JPH1156343A - Fixed bed reactor for culturing animal cell and sowing of cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a fixed bed reactor for culturing animal cells, capable of uniformly sowing a cell into a reactor for culturing animal cells and provide a method for sowing cells by utilizing the reactor. SOLUTION: A liquid port opening 5 is provided in the center part in the flow path direction through which culture medium of a container 1 for housing cells in which culture medium can be passed through the interior is passed and the port opening 5 is used as a sowing port.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a fixed bed reactor for culturing animal cells and a method for seeding cells using the reactor.

[0002]

2. Description of the Related Art As a fixed bed reactor for culturing animal cells, a hollow fiber reactor or a column reactor having an immobilized carrier sealed in a column is used. The former is to supply oxygen and nutrients to the cells through the wall of the fiber by arranging a plurality of hollow fibers in the space where cells are present and supplying fresh medium into the hollow fibers, and the latter is a porous glass or A carrier for immobilizing cells such as a nonwoven fabric coated with cellulose or collagen is sealed in a column, and a fresh medium is supplied and used here. To these fixed bed reactors, a medium is fed from a medium preparation tank. When seeding cells into these reactors, in hollow fiber reactors, a syringe or pump was connected to one of the nozzles located at both ends of the space where cells should exist (cell chamber), and the cell solution was pushed in and pushed in from the other nozzle A method is employed in which an equal amount of medium is extracted from the cell solution. In a column-type reactor, cells may be pushed into the column from a liquid sending port for sending a medium to the column. In these methods, a gradient or bias of the seeding concentration occurs from one end to the other end of the reactor, which causes cells to not grow uniformly in the reactor in subsequent culture.

[0003]

SUMMARY OF THE INVENTION In view of the above-mentioned conventional disadvantages, the present invention seeks to reduce the gradient or bias of the seeding concentration in a reactor.

[0004]

Means for Solving the Problems The present inventors have made intensive studies to solve the above-mentioned problems, and have obtained the following findings.
That is, when the cell solution was colored with ink or the like and seeded in a fixed-bed reactor, the cell solution tended to sink to the bottom of the reactor because the specific gravity of the cell solution was slightly larger than that of the culture solution. When a cell solution is inserted from one of the nozzles at both ends of the cell compartment and the culture solution is removed from the other, as in a hollow fiber type reactor, the cells do not spread throughout the cell compartment, and a flow path is created at the shortest distance between the nozzles. It is difficult to inoculate the whole cells uniformly. From these results, the present inventors have come to realize that if the cells are seeded from the central part of the reactor and evenly distributed to the left and right, the bias can be reduced, and the present invention has been completed. That is, the present invention proposes a fixed bed reactor for culturing an animal cell, which has a liquid inlet / outlet at the center of a container for accommodating cells through which the medium can pass, in the direction of the flow path through which the medium passes.
In addition, a cell seeding method for seeding cells from the liquid inlet / outlet is proposed.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The above fixed bed reactor is a container capable of storing and culturing animal cells, and is applicable to both adhesive cells and floating cells. A function of supplying the medium to the container and a function of extracting the medium are additionally provided. Typically, there are hollow fiber type reactors and column type reactors, but a container in which fibers are arranged as a medium supply path instead of hollow fibers may be used. The container has a liquid inlet / outlet at the center in the flow direction through which the culture medium passes. This doorway is a port for seeding cells, and can also be used as a medium doorway.
Cells seeded from the entrance are equally distributed in the left and right sides of the container. Also, when the medium is supplied or withdrawn from the inlet / outlet, and withdrawn or supplied from both ends of the container, the movement distance of the medium is halved as compared with the case where the medium is moved in and out from both ends of the container. .

[0006]

Embodiment 1 The present invention will be described in detail with reference to embodiments.
FIG. 1 is a partial cross-sectional view of a hollow fiber reactor according to a first embodiment of the present invention. The container 1 for storing animal cells is composed of a hollow fiber 4 and a cell chamber 7. At both ends of the container 1, a medium inlet 2 and an outlet 3 are fixed, and the medium entering from the inlet passes through the hollow fiber 4 and reaches the outlet. A liquid port 5 is arranged at the center of the container 1 in the flow direction, and is normally closed with a rubber stopper 6.

