The cultivation of cells in an artificial environment has become a versatile tool in cellular and molecular biology, especially for cell research. Cultured primary cells and continuous cell lines are indispensable in investigations of basic, biomedical, and translation research.  In primary cell culture, some common problems can be encountered due to various factors, including environmental conditions and contamination, and there are strategies to solve these issues.

1 pH Shift in Medium:

Cause: Incorrect carbon dioxide (CO₂) tension.

Solution: Adjust the CO₂ levels in the incubator based on the concentration of sodium bicarbonate in the medium. For sodium bicarbonate concentrations of 2.0 to 3.7 g/L, 5% to 10% CO₂ should be used, respectively. Alternatively, switch to CO₂-Independent Medium or add HEPES buffer to the medium.

2 Contamination by Bacteria, Fungi, and Yeast:

Cause: Contamination can occur from laboratory equipment, reagents, and the operator through various means such as hair, hands, breath, or clothing.

Solution: Discard contaminated cultures and medium. Use good aseptic techniques, reduce accidents like spillages, keep work areas clean, and perform routine quality checks for contamination.

3 Mycoplasma Contamination:

Cause: Mycoplasmas can alter the host culture's cell function.

Solution: Segregate and test the culture for mycoplasma infection. If confirmed, discard the culture and decontaminate the work area.

Chemical Contamination:

Cause: Incorrect reagent constitution or substandard quality of water, sera, or containers.

Solution: Ensure the quality of reagents and materials used in the culture. Routine checks for endotoxins and other contaminants are essential.

4 Cells Not Adhering to Culture Vessel:

Cause: Over-trypsinization, mycoplasma contamination, or lack of attachment factors.

Solution: Trypsinize cells for a shorter time or use less trypsin. For mycoplasma contamination, test and discard if necessary. Ensure that serum-free media contain necessary attachment factors.

5 Decreased Growth of Culture:

Cause: Changes in medium or serum composition.

Solution: Compare media formulations and serum lots for differences, increase the initial cell inoculum, and adapt cells sequentially to new media.

6 Nutritional Requirements:

Problem: Primary cells are sensitive and may require nutrients not present in classical media.

Solution: Use specialty media tailored to the specific nutritional needs of each cell type. Customized media can be formulated with reduced serum levels to prevent unwanted differentiation or the growth of contaminating cells like fibroblasts.

7 Substrate for Cell Attachment:

Problem:  Standard synthetic polymers may not promote optimal cell attachment and growth.

Solution: Seed primary cells on more physiologically relevant substrates to enhance cell attachment, growth, and purity.