According to the types of animal cells, monolayer culture, suspension culture and immobilized culture can be used for large-scale culture.

I. Growth characteristics and culture temperature of animal cells

1, cells grow slowly and are easy to be polluted, and antibiotics are needed for culture.

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

It needs less oxygen and can't stand strong ventilation and stirring.

4. Population growth effect, attached growth (anchoring dependence)

5. The products in the process of culture are distributed inside and outside the cell, and the cost is high.

6. Primary cultured cells usually degenerate and die after 50 generations.

Accord to different dependence on growth substrates in vitro culture, animal cell can be divided into two types:

Adhesion-dependent cells: They need to be attached to a solid or semi-solid surface with an appropriate charge to grow. Most animal cells, including non-lymphoid tissue cells and many aneuploid cells, belong to this category.

Adhesion-independent cells: They can grow without adhering to solid surfaces, including blood, lymphoid tissue cells, many tumor cells and some transformed cells.

The optimum temperature of cultured cells is equivalent to the normal temperature of various cells or tissues. The optimum temperature for human and mammalian cell culture is 35~37℃. Deviating from this temperature, the normal metabolism and growth of cells will be affected and even die. Generally speaking, the tolerance of cultured cells to low temperature is higher than that to high temperature. When the temperature does not exceed 39℃, the cell metabolic intensity is directly proportional to the temperature; The cells were cultured at 39~40℃ for 65438 0 h, which was damaged to some extent, but still recovered. When the temperature reaches above 43℃, many cells will die. When the temperature drops to 30~20℃, the cell metabolism decreases, so the material exchange with the culture medium decreases. The first thing to see is the morphological changes of cells and the shedding of cells from the matrix. When the cultures return to the initial culture temperature, their original morphology and metabolism also return to the original level.

Second, monolayer culture (attachment culture)

It refers to the culture of cells attached to a solid surface.

1. growth characteristics: adherent cells should be attached to the wall of the culture container (bottle) during culture. Once the cells adhere to the wall, they will spread rapidly, then start mitosis and quickly enter the logarithmic growth period. After a few days, the culture surface is covered, forming a dense cell monolayer.

2. Advantages of monolayer culture:

Easy replacement of culture solution; Cells are closely attached to the surface of solid phase, and the old culture solution can be directly dumped, and the new culture solution can be directly added after cleaning.

Perfusion culture is easy to use, thus achieving the purpose of improving cell density; Because the cells fix the surface, there is no need for a filtration system.

When cells adhere to the growth substrate, many cells will express a product more effectively.

The same equipment can use different medium/cell ratios.

Suitable for all types of cells.

3. Disadvantages of monolayer culture: Compared with suspension culture,

Outward bound training is difficult and costly;

Covers a large area;

Can not effectively monitor the growth of cells;

4. Cell adhesion surface: net positive charge and high surface activity are required. For microcarriers, a certain charge density is also needed; If it is an organic surface, it must be hydrophilic and positively charged.

5. Single-layer culture system: it mainly includes rotating bottle, hollow fiber (introduced later), glass beads and microcarrier system (introduced later).

Shake flask culture system: firstly, the adherent cells were cultured by shake flask system. Rotating flask culture is generally used in the transition stage from small-scale culture to large-scale culture, or as a way to prepare cells for bioreactor inoculation. Cells are inoculated in a rotating cylindrical incubator-rotating flask, and the rotating flask rotates continuously during the culture process, so that cells contact with culture solution and air alternately, thus providing better mass and heat transfer conditions.

Rotating bottle culture has the advantages of simple structure, low investment, mature technology, good repeatability, and simply increasing the number of rotating bottles for amplification. However, it also has its disadvantages: high labor intensity, large space occupation, small surface area per unit volume for cell growth, low cell growth density, and limited monitoring and control of environmental conditions during culture. At present, the rotary bottle culture system includes two types: carbon dioxide incubator and rotary bottle machine.

Single-layer culture in reactor In this culture mode, cells grow on a fixed surface and will not flow with the culture solution because of stirring, so it is easier to replace the culture solution, and no special equipment is needed to separate cells from the culture solution, so perfusion culture can be used to obtain high cell density and products can be obtained effectively; However, it is difficult to expand the scale and can not directly monitor the growth of cells, so it is mostly used to prepare low-dose and high-value biopharmaceuticals.

CelliGen, CelliGen PlusTM and Bioflo3000 reactors are commonly used single-layer culture bioreactors, which can be used for cell single-layer culture, with basket stirring system and disc-shaped carrier. This carrier is a nonwoven polyester fiber disc with a diameter of 6 mm, and has a high surface area/volume ratio (1200cm2/g), which is beneficial to obtain high cell density. Basket stirring system and carrier culture are the most commonly used methods for adherent cell culture at present, which are used for hybridoma cells, Hela cells, 293 cells, CHO cells and other cells. Cells cultured in this way can adhere to the wall quickly after inoculation.

Three, suspension culture (suspension culture)

Refers to the process of free suspension growth of cells in a reactor. Mainly used for cell culture without attachment dependence, such as hybridoma cells; It is developed on the basis of microbial fermentation.

Serum-free suspension culture is a cell culture method in which protein or hormones from human or animals are used instead of animal serum. It can reduce the later purification work and improve the product quality, and is gradually becoming a new research direction of large-scale animal cell culture.

Four. Immobilized culture.

Animal cells are combined with water-insoluble carriers and then cultured. These two types of cells are suitable, and have the advantages of high cell growth density, strong shear resistance and strong pollution resistance, and cells are easy to separate products, which is beneficial to product separation and purification. There are many preparation methods, including adsorption method, valence attachment method, ion/valence crosslinking method, embedding method, microcapsule method and so on.

1. adsorption method: the method of fixing cells by adsorbing them on their surfaces with solid adsorbents is called adherent method. Simple operation and mild conditions are the earliest methods for animal cell immobilization. Disadvantages are: the carrier load is low and the battery is easy to fall off. Microcarrier culture and hollow fiber culture are representative of this method, which will be introduced later.

2.* * * valence attachment method: The immobilization method of animal cells combined with solid carriers through * * * valence bonds is called * * * valence covalent attachment method. This method can reduce the leakage of cells, but the introduction of chemical reagents affects the activity of cells, and the cells are not protected because of the small adhesion and diffusion restrictions.

3. Ion/* * * valence crosslinking method: When cell suspension is treated with bifunctional reagent, bridges will be formed between cells, and flocculation will produce crosslinking. This method of fixing cells is called ion/* * * valence crosslinking. Cross-linking agent will cause some cell death and diffusion restriction.

4. Embedding method: The method of embedding cells in porous carriers to make immobilized cells is called embedding method. Its advantages are simple steps, mild conditions, large load, less cell leakage and mechanical shear resistance. Disadvantages are: diffusion limitation, not all cells are in the optimal matrix concentration, and macromolecular matrix can not penetrate into the polymer network. Generally speaking, it is suitable for the immobilization of non-attachment dependent cells. Common carriers are porous gels, such as agarose gel, calcium alginate gel and fibrin.

5. Microencapsulation: The cells are wrapped in beads with hydrophilic semipermeable membrane, so that the cells cannot escape, but small molecules and nutrients can freely enter and exit the semipermeable membrane; Capsule is a tiny culture environment, similar to liquid culture, which can protect cells from damage, so cells grow well and have high density. The diameter of microcapsules should be controlled at 200-400 μ m, and attention should be paid to:

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

The used reagents and membrane materials are non-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 in that culture proces.