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A method for efficient passage of human diploid cell microcarrier culture

A technology for culturing human diploid cells and microcarriers, applied in the field of effective subculture, can solve the problems of uneven adhesion, cell viability, low harvest rate and adhesion rate, etc., and achieve the goal of improving digestion efficiency and increasing contact adhesion Chance, effect of reduced culture volume

Active Publication Date: 2020-05-12
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to solve the problems of low cell viability, harvest rate and adhesion rate and uneven adhesion in the digestion and subculture method of culturing human diploid cells in existing bioreactors and microcarriers, and to provide a human diploid A method for effective subculture of ploidy cells in the process of amplified culture in bioreactors and microcarriers. This method can improve the viability, adhesion rate and adhesion uniformity of cells during the subculture process, so that the subculture efficiency can be improved. It is widely used in vaccine scale chemical production

Method used

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  • A method for efficient passage of human diploid cell microcarrier culture
  • A method for efficient passage of human diploid cell microcarrier culture

Examples

Experimental program
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Effect test

Embodiment 1

[0040] Implement 1 Effective subculture of microcarriers of MRC-5 cells

[0041] ⑴ Microcarrier culture of MRC-5 cell seeds in a 1.5 liter bioreactor:

[0042] Add dry 2.0 g cytodex1 microcarriers to a suitable siliconized glass vial and add 200 ml Ca-free 2+ , Mg 2+ After mixing with PBS, it was hydrated overnight at room temperature, washed three times with the above PBS, and sterilized at 121° C. for 30 minutes by high-pressure damp heat before use.

[0043] Allow the sterile microcarriers to settle, pour off the supernatant, rinse and replace twice with warm MEM medium. Transfer the microcarriers to a 1.5-liter bioreactor, add 500 milliliters of MEM complete medium, and set the culture parameters: temperature 37 ° C, pH 7.2, DO50%, rotation speed 30 rpm to balance overnight.

[0044] After digesting the MRC-5 cells in 7 T225 cell flasks, the cell suspension was added to the bioreactor, and the volume of the reserved medium in the bioreactor was adjusted according to the...

Embodiment 2

[0057] The effective subculture of the microcarrier of embodiment 2 KMB17 cells

[0058] (1) Microcarrier culture KMB17 cell seed in 5.0 liters of bioreactors:

[0059] Add dry 10.0 g cytodex1 microcarriers to a suitable siliconized glass vial and add 1000 ml Ca-free 2+ , Mg 2+ After mixing with PBS, it was hydrated overnight at room temperature, washed 5 times with the above PBS, and sterilized by high pressure at 121°C for 30 minutes before use.

[0060] Allow the sterile microcarriers to settle, pour off the supernatant, rinse and replace twice with warm M199 medium. Transfer the microcarriers to a 1.5-liter bioreactor, add 500 milliliters of M199 complete medium, and set the culture parameters: temperature 37 ° C, pH 7.2, DO 50%, rotation speed 30 rpm to balance overnight.

[0061] The 5.0-liter bioreactor was added to the M199 basal medium 5 days in advance to carry out the sterility test according to the culture parameters (37°C, pH7.2, DO50%). After the sterility te...

Embodiment 3

[0073] Example 3 Determination and calculation of cell viability, cell shedding percentage, cell adhesion rate and cell recovery rate during subculture

[0074] (1) Before the cell digestion step, take 1 ml of uniformly suspended culture, centrifuge at 1000 rpm for 5 minutes to settle the carrier cells, carefully remove the supernatant, add 0.1 mol / L citric acid solution containing 0.1% crystal violet to resuspend Precipitate, incubate at 37°C for 10 minutes, shake on a vortex mixer for 5 minutes, then take a sample and count the stained released nuclei on a hemocytometer. The measured nucleus concentration multiplied by the culture volume is the total number of cells before digestion;

[0075] (2) When sampling every 5 minutes during digestion, absorb the supernatant, add complete serum-containing medium to neutralize the trypsin, sample 1:1 and add trypan blue staining solution, and count the cells automatically on TC20 after 5 minutes of action Cell viability was detected o...

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Abstract

The invention relates to the technical field of biology, in particular to an effectively passage method for human diploid cell microcarrier culture in a bioreactor microcarrier scale-up culture process. According to the method, a microcarrier is utilized to culture a human diploid cell seed, a blank microcarrier is prebalanced in a seed culture system, a seed cell is digested by trypsin digestive juice containing sodium citrate, the seed cell is moderately stirred and cultured discontinuously for 3 to 6 hours in a seed reactor, and then the seed cell is transferred to an amplification reactor to be amplified and cultured to obtain effective passage in the human diploid cell microcarrier scale-up culture process. The effective passage method disclosed by the invention has effects of improving vigor, adherence rate and adherence evenness of cells in a passage process, obviously improving passage efficiency and solving the problem of difficulty in scale-up culture of the human diploid cells on the bioreactor and the microcarrier.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an effective subculture method for human diploid cells in the microcarrier amplification culture process of a bioreactor. Background technique [0002] MRC-5, KMB17, WI-38 and other diploid cells are in vitro cultured cells derived from normal human tissues. They have no potential carcinogenicity, no contamination by exogenous factors, and have a wide spectrum of virus sensitivity. They are currently considered the most The safe and most suitable cell matrix has been widely used in the production of vaccines. [0003] The large-scale culture technology of animal cells is widely used in the production of biological products. Since the 1980s, a culture technology integrating bioreactors and microcarriers has been gradually established. This technology has the advantages of both single-layer adherent culture and suspension culture. The specific surface area of ​​microcarriers is large ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071
CPCC12N5/0602C12N2531/00
Inventor 姜云水李剑波包佳源王平唐彩华顾春燕张峰吴洁毛子安庄昉成毛江森
Owner ZHEJIANG PUKANG BIOTECH
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