Cell technology animal cell large-scale culture method

According to the type of animal cells, three culture methods, such as adherent culture, suspension culture, and immobilization culture, can be used for large-scale culture.

First, animal cell growth characteristics and culture temperature

1. The cells grow slowly, are easy to be contaminated, and need antibiotics for culture.

2. Large cells, no cell wall, low mechanical strength, poor environmental adaptability

3. Less aerobic, not able to withstand strong ventilation and agitation

4. Population growth effect, adherent growth (anchor dependence)

5. The culture process product is distributed inside and outside the cell, and the cost is high.

6. Primary cultured cells generally breed for 50 generations and degenerate and die.

Animal cells can be divided into two categories based on differences in growth matrix dependence when cultured in vitro:

• Adherent-dependent cells: need to adhere to a solid or semi-solid surface with a moderate amount of charge to grow, most animal cells, including non-lymphoid tissue cells and many aneuploid cells belong to this category.

Non-adherent-dependent cells: can grow without attaching to the surface of the solid phase, including blood, lymphoid tissue cells, many tumor cells, and certain transformed cells.

The optimum temperature for culturing cells is equivalent to the normal temperature of the various cells or tissues. The optimum temperature for human and mammalian cell culture is 35-37 °C. Deviating from this temperature, the normal metabolism and growth of cells will be affected and even die. In general, cultured cells have higher endurance to low temperatures than high temperatures. When the temperature does not exceed 39 °C, the cell metabolism intensity is proportional to the temperature; the cell culture is placed in the environment of 39~40 °C for 1 h, which is subject to certain damage, but can still recover; when the temperature reaches above 43 °C, many cells will die. When the temperature drops to 30 to 20 ° C, the metabolism of the cells is lowered, and thus the exchange of substances with the medium is reduced. The first thing that is seen is the change in cell morphology and the detachment of cells from the matrix. When the culture returns to the initial culture temperature, their original morphology and metabolism are restored to their original levels.

2. Attachment culture refers to the cultivation of cells attached to a certain solid surface.

1. Growth characteristics: Adherent-dependent cells should be attached to the culture (bottle) vessel wall during culture. The cells spread rapidly as soon as they adhere to the wall, then begin to mitosis and quickly enter the logarithmic growth phase. After a few days, the culture surface is covered and a dense cell monolayer is formed.

2. Advantages of adherent culture:

● It is easy to change the culture solution; the cells adhere closely to the surface of the solid phase, and the old culture solution can be directly poured out, and the new culture solution is directly added after washing.

● It is easy to use perfusion culture to achieve the purpose of increasing cell density; because the cells are fixed on the surface, no filtration system is needed.

• Many cells will express a product more efficiently when the cells are attached to the growth substrate.

● Different culture medium/cell ratios can be used for the same device.

● Suitable for all types of cells.

3. Disadvantages of adherent culture: compared with suspension culture

● Expanding the training is more difficult and the investment is large;

● Large area;

● can not effectively monitor the growth of cells;

4. Cell-attached surface: requires a net positive charge and a high surface activity. For microcarriers, it is also required to have a certain charge density; if it is an organic surface, it must be hydrophilic and positively charged.

5. Adherent culture system: mainly rotary bottles, hollow fibers, glass beads, microcarrier systems.

● Rotating bottle culture system: Culture of adherent-dependent cells was initially cultured using a spinner system. Rotary flask culture is generally used in the transitional stage of small-scale cultivation to large-scale culture, or as a pathway for inoculating cells in a bioreactor. The cells are seeded in a rotating cylindrical incubator-rotary bottle. During the culture, the rotating bottle is continuously rotated, so that the cells alternately contact the culture solution and the air, thereby providing better mass transfer and heat transfer conditions.

The rotary bottle culture has the advantages of simple structure, low investment, mature technology, good repeatability, and simple enlargement of the number of bottles. However, it also has its shortcomings: it has large labor intensity, large space occupation, small surface area for cell growth per unit volume, low cell growth density, and limited environmental conditions for monitoring and control during culture. The rotary bottle culture system currently used includes two types of carbon dioxide incubators and roller bottles.

●Reactor adherent culture: In this culture method, cells are attached to a fixed surface for growth, and do not flow together with the culture solution because of agitation, so it is relatively easy to change the culture solution, and no special device for separating cells and culture liquid is required. It can be used to obtain high cell density by perfusion culture, and can effectively obtain a product; however, it is difficult to expand the scale and cannot directly monitor the growth of cells, so it is often used to prepare biopharmaceuticals with small dosage and high value.

The CelliGen, CelliGen PlusTM and Bioflo 3000 reactors are commonly used in adherent culture bioreactors for basket mixing systems and disc-shaped carriers for cell-adherent cultures. This carrier is a 6 mm diameter non-woven polyester fiber disc with a high surface area to volume ratio (1200 cm2/g) for high cell density. Basket mixing system and vector culture are currently the most widely used methods for adherent cell culture, and are used for hybridoma cells, Hela cells, 293 cells, CHO cells and other cell cultures. The cells are cultured in this way, and the cells are fastened after inoculation.

3. Suspension culture: refers to the process in which cells are freely suspended in a reactor. It is mainly used for non-adherent-dependent cell culture, such as hybridoma cells; it is developed on the basis of microbial fermentation.

Serum-free suspension culture is a cell culture method that replaces animal serum with known human or animal-derived proteins or hormones. It can reduce post-purification work and improve product quality, and is gradually becoming a new research direction for large-scale culture of animal cells.

