JPWO2017104558A1 - Hollow fiber membrane and hollow fiber module for cell culture - Google Patents

Abstract

An object of the present invention is to provide a hollow fiber membrane that does not require coating of a cell adhesion factor and can adhere and culture cells, and a cell culture method using the hollow fiber membrane.
A hollow fiber membrane for seeding and culturing cells on an inner surface and / or an outer surface, wherein the hollow fiber membrane is composed of only a hydrophobic polymer. A hollow fiber module storing a yarn membrane and a hollow fiber bundle obtained by bundling a plurality of hollow fiber membranes, and a cell culture method using the hollow fiber module as a cell culture container.
[Selection figure] None

Description

  The present invention relates to a hollow fiber membrane and a hollow fiber module used as a culture substrate for culturing cells.

  Stem cells are cells that can form organs and tissues, and are considered to be present in most organs and tissues even in adults. Among the stem cells, embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are all-purpose cells and have the ability to differentiate into all tissues and organs. On the other hand, somatic stem cells cannot differentiate into all organs and tissues, but differentiate into specific tissues and organs. Somatic stem cells that can be collected from human tissues can be collected from patients themselves, and since there is no fear of rejection, they have been attracting attention as cells for transplantation used in cell transplantation therapy.

  Somatic stem cells in humans are known to date such as mesenchymal stem cells, hematopoietic stem cells, neural stem cells, cardiac muscle stem cells, pancreatic stem cells, skin stem cells, bone marrow stem cells, retinal stem cells, corneal endothelial stem cells, and the like. However, there are very few somatic stem cells in the tissue.

  Therefore, cell transplantation treatment research that advances somatic stem cells obtained from living tissue in vitro, amplifies the number of cells necessary for treatment, and uses it for the treatment of the same person or others, has been put to practical use. Being started.

  However, in general cell culture work, there are high risks of biological contamination and high costs due to labor costs, so it is safe and low-cost for further development of cell transplantation therapy. To culture stem cells.

  By the way, cells that are adhesive in vivo cannot grow or survive unless they adhere to the culture substrate even in vitro. For this reason, in order to culture an adherent stem cell, the presence of a cell adhesion factor such as collagen or fibronectin that promotes adhesion to a culture substrate is required.

  Patent Document 1 discloses a cell culture system using a hollow fiber membrane. Two types of hollow fiber membranes are shown as substrates for seeding cells, Desmopan® (0.5% thermoplastic polyurethane) and Polyflux (trade name) (polyamide + polyarylethersulfone + polyvinylpyrrolidone) The mixture) has been tested. Although these membrane characteristics are unknown, the polyflux is a hydrophilic membrane because it is a mixture of polyvinyl pyrrolidone. Patent Document 2 discloses a cell culture system using a hollow fiber membrane composed of 65-95% hydrophobic polymer and 5-35% hydrophilic polymer, and uses the polyflux described above. An example is shown. These documents indicate that efficient expansion culture can be carried out with a hollow fiber membrane type cell culture device. However, according to this document, it is shown that the desired culture can be performed by performing a surface treatment such as platelet lysate, plasma, and fibronectin prior to the use of the membrane. It has been shown that in cell culture, it is necessary to coat a substrate with some cell adhesion factor in advance. Patent Document 3 discloses a technique using a hollow fiber membrane in suspension cell culture. This document shows that gas supply and medium exchange are performed by using a hydrophobic hollow fiber membrane, but the hollow fiber membrane is not used as a substrate for cell culture. Patent Document 4 discloses a gamma ray or a beta ray of 12.5 to 175 kGy in a membrane containing polysulfone, polyethersulfone or polyarylethersulfone, and polyvinylpyrrolidone in the presence of oxygen at a concentration of 4 to 100 vol%. Alternatively, a technique is disclosed in which adherent cells can be cultured without any pretreatment with a surface modifying substance by irradiation with an electron beam.

However, coating such a cell adhesion factor onto a culture substrate results in increased material costs and work costs for the adhesion factor. Furthermore, when the cell adhesion factor is an animal-derived component, a new risk of infection is newly created, in addition to the problem of such cost increase. In recent years, the risk of infection has been reduced by using cell adhesion factors produced by genetically modified E. coli, but in such production, each of the medium components used in the cultivation process of recombinant E. coli In some cases, complicated work such as origin tracking is required.
Therefore, in order to solve these problems, it is necessary to provide a culture substrate capable of adhering cells and culturing and growing them without requiring a coating of a cell adhesion factor.

Special table 2009-540865 Special table 2010-523118 JP 2014-117190 A Special table 2012-503688 gazette

  The present invention provides a hollow fiber membrane capable of cell adhesion and culture, and a cell culture method using the hollow fiber membrane, which does not require a pretreatment such as a cell adhesion factor coating treatment or surface modification with an electron beam or the like. This is the issue.

  As a result of intensive studies to solve the above problems, the present inventor has found that the above problems can be solved by the following means, and has reached the present invention.

That is, this invention consists of the following structures.
1. A hollow fiber membrane for culturing cells by seeding and culturing cells on the inner surface and / or outer surface, wherein the hollow fiber membrane is composed only of a hydrophobic polymer.
2. 2. The hollow fiber membrane according to 1, wherein the hydrophobic polymer is a polymer having a contact angle of 70 degrees or more and 93 degrees or less.
3. The hollow fiber membrane according to 1 or 2, wherein the hydrophobic polymer is one or more selected from the group consisting of polyethersulfone, polysulfone, polyvinylidene fluoride, and polyethylene.
4). A hollow fiber module for cell culture in which a hollow fiber bundle obtained by bundling a plurality of hollow fiber membranes according to any one of 1 to 3 is stored.
5. A cell culture method using the hollow fiber module according to 4 as a cell culture container.
6). A cell culture apparatus comprising the hollow fiber module according to 4 as a cell culture container.

