Cell Death Troubleshooting in Cell Culture

• Cell Death: an Overview
• Accessing and Troubleshooting Cell Death
• Related Products

Cell death: an overview

There is perhaps nothing more frustrating to a cell culturist than removing a dish from the incubator only to find that it’s full of dead cells. Getting to the bottom of the problem can be very challenging and time consuming, but with diligence and proper recordkeeping, the death of cells in culture can be diagnosed and avoided.

Cell death occurs primarily in two forms: passive and programmed. Passive cell death, or necrosis, is an ATP-independent process that results from sudden and severe environmental stress that leads to cell swelling, and eventually cell lysis. In contrast, programmed cell death is ATP-dependent. One form—apoptosis—can be triggered externally or internally, and leads to cell shrinkage, increased cytosolic Ca²⁺ concentrations, and membrane blebbing. Another form of programmed cell death—autophagy—is a process which normally promotes cell survival by degrading and recycling cellular components through the cell’s lysosomal system. Under extreme conditions of stress, however, autophagy can lead to cell death, and is characterized by the presence of a vesicle that encompasses the mitochondria, endoplasmic reticulum, and ribosomes, and then delivers them to the lysosomal system. Recent research suggests that apoptosis and necrosis are two extremes among as many as ten or more different forms of cell death, and that there is a significant amount of crosstalk between these two processes.

Figure 1. Live/Dead Cell Double Staining.

So Many Ways to Go: A Cell Death Glossary

Anoikis: apoptosis in adherent cells that occurs due to the loss of matrix interactions.  May be a major mechanism for tumor-suppression.

Apoptosis, extrinsic: programmed cell death induced by extracellular signals that start with the ligation of transmembrane receptors, including FAS

Apoptosis, intrinsic: programmed cell death that may or may not be dependent on caspase, characterized by altered mitochondrial membrane potential

Autophagic cell death: cytoplasmic vacuolization with indiscriminate catabolism of cytoplasmic contents

Ferroptosis: Iron-dependent, nonapoptotic, oxidative cell death, triggered by cysteine uptake

Necroptosis: a programmed form of necrosis, characterized by TNFR1 signaling through RIP1, when caspase-8 is inhibited

Necrosis: premature cell death, often resulting from infection or injury

Parthanatos: depletion of ATP and NAD+ by excess polymerase activity

Partial demolition/cornification: permanent modification of cell structure and function, usually caspase-dependent.  Examples include erythrocyte enucleation, lens fiber formation, and skin cell keratinization.

Pyroptosis: caspase-1-mediated apoptosis observed under inflammatory conditions—responsible for attrition of T cells in HIV/AIDS

Secondary necrosis: necrosis that occurs as a consequence of apoptosis, often observed in cell culture

Accessing and Troubleshooting Cell Death

Careful microscopic examination of culture vessels may reveal obvious cell death characterized by cell crenation, blebbing, and debris consisting in part of cell ‘ghosts’, or membranous remains. For more subtle cases, there are a variety of assays available for assessing cell death in cultures. Perhaps the most accessible of these is the trypan blue exclusion assay that is based on the principle that healthy cells with intact membranes will exclude the dye, and is quickly performed on detached or suspension cells by hemacytometer. Viability assays can be used to distinguish and quantify live cells. These assays assess cell viability by measuring cellular functions such as enzyme activity, ATP production, plasma membrane function, and cell adherence.

Figure 2. Cell Counting Using a Hemocytometer and Trypan Blue.

