Practical Mammal Cell Culture Manual (on)

Cell culture medium concept

Cell culture refers to a culture technique in which cells are taken out from tissues in vivo to mimic the in vivo environment, to grow and reproduce, and to maintain their structure and function. The culture of the cell culture may be a single cell or a cell population.

Cell culture purpose and use

1. Scientific research: drug research and development and basic research drug research and development

(1) Screening of new drugs: such as the study of the efficacy of chemical synthetic drugs, the screening and identification of active ingredients of traditional Chinese medicine.

(2) Vaccine research and development: research and development of viral vaccines (hepatitis virus vaccine, AIDS vaccine, etc.), tumor vaccine (polypeptide vaccine), etc.

(3) Research and development of genetic engineering drugs: such as interferon research and development, cell growth factor research and development.

(4) Research and development of cell engineering drugs: research and development of biologically active peptides, research and development of bioactive components such as ginsenosides and paclitaxel.

(5) Preparation of monoclonal antibodies: including monoclonal antibodies for diagnosis and monoclonal antibodies for treatment.

basic research

(1) Mechanism of drug action

(2) Gene function

(3) Mechanism of disease

2. Biopharmaceutical

(1) Vaccine production: such as viral vaccine (hepatitis virus vaccine, AIDS vaccine, etc.), tumor vaccine (polypeptide vaccine)

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(2) Gene engineering drug production: Some cell growth factors such as interferon, granulocyte growth factor, thymosin, etc., which have therapeutic value in clinical medicine.

(3) Production of diagnostic and pharmaceutical monoclonal antibodies

(4) Cell engineering drug production: some biologically active peptides, biologically active substances, etc. in biological cells

Cell culture medium

1. Suitable cell culture medium

A suitable cell culture medium is one of the most important conditions for cell growth and proliferation in vitro. The medium not only provides cell nutrition and a basic substance for promoting cell growth and proliferation, but also provides a living environment for culturing cell growth and reproduction.

2, high quality serum

Currently, most synthetic media require the addition of serum. Serum is one of the most important components in cell culture fluids, and contains various growth factors and other nutrients required for cell growth.

3. Sterile non-toxic cell culture environment

Sterile and non-toxic operating environment and culture environment are the primary conditions for ensuring the success of cells in vitro. Cells cultured in vitro, due to lack of defense against microorganisms and toxic substances, may be caused by cell poisoning death once they are contaminated by microorganisms or toxic substances, or their own metabolic substances accumulate. Therefore, when cells are cultured in vitro, the cell survival environment must be kept sterile and non-toxic, and the cell metabolites should be removed in time.

4, constant cell growth temperature

To maintain the vigorous growth of cultured cells, there must be a constant and appropriate temperature.

5. Suitable gas environment

Gas is one of the necessary conditions for mammalian cell culture to survive. The required gases are mainly oxygen and carbon dioxide.

Cell culture medium and basic components

There are many kinds of cell culture media, and they are divided into a synthetic medium and a natural medium according to their sources (mostly the medium used in the present invention is a synthetic medium), and are classified into a dry powder medium and a liquid medium according to their material states. The dry powder medium needs to be prepared and sterilized by the experimenter himself. The liquid medium is provided by a professional merchant, and the user can directly use it, which is very convenient. Each cell has its own suitable medium, as detailed in Schedule 1.

1. The main components of synthetic medium are: Amino Acids, carbohydrates, inorganic salts, Vitamins and other auxiliary substances.

Amino acid

Amino acids are the basic unit of protein. Different kinds of cells have different amino acid requirements, but several amino acid cells cannot be synthesized by themselves, and must be provided by a culture medium. These amino acids are called essential amino acids. Glutamine

Amides are essential amino acids for the synthesis of nucleic acids and proteins by cells. In the absence of glutamine, cells die poorly and die.

Therefore, a large amount of glutamine is present in various culture solutions. However, since glutamine is very unstable in solution, it should be stored in a refrigerator at -20 ° C and added to the culture solution before use. When the glutamine-containing culture solution is stored in a refrigerator at 4 ° C for more than 2 weeks, the original amount of glutamine should be re-added.

Carbohydrate

Carbohydrates are the main source of energy for cell growth, some of which are components of synthetic proteins and nucleic acids. There are mainly glucose, ribose, deoxyribose, sodium pyruvate and acetic acid.

Inorganic salt

The main function of the inorganic salts in the culture fluid is to help the cells maintain the osmotic pressure balance. In addition, cells help regulate cell membrane function by providing sodium, potassium and calcium ions. The osmotic pressure of the culture medium is a very important factor, and the cells can usually tolerate 260mOsm/kg −320 mOsm/kg. The osmotic pressure of the standard culture solution fluctuates within this range. Special Note: Adding other substances to the culture solution may significantly change the osmotic pressure of the culture solution, especially those dissolved in strong acids or bases. When adding HEPES to the culture solution, the sodium ion concentration should be adjusted as follows.

Buffer system

Most cells require a pH of 7.2 - 7.4. However, the optimum pH for cell culture varies with the type of cell cultured. Fibroblasts prefer a higher pH (7.4 - 7.7), whereas passaged transformed cell lines require a partial acid pH (7.0 - 7.4). Since most of the culture solution is buffered by sodium bicarbonate (NaHCO3) and CO2 system, the concentration of CO2 in the gas phase should be balanced with the concentration of sodium bicarbonate in the culture solution. If the CO2 concentration in the gas or incubator air is set at 5%, the amount of NaHCO3 added to the culture solution is 1.97 g/L; if the CO2 concentration is maintained at 10%, the amount of NaHCO3 added to the culture solution is 3.95 g/L. Cell culture caps should not be tightened too tightly to ensure gas exchange.

