The animal cell bioreactor is a system that simulates the internal environment of animals and conducts biological culture in vitro. It is a high-tech product integrating mechanics, fluids, control, biology and other disciplines. The ultimate goal is to achieve high-density cell growth and efficiently produce target products such as enzymes, monoclonal antibodies, and vaccines with medical value. Compared with the traditional biological products production process, which has many shortcomings such as long production cycle, cumbersome operations, heavy workload, and easy contamination, the bioreactor system has better stability and safety, saving a lot of labor, production space and energy consumption. , reducing production costs and having obvious advantages.
◆Stirred bioreactor
The stirring paddle rotates to drive the liquid flow, thereby providing power for liquid phase stirring. Its structure is similar to a traditional microbial fermentation tank, with the main difference being the structure of the agitator. Since animal cells have no cell walls, they are very sensitive to shear force. In order to avoid cell damage, the stirred reactor has been improved, including improving the stirring paddle, gas supply method, adding accessories, etc., to further optimize the conditions for suitable cell growth. environment.
❶Mixer
The form of the mixer has a great impact on cell growth. The choice of mixer paddles not only requires efficient mixing characteristics, but also reduces the shear force on the cells. The main purpose of this improvement is to culture cells in a low-shear flow field environment. Currently, the widely used agitator types include propeller blades, Rushton blades and ElephantEar blade agitators. A stirrer whose propeller blades mainly produce axial flow. Rushton blades mainly produce circumferential flow. Due to the large shear force and poor mixing effect, auxiliary baffles are installed to generate axial flow or a double-layer Rushton blade agitator is installed. The ElephantEar blade is a mixed flow blade. It can generate circumferential and axial flow fields by itself during stirring, which is conducive to the full dissolution and mixing of gas, liquid and materials, and the shear force of the flow field is small. On the basis of the mixing paddle, different types of mixers have been developed by adding accessories and other improvements. Negative pressure is generated in the hollow shaft of the stirrer, so that the culture medium is sucked in from the bottom of the reactor and discharged from the top; a rotating filter is added and the cell-free culture medium is collected using the rotating filter.
❷Bubble distributor
The structural form of the bubble distributor is closely related to the effect of dissolved oxygen in the culture medium. Bubble distributors commonly used in animal cell culture can be divided into L-shaped, ring-shaped, and microporous types. The L-type distributor has a simple structure and is easy to process. It is commonly used in reactors with small capacity. Due to the problem of uneven distribution of dissolved oxygen, it is not suitable for large-scale reactors. Annular bubble distributors are the most widely used, using 5 to 6 orifices with apertures between 0.5 mm and 1 mm. The microporous bubble distributor is a gas filter developed by the American Mott Company. Its structure is pressed and sintered using special metal powder. This structure can break the bubbles passing through the sintered layer multiple times to form a mist-like micron Bubble. During the gas-liquid-solid transfer process, the transfer resistance of the liquid film around the bubble hinders the cells’ ability to obtain oxygen. This liquid film resistance is a key factor in oxygen supply needs. The mild hydrophobicity of the cells prompts the cells to gather toward the air-liquid interface during the rise of the bubbles, and break as the bubbles rise to the liquid surface. This process will cause serious damage to the cells; if the bubbles are stable, it will lead to a lack of nutrients in the cells around the bubbles.
❸Heating method
The culture temperature of animal cells is usually around 37°C, and the allowable control error range is ±0.25°C. The heat source mainly consists of biological reaction heat, stirring heat, radiation heat and ventilation heat. Heating methods include overall wall heating, bottom heating and side wall heating. Generally, jacketed water circulation, electromagnetic heat generation, and electric blankets are used to control the temperature in the reactor. No matter which method is used to control the temperature, the temperature adjustment must be gradually increased to avoid excessive temperature damage and cell apoptosis.
◆Non-stirred bioreactor
The biggest disadvantage of stirred bioreactors is cell damage caused by shear force. Non-stirred reactors produce smaller shear forces and have outstanding advantages in animal cell culture.
❶Airlift bioreactor
LeFranios was first developed in 1956. Its principle is to use a circulating airlift central air inlet, no stirring device, and a guide tube added to a traditional bubble tower. Its advantages are simple structure, convenient operation, relatively mild turbulence, little damage to cells due to shearing force, and easy realization of high-density culture of animal cells. Since the enlarged airlift reactor cannot determine the operating range that is not affected by shear force, it is rarely used on a large scale.