  According to the present invention, it is possible to provide a hollow fiber membrane as a culture substrate that does not require a cell adhesion factor coating treatment. Further, by using the hollow fiber membrane of the present invention, it is possible to construct and provide a cell culture system that can culture various cells such as mesenchymal stem cells safely, simply and with high efficiency.

It is a schematic diagram which shows an example of the hollow fiber module of this invention. It is a schematic diagram which shows an example of the cell culture apparatus of this invention.

  In the present invention, “hydrophobic” means difficulty in conforming to water (in the case of “hydrophilic”, facilitating conformity to water). For example, a contact angle may be mentioned as an example of an index that objectively indicates the hydrophobicity and hydrophilicity of a polymer and water. The contact angle is defined as the angle formed by the tangent line and the solid surface when the tangent line is drawn on the curved surface of the liquid from the contact area of the solid, liquid and gas of the water droplet placed on the surface of the polymer sheet, film or membrane. For example, it is a value measured based on the sessile drop method of JIS R3257 (1999). The smaller the contact angle, the higher the hydrophilicity, and the larger the contact angle, the higher the hydrophobicity.

(Hollow fiber membrane)
In the present invention, the polymer constituting the hollow fiber membrane is not particularly limited as long as it can hold cells on the surface of the hollow fiber membrane and can take a structure that allows a solution or a low-molecular substance to permeate. , Polytetrafluoroethylene (contact angle: 104 degrees), polyurethane (same: 96 degrees), polyethersulfone (same: 85-90 degrees), polyvinylidene fluoride (same: 82 degrees), polysulfone (same: 77 degrees) , Polyethylene (73:88 degrees), cellulose triacetate (67 degrees), ethylene vinyl alcohol copolymer (55:65 degrees), and derivatives thereof. However, if the hydrophobicity is too strong, the cell adhesion is good, but there is a problem that the wettability with respect to a liquid medium (hereinafter sometimes simply referred to as a medium) is lowered. On the other hand, if the hydrophilicity is too strong, the wettability to the medium is good, but there is a problem that the cell adhesiveness is lowered. Therefore, in the present invention, it is preferable to select a polymer having a contact angle of generally 70 degrees or more and 93 degrees or less in order to achieve both cell adhesion and wettability to a medium. When the medium comes into contact with the surface of the hollow fiber membrane produced using such a polymer, fibronectin and other proteins are adsorbed in a short time. Thereby, since a cell can adhere | attach to a hollow fiber membrane surface rapidly, it can be made to transfer to proliferation after that.

  In the present invention, the hollow fiber membrane is preferably composed only of the hydrophobic polymer, but as other components, a low molecular component such as glycerin for the purpose of imparting water wettability, a solvent used for film formation, Residual components of the solvent, low molecular additives such as crosslinking inhibitors and antioxidants may be included.

  In the present invention, the size of the hollow fiber membrane is not particularly limited, but the inner diameter is preferably 100 to 1000 μm, more preferably about 150 to 500 μm. The film thickness is not particularly limited, but may be set in a range in which the hollow fiber membrane maintains an appropriate strength and does not have a significant problem with the permeability of the substance, and is preferably about 10 to 150 μm, for example. When seeding cells inside the hollow fiber membrane and culturing to perfuse the medium, the inner diameter of the hollow fiber membrane is not only a two-dimensional effect, but also the seeding of cells, the area to proliferate, the cell density, Advantages of hollow fiber culture because it is a matter related to design and operating conditions that affect not only compactness, which is a three-dimensional effect, but also the contact volume with the medium, the flow of the medium, the linear velocity, and the shear force. It becomes a requirement to make use of.

  In the present invention, the pore diameter of the hollow fiber membrane is not particularly limited as long as it is a pore that does not allow cells to pass through but allows medium components such as water, salts, and proteins to pass through. It is desirable to have a relatively large pore size with good exchange efficiency, and the average pore size is preferably 0.001 to 0.5 μm, more preferably about 0.01 to 0.1 μm. The molecular weight cut off (molecular weight at which the sieve coefficient is less than 0.1) is preferably 1 to 1,000,000, and more preferably about 2 to 200,000. Furthermore, the pore size of the membrane is also affected by the adsorption and clogging of various biological components that accompany the culture. That is, the optimum design should be implemented in view of the interaction with these materials.

In the present invention, the water permeability of the hollow fiber membrane is not particularly limited, but is preferably 10 to 1000 mL / m ² / hr / mmHg, more preferably 20 to 500 mL / m ² / hr / mmHg. If the water permeability is too small, sufficient material movement cannot be expressed. Also, if the water permeability is too large, a pressure difference between the membranes will occur when a medium or the like is passed through the inner and outer cavities of the hollow fiber membrane, and filtration may occur on its own, or the length of the hollow fiber membrane This is not preferable because a flow (medium component) distribution (concentration difference) can be generated.

  As described above, the hollow fiber membrane of the present invention rapidly adsorbs the protein contained in the medium by contacting the medium, but this impairs the water permeability and substance permeability of the membrane during cell culture. That is not preferable. The hollow fiber membrane of the present invention efficiently exchanges substances such as supply of nutrients and gas and removal of waste products to cells in culture together with the adhesion of proteins that promote cell adhesion. It is possible to implement.

