Human embryonic lung fibroblastic cell SV-7 and application thereof

A technology of fibroblasts and SV-7, applied in the biological field, can solve problems such as poor growth status, increased production costs, and impact on virus production, and achieve good adaptability and high adaptability

Active Publication Date: 2013-11-13
SINOVAC RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The generation of original cell seeds obtained from official or other formal channels is close to 20 generations, and it is difficult to expand the cells to the ideal production scale. At the same time, the higher the cell generation, the more difficult it is to cultivate, and the worse the growth state of the cells, all of which will directly Affect the yield of virus and increase production cost
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Method used

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  • Human embryonic lung fibroblastic cell SV-7 and application thereof
  • Human embryonic lung fibroblastic cell SV-7 and application thereof
  • Human embryonic lung fibroblastic cell SV-7 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045]The acquisition and karyotype analysis of embodiment 1SV-7 cell line

[0046] Obtaining of 1SV-7 cell line

[0047] from having as Figure 1A and Figure 1B The edge of the lung tissue was cut into pieces of about 1mm from a 14-week-old male embryo with the indicated karyotype 3 Rinse the tissue pieces with PBS solution (pH 7.2) for 2 minutes, discard the supernatant, and repeat 3 times. Digest with tissue digestion solution containing 0.2% type Ⅰ collagenase at 37°C for 30 minutes. Oscillate once every 8 minutes, pipette with cell growth medium containing 15% fetal calf serum, and separate the cells until no obvious visible tissue pieces. Filter the cell suspension through a 100-mesh stainless steel screen and centrifuge at 1000-1200 rpm for 5 minutes. Resuspended to a cell density of 5×10 5 / mL, transfer to T75 cell culture flask, place at 36.5±0.5℃, 5%CO 2 Cultured in an incubator. After treatment with 0.25% trypsin, the cells were continuously cultured. The ...

Embodiment 2

[0061] Example 2 Adaptability of varicella-zoster virus in SV-7 cell line

[0062] Inoculate the working seeds of Oka strain varicella zoster virus (from ATCC, No. VR-795) to SV-7 and MRC-5 cells at an MOI of 0.001, and place the cell bottle inoculated with the virus at 35.0±1.0°C for 1 hour to adsorb , shake once every 20 minutes; after the adsorption is completed, add virus maintenance solution, and place in a 35.0±1.0°C incubator to continue culturing. In order to ensure the growth of cells and viruses, the virus culture medium was replaced every 48±4 hours during the virus culture process, and the virus culture medium was changed on the 12th, 24th, 36th, 48th, 60th, 72nd, 84th, 96th, 108th, 120th, and 132nd days after inoculation respectively. , 144, 168, 192 and 216 hours under the microscope to observe and record the disease state, discard the culture medium and wash the cell surface with PBS solution, then add PBS solution, freeze and thaw the cells, and use the microcy...

Embodiment 3

[0063] The adaptability of embodiment 3 rabies virus in SV-7 cell line

[0064] 1. Culture the human diploid cell line SV-7 obtained in Example 1 into a dense monolayer, digest with 0.25% trypsin, and use medium such as MEM, EMEM, DMEM, M199 and L60 containing 10% bovine serum collected and prepared into 3 x 10 5 cells / mL cell suspension, inoculate the rabies virus CTN-1V strain into the cells according to the MOI of 0.01-0.1, add virus maintenance solution, culture at 33°C for 13 days, observe cell changes, replace the virus maintenance solution every 2 days, and harvest The cell supernatant was used to detect the virus titer. Table 3 is the titer result of rabies virus inoculated with SV-7 cells of different passages.

[0065] The titer result of the rabies virus cultured by the SV-7 cells of different passages in table 3

[0066]

[0067] 2. Virus passaging stability: After the virus was cultivated for 9 days, the virus liquid was harvested and used as rabies virus se...

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Abstract

The invention provides a human embryonic lung fibroblastic cell SV-7 with collection number CGMCC No.6956 and an application thereof. The SV-7 cell has obvious characteristics, vigorous growth and long life cycle; the average population doubling level is the 60th generation; the SV-7 cell is pure without pollution, and the culture method is simple; and moreover, the SV-7 cell has adaptability to multiple viruses, can be used for culturing varicella-zoster virus, enterovirus type 71, poliovirus, coxsackie virus A16, rubella virus, hepatitis A virus, rabies virus, rotavirus and measles virus, and can be further used for developing and preparing vaccines against the viruses. The cell culture cost is low, and the SV-7 has good economic values and broad application prospects.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to human embryonic lung fibroblast SV-7 and application thereof. Background technique [0002] Human diploid cells are derived from normal human tissues. They can be subcultured in vitro for limited generations and establish a stable cell bank system. They have normal human diploid chromosome numbers and have no potential tumorigenicity. For human vaccines, human diploid cells are used as the matrix, and there is no need to consider the influence of foreign cell proteins and foreign DNA residues. At present, human diploid cells used for vaccine production at home and abroad mainly include WI-38, MRC-5, 2BS, KMB-17 and so on. More than 40 years of clinical experience have shown that human diploid cells have good safety and efficacy as a cell matrix for vaccine production. [0003] Human diploid cell fluid has some shortcomings, which affect its wide application in vaccine production, s...

Claims

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Application Information

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IPC IPC(8): C12N5/073C12N7/00
Inventor 王琳张燕吕哲姚志东姜德玉李育蓉高强尹卫东
Owner SINOVAC RES & DEV
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