What is Cell Culture?

Cell culture is a laboratory technique used to grow and maintain cells in aseptic conditions. This process involves using a cell line, which includes a population of cells with uniform phenotypes and characteristics originating from a single progenitor cell.

In Vitro Cell Culture

In vitro cell culture allows for a greater degree of control over experimental conditions like medium composition, temperature, CO2 and humidity levels. As cells proliferate, they must be regularly passaged and monitored for signs of contamination to ensure quality and integrity of the culture.

Under optimal growth conditions, cell cultures can be harnessed to produce large quantities of therapeutics like recombinant proteins and monoclonal antibodies, to name a few.

Types of Stable Cell Line Types

Stable cell lines are derived from immortalized cells that have undergone genetic modification. These modifications often optimize expression of target genes with the purpose of expressing and secreting biological by-products with therapeutic implications. They are favored for their ability to rapidly grow and scale within controlled bioreactor conditions. Below are the 2 most widely used stable cell lines.

CHO Cells: Powerhouses of Therapeutic Protein Production

CHO (Chinese hamster ovary) cells are commonly used for the large-scale production of monoclonal antibodies (mAbs) and recombinant proteins (rProteins). These cells can grow and divide rapidly under controlled bioreactor conditions making them well-suited for the high-yield production of therapeutic proteins.

HEK 293 Cells: Pioneers in Viral Vector Production

HEK 293 (Human Embryonic Kidney 293) cells are widely used for viral vector production. Viral vectors like Adeno-associated virus (AAV) have therapeutic application for vaccines and gene therapies.

Cell Culture Optimal Growth Conditions

Monitoring and controlling growth conditions like temperature, pH, dissolved oxygen, CO2, media composition and cell density are critical because they directly impact cell health.

Temperature:

The optimal temperature for most cell cultures is 37°C. However, some cells may require special incubation conditions (e.g., lower or higher temperature). Incubators are regularly calibrated to ensure that the correct temperature is maintained.

pH:

The pH of the culture media is important for several reasons. First, it can affect the solubility of certain compounds in the media, which can impact cell growth or metabolism. Second, changes in pH can indicate contamination of the culture by microorganisms. The pH of the cell culture environment is usually maintained between 7.0 and 7.5.

Dissolved Oxygen:

Dissolved oxygen (DO) levels in the culture media are another critical parameter. Oxygen is required by cells for respiration, and too little oxygen can result in poor growth or death of the cells, while too much oxygen can be toxic. DO levels are monitored within a narrow range that is optimal for the cell type being cultured.

CO2:

Carbon dioxide (CO2) plays a critical role in maintaining the proper pH and oxygen levels for mammalian cell culture. The optimal CO2 level for cell culture varies depending on the type of cells being grown but is typically between 5% and 10%. High CO2 levels can lower the pH of the culture medium and cause it to become acidic. Low CO2 levels can turn the culture medium basic and more alkaline. Both situations can be toxic to cells.

Media Composition:

Cell culture media typically contains a mixture of nutrients, amino acids, growth factors and hormones that support the proliferation and survival of cells. The composition of the cell culture medium can vary depending on the type of cells being cultured and the desired outcome of the culture.

Cell Culture Considerations

Significance of Cell Counting

Cell counting is needed because it allows researchers to determine the number of cells in a culture to monitor the growth and health of the cells over time. Knowing the number of cells in a culture is important for several reasons:

1. Cell density: Cell density refers to the number of cells per unit area or volume. It can have a significant impact on cell behavior, such as growth, differentiation and apoptosis. Knowing the cell density allows for optimizing the conditions for cell growth and ensuring that the cells are not overcrowded.
2. Cell growth and proliferation: Cell counting allows for monitoring the growth and proliferation of cells over time. By counting cells at regular intervals, determinations can be made of the rate of cell growth, the doubling time and the saturation density of the culture.
3. Cell viability: Determining the percentage of viable cells in a culture is essential, as cell viability can be affected by various factors, such as the age of the culture, the quality of the growth medium and the presence of contaminants.
4. Experiment design: Knowing the number of cells in a culture is useful for designing and conducting experiments, such as drug screenings, cell-based assays and gene expression studies.
5. Automation: Overall, automation in cell counting allows for a faster, more accurate, and repeatable process, which can save time and reduce the potential for human error.

Cellular Confluence

Confluence is an important parameter when growing cells in culture, as it can impact the rate of cell growth and the yield of desired by products such as mAbs or rProteins. The number of cells per surface area or volume of culture system (e.g., dish, flask, bioreactor) and rate of cell growth all impact confluency.

Confluence Monitoring Methods and Automation

A microscopeis used to monitor confluence by checking the percentage of the surface area covered by cells or by measuring the optical density of the culture. This provides a relative measure of the number of cells present.

Automation can be used to measure confluency by using image analysis to automatically count the number of cells in a culture dish or flask and then calculate the confluency as a percentage of the total available space. Additionally, automation can be used to control the growth conditions of the cells, such as temperature, humidity and the addition of nutrients, to ensure optimal growth and confluency.

Maintenance and Subculture

• Cells in culture require regular maintenance to remain healthy and proliferate. This includes replacing the media which provides nutrients and other factors necessary for cell growth, as well as removing waste products from the vessel.
• Additionally, cells may need to be subcultured or transferred to fresh media when they reach a certain density to prevent overcrowding and ensure that all cells have access to the necessary resources

Cell Culture: Future Developments

There are several exciting developments in cell culture technology that are moving the field forward. Some of these include:

• 3D cell culture: There is a growing interest in using three-dimensional (3D) cell culture systems, as they more closely mimic the in vivo environment and can provide physiologically relevant results. These systems can include scaffolds, hydrogels, or microfluidic devices that can be used to create 3D structures in which cells can grow and differentiate.
• Automation: New developments in automation are becoming increasingly important in cell culture, as they allow for the efficient and consistent growth of a larger number of cells. Automated systems include liquid handling robots, cell counters and imaging systems.

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