JP6952297B2 - Hollow fiber membrane for cell culture and cell culture method - Google Patents

Description

The present invention relates to a hollow fiber membrane for cell culture. More specifically, the present invention is a hollow fiber membrane that can be cultured with almost no decrease in cell viability and activity, and is manufactured in the fields of pharmaceuticals, medical care, foods, chemical industry, and /. Alternatively, the present invention relates to a hollow fiber membrane preferably used in research and development.

Conventionally, hollow fiber membranes have been used for removing bacteria and protozoa in the water purification field, removing impurities in the industrial field, separating or concentrating heat-sensitive substances such as proteins and enzymes, artificial dialysis in the medical field, and manufacturing pharmaceuticals and medical water. Virus and protein removal, ultra-pure water production, electrodeposition paint recovery, yarn and pulp factory sewage treatment, oil-containing wastewater treatment, building wastewater treatment, fruit juice clarification, sake production, cheese whey concentration -It has been put to practical use in various fields such as desalination, production of concentrated milk, concentration of egg white, use in bioreactors, removal of fine particles in gas, and water treatment of nuclear power plants.

On the other hand, there have been reports of applications of hollow fiber membranes to cell culture by utilizing the substance permeation performance of membranes, in addition to the above-mentioned usage methods for the purpose of removing foreign substances and separating and purifying. For example, Patent Document 1 discloses a method of culturing cells with nanofibers located between two types of hollow fiber membranes, one for gas supply and the other for medium supply. In this case, the cells were located outside the hollow fiber membrane, which was used only as part of the nutrient and oxygen supply and scaffolding material.

Further, Patent Document 2 discloses a method of filling and culturing hepatocytes inside a hollow fiber membrane. Although this method is an efficient culturing method, there is a problem that the activity is remarkably lowered with the lapse of culturing time. Patent Document 3 discloses a method for culturing stem cells in the same manner as described above, and Patent Document 4 discloses a method for culturing iPS cells. These are methods for culturing a large amount of pluripotent cells while maintaining the undifferentiated state of the cells.

As described above, the method of culturing cells in the hollow fiber membrane is to use the substance produced by the cells as an artificial organ such as an artificial liver in the form of a hollow fiber membrane bundle or a module because the substance produced by the cells can be taken out from the hollow fiber membrane. It is a very useful technology in the medical and industrial fields. In addition, the cells cultured in the hollow fiber membrane can be taken out as a columnar cell aggregate.

However, the hollow fiber membrane used for the above cell culture has a large inner diameter of 285 μm or 330 μm, and since the inner diameter area of the part where the cells are filled is large, sufficient oxygen and nutrients can be supplied to the central part. There was a problem that the cells inside did not spread and died. In order to solve such a problem, Patent Document 5 discloses a method of increasing the cell viability by crushing the hollow fiber membrane to form a deformed cross section. However, there is a problem that the cost is increased by the process of making the deformed cross section, and there is a problem that it is difficult to efficiently take out the cell aggregate inside by making the deformed cross section.

Further, it has been considered that the hollow fiber membrane used for such cell culture should have high hydrophilicity. Therefore, for example, in Patent Documents 4 and 5, polyethylene and an ethylene / vinyl alcohol copolymer are used as the material of the hollow fiber membrane. Coated ones have been used. In this case, although it is made hydrophilic by the ethylene / vinyl alcohol copolymer, the degree of hydrophilicity is low and there is a concern that the coating agent may be peeled off or eluted.

Japanese Unexamined Patent Publication No. 2010-148496 Japanese Unexamined Patent Publication No. 2004-166717 Japanese Unexamined Patent Publication No. 2014-60991 Japanese Unexamined Patent Publication No. 2006-7207 International release WO2004 / 20614

An object of the present invention is to solve the above-mentioned problems and to provide a hollow fiber membrane for cell culture that maintains a high cell viability and is easy to handle.

The present inventors have diligently studied to solve the above-mentioned problems, and found that it is a polymer hollow fiber membrane for introducing and culturing cells into a hollow portion, and has an inner diameter in the range of 20 μm to 150 μm. By using a hollow fiber membrane for cell culture, which has a film thickness in the range of 30 μm to 250 μm and is porous so that a liquid can pass through the hollow fiber membrane wall, cells can be suitably cultured and high. We found that survival rates could be achieved.

