JPS6125476A - Cell culture device packed with dispersed hollow fiber - Google Patents

Abstract

PURPOSE:The titled cell culture device providing cells with very small environment, promoting cultivation and multiplication, contributing to large-scale cultivation, obtained by dispersing uniformly semipermeable, porous hollow fibers having a specific effective length between partition walls, having one end of the fibers opening into an outer face of the partition wall. CONSTITUTION:The cell culture device 14 consists of plural semipermeable or porous hollow fibers 1, the container 2 and the partition walls 3 to fix both ends of the hollow fibers 1 to both the ends of the container 2. This cell culture device 14 is a desired cell culture device packed with dispersed hollow fibers wherein the effective length of the hollow fibers 1 between the partition walls 3 is substantially longer than the distance between the partition walls, the hollow fibers 1 are uniformly dispersed and packed between the partition walls, at least one end of the hollow fibers 1 opens into an outer face of the partition wall, and cells are cultivated in the hollow fiber dispersed space 6 or on the surface of the hollow fibers. The container 2 is provided with the end conduit 5 connected to the inside of the hollow fibers 1 and with the side conduit 7.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a culture vessel for culturing and proliferating cells. More specifically, the present invention relates to a hollow fiber cell culture device suitable for carrying out cell culture on a large scale.

[Prior art] Mass culture of cells on a large scale is an example of a virus.

Vaccines, antiviral drugs such as interferon.

It is also essential for the production of biological drugs such as hormones.

Particularly in recent years, the production of monoclonal antibodies targeting specific proteins has been based on mass culture of hybridomas using antibody-producing cells and myeloma, and solving this technology is an industrially important theme.

Conventional cell culture includes in vivo culture, which is carried out in the peritoneal cavity of an animal, and in vitro culture, which is carried out in a laboratory vessel. On a laboratory scale, Shunja is like a citrine or a test tube.

Alternatively, it is carried out using a culture bottle, but if it can be made on a larger scale, it would have high industrial value.

In recent years, several proposals have been made as methods for mass culturing cells and devices for the same. In principle, various improvements have been made to increase the area on which cells attach and to efficiently exchange nutrients, waste products, and gaseous substances. on the other hand,
Even in planktonic cells, maintenance of suspension state and mass exchange are i1
It is essential.

A method and apparatus for culturing cells in vitro by placing them on semipermeable hollow fibers was proposed by Nazek et al. A liquid medium is supplied to the medium, and oxygen is supplied to the medium (
Supplied by tax law. Next, Mr. Plant developed a cell growth method in which cells were attached to the outer wall of a hollow fiber in a culture medium, and a gaseous oxygen carrier was supplied to the inner wall of the hollow fiber, thereby supplying oxygen to the cells of C through the hollow fiber membrane. (Japanese Unexamined Patent Application Publication No. 1973-
36684). Furthermore, Hayano et al. have proposed a vessel in which semipermeable hollow fibers are covered with a gas-permeable thin film, and cells are cultured outside the hollow fibers inside the thin film (Japanese Patent Application Laid-open No. 5-1111).
1-98382). Regarding planktonic cells, Mr. Furuta et al. proposed a method in which a porous membrane hollow fiber is covered with a shell, a medium is poured into the hollow fiber, and II cells are cultured between the shell and the hollow fiber (Special 111 Publication No. 56-42584).

However, even with these prior art techniques, it is difficult to say that the technology for culturing human cells has been sufficiently established. In particular, it is essential that the hollow system be semi-permeable or porous and work to supply and exchange nutrients, produced metabolites, waste products, and gas (oxygen) in the medium, but if cells are to attach, it is necessary to There is not enough contribution to floating.

In other words, because the creation of a microenvironment for cells to grow is insufficient, the advantages of hollow fiber culture vessels, namely the above-mentioned material exchangeability and large surface area, cannot be utilized.

[Purpose of the invention] In view of this situation, the purpose is to take advantage of the characteristics of the hollow fiber type cell culture device to provide a better microenvironment to cells, promote their culture, and thereby contribute to large-scale culture. As a result of extensive research, we have completed the cell culture device of the present invention.

[Configuration of the Invention] That is, the present invention provides a cell culture device comprising a plurality of semipermeable or porous hollow fibers, a container, and a partition wall that fixes both ends of the hollow fibers to both ends of the container. J3, wherein the effective length of the hollow fibers is substantially longer than the distance between the partition walls, and the hollow fibers are uniformly distributed and packed between the partition walls;
At least one end of the hollow fiber is open to the outer surface of the partition, and Ill! This is a hollow fiber dispersion-filled cell culture device characterized in that the cell culture device 11 is cultured in the hollow fiber dispersion gap or on the outer surface of the hollow fibers.

