CHO cell serum-free culture entry technology manual
- Shenzhen hundred Enver biological
1, CHO cell background
      CHO cells are Chinese hamster ovary cells (Chinese Hamster Ovary, CHO), 1957 Nian Dr. Theodore T. Puck University of Colorado is obtained from a separate adult female hamster ovary, adherent to the epithelial-type cells, is widely used in biological engineering Cell line. CHO-K1 cells are widely used in industrial production. They are transformed cell lines with a cell chromosome distribution frequency of 2n = 22 , which is a subdiploid cell. The ATCC preserves the CHO-K1 cell line, numbered CCL-61 , and is widely used for expression of recombinant DNA proteins. Due to the presence of genetic defects the cells, no gene synthesis proline, glutamic acid can not be converted to glutamic acid - γ - semialdehyde, L- proline should be added to the process to grow in the culture medium. And since the cells have been adapted to cholera toxin, morphology has changed. The initial cells are adherent cells, which can also be suspended in growth after multiple passages.
2 , CHO cell expression system advantages and characteristics
     Due to the complexity of mammalian cell structure, function and gene expression regulation, the expression of foreign genes in mammalian cells is significantly different from that in prokaryotes, so that foreign genes need to be efficiently expressed in mammalian cells. The elements are also different from the elements required for expression in prokaryotic cells. The expression of foreign genes in mammalian cells includes transcription of genes, translation of mRNAs, and processing of post-translational proteins, such as glycosylation, phosphorylation, formation of oligomers, and intramolecular or intermolecular disulfide of proteins. The formation of a bond or the like is complicated, and expression of a biologically functional protein such as a membrane protein, an antibody, and an enzyme having a specific catalytic function needs to be carried out in mammalian cells. The basic principle of mammalian gene expression host cell selection is rich source, high transformation efficiency and good expression effect. CHO cells as an expression system have the following advantages in addition to the above characteristics:
1 ) After the foreign gene is integrated into the host chromosome, it can be stably maintained without selection pressure;
2 ) It is suitable for the secretory expression and intracellular expression of a variety of proteins, and rarely secretes its own endogenous protein, facilitating the separation and purification of downstream products;
3 ) The requirements for the medium are low and can be cultured in serum-free medium;
4 ) The cells can be cultured in adherence or suspension culture, and have high tolerance to shear and osmotic pressure;
5 ) It can be cultured in high density and can be expanded to more than 20,000L when it is produced on a large scale. It is one of the most widely used mammalian gene expression receptor cells.
    Other researchers have tried to transfer the insulin-like growth factor IGF gene and the transferrin gene into CHO cells to obtain the " super CHO" that can secrete essential proteins by itself . There is no need to transfer transferrin and insulin in the medium, and the cells can be in serum-free medium. Good growth in ( SFM ).
3 , CHO cell serum-free and animal-free component medium
     The serum-containing cell culture medium is inferior in controllability and reproducibility because of the uncertain composition of serum, which is not conducive to industrialization and scale. The composition of serum-free and animal-free medium is determined, and the reproducibility and controllability are relatively good. Therefore, whether it is a research client or a clinical treatment client, it is increasingly inclined to use a serum-free and animal-free medium to culture CHO cells. . At present , most CHO cell cultures can be carried out in serum- free medium . The use of serum-free culture instead of traditional serum culture has become an inevitable trend in the development of CHO cell culture . Serum-free culture has been widely used in cell biology, pharmacology, oncology , cell engineering and clinical treatment .
     Based on the research and development team with rich experience in cell culture medium research and large-scale cultivation of animal cells, Shenzhen Bainwei Bio has devoted research and development of a variety of personalized culture media. It has been gratifying through its application in production practice. The results, such as CHO cell serum-free animal component cell culture medium ( BW12009 ) is one of them.
4 , the characteristics of Baienwei CHO cell serum-free medium ( BW12009 )
     BW12009 is a new-generation CHO cell serum-free and animal-free cell culture medium developed by Shenzhen Bainwei Biotechnology . It can support the growth and maintenance of various CHO cells. It can be widely used in serum-free suspension culture of CHO cells and antibodies, recombinant proteins , The production process of the vaccine . The production and inspection of cell culture media products are strictly implemented in GMP management and ISO9001 management system, in line with the requirements of biopharmaceutical raw materials, and the quality is stable and reliable.
1 ) It is a serum-free and animal-free medium, and its chemical composition is clear, which ensures the safety of the use of raw materials in cell culture;
2 ) Support the growth of a variety of CHO cell lines , including adherent and suspension cell culture, with a wide range of applicability;
3 ) It can maintain high-density culture of CHO cells , and the cell proliferation rate is fast, which is suitable for experimental research and large-scale culture application;
4) Depending on the user, the media can be customized to meet the application requirements of various different systems and the like;
5 ) There are a variety of packaging specifications to choose from to meet the needs of different users.
5 , custom CHO cell serum-free medium
     The presence of multiple clonal cell line CHO cell, ATCC preserved CHO cells as many as 38 species plus one hundred cell research unit or similar companies Enver constructed, the number of cloned cells much more. For serum-free culture of CHO cells, due to the high performance of the different constructs CHO cell lines, clonal cell different nutritional requirements are also very different, often on the nutritional requirements of the individual. In addition, the different culture stages of the CHO cell culture process and the various stages of the recombinant CHO cell line require different nutrients or limiting factors for cell metabolism, that is, the same CHO cell line requires different culture media throughout the culture process, and requires A personalized medium corresponding to it . Bai Enwei has provided personalized CHO cell serum-free media to many world-renowned biopharmaceutical companies .
6 , CHO cells serum-free domestication
     CHO cells grown in serum-containing medium can undergo a certain cell acclimation process to adapt  Cell culture medium for serum-free culture of cells. Enver provide one hundred have been domesticated CHO cell lines. If necessary, the investigator can also perform serum-free domestication of CHO cells as follows .
     CHO cells that have been adapted to serum-free culture can be cultured in serum-free culture directly in BW12009 . It is recommended that the cell density of the first two generations be no less than 4 × 10 5 cells/ml .
The serum-free domestication process of cells is as follows:
1. Take adherent CHO cells in logarithmic growth phase , the viability is greater than 95% , and start serum-free domestication.
2. Cell domestication can be carried out in a T-flask , a shake-flask or a spinner-bottle .
3. Mix serum-free cell culture medium and serum-containing medium in a ratio of 1 : 1 ( V/V ) to inoculate cells with a density of 2 - 4 × 10 5 cells/ml at 37 ° C, 5 % CO 2 The incubator is cultured.
The cell growth and viability, the drop in each stage can be stably passaged in serum 1--3 generation inoculum density is maintained at 2- 4 × 10 5 cells / ml .
5. Gradually increase the ratio of serum-free cell culture medium in the mixture ( V/V ), that is, reduce the serum content in the mixture, and maintain the cell seeding density of 2 - 4 × 10 5 cells/ml during the passage . Until the serum concentration in the mixture is reduced to 0.1-0.2% , and the cell viability of each generation is greater than 90 %, the cells can be completely cultured in the CHO serum-free animal-free medium at this time.
6. Enlarged culture in CHO serum-free and animal-free medium to establish a CHO seed cell bank adapted to serum-free and animal-free culture .
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