That is, the present invention provides a hollow fiber membrane for cell culture and a hollow fiber membrane module according to the following aspects.
Item 1. A polymer hollow fiber membrane for introducing and culturing cells into a hollow portion, the inner diameter is in the range of 20 μm to 150 μm, the film thickness is in the range of 30 μm to 250 μm, and the hollow fiber membrane wall is covered with liquid. A hollow fiber membrane for cell culture that is porous and can pass through.
Item 2. Item 2. The hollow fiber membrane for cell culture according to Item 1, wherein the hollow fiber membrane has a pore size in the range of 0.02 μm to 0.5 μm.
Item 3. Item 2. The hollow fiber for cell culture according to Item 1 or 2, wherein the contact angle of the hollow fiber membrane with water is 60 ° or less, and the amount of pure water permeated at 25 ° C. is 300 L / (m ^{2 · atm · h) or more.} film.
Item 4. Item 2. The hollow fiber membrane for cell culture according to any one of Items 1 to 3, wherein the hollow fiber membrane is a polyamide and / or a copolymer of polyamide.
Item 5. Item 2. The hollow fiber membrane for cell culture according to any one of Items 1 to 4, wherein the inner diameter of the hollow fiber membrane is in the range of 90 μm to 150 μm.
Item 6. Item 2. A plurality of hollow fiber membranes for cell culture according to any one of Items 1 to 5 are arranged in the same direction, one end or both ends are sealed with an adhesive, and at least one end. Is an open hollow fiber membrane module for cell culture.
Item 7. Item 2. The cell culture hollow filament membrane according to any one of Items 1 to 5 is filled with cells, the cell culture hollow filament membrane is immersed in the culture solution of the cells, and the temperature and humidity and gas suitable for the cells are used. A cell culture method in which cells are cultured in an atmosphere.

Since the hollow fiber membrane for cell culture of the present invention maintains a high level of cell viability and metabolic activity, cell proliferation and production of useful substances can be efficiently performed. Further, by using human cells, it can be used in the form of, for example, an artificial liver, and can be effectively used as a medical device for supplementing a functionally deteriorated organ. From this, the hollow fiber membrane for cell culture of the present invention is suitably used in manufacturing, medical treatment, and / or research and development in the fields of pharmaceuticals, medical fields, food fields, and chemical industries.

It is the schematic of the apparatus for measuring the pure water permeability of the polyamide hollow fiber membrane of this invention. It is a graph which shows the relationship between the cell culture period and the cell viability in Examples 1, 2 and Comparative Example 1. It is a graph which shows the relationship between the cell culture period and the ammonia removal ability in Examples 1 and 2 and Comparative Example 1. It is a graph which shows the relationship between the cell culture period and the albumin secretory ability in Examples 1 and 2 and Comparative Example 1. In Example 1, it is a micrograph which confirmed the expression of the hypoxia-inducing factor of the cell in the hollow fiber membrane after 7 days of culture by immunofluorescence staining. In Comparative Example 1, it is a micrograph which confirmed the expression of the hypoxia-inducing factor of the cell in the hollow fiber membrane after 7 days of culture by immunofluorescence staining. It is a graph which shows the relationship between the culture days and the cell density in Example 3 and Comparative Example 3. It is a graph which shows the relationship between the culture days and the ammonia removal ability in Example 3 and Comparative Example 3. It is a graph which shows the relationship between the culture days and the urea-producing ability in Example 3 and Comparative Example 3.

