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Serum-free medium and culture method suitable for adherent cho cells
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A technology of serum-free medium and culture method, applied in the field of serum-free medium and culture of adherent CHO cells
Active Publication Date: 2020-11-13
GUANGDONG SHUNDE IND DESIGN INST GUANGDONG SHUNDE INNOVATIVE DESIGN INST
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Problems solved by technology
At present, the development of serum-free medium for CHO cells is mainly focused on suspension culture, and there are few serum-free medium suitable for adherence
Method used
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Embodiment 1
[0048] Embodiment 1 is applicable to the preparation of the serum-free medium of adherent type CHO cell
[0049] The basal medium suitable for the adherent growth of CHO cells has changed the trace elements, vitamins and amino acids of the DMEM / F12 medium, and the rest of the components are basically the same as the DMEM / F12 medium. The prepared medium is called "basic medium" .
[0050] When preparing the basal medium, firstly, each trace element component is prepared into a 1000-fold concentrated solution, and the composition and content are as follows:
[0051]
[0052] Each vitamin component is prepared into a 1000-fold concentrated solution, in which inositol is added separately when used, and the concentration is 13.8mg / L. Other specific compositions and contents are as follows:
[0053]
[0054]
[0055] Divide each amino acid component into 100 times concentrated solution, the specific composition and content are as follows:
[0056]
[0057] The inorgani...
Embodiment 2
[0068] The basal medium suitable for the adherent growth of CHO cells, the "basic medium" is as described in Example 1.
[0070] The composition and content of hormone substances are as follows: 5 μg dexamethasone, 15 μg hydrocortisone, 3 mg ethanolamine, 0.5 mg putrescine, 10 mg insulin, 0.5 mg progesterone, and 1 mg thyroxine are added to each liter of basal medium.
[0071] The composition and content of the growth factors are as follows: 0.5 mg EGF, 1 mg / L FGF, and 1 mg / LCSF are added per liter of basal medium.
[0072] The composition and content of the ...
Embodiment 3
[0074] The basal medium suitable for the adherent growth of CHO cells, the preparation of the "basic medium" is as described in Example 1.
[0075] Lipid substances are prepared into a concentrated solution, 10mg stearic acid, 10mg myristic acid, 10mg palmitic acid, 10mg linolenic acid, 10mg linoleic acid, 10mg oleic acid, 10mg palmitic acid, 5mg arachidonic acid, after being completely dissolved Add to 1L 0.2% Tween solution, add 0.2% BSA, and heat in a water bath at 37°C for 30 minutes. Add 1mL of this concentrate per liter of basal medium when in use.
[0076] The composition and content of hormone substances are as follows: 20 μg of hydrocortisone, 6 mg of ethanolamine, 1.5 mg of putrescine, 10 mg of insulin, 1 mg of progesterone, 0.2 mg of thyroxine, and 0.2 mg of epinephrine are added to each liter of basal medium.
[0077] The composition and content of growth factors are specifically 1 mg EGF, 1 mg IGF, 0.2 mg / LFGF, 0.2 mg / L CSF per liter of basal medium.
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Abstract
The invention discloses a serum-free culture medium and a culture method applicable to anchorage-dependent CHO (Chinese hamsterovary) cells. The serum-free culture medium comprises basic culture media, lipid substances, hormone substances, growth factors and anchoring factors. The lipid substances, the hormone substances, the growth factors and the anchoring factors are added into the basic culture media. The serum-free culture medium and the culture method applicable to the anchorage-dependent CHO cells have the advantages that the lipid substances, the hormone substances, the growth factors and the anchoring factors which are in specific concentration ranges are added into the basic culture media, accordingly, the serum-free culture medium can be directly used for anchoring growth of the CHO cells instead of the traditional serum-containing culture media, continuous passage can be carried out, and the CHO cells are in stable states when cultured; the contents of various components in the basic culture media are further adjusted, certain trace element components and the like are added into the basic culture media, accordingly, the serum-free culture medium is applicable to anchoring growth of the CHO cells, and the anchoring growth stability and the growth rate of the CHO cells further can be guaranteed.
Description
technical field [0001] The invention relates to the field of cellengineering, in particular to a serum-free medium and a culture method suitable for adherent CHO cells. Background technique [0002] Cell culture medium is not only the basic material for supplying cellnutrition and promoting cellreproduction and proliferation in cultured cells, but also the living environment for cultured cells to grow and reproduce. It is the most important part of biomedical engineering. Cell culture medium is divided into basal medium and serum-free medium according to the way of use. The main components of the basic medium are: inorganic salts, amino acids, carbon sources, vitamins, buffer systems and other types of micronutrients, and the components are clear and clear. The basal medium cannot promote the growth and proliferation of cells and needs to be used in conjunction with serum. The main function of serum is to provide cells with the substances needed for growth and prolifera...
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