  In the present invention, it is preferable that the hollow fiber membrane does not pass a protein having a large molecular weight (about 400,000) such as fibronectin. Cells adhere to the membrane using the protein attached to the membrane, and a sufficient extracellular matrix is formed with the addition of the protein produced by the cell itself, enabling the formation and maintenance of a favorable cell growth environment suitable for culture. It becomes.

  In the present invention, the merit of using a hollow fiber membrane as a culture substrate is that material exchange is possible through a membrane that cannot be realized by ordinary petri dish or flask culture, and supply (concentration adjustment) of nutrients, oxygen, and carbon dioxide and waste An object can be efficiently removed. However, at the same time, there is a concern that necessary components may be excessively removed. Therefore, by controlling the pore diameter optimally, the culture environment in the vicinity of the cells can be improved and the culture efficiency can be improved. However, the pore size (substance permeability) is very susceptible to the adsorption of proteins in the medium and cell secreted proteins (mainly fibronectin that forms a cell-adhesive extracellular matrix). Therefore, even under these influences, it is necessary that a sufficient mass exchange be performed. In the prior art, in order to make up for this point, the membrane has been preferably hydrophilized. However, in order to improve the adhesiveness of the cells, a treatment for coating the substrate with a highly purified cell adhesion factor for a long time (1 night to 1 day) was required. In the present invention, by using a hollow fiber membrane made of a polymer having a contact angle in a specific range, the protein quickly adheres to the membrane surface, and the adhesiveness of cells is improved.

(Hollow fiber module)
In the present invention, for example, a hollow fiber module that is a cell culture container can be produced by storing several tens to several tens of thousands of hollow fiber bundles in a cylindrical container. Depending on the size of the hollow fiber membrane, such a hollow fiber module can take a large culture area per unit volume and can simplify the culture operation. High cell culture.

  Although the structure of the module using such a hollow fiber membrane is not specifically limited, For example, as shown in FIG. 1, the hollow fiber membrane 4 is provided in the module case 3 having four openings (end conduit and side conduit). Can be bundled and filled as necessary. Of the four openings, two end conduits 1a or 1b are formed in the hollow fiber membrane in a state in which the lumen (hollow part) and the outer cavity of each hollow fiber membrane are separated at both ends of the hollow fiber bundle. Adhering and fixing to the end of the module case with an appropriate sealing material (for example, polyurethane-based potting agent) so as not to block the hollow portion, the liquid introduced from one of the end conduits 1a or 1b is a hollow fiber membrane. It is configured to be led out from one end conduit 1b or 1a (that is, to flow in one direction) through the hollow portion. On the other hand, of the openings, the remaining two side conduits 2a or 2b are inside the module case 3 and outside the hollow fiber bundle (hereinafter also simply referred to as “external cavity”). ) And the liquid introduced from one of the side conduits 2a or 2b is led out from the other side conduit 2b or 2a that passes through the outer lumen of the module (ie, flows in one direction). ) Is configured as follows.

  In the present invention, when the hollow fiber module is used as a cell culture container, the cell culture may be performed in either the lumen or the outer lumen of the hollow fiber membrane, but the culture is preferably performed in the lumen. For example, when culturing cells in the lumen, cells are seeded on the inner surface of the hollow fiber membrane by injecting a cell suspension from the end conduit and filling the lumen, and after seeding, The suspension is perfused by switching to a culture medium and cultured for a certain period of time. During this time, it is preferable that the medium is injected into the outer space from the side conduit and perfused at the same time.

  The liquid medium has a role of supplying nutrients necessary for the cells and gases such as oxygen and carbon dioxide. During the culture period, it is preferable to continue supplying the medium in one direction in both the inner and outer lumens for gas exchange, nutrient supply to cells, and removal of waste products. At that time, by using a pump or the like, it can be circulated at an appropriate speed, supplied or discharged.

  The hollow fiber module is sterilized by an appropriate method and supplied to the market. The sterilization method is not particularly limited, and examples thereof include high-pressure steam sterilization, electron beam sterilization, radiation sterilization, and ethylene oxide gas sterilization.

  In the present invention, by using a hollow fiber module as a culture container, it is possible to always supply a fresh medium to the cells, so that the medium replacement work required when using a petri dish or a multistage flask as a culture container is not required. , The operator's restraint time and contamination risk can be reduced.

(Cells to be cultured)
In the present invention, the cells to be cultured are not particularly limited, but adhesive animal cells are preferable. The origin of the cells is not particularly limited, and those derived from any animal such as humans, pigs, dogs and mice can be used. Adhesive animal cells can target both primary cultured cells and established cells. In addition, primary cells such as epidermal keratinocytes, vascular endothelial cells, fibroblasts and hepatocytes, stem cells such as embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, adipose precursor cells, hepatic stem cells, progenitors It may be a cell. In addition, these cells may be cells into which a foreign gene has been introduced before culturing, or may be cells that have been previously stimulated and processed with stimulating factors such as antibodies and ligands.

(Cell culture device)
FIG. 2 shows one configuration example of the cell culture device of the present invention. In FIG. 2, 5 and 6 are liquid medium storage containers (culture bags etc.), respectively. A circuit is connected from the liquid medium storage container 5 to the end conduit (1a in FIG. 1) of the cell culture container 10 (hollow fiber module) via the aseptic connection connector 41 and the valve 21, and the liquid medium storage container 5 The contained medium can be transferred to the lumen of the cell culture container 10. On the other hand, a circuit is connected from the liquid culture medium storage container 6 to the side conduit (2a in FIG. 1) of the cell culture container 10 via the aseptic connection connector 42 and the valve 23, and enters the liquid culture medium storage container 6. The culture medium can be transferred to the outer space of the cell culture container 10.

