JPS6317685A - Device and method for cell culture - Google Patents

Abstract

PURPOSE:To achieve high growth performance in cell culture, by culturing a cell in a culture device consisting of a porous membrane having homogeneous pectinated network structure having pectinated rectangular shape encircled with micro-fibril and a knot part composed of stacked lamellas. CONSTITUTION:A porous membrane made of a hollow fiber having a wall thickness of 1-1,000mum and inner diameter of 0.01-10mm is produced from a polymer such as crystalline polyethylene, polypropylene, etc., by draw pore- forming process. Preferably, the core diameter of the membrane is 0.1-2.0mum and the porosity of the membrane is 50-80%. When the membrane is observed by a scanning electron microscope, the areal ratio of the knot part in the field is preferably 10-30%. The average length and diameter of the micro-fibril are controlled to 0.1-6.0mum and 0.02-0.3mum, respectively, A cell is placed outside of a hollow fiber partitioned with said porous membrane and a culture liquid is circulated through the hollow part from one opening to another opening to enable the supply of nutrients and oxygen or the removal of product or waste produced by the cultured cell.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Application Field) The present invention relates to a cell culture vessel comprising a porous membrane and a cell culture method using the porous membrane.

(Prior art and its problems) Since KNAZEK et al.'s report on a cell culture method using hollow fibers (Science, 178:65, 1972), attempts at cell culture using hollow fibers (hereinafter referred to as the hollow fiber method) have been made. Many cases have been reported.

The hollow system method can secure a large amount of liquid-permeable membrane surface, which is important for substance exchange in a limited volume, and which also serves as a substrate for adherent cells to grow. Therefore, nutrients and oxygen can be supplied and products and waste products can be removed extremely effectively. Therefore, it is expected to be a powerful method for producing useful substances by cell culture.

Known hollow fiber methods include examples of using hollow fibers such as polysulfone and cellulose diacetate.

Among these, polysulfone hollow fibers have superior performance as a substrate compared to cellulose-based hollow fibers, and are widely used. However, according to the research conducted by the present inventors, it was found that the initial adhesion of cells to the polysulfone hollow fibers was not satisfactory, and the cell proliferation rate was far inferior to that of polystyrene dishes. Ta.

According to the findings of the present inventors, (1) Polysulfone hollow fibers consist of a parenchymal portion and a hollow portion, and this partial structural difference causes non-uniformity in liquid permeation.

(The structure of the riborisulfone hollow fiber parenchyma is a sponge-like structure in which particulate polysulfone is connected, and the porosity is low, and the pores are not through holes, so the liquid permeability is low. A bypass occurs in the flow of the liquid, resulting in a partial culture medium supply shortage, and cells proliferate only locally, or as the number of cells increases, the partial culture medium supply shortage is promoted, Therefore, as a result, the cell growth rate becomes low.Furthermore, in general, even if the membrane is porous, the membrane surface is relatively flat, and if cells grow uniformly on the membrane surface, the membrane This blocks the pores on the surface, resulting in a similar situation where the supply of culture fluid is insufficient.

The inventors have obtained the knowledge that the membrane and pore wall structure are important for the performance of hollow fibers for cell culture, and based on this knowledge, they have identified a hollow fiber that can achieve a proliferation rate comparable to that of polystyrene dishes. It was discovered that it has a membrane wall structure.

(Means for solving the problem) According to the research of the inventors of the tree, the pore wall structure of hollow fibers for cell culture necessary to obtain a high proliferation rate is: ■Through holes with high porosity. (2) The pore wall structure is substantially homogeneous in the membrane wall, so that the liquid permeability is uniform on the membrane surface.

This prevents a partial shortage of culture medium supply and allows cells to proliferate uniformly. Furthermore, (3) the membrane surface has a network-like fine unevenness; this is because even if cells proliferate uniformly on the membrane surface, the pores on the membrane surface are not completely blocked; This is because you will not fall into a situation where there is a lack of supply of culture fluid, and
This is because the network-like fine irregularities on the membrane surface help the cells to be physically caught when cells are added, increasing the initial adhesion of the cells.

The present inventors have made the present invention as a result of intensive research to obtain a hollow fiber for cell culture having such a pore wall structure. That is, the present invention provides a cell culture device using a porous membrane having a homogeneous comb-like rectangular network structure surrounded by microfibrils extending in the longitudinal direction and nodules consisting of stacked lamellae. This is a cell culture method using the porous membrane. As a result, cells to be cultured on the porous membrane exhibit much higher proliferative properties than on conventionally known hollow fibers for cell culture.

