Bovine kidney cell capable of being subjected to suspension culture, suspension culture method for bovine kidney cell and application thereof

[0030] Embodiment 1, acquisition and preservation of suspension bovine kidney cell line (suspension MDBK cell line)

[0031] The suspension MDBK cell strain in the present invention is a MDBK cell strain capable of suspension culture obtained by screening ordinary adherent MDBK cells through procedures such as low-serum adherent culture, low-serum suspension culture, and serum-free suspension culture.

[0032] The specific screening process is as follows:

[0033] 1. Adherent MDBK Cell Culture

[0034] Usually, the adherent MDBK cells are cultured at 37°C with 10% (volume percentage, V / V) newborn bovine serum (purchased from: Jinyuankang Bioengineering Co., Ltd.) in commercially available DMEM medium (purchased from: GIBCO), and the cells are After the monolayer is overgrown, digest and disperse with 0.25% (V / V) trypsin, dilute and subculture according to the ratio of 1:3 to 1:5, and expand the culture.

[0035] 2. Low serum adherent culture

[0036] Adherent MDBK cells co

[0059] Embodiment 2, the acquisition of bovine kidney cell MDBK-S CGMCC No.11795 kinds of cell suspensions

[0060] The method for obtaining bovine kidney cell MDBK-S CGMCC No.11795 kinds of cell suspensions comprises the following steps:

[0061] 1) Recovery of bovine kidney cells MDBK-S CGMCC No.11795

[0062] Take out a suspension of frozen bovine kidney cell line MDBK-S CGMCC No.11795 from liquid nitrogen, thaw quickly in a water bath at 37°C-40°C, centrifuge at 1000rpm for 5min, discard the supernatant, and add the bovine kidney cell suspension above Resuspend the cells in the culture medium, transfer them to the Erlenmeyer shaker flask, add special suspension medium for bovine kidney cells, and adjust the cell density to 0.3-0.5×10 6 Cells / mL, placed in a shaker at 37°C, 100rpm for suspension culture.

[0063] 2) Passaging of bovine kidney cells MDBK-S CGMCC No.11795

[0064] Step 1) Sampling and counting after 48-72 hours of cell culture, if the cell density is ≥1.0×1

[0065] Example 3. Reactor fed-batch culture of bovine kidney cells MDBK-S CGMCC No.11795

[0066]Fed-batch culture of bovine kidney cells MDBK-S CGMCC No.11795, including the following levels and scale-up culture:

[0067] 1) Fed-batch culture in 5L reactor

[0068] First use bovine kidney cell-specific suspension medium to dilute the seed cell suspension (prepared in Example 2) of bovine kidney cell MDBK-S CGMCC No.11795 to a density of 0.3-0.5×10 6 cells / mL, then transfer 1000-2000mL of diluted cell suspension (according to the reactor conditions) to a 5L reactor, and set the cell culture conditions: temperature 37°C, dissolved oxygen 40-60% (saturation percentage), pH value 7.0-7.2, rotation speed 40-60rpm, until the cell density reaches 0.8×10 6 When cells / mL (judging that the cells are growing vigorously), start feeding the special suspension medium for bovine kidney cells, the flow rate is 1000mL / day, stop feeding when the culture volume reaches 5L, and take it out