BHK-21 cell strain and culture method and purpose thereof

A BHK-21, culture method technology, applied in the culture process, tissue culture, animal cells, etc., can solve the problems of downstream virus purification, reducing serum concentration, etc., and achieves a simple method for cell recovery, simplified cryopreservation steps, and adaptability. strong effect

Inactive Publication Date: 2018-08-14
CANVEST WUHAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Furthermore, the addition of high concentration (10-15%) animal-derived serum during cell adherence culture will cause difficulties in downstream virus purification
For a long time, researchers have used the domestication method of serum-free suspension culture of CHO cells to try to gradually reduce the serum concentration; add growth factors, hormones, amino acids and other nutrients for serum-free selection; continuous culture selection and other methods to domesticate BHK-21 Cells, change the culture state of cells (suspension culture), so that they can be used in the industrial production of vaccines, but no successful examples have been seen yet

Method used

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  • BHK-21 cell strain and culture method and purpose thereof
  • BHK-21 cell strain and culture method and purpose thereof
  • BHK-21 cell strain and culture method and purpose thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 recovery of BHK-21 cell line

[0040] The BHK-21 cell line (purchased from the China Center for Type Culture Collection (CCTCC)) preserved in the liquid nitrogen tank was taken out, and transferred to a water bath at 37°C for rapid thawing (within 0-2 minutes). Cells were inoculated into T25 cell culture flasks after lysis, and 7ml of MEM medium with 10% (v / v) fetal calf serum was added thereto, gently blown and mixed, and after cultivating for 4-6hr, the cell culture medium was removed. Replace with fresh MEM medium containing 10% (v / v) fetal bovine serum, and culture in a 5% (v / v) carbon dioxide incubator at 37° C. for 2-3 days for sorting single cells.

Embodiment 2

[0041] Sorting and clone cell culture expansion of embodiment 2BHK-21 cells

[0042] (1) Aseptically remove peritoneal cells (mainly macrophages and other immune cells) from the peritoneal cavity of Kunming mice, centrifuge at 1000 rpm for 10 min, discard the supernatant, and use fresh commercially available (Gibco company) MEM medium Suspend cells, count, adjust cell concentration to 10 6 cells / ml, inoculated cells into 96-well cell culture plate, 0.1ml / well. The 96-well cell plate was placed in a 37°C, 5% carbon dioxide incubator and cultured for 24-36hr to prepare the feeder cells to obtain a 96-well feeder cell culture plate containing 1.0% (v / v) fetal bovine serum.

[0043] (2) Take a bottle of BHK-21 cell line prepared in step S1 (cells are required to grow to 95% confluency, culture for 2-3 days), discard the culture supernatant, add 1.0ml, 0.25% (W / V) trypsin Digest the dispersed cells and observe under the microscope to ensure that all cells are in the state of sing...

Embodiment 3B

[0046] Serum-free suspension culture acclimation of embodiment 3BHK-21 clone cell population

[0047] (1) The cryopreserved BHK-21 clone cell population was revived for culture. Commercial serum-free media from different sources were divided into B, Q, X, Y, Z groups. Add 2.0% (v / v) fetal bovine serum to the serum-free medium, inoculate 10 4 cells / ml of BHK-21 clone cells, after one week of culture, they were transferred to a serum-free medium containing 1.0% (v / v) fetal bovine serum for acclimatization, and the serum concentration was gradually decreased from 0.5% to 0.2% in this order of operation. to 0, repeated domestication of BHK-21 cloned cells (the purpose of domestication is the serum-free culture of cells, repeated domestication is to gradually reduce the serum concentration of the cell culture medium until the cells can grow normally without adding serum, in fact, a continuous screening the process of).

[0048] (2) After repeated domestication of BHK-21 cloned c...

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Abstract

The invention relates to a method for culturing a BHK-21 cell strain suspendedly grown in a plurality of serum-free mediums. The method comprises the following steps: S1) recovering a BHK-21 cell line; S2) sorting the BHK-21 cell line, and cloning cell and performing culture amplification; and S3) performing serum-free suspension culture on a BHK-21 clone cell colony. The invention also provides the BHK-21 cell strain suspendedly grown in a plurality of serum-free mediums and a purpose thereof. The method does not require a microcarrier as an adhesion carrier, bottle rotation is not required for keeping a rolling culture mode, the medium does not contain serum, and no protein component is contained, and the medium is the CD-grade commercial serum-free medium composed of inorganic salt anda nutriment. The serum-free suspension culture BHK-21 cell strain can directly add DMSO by the serum-free medium for cryopreservation, and the serum-free medium can be directly used for recovery. TheBHK-21 cell strain provides novel thinking and the method for industrial-grade expanded production of a virus vaccine.

Description

technical field [0001] The invention relates to a method for cultivating animal cells, in particular to a method for culturing and application of a BHK-21 cell strain capable of growing in suspension in various serum-free mediums. Background technique [0002] The serum-free suspension culture of animal cells has always been the goal pursued by large-scale production of genetically engineered drugs and virus vaccines, but so far there are few successful examples. CHO cells (Chinese hamster ovary cells) are the most successful example. They can be cultured continuously in suspension in hundreds of liters of containers with serum-free medium for 2-3 weeks, and the cell growth density can reach 10 8 cells / ml, which solves the technical problem of mass expression of genetically engineered drugs and efficient amplification of viruses. The cells have been used in large-scale production of genetically engineered drugs (protein drugs, polypeptide drugs, and human-derived therapeutic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/0686C12N2500/90
Inventor 郑从义桂哲徐国东
Owner CANVEST WUHAN BIOTECH
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