(Structure 1 &) A porous membrane having a comb-like homogeneous network structure of a comb-like rectangle surrounded by microfibrils extending in the longitudinal direction and nodules consisting of stacked lamellae is, for example, a polyolefin or the like. It has an oriented structure obtained from crystalline polymers such as polysulfone, polyacrylonitrile, etc., and is different from the random sponge-like structure without directionality as a precipitated aggregate known from polysulfone and polyacrylonitrile. In addition to polyolefin, Teflon, polyacetal, polyphenyl sulfide, and the like are known as crystalline polymers forming the network structure.

This porous membrane has a film thickness of 1 to 110001L and an inner diameter of 0.01L.
It is a hollow fiber of ~10 mm, and for polyolefins, a hollow filament is formed by melt extrusion of a polymer of crystalline polyethylene or polypropylene, and by stretching it, fibril cleavage is generated in the hollow wall. It can be prepared by a method. These manufacturing methods are disclosed in JP-A-57-42919 or JP-A-57-66114.
It is already known as.

The porous membrane used in the present invention should have a pore size large enough to allow a sufficient flow of the culture medium, and should be suitable for cells (approximately several tens of μm).
m) should not leak, specifically 0.02 to 4.0
The final length is preferably 0°1 to 2.04 m. The porosity of the porous membrane is such that a homogeneous comb-like rectangular network structure surrounded by nodules consisting of crystalline microfibrils and stacked lamellae is maintained, and the porosity of the porous membrane is 1=1 in practical use. It is sufficient to have sufficient strength, specifically 30 to 90%
, More preferably, the thickness and inner diameter of the porous membrane should be 50 to 80%, so that a sufficient flow of the culture solution can be obtained.
There is no particular restriction as long as the size allows the cells to exist on the surface, but if the cells are fried, the film thickness and inner diameter will each be l
l-1O00JL, 0.01 to 10 mm can be used. According to the experience of the present inventors, hollow fibers having a membrane thickness and an inner diameter of 10 to 1004 mm and 0.1 to 1.0 mm, respectively, are particularly suitable for practical use.

When the network structure of the present porous membrane is observed using a scanning electron microscope, it is desirable that the area covered by the nodules in the image is 5 to 50%, more preferably 10 to 30%. When cells attach to this porous membrane,
It is thought that it mainly binds strongly to the nodules, and therefore, if the ratio of the area covered by the nodules is lower than 5%, the binding of cells to the present porous membrane will be weakened, affecting cell proliferation. or.

If the ratio is too high than 50%, it will affect the mass exchange capacity of the porous membrane. Similarly, when observed with a transmission electron microscope, the average length and thickness of microfibrils are 0.1 to 0.1, respectively, in terms of pore size or mass exchange ability.
The thickness is preferably 6.0 μm, preferably 0.02 to 0.3 μm.

To explain in detail the method of carrying out the present invention, the cell addition port is located in one space of a container which is provided with at least one of a cell addition port and a culture medium inlet and outflow port separated by the cell addition port and the porous membrane. Inject more cells.

A cell culture device for culturing cells by supplying nutrients and oxygen necessary for the growth of cultured cells or removing products and waste products produced by cultured cells by diffusion or forcibly through a membrane wall. It's a method. To give a more specific example, cells are placed on the outside of the hollow fiber separated by the porous membrane, and nutrients and oxygen are circulated inside the hollow fiber from one opening of the hollow fiber to the other. A cell culture device and method for supplying cells or removing products and waste products produced by cultured cells. Alternatively, there is a cell culture device and method for supplying nutrients and oxygen or removing products and wastes produced by cultured cells by forcibly creating a flow of the culture solution in the thickness direction of the hollow fiber membrane. Furthermore, even if the porous membrane is a flat membrane, by fixing the flat membrane so as to divide the inside of the container into two spaces, it can function in the same way as a hollow fiber, and therefore can be used in the same manner as above. Is possible.

Next, a more specific explanation will be given with reference to examples.

The various physical properties were determined as follows.

"Porosity (%)" It was determined using a mercury porosimeter.

"Average pore diameter (lm) J" A pore diameter that indicates a pore volume that is 1/2 of the total pore volume on a pore diameter-pore volume integral curve determined by a mercury porosimeter.

"Tensile strength at break (K g f / cm'), tensile elongation at break (%)" Measured with an Instron type tensile tester at a strain rate of 200 for 57 minutes.