  A circuit is connected from the end conduit (1b in FIG. 1) of the cell culture container 10 to the waste liquid collection container (or cell collection container) 7 via the pump 31, and the medium passing through the cell culture container 10 is discarded. It can be done. The pump 31 provided in the circuit supplies liquid (such as a medium) from the liquid medium storage container 5 to the cell culture container 10, discharges the liquid from the cell culture container 10, and discards the liquid. It is possible to control the flow rate and the like. On the other hand, a circuit is connected from the side conduit (2b in FIG. 1) of the cell culture container 10 to the waste liquid collection container 7 via the pump 30, so that the medium that has passed through the cell culture container 10 can be discarded. ing. The pump 30 provided in the circuit supplies liquid (such as a medium) from the liquid medium storage container 6 to the cell culture container 10, discharges the liquid from the cell culture container 10, and discards the liquid. It is possible to control the flow rate and the like.

In FIG. 2, at least the liquid medium storage containers 5 and 6 and the cell culture container 10 are preferably accommodated in a CO ₂ incubator 50. In addition, a work controller (work panel) 60 may be added to perform the above operations and process control and monitoring.

(Cell culture)
When the hollow fiber membrane of the present invention is used, for example, when cells are cultured in the lumen of the hollow fiber membrane, the above-described hollow fiber module is used, and the liquid in which the cells are suspended from one of the end conduits is contained in the hollow fiber membrane. Cells can be seeded by flowing into the cavity. After allowing to stand for a certain period of time and allowing the cells to adhere to the surface of the hollow fiber membrane, the cells can be cultured and proliferated by continuously or intermittently feeding the medium to the hollow fiber module installed in the incubator. The medium has a role of supplying nutrients necessary for the cells and gases such as oxygen and carbon dioxide. In addition, the medium may be determined according to the type of the cultured cell, and may be any medium that is normally used as the medium for the cell. The culture method of the present invention can also be used for serum-free culture.

  In addition, when supplying the culture medium, separate liquid culture medium storage containers may be used for the lumen and the outer cavity, and the medium may be distributed and supplied from one container to both the lumen and the outer cavity. . In this case, the medium composition of the inner cavity and the medium composition of the outer cavity may be the same or different. The medium flow rate in the lumen and the medium flow rate in the outer lumen may be the same or different, but it is more preferable to increase the medium flow rate in the outer lumen.

  The flow rate of the medium during cell culture is not particularly limited, but it is preferable to appropriately adjust the flow rate according to cell growth. In particular, in the initial stage of culture until entering the logarithmic growth phase, it is necessary to strictly control the flow rate in order to supply nutrients and maintain the microenvironment around the cells. That is, if the flow rate is too slow, nutrient supply to the cells is not sufficient, and the cells are difficult to grow. Conversely, if the flow rate is too fast, the environmental changes around the cells will increase. The flow rate is preferably adjusted based on monitoring changes in glucose and lactic acid concentrations in the medium.

  In the present invention, the medium flow in the hollow fiber module is preferably unidirectional. Specifically, a form in which the culture medium is always introduced from one end conduit and flows toward the opposite conduit (outlet) can be mentioned. A so-called circulation system in which the medium derived from the hollow fiber module is once again introduced from the introduction port may be used. In addition, at least one of the inner and outer cavities of the hollow fiber membrane may be circulating, or both may be circulating.

  In the present invention, the hollow fiber module may be rotated, shaken, rolled, or the like after cell seeding and / or during cell culture. Such an operation can be performed, for example, by uniformly dispersing and adhering the cells seeded in the lumen of the hollow fiber membrane to the surface of the hollow fiber membrane, or by removing easily when bubbles are generated, It is preferable in terms of spreading the nutrients of the medium evenly.

(Recovery of cells)
In the present invention, the means for recovering cells cultured using the hollow fiber membrane is not particularly limited. For example, when cells are cultured in the lumen of the hollow fiber membrane, the perfusion of the medium is stopped, and then the medium existing in the lumen and the outer cavity of the hollow fiber membrane is removed. Phosphate buffered saline (PBS) is perfused for a certain period of time, and the medium is thoroughly replaced with PBS. Next, PBS is removed, and a protease solution such as trypsin is filled into the inner and outer cavities of the hollow fiber membrane and incubated for a certain period of time. By such treatment, the cultured cells are detached from the hollow fiber membrane, and then the medium is flowed into the lumen of the hollow fiber membrane to flow out of the hollow fiber membrane, thereby collecting the cells.

