Different cell lines have different characteristics and optimal growth conditions. Identifying and understanding these will be necessary for developing a suitable method for culturing cells and adopting the appropriate cell culture technology for your application. Typically, these characteristics are:
- Sensitivities to temperature
- Potential to damage via shear
- Oxygen and nutrient demand
- Growth rate
- Metabolic profile
Having defined goals and knowing the desired results in terms of cell viability, cell density, productivity, and titers (if applicable) are influential factors that help shape cell culture process development. Further considerations for selection of appropriate cell culture technologies and bioreactors may take place when a process is developed, and the basic process flow and steps are defined.
Adherent and suspension cell culture/HEK and CHO cells
Different bioprocess applications warrant important considerations regarding cell cultures and cell culture technologies.
Adherent cell culture is easy to setup and control and offers a flexible platform for many different cell types, as most un-modified mammalian cells thrive best in adherent-dependent growth conditions. Lentiviruses, adenoviruses, and adeno-associated viruses (AAV) are broadly employed for gene and cell therapy applications. These viruses are commonly produced using human embryonic kidney (HEK) cells in adherent conditions. Historically, adherent cell culture was the preferred method for the biomanufacturer, using HEK cells due to the favorable characteristics during the early development stages. Adherent cell culture is however, limited by growth surface area, and flatware, typically used during development, struggles to scale efficiently to industrial-scale solutions. The use of cell culture microcarriers in suspension offers a solution to this limitation. Microcarriers are small beads (diameter of around 150 µm) that are kept in suspension in culture media and allow adherent cells to grow attached to their surface. This modality allows higher cell densities of adherent-dependent cells thanks to the significantly increased growth surface. This is particularly beneficial for applications that require higher product titers and process scale-up.
Some cells can be adapted to suspension cell culture. Chinese Hamster Ovary (CHO) cells for instance are very robust, versatile cells that deliver high productivity and desirable yields. They are commonly grown in suspension for production of antibodies and recombinant proteins. Similar to CHO cells, multiple clones of HEK cells can be selected for their ability to grow in suspension. HEK cell suspension culture is advantageous for manufacture of recombinant proteins or monoclonal antibodies, thanks to their proficiency in generating precise and very specific human glycosylation patterns, essential for the effectiveness of these classes of therapeutic agents. Due to the simpler process of cell recovery, suspension culture is a valuable platform for applications where the cells themselves represent an intermediate or final product, for example hematopoietic stem cells or CAR-T cell production/expansion. Suspension vessels like stirred tank or rocking platform bioreactors can be employed in the seed train process as an intermediate step of cell expansion before the large-scale production run.
Considerations based on the desired product type, product purpose and required production scale are crucial in defining the appropriate cell types, cell culture modalities and processes.
Cell culture and the vessels we choose
It is evident that different cell culture modalities, manufacturing purposes and scales require distinct cell culture vessels. In early research and development phases polymeric flatware is commonly employed for adherent cell culture at small scale. Similarly, glass, or polymeric shaker flasks are utilized for suspension cell culture at small scale and as part of the seed train process at larger scales. Moving to production either at small or large scale, there is a demand for better-suited vessels like cell factories or bioreactors. In flasks and cell factories, it is only temperature and CO2 concentration that are usually regulated. These types of vessels are typically enclosed in incubators where the temperature is usually set to 37°C and CO2 concentration between 5% and 10%. Both temperature and CO2 are maintained constant, whereas other key parameters cannot be controlled; pH, for example cannot be adjusted and it is fated to drift over time.
Bioreactors, instead, allow for a dynamic, fine regulation of temperature, pH, oxygen flow and CO2 concentrations. Precisely controlling these parameters is crucial for efficient cell growth and product yield. Different types of bioreactors can be employed for adherent or suspension cell culture applications.
It should be noted that cell growth and production phases can require different optimal parameters. For example, when using HEK cells to produce lentiviral particles, the sensitivity of both should be considered when defining the optimal temperature and pH for cell growth and virus production. In addition to determining optimal temperature and pH setpoints, extensive early development investigations are required to define process specific optimal cell culture conditions such as media composition, oxygen demand and nutritional needs. Furthermore, proper process transfer is needed for efficient scale-up and successful product manufacture.