’t reached the expiration date. Media colour changes in your cultures can also be a great indicator that it is time to passage your cells or swap out the media. Certain additives, such as L-glutamine, are unstable and will convert to an unusable form over time, these should be stored frozen and only added to your media right before use6. 

 

Cryopreservation

Cryopreservation of cell lines allows for maintenance of a stock to continue deriving future cultures from the same line without over-passaging3. 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. Using a cryoprotective agent such as DMSO is important to prevent the formation of damaging ice crystals7. Slowing the cooling rate using insulated containers, such as a Mr. Frosty (https://www.thermofisher.com/order/catalog/product/5100-0001), will ensure a gradual and controlled rate of freezing3.  

When thawing, warm rapidly to 37°C and dilute gently with pre-warmed medium3. 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. 

Stress from dissection and passaging

Cells are especially vulnerable during stressful manipulations such as dissection and passaging. Taking extra care during these procedures can improve the rate of cell survival. When passaging cells or processing dissected tissue, all components, including trypsin, serum, and media, should be warmed to at least room temperature to minimize cell stress3. Trypsin, used to dissociate tissue and detach adherent cells can be toxic and damaging3,8,9. If you think this step could be a problem, consider decreasing the time your cells spend in trypsin, or finding an alternative agent, such as papain (for dissociation). Pipette cells gently and prevent bubbles, to avoid shearing cells by surface tension3,10. Centrifugation at high speeds can also damage cells. Although centrifugation times and speeds vary depending on cell type, the speed should generally not exceed 100g3. 

Over-passaging and density

The number of times a cell line has been passaged has significant impacts on cellular function and genetic expression11,12. 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. 

Actively 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. There is a fine balance to strike here, as most cells also hate growing too sparsely. A good guide for seeding density of proliferative cells can be found here: https://www.thermofisher.com/ca/en/home/references/gibco-cell-culture-basics/cell-culture-protocols/cell-culture-useful-numbers.html
When passaging cells around 80% confluency, a typical split ratio is between 1:3 and 1:8 once a week to maintain a healthy density after passaging3. 

For non-proliferative cells such as neurons, the optimal seeding density will vary based on cell type and experiment, but should be relatively high for healthy cultures8. 

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 degraded14. The most commonly used coating substrates are poly-lysine (PLL and PDL) and poly-ornithine (PLO) which are homopolymeric chains of the positively charged amino acid lysine and ornithine, respectively8,10,15,16. PDL is formed from the D-lysine enantiomer and is more resistant to enzymatic degradation by proteases such as trypsin17,18 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 degradation19. 

Contamination

A natural consequence of creating an ideal environment for cell culture is that this environment is also perfect for the growth of undesirable contaminants such as bacteria, yeast, and fungi. Sometimes the signs are obvious as unusual growths, cloudy media, or drastic changes in media color, however contamination can often be more subtle3,9,13. Mycoplasmas are tiny, bacteria-like microbes that lack a cell wall and can grow to extremely high density in cell cultures without any visible signs13. This can also occur with slow growing, antibiotic resistant bacterial contaminations that are difficult to detect under the microscope. In either case, these contaminations can drastically alter cellular function. If you observe slow cell growth, high rates of detachment, or unexplainable cell death, a mycoplasma or bacterial contamination could be to blame3. There are a number of ways to test indirectly for mycoplasma contaminations, including PCR kits and ELISA assays, but the most recommended test is a DNA fluorochrome stain13. With this test, cells are fixed and stained so that DNA fluoresces under UV light. When compared to positive and negative control slides, mycoplasma contamination can be identified. This test can also be used to identify any other microbial contaminants, including subtle bacterial contaminations. Any contaminated cultures should be immediately discarded. If you encounter consistent issues with contamination, review your media, aseptic technique, and workspace to identify potential sources of contaminants13. 

 

References

  1. Ryan, J. A. Corning Guide for Identifying and Correcting Common Cell Growth Problems. (2008).
  2. Chi, H.-J. et al. Effect of evaporation-induced osmotic changes in culture media in a dry-type incubator on clinical outcomes in in vitro fertilization-embryo transfer cycles. Clin Exp Reprod Med 47, 284–292 (2020).
  3. Masters, J. R. & Stacey, G. N. Changing medium and passaging cell lines. Nat Protoc 2, 2276–2284 (2007).
  4. 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.
  5. 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).
  6. Arora, M. Cell Culture Media: A Review. Materials and Methods (2023).
  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. Sahu, M. P., Nikkilä, O., Lågas, S., Kolehmainen, S. & Castrén, E. Culturing primary neurons from rat hippocampus and cortex. Neuronal Signal 3, NS20180207 (2019).
  9. Segeritz, C.-P. & Vallier, L. Cell Culture. Basic Science Methods for Clinical Researchers 151–172 (2017) doi:10.1016/B978-0-12-803077-6.00009-6.
  10. Brewer, G. J. & Torricelli, J. R. Isolation and culture of adult neurons and neurospheres. Nat Protoc 2, 1490–1498 (2007).
  11. 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).
  12. 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).
  13. Ryan, J. A. Understanding and Managing Cell Culture Contamination. Corning Life Sciences (2002).
  14. 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).
  15. 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.
  16. McKeehan, W. & Ham, R. Stimulation of clonal growth of normal fibroblasts with substrata coated with basic polymers. J Cell Biol 71, 727–734 (1976).
  17. Tsuyuki, E., Tsuyuki, H. & Stahmann, M. A. THE SYNTHESIS AND ENZYMATIC HYDROLYSIS OF POLY-d-LYSINE. Journal of Biological Chemistry 222, 479–485 (1956).
  18. 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.
  19. Clément, J.-P. et al. Dendritic Polyglycerol Amine: An Enhanced Substrate to Support Long-Term Neural Cell Culture. ASN Neuro 14, 17590914211073276 (2022).