Table 1. Common causes of cell death, and solutions for maintaining viable cultures

Root / Cause	Solution
No/few viable cells after thawing from stock
Stock culture was of poor quality	Ensure starting culture used to generate stock has been properly identified, and is healthy, free of microbial contamination, and late in the log phase of growth (but not 100% confluent). Harvest cells gently to prevent damage.
Stock was stored incorrectly	Stock should be stored at temperatures below -130 °C, ideally in liquid nitrogen, at all times to ensure maximum viability. Storage in the vapor phase above the liquid nitrogen is preferable to storage in the liquid itself due to the risk of vial explosion during thawing if liquid nitrogen has leaked into the vial.
Stock was thawed incorrectly	Follow the supplier’s recommended protocol when thawing cells. In general, cells should be thawed rapidly. Use pre-warmed media. Remove media containing cryoprotectant as soon as possible to prevent a reduction in viability. Ensure cells are handled with care. Do not vortex or centrifuge at high speeds as cells are particularly vulnerable to damage following cryopreservation.
Poor cell attchment after thawing from stock or passaging	For a list of possible causes of cell detachment, visit here
Cell death in previously healthy culture
Cells have been passaged too many times	Obtain a new stock of cells that has been subcultured fewer times.
Temperature fluctuation in incubator	Manually check incubator temperature with independent thermometer. Because even a few minutes of high temperatures can lead to cell death, record temperature over time.
CO2 is contaminated with cytotoxic components	Use CO2 graded for cell culture.
CO2 levels do not match what is needed by the bicarbonate-based buffering system of the media being used, leading to inadequate control of pH	Ensure that incubator CO2 levels match what is required by the buffering system. In general, the higher the level of bicarbonate in the buffer, the greater the concentration of CO2 needed.
Microbial contamination	See Cell Contamination for an extensive list of ways to prevent and eliminate microbial contamination. Note that while fungal, viral, and many types of bacteria may be to blame, mycoplasma contamination rarely causes cell death.
Exposure of cultures to fluorescent light causes light sensitive media components (riboflavin, tryptophan, and HEPES) to be converted to cytotoxic free radicals and H2O2	Store cells and media in the dark away from fluorescent light.
Cells have become too confluent	Cell death occurs when cultures become overcrowded. Passage cells late in the log phase of growth; do not allow confluence to exceed 80%.
Media, serum, buffers, etc. are of poor quality or incorrectly formulated.	Discard current reagents and use new lots. If cell death results following use of new reagent(s), identify possible suspicious lots. Test all new lots of reagents in culture prior to introduction to valuable or irreplaceable cultures.

Related Products

Culture Media	Trypsin	Fetal Bovine Serum (FBS)	Antibiotics
Culture Flasks	Authenticated Cell Lines	Pipette Tips	Sterile Filtration
Pipettors	Scepter™ 3.0 Automated Cell Counter	Mycoplasma Detection	Cell Viability Assays
Hemocytometer	Trypan Blue Exclusion Assay	MilliSentials™ Aliquoting Pipette Controller	MilliSentials™ Lab Labeling System

References

1. Krampe B, Al-Rubeai M. 2010. Cell death in mammalian cell culture: molecular mechanisms and cell line engineering strategies. Cytotechnology. 62(3):175-188. https://doi.org/10.1007/s10616-010-9274-0

2. Cummings BS, Wills LP, Schnellmann RG. 2012. Measurement of Cell Death in Mammalian Cells. Current Protocols in Pharmacology. 56(1):12.8.1-12.8.24. https://doi.org/10.1002/0471141755.ph1208s56

3. Méry B, Guy J, Vallard A, Espenel S, Ardail D, Rodriguez-Lafrasse C, Rancoule C, Magné N. 2017. In Vitro Cell Death Determination for Drug Discovery: A Landscape Review of Real Issues. Journal of Cell Death. 10117967071769125. https://doi.org/10.1177/1179670717691251

4. Fundamental Techniques in Cell Culture: Laboratory Handbook. . 3rd Edition. ECACC and Sigma Aldrich®

5. ATCC Animal Cell Culture Guide: Tips and Techniques for Continuous Cell Lines..

6. Ryan, JA.. Corning Guide for Identifying and Correcting Common Cell Growth Problems..

7. Cryogenic Storage of Animal Cells. ATCC. Tech Bulletin No. 3..

8. Primary Cell Culture. Sigma Aldrich®.