4. Immobilization culture: It combines animal cells with water-insoluble carriers and then cultures them. The above two types of cells are suitable for use, and have the advantages of high cell growth density, strong shear resistance and strong anti-pollution ability, and the cells are easy to separate products, which is beneficial to product separation and purification. There are many preparation methods, including adsorption method, covalent attachment method, ion/covalent crosslinking method, embedding method, microcapsule method and the like.

1. Adsorption method: A method in which a cell is adsorbed on a surface thereof by a solid adsorbent to immobilize a cell is called an adsorption method. The operation is simple, the condition is mild, and it is the earliest research method used in animal cell immobilization. The disadvantage is that the carrier has low load capacity and the cells are easy to fall off. Microcarrier culture and hollow fiber culture are representative of this method and will be specifically described later.

2. Covalent attachment method: An immobilization method in which an animal cell is combined with a solid phase carrier by a covalent bond is called attachment by covalent bonding. This method can reduce cell leakage, but chemical agents must be introduced, which have an effect on cell activity, and the diffusion limit is small due to attachment, and the cells are not protected.

3. Ion/covalent cross-linking method: The bifunctional reagent treats the cell suspension, which forms a bridge between cells and flocculates to produce cross-linking. This method of immobilized cells is called cross-linking. By covalent bonding). Cross-linking reagents cause some cells to die and also have diffusion limitations.

4. Embedding method: A method of embedding a cell inside a porous carrier to form an immobilized cell is called an entrapment method. The advantages are: simple steps, mild conditions, large load, less cell leakage, and resistance to mechanical shearing. The disadvantage is that diffusion limits, not all cells are at the optimal substrate concentration, and the macromolecular matrix cannot penetrate into the polymer network. It is generally suitable for the immobilization of non-adherent-dependent cells. The commonly used carriers are porous gels such as agarose gel, calcium alginate gel and fibrin.

5. Microencapsulation: a layer of hydrophilic semi-permeable membrane is used to surround cells in bead-like microcapsules, cells can not escape, but small molecules and nutrients can freely enter and exit the semi-permeable membrane; The inside is a kind of micro culture environment, which is similar to liquid culture and can protect cells from damage, so the cells grow well and have high density. The diameter of the microcapsules is preferably controlled at 200-400 μm. Should pay attention to the preparation:

● Mild, fast, no damage to cells, try to operate under liquid and physiological conditions;

● The reagents and membrane materials used are not toxic to cells;

● The pore size of the membrane can be controlled, and nutrients and metabolites must pass freely;

● The membrane should have sufficient mechanical strength to resist agitation during culture.

Fifth, the application of anti-apoptosis strategy in large-scale cell culture

In the large-scale production of bioreactor animal cells, apoptosis is a major part of cell death. Recent studies have shown that 80% of cell deaths in large-scale culture bioreactors are caused by apoptosis, rather than previously thought to be necrotic. In large-scale cell culture, cell death is the biggest obstacle to maintaining high activity and high density of cells. In theory, preventing or prolonging cell death can greatly increase the production of recombinant proteins produced by bioreactors.

Apoptosis is precisely regulated by a series of genes and is a physiological phenomenon necessary for the development of multicellular organisms and the maintenance of homeostasis. The ultimate performers of apoptosis are known to be the Caspase family, which are all cysteine ​​proteases, each recognizing a 4 amino acid sequence and cleaving the substrate at the C-terminal aspartate residue of the recognition sequence. Caspase contains a sequence that can be recognized by itself, which can cleave and activate itself to cause signal amplification and act on downstream Caspase members, thereby forming a cascade of Caspase family, which ultimately acts on effector proteins and causes apoptosis.

Therefore, in large-scale culture, it interferes with the occurrence of apoptosis in culture, and enhances the ability of cells to specifically resist the pressure caused by apoptosis, which is beneficial to increase the cell culture density, prolong the cell culture cycle, and thereby improve the target product. The yield is 2-3 times.

1. Nutrients resist apoptosis

In conventional bioreactor configurations, nutrient depletion or lack of specific growth factors in the medium causes apoptosis, such as depletion of serum, sugar or specific amino acids. The addition of amino acids or other key nutrients to the culture medium can inhibit apoptosis and prolong the culture time to improve product production. Apoptosis in large-scale culture is mainly caused by depletion of nutrients or accumulation of metabolites. For example, glutamine depletion is the most common cause of apoptosis, and once apoptosis occurs, supplementation with glutamine can not reverse apoptosis. . In addition, when animal cells are cultured in serum-free, protein-free medium, the cells become more fragile and more prone to apoptosis.

2. Gene anti-apoptosis

A series of gene products related to apoptosis can be positively and negatively regulated, so the mechanism of apoptosis can be regulated by introducing the corresponding gene. The Bcl-2 gene is currently the most potent anti-apoptotic gene and exhibits strong anti-apoptotic activity in various cell lines.

3. Chemical methods for anti-apoptosis

Biochemical changes occur in many parts of the cell when apoptosis occurs. Some changes such as changes in cellular redox conditions produce reactive oxygen species in the apoptotic signal phase. Others such as disruption of mitochondrial membrane potential and activation of caspase occur during the apoptotic effect phase. It is the same in most cell deaths. Therefore, preventing these biochemical changes may prevent or at least delay the occurrence of apoptosis, and the use of chemicals to inhibit the occurrence of signal effects is considered to be one of the anti-apoptotic strategies.