Furthermore, in the cell culture device of the present invention, the container is provided with at least one end conduit that communicates with the inside of the hollow fiber and at least one side conduit that communicates with the hollow fiber dispersion gap; The outer surface of the hollow fiber is substantially filled with no excess space.

FIG. 1 shows the basic configuration of the present invention. A cell culture device comprising a plurality of semipermeable or porous hollow fibers, a container 2, and a partition wall 3 that fixes the ends of the hollow fibers to the ends of the container,
The length of the region with a free surface between the two partition walls of the hollow fiber (hereinafter referred to as @decrease length) is the shortest path 11 between the two corner walls.
11 (hereinafter referred to as the distance between partition walls), and the hollow fibers are uniformly dispersed. These configurations are necessary to form the microenvironment described below. Furthermore, at least one end of the hollow fiber is open on the outer surface of the partition wall and communicates with the end chamber 4 and the end conduit 5.
leading to. The container 2 is therefore provided with at least one end conduit 5 which communicates with the interior of the hollow fiber. 1st
In the case of figure (a), the end conduit 5a, one end chamber 4a-the hollow fiber 1
It is connected to interior-4b-5b and is generally supplied with a liquid medium.

At least one lateral conduit 7 is attached to the container 1 which communicates with the hollow fiber dispersion gap 6 (i.e. the microenvironment under artificial conditions for cell culture) and is generally used for inoculation of a cell suspension, Used for collecting products, etc. Figure 1 (0)
As shown in the figure, hollow fibers 2 are uniformly dispersed in the container 1.
It is important that the hollow fiber bundle is substantially full and does not have excess void space. In other words, the hollow fiber dispersion gap 6
is uniformly distributed within the container 1, aiming at improving cell culture volumetric efficiency.

FIG. 2 is a schematic diagram showing cells being cultured in the hollow fiber dispersion gap 6 or on the surface of the hollow fiber 1. A indicates adherent cells and S indicates planktonic cells. However, A.

The relative sizes of S and 1 are of no significance in this figure.

In order for cells to grow normally, the above-mentioned mass balance is insufficient, and the distance between the cells and the support (hollow fiber surface), the distance between the fine saturated phases, the density of the cells, and even the intercellular The microenvironment of each cell, including the cooperative effects and information transmission, is essential9, and the microenvironment and its artificial endophyte (' Although many aspects of l are not clear, it is true that at least the hollow fiber surface, its vicinity, and the hollow fiber dispersion gap are one of the artificial conditions for the formation of a microenvironment.

Furthermore, it is preferable that the microenvironment is dynamic in terms of mass exchange and balance, and the hollow fiber network structure of the present invention allows microcurrents to flow near the cells. It can be made from a volumetric flow of medium. Furthermore, in the case of floating cells, the suspended state of the cells can be maintained by a minute flow.

If the cell culture vessel shown in FIG. 1 has excessive space, the cell culture volumetric efficiency will decrease, which is undesirable. In other words, cells in excess space may die due to insufficient material exchange, and there are disadvantages such as uneven flow of the exchange medium and reduced usage efficiency.

Aspects of the present invention will be explained using the drawings. FIG. 3 shows a cell culture device of the present invention in which the hollow fibers 1 are crimped and the effective length is longer than the distance between the partition walls. The hollow fibers may be crimped during spinning, for example, by setting the discharge speed of semi-dry, semi-wet spinning faster than the take-up speed during coagulation (helical yarn), or it may be applied mechanically later (plane wave yarn). . For example, if the crimp size is given by a sinusoidal curve, the period is preferably 10ffi or less, preferably 5 ffi or less. In order to obtain a finer structure, a period of 3 seconds or less is preferable. By bundling the crimped fibers, the entire hollow fiber bundle becomes bulky, making it possible to form the uniform dispersion of the present invention.

FIG. 4 shows a cell culture vessel of the present invention in which the hollow fibers 1 are at an angle to the longitudinal axis of the container and intersect with each other, so that the effective length is longer than the distance between the partition walls. The intersecting angle represented by the hollow fibers 1a and 1b is preferably 2 to 90 degrees, preferably 5 to 60 degrees. In order to obtain a more uniform network structure, the angle is preferably 10 to 40 degrees. Cross-overlapping of hollow fibers can be achieved by winding one to a desired number of hollow fibers while traversing them. The cross-superimposed layers can be bundled as they are, or they can be wound to obtain a desired cross-hollow fiber aggregate.

