Fundamental techniques used in cell culture
Cell culture involves the cultivation and growth of cells outside their natural environment, in a laboratory setting.
The following are some fundamental techniques used in cell culture:
1. Aseptic technique: Aseptic techniques are essential to maintain a sterile environment and prevent contamination. This includes working in a laminar flow hood, sterilizing equipment and media, and using proper sterile techniques, such as wearing personal protective equipment and using sterile instruments.
2. Cell line selection: Choosing the appropriate cell line for the experiment is crucial. Factors to consider include the origin of the cell line, its growth requirements, and its suitability for the intended research purpose.
3. Cell culture media: Cells require a suitable growth medium and serum providing essential nutrients, growth factors, and hormones. Cell culture media typically comprise a basal medium supplemented with fetal bovine serum (FBS) or other serum substitutes, amino acids, vitamins, and antibiotics.
4. Subculturing: Cells have a limited lifespan and will eventually stop dividing or undergo senescence. To maintain cell viability and expand the cell population, cells are subcultured by detaching them from the culture vessel, usually by trypsinization, and seeding them into new culture vessels at an appropriate density.
5. Cryopreservation: Cryopreservation allows long-term storage of cells by freezing them at very low temperatures, typically using liquid nitrogen. This technique typically involves using liquid nitrogen and adding a cryoprotective agent (e.g., dimethyl sulfoxide) to the cell suspension before freezing, which helps protect cells from damage caused by ice crystal formation.
6. Contamination and quality control: Contamination by bacteria, fungi, and mycoplasma can significantly impact cell culture experiments.
7. Monitoring Cell Growth and Viability: Regularly monitoring cell growth and viability is crucial to assess cell health and optimize experimental conditions. Techniques such as trypan blue exclusion, cell counting using a hemocytometer or automated cell counters, and measuring metabolic activity (e.g., MTT assay) are commonly employed.
8. Ethical Considerations: When working with cell culture, it is important to adhere to ethical guidelines and regulations, especially when using human or animal-derived cells. Obtain appropriate ethical approvals and ensure proper consent and compliance with relevant laws.
References
1. Philippeos C, Hughes RD, Dhawan A, Mitry RR. Introduction to cell culture. Methods Mol Biol. 2012;806:1-13. doi: 10.1007/978-1-61779-367-7_1. PMID: 22057441.
2. Honegger P. Overview of cell and tissue culture techniques. Curr Protoc Pharmacol. 2001 May;Chapter 12:Unit12.1. doi: 10.1002/0471141755.ph1201s04. PMID: 21965066.
3. Hudu SA, Alshrari AS, Syahida A, Sekawi Z. Cell Culture, Technology: Enhancing the Culture of Diagnosing Human Diseases. J Clin Diagn Res. 2016 Mar;10(3):DE01-5. doi: 10.7860/JCDR/2016/15837.7460. Epub 2016 Mar 1. PMID: 27134874; PMCID: PMC4843260.
