Cell culture is a technique to isolate and culture cells of interest in laboratories. The term cell generally refers to mammalian cells, although the principles apply to prokaryotes and other eukaryotic cells.
In cell cultures, animal cells are isolated from tissue sections and cultured in optimal conditions, including temperature, pH, and carbon dioxide to oxygen ratio. They feed on nutrients and growth factors in the culture medium.
To learn more about the medium components and their roles, visit our article on culture media preparation!
Type of Cells in Cell Culture
1. Primary Cells
Primary cells are individualized cells from tissue sections that have been dissociated or digested by enzymes.[2-4]
Non-cancerous primary cells are typically slow-growing. When they proliferate to 80% confluency, primary cells are subcultured or passaged so they can continue to divide. Most primary cells are finite – they can divide after being subcultured to a certain generation before senescence. In contrast, cancerous primary cells can be indefinitely subcultured.[1-3]
Primary cells are regarded as the closest representation of the originating tissue. Initially, their genetic and phenotypic identities are heterogeneous because the cell composition in tissue sections is mixed. Hence, primary cells’ identities can change after every passage. However, primary cell identities can become more homozygous if one cell type dominates the other in its growth and proliferation rates.[1-2]
2. Secondary Cells
Secondary cells are primary cells that have been passaged and cultured in a fresh medium several times.
Oftentimes, secondary cells are immortal. They can be cultured indefinitely when nutrients and fresh medium are regularly provided. Compared to primary cells, they are fast-growing and have more homogeneous genotypic identities that are consistent over generations. However, secondary cells tend to differentiate into aberrant cells when grown for a prolonged period.
Secondary cells are easier to handle than primary cells. They are typically cultured to produce a population of cells with similar identities for viral, immunology, and toxicology tests.
3. Cell Lines
Cell lines are cells from an aggregated or monolayer of primary cells that have been passaged at least once.
The cell lines’ genotypic and phenotypic identities are derived from their sourced primary cells. Their identities are more homogeneous because cell aggregate and monolayer typically represent a group of cells with similar identities.[1-2]
- Immortalized cell lines can indefinitely proliferate. Often, these originate from stem cells or cancerous primary cells. They can also be created by genetic transformation with viral elements, mutated genetic elements, or fusion with cancerous cells.
- Cell strains are selected subpopulations of a specific cell line, which has been genetically engineered to possess additional characteristics. Genetically manipulated cell strains are called transformed cell lines.
Cell lines are the go-to cells in research and development, while primary cells are for confirmation studies. Examples of commercially available cell lines are human epithelial cell lines from cervical cancer cells (HeLa) and hamster ovary cell lines (CHO).
Cell Culture Applications
Cell culture enables users to multiply specific cells for tissue regeneration or to obtain cellular products such as genetically engineered proteins and attenuated vaccines.
In addition, cell culture can be adapted into a testing or investigative platform. For example, the in vitro evaluation of candidate compounds is performed in the relevant cell culture before animal studies and clinical trials.
It screens for the compound toxicity and the target cells’ response to the candidate compounds, allowing researchers to select only promising ones for further testing. Similarly, cell culture can be used to model and demonstrate the hypothesized gene function, cancer, hereditary or viral disease pathology, and pathogenesis.
What To Consider When Setting Up and Maintaining Cell Culture Laboratories
Aseptic & Biosafety Practices
Aseptic and biosafety practices are keys to running a safe and efficient cell culture laboratory. Aseptic practices keep the cell culture sterile and prevent contaminations from workers and the working areas. Biosafety practices create a safe environment by reducing threats during exposure to cell cultures, which can be infectious.[1-2]
Most cell culture works are performed in laboratories with at least biosafety level-2 (BSL-2), where access to the working area is restricted to trained personnel. It is sufficient for non-contagious cell lines but not for works involving primary cells. Most lab works involving primary cells are handled in a BSL-3 or BSL-4 laboratories, depending on the potential risks, availability of relevant therapies, and vaccines.
To learn more about biosafety principles and the meaning of each biosafety level, read our article on biosafety in microbiology laboratories.
Equipment for Aseptic & Biosafety Practices
1. Biosafety Cabinet
- A biosafety cabinet, also known as cell culture and laminar flow hood, is the must-have equipment in cell culture laboratories. It provides an aseptic working area and prevents contaminations in the cell culture.
- The class II biosafety cabinet is the recommended class for cell culture. It has a built-in fan and a high-efficiency particulate air (HEPA) filter that keeps bacterial, fungal, and viral particles from contaminating the cell culture and working surface.
- Disinfect the working surface inside the cabinet, equipment, containers, and tools used to work with the cell culture before and after use. Wipe them clean with 70%(v/v) ethanol and other disinfectants or use a UV lamp before and after work.
