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Human diploid cell ZFB as well as construction method and large-scale culture method thereof

A technology of human diploid cells and culture methods, applied in the fields of human diploid cells ZFB cells and their construction, and large-scale culture, can solve the problem of limited generation of diploid cells, limited antigen production, and unresolved virus titers. Improvement and other issues

Inactive Publication Date: 2020-04-14
ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, considering that WI-38 cells have been detected to be contaminated, they are no longer used for vaccine production
Due to the scarcity of MRC-5 cells authorized by WHO and the limited generation of diploid cells themselves, the current production is generally up to 35 generations, resulting in a severe limitation in the production of antigens, and a variety of viruses that adapt to this type of cells The titer needs to be improved, especially for Japanese encephalitis virus

Method used

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  • Human diploid cell ZFB as well as construction method and large-scale culture method thereof
  • Human diploid cell ZFB as well as construction method and large-scale culture method thereof
  • Human diploid cell ZFB as well as construction method and large-scale culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1——ZFB cell line establishment

[0037] experimental method

[0038] The present invention successfully applied for the "Ethical Review Approval Document for the Establishment of ZFB Strain of Human Diploid Cells" from the Clinical Medical Research Ethics Committee of the First Affiliated Hospital of Anhui Medical University. After various physical examinations and family genetic medical history investigations on the parents of the donated embryos, two suitable embryos were screened out, namely Embryo No. 2 and Embryo No. 3.

[0039] 1. Primary cell acquisition

[0040] Doctors from the First Affiliated Hospital of Anhui Medical University used the method of water bag induction to obtain the fetus, and took out the embryonic lung tissue, which was placed in a sterile transport box and transported to the laboratory. In a biosafety cabinet, cut the tissue crosswise to 1 mm using surgical scissors 3The small pieces were rinsed 3 times with sterile phosphate-buff...

Embodiment 2

[0054] Embodiment 2——ZFB cell line test

[0055] experimental method

[0056] 1. Cell morphology observation and blood adsorption test

[0057] 1.1 Preparation of red blood cell suspension

[0058] Guinea pig blood and chicken blood were collected and stored in cell preservation solution respectively. The blood cells were centrifuged at 2000rpm for 5min, the supernatant was discarded, an appropriate amount of normal saline was added and mixed with a pipette repeatedly, washed twice continuously; 2000rpm was centrifuged for 10min, the supernatant was discarded, and a 1% erythrocyte suspension was prepared with normal saline, 2 Store at -8°C for later use. Fresh red blood cells should not be stored for more than 7 days at 2-8°C, and the solution does not contain calcium and magnesium ions.

[0059] 1.2 Observation of cell morphology and blood adsorption test

[0060] Inoculate an appropriate amount of the cells to be tested in 6 T25 cell culture flasks, observe the cell mor...

Embodiment 3

[0103] The present invention has entrusted the China Institute for Food and Drug Control to carry out the identification test of ZFB-2 and ZFB-3 cells in the 8th to 12th generation, the inspection of endogenous virus factors, the inspection of tumorigenesis, and the inspection of chromosome karyotype, and issued a report. The experimental results prove that both ZFB-2 and ZFB-3 cells are qualified.

[0104] Among them, the qualified numbers of ZFB-2 are: SH201804814(P11), SH201804813(P22), SH201804812(P31), SH201804811(P41), SH201804810(P52);

[0105] ZFB-3 qualified numbers are: SH201804801(P10), SH201804802(P21), SH201804803(P31), SH201804804(P42), SH201804805(P50); SH201804806(P60).

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Abstract

The invention discloses a human diploid cell ZFB as well as a construction method and a large-scale culture method thereof, a classification name of the cell is ZFB-2, and a preservation number is CGMCC No. 17481; the preservation number of the ZFB-3 is CGMCC No.17482; the construction method of a ZFB cell strain comprises the following steps: taking embryonic tissue blocks for adherent culture; when the cells grow into a compact monolayer, culturing the monolayer cells by adopting an MEM culture solution containing 10% of fetal calf serum, and carrying out passage; discarding a cell culture supernatant, and digesting and cryopreserving; the large-scale culture method of the ZFB cells comprises the following steps: inoculating a ZFB cell strain of a working cell bank into a primary bioreactor for cell amplification; taking a microcarrier covered with cells in the primary bioreactor, washing, adding trypsin containing 0.1% of collagenase, and digesting; and then inoculating into a secondary bioreactor for culturing. The ZFB-2 cell and the ZFB-3 cell provided by the invention are good in state, rapid in growth, long in passage life, high in sensitivity to viruses and convenient to widely use.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a human diploid ZFB cell, a construction method thereof, and a large-scale culture method. Background technique [0002] Human diploid cells are isolated from human normal tissues (usually derived from embryos) and cultured in vitro to establish cell lines. Human diploid cells (such as WI-38, MRC-5, KMB-17, 2BS, etc.) have limited growth generations, and the passage life is generally 40-60 generations. After a certain number of generations, the cells will stop proliferating and Gradually grow old and die. Human diploid cells have broad viral susceptibility, good stability, and non-tumorigenicity throughout their passage lifespan. Except that the cells of the final generation will have a low probability of chromosome number and structural abnormalities, the human diploid cells of other generations can basically maintain the diploid karyotype. Compared with the primary ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/073
CPCC12N5/0603C12N2509/00C12N2511/00C12N2531/00
Inventor 蒋仁生徐文龙沈慧莉靳正伟陶立峰谭小东杨世龙
Owner ANHUI ZHIFEI LONGCOM BIOPHARM CO LTD
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