Microfiber material for fixing cells

Abstract

The purpose of the present invention is to provide a microfiber material for fixing cells that makes it possible to fix cells up to a deep portion of the microfiber material in a short amount of time without loss by simply dripping cells onto the microfiber material, and that can increase the density of cells.　The present disclosure relates to a microfiber material for fixing cells comprising a microfiber that contains both (A) a non-water-soluble polymer material that is swellable when made into microfibers and (B) a cell-adhesive polymer material.

Description

Microfiber materials for cell immobilization

[0001] This disclosure relates to microfiber materials for cell immobilization, etc.

[0002] In recent years, research into regenerative medicine technologies using cells such as stem cells and iPS cells has advanced, and there is a need for cell scaffold materials (immobilization carriers) suitable for living organisms. For example, regenerative medicine using exosomes is attracting attention. Exosomes are microvesicles released from cells, and their involvement in various bodily functions has been suggested, leading to expectations for applications in the treatment or prevention of diseases. Normal cells have a low proliferation rate, and the amount of exosomes released from them is less than 1 μg of protein per 1 mL. In contrast, the amount of cells required for a single treatment is thought to be several hundred to several thousand times that amount, so it is necessary to immobilize animal cells and continuously culture them in large quantities. Furthermore, there is also interest in performing permanent cell therapy by immobilizing cells differentiated from somatic cells or pluripotent stem cells such as iPS cells onto cell sheets and implanting them in the body. In this case, it is necessary to rapidly immobilize these valuable therapeutic cells onto immobilization carriers without loss, culture them in large quantities, and implant or administer them into the body as quickly as possible.

[0003] To date, methods for immobilizing and mass-culturing these animal cells have been reported, such as using hollow fibers, porous microparticles, and nanoparticles (for example, Patent Document 1).

[0004] However, conventional methods have several problems, including (1) only a portion of the donated cells bind to the carrier during immobilization, resulting in the loss of many cells that cannot be immobilized; (2) the cell immobilization and production operations are complicated; and (3) the cell immobilization density is not high. Furthermore, when cells are immobilized on these carriers to achieve high cell density, if the void structure between the microfibers is not appropriate, (4) the supply of oxygen and nutrients to the inside of the microfiber material becomes insufficient, leading to decreased cell viability (cell death); and (5) the metabolic activity of the cells decreases, which inhibits the production of useful substances such as exosomes. Therefore, an optimal method for mass-culturing cells to produce exosomes, or an optimal method for using cells in cell sheets, has yet to be developed.

[0005] On the other hand, while fine fibers (nanofibers or microfibers) have long been studied as cell scaffolding materials, there are few reports on the degree of cell immobilization using these scaffolding materials. For example, Patent Document 2 describes a cell scaffolding material containing nanofibers made of an organic polymer with a number-average single fiber diameter of 1 to 200 nm. However, since the diameter of animal cells is generally 1 to 10 μm, they hardly adhere to nanofibers with a diameter of 1 to 200 nm, and instead proliferate in clumps in the gaps between the nanofibers, resulting in a significantly slower proliferation rate. Therefore, it is difficult to use nanofibers with a diameter of 1 to 200 nm as a scaffolding material. Furthermore, Patent Document 3 describes a cell scaffolding material containing a sponge-like three-dimensional structure made of synthetic polymer nanofibers with a number-average diameter of 1 nm or more and less than 1000 nm, and a porosity of 80% to 99.99%.

[0006] Furthermore, Patent Document 3 aims to increase the amount of cells immobilized by using nanofibers with high porosity. However, Patent Document 3 only discloses that the maximum cell immobilization rate (cell retention rate) of the structure is 88%. Examples 1 to 4 of Patent Document 3 describe porosity of 98.1 to 99.1%, but since the maximum cell retention rate is the aforementioned 88%, it is not the case that increasing the porosity improves the cell retention rate, and it is thought that if the porosity is too high, cells may even leak out.

[0007] Non-patent document 1 states that if you use Cytodex-1 (registered trademark), which is currently one of the best porous micro-immobilization carