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Serum-free media (Serum-Free Media)

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In general, the growth of animal cells depends on the presence of serum. In normal media, most cells cannot proliferate without serum. Currently, the better serum that is usually added to the serum medium is bovine serum. Studies have found that the use of bovine serum in cell culture poses a risk of contaminating foreign viruses and pathogenic factors: due to inconsistencies in biological activity and factors between different batches of bovine serum, product and experimental results are poorly reproducible: residues in the product Bovine serum is susceptible to an allergic reaction to the serum by the vaccinator.

Serum-Free Media, usually expressed as SFM, as the name implies, does not require the addition of serum in cell culture, but in some applications it may be necessary to add growth factors or cytokines. The serum-free medium contains the main components of serum: adhesion factors, growth factors, essential nutrients and hormones, which can reduce the adverse factors caused by serum and make the conditions of cell culture more stable. After experiencing natural and synthetic media, serum-free media and serum-free culture have become a major trend in cell culture today. The use of serum-free culture simplifies the process of purifying and identifying various cell products to avoid the hazards of viral contamination.

1. Serum-free cell culture medium and its advantages and disadvantages

(1) serum-free medium and classification

The serum-free medium is the third type of medium following the natural medium and the synthetic medium. Compared with traditional culture media, serum-free media is a medium that does not contain animal serum or other biological extracts, but can still maintain cells for growth and reproduction in vitro for a long time. The serum-free medium is widely used in modern biotechnology because of its relatively clear composition and simple preparation process. Serum-free culture techniques are also powerful tools for elucidating basic research issues in cell growth, proliferation, differentiation, and regulation of gene expression.

At present, the study of serum-free medium has two directions: one is that the medium does not contain any animal-derived additive components; the other is that the medium does not contain undefined additive components. According to this, the serum-free medium with more current applications can be classified into the following four types:

1. Serum-free medium in a general sense, using various biological materials that can replace serum function to prepare cell culture media, such as bovine serum albumin (BSA), transferrin, insulin and other biological macromolecular substances, and from serum. Extracted protein-bound mixed lipids, hydrolyzed proteins, and the like. It is characterized by a high protein content in the medium, the chemical composition of the added substances is not clear, and contains a large amount of animal-derived protein.

2. Animal-free medium: Many animal-derived media developed by commercial companies are based on the safety considerations of producing recombinant drugs. The added components in the medium are not of animal origin, and the required proteins are derived from recombinant proteins or protein hydrolysates. These components ensure the need for cell growth and proliferation.

3. Animal-free protein medium: The medium is completely free of animal-derived proteins, but some of the additives are derived from hydrolyzed fragments of plant proteins or other derivatives such as synthetic polypeptide fragments. Such media components are relatively stable, but steroid hormones and lipid precursors must be added and are highly specific to the cultured cells.

4. Chemical composition defined medium, which is the safest and most ideal medium at present, firstly ensuring the consistency between the batches of the medium, and adding a small amount of animal-derived protein hydrolysate and protein. They are all well-defined components. It is characterized by the nature of the medium, and it is also convenient to mix the medium.

(B) the advantages and disadvantages of serum-free medium

Advantages of serum-free medium:

1. It can avoid the quality change between serum batches and improve the cell culture and the repeatability of experimental results.

2. Avoid serum cytotoxicity and serum-derived contamination.

3. Avoid the effects of serum components on experimental studies.

4. Conducive to the differentiation of cultured cells in vitro.

5. Improve product expression levels and make cell products easy to purify.

6. The components are stable and can be mass produced.

7. Does not contain mitogen inhibitors, can promote cell proliferation.

Disadvantages of serum-free medium:

1. Cells in serum-free medium are susceptible to certain mechanical and chemical factors, and the preservation and application of the medium is not as convenient as traditional synthetic media.

2. The cost is higher.

3. Targeted, a serum-free medium is only suitable for the culture of a certain type of cells.

4. Cells at different stages of development (eg, stem cells compared to oriented precursor cells) require different formulations, and selection of growth factors and cytokines is particularly important. Moreover, while removing serum, some serum proteins are also protected and detoxified, so the requirements for reagents, water purity and instrument cleanliness are higher.

Second, serum-free medium composition and main addition factors

The serum-free medium consists of a nutrient-complete basal medium supplemented with hormones, growth factors, adherent factors and binding proteins.

(1) Basic medium

Early basal media for cell culture were natural media such as plasma clots, lymph, soy peptone and embryo extracts. In 1950, Morgan et al. studied 199 medium based on previous studies, which was the development of animal cell culture medium to a new stage and synthetic medium stage. The synthetic medium is a basic medium in which a certain proportion of amino acids, vitamins, inorganic salts, glucose, and the like are combined in order to grow cells. There are currently hundreds of synthetic media products on the market. Among the numerous synthetic media, MEM, DMEM, RPMI 1640, F12, and TC100 are the most widely used.