A method of using the reactor of Example 1 will be described. A cell solution to be seeded in a syringe with a needle is previously collected. With the medium in the container 1, the injection needle is passed through the rubber stopper 6 to inject the cell solution into the cell chamber 7. By this operation, the cells are seeded around the center of the cell room.
The medium with the same volume as the seeded cell solution passes through the hollow fiber wall and penetrates into the inside. As the cell fluid is exchanged, the cells are easily dispersed throughout the cell compartment. By adjusting the seeding speed and the amount of liquid, seeding can be performed uniformly in the cell compartment.

Using the reactor of Example 1, cells were seeded by the above method and CHO cells were cultured. As a result, the cell density reached 6 × 10 ⁷ cells / ml, and was 3 × 10 ⁷ cells / ml in the conventional method. Cell density was improved. Cell bias was also small.

In this embodiment, the medium corresponding to the seeded cell solution was removed by penetrating the hollow fiber wall. However, even if the nozzle was disposed at both ends 8 of the cell chamber as in the prior art and the medium was removed from the nozzle, the conventional method was adopted. It is obvious that the dispersion is more uniform than the method described in the above. It is also an effective means to uniformly disperse the seeding while pulling out the injection needle deeply at the time of sowing.

[0010]

[Embodiment 2] FIG. 2 is a sectional view of a column type reactor of Embodiment 2. The medium inlets and outlets 11 and 12 are fixed to a plastic container 13 with silicone packings 14 and 15 keeping the airtight. At the end of the container 13 is a filter 1
0.5 having a diameter of 0.5 filled with the containers 8 and 19
mm porous cell carrier. A liquid port 20 is provided on a side surface of the container 13.

A method for using the reactor of Example 2 will be described. The container 13 contains cell carriers such as glass or cellulose beads which are porous and can also carry cells therein, surface-adsorbed apatite or gelatin beads, collagen-coated nonwoven fabrics, and mixtures thereof. I have. Filters 18 and 19 have the property that they do not leak these carriers out of the container but allow the culture medium to pass through. When the cell solution is injected from the liquid inlet / outlet 20, the cells are dispersed in the container.
The same amount of culture medium as the injected cell solution was applied to the medium inlet and outlet 1
Emitted from 1 and 12. The inlet and outlet of the culture medium may be at the medium inlet and outlet 11 and 12 or at the liquid inlet and outlet 20.

CHO cells were seeded from the liquid port 20 using the porous glass as a carrier and allowed to stand. After 1 hour, the medium was supplied from the liquid port 20 and extracted from the medium ports 11 and 12, and cultured. The density reached 10 ⁸ cells / ml, and the bias of the cells was smaller than that in the case where the medium was flowed in the direction from 11 to 12.

[0013]

According to the present invention, the deviation of the seeding concentration of cells can be reduced, and when used as a culture medium supply port, the flow path of the culture medium can be shortened, and accordingly, the denaturation of the culture medium passing through the reactor can be reduced.

[Brief description of the drawings]

FIG. 1 shows a partial cross-sectional view of a hollow fiber reactor of Example 1.

FIG. 2 shows a cross-sectional view of a column reactor of Example 2.

[Explanation of symbols]

 1. Container 2,3. 3. Media inlet and outlet Hollow fiber 5. Liquid inlet / outlet 6. Rubber stopper 7. Cell compartment 8. Nozzle positions at both ends of cell chamber 11, 12. Medium inlet and outlet 13. Container 14,15. Silicone packing 16, 17. Cap nut 18,19. Filter 20. Liquid port

Claims (2)

[Claims] 1. A fixed bed reactor for culturing animal cells, characterized in that a container for accommodating cells through which a culture medium can pass has a liquid inlet / outlet at a center in a flow path direction through which the culture medium passes. 2. A method for seeding cells, comprising seeding cells from said liquid port.