❷Hollow fiber reactor
Hollow fiber membranes are used to isolate cells from the culture medium, and cells grow on the inner and outer walls of the hollow fiber. Its principle is designed to simulate the structure and function of capillaries. The inner diameter is about 200 μm, and the wall thickness is 50 μm to 70 μm. The tube wall is a porous membrane, and small molecules such as O2 and CO2 can freely diffuse through the membrane. Its advantages include reduced shear force, increased oxygen transfer coefficient, selective penetration of nutrients, and high-density culture of cells. However, the culture environment is uneven, the product quality is unstable, it is difficult to scale up the culture, and it is difficult to clean and reuse.
❸Torrent bioreactor
It uses a new type of non-bubble oxygen transfer mechanism. The principle is that the torrent oscillator mechanically shakes to generate rapids, causing the culture medium to repeatedly wash away the oxygen molecular layer on the inner surface of the cell culture bag, accelerating the dissolution of oxygen molecules into the culture medium to meet the normal needs of cells. growth and metabolic needs. The advantage is that it avoids the shear force generated by bubbling and stirring, greatly improves the oxygen transfer coefficient, thereby improving the cell survival rate and solving the problem of linear amplification culture. Compared with tank-type bioreactors, complex process steps such as cleaning and sterilization are eliminated. At present, it has been widely used in large-scale culture of various animal cells, but its investment cost is relatively high.
◆New bioreactor
Based on the complexity of the animal cell culture process and culture methods, different types of bioreactors are suitable for the growth requirements of different types of animal cells. For this reason, a variety of new bioreactors have been developed at home and abroad in recent years. The design of new bioreactors tends to solve problems related to foam, shear force, etc.
❶Packed bed bioreactor
The reactor is filled with a medium of a certain material for cell adhesion growth, and the cells are directly retained in the packed bed. Among them, sheet carrier culture is widely used. Provide sufficient nutrients and take away metabolic by-products through continuous perfusion of culture medium. At the same time, cells are trapped in the reactor and high cell densities can be achieved. The advantage is to further increase cell productivity and greatly reduce labor consumption. Filling media include: ceramic beads with micropores, polyurethane and cellulose foam, glass fiber, microcarriers, sheet carriers, etc. According to the flow form of the filling medium, it is divided into fluidized bed and fixed bed bioreactors. The basic principle of the fluidized bed bioreactor is that the culture fluid circulates vertically upward through ventilation and stirring, and uses a membrane separation system with a special structure to ensure sufficient exchange of gases during the perfusion culture process. The reactor can be bubble-free aerated, has good mass transfer performance and low shear force. The fixed bed bioreactor is formed by adding fixed media accessories to the fluidized bed bioreactor. The biggest disadvantage is that cell growth density and viability cannot be measured directly. However, the culture environment for cell growth and product synthesis has been optimized, and it is currently widely used in the expression and production of recombinant proteins.
❷Pulsating laminar flow bioreactor
Developed by Thompson et al., it can generate a pressure waveform that approximates physiology by introducing pulsatile laminar flow into a fluid column using a mechanical ventilator to produce a pressure waveform similar to mammalian physiology. Placing vascular structures in semi-compliant tubes to facilitate additional circumferential stretch variables serves as a potential signaling mechanism.
❸Shaking bed bioreactor
The principle of the shaking table bioreactor is that the shaking plate replaces the stirring paddle, and the culture bag generates waves to provide liquid phase stirring power. Its advantages are suitable for the culture of a variety of cells, easy linear amplification, high recombinant protein yield, saving space and labor, etc. advantage. GE’s WAVE bioreactor is widely used. It uses a plastic bag made of multi-layer composite walls to replace the stainless steel (or glass) tank of the traditional fermentation tank. The plastic bag of the fermentation tank is composed of three layers of plastic. It is fixed on the vibration platform of the host machine and performs “saw” reciprocating motion, causing the culture medium liquid in the plastic bag to produce periodic motion similar to “waves” to achieve uniform mixing.
❹Space bioreactor
Because it simulates a microgravity environment, it is called a space bioreactor. The structure consists of two inner and outer cylinders. The outer cylinder is fixed and the inner cylinder rotates, which reduces the gravity of the culture to a certain extent. The advantage is that there is no stirring shear force, the cells are protected from mechanical damage, and the cells are in a three-dimensional growth environment. Space bioreactors provide a new way to reconstruct human tissues in vitro.