  Hereinafter, the effectiveness of the present invention will be described with reference to examples, but the present invention is not limited thereto. In addition, regarding manufacture of the hollow fiber membrane in an Example, the typical manufacturing method is described below. The hollow fiber membrane in the present invention is produced by a non-solvent induced phase separation method or a thermally induced phase separation method. Film formation by non-solvent-induced phase separation is performed by preparing a spinning stock solution in which a polymer is dissolved in a solvent and an internal coagulating liquid mainly composed of water, and spinning from the central coagulating liquid from the center port of the cylindrical double tube nozzle and from the outer slit. After discharging the stock solution and passing through the air gap part, it is immersed in an external coagulation liquid, causing film formation by phase separation and desolvation, then washing, applying glycerin etc. if necessary, and then drying. is there. In addition, the film formation by the heat-induced phase separation method produces a spinning stock solution in which a polymer and a latent solvent are melted at a high temperature, and a cylindrical double tube nozzle together with an internal solution (or gas) for forming a hollow portion. The internal solution is discharged from the center port, and the spinning dope is discharged from the outer slit, and after film formation by phase separation by rapid cooling in the air gap part, it is washed and, if necessary, glycerin is applied and then dried. . Glycerin does not affect the hydrophobicity or hydrophilicity of the hollow fiber membrane itself. By filling the pores of the hollow fiber membrane with glycerin, pore deformation due to drying can be suppressed and culture can be performed. It functions as a material for introducing water into the pores for easy replacement with water or a medium immediately before use. In the case of a hydrophobic membrane, it may be difficult to easily replace the inside of the pores with water due to surface tension only by contact with water, so it is necessary to perform some water replacement treatment. . An operation such as substitution from water by alcohol treatment can also be employed.

(Measurement of inner diameter and film thickness)
The hollow fiber membrane has an inner diameter, an outer diameter, and a film thickness. The hollow fiber membrane is cut through a razor on the top and bottom surfaces of the slide glass so that the hollow fiber membrane does not fall out into a hole of φ3 mm in the center of the slide glass. And after obtaining the hollow fiber membrane cross-section sample, it is obtained by measuring the minor axis and major axis of the hollow fiber membrane cross section using a projector Nikon-V-12A. For the hollow fiber membrane close to a perfect circle, the minor axis and the major axis in two directions were measured, and the arithmetic average values thereof were defined as the inner diameter and outer diameter of one cross section of the hollow fiber membrane. The film thickness was calculated by (outer diameter−inner diameter) / 2. Measurements were similarly performed on five cross sections including the minimum and maximum, and the average values were defined as the inner diameter, outer diameter, and film thickness.

[Example 1]
(Production of hollow fiber membrane)
Polyethersulfone (4800P, manufactured by Sumitomo Chemical Co., Ltd.) 20% by mass, N methylpyrrolidone (Mitsubishi Chemical Co., Ltd.) 36% by mass, and triethylene glycol (Mitsui Chemicals Co., Ltd.) 44% by mass are uniformly dissolved. Produced. The film-forming stock solution is discharged from the outer slit of a cylindrical double tube nozzle heated to 70 ° C., and at the same time, 13.5% by mass of N-methylpyrrolidone, 16.5% by mass of triethylene glycol, 70 of water from the center hole of the nozzle. A hollow forming agent (internal coagulation liquid) consisting of mass% was discharged, passed through a dry portion of 300 mm, immersed in 75 ° C. water and solidified, washed sufficiently with water, and then wound into a bundle. After cutting, the yarn bundle was immersed in a 50% glycerin aqueous solution at 50 ° C., and then the excess glycerin aqueous solution was removed, followed by ventilation drying at 60 ° C. The resulting hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 300 μm, and a film thickness of 50 μm.

(Production of hollow fiber module 1)
The test hollow fiber module 1 was produced as follows. After inserting 100 obtained polyethersulfone hollow fiber membranes into a cylindrical polycarbonate module case with a diameter of 1 cm and a length of 10 cm, a polyurethane potting agent is used so as not to block the hollow portion of the hollow fiber membrane. Both ends were fixed to a module case to produce a hollow fiber module having a shape as shown in FIG.

[Example 2]
(Production of hollow fiber membrane)
Polysulfone (Amoco P-3500) 30% by mass, N, N-dimethylacetamide 45% by mass, and ethylene glycol 25% by mass were uniformly dissolved to prepare a membrane forming stock solution. The film-forming stock solution is discharged from an outer slit of a cylindrical double tube nozzle heated to 110 ° C., and 51.4% by mass of N, N-dimethylacetamide, 28.6% by mass of ethylene glycol, water from the center hole of the nozzle A hollow forming agent composed of 20% by mass was discharged, passed through a 300 mm dry part, immersed in water at 50 ° C. to be solidified, sufficiently washed with water, and wound around a bundle. After cutting, the yarn bundle was immersed in a 50% glycerin aqueous solution at 50 ° C., and then excess glycerin solution was removed, followed by ventilation drying at 60 ° C. The resulting hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 300 μm, and a film thickness of 50 μm.

(Production of hollow fiber module 2)
Using the obtained hollow fiber membrane (bundle), a test hollow fiber module 2 was produced in the same manner as in Example 1.

[Example 3]
(Production of hollow fiber membrane)
Polyvinylidene fluoride (SOLVAY: SOLEF6020 (registered trademark)) 20% by mass and diethylene glycol monoethyl ether acetate 80% by mass were uniformly dissolved at 148 ° C. to prepare a film forming stock solution. It is discharged from the outer slit of a cylindrical double tube nozzle heated to 148 ° C., triethylene glycol is discharged as a hollow forming agent from the central hole of the nozzle, and after passing through a 20 mm dry section, 10 ° C. diethylene glycol monoethyl ether It was immersed in a cooling bath comprising a 60% by mass aqueous solution of acetate and solidified, and after sufficient water washing, it was wound up into a bundle. After cutting, the yarn bundle was immersed in a 50% glycerin aqueous solution at 50 ° C., and then excess glycerin solution was removed, followed by ventilation drying at 60 ° C. The obtained hollow fiber membrane had an inner diameter of 250 μm, an outer diameter of 350 μm, and a film thickness of 50 μm.

(Production of hollow fiber module 3)
Using the obtained hollow fiber membrane (bundle), a test hollow fiber module 3 was produced in the same manner as in Example 1.