FIG. 5 shows a cell culture device of the present invention in which a plurality of hollow fibers are twisted and the effective length is longer than the distance between partition walls.

Twisting can be applied at the time of yarn spinning, and if necessary, loosening and filling can provide a uniformly distributed bundle.

FIG. 6 shows a cell culture vessel of the present invention in which hollow fibers are woven together and the effective length is better than the distance between partition walls. A woven fabric is made from the hollow fibers and assembled to obtain a hollow fiber woven bundle. .

In the embodiments of FIGS. 5 and 6, the twisting period and weaving period are preferably 1 (l mtn or less), and preferably 5M or less.

In order to obtain a finer structure, a period of 3a or less is preferable. The hollow fibers to be twisted or woven are formed by combining one to a plurality of hollow fibers, but if necessary, non-hollow fibers may be mixed. The mixed yarn contributes to bulkiness and uniform dispersion, and also serves as a turbulence element for forming microflows, and can also strengthen the yarn bundle.

The semipermeable or porous hollow fiber used in the present invention is preferably made of a polymeric material in which the membrane itself is not cytotoxic and is not subject to alteration or decomposition by sterilization or culture medium. Examples of the polymeric material include cellulose ester, acrylic polymer, polysulfone, polyethersulfone, and fluoropolymer. Hollow fiber dimensions: inner diameter 10-2000μ, membrane thickness 5-1000μ, preferably inner diameter 30-1000μ, membrane thickness 10-500μ, more preferably inner diameter 50-500μ. The film thickness is 15 to 200μ.

Hollow fibers are crimped, cross-overlapping, twisted, #1, etc.
It is better to have a certain degree of strength and flexibility. The number of condensed tubes varies depending on the size of the cell culture vessel and the area of the filled membrane, but can be appropriately selected within the range of 50 to 100,000.

100 to 10,000 is practical. The packing density of the hollow fibers is within the range of 20-65%, preferably 30-55%.
%, it is preferable that the filling is uniformly dispersed.

A cell culture vessel having the structure of the present invention is assembled by filling the container 2 with a bundle of hollow fibers, molding the 11ii wall by a known method, and opening at least one end of the hollow fibers to the outer surface of the partition wall. The shape, dimensions, and number of parts of the container may be designed as desired, and the end conduit 5 and side conduit 7 may take any form as long as they have a communication function. The example shown in FIG. 7 is an incubator in which crimped hollow fibers 1 are bundled in a U-shape and housed in a container 2 having an internal shell wall 8. 8 has a guide hole 9 and an internal culture chamber 1
0 is added. 2 is provided with conduits 11, 12 and stirring means 13. The example in Fig. 8 is the side conduit 7b.
is extended into the container 2 through the partition wall 3C to form a core tube 14, and the hollow fiber 1 is wound crosswise over the core tube 14 to form an incubator. The intersection angle of the hollow fibers is 10~170°, preferably 30~
150", more preferably 60-120°.

In the examples shown in FIGS. 7 and 8, culture medium exchange and circulation may be performed through the conduit 7a--gap 6--conduit 7b system.

The material for the container is selected based on mechanical strength, safety, and moldability, and may include uncast steel, glass, hard polymer, and the like. In the case of polymers, we use polycarbonate and polysulfone due to their transparency and heat resistance.

Poly4 methylpentene 1 and polypropylene are preferred.

The materials for the partition walls are moldable silicone rubber and epoxy resin, which require flexibility and sealing properties.

Urethane resin or the like can be used.

[Effects of the Invention] According to the hollow fiber dispersion-packed cell culture device of the present invention, animal cells, plant cells, microbial cells, etc. can be cultured continuously and at high density. The uniformly dispersed hollow fiber interstices provide a suitable microenvironment for, for example, animal adherent cells, allowing efficient culture. It also maintains suspension and provides a dynamic microenvironment for the animal's planktonic cells. These cells may be modified artificially or by genetic manipulation. By using one or more of the apparatuses of the present invention, it can be applied to large-scale culture.

The present invention will be explained below using Examples, but the present invention is not limited to these Examples.

