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Monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3 and application of monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3

A 293t-clone3, epithelial cell technology, applied to embryonic cells, urinary tract/kidney cells, artificial cell constructs, etc., can solve the problem of low AAV production efficiency

Active Publication Date: 2020-05-15
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Since the packaging cell lines used in the existing AAV production are generally obtained from ATCC (American Type Culture Collection Center), they are used directly or after other modifications, but the cells used are still composed of a group of cells with heterogeneity A cell line, that is, a group of cells with different AAV packaging efficiencies, may result in an overall inefficient production of AAV, and clinical treatment requires sufficient quantities of AAV, i.e. high titers

Method used

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  • Monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3 and application of monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3
  • Monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3 and application of monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3
  • Monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3 and application of monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3

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Experimental program
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Effect test

Embodiment 1

[0030] Obtaining 293T-Clone3 Monoclonal Cell Line of Human Embryonic Kidney Epithelial Cells

[0031] (1) Culture the 293T cells purchased from ATCC, and divide them into multiple cell culture flasks for culture when they are full.

[0032] (2) When the cell density of one of the bottles is about 70-80%, discard the supernatant, wash it once with PBS, and then add an appropriate amount of trypsin digestion solution to digest in an incubator to disperse them into single cells.

[0033] (3) After the digestion to a single cell was observed under a microscope, complete medium was added to terminate the digestion, and then centrifuged at 1000 rpm for 5 min.

[0034] (4) Discard the supernatant, resuspend the cells with complete medium and count with trypan blue.

[0035] (5) Dilute the cells with cell culture medium (the dilution ratio is based on the fact that each well of the 96-well plate contains at most 1 cell) and add them to the 96-well plate for culture;

[0036] (6) On ...

Embodiment 2

[0039] 1. Virus packaging test of human embryonic kidney epithelial cell 293T-Clone3 monoclonal cell line

[0040] A human embryonic kidney epithelial cell 293T-C3 monoclonal cell strain (hereinafter referred to as 293T-C3 cell) obtained in Example 1 and AAV293 are used to package serotypes AAV1, AAV2, AAV3, AAV5, AAV6, and pDG8 respectively, and measure the packaging. Viral titers of AAV. Specific steps are as follows:

[0041] (1) Plate 293T-C3 in five 150mm dishes, and then put AAV packaging plasmids (pAAV1, pAAV2, pAAV3, pAAV5, pAAV6 and pDG8 plasmids), helper plasmids (pHelper plasmids) and expression plasmids (pAAV-CB-EGFP Plasmids) were mixed at a molar ratio of 1:1:1 to obtain a mixed plasmid, which was then co-transfected into 293T-C3 cells with PEI transfection reagent, and the mass ratio of PEI transfection reagent to mixed plasmid was 1:2, 8- After 10 hours, the cells were replaced with medium, and the culture was continued until 48 hours before taking pictures u...

Embodiment 3293

[0065] Example 3 Establishment of 293T-C3 tertiary cell bank

[0066] 1. Establishment of tertiary cell bank

[0067] (1) if Figure 5 As shown, the 293T-C3 cells screened above were expanded and cultured until 1x10 7 Freeze about 30 tubes per tube, then discard the supernatant of the cells, wash with PBS, add digestion solution and digest in the incubator for 1 minute; add medium to stop digestion, resuspend and count, and centrifuge at 1000rpm for 5 minutes; 1x10 7 The cells were resuspended by adding the corresponding amount of cryopreservation solution, and then 1mL cells were added to each cryopreservation tube, placed in a programmed cooling freezer box, placed at -80°C overnight, then transferred to a gas-phase liquid nitrogen tank as a seed bank and made good record.

[0068] (2) One cell was taken out from the above-mentioned frozen seed bank cells, expanded and cultured, and then expanded and frozen according to the above method, and 50 cells were frozen as the m...

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Abstract

The invention relates to the technical field of biology, in particular to a monoclonal cell strain of human embryonic kidney epithelial cells 293T-Clone3 and application of the monoclonal cell strainof the human embryonic kidney epithelial cells 293T-Clone3. A preservation number of the monoclonal cell strain is CCTCC NO: C2019262, the monoclonal cell strain has high transfection efficiency, thetransfection efficiency is higher than that of commercial AAV293, the transfection efficiency all reaches 90% or above, both the total virogene copy numbers of supernatant and cells of AAV packaged ineach 150-mm vessel are 1011 or above, and can be up to 1012, the virogene copy numbers in supernatant are all 1011 or above, and the virogene copy number of AAV packaged by the AAV293 ranges from 109to 1011. Compared with the AAV293, the monoclonal cell strain of the human embryonic kidney epithelial cells 293T-Clone3 can meet the experiment requirements more.

Description

technical field [0001] The invention relates to the technical field of monoclonal cell lines, in particular to a human embryonic kidney epithelial cell 293T-Clone3 monoclonal cell line and applications thereof. Background technique [0002] Gene therapy is based on changing human genetic material, introducing exogenous normal genes or therapeutic genes into target cells to correct or compensate for diseases caused by gene defects and abnormalities, so as to achieve the purpose of treating diseases. However, AAV has the advantages of wide host range, high safety, low immunogenicity, and long expression time. At the same time, AAV has multiple serotypes, and the capsid proteins of different serotypes recognize different receptors on the cell surface, so in different tissues Infection efficiencies vary among cells, enabling efficient transduction of specific cell types. Therefore, AAV has been widely used in basic research and clinical trials, and has become one of the most co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N5/073C12N15/864C12N7/00C12R1/91
CPCC12N5/0603C12N5/0686C12N15/86C12N7/00C12N2750/14121C12N2750/14143C12N2750/14152
Inventor 王志民连雪琪钟晶晶赵晓燕褚涌超卢双双秦斌张成林王尧河
Owner ZHENGZHOU UNIV
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