[Comparative Example 1]
(Production of hollow fiber membrane)
Polyethersulfone (PES) 20% by mass, polyvinylpyrrolidone (PVP, Luvitec (trade name) K90PH manufactured by BASF) 3.5% by mass, N, N-dimethylacetamide 72% by mass, pure water 4.5% by mass 50 It melt | dissolved uniformly at ° C and produced the film forming stock solution. The film-forming stock solution is discharged from an outer slit of a cylindrical double tube nozzle heated to 75 ° C., and an aqueous solution of 55% by mass of N, N-dimethylacetamide is discharged as a hollow forming agent from the center hole of the nozzle. After passing through the dry section, it was immersed and solidified in a 25% by weight aqueous solution of N, N-dimethylacetamide at 60 ° C., sufficiently washed with water, and wound up into a bundle. The yarn bundle was cut, and after excess water was removed, it was dried by ventilation at 60 ° C. The resulting hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 280 μm, and a film thickness of 40 μm. The film thus obtained was spontaneously wetted by water even without glycerin or alcohol treatment due to the inclusion of PVP.

It was 24.4% when the content rate of PVP of the inner surface of the produced PVP containing PES hollow fiber membrane was measured by X-ray photoelectron spectroscopy (ESCA).
In the measurement, one hollow fiber membrane was obliquely cut with a razor so that a part of the inner surface was exposed, and was attached to a sample stage so that the inner surface could be measured.
The measurement conditions are as follows.
Measuring device: ULVAC-Phi ESCA5800
Excitation X-ray: MgKα ray X-ray output: 14 kV, 25 mA
Photoelectron escape angle: 45 °
Analysis diameter: 400μmφ
Pass energy: 29.35 eV
Resolution: 0.125 eV / step
Degree of vacuum: about 10 ⁻⁷ Pa or less From the measured value of nitrogen (N) and the measured value of sulfur (S), the PVP content on the inner surface of the hollow fiber membrane was calculated by the following formula.
PVP content (Hpvp) [%]
= 100 × (N × 111) / (N × 111 + S × 232)

(Production of hollow fiber module 4)
Using the obtained hollow fiber membrane (bundle), a test hollow fiber module 4 was produced in the same manner as in Example 1.

[Comparative Example 2]
(Hollow fiber membrane)
A commercially available hollow fiber dialyzer (manufactured by Nipro Corporation, FB-210Uβ) was disassembled, and the hollow fiber membrane was taken out and used for the test. The hollow fiber membrane was made of cellulose triacetate and was given glycerin. The hollow fiber membrane had an inner diameter of 200 μm, an outer diameter of 230 μm, and a film thickness of 15 μm.

(5 production of hollow fiber modules)
Using the hollow fiber membrane (bundle), a test hollow fiber module 5 was produced in the same manner as in Example 1.

[Comparative Example 3]
(Hollow fiber membrane)
A commercially available hollow fiber type plasma component separator (manufactured by Asahi Kasei Medical Co., Ltd., Cascade Flow EC-20W) was disassembled and used for the test. The membrane was made of an ethylene vinyl alcohol copolymer and had water attached to it. The hollow fiber membrane had an inner diameter of 175 μm, an outer diameter of 255 μm, and a film thickness of 40 μm. Even after drying, rewetting was possible.

(Production of hollow fiber module 6)
Using the hollow fiber membrane (bundle), a test hollow fiber module 6 was produced in the same manner as in Example 1.

(Measurement of water permeability)
For the module in which glycerin is applied to the hollow fiber membrane, pure water is introduced from one end (1a in FIG. 1) of the liquid passage port into the lumen of the hollow fiber membrane, and is allowed to flow out from the other end (1b). The membrane was thoroughly washed to remove glycerin and replace membrane pores and hollow portions with pure water. Thereafter, the outer space of the hollow fiber membrane was similarly washed by passing water from 2a to 2b in FIG. The outlet circuit (the outlet side from the pressure measurement point) was sealed with forceps. Pure water kept at 25 ° C. is put into a pressurized tank, and while controlling the pressure by a regulator, pure water is sent to the lumen (1a) of the module kept at a constant temperature bath at 25 ° C. By closing (2b), the amount of filtrate flowing out from the permeate side (2a) of the membrane was measured for a certain period of time. The intermembrane | transmembrane pressure difference was made into the pressurization pressure (same as pressurization to a tank) to the module entrance side, and was measured by 100 mmHg. The water permeability (mL / m ² / hr / mmHg) of the hollow fiber membrane was calculated using the area of the membrane permeation portion of the module used (membrane area based on the inner diameter of the hollow fiber membrane).

(Filtration experiment)
(1) A test solution was prepared as follows. That is, inulin (molecular weight 5,500, Nacalai Tesque), dextran T10 (molecular weight 10,000, Sigma-Aldrich), dextran T40 (molecular weight 40,000, Sigma-Aldrich) are distilled water for injection (Otsuka Pharmaceutical Co., Ltd.). ) To prepare an aqueous solution having a concentration of 1,000 ppm, respectively, and used as a test solution in subsequent experiments.
(2) After each test solution was passed through the hollow fiber membrane lumen of the hollow fiber module and sufficiently substituted, distilled water kept at 25 ° C. or each test solution was adjusted to a pressure of 100 mmHg in the same manner as the water permeability measurement. In addition, the mixture was filtered for a certain time, and each sieve coefficient (Sieveing Coefficient, hereinafter abbreviated as SC) was obtained. Sampling of the liquid started from the time when the filtrate started to appear, and sampling was started 2 minutes later, and the entire amount of the filtrate was recovered for several minutes (3 to 4 minutes). SC is defined as the ratio of the concentration of dextran, etc. in the permeate to the concentration of dextran, etc., in the feed. The concentration of dextran and the like is quantified by a conventional method such as GPC method or colorimetric method (anthrone sulfate method), but it is convenient to use a monodisperse dextran reagent with a defined molecular weight as in this method. Any colorimetric method can be used. Further, during the experiment, the water permeability in each test solution was also measured.
(3) Next, the lumen of the hollow fiber membrane of each hollow fiber module was filled with Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum so as to be 10 vol%, and allowed to stand at 25 ° C for 30 minutes. After removing the filling liquid and washing with pure water 100 times the volume of the hollow portion of the hollow fiber membrane, each test liquid was filtered in the same manner as in (2) to determine SC and water permeability. The rate of change is a value after contact / a value before contact.