The hollow fiber membrane for cell culture of the present invention is a polymer hollow fiber membrane for introducing and culturing cells into a hollow portion. Any polymer can be used as long as the effects of the present invention are not impaired. For example, polyamide, polyethylene, polyarylate, polysulfone, polyethersulfone, polyester, polyvinyl alcohol, polyvinyl acetate, ethylene / vinyl alcohol copolymer, etc. Examples thereof include polyvinylidene fluoride, polytetrafluoroethylene, cellulose acetate, polyacrylonitrile and the like. These polymers may be used alone, or may be two or more types of copolymers, or may be used in combination. Among these, polyamide and / or a copolymer of polyamide is preferable. Examples of the polyamide include polyamide 6, polyamide 66, polyamide 46, polyamide 610, polyamide 612, polyamide 11, polyamide 12, polyamide MXD6, polyamide 4T, polyamide 6T, polyamide 9T, and polyamide 10T. Specific examples of the polyamide copolymer include a copolymer of polyamide and a polyether such as polytetramethylene glycol or polyethylene glycol. The ratio of the polyamide component in the polyamide copolymer is not particularly limited, but for example, the ratio of the polyamide component is preferably 70 mol% or more. When the ratio of the polyamide component in the polyamide copolymer satisfies the above range, a relatively tough and strong hollow fiber membrane is obtained. Of these, polyamide 6, polyamide 66, polyamide 46, polyamide 610, and polyamide 11 are more preferred, with polyamide 6 being the most preferred.

The inner diameter of the hollow fiber membrane of the present invention is in the range of 20 μm to 150 μm, preferably 50 μm to 150 μm, more preferably 80 μm to 150 μm, and particularly preferably 90 to 150 μm. If it is smaller than this range, it becomes difficult to fill the cells, and if it is larger than this range, there is a problem that the survival rate of cells is lowered due to lack of oxygen and nutrition.

The film thickness of the hollow fiber membrane of the present invention is in the range of 30 μm to 250 μm, preferably 30 μm to 150 μm, more preferably 50 μm to 120 μm, and particularly preferably 80 μm to 100 μm. If it is smaller than this range, there is a problem that the strength of the hollow fiber membrane is weakened, and if it is larger than this range, the mass transfer of the hollow fiber membrane wall is not smooth, so that the cell viability may decrease. ..

In the hollow fiber membrane of the present invention, the hollow fiber membrane wall is porous, and a liquid can permeate from the outside to the inside or from the inside to the outside. As a result, fresh medium is supplied from the outside and waste products are excreted from the inside. The pore size of the hollow fiber membrane is preferably in the range of 0.02 μm to 0.5 μm, more preferably 0.05 μm to 0.3 μm, further preferably 0.05 μm to 0.2 μm, and 0.08 μm to 0.12 μm. Is particularly preferable. If it is smaller than this range, mass transfer may not be smooth, and if it is larger than this range, the strength of the hollow fiber membrane may decrease. The pore diameter referred to here is the diameter of the polystyrene latex fine particles when 90% or more is blocked when the aqueous suspension of the polystyrene latex fine particles is flowed from the inside to the outside of the hollow fiber membrane.

In the present invention, the particle blocking rate is determined by adding 1 ml of polystyrene fine particles having a predetermined pore size manufactured by Duke Scientific Co., Ltd. to 299 ml of a 0.1% Triton X-100 aqueous solution, stirring and dispersing for 3 hours, and the amount of water permeation shown in FIG. The liquid was passed through a measuring device, the liquid that had passed through the membrane was collected, and the absorbance of the liquid before and after the membrane permeation at 380 nm was measured and calculated by the following formula.
Particle blocking rate = (initial absorbance-permeate absorbance) / initial absorbance x 100

The material itself of the hollow fiber membrane for cell culture of the present invention is excellent in hydrophilicity. In the present invention, the contact angle with water is adopted as an index of hydrophilicity. The contact angle with water is the angle between the tangent line at the end point of the water droplet formed on the surface of the membrane and the surface of the membrane by gently contacting the surface of the membrane with water droplets of 0.1 to 2.0 μl. It was obtained by measuring with a meter. Therefore, it can be said that the smaller this value is, the higher the hydrophilicity is. The measured value of the contact angle in the present specification is a value using an automatic contact angle meter DM-500 manufactured by Kyowa Interface Science.

The contact angle with water is usually 65 ° or less, preferably 60 ° or less, and more preferably 55 ° or less. If it is higher than this value, the hydrophilicity is low, and there are problems such as not smooth mass transfer, poor cell proliferation, and poor acquisition of cell mass after proliferation.

