Cell culture is a technique that involves the cultivation of cells under carefully controlled laboratory conditions. Since its introduction in the early 20th century, it has played an indispensable role in studying tissue development, elucidating disease mechanisms, and facilitating the production of biopharmaceuticals. This method allows for the precise manipulation of genes and molecular pathways, making it an ideal platform for disease modeling and drug toxicity testing.

The controlled environment, along with the use of homogeneous cell populations, ensures that cell culture experiments yield reproducible and consistent results, in contrast to studies involving whole organs. As such, cell cultures are indispensable for advancing research in cell biology, understanding disease mechanisms, investigating drug effects, and developing tissue engineering techniques. They are integral to preclinical studies, cancer research, and functional genomics.

However, to maintain the reliability and validity of cell culture experiments, adherence to Good Cell Culture Practices (GCCP) is critical. Any deviation from these standards can lead to experimental errors, undermining the reproducibility and accuracy of the findings.

Essentials of cell culture

Along with a controlled environment, cell culture involves using specialized media that supply essential nutrients, pH buffering, and growth factors for cultivating cells. The success of cell culture relies on the careful selection of cell lines, appropriate culture systems, and suitable media supplements.

Cell line selection

Immortalized or continuous cell lines are extensively used in research owing to their cost-effectiveness, ease of handling, and ability to provide consistent, reproducible results. These cell lines serve as valuable models for investigating disease mechanisms and drug development. However, they may not fully recapitulate the complex behavior of primary cells, necessitating cautious interpretation of experimental outcomes.

A study published in the International Journal of Cancer highlighted that over 5% of submitted manuscripts were based on misidentified cell lines. This underscores the importance of stringent quality control, thorough staff training, and active collaboration among journals, institutions, and funding agencies to uphold rigorous cell culture practices.

Culture systems

The selection of culture systems is driven by the specific requirements of each experiment. Two-dimensional (2D) cell cultures are simple, cost-effective, and well-suited for functional assays. However, they often fail to preserve critical cellular interactions, leading to altered cell morphology and division. These limitations have prompted the development of three-dimensional (3D) culture models, which offer a more accurate representation of in vivo conditions. Optimizing 3D cell culture conditions can significantly enhance our understanding of cancer biology, facilitate biomarker discovery, and support the study of targeted therapies.

Cell cultures may be maintained under adherent conditions, where cells attach to surfaces such as glass or plastic dishes, or in suspension, which better mimics the natural environment of certain cell types, such as lymphocytes. Suspension cultures are particularly advantageous when studying cells in a more physiologically relevant context.

Media formulation and supplements

The formulation of cell culture media is critical for supporting cell growth and ensuring reproducibility. Some cell types require non-essential amino acids to optimize growth and alleviate metabolic stress. Widely used media, such as modified Eagle’s medium, are formulated to provide essential nutrients, including carbohydrates, amino acids, vitamins, and salts, necessary for maintaining various mammalian cell types.

In certain instances, growth factors, hormones, and antibiotics are incorporated into specialized media to support specific cellular functions and prevent contamination.

Aseptic cell culture practice

To prevent contamination, a biosafety cabinet is essential for maintaining a sterile environment during cell culture procedures. It should be placed in a draft-free area and undergo a warm-up period prior to use. The work surface must be decontaminated with antifungal detergent followed by 70% ethanol, and all equipment entering the cabinet must be sanitized. Regular maintenance, including cleaning, servicing, and ensuring proper airflow, is crucial for the cabinet’s optimal performance.

Commercially available sterile media and filter sterilization techniques help minimize contamination risks, while autoclaving is used to sterilize equipment that comes into contact with cultured cells. Although antibiotics such as penicillin/streptomycin can inhibit bacterial growth, their routine use may lead to antibiotic resistance and can interfere with cell metabolism and experimental results. Therefore, their use should be judicious and limited to specific circumstances.

Step-by-step protocols

The following guide outlines the essential protocols for cell culture.

Thawing cells

Successful mammalian cell culture requires key techniques, including thawing frozen stocks, plating cells, changing media, passaging, and cryopreservation. Cells from previously cultured lines can generally be thawed directly into a new culture medium. It is critical not to thaw cells from different stem cell lines simultaneously. When thawing cells from the same line and lot, no more than two vials should be thawed at once to minimize the exposure of thawed cells to the cryopreservation medium, thereby reducing potential damage.

