Chinese Hamster Ovary (CHO) cells are a cell line derived from the Chinese hamster, widely used in biotechnology and biomedical research. They play a significant role in producing therapeutic proteins and recombinant biopharmaceuticals.
The Role of CHO Cells in Biotechnology
CHO cells are extensively utilized in biotechnology due to several advantageous characteristics that make them well-suited for large-scale production of therapeutic proteins. A primary reason for their widespread adoption is their ability to perform human-like post-translational modifications, particularly glycosylation. Glycosylation, the process of attaching carbohydrates to proteins, is important for the stability, activity, and proper function of many therapeutic proteins. Producing these complex modifications in a human-like manner is something bacterial systems cannot achieve, making CHO cells more suitable for human therapeutics.
These cells exhibit robustness and resilience, allowing them to tolerate the stresses encountered during industrial-scale production processes, including variations in shear stress and oxygen levels within bioreactors. Their ability to grow in suspension cultures is also beneficial for large-scale bioreactor production, facilitating scalability and consistency.
CHO cells can be cultivated in serum-free and chemically defined media, which helps reduce the risk of contamination from animal-derived components and enhances the overall safety of biological products. Genetic engineering of CHO cells is relatively straightforward, enabling manipulation to express a wide variety of proteins. This flexibility has led to numerous CHO cell lines, each optimized for specific protein production, and their long history of safe use and regulatory acceptance further solidify their position as a preferred host for manufacturing biological drugs, including monoclonal antibodies, recombinant proteins, and certain vaccines.
Setting Up a CHO Cell Culture Environment
Establishing a proper environment is important before commencing any CHO cell culture work. A sterile workspace, typically a biological safety cabinet or laminar flow hood, is necessary to prevent contamination. This cabinet provides HEPA-filtered, laminar airflow, protecting both cells and the operator.
A CO2 incubator maintains a stable temperature (usually 37°C) and CO2 concentration (typically 5%) to mimic physiological conditions for optimal cell growth and pH regulation.
A water bath warms media and reagents to 37°C, and a centrifuge is used for cell harvesting and pelleting. Essential media and reagents include basal media, such as Ham’s F-12 or DMEM/F-12, which provide basic nutrients. These are often supplemented with serum or serum-free alternatives, antibiotics to prevent bacterial contamination, and L-glutamine for energy production and biosynthesis. Strict aseptic technique, which involves creating a barrier between environmental microorganisms and the sterile cell culture, is important throughout all preparation and handling steps to minimize contamination risk.
Standard CHO Cell Culture Procedures
Thawing Cells
Thawing frozen CHO cells requires a rapid and gentle approach. A cryovial containing frozen cells should be removed from liquid nitrogen storage and immediately placed into a 37°C water bath. Swirl the vial gently until only a small ice crystal remains, typically within 60 seconds. Once thawed, quickly transfer the contents to a sterile tube containing pre-warmed culture medium. Gently centrifuge the cells to pellet them and remove residual cryoprotectant like DMSO.
Subculturing (Passaging) Cells
Subculturing CHO cells is performed when they reach an appropriate confluency, usually 70-90% for adherent cells. Remove the old culture medium and rinse the cell monolayer with a balanced salt solution without calcium and magnesium to remove traces of serum that could inhibit trypsin. Add a dissociation agent like trypsin-EDTA to detach the cells from the vessel surface. Once detached, neutralize the trypsin with fresh, serum-containing medium. Gently pipette the cell suspension to ensure single-cell dispersal before inoculating new culture vessels at a desired density.
Cell Counting
Cell counting determines cell density and viability, often using a hemocytometer or an automated cell counter. Mix a small sample of the cell suspension with trypan blue dye, which live cells exclude but dead cells take up. Load the mixture onto a hemocytometer, and count both viable (unstained) and non-viable (blue-stained) cells under a microscope. This allows for calculating total cell count and the percentage of viable cells.
Cryopreservation (Freezing Cells)
Cryopreservation is done for long-term storage. Healthy, rapidly dividing cells are prepared for freezing, typically at a concentration of 1-5 x 10^6 cells/mL. A cryoprotectant such as dimethyl sulfoxide (DMSO) is added to the freezing medium to prevent ice crystal formation that can damage cells. The vials are then subjected to controlled-rate freezing until they reach -80°C, before being transferred to liquid nitrogen for storage at -196°C.
Ensuring Culture Health and Viability
Maintaining CHO cell culture health and viability requires continuous monitoring and quality control. Regularly observing cell morphology under a microscope provides early indications of cell stress, contamination, or changes in cell behavior. Adherent cells appear flattened or spindle-shaped, while suspension cells remain rounded; deviations like cytoplasmic granularity or membrane blebbing signal distress.
Contamination by bacteria, fungi, or mycoplasma is a common challenge that can compromise experimental integrity. Bacterial and fungal contamination often manifest as turbidity or visible particulates in the medium. Mycoplasma, though harder to detect without specialized testing, can significantly alter cell appearance and metabolism.
If contamination is suspected, immediate action, such as quarantining affected cultures and using elimination strategies, is necessary to prevent spread. Troubleshooting poor growth or unexpected cell behavior involves assessing issues like nutrient depletion or pH shifts. Accumulation of metabolic byproducts or changes in medium osmolality can impact cell growth and productivity.
Regular monitoring of parameters like glucose, lactate, and pH is important to ensure optimal conditions for cell proliferation and protein production. Consistent record-keeping of all culture parameters and routine quality control checks are also important for reproducibility and reliability.