The difference between microbial cells, plant cells and animal cells in culture

Microbial Cell Culture, referred to as microbial culture, includes prokaryotic cell culture (bacteria culture) and eukaryotic cell culture (mold culture and yeast culture). These cells are small, have cell walls, and have a very short cell cycle, such as bacteria multiplying once every 20-30 minutes.

Plant cell culture refers to protoplast culture. Plant cell cultures that do not take into account the isolation of protoplasts, whether isolated single cells or tissue pieces, are called plant tissue cultures.

Animal tissue cell culture includes culture by inoculation of single cell suspension and culture by inoculation of tissue blocks. Passage cells, such as cell lines and cell lines, are usually cultured by inoculating single-cell suspensions, which is a common way for animal cell culture. Primary cells refer to cell aggregates derived from animal tissues, often containing a variety of cell types, and the plasma membranes between cells are tightly connected. If the cells in the tissue do not need to be separated during the culture, the culture directly using the tissue block is tissue culture. If it needs to be prepared into a single cell suspension and then inoculated and cultured, it belongs to a primary cell culture.

1. Cellular microorganism culture

Cellular microorganisms include prokaryotic microorganisms and eukaryotic microorganisms. Bacteria, actinomycetes, spirochetes, mycoplasma, chlamydia, and rickettsia generally belong to single-celled prokaryotic organisms, and actinomycetes grow in hyphae, which are composed of multiple cells, but each cell often contains multiple sets of nuclei Substance, belonging to non-septal hyphae. Yeasts are generally single-celled eukaryotes, and molds are often multicellular eukaryotes. Most of the fungal hyphae are septated hyphae, and each cell generally contains 1 nucleus or more than 2 nuclei.

(1) Training purpose

According to the difference in the physical properties of the culture medium, it can be divided into three types: ① liquid culture, which is used for the purpose of enrichment culture and identification culture; Semi-solid culture, generally used for identification and enrichment purposes.

(2) Training form

Among the liquid culture types, fermentation tube culture, fermenter tank culture, shake flask culture, multi-well plate culture and other forms are common. In the solid culture type, Petri Dish plates, Kjeldahl (flat bottle) plates, and test tube slopes are common forms.

(3) Cultivation instruments

The incubator used for prokaryotic cell culture is a biochemical incubator or anaerobic incubator. The former is used for aerobic bacteria culture, and the latter is used for anaerobic bacteria culture. Anaerobic culture requires food-grade nitrogen provided by steel cylinders. The mold incubator is used for mold cultivation, which can provide high relative humidity and high air tightness.

2. Plant tissue cell culture

Plant tissue culture refers to the process of isolating a part of a plant, that is, an explant, by aseptic operation, inoculating it on a medium, and cultivating it under artificially controlled conditions to produce a complete plant. Plant tissue culture mainly includes protoplast culture, suspension cell culture, anther culture, tissue culture, organ culture and other forms, the most common of which is callus culture.

(1) Training purpose

Due to the totipotency of plant cells, various types of plant tissue cell cultures can be induced to differentiate to form callus and develop into new plants. Plant tissue culture technology is not only used for rapid propagation, but also has a wide range of uses. These applications include haploid breeding, germplasm conservation, physiological studies, and gene transformation, among others.

(2) Types of training

1. Plant protoplast culture

Plant cell culture refers only to plant protoplast culture in a narrow sense, and includes plant protoplast culture and plant tissue culture in a broad sense.

A plant cell from which the cell wall has been removed is called a protoplast. Plant protoplast culture is to use cellulase or pectinase to degrade the cell wall of cells in a tissue block derived from a certain plant organ, centrifuge to collect a single cell suspension, and then inoculate and culture. Its characteristics are: ① It is relatively easy to absorb foreign genetic material, such as DNA; ② It is convenient for cell fusion to form hybrid cells; ③ It is as totipotent as a complete cell, and can still produce a cell wall, which can be induced to differentiate into a complete plant.

2. Plant tissue culture

Including shoot apical meristem or callus culture. Among them, the induced callus can produce regenerated plants under suitable conditions. It is used for the following purposes: ① to study the growth and development, differentiation and genetic variation of plants; ② to clone specific varieties, that is, asexual reproduction; ③ to obtain metabolites of specific plant tissue cells.

3. Suspension plant cell culture

A culture technique developed on the basis of callus culture techniques. It is suitable for industrialized large-scale cell culture and the preparation of plant metabolites.

4. Anther culture

Also called haploid culture. Haploid plants can be obtained through anther or pollen culture, and the most homozygous plants or varieties can be obtained after artificial doubling.

5. Plant organ culture

Plant organ culture refers to the culture of embryos, anthers, ovaries, roots or stems of plants in vitro.

(3) Culture conditions

Artificial culture conditions, that is, artificial environment, include: ① light conditions that provide energy sources; ② temperature conditions that ensure metabolism; ③ humidity conditions that provide air-water balance; ④ culture media that provide sources of biological raw materials; sterile environment. The artificial environment mimics the plant's natural environment, i.e. light, temperature, humidity, soil and the sterile environment inside the intact plant.

The selection of light and temperature is usually based on the ecological habits of the plants to be cultivated. It is customary to use a combination of dark culture and light culture (8h/d, 30001x) to alternately carry out the whole process of cultivation, and the temperature is controlled within the range of 25-28°C. Humidity does not need to be specially adjusted in tissue culture, because the relative humidity in the culture container is almost 100%. Therefore, the key and difficulty of how to control the artificial culture environment lies in the selection and preparation of the medium.

1. Medium

The plant medium is developed from the nutrient solution of soilless cultivation, which can be understood as simulated soil in a sense. The initial medium is potato extract. With the deepening of plant physiology and biochemical research, the composition of the medium becomes more and more complex, with more and more components.

When agar and gelatin supports were discovered and applied to culture media, solid media were born. Solid media is a type of media commonly used in tissue culture media. Different plants have different requirements for the optimum pH value of the medium, most of which are in the range of 5-6.5. Generally, the pH of the medium is controlled at 5.8, which can basically meet the needs of most plant cultures. The osmotic pressure can be adjusted by adding substances such as salt, sucrose, mannitol and ethylene glycol to the medium. Usually 1-2 atmospheric pressure can promote the growth of plant tissue, when the pressure is above 2 atmospheric pressure, growth disorder occurs, and when the pressure is 6 atmospheric pressure, the plant tissue cannot survive. Regardless of whether the culture target is designed for cell growth or for the accumulation of metabolites, the culture medium is mainly composed of carbon sources, organic nitrogen sources, inorganic salts, plant growth hormones, organic acids and some compound substances.

(1) Carbon source Sucrose or glucose is a commonly used carbon source, and fructose is worse than the former two. Other carbohydrates are not suitable as a single carbon source. Generally, increasing the content of sucrose in the medium can increase the amount of secondary metabolites of cultured cells.

(2) Organic nitrogen sources The organic nitrogen sources usually used include protein hydrolyzate (including casein hydrolyzate), glutamine or amino acid mixture. Organic nitrogen sources are beneficial in the early growth stages of primary cultures of cells. L-Glutamine can replace or supplement certain protein hydrolysates.

(3) Inorganic salt macroelements and trace elements are usually replaced by inorganic salts, and iron salts usually appear in the form of chelated iron. For different culture forms, the optimal concentration of inorganic salts is not the same. Usually, the concentration of inorganic salts containing a large number of elements in the culture medium is controlled at about 25mmol/L. The concentration of nitrate is generally 25-40mmol/L. Although nitrate can be used as an inorganic nitrogen source alone, adding ammonium salt is beneficial to cell growth. Ammonium salts can also act as nitrogen sources alone if some succinic acid or other organic acids are added. Potassium must be added to the culture medium at a concentration of 20mmol/L. The concentration of trace elements such as phosphorus, magnesium, calcium and sulfur is between 1-3mmol/L.

(4) Plant growth hormone Most plant cell culture medium contains natural and synthetic plant growth hormone. Plant hormones have an unshakable core position in tissue culture, including five categories, namely, auxins, cytokinins, gibberellins, ethylenes and somatostatins. Commonly used in plant tissue culture are auxins and cytokinins. Mitogens and auxins are usually used together to promote cell division and growth. Auxin can promote the formation of roots in plant cells and tissue culture. Effective and commonly used are indole butyric acid, indole acetic acid and naphthalene acetic acid, and mitogens are usually adenine derivatives.

