Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells

A serum-free culture medium and culture medium technology, applied in animal cells, culture process, tissue culture, etc., can solve the problems of unclear serum composition and high requirements for culture conditions

Inactive Publication Date: 2016-05-04
马忠仁 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Serum-free medium uses serum substitutes with clear components, which not only simplifies the downstream process, but also avoids the risk of biological contamination. However, the serum components are still unclear, and MDCK cells themselves have high requirements for culture conditions. The development of this serum-free medium has always been the focus and difficulty of research in this field

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  • Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells
  • Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells
  • Serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] The serum-free medium for full suspension culture of MDCK cells of the present invention is prepared by adding the following components (in g / L) on the basis of the DMEM / F12 medium containing NaCl of 5g / L:

[0163] Trace elements:

[0164] Sodium stannate (Na 2 SeO 3 -5H 2 O) 0.000005

[0165] Growth Factors:

[0166] Recombinant human epidermal growth factor (EGF) 0.0001

[0167] Hydrocortisone 0.000036

[0168] Recombinant whole chain insulin 0.005

[0169] Prostaglandin E10.000025

[0170] Human transferrin 0.0047

[0171] Thyroxine (T3) 4.0×10 -10

[0172] Lipid complex:

[0173] Vitamin E0.00005

[0174] Cholesterol 0.00005

[0175] Ethanolamine 0.000925

[0176] β-mercaptoethanol 0.000975

[0177] Tween-800.00625 (density is 1.09g / ml, weighed as 1g / ml)

[0178] Plant Hydrolyzate:

[0179] HyPep15105

[0180] Recombinant Human Albumin:

[0181] ACF1

[0182] other:

[0183] Mannitol sugar 1.

[0184] Emulsification method: take 2.5ml Tween-80 to...

Embodiment 2

[0189] The difference between this embodiment and embodiment 1 is:

[0190] In the medium formula, the concentration of recombinant human serum albumin ACF is 0.5g / L.

[0191] All the other parts are the same as in Example 1.

[0192] Apply the culture medium of this example to suspend MDCK cells without adding serum, and the cell seeding density is: 3.0×10 5 / ml, culture temperature: 37°C, rotation speed: 120rpm. See results Figure 10 , Cell map of cultured for 72h.

Embodiment 3

[0194] The serum-free medium for full suspension culture of MDCK cells of the present invention is prepared by adding the following components (in g / L) on the basis of the DMEM / F12 medium containing NaCl of 5g / L:

[0195] Trace elements:

[0196] Sodium stannate (Na 2 SeO 3 -5H 2 O) 0.000001

[0197] Growth Factors:

[0198] Recombinant human epidermal growth factor (EGF) 0.00005

[0199] Hydrocortisone 0.000012

[0200] Recombinant whole chain insulin 0.001

[0201] Prostaglandin E10.00001

[0202] Human transferrin 0.002

[0203] Thyroxine (T3) 1.0×10 -10

[0204] Lipid complex:

[0205] Vitamin E0.0001

[0206] Cholesterol 0.0001

[0207] Ethanolamine 0.001

[0208] β-mercaptoethanol 0.001

[0209] Tween-800.01 (density is 1.09g / ml, weighed as 1g / ml)

[0210] Plant Hydrolyzate:

[0211] HyPep15101

[0212] Recombinant Human Albumin:

[0213] ACF0.5

[0214] other:

[0215] Mannitol sugar 5.

[0216] The preparation method of the medium in this example ...

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Abstract

The invention provides a serum-free culture medium used for full-suspension culture of MDCK (Madin Darby Canine Kidney) cells. The culture medium is prepared from a DMEM/F12 culture medium containing 5g/L of NaCl and the following components: sodium stannite, recombinant human epidermal growth factors, hydrocortisone, recombinant full-chain insulin, prostaglandin E1, human transferrin, thyroxine (T3), vitamin E, cholesterol, ethanol amine, beta-mercaptoethanol, Tween-80, Hypep1510, recombinant human serum albumin ACF and mannitol. With the adoption of the culture medium provided by the invention, the full-suspension culture of the MDCK cells can be carried out very well under the condition that serum is not added; after the MDCK cells are continuously cultured for 20 generations, the average proliferation concentration of the MDCK cells is 2.308*10<6>/mL, the average cell viability is 97.4 percent and the average doubling time is 34.48 hours, so that the serum-free culture medium provides technical supports for developing influenza vaccines of cell matrixes of mammals in China.

Description

technical field [0001] The invention belongs to the technical field of biological products, and in particular relates to a serum-free culture medium for full suspension culture of MDCK cells. Background technique [0002] Influenza (bird flu) is an important infectious disease that endangers human health and the development of the poultry industry. Vaccination is currently one of the most important means of preventing influenza (bird flu). Traditional influenza vaccines are produced with chicken embryos. There is a preparation process in this method. Due to the high probability of microbial contamination in the medium, the high content of endotoxin in the product, the large injection side effects, the high cost of environmental pollution treatment of waste, the difficulty in the supply of chicken embryos when the influenza pandemic breaks out, and the long production cycle, etc., it is necessary to develop safe and efficient animal cells. A matrix influenza vaccine is impera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2500/05C12N2500/25C12N2500/34C12N2500/36C12N2500/38C12N2500/44C12N2500/46C12N2500/50C12N2501/02C12N2501/11C12N2501/395C12N2501/998C12N2501/999
Inventor 马桂兰乔自林
Owner 马忠仁
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