(Fibronectin adsorption test)
The DMEM to which serum was added so that the volume of the hollow fiber modules of Example 1 and Comparative Example 2 was 1 vol% was filled and allowed to stand for 60 minutes, and then the medium was collected. The amount of fibronectin adsorbed to the hollow fiber membrane was calculated from the fibronectin concentration in the medium before filling and the fibronectin concentration in the recovered solution. Fibronectin was measured using Fibronectin EIA Kit (Takara Bio Inc.).

The results of the filtration experiment are shown in Tables 1 and 2. The results of the fibronectin adsorption test are shown in Table 3. Table 1 shows changes in the sieving coefficient (SC) before and after contacting the medium described in (3) above. On the other hand, Table 2 shows changes in water permeability before and after contacting the medium described in (3) above. The hollow fiber membranes of the present invention tend to be slightly less permeable to polymers having a molecular weight of 40,000 or more compared to the hollow fiber membranes of the comparative examples, but all show good substance permeability. Yes.
When these hollow fiber membranes were brought into contact with DMEM to which fetal bovine serum, which is a general medium, at 10 vol% was brought into contact at 25 ° C. for 2 hours, the membrane characteristics of the hollow fiber membranes were changed. That is, the sieving coefficient of the hollow fiber membranes of Examples 1 to 3 varies greatly compared to the hollow fiber membranes of Comparative Examples 1 to 3. This is because the protein contained in the medium is more efficiently adsorbed to the surface of the hollow fiber membrane of the present invention as shown in Table 3. Moreover, since the substance permeability of a certain level or more is maintained even after the protein is adsorbed, the cell culture is not affected at all. Thus, it was shown that the hollow fiber membranes (hollow fiber modules) of Examples 1 to 3 have characteristics that become a culture substrate (container) excellent in cell adhesion.

  From the results in Table 3, the hollow fiber membrane of Example 1 was found to adsorb 4 times or more fibronectin in a short time with respect to the hollow fiber membrane of Comparative Example 2. That is, the hollow fiber membrane of the present invention can easily adsorb an extracellular matrix or the like necessary for culturing mesenchymal stem cells, and can quickly prepare a favorable environment as a scaffold for cell proliferation. It is suggested that it can exert an excellent effect on sex and subsequent proliferation.

(Cell culture experiment using hollow fiber module 1: using general medium)
FIG. 2 shows a simplified configuration of the culture apparatus used for the cell culture experiment. Hollow fiber module 1-6 was used for the cell culture experiment. The cells used were primary human mesenchymal stem cells purchased from Takara Bio Inc. The cell seeding density was 1900 cells / cm ² . The flow rate of the medium was 0.33 mm / min for the inner lumen of the hollow fiber membrane and 3.46 mm / min for the outer lumen of the hollow fiber membrane. In addition, a culture bag 1L (manufactured by Nipro Co., Ltd.) (5 (for inner lumen) and 6 (for outer lumen) in FIG. 2) was used for medium supply. The medium used was Dulbecco's modified Eagle medium (DMEM) supplemented with fetal bovine serum at 10 vol%. As a medium perfusion pump, a total of two Perista Biomini pumps (manufactured by Ato) (for Alumina) and 30 (Ambient) in FIG. And culturing at 37 ° C. for 7 days in a CO ₂ incubator. The medium supply method was unidirectional for both the hollow fiber membrane lumen and the hollow fiber membrane outer lumen. Mesenchymal stem cells were seeded in the lumen of the hollow fiber membrane and allowed to stand for 2 days, and then perfusion of the lumen of the hollow fiber membrane was started. After culturing for 7 days, the medium perfusion was stopped and the cells grown in the hollow fiber module were collected. At the time of cell recovery, a cell dissociation reagent, 0.25% trypsin solution (manufactured by Life Technologies) was used.

(Cell culture experiment 2 using hollow fiber module: using low serum medium)
Cell culture experiment 2 was performed using the same cell culture apparatus as cell culture experiment 1. Using hollow fiber module 1-6, primary human mesenchymal stem cells purchased from Takara Bio Inc. were used. In this experiment, MF-medium (registered trademark) mesenchymal stem cell growth medium (Toyobo Co., Ltd.) supplemented with fetal bovine serum at 1 vol% was used as the medium. Other conditions were the same as those in the cell culture experiment 1.

(Cell culture experiment 3 using hollow fiber module: using serum-free medium)
Using the same cell culture apparatus as in cell culture experiment 1, cell culture experiment 3 was performed. Using hollow fiber module 1-6, primary human mesenchymal stem cells purchased from Takara Bio Inc. were used. In this experiment, serum-free MF-medium (registered trademark) mesenchymal stem cell growth medium (manufactured by Toyobo Co., Ltd.) was used. Other conditions were the same as those in the cell culture experiment 2.