"Percentage of nodule area (%)" Calculated by cutting out the nodule from a scanning electron wJ microphotograph of the outer surface (16 μm in height and 24 pm in width) and calculating the weight ratio with the original photograph.

"Average length and thickness of microfibrils" Scanning electron W
4 Using a microscopic image, photograph the cut surfaces at 3 points on the outer surface, inner surface, and approximately the center of the membrane thickness at a magnification of 10,000 times, and then measure the length of each section at 50 points. death,
The average value was calculated.

(Example 1) High density polyethylene (density 0.968. MI value 5.5,
(trade name HIZEX 2208J) was spun using a circular double spinneret at a spinneret temperature of 150°C, and the resulting hollow fiber was
After annealing at 0° C. for 2 hours, hot stretching was performed at room temperature for 30% and then at 105° C. for 350% to obtain a hollow fiber polyethylene membrane (porous polyethylene membrane). The polyethylene porous membrane has an inner diameter of 320 lm, a film thickness of 45 pm, a tensile strength at break of 485 K g f/c when dry, a tensile elongation at break of 32%, a knot area ratio of 21.3%, a micro Average length of fibrils: 1.7 pm, average thickness: 0.051
FIG. 1 shows a scanning electron microscope image of the 0-wire porous polyethylene rod that was Lm. FIG. 1 clearly shows the nodules, microfibrils, and pores that form the porous membrane.

The polyethylene porous membrane was covered with a glass cover glass (2
4 x 32 mm) in the long side direction, and apply silicone adhesive (
It was fixed with Silastic (manufactured by Dow Corning, USA). The fixed porous polyethylene membrane was soaked in 70% ethanol water for 1 hour to moisten and sterilize it, and then thoroughly washed with sterile double-distilled water. The polyethylene porous membrane was placed in a cell culture dish (Falcon 2003, Becton Dickinson, USA), 2 x 10 adherent HeLa cells were added, and penicillin 100 IU/ml and streptomycin were added. 50 bodies g/m1. kanamycin 50p,
g/ml Eagle M with 10% fetal bovine serum
The cells were cultured in EM culture solution at 37°C under 5% CO2 for 24 hours. After culturing, the cells were fixed with methanol, stained with Giemsa, and observed under a WJ microscope. This result was used as a test of initial cell adhesion.

The microscopic image obtained from this poem is shown in Figure 2. As is clear from Figure 2, HeLa was evenly spread and attached to the polyethylene porous membrane, and the initial adhesion of the cells was high. .

(Example 2) Cells were cultured using the polyethylene porous membrane obtained in Example 1 by the hollow fiber method. The circuit used for culture is
The 150 polyethylene porous membranes shown in the figure (2 in the figure)
) is open at both ends, and there are two cell injection/extraction ports (
A glass tube module (hereinafter referred to as cell culture vessel, 1 in the figure) for cell culture for hollow system method is fixed to a glass tube (φ8.4 x 94 mm) equipped with 3) in the figure using silicone as a botting material. It was created.

The cell culture vessel was sterilized with ethylene oxide gas, moistened with 70% ethanol, thoroughly washed with sterile double-distilled water, and then used for experiments. Separately, culture clear water (reservoir, 4 in the figure),
Connect the squeeze tube (6 in the figure) attached to the squeeze pump (5 in the figure) with the silicone tube (7 in the figure) to form a circuit. , sterilized in an autoclave for 20 minutes, and connected to the cell culture vessel. HeLa was added to the outside of the polyethylene porous membrane of this cell culture vessel, and culture was performed. Initial number of added cells: 5 x 10
Cells and culture medium contained 100 IU/ml of penicillin, 5014-g/ml of streptomycin, and 5-g/ml of kanamycin.
Eagle MEM medium containing 0.04 g/ml, 10% fetal bovine serum was used. Culture was carried out after addition of HeLa.
After standing still for a period of time, the flow rate was 1 ml/min.

After culturing for 3 days, the cell culture vessel was broken and the polyethylene porous membrane was taken out, stained with Giemsa in the same manner as in Example 1, and observed under a microscope. The results are shown in Figure 4. As is clear from Figure 4, HeLa was deposited on the polyethylene porous membrane.
are spreading uniformly and proliferating, and individual HeLa
The extensibility was also good. That is, the polyethylene porous membrane exhibited excellent cell adhesion and cell proliferation.

