JPS63317076A - Bioreactor using hollow yarn - Google Patents

Abstract

PURPOSE:To improve rate of multiplication, metabolite production rate and separation efficiency of metabolite, by using hollow yarn having extremely larger hole diameter of inner surface than that of outer surface as hollow yarn to be packed in a reaction tube of a bioreactor. CONSTITUTION:A reaction tube 2 provided with a substrate solution feed pipe 9 and a substrate pipe discharge pipe 10 at the side wall and with headers 3 and 4 at both the ends is packed with a great number of porous hollow yarn 5 (e.g. porous ceramic) having different hole diameters of about 10-20mum pore diameter at the inner surface and about 0.1-0.2mum at the outer surface in parallel with the reaction tube 2 and both the end of the yarn is opened to the headers 3 and 4. A cell solution tank 8 is set between a cell solution discharge pipe 6 attached to the header 3 and a cell solution feed pipe 7 fixed to the header 4 and the cell solution is circulated by a pump 2. Simultaneously a substrate solution in the substrate solution tank 11 is circulated in the reaction tube 2 by a pump, cells are multiplied and a metabolite is produced.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention supplies cell fluid into hollow fibers whose inner pore diameter is extremely larger than the outer pore diameter, thereby increasing proliferation efficiency and reducing metabolic products. This article relates to a bioreactor using hollow fibers with different pore sizes to improve separation performance.

[Conventional technology]

Conventionally, in cell proliferation using a module using hollow fibers, cell fluid was supplied to the gap outside the hollow fibers. As the hollow fiber, a porous hollow fiber such as a polymer material such as polysulfone or polyvinylidene fluoride has been used, which has a pore size through which cells cannot pass through and a substrate solution can leached out.

[Problem that the invention seeks to solve]

However, the site where cells actually proliferate and metabolize is the hollow fiber wall surface that comes into contact with the substrate solution. Since the hollow fibers have a smaller pore size than the cells to prevent cells from passing through, the contact surface of the cells with the matrix solution was limited. Furthermore, when recovering cell metabolites, it is difficult to apply pressure to the outside of the hollow fiber, which reduces the efficiency of separating the metabolites.

[Means of problem solving]

The present invention aims to solve the above problem, and has a structure in which a substrate liquid supply pipe and a substrate liquid discharge pipe are provided on the side wall, and a large number of porous hollow fibers are placed parallel to the reaction tube in a reaction tube with both ends closed. A bioreactor using a module that is housed with both ends opened in a header provided at the end of the reaction tube and that leads the openings to a cell fluid supply pipe and a cell fluid discharge pipe, the pore diameter of the inner surface of the hollow fiber being is about 10 to 20 μm, and the outer surface has a pore diameter of about 0.1 to 0.2 μm.

The hollow fiber material according to the present invention is made of ceramics or polymeric materials such as polysulfone, polyvinylidene fluoride, etc. When ceramics are used, it can be sterilized at 121°C, IKg/cnl, and for 15 minutes, which is the standard for sterilization. It is a preferred material because it can withstand autoclave sterilization conditions and can be used repeatedly. As the ceramic, a sintered body of alumina, alumina ceramic, silica, etc. is used.

The pore size on the inner surface of the hollow fiber is preferably large in order to provide a place where cells can enter and absorb and metabolize the substrate solution exuding from the outside, but there is a limit in terms of strength. In reality, it is about 10 to 20 μm. The pore size on the outer surface of the hollow fiber must be such that the substrate solution can penetrate into the hollow fiber, but cells cannot pass through. Specifically, the pore diameter is approximately 0.1 to 0.2 μm, but it may be larger than this if the cells are large enough to prevent cells from passing through. The thickness of the hollow fibers varies depending on the type of cell, and in general, thinner fibers are used for microorganisms, and thicker ones are used for animal cells.

Moziere is made by adhering both ends of such hollow fibers together with adhesive, storing them in a reaction tube with both ends closed, parallel to the reaction tube, with both ends open to the lugs at the ends of the reaction tube. The openings are guided to the inlet and outlet collecting pipes of the hollow fiber feed liquid. The side wall of the reaction tube is provided with a supply port and a discharge port for a liquid to be supplied between the hollow fibers, in the present invention a substrate solution.

The cells used in the present invention can be preferably used as animal cells or microorganisms that perform extracellular secretion.

