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Method for culturing mdck cells

A culture method and cell culture technology, applied in the field of MDCK cell culture, can solve problems such as concerns about serum safety and adaptability

Inactive Publication Date: 2018-11-09
THE RES FOUND FOR MICROBIAL DISEASES OFOSAKA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of serum has concerns regarding safety and suitability for industrial-scale production

Method used

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  • Method for culturing mdck cells
  • Method for culturing mdck cells
  • Method for culturing mdck cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] (Example 1) Preparation and selection of cloned MDCK cells

[0062] 1. Single cell cloning

[0063] (1) Cultivation using serum-containing medium

[0064] Thaw the MDCK cells (ATCC No.CCL-34, Lot 1166395, passage number 53) obtained from ATCC, add Eagle MEM medium (Nissui Pharmaceutical) containing 10% fetal calf serum (hereinafter referred to as FCS) and perform centrifugal washing . Eagle MEM medium containing 10% FCS was added to the pellet to suspend it, and it was cultured in a T75 flask. After the cells cultured at 37°C±1°C for five days were washed with a PBS solution containing 1 mM EDTA-4Na, trypsin was added to detach the cells from the culture vessel. Add Eagle MEM medium containing 10% FCS to neutralize trypsin, and pass in T225 flasks. After culturing the cells at 37°C±1°C for four days, the cells were also passaged in T225 flasks. After cells cultured at 37° C.±1° C. for six days were recovered by trypsin treatment, Eagle MEM medium containing 10% FCS...

Embodiment 2

[0098] (embodiment 2) use the cultivation of the clone MDCK cell line of microcarrier

[0099] Cell line A was cultured in serum-free medium OptiPRO SFM (Thermo Fisher Scientific) supplemented with 4 mM glutamine. Cell line B and pre-cloned cells were used as control cells. Cells at 2.3 x 10 4 cells / cm 2 inoculation density. Microcarriers used Cytodex 1 (GE Healthcare Life Sciences) at a density of 3.5 g / L. The culture conditions were as follows: the stirring speed before the 48th hour of culture was 15 rpm, the stirring speed after 48 hours was 30 rpm, the pH was 7.0, the temperature was 37.0° C., and the dissolved oxygen concentration (DO) was 3.00 ppm. Vent through perforated tubes. In addition, a bioreactor with a capacity of 3 L was used as a culture container.

[0100] The number of cells in the culture solution was confirmed by extracting a part of the culture solution and dispersing the cells adhered to the microcarriers using trypsin. For the measurement of the...

Embodiment 3

[0110] (Example 3) Confirmation of the expansion rate of the cloned MDCK cell line

[0111] In the same manner as in Example 2, the cell line A was inoculated at the following cell inoculation densities a to d, and cultured. Use precloned cells as controls.

[0112] a: 2.0×10 4 cells / cm 2 (17.6×10 4 cells / mL)

[0113] b: 1.0×10 4 cells / cm 2 (8.8×10 4 cells / mL)

[0114] c: 0.6×10 4 cells / cm 2 (5.3×10 4 cells / mL)

[0115] d: 0.3×10 4 cells / cm2 (2.6×10 4 cells / mL)

[0116] In addition, the culture capacity is 400mL, at 37.0°C, 5% CO 2 In the presence, stirring culture was carried out at 60 rpm. Cytodex 1 was used as microcarrier. The density of the microcarriers is 2.0 g / L.

[0117] The result is as Figure 5 shown. The values ​​near each point in the graph represent the value of the amplification factor at each time point. From this, it can be seen that the expansion rate of the cell line A is higher than that of the pre-cloned cells when about 30 hours to 1...

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Abstract

The present invention pertains to: cloned MDCK cells which exhibit an expansion factor of 4.5-fold or more when being cultured using microcarriers; a method for culturing the MDCK cells; and a methodfor multiplying a virus by using the MDCK cell culturing method.

Description

technical field [0001] The invention relates to a method for culturing MDCK cells using microcarriers and MDCK cells suitable for culturing microcarriers. In addition, the present invention also relates to a virus propagation method achieved by culturing MDCK cells using microcarriers. [0002] This application claims the priority of Japanese application Japanese Patent Application No. 2016-65251 incorporated herein by reference. Background technique [0003] In recent years, in various fields such as pharmaceutical production and regenerative medicine, it is required to efficiently cultivate cells, tissues, microorganisms, etc. in large quantities. In particular, in order to realize the practical application of biopharmaceuticals and regenerative medicine, it is important to establish a mass culture technique for cells. The large-scale cultivation of cells enables the transformation to large-scale production of monoclonal antibodies, recombinant proteins, membrane protein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12N7/00
CPCC12N7/00C12N2760/16151C12N2760/16134C12N2760/16234C12N2760/16251C12N5/0075C12N5/0686C12N2760/16051A61K39/145C12N5/0037C12N2510/02C12N2531/00C12N2760/16334
Inventor 鱼谷多惠桑原宗一郎
Owner THE RES FOUND FOR MICROBIAL DISEASES OFOSAKA UNIV
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