The composition of the basal medium is completely known, so that certain components of the basal medium can be adjusted correspondingly when culturing different cell lines to better meet the nutritional requirements of the cell strain or to enhance the protein of interest. The amount of expression.

(2) Main addition factors

Also known as supplemental factor, it is a general term for various factors that replace serum. Most serum-free cultures must be supplemented with 3-8 factors, and no single factor can replace serum. More than 100 such factors are known, some of which are essential supplements, such as insulin, sodium selenite, and transferrin, most of which are auxiliary factors.

According to their functions, we can divide the supplementary factors into four categories:

Hormone and growth factor

Many cells need to be supplemented with hormones such as insulin, growth hormone, glucagon, etc. when cultured in serum-free. Almost all cell lines require insulin, a polypeptide that binds to insulin receptors on cells to form complexes that promote the synthesis of RNA, proteins and fatty acids, and consistent apoptosis, an important cell survival factor. In serum-free culture, insulin is used at a concentration of 0.1-10 μg/ml. Jan et al believe that rapid depletion of insulin in batch culture is the main reason for the decrease in cell growth rate. In addition, progesterone, hydrocortisone, estradiol, etc. in thyroxine and steroid hormones are also commonly used supplement factors for serum-free cell culture. Different cell lines have different requirements for the type and quantity of hormones.

Growth factors are complementary factors necessary to maintain cell survival, proliferation and differentiation in vitro. According to chemical properties, it can be divided into polypeptide growth factors and steroid growth factors. The growth factors added in serum-free medium are mainly polypeptide growth factors. In recent years, there have been 20-30 kinds of polypeptide growth factors identified, and more than half of them can obtain corresponding recombinant growth factors by means of genetic recombination. The most common growth factors in serum-free medium are epidermal growth factor (EGF), fibroblast growth factor (FGF) and nerve growth factor (NGF). Growth factors are potent mitogens that shorten the doubling time of the cell population.

Binding protein

There are two types of binding proteins, one is transferrin and the other is albumin.

The specific transferrin receptor is present on most mammalian cells. The binding of the receptor to the complex of transferrin and iron ions is the main source of essential trace iron for cells. In addition, transferrin also has the properties of growth factors. And can be combined with other trace elements such as vanadium. The amount of transferrin required by different cells is also different.

Albumin is also a commonly added additive in serum-free media. It stabilizes and regulates the activity of the above substances in serum-free medium by combining with vitamins, lipids, hormones, metal ions and growth factors, in addition to binding toxins and alleviating the effects of proteases on cells.

3. Adherence factor

The vast majority of eukaryotic cells need to be affixed to a suitable substrate when grown in vitro. Cell fixation is a complex process involving the attachment of adherent factors to the surface of a vessel or carrier. The combination of cells and adherent factors. The adherent factors commonly used in serum-free culture include interstitial and serum components such as fibronectin, collagen, laminin and polylysine.

4. Other addition factors

The serum-free culture of some cell lines also needs to supplement certain low molecular weight chemicals, such as trace elements, vitamins, lipids, etc. Vitamin B is mainly involved in cell metabolism in the form of coenzyme, and vitamin C and vitamin E have antioxidant effects. Butane diamine and linoleic acid provide lipids required for cell membrane synthesis and water-soluble lipids required for cell growth.

Third, the application of serum-free medium

It has been generally applied to serum-free medium in the cultivation of large-scale animal cells. In the field of application of biological products such as vaccine growth, monoclonal antibodies and various biologically active proteins, optimizing the components of the serum-free medium allows different cells to maintain high-density culture in an environment that is most conducive to cell growth and expression of the desired product. reduce manufacturing cost. In human cell culture, the use of serum-free medium can also selectively control and avoid excessive growth of fibroblasts. Under serum-free culture conditions, the growth of certain cells and the production of antibodies are even several times higher than when serum is present.

In addition to the production of biological products, serum-free media is also widely used in the field of cell biology, pharmacology, oncology:

(1) Studying the differentiation conditions of cells: The composition of the serum-free medium can be a completely known chemical substance, and thus the type and amount of important biologically active substances can be increased or decreased according to the needs of the research. This provides an effective means for studying the conditions of cell differentiation.

(2) Research on interactions with cells such as hormones, growth factors and drugs.

(3) For selecting cells of interest from a variety of cell-mixed cultures: by exchanging certain components in serum-free culture, it is possible to inhibit excessive growth of non-target cells in primary tissue culture to achieve selection of cells of interest. purpose.

(4) Tumor pathology and etiology studies: For studying the effects of oncogenic factors on cells, studying the ability of tumor cells to respond to peripheral signals that may trigger normal cell terminal differentiation, or for studying normal cells and tumor cells. The relationship between growth and migration and the basement membrane signal.

(5) Serum-free medium has been widely used in the production of biological products. Many production units at home and abroad are working on how to combine serum-free medium with fermenter culture technology.

In addition, serum-free media is also widely used in the research and clinical treatment of stem cells and immune cells. And the demand for serum-free medium is the largest and most urgent in this field.