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
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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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