Further, the hollow thread membrane for cell culture of the present invention has a water permeation amount of pure water at 25 ° C. of preferably 300 L / (m ² · atm · h) or more, more preferably 300 L / / as one of the substance transfer performances. It is (m ² · atm · h) to 2500 L / (m ² · atm · h), more preferably 800 L / (m ² · atm · h) to 2000 L / (m ² · atm · h), and in particular. preferably from ^{1200L / (m 2 · atm ·} h) ~1800L / (m 2 · atm · h).

Here, the amount of water permeation in the present invention is a value measured by internal pressure filtration. Specifically, the hollow fiber membrane is cut to 10 to 20 cm, and injection needles having a diameter matching the inner diameter are inserted into the hollow portions at both ends. Then, after setting in the device as shown in FIG. 1, pure water is passed through the liquid feed pump 13 for a predetermined time (minutes), and the volume (L) of water that has passed through the membrane and stored in the saucer 18 is defined as the amount of permeated water. It was calculated by the following formula.

Permeable amount = Permeable water amount (L) / [Inner diameter (m) x 3.14 x Length (m) x {(Inlet pressure (atmospheric pressure) + Outlet pressure (atmospheric pressure)) / 2} x time]

If the amount of water permeation is ^{less than 300 L / m 2} · atm · h, mass transfer may not proceed smoothly and cell proliferation and activity may decrease.

The hollow fiber membrane for cell culture of the present invention can be used as it is, but it can be made into a modular shape for more efficient use. The module may have any shape as long as the effects of the present invention are not impaired, but a plurality of hollow fiber membranes are arranged in the same direction, and at least one end or both ends are sealed with an adhesive. , It is preferable that at least one end is open. The length of the hollow fiber membrane can be changed depending on the application. The end portion to be opened can be satisfactorily opened by adhering a plurality of hollow fiber membrane bundles with an adhesive and cutting the hollow fiber membrane bundles in the vertical direction together with the adhesive. The type of adhesive used at this time is not particularly limited, but epoxy resin, urethane resin, polyethylene resin and the like are preferable, and epoxy resin and urethane resin are more preferable.

The end portion to be not opened can be fused by heat without using an adhesive to close the mouth, or can be adhered by hardening with an adhesive as described above. A plurality of hollow fiber membranes may be solidified together or separated at the end portion to be not opened.

The hollow fiber membrane for cell culture of the present invention introduces cells into the hollow fiber membrane for culturing. The method for introducing cells can be any method as long as the effects of the present invention are not impaired. In the case of a small module, a method of press-fitting using a syringe or the like can be adopted. In the case of medium-sized and large-sized modules, a method can be adopted in which cells are pumped together with the medium and press-fitted. After the cells are introduced into the hollow fiber membrane by the above method or the like, the cells can be brought to the end of the hollow fiber membrane by a method such as centrifugation or suction.

The hollow fiber membrane into which the cells have been introduced is preferably placed in an environment in which the cells can preferably proliferate, differentiate, and express functions, and the cells are preferably cultured. Examples of the environment include the composition / type of medium, temperature / humidity, gas composition, presence / absence of vibration, and the like. For example, in the case of culturing human cells, it is preferable to culture them in an environment of a temperature of 37 ° C., a humidity of 90% to 95%, a gas composition of 5% carbon dioxide, and 95% air. The hollow fiber membrane into which cells have been introduced exchanges nutrients, gas, and waste products through the hollow fiber membrane, and therefore is preferably cultured in a medium.

The cells cultured in the hollow fiber membrane for cell culture of the present invention are not particularly limited, but are preferably animal cells. Examples of such cells include metabolic and storage cells such as hepatocytes, fat cells and Ito cells, barrier function cells such as pararenal cells, glomerular epithelial cells and conduit cells, exocrine epithelium such as mammary gland cells, salivary gland mucous cells and prostate cells. Cells, pituitary anterior lobe cells, thyroid cells, adrenal cells, luteinizing cells and other hormone-secreting cells, epidermal basal cells, capillary cells and other keratinized epithelial cells, various nerve cells, corneal fibroblasts, cartilage cells, osteoblasts Matrix cells, skeletal muscle cells, myocardial cells, muscle satellite cells, smooth muscle cells such as muscle cells, leukocytes, erythrocytes, neutrophils, obesity cells, natural killer cells, etc. Examples thereof include embryo cells such as mother cells, nurse cells, ES cells, stem cells such as iPS cells and the like. Among these, hepatocytes, adipocytes, chondrocytes, muscle cells and stem cells are preferable.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

In the examples, the water permeability of the hollow fiber membrane was measured by the method described above.

