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Serum-free culture medium suitable for adherent growth of hybridoma cells

A serum-free culture medium, hybridoma cell technology, applied in the field of biomedicine, can solve the problems of complex bovine serum composition, unsecured supply, long delivery period, etc., to eliminate the risk of mycoplasma contamination, maintain cell activity, and control batches. poor effect

Pending Publication Date: 2022-05-13
SHANGHAI JIEMEN BIO TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these culture media have the following defects: (1) bovine serum, an important component of the culture medium, generally needs to be imported from abroad. It is large, and sampling inspection is required before each use, and can only be purchased and used after passing the test, which not only takes a long time, but also increases the workload of the inspection, and the quality of raw materials is not guaranteed; It will inhibit the growth of cells, and there is also the risk of mycoplasma contamination; (4) bovine serum, especially fetal bovine serum, is expensive, and 10-15ml of bovine serum is required for every 100ml of medium, which is expensive

Method used

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  • Serum-free culture medium suitable for adherent growth of hybridoma cells
  • Serum-free culture medium suitable for adherent growth of hybridoma cells
  • Serum-free culture medium suitable for adherent growth of hybridoma cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] A serum-free medium suitable for the adherent growth of hybridoma cells, comprising the following concentrations of components: phenylalanine 45mg / L, threonine 30mg / L, tryptophan 35mg / L, tyrosine 45mg / L , Valine 24mg / L, Methionine 18mg / L, Lysine 50mg / L, Leucine 35mg / L, Histidine 40mg / L, Cystine 20mg / L, Arginine 35mg / L, Serine 28mg / L, sodium bicarbonate 2000mg / L, sodium dihydrogen phosphate 200mg / L, disodium hydrogen phosphate 190mg / L, magnesium chloride 40mg / L, sodium chloride 5000mg / L, magnesium sulfate 500mg / L, potassium chloride 500mg / L , calcium chloride 150mg / L, sodium hydroxide 1000mg / L, copper sulfate 6mg / L, manganese chloride 5mg / L, ferric citrate 5mg / L, calcium pantothenate 7mg / L, folic acid 10mg / L, nicotinamide 6mg / L L, vitamin B2 8mg / L, thiamine 8mg / L, biotin 8mg / L, vitamin B12 5mg / L, vitamin C 10mg / L, vitamin A 6mg / L, reduced glutathione 6mg / L, breast Glycoside 5mg / L, Adenosine Triphosphate 5mg / L, Xanthine 8mg / L, Uracil 3mg / L, Zinc Sulfate 5mg / L, Hydrocort...

Embodiment 2

[0029]A serum-free medium suitable for the adherent growth of hybridoma cells, comprising the following concentrations of components: phenylalanine 15mg / L, threonine 12mg / L, tryptophan 15mg / L, tyrosine 15mg / L , Valine 10mg / L, Methionine 10mg / L, Lysine 15mg / L, Leucine 20mg / L, Histidine 10mg / L, Cystine 10mg / L, Arginine 15mg / L, Serine 10mg / L, sodium bicarbonate 1000mg / L, sodium dihydrogen phosphate 100mg / L, disodium hydrogen phosphate 100mg / L, magnesium chloride 15mg / L, sodium chloride 3000mg / L, magnesium sulfate 200mg / L, potassium chloride 200mg / L , calcium chloride 100mg / L, sodium hydroxide 100mg / L, copper sulfate 2mg / L, manganese chloride 1mg / L, ferric citrate 1mg / L, calcium pantothenate 1mg / L, folic acid 5mg / L, nicotinamide 2mg / L L, vitamin B2 2mg / L, thiamine 1mg / L, biotin 1mg / L, vitamin B12 2mg / L, vitamin C 5mg / L, vitamin A1mg / L, reduced glutathione 3mg / L, thymidine 2mg / L, adenosine triphosphate 2mg / L, xanthine 3mg / L, uracil 1mg / L, zinc sulfate 1mg / L, hydrocortisone 2mg / L,...

Embodiment 3

[0031] (1) Prepare 100ml of serum-free medium: according to the formula of serum-free medium in Example 1, accurately weigh each component in turn, add 100ml of purified water to dissolve, stir evenly, then add 1ml of 100× penicillin (Gibco), 1ml of 100× Streptomycin (Gibco), mixed well, adjusted the pH value to 7.2-7.4 with 1M HCl / NaOH, and sterilized and filtered with a 0.2 μm filter membrane;

[0032] (2) Prepare 100ml of bovine serum medium: Take 85ml of RPMI-1640 medium (Sigma), add 15ml of fetal bovine serum (Gibco), 1ml of 100×penicillin (Gibco), 1ml of 100×streptomycin (Gibco), mix well ;

[0033] (3) Take 25ml each of serum-free medium and bovine serum medium, and add them to two T75 cell culture flasks (Corning); revive 2 ferritin hybridoma cells (Fer 88-5, Shanghai Jiemen Biology), and the cell density About 1×10 6 cells / ml, respectively added to T75 culture flasks; 5% CO 2 The incubator was cultured at 37 degrees for 3 days, and the growth status of the cells wa...

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Abstract

The invention discloses a serum-free culture medium suitable for adherent growth of hybridoma cells. The serum-free culture medium comprises various nutrient substances such as various amino acids, vitamins, metal salts, coenzymes, hormones, glycoproteins and basic nutrients. The serum-free culture medium is completely free of serum components, chemical components are clear and convenient to prepare, raw materials are common and easy to obtain, and the cost is low; the serum-free culture medium is simple in preparation method, large-scale stable production can be realized, batch difference can be easily controlled, high-density cells can be rapidly obtained, cell activity can be maintained for a long time, the serum-free culture medium can completely replace a bovine serum culture medium to be used for adherent culture of hybridoma cells, the hybridoma cell culture and monoclonal antibody preparation processes are greatly simplified, the batch difference is reduced, and the production cost is reduced. The cost is reduced, and the risk of mycoplasma pollution is also eliminated.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a serum-free culture medium suitable for the adherent growth of hybridoma cells. Background technique [0002] Most hybridoma cells are currently cultured in media containing fetal bovine / newborn calf / calf serum. However, these culture media have the following defects: (1) bovine serum, an important component of the culture medium, generally needs to be imported from abroad. It is large, and sampling inspection is required before each use, and can only be purchased and used after passing the test, which not only takes a long time, but also increases the workload of the inspection, and the quality of raw materials is not guaranteed; It can inhibit the growth of cells, and there is also the risk of mycoplasma contamination; (4) bovine serum, especially fetal bovine serum, is expensive, and 10-15ml of bovine serum is required for every 100ml of medium, which is expensive. ...

Claims

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Application Information

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IPC IPC(8): C12N5/12
CPCC12N5/12C12N2500/90
Inventor 付理文
Owner SHANGHAI JIEMEN BIO TECH
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