Example 1 Hilulosatate hollow fibers with an inner diameter of 250 μm and a film thickness of 50 μm were mechanically crimped (period: 7 mm), and 2,500 fibers were bundled and packed into a polycarbonate cylindrical container (filling rate: 34%).
, mold the septum using urethane resin and assemble the cell culture vessel as shown in Figure 3. The molecular fraction m of the membrane is 50,000, the area n9 is 0.4, and the volume of the hollow fiber gap fi is 70II11. Using this cell culture vessel 14, the system shown in the flowchart shown in FIG. 9 is assembled. 15, 17, 10, and 23 are tubes, and 20 is an exchange medium tank. Oxygen is blown into the exchange medium 2) using pump 1.
8 circulated the medium through an in-line filter (0.22μ) 16 into the cell culture vessel 14. The fused cells (mouse hybridoma 4C108B cell line whose parent strain is mouse miniloose cell P3U1) were inoculated into the gap 6 using an aqueous system, and the medium supplemented with 5% bovine supernatant (RPM I 1640) was added.
), the cell density increased from 5 x 105 cells/〆 to 1 x 107 cells/-, and the cells could be cultured for 2 weeks.

In a stirred culture tank at 70°C without using this device, the growth remained at 2 x 106 cells/III.

Example 2 A cell culture vessel as shown in Fig. 4 was assembled by stacking and winding 3,000 polysulfone hollow fibers (fractionated molecular weight: 30,000) with an inner diameter of 300μ and a membrane thickness of 80μ crosswise at an angle of 30°.

This device was subjected to cell culture in the same manner as in Example 1.

Example 3 One 8-filament polyethersulfone hollow fiber (fraction 1i 10,000) with an inner diameter of 280μ and a film thickness of 80μ was spun, and two fibers were twisted (3 times/α) and bundled (3200 fibers). Assemble the cell culture vessel as shown in Figure 5.

This device was used for cell culture in the same manner as in Example 1.

Example 4 8 filaments of polyvinylidene fluoride hollow fibers (molecular weight cut off: 2,000,000) with an inner diameter of 280μ and a film thickness of 85μ
2000 threads are spun and woven with a mesh size of 4aw), No. 6
Assemble the cell culture vessel as shown in the figure. This device was subjected to cell culture in the same manner as in Example 1.

[Brief explanation of drawings]

Figure 1 shows the basic configuration of the present invention.1 (A) Longitudinal section and (0
) Figures 1 and 2, which are cross-sectional views (A-A plane), are schematic diagrams of hollow fibers and cells. 3 to 8 are longitudinal sectional views showing aspects of the present invention. FIG. 9 is a flowchart of cell i8 cultivation. 1... Hollow fiber, 2... Container, 3... Partition wall, 4...
・End chamber. 5... End conduit, 6... Hollow fiber dispersion gap. 7... Lateral duct, Δ... Adherent cell, B... Floating cell. 14...Cell culture vessel, 20...Exchange medium tank. 'JiI Ki (I) (B) 31st yA4 En No. 50 51!6 Kuchiro 7 Tato 8111 9gIJ Ding V Ren Neyu j 10 Me October 1985 9F1'

Claims (9)

[Claims] (1) In a cell culture device consisting of a plurality of semipermeable or porous hollow fibers, a container, and a partition wall that fixes both ends of the hollow fibers to both ends of the container, the effective length of the hollow fibers between the partition walls. is substantially longer than the distance between the partition walls, and the hollow fibers are evenly distributed and filled between the partition walls, and at least one end of the hollow fibers is open to the outer surface of the partition wall. and a hollow fiber dispersion-filled cell culture device, characterized in that cells are cultured in the hollow fiber dispersion gap or on the outer surface of the hollow fibers. (2) The container is provided with at least one end conduit communicating with the inside of the hollow fiber and at least one side conduit communicating with the hollow fiber dispersion gap. Cell culture vessel as described. (3) The cell culture device according to claim 1, wherein the container is substantially filled with the dispersed hollow fiber bundle and has no excess space. (4) The cell culture device according to claim 1, wherein the hollow fibers are crimped. (5) The cell culture device according to claim 1, wherein the hollow fibers are at an angle to the long axis of the container and intersect with each other. (6) The cell culture device according to claim 1, wherein the hollow fibers are twisted in plurality. (7) The cell culture device according to claim 1, wherein the hollow fibers are woven together. (8) The cell culture device according to claim 1, wherein the cells are floating cells. (9) The cell culture device according to claim 1, wherein the cells are adherent cells.