(Example 3) HeLa was cultured in a cell culture vessel in the same manner as in Example 2. After addition of HeLa, the flow rate was approximately 0.15 ml/min after standing for 3 hours, and the flow rate was increased to approximately 1 ml/min from the 4th day of culture. The culture solution was replaced as appropriate. Cells were collected using Dulbecco's PBS(-) (Dulbecco'5PBS).
After washing with trypsin solution (Dulbetzco's PBS)
(-) was treated with a solution containing 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid (EDTA).

As a comparative example, culture was performed using polysulfone hollow fibers. The polysulfone hollow system was prepared by defoaming a mixture of 10% by weight of aromatic polysulfone (Union Carbide Co., Ltd. YP-T700, USA), 70% dimethylacetamide (Q%), and 20% by weight of triethylene glycol to obtain a stock solution.

The stock solution was mixed at 3.3 ml/min using a circular double spinneret at 30 ml/min.
It was spun into a fluorocarbon liquid at °C (1.5 m vertically upward). At this time, water was introduced as a hollow forming agent at a rate of 3.4 ml/min. After passing through a water tank, it was wound up at a winding speed of 20 m/min, washed with water, and autoclaved at 125° C. for 1 hour.

The polysulfone hollow fiber thus obtained had an inner diameter of 360 mm.
pm, and the film thickness was 80 JLm. The polysulfone hollow fibers were incorporated into a glass tube module in the same manner as the polyethylene porous membrane (voresulfone module).
This polysulfone module was used as a comparative example and cultured using the same method as above. However, the flow rate was determined from preliminary experiments at an optimal flow rate of 5 m.
The growth curve of A is shown in FIG. 5 as curve A and white line B.

Judgment of life and death was carried out using trypan blue. In the cell culture vessel, HeLa proliferated logarithmically after the start of culture, and after 12 days of culture.
On the second day, the number of viable cells reached approximately 4.4 x 10, whereas in the comparative example it was only 8.3 x 10', indicating a clearly high proliferation rate in this cell culture device.

(Effects of the Invention) The network structure of the present porous membrane exhibits excellent performance in initial cell adhesion, cell spreadability, and cell proliferation. Furthermore, this porous membrane has high strength and is much more practical than polysulfone membranes. Therefore, a cell culture device made of the present porous membrane having a network structure and a cell culture method using the cell culture device exhibit far superior cell culture performance than conventional methods.

[Brief explanation of the drawing]

FIG. 1 is a scanning electron microscope image showing the shape of ia m of a porous polyethylene membrane. Figure 2 shows H in Example 1.
It is a micrograph showing the morphology of an organism when eLa cells are cultured. FIG. 3 shows an example of a culture system using a hollow fiber method using a porous polyethylene membrane. FIG. 4 is a micrograph showing the morphology of the organism when HeLa was cultured in Example 2. FIG. 5 shows a growth curve (A in the figure) when HeLa was cultured in Example 3, and a growth curve (B in the figure) of a comparative example. 1. Cell culture vessel 5. Shigoki bomb 2, polyethylene porous membrane 6. Shigogi tube 3, cell injection/
Outlet 7. Silicone tube 4, culture solution reservoir

Claims (7)

[Claims] (1) A cell culture vessel having a porous membrane having a homogeneous comb-like rectangular network structure surrounded by microfibrils extending in the longitudinal direction and nodules consisting of stacked lamellae. (2) The pore diameter and porosity of the porous membrane measured with a mercury porosimeter are 0.02 to 4.0 μm and 30 μm, respectively.
90%. The cell culture device according to claim 1. (3) The cell culture device according to claim 1 or 2, wherein the porous membrane is made of polyolefin. (4) A cell culture method using a porous membrane having a membrane wall consisting of a comb-like homogeneous network structure of a comb-like rectangular shape surrounded by microfibrils extending in the longitudinal direction and nodules consisting of stacked lamellae. . (5) Supplying nutrients and oxygen for cultured cells from one space separated by a porous membrane to the other space separated by the porous membrane where cultured cells exist, or cultured cells 5. The cell culture method according to claim 4, wherein the products or waste products are removed from the cultured cell side space to the other space through a membrane wall. (6) The pore diameter and porosity of the porous membrane measured with a mercury porosimeter are 0.02 to 4.0 μm and 30 μm, respectively.
90% of the cell culture method according to claim 4 or 5. (7) The cell culture method according to any one of claims 4 to 6, wherein the porous membrane is made of polyolefin.