Medium combination for suspension culture of human-derived cells and preparation method for oncolytic vaccinia virus
A technology of cell suspension and vaccinia virus, which is applied in the direction of cell culture active agents, biochemical equipment and methods, viruses, etc., can solve the problems of drop, drop of cell viability, low viability, etc., and achieve reduction of culture volume, simplification of operation, The effect of reducing labor intensity
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0040] Embodiment 1. Serum-free suspension culture of virus host cell HeLa S3
[0041] 1.1 HeLa S3 cell adherent subculture
[0042] (1) Cell source: cervical cancer cells, purchased from the American Type Culture Collection, HeLa S3 (ATCC®CCL2.2™), amplified after acquisition, subpackaged and frozen.
[0043] (2) Medium: DMEM (5% FBS), refers to DMEM medium containing 5% fetal bovine serum.
[0044] (3) Resuscitation: Resuscitate one HeLa S3 cell from liquid nitrogen, transfer it into a T25 square flask, and culture it in DMEM (5% FBS).
[0045] (4) After 2-4 days of culture, when the confluence of HeLa S3 cells grows in T25 square flasks to 80%~100%, rinse the cells with D-PBS solution, add trypsin to digest, resuspend with medium, gently Pipette the cells to disperse, passage to T75 square flask at 1:3~1:5 for further culture. After 2 to 4 days, the cells were subcultured into T175 square flasks to continue culturing in the same way.
[0046] 1.2 Suspension acclimation ...
Embodiment 2
[0050] Example 2 Culture medium combination
[0051] There are not many serum-free media available in the market for HeLa S3 cell culture, and most of them can only support the adherent growth of HeLaS3 cells, and very few support the suspension growth of HeLa S3 cells, even the cells that support the suspension growth of HeLa S3 cells Medium, cell growth is not good.
[0052] In this example, with EX-Cell ® 293, DMEM, VirusPro ® Two or three of CD HeLa were combined in equal proportions to form a new serum-free medium, and HeLa S3 cells were cultured in suspension in 125ml shake flasks, initial cell seeding density: 7.5×10 5 cells / ml, 37℃, 5%CO 2 , 110rpm shaking culture. After 3 days of culture, the cell seeding density was about 0.8×10 6 Cells / ml was subcultured, and after continuing to culture for 3 days, the cell density and viability were detected. The results are shown in Table 1:
[0053] Table 1 Cell density and viability after culture
[0054]
[0055] Th...
Embodiment 3
[0059] Embodiment 3 different culture medium combination expands virus
[0060] 3.1 Experimental method
[0061] Cell line: HeLa S3 (serum-free suspension culture)
[0062] Vaccinia virus: Lister strain, a genetically modified recombinant vaccinia virus strain
[0063] Medium:
[0064] ①EX-Cell ® 293+ DMEM (1:1)
[0065] ② Virus Pro ® CD HeLa + DMEM (1:1)
[0066] ③VirusPro ® CD HeLa + EX-Cell ® 293 (1:1)
[0067]④EX-Cell ® 293+ VirusPro ® CD HeLa+DMEM (1:1:1)
[0068] Cell culture: use the above four medium combinations to culture HeLa S3 cells respectively, 25ml / bottle, when the growth reaches the third day, take samples respectively to detect the cell density. Dilute to about 4×10 with the corresponding medium 6 cells / ml, ready to receive poison.
[0069] Virus amplification: Calculate the amount of virus seed incorporation according to the MOI of 0.02 pfu / cell, and inoculate Lister virus into the cells cultured in the four groups of medium. After conti...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More