How to Maintain Cultured Cells: A Basic Overview

Maintaining cultured cells is a crucial skill for any life science researcher who works with cell-based assays or experiments. Cultured cells are living organisms that require optimal conditions and care to grow and function properly. In this blog post, I will outline some of the key aspects and tips for maintaining cultured cells.

Types of cultured cells

There are different types of cultured cells that have different characteristics and requirements. The most common types are:

• Primary cells: These are cells that are isolated directly from a tissue or organ of an animal or human. They have a limited lifespan and can only be passaged a few times before they stop growing or undergo senescence. They retain most of the features and functions of their original tissue, making them more physiologically relevant and representative of in vivo conditions.
• Cell lines: These are cells that have been immortalized or transformed to enable indefinite proliferation in culture. They can be derived from primary cells, tumors, or genetic engineering. They are more stable and consistent than primary cells, making them easier to manipulate and scale up. However, they may also acquire mutations or alterations that affect their behavior and response to stimuli.
• Stem cells: These are cells that have the ability to self-renew and differentiate into various cell types. They can be derived from embryonic sources, adult tissues, or induced pluripotent stem cells (iPSCs). They have great potential for regenerative medicine and disease modeling, but they also pose ethical and technical challenges.

Depending on the type of cultured cells, you may need to use different media, supplements, growth factors, substrates, and protocols to maintain them.

Culture conditions

Cultured cells require specific environmental conditions to survive and thrive. Some of the main factors that affect culture conditions are:

• Temperature: Most mammalian cells grow best at 37°C, which is the normal body temperature of humans and other warm-blooded animals. However, some cells may prefer lower or higher temperatures depending on their origin or adaptation. For example, insect cells typically grow at 25-28°C, while some thermophilic bacteria can grow at 60°C or above. You should use a reliable incubator that can maintain a constant and uniform temperature for your cultured cells.
• Gas mixture: Most mammalian cells require a mixture of oxygen and carbon dioxide to support their metabolism and pH regulation. The optimal gas mixture depends on the type of medium and buffer system used, but generally ranges from 5% to 10% CO2 in air. Some cells may also require additional gases, such as nitrogen or hydrogen, to create anaerobic or hypoxic conditions. You should use a gas-controlled incubator or a gas delivery system that can provide the appropriate gas mixture for your cultured cells.
• Humidity: Most mammalian cells require a high level of humidity to prevent evaporation and dehydration of the culture medium. The optimal humidity level depends on the type of culture vessel and the volume of medium used, but generally ranges from 80% to 95%. You should use a humidified incubator or a sealed culture vessel that can maintain a sufficient level of humidity for your cultured cells.

Culture medium

Cultured cells require a suitable culture medium that provides them with the necessary nutrients, growth factors, hormones, and other molecules to support their growth and function. The composition and quality of the culture medium can have a significant impact on the health and performance of the cultured cells. Some of the main components and considerations of the culture medium are:

• Base medium: This is the liquid component that contains the basic salts, sugars, amino acids, vitamins, and minerals that are essential for cell survival and metabolism. There are many types of base media available for different types of cultured cells, such as DMEM, RPMI-1640, MEM, F-12, etc. You should choose the base medium that is compatible with your cell type and application.
• Serum: This is the liquid component that is derived from animal blood, such as fetal bovine serum (FBS), newborn calf serum (NCS), horse serum (HS), etc. It contains a complex mixture of proteins, lipids, hormones, growth factors, and other molecules that enhance cell growth, attachment, differentiation, and function. However, it also introduces variability, contamination risk, ethical issues, and high cost. You should use high-quality serum that is tested for sterility, endotoxin level, mycoplasma contamination, virus presence, etc. You should also use the appropriate amount of serum for your cell type and application, usually ranging from 2% to 20%.
• Supplements: These are additional components that are added to the base medium and serum to provide specific functions or benefits for the cultured cells. Some common supplements include antibiotics (e.g., penicillin-streptomycin), antifungal agents (e.g., amphotericin B), glutamine, sodium pyruvate, non-essential amino acids, etc. You should use the supplements that are suitable for your cell type and application, and follow the manufacturer’s instructions for preparation and storage.
• pH: This is the measure of the acidity or alkalinity of the culture medium, which affects the enzyme activity, membrane permeability, and ion balance of the cultured cells. The optimal pH range for most mammalian cells is 7.2 to 7.4, which is maintained by the buffer system of the medium and the gas mixture of the incubator. You should monitor the pH of the culture medium regularly using a pH meter or a pH indicator dye, such as phenol red. You should also adjust the pH of the medium if necessary using sodium bicarbonate or hydrochloric acid.

Subculture and passaging

Subculture and passaging are the processes of transferring cultured cells from one culture vessel to another to maintain their optimal growth and prevent overcrowding, nutrient depletion, or senescence. Subculture and passaging are essential for maintaining continuous cell lines and expanding primary cells. The frequency and method of subculture and passaging depend on the type, growth rate, and density of the cultured cells. Some of the main steps and tips for subculture and passaging are:

• Aspirate: This is the step of removing the old culture medium from the culture vessel using a sterile pipette or a vacuum system. You should aspirate gently and carefully to avoid damaging or losing the cultured cells, especially if they are adherent or fragile.
• Wash: This is the step of rinsing the cultured cells with a sterile solution, such as phosphate-buffered saline (PBS) or Hank’s balanced salt solution (HBSS), to remove any residual medium, serum, or supplements that may interfere with the next step. You should wash once or twice depending on the type and condition of the cultured cells.
• Detach: This is the step of detaching adherent cultured cells from the surface of the culture vessel using a mechanical or enzymatic method. For mechanical detachment, you can use a sterile scraper or a pipette to gently scrape or pipette up and down the cultured cells until they are detached. For enzymatic detachment, you can use a sterile solution that contains an enzyme that breaks down the proteins that bind the cultured cells to the surface, such as trypsin, collagenase, accutase, etc. You should use the appropriate amount and exposure time of the enzyme for your cell type and application, and neutralize it with serum or an inhibitor after detachment.
• Resuspend: This is the step of resuspending detached cultured cells in fresh culture medium using a sterile pipette or a vortex mixer. You should resuspend thoroughly and gently to ensure a homogeneous cell suspension and avoid cell clumping or damage.
• Count: This is the step of counting the number of viable cultured cells in a given volume of cell suspension using a manual or automated method. For manual counting, you can use a hemocytometer and a microscope to count the number of live and dead cells in a grid area after staining them with trypan blue or another dye that excludes live cells. For automated counting, you can use a device that uses optical or electrical methods to count and measure the cultured cells without staining them.
• Seed: This is the step of seeding a desired number of cultured cells into a new culture vessel with fresh culture medium using a sterile pipette. You should seed according to your cell type and application, usually ranging from 1 x 10³ to 1 x 10⁶ cells per cm². You should also distribute the cell suspension evenly over the surface of the culture vessel by gently swirling or rocking it.

Conclusion

Maintaining cultured cells is a vital skill for any life science researcher who works with cell-based assays or experiments. Cultured cells are living organisms that require optimal conditions and care to grow and function properly. By following the aspects and tips outlined in this blog post, you can improve your chances of successful maintenance and performance of your cultured cells.