Example 1
Polyamide 6 hollow fiber membrane with a pore size of 0.1 μm (inner diameter 118 μm, film thickness 85 μm, water permeability 1500 L / (m ² · atm · h), water contact angle 51 °, Unitika Ltd.) The module was manufactured so that the effective length was 26 mm by bundling nine of them. Both ends of the module were glued with silicone rubber, and one end was structured so that it could be connected to a syringe barrel. The effective culture volume inside the hollow fiber membrane was 2.6 mm ² . 2.2 × 10 ⁶ rat primary hepatocytes were introduced into this module and centrifuged at 200 G × 180 seconds with a centrifuge to fill the cells. This was placed in a medium and cultivated by swirling at 45 rpm under the conditions of a temperature of 37 ° C., a humidity of 95%, and a carbon dioxide concentration of 5%. As a result, as shown in FIG. 2, the cell viability showed a high value of 40% or more even after culturing for 7 days. In addition, as shown in FIG. 3, the ammonia removal rate was about 400 μmol / cm ³ / day, and as shown in FIG. 4, the albumin secretion rate was about 2 mg / cm ³ / day, both showing high values, and high function as hepatocytes. Was found to have been expressed.
When the expression of hypoxia-inducible factor was confirmed by immunofluorescence staining in the cells in the hollow fiber membrane 7 days after culturing in order to grasp the oxygen deficiency, as shown in FIG. 5, there were almost no green cells up to the center. It was confirmed that oxygen was not deficient.

Example 2
A polyamide 6 hollow fiber membrane having a pore diameter of 0.1 μm, an inner diameter of 148 μm, a film thickness of 94 μm, a water permeability of 1500 L / (m ² · atm · h), and a water contact angle of 51 ° was used. Rat primary hepatocytes were cultured in the same manner as in Example 1 except that the module was prepared to be 19 mm. The effective culture volume of the module at this time was 2.6 mm ² . As a result, as shown in FIG. 2, the cell viability showed a high value of 40% or more even after culturing for 7 days. As shown in FIG. 3, the ammonia removal rate after culturing for 7 days was about 350 μmol / cm ³ / day, and as shown in FIG. 4, the albumin secretion rate after culturing for 7 days was about 2 mg / cm ³ / day. It showed a high value, and it was found that it expressed a high function as a hepatocyte.

Comparative Example 1
A polyethylene plasma separation membrane (pore diameter 0.3 μm, inner diameter 330 μm, film thickness 50 μm) coated with ethylene-vinyl alcohol was used for the hollow fiber membrane, and two of these membranes were bundled to prepare a module so that the effective length was 15 mm. Cultured rat primary hepatocytes in the same manner as in Example 1. The effective culture volume of the module at this time was 2.6 mm ² . As a result, as shown in FIG. 2, the cell viability was remarkably reduced to about 15% after culturing for 7 days. As shown in FIG. 3, the ammonia removal rate after culturing for 7 days was about 200 μmol / cm ³ / day, and as shown in FIG. 4, the albumin secretion rate after culturing for 7 days was about 0.8 mg / cm ³ / day. Both became low.
When the expression of hypoxia-inducing factor was confirmed by immunofluorescence staining in the cells in the hollow fiber membrane 7 days after culturing in order to grasp the oxygen deficiency, as shown in FIG. 6, most of the cells inside the hollow fiber membrane were green. It was confirmed that most of the cells were deficient in oxygen.

Comparative Example 2
Polyamide 6 hollow fiber membrane (pore diameter 0.1 μm, inner diameter 366 μm, film thickness 105 μm, water permeability 4000 L / (m ² · atm · h), water contact angle 51 °, manufactured by Unitika Co., Ltd.) was used as the hollow fiber membrane. Rat primary hepatocytes were cultured in the same manner as in Example 1 except that a module was prepared so as to have an effective length of 12 mm by bundling two of them. The effective culture volume of the module at this time was 2.5 mm ² . As a result, the cell viability was remarkably reduced to about 20% after culturing for 7 days.