Following thawing, cells are plated into containers suitable for either adherent or suspension culture, with the necessary nutrients and supplements.

Maintenance of proper temperature and pH

The optimal temperature for cell cultures depends on the species’ body temperature and the cells’ natural environment. Most human and mammalian cells thrive at 36–37°C, whereas cells from cold-blooded animals can tolerate temperatures between 15–26°C. Human and mammalian cultures typically require a tightly regulated pH range of 7.2–7.4.

To maintain pH levels and simulate specific conditions such as hypoxia, CO₂ and O₂ levels can be controlled in specialized incubators. In addition, alternative carbon sources such as galactose or fructose may be used to reduce acid accumulation.

Subculturing (passaging)

Cell cultures consume nutrients, produce toxic by-products, and proliferate, necessitating regular passaging to maintain healthy growth. Passaging is performed when cultures reach approximately 80% confluency, transferring cells to new vessels. This process involves detaching cells by using enzymes or mechanical methods. For adherent cultures, surface area limits cell growth, while suspension cultures are constrained by cell concentration in the medium. Monitoring growth rates is essential for maintaining healthy suspension cultures.

For adherent cells

  • Adherent cells are passaged when they approach optimal confluency, using enzymatic or mechanical methods for detachment.
  • In a biosafety cabinet, cells are washed with PBS (without Mg²⁺ and Ca²⁺), then treated with enzymes such as trypsin, dispase, and EDTA at 37°C to aid detachment.
  • The detachment process is monitored microscopically and typically takes 1–60 minutes, depending on the cell type and enzyme used.
  • Detached cells are collected, enzyme activity is neutralized, cells are counted, and reseeding is performed at concentrations suitable for their growth.

For suspension cells

  • Suspension cultures are subcultured by aseptically removing one-third of the cell suspension and replacing it with prewarmed complete medium.
  • The suspension is centrifuged at 300 × g for 10 minutes, and the supernatant is discarded to concentrate cells for transfer or experiments.
  • The resulting pellet is gently resuspended in the desired medium by pipetting up and down three times.
  • High centrifugation speeds or vigorous pipetting should be avoided, as these may damage fragile single cells

Determination of cell viability

Cell viability is determined during culture and handling, especially when specific cell counts are required for assays or new cultures. A vital dye exclusion assay, such as with a blue dye that stains nonviable cells, is commonly used to mark dead cells. The cells are then counted using a hemacytometer under a microscope. The total number and percentage of viable cells are calculated, with healthy cultures typically showing 80–95% viability.

Cryopreservation

Mammalian cells are preserved in liquid nitrogen for extended periods, halting all biological activity at ultra-low temperatures. The frozen vial is thawed carefully in a 37°C water bath to revive the cells. After thawing, the cells are gradually mixed with prewarmed medium to reduce osmotic shock, followed by centrifugation and washing to remove cryoprotectants. The cells are resuspended in fresh medium, transferred to a culture vessel, and typically attached within 24 hours.

Troubleshooting and optimization

  • Cell viability issues: Proper storage and thawing protocols should be followed to minimize cell damage. Because glycerol is commonly used as a cryoprotectant to prevent ice crystal formation during freezing, it should not be exposed to light during storage, and cells should be handled with care to avoid mechanical stress that could compromise their integrity.
  • Attachment problems: To improve cell attachment, it is crucial to thoroughly wash cells to remove any residual enzymes from the digestion process. Excessive enzymatic digestion should be minimized, and fetal bovine serum (FBS) should be used to inhibit further enzymatic activity, promoting better attachment.
  • Slow growth and contamination: Ensure the correct selection of growth medium and serum for the specific cell type. Regular quality control of culture components should be conducted to verify their stability and suitability. Routine Mycoplasma testing should also be performed to prevent contamination that can hinder cell growth.
  • Confluency and passage issues: Excessive passaging should be avoided to prevent cellular stress and senescence. If confluency is too low, plating density should be adjusted accordingly to encourage proper cell growth.
  • Environmental conditions: Regular monitoring of environmental parameters such as CO₂, temperature, and pH in the incubator is essential for maintaining ideal growth conditions. Ensuring adequate gas exchange and regularly replacing the culture medium helps reduce the accumulation of metabolic waste and toxins that may hinder cell growth.