- An autoclave sterilizes buffers, equipment, and consumables before they come into contact with the cell culture, reducing contamination risks. Wastes created while working with the cell culture should be separated in biohazard waste containers and autoclaved to decontaminate before disposal.[1-3]
- You can learn more about autoclaves and alternative sterilizing technologies here!
3. Personal protective equipment (PPE)
- Personal protective equipment (PPE) and good hygiene practice are the last line of defense between workers and threats from hazardous agents. It is recommended to wear PPE before entering the cell culture lab.
- When PPE is contaminated with cell cultures, it should be immediately removed, disposed of in biohazard waste containers, or kept in a closed container before decontamination. Reusable PPE should be taken off inside the lab before leaving. For more details, check out our article on laboratory safety rules and guidelines.
Equipment for Cell Culture Laboratories
Aside from equipment for biosafety and aseptic practices, cell culture laboratories also require the following equipment:
- A CO2 Incubator provides an environment for cultured cells to proliferate. Air-jacketed CO2 incubators are resistant to temperature fluctuation, making them suitable for multi-user cell culture laboratories that usually deal with adherent cell culture. In cases of suspension cell culture, CO2 shaker-incubators are more fitting. If you want more information on laboratory incubators, read our article on how to choose the right incubator for your research/lab work!
- An inverted light microscope is used to observe and record morphology, attachment, and contaminations in cell culture vessels.
- A hemacytometer is a set of microscopic slides and glasses used to estimate the cell concentration in a culture vessel based on the number of sampled cells. In many cases, the sampled cells are stained with dyes such as trypan blue and safranin to help distinguish between cultured cells and contaminations.
- A temperature-adjustable water bath or dry bath is necessary to warm the cell culture media and other buffers to the culturing temperature.
- A vacuum pump is connected with a silicone hose and a Pasteur pipette to aspirate the cell culture medium. The vacuum pump can be replaced with sterile pipettes and pipette fillers.
In addition, cell culture laboratories also need general equipment for life science laboratories such as microcentrifuges, laboratory refrigerators, freezers, etc.
Consumables for Cell Culture Laboratories
Most consumables in cell culture laboratories are generic, except for some that come in contact with the cell culture. The plastic consumables and glassware are used and handled like those found in most life science laboratories.
- Cell culture flasks are vessels for suspension cell cultures. The flask bodies are made from sterile polycarbonate or polystyrene plastics shaped into an Erlenmeyer flask or a rectangular box with a canted neck sealed with a solid or vent cap. The latter contains a hydrophobic filter that clarifies potential external contaminants from the vessel, facilitating a better gas exchange without compromising contamination risks.
- Cell culture dishes and plates are culturing vessels for adherent cell cultures, which require a uniformly flat and non-hydrophobic surface for cell attachment and monolayer Plasticware compatible with cell cultures must be TC-treated, which signifies that the surface is modified to become more hydrophilic than typical plastics.
- Cell culture towers are vessels for scaling-up adherent cell cultures. They must also be TC-treated to decrease the hydrophobicity of the vessel’s surface.
- Cryogenic vials and storage boxes are used to cryogenically store/preserve cell culture portions. The vials are made from polycarbonate that withstands pressure and frost from prolonged exposure to liquid nitrogen. Most of them are sterile or autoclavable before use. Similarly, cryogenic storage boxes are made from similar materials or water-repellent cardboard paper so they can withstand water and liquid nitrogen.
Cell culture is a technique that isolates and cultivates animal cells in laboratories. It requires specialized equipment such as class II cabinets, CO2 incubators, and inverted microscopes. The crucial part of running and maintaining cell culture laboratories are aseptic and biosafety practices, which prevent contamination and create a safe working environment for workers.
- Segeristz, Charis P. and Vallier, Ludovic. “Chapter 9 Cell Culture: Growing Cells as Model Systems In Vitro” Basic Science Methods for Clinical Researchers, 2017, pp.151-172 http://dx.doi.org/10.1016/B978-0-12-803077-6.00009-6
- Verhoeckx, K., Cotter, P., López-Expósito, I., et al., editors, “Part II General Introduction to Cells, Cell Lines and Cell Culture” The Impact of Food Bioactives on Health: in vitro and ex vivo models, 2015, pp. 83-92, https://www.ncbi.nlm.nih.gov/books/NBK500160/
- Verma, A., Verma, M., and Singh, A. “Animal tissue culture principles and applications” Animal Biotechnology, 2017, pp. 269-293 https://doi.org/10.1016/B978-0-12-811710-1.00012-4
- Phelan, Katy, and May, Kristin M. “APPENDIX 3F Mammalian Cell Tissue Culture Techniques” Current Protocols in Molecular Biology A.3F.1-A.3F.23, 2017, https://doi.org/10.1002/cpmb.31