(5) The addition of organic acids to pyruvate or tricarboxylic acid cycle intermediates such as citric acid, succinic acid, and malic acid can ensure the growth of plant cells on the medium with ammonium salt as a single nitrogen source and the ability to tolerate potassium salt At least increase to 10mmol/L. Krebs cycle intermediates also enhance the growth of cells and protoplasts at low inoculum sizes.

(6) Compound substances are usually used as cell growth regulators, such as yeast extract, malt extract, coconut water and fruit juice. Currently these substances have been replaced by nutrients of known composition. It was also found in many instances that some extracts were toxic to cells. Coconut juice is still widely used, and its concentration in the culture medium is 1-15mmol/L.

2. Culture vessel

Culture vessels emphasize the shape of plant tissue cell culture, including test tubes, Erlenmeyer Flasks, canning bottles, Petri Dishes, flat culture bottles, concave glass slides, L-shaped tubes and T-shaped tubes.

3. Cultivate the external environment

Plant tissue cell culture requires light, temperature, humidity and other conditions. The incubators that meet these conditions are light incubators, large artificial climate chambers, artificial climate chambers or plant bioreactors.

3. Animal cell culture

(1) Type of training

1. According to the animal classification of cultured cells

Divided into invertebrate cell culture and vertebrate cell culture.

(1) Invertebrate cell culture refers to cell culture derived from the phylum Porzoa, Coelenterate, Annelida, Arthropoda, Mollusca and Echinodermata.

The cell culture research of invertebrates is later than that of mammals. Due to the particularity of invertebrate cells in terms of morphology, structure, function and nutritional requirements, most of the cell cultures stay at the level of primary culture and limited cell lines.

The phylum Arthropoda can be divided into 3 subphylums and 7 classes. Among them, there are many types of Crustacea, Arachnida, Myriapoda, and Insecta. They are widely distributed. Some types are available for medicinal purposes, mainly represented by shrimps and crabs. ,insect. Since 1961, more than 800 insect cell lines have been established.

(2) Vertebrate cell culture These vertebrates from low to high include fish, amphibians, reptiles, birds and mammals.

Vertebrate cell culture was carried out earlier, and the establishment of cell lines was relatively complete. Mammalian cell culture research started early, and there are many studies. Its biological characteristics are closer to human natural products, and it is the focus of research and development of biotechnology products.

In the 1980s, after WHO clarified that mammalian passage cells could be used for the production of biological products, cell culture in this field has achieved considerable development in the research and development of biotech drugs. According to literature reports, 70% of approved biotechnology drugs are produced by mammalian cells.

2. Classification according to cell growth characteristics

It is generally divided into adherent culture and suspension culture. Please refer to Task 31.

3. Classification according to training purpose

Primary culture, subculture, clone culture, expansion culture, etc. For details and operation methods, refer to task 33.

(2) Culture conditions

1. Medium

Commonly used are serum-containing medium and serum-free medium, which are generally synthetic medium, and various types of commercial medium are available, such as dry powder medium and liquid medium. Serum-containing medium usually uses fetal calf serum, newborn calf serum or calf serum, and the general addition amount is 5%-20%.

2. Culture vessel

Commonly used culture vessels include culture dishes, square culture flasks, porous cell Culture Plates, etc. For details, please read Task 19 and Task 20.

3. Culture equipment

The open culture uses a professional CO2 incubator to provide temperature, humidity and pH guarantees for the growth of animal cells. Among them, the constant concentration of CO2 is crucial to stabilizing the pH of the culture solution.

In addition to the commonly used CO2 incubator, animal cell bioreactors, cell factories or roller bottle machines and other instruments or large-scale equipment are also required in production.

4. Microscopic examination equipment

The inverted biological microscope is a professional microscopic inspection instrument, which is used to observe the growth status of the culture, check whether there is microbial contamination, and preliminarily determine what kind of cell type microbial contamination is.

4. The main differences in the cultivation of microbial cells, plant cells and animal cells

In biotechnology, people have used the mass culture of bacteria and filamentous fungi to produce various enzymes, antibiotics, proteins, amino acids and other products, but many biological substances with important value, such as toxins, vaccines, interferons, monoclonal antibodies, etc. , pigments, fragrance substances, etc. must be prepared by means of large-scale cultivation of animal cells and plant cells.

If you have a certain understanding of the similarities and differences in the cultivation of microbial cells, plant cells or animal cells, it will help learners to better grasp the basic conditions and requirements of animal cells, and establish a certain theoretical basis for mammalian cell culture.