[Effect]

In the present invention, the pore diameter on the inner surface of the hollow fiber is extremely large and a gap is created that allows a large number of cells to enter. This increases the contact area between the liquid inside the hollow fiber and the liquid outside the hollow fiber, allowing cells to grow efficiently. can be done. In addition, by supplying the cell fluid into the hollow fibers, it becomes easy to pressurize the cell fluid, and the efficiency of separating metabolites from the cell fluid improves. Furthermore, if ceramic hollow fibers are used, they can withstand autoclave sterilization and can be used repeatedly.

〔Example〕

FIG. 1 is a schematic diagram showing the bioreactor of the present invention.

1 is a module in which headers 3.4 are provided at both ends of the reaction tube 2. 5 is a hollow fiber whose main component is alumina,
Inner diameter 0.5mm, outer diameter 1■, inner hole diameter 10-20μm1
The one with an outer pore diameter of 0.1 μm and a length of 90 mm was used. The hollow fibers are bonded together with adhesive in the header, and a cell fluid tank 8 is provided between the cell fluid discharge pipe 6 provided in the header 3 and the cell fluid supply pipe 7 provided in the header 4, and the cell fluid is circulated by a pump. Ta.

9 is a substrate liquid supply pipe provided on the side wall of the reaction tube, and a substrate liquid tank 1) is provided between it and the substrate liquid discharge pipe 10 provided on the side wall of the reaction tube.
A pump was installed to circulate the cell fluid.

The hollow fibers 5 are made by sintering a hollow fiber molded body with a pore size of 10 to 20 μm using a dry-wet method using ceramic raw material powder with a particle size of 10 to 20 μm, and the surface of the formed body is sintered with a pore size of 0.1 to 0.2 μm.
It was obtained by coating the surface so that

In this example, He1a 229 was used as the animal cell.
A solution in which cell lines (cervical cancer-derived cells) were suspended in MEM medium was used. As the substrate solution, MEM medium containing 10% FBS and various amino acids was used.

The relationship between cell number and culture days is shown in Figure 2. In addition, in order to measure the transmittance of the produced protein, 2Kg/co!
The relationship between the pressurization time and the amount of protein permeation was calculated as follows.
Shown in the figure. The amount of transmission is determined by the absorbance of the medium (0, D, 280
nm).

As a comparative example, an experiment was conducted in the same manner as in the example except that a hollow fiber having a pore diameter of 0.1 μm was used throughout, and the results are shown in FIGS. 2 and 3.

〔effect〕

The present invention improves cell proliferation rate, metabolite production rate, and metabolite separation efficiency. Furthermore, using ceramics for the hollow fibers improves durability and allows repeated use.

[Brief explanation of drawings]

The drawings show examples of the present invention; Fig. 1 is a schematic diagram showing a bioreactor, Fig. 2 is a graph showing the relationship between cell number and culture time, and Fig. 3 is a graph showing the relationship between metabolite permeation amount and pressurization time. It is a graph showing the relationship between. In the drawings, symbol ■ is a module, 2 is a reaction tube, 3.4 is a header, 5 is a hollow fiber, 6 is a cell liquid outlet, 7 is a cell liquid supply port, 8 is a cell liquid tank, 9 is a substrate liquid supply port, 10 is a substrate liquid outlet; 1
) is the substrate liquid tank. Patent applicant: NOK Co., Ltd. Agent Patent attorney: Yoshi 1) Toshio (1 other person) Transmission amount (0, D, 280 nm) Number of cells (cells/ml)

Claims (2)

[Claims] (1) A substrate liquid supply pipe and a substrate liquid discharge pipe are provided on the side wall, and a large number of porous hollow fibers are placed in a reaction tube that is closed at both ends, parallel to the reaction tube, and both ends are opened to a header provided at the end of the reaction tube. A bioreactor using a module that is housed in a hollow fiber and that leads the openings to a cell fluid supply pipe and a cell fluid discharge pipe, wherein the inner surface of the hollow fiber has a pore diameter of approximately 10 to 20 μm, and the outer surface has a pore diameter of approximately 0 μm. A bioreactor using hollow fibers with different pore sizes ranging from .1 to 0.2 μm. (2) A bioreactor using hollow fibers according to claim 1, wherein the hollow fibers are made of porous ceramics.