The results of cell culture experiment 1-3 are summarized in Table 4.
In the cell culture experiment 1, Dulbecco added with fetal bovine serum, which is a general medium, to 10 vol% by using a hollow fiber module storing a hollow fiber membrane composed only of the hydrophobic polymer of the present invention. A good cell growth rate was obtained by culturing using the modified Eagle medium (DMEM).
Moreover, in the cell culture experiment 2, the culture experiment was carried out using a low serum concentration (1 vol%) mesenchymal stem cell-dedicated medium specialized for mesenchymal stem cell culture. Also in this culture experiment, the hollow fiber module storing the hollow fiber membrane composed only of the hydrophobic polymer of the present invention showed a good cell growth rate.
Furthermore, in the cell culture experiment 3, a culture experiment was performed using a serum-free medium in which no serum was added to the medium exclusively for mesenchymal stem cells. Also in such cell culture experiments, the hollow fiber module storing the hollow fiber membrane composed only of the hydrophobic polymer of the present invention showed a good cell growth rate.
On the other hand, when the hollow fiber module of the comparative example was used, cell proliferation was hardly observed.
That is, it can be said that what is excellent as a cell culture substrate is a hollow fiber membrane composed only of a hydrophobic polymer not containing a hydrophilic polymer.

(Measurement of cell growth rate)
From the cell culture container (hollow fiber module) after completion of the culture, the cell growth rate was calculated by the following formula using the number of recovered living cells and the initial number of seeded cells.
Cell proliferation rate (%) = (number of viable cells recovered−number of seeded cells) / number of seeded cells × 100

(Measurement of cell number)
The collected liquid containing the cells was finally suspended in 1 ml of culture solution by centrifugation. A solution obtained by mixing this suspension and trypan blue staining solution at a ratio of 1: 1 was added to a hemocytometer, and the number of cells was measured under a microscope.
1. The surface of the hemocytometer and cover glass is washed with 70% isopropanol, excess isopropanol is wiped off and air-dried.
2. Wet the side of the cover glass with Reagent grade water and attach it to the hemocytometer.
3. After thoroughly agitating the cell suspension with a Pasteur pipette, immediately pour it into a hemocytometer and fill the groove.
4. Perform steps 1 to 3 using a separate hemocytometer (measure twice and average).
5. Place a hemocytometer on the microscope and focus on the grid lines (10 × objective).
6). Using a counter, rapidly measure the number of cells in a 1 mm ² area.
* Since errors are likely to occur, at least 100-500 cells are counted for accurate counting.
Method of calculation:
C = N × 10 ⁴
C: Number of cells per ml N: Average number of measured cells 10 ⁴ : Conversion value of volume for 1 mm ² Total number = C × V
V = volume of the liquid in which the cells are suspended

(Cell surface marker measurement)
In order to confirm that the performance of the mesenchymal stem cells was maintained even after the culture, surface marker measurement was performed before the culture and after the culture for 7 days. Surface markers are based on ISCT (International Society for Cellular Therapy) statement, CD105, CD73, CD90 are positive (≧ 95%), CD45, CD34, CD11b, CD19, HLA-DR are negative (≦ 2%) It was based on that.
The surface marker expression was measured using a flow cytometer (BD FACSCalibur, manufactured by BD). As an antibody, Human MSC Analysis Kit (manufactured by BD) was used.
Table 5 (before culturing) and Table 6 (after culturing) show the results of measuring the surface marker of mesenchymal stem cells before and after culturing carried out in cell culture experiment 1. Since there was no change in the expression pattern of the cell surface marker before and after the culture, it was confirmed that the properties of the mesenchymal stem cells were maintained.
From this, it can be said that the hollow fiber membrane consisting only of the hydrophobic polymer of the present invention is excellent as a cell culture substrate for adhesive cells, particularly adhesive stem cells.

  According to the present invention, it is possible to provide a hollow fiber membrane as a culture substrate that does not require a cell adhesion factor coating treatment. Furthermore, by using the hollow fiber membrane of the present invention, it is possible to construct and provide a cell culture system that can easily and efficiently culture various cells such as mesenchymal stem cells.

1a, 1b: End conduit 2a, 2b: Side conduit 3: Module case 4: Culture substrate (hollow fiber membrane)
5, 6: Liquid medium storage container 7: Waste liquid recovery solution or cell recovery container 21, 22, 23, 24, 25, 26, 27, 28: Valves 41, 42, 43: Aseptic connection connector 8: Cell culture container (hollow Thread module)
30, 31: Pump 50: CO ₂ incubator 60: Work controller (work panel)

Claims (6)

  A hollow fiber membrane for culturing cells by seeding and culturing cells on the inner surface and / or outer surface, wherein the hollow fiber membrane is composed only of a hydrophobic polymer.   The hollow fiber membrane according to claim 1, wherein the hydrophobic polymer is a polymer having a contact angle of 70 degrees to 93 degrees.   The hollow fiber membrane according to claim 1 or 2, wherein the hydrophobic polymer is at least one selected from the group consisting of polyethersulfone, polysulfone, polyvinylidene fluoride, and polyethylene.   A hollow fiber module for cell culture in which a hollow fiber bundle obtained by bundling a plurality of hollow fiber membranes according to any one of claims 1 to 3 is stored.   A cell culture method using the hollow fiber module according to claim 4 as a cell culture container. A cell culture apparatus comprising the hollow fiber module according to claim 4 as a cell culture container.

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