Example 3
10 of the polyamide 6 hollow fiber membranes (inner diameter 118 μm, film thickness 85 μm, water permeability 1500 L / (m ² · atm · h), water contact angle 51 °, manufactured by Unitika Ltd.) used in Example 1 with a pore diameter of 0.1 μm. The modules were bundled and produced so as to have an effective length of 22 mm. Both ends of the module were glued with silicone rubber, and one end was structured so that it could be connected to a syringe barrel. The effective culture volume inside the hollow fiber membrane was 2.4 mm ² . In this module, a cell line established to express eight hepatic transcription factors using a drug-induced gene expression-inducing system was introduced into mouse liver cancer-derived hepatoma cells (Hepa1-6), and Example 1 Similarly, the cells were filled by centrifuging at 200 G × 180 seconds with a centrifuge. The transfer transcription factors introduced into the cells are hepatocyte nuclear factor (HNF) -1α, -1β, -3β, -4α, 6 and CCAAT / enhancer binding protein (C / EBP) -α, -β, -γ. (Biochemical Engineering Journal 60,67-73, 2012). This module was grown and cultured in DMEM and 10% FBS medium until the 5th day of culture, and from the 5th day, functional expression was induced in a medium supplemented with 0.1 μg / mL of doxycycline. As a result, as shown in FIG. 7, the number of cells steadily proliferated until the 5th day, and gradually decreased after the induction of function expression. ^{The ammonia removal rate showed a high value of about 700 μmol / cm 3} / day after 12 days as shown in FIG. 8, and the urea production rate also showed a high value of about 30 mg / cm ³ / day after 12 days as shown in FIG. rice field. From this, it was found that even in a transgenic cell line, effective proliferation and functional expression can be achieved by using the hollow fiber membrane of the present invention.

Comparative Example 3
Examples except that a plasma separation membrane made of cellulose triacetate (pore size 0.2 μm, inner diameter 285 μm, film thickness 51 μm) was used as the hollow fiber membrane, and six of these were bundled to prepare a module so as to have an effective length of 30 mm. Similar to No. 3, a cell line established to express eight hepatic transcription factors using a drug-induced gene expression-inducing system was proliferated and cultured on mouse liver cancer-derived hepatoma cells (Hepa1-6) to induce functional expression. bottom. The effective culture volume of this module was 11.5 mm ² . As a result, as shown in FIG. 7, the cells proliferated steadily until the 5th day, and gradually decreased after the induction of function expression. However, as shown in FIGS. 8 and 9, almost no functional expression was observed. From this, it was found that this hollow fiber membrane cannot express the function in the transgenic cell line.

1: Liquid feed pump 2: Hollow fiber membrane 3: Reservoir 4: Pressure adjustment valve 5: Inlet pressure gauge 6: Outlet pressure gauge

Claims (3)

A polymer hollow fiber membrane for introducing cells into a hollow portion and culturing them (excluding hollow fiber membranes having an irregular cross section) .
The hollow fiber membrane is a polyamide and / or a copolymer of polyamide, and is
The polyamide copolymer has a polyamide component ratio of 70 mol% or more.
The inner diameter is in the range of 90 μm to 150 μm,
The film thickness is in the range of 30 μm to 250 μm.
The pore diameter is 0.05 μm to 0.2 μm,
It is porous so that liquid can pass through the hollow fiber membrane wall.
A hollow fiber membrane for cell culture, characterized in that the contact angle of the hollow fiber membrane with water is 60 ° or less, and the amount of pure water permeated at 25 ° C. is 300 L / (m ^{2 · atm · h) or more.} A plurality of hollow fiber membranes for cell culture according to claim 1 are arranged in the same direction, one end or both ends are sealed with an adhesive, and at least one end is open. Hollow fiber membrane module for cell culture. The hollow filament membrane for cell culture according to claim 1 is filled with cells, the hollow filament membrane for cell culture is immersed in a culture solution of the cells, and placed in a temperature and humidity suitable for the cells and a gas atmosphere. A method of culturing cells.

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