Animal vaccine cultivation and fermentation production

The in vitro culture of animal cells has always been a research field that scientists attach great importance to. Nearly a century after American Harrison successfully cultured the neural tissue of tadpoles in vitro in 1907, animal cell culture played a very important role in animal virus research and vaccine production. Through large-scale cultivation of animal cells, many valuable biological substances have been obtained, such as vaccines, diagnostic reagents, interferons, and monoclonal antibodies, providing favorable guarantees for human health. The concept of "tissue engineering" was proposed in the late 1980s, and the three-dimensional culture of animal cells became an important research topic, gradually making it possible for scientists to achieve their dream goal of reconstructing human tissue in vitro.

The cultivation of animal cells generally includes methods such as adherent culture, suspension culture, and microcarrier suspension culture. In the process of animal cell culture, the cell culture bioreactor is a key equipment that provides a suitable growth environment for cells and determines the quality and yield of cell culture. According to the growth requirements of animal cells, the principles that must be followed in the design or improvement of such reactors include low shear effects, good transfer effects, and fluid dynamics properties.

1. Stirring bioreactor

Stirring reactors rely on stirring blades to provide power for liquid-phase agitation, and they have a wide operating range, good mixing performance, and concentration uniformity, making them widely used in biological reactions. However, due to the lack of cell wall protection, animal cells are highly sensitive to shear forces, and direct mechanical stirring can easily cause damage to them. Traditional stirred reactors used for microbial cultivation are clearly not suitable. Therefore, the stirred reactors used in animal cell culture have been improved, including improved oxygen supply methods, the form of stirring blades, and the installation of auxiliary components inside the reactor.

1.1 Improvement of Oxygen Supply Method

In general, stirred reactors are often accompanied by bubbles, providing the necessary oxygen for cell growth. Due to the sensitivity of animal cells to the shearing of bubbles, many efforts have been made to improve the oxygen supply method.

Cage oxygenation is a type of oxygen supply method in stirred animal cell reactors, where bubbles are separated by a mesh and do not come into direct contact with the cells. The reactor can ensure both mixing efficiency and minimal shear force to meet the requirements of cell growth. Akira Kitano reported an improved stirred animal cell reactor, which is pear shaped as a whole and stirred at the bottom of the reactor. A conical stainless steel wire mesh is installed outside the stirring shaft to rotate together with the stirring shaft. The bubble tube at the axis bulges inside the wire mesh, while the cells outside the wire mesh do not directly contact the bubbles.

Gelligen reactor is a cell reactor suitable for microcarrier systems produced by New Brunswick Scientific Co. in the United States. There is a hollow guiding stirrer in the reactor, and the culture medium and cells are circulated up and down through the hollow guiding stirrer. The reactor adopts cage type oxygen supply, and the oxygen dissolved in the liquid is evenly distributed into the reactor through the convection of the liquid outside the wire mesh. The reactor also comes with a gas regulation system to control the dissolved oxygen concentration and pH value. Since the bubbles do not directly contact with the cells, the ventilation volume is not limited, and the foam is less without defoamer, and the shear force on the cells in the circulation process is also very small. The CellCul-20 animal cell culture reactor developed by the Institute of Biochemical Engineering at East China University of Science and Technology has a similar working principle to the Gelligen reactor, but uses a double-layer cage oxygen supply to improve the oxygen transfer coefficient. Vero cells were cultured using perfusion technology in a 20L reactor, and the number of cells increased by 37 times and the density exceeded 1 × 107 cells/mL after continuous cultivation for 5 days.

There are also other improved oxygen supply methods. Lavery et al. measured oxygen transfer in animal cell culture media using a reactor with double-layer agitation. The stirring shaft is hollow, and gas enters from the bottom of the shaft and is evenly dispersed in the liquid through the lower blade stirring. The upper blade suppresses the splashing of bubbles at the liquid surface, thereby reducing damage to the cells.

Suker et al. introduced a stirred bubble reactor that can minimize the damage of bubbles to animal cells in culture. There is a gas-liquid mixing tube below the liquid level in the center of the reactor, which is equipped with a bubbler and a stirrer. The liquid and the gas coming out of the bubbler are mixed in reverse flow in the mixing tube, and then flow out of the mixing tube under the action of the stirring blade to form a circulation in the reactor. They designed three different forms of mixing tubes to ensure that the bubbles inside the mixing tubes can only float at the tube mouth and do not enter the culture medium as much as possible. The cultivation effect in a 2.4L reactor in the experiment was similar to that in a 100mL spinner bottle.