HEPES is a non-ionic buffer with good buffering capacity in the pH range of 7.2 - 7.4, but it is very expensive and may be toxic to some cells at high concentrations. HEPES buffer can be used with low levels of sodium carbonate (0.34 g/L) to counteract the increase in osmotic pressure caused by the additional addition of HEPES. Under such culture conditions, the lid of the cell culture flask should be tightened to prevent the small amount of carbonate required in the culture solution from being dissipated into the air. Most of the culture medium contains phenol red as a pH indicator, the acidic medium is orange-yellow, and the alkaline medium is dark red.

Vitamin

In cell culture, although serum is an important source of vitamins, various media have been added with various vitamins to accommodate more cell line growth.

Other ingredients

Some other ingredients are also included in some of the more complex cultures. For example, DMEM culture medium commonly used in hybridoma technology requires the addition of sodium pyruvate and 2-mercaptoethanol (2-Me) when used. 2-Me plays an important role in cell growth. Some people think that it is equivalent to fetal bovine serum, which directly stimulates cell proliferation. The active part of 2-Me is a sulfhydryl group. One of the important functions is to reduce the sulfur-containing compound in serum to glutathione, which can induce cell proliferation and is a non-specific activation. At the same time, the damage of the cultured cells by the peroxide is avoided. Another important role is to promote mitogen reaction and DNA synthesis, increase the transformation of plant lectin (PHA) on lymphocytes, has been widely used in hybridoma technology, and has also begun to be used in some cells that are difficult to culture. 2-Me is a small molecule reducing agent that is highly oxidizable. The molecular weight is 78.13. Pure 2-Me is a colorless and irritating liquid with a specific gravity of 1.110-1.120 (Do20). The final concentration is usually 5×10-5M. It is usually formulated into a 0.1 M stock solution, and 0.5 ml per liter of the culture solution is used.

Liquid medium storage:

The liquid medium should be stored in a refrigerator at 4 ° C in the dark, and preheated at 37 ° C before the experiment. The serum-free medium was valid for 12 months. L-glutamine in the liquid medium will slowly decompose as the storage time increases. If the cells are poorly grown, an appropriate amount of L-glutamine can be added.

Dry powder culture: Store in a refrigerator at 4 °C in the dark, valid for 36 months.

serum

The serum added to the cell culture solution includes bovine serum, horse serum, human serum, and the like, and bovine serum is the most commonly used serum, and is classified into fetal bovine serum and newborn calf serum. Fetal bovine serum is a serum isolated from fetal calves taken from a cow's abdomen and is expensive. Fresh calf serum is a serum that has been isolated from newly born, unfed calves. If the manufacturer can do this, the quality of the newborn calf serum is not much different from that of fetal calf serum. If the calf is breast-fed after birth, the serum taken from the calf may contain more bioactive substances, and its quality is obviously not as good as the first two.

The quality, type and concentration of serum may affect the growth of cells, and the ability of different batches of serum to support cell growth is different, especially for the growth of clonal cells, some batches of serum may contain toxic or inhibiting cells. The substance that grows. Therefore, before purchasing a large amount of serum, the serum support cell growth ability must be tested, and then, in large quantities, the same batch of serum of good quality must be purchased, and the following points should be noted:

(1) Serum that requires long-term storage must be stored in a -20 ° C – 70 ° C low temperature freezer. Do not store in a refrigerator at 4 °C for more than 1 month. Since the volume of the serum will increase by about 10% when frozen, the serum must be reserved for a certain volume before it is frozen into the low-temperature refrigerator, otherwise it is prone to contamination or glass bottle cracking.

(2) The serum supplied by the general manufacturer is sterile and does not need to be filtered and sterilized. If serum is found to be suspended, the serum can be added to the culture medium for filtration. Do not directly filter the serum.

(3) The thawing of bottled serum requires a gradual thawing method: -20 ° C to -70 ° C The serum in the low temperature refrigerator is dissolved in a refrigerator at 4 ° C for 1 day. Then, it was transferred to room temperature, and then completely dissolved before being dispensed. During the dissolution process, it should be gently shaken evenly (be careful not to cause bubbles) to make the temperature and composition uniform, reducing the occurrence of precipitation. Do not directly thaw the serum from -20 ° C into 37 ° C, so that because the temperature changes too much, it is easy to cause protein aggregation and precipitation.

(4) Heat inactivation refers to the serum that has been completely thawed at 56 ° C for 30 minutes. Shake it regularly during heating. The purpose of this heat treatment is to inactivate the complement in the serum. This heat treatment is generally not recommended unless necessary, as heat treatment can cause a significant increase in serum deposits and can also affect serum quality. Complement participation reactions include: cytotoxicity, contraction of smooth muscle cells, histamine release from mast cells and platelets, enhanced phagocytosis, and chemical chemotaxis and activation of lymphocytes and macrophages.

(5) Do not leave the serum at 37 °C for too long, otherwise the serum will become cloudy, and the active ingredients in the serum will break and affect the serum quality.

(6) Precipitate floc in serum: mainly caused by denaturation of lipoprotein in serum and fibrin in serum after thawing, these flocs do not affect the quality of serum itself. It can be removed by centrifugation at 3000 rpm for 5 minutes or without treatment.

"Little black spots" under the microscope: After heat-treated serum, the formation of precipitates is significantly increased. Some precipitates are observed under a microscope like "small black spots" and are often mistaken for serum contamination. In general, this small black spot does not affect cell growth, but if serum quality is suspected, stop using it immediately and replace another batch of serum.