What are the common issues in cell culture?

Common issues encountered during cell culture primarily fall into three main categories: culture medium management, contamination control and abnormal cell growth. BUNSEN BUNSEN has compiled a list of these issues and the corresponding key points based on practical scenarios, as follows:

culture medium Frequently Asked Questions

Abnormalities caused by improper storage: Liquid culture medium—if not stored as required at 2–8 °C away from light, freezing may cause salt precipitation and abnormal osmotic pressure; prolonged exposure to air after opening may result in CO₂ release, causing culture medium to become alkaline and its colour to darken.

Abnormal pH levels: A mismatch between the CO₂ incubator setpoint and the culture medium buffer system, as well as microbial contamination, can cause the pH to remain persistently below 6.8, thereby inhibiting cellular metabolism, or to rise above 7.6, thereby destroying enzyme viability, ultimately reducing cell viability.

Degradation of key nutrients: Free L-glutamine degrades readily in solution; at 37 °C, the daily degradation rate can reach 10 per cent. The ammonia produced is toxic to cells and impairs their normal proliferation.

Cell condition deteriorates after replacing culture medium: As the formulations of culture medium vary between manufacturers, it is more difficult to acclimatise passaged cell lines to a new medium; replacing culture medium directly in fragile, freshly thawed cells can lead to a sharp drop in viability.

Frequently Asked Questions on Pollution

Bacterial contamination: Outbreaks occur rapidly; within 1–2 days, culture medium becomes cloudy and develops an unpleasant odour. Under the microscope, regular spherical or rod-shaped motile particles are visible; these consume large amounts of nutrients and secrete toxins, leading to widespread cell death.

Fungal contamination: In the early stages culture medium, it is difficult to detect; in the later stages, white or yellow, flocculent hyphae become visible to the naked eye. Spores can be transmitted through the air and are highly likely to spread and contaminate the entire laboratory.

Mycoplasma contamination: Cannot be directly observed under a light microscope; culture medium the culture remains clear at all times, manifesting only as abnormal cell morphology and slowed proliferation. This is the most insidious type of contamination and can persistently compromise the reliability of experimental results.

Misinterpretation of ‘black spot contamination’: Black spots observed under the microscope are not necessarily contamination; they may also be cellular metabolic by-products, cell debris, serum cold-induced precipitates or apoptotic bodies. A comprehensive assessment must be made in conjunction with the cells’ growth status to avoid mistakenly discarding normal cells.

Frequently Asked Questions on Abnormal Cell Growth

Improper subculturing techniques: Excessive digestion or excessive force when pipetting can cause significant mechanical damage to cells, resulting in a large amount of cell debris and a substantial reduction in cell viability in subsequent cultures.

serum Batch-to-batch variation: There are fluctuations in the nutritional composition of different batches of serum; switching batches without first conducting a pilot test is likely to result in a precipitous decline in cell growth.

Cell cross-contamination: When cells from different species or strains are cultured together, they cannot be accurately identified by morphological observation alone, which may lead to experimental results deviating from expectations; confirmation via STR analysis is therefore required.

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