1.2 Improvement of mixing blade

The form of the stirring blade has a significant impact on cell growth, and improvements in this area mainly consider how to reduce the shear force on cells. Kaman et al. improved the form of the stirring blade and added accessories to the reactor. The experiment proved that the improved reactor is suitable for high-density cultivation of shear sensitive cells. The reactor adopts a double helix ribbon impeller, and three surface baffles are installed on the top flange cover. Each baffle has an angle of 30 ° relative to the radial direction and is inserted vertically into the liquid surface. The presence of baffles reduces vortices on the liquid surface. This reactor maintained a small shear force and was used for the cultivation of insect cells in the experiment. The final cultivation density reached 6 × 106 cells/mL, with a survival rate of over 98%.

2 Non Stirred Bioreactors

The biggest disadvantage of using stirred bioreactors for animal cell culture is the high shear force, which can easily damage cells. Although various improvements have been made, this problem is still difficult to avoid. In contrast, non stirred reactors generate less shear force and exhibit strong advantages in animal cell culture.

2.1 Filled bed reactor

A packed bed is a type of material used to fill a reactor with a certain amount of filling material for cell adhesion and growth. Nutrient solution is provided through circulating perfusion and can be continuously replenished during the circulation process. The oxygen required for cell growth can also be carried by circulating nutrient solution outside the reactor, so there will be no bubble damage to the cells. This type of reactor has low shear force and is suitable for high-density cell growth.

Park et al. cultured animal cells in a continuous flow culture system consisting of a packed bed reactor and an external circulation device. The packing material in the reactor is ceramic beads with micropores, where cells grow and the culture medium can also diffuse within the micropores. The experiment proved that the reactor is suitable for high-density cultivation of animal cells, with a final cell density of 5 × 108 cells/mL.

Chiou et al. cultured insect cells in a packed bed reactor filled with polyurethane and cellulose foam, proving that these two microporous materials are suitable for the growth of insect cells and are not easy to fall off. The final average density of cells cultured at high density in two types of packed beds reached 4.3 × 107 and 5.2 × 107 cells/mL.

John et al. simulated the growth environment of mouse bone marrow cells in an air lift packed bed reactor with glass fiber as the annular packing, demonstrating that this type of reactor can be used for large-scale cultivation of animal cells. Cong et al. used microcarriers to cultivate seed cells and successfully achieved large-scale cultivation of CHO cells in a packed bed reactor, with a maximum cell density of 2 × 107 cells/mL.

2.2 Hollow fiber reactor

Hollow fiber bioreactor is widely used for the cultivation of animal cells due to its low shear force. This type of reactor consists of hollow fiber tubes, each with an inner diameter of approximately 200 μ m and a wall thickness of 50-70 μ m. The tube wall is a porous membrane, through which small molecules such as O2 and CO2 can freely diffuse. Animal cells attach to the outer wall of the hollow fiber tube and grow, making it easy to obtain oxygen.

John et al. reported a radial flow hollow fiber reactor for large-scale cultivation of animal cells. There is a vertical central distribution tube inside the reactor, and a circular bed is formed by hollow fiber tubes parallel to the distribution tube on the outside. The culture medium flows through the hollow fiber bed from the central distribution tube, and cells adhere and grow on the outer wall of the hollow fibers. The mixed gas of air and CO2 flows through the bed layer in a cross flow with the culture medium between the hollow fibers, providing oxygen to the cells and maintaining a certain pH environment. The cell metabolites are carried out with the airflow. The surface density of cell growth in this reactor can reach 7.3 × 106 cells/cm2.

Guinn invented a bioreactor device for animal cell culture, consisting of a hollow fiber reactor and a perfusion system. The liquid is circulated in the reaction system through a pump, and the perfusion system replenishes or replaces the culture medium and removes metabolites. Cells grow on one side of a hollow fiber membrane, while the culture medium diffuses nutrients to the cells on the other side of the membrane. This reactor can provide a mild growth environment for cells.

Gebhard's invention is also a bioreactor for animal cell culture, which uses a pump to control the circulation of culture medium inside the reactor. The culture medium flows inside the hollow fiber cavity and supplies the nutrients required for cell growth to the other side through the hollow fiber membrane. This reactor can control dissolved oxygen, medium composition, temperature, and pH value, making it suitable for high-density suspension culture of cells, especially for animal cells.

Gramer introduced a small hollow fiber animal cell culture reactor, which consists of oxygen permeable membrane tubes and hollow fiber bundles to form an inner and outer space for cell growth and provide nutrients to the cells. The cell culture density can reach almost 2 × 108 cells/mL. This reactor can be used to predict the possible effects of different media, culture conditions, and other factors on cell growth for large-scale hollow fiber culture systems.