Cell Culture in Bioreactors

In bioreactors, mixing of the culture is essential to ensure the homogenous transfer of oxygen and nutrients to the cells, for maximal cell growth and productivity. However, mixing generates shear forces, which, if too high, can damage “shear-sensitive” cells like mammalian and stem cells.To prevent potential drops in product yield and quality, the shear force on the cells must be considered. There are several ways to reduce the shear force imparted on the culture, while still maintaining effective mixing. This includes the type of impeller and the method of gassing.

Impellers are agitation devices that are equipped with blades and are used in bioreactors to mix cell cultures. The orientation of impeller blades influences the shear forces on the bioreactor culture:

• Blades that are axial, or have a 45°-degree angle, provide gentle yet effective mixing that is suitable for sensitive cell cultures. Examples are pitched-blade impellers and marine impellers.
• Radial blades are suitable for more robust cell cultures, hence are more commonly used for microbial fermentation. An example is the Rushton impeller.

Other aspects related to the impeller design can also affect the shear force on the culture, including the number of blades, the impeller speed and diameter.

The addition of gasses (referred to as ‘gassing’) is an essential aspect of bioprocessing, required to add oxygen into respirating cells. Gas can either be added into the bioreactor headspace, or sparged into the culture suspension, which influences the shear force on the cells. Gassing into the headspace is recommended for shear-sensitive cultures, as gassing within the culture introduces bubbles, which can cause shear forces when breaking up and potentially damage sensitive cells.

Critical process parameters (CPPs) are essential for cell growth, survival, and product formation. These can include, but are not limited to temperature, the dissolved oxygen concentration (DO), pH, and metabolite concentrations. Within cell culture in bioreactors, the critical parameters and their optimal setpoints must be identified before setting up effective monitoring and control systems to achieve maximum yields and productivity.

The regulation of CPPs is important for the yield and quality of biologics like monoclonal antibodies. Bioprocess monitoring and control is also particularly important for culturing high-quality stem cells, with the strict maintenance of an optimal culture environment required to support the reproducible and scalable cultivation.

Bioreactor controllers can greatly improve the efficiency of parameter monitoring and control. These systems use real-time, in-process sensors to detect changes in parameters. This data is transmitted to integrated bioprocess control software that precisely regulates the adjustment of pumps, valves, or motors to maintain optimal parameter concentrations. This achieves automated and continuous parameter control, reducing the need for human intervention and providing a more constant culture environment to maximize product yield and quality.

When working with cell culture in bioreactors, the operation mode is an important consideration and refers to the strategic addition of substrates essential for cell growth and productivity. Several operation modes have been developed for bioreactor cultures, including batch, fed-batch, and perfusion culture. In batch fermentation, the substrate is added only at the start of the bioprocess. In fed-batch fermentation, batch fermentation is conducted for a period of time, followed by the incremental addition of substrate towards the end of the bioprocess to extend the culture’s productivity. However, the highest cell densities and yields can be achieved with perfusion culture, with continuous substrate addition.

Perfusion is a continuous culture technique with constant substrate addition, where cells are retained or recycled within the bioreactor, while the medium is exchanged to replenish nutrients and remove secreted products and by-products. This allows for a steady culture environment to be maintained to improve product yield and quality. Perfusion culture requires the use of a cell retention device. There are different options available, including (alternate) tangential flow filter devices, spin filters, and packed-bed bioreactors.

Contamination is a challenge for cell cultures in bioreactors. The cellular environment is a perfect balance of nutrients, oxygen, pH and temperature, which unfortunately allows unwanted microbes to thrive. Within just a few hours, microbial contaminants can take over a culture, resulting in wasted time, efforts, and money. Contamination can occur in all types of bioreactor cultures, although human and animal cell cultures are at a higher risk. These cells have a slower growth rate and so are less able to compete with faster growing microorganisms that may unintentionally be introduced into the culture.

Traditional, reusable bioreactors face an increased risk of microbial contamination. In these vessels, contamination can arise from several sources, including:

• The integration of equipment such as sensors
• Improperly installed or damaged seals
• Improper cleaning between uses

These bioreactors also have an increased risk of cross-contamination, if the equipment is used to culture different types of cells or the production of different types of proteins.

Single-use bioreactors , made from disposable plastic rather than glass or steel, can be used to reduce the risk of contamination. These bioreactors remove the need for cleaning procedures between bioreactor runs to reduce the risk of microbial contamination and cross-contamination. Sterilization can be conducted after equipment assembly and after the integration of sensors to further reduce contamination risk.

Typically, cell culture in bioreactors starts off as small-scale, with the gradual increase in working volumes until pilot- or production-scale is reached. However, this is no easy feat, with an increase in bioreactor size often resulting in less efficient culture mixing that causes reduced oxygen and nutrient transfer and negatively impacts the bioprocess. Many parameters influence the efficiency of cell culture in bioreactors and should be optimized to prevent a drop in productivity when scaling up. This includes mixing time, bioreactor and impeller design, impeller tip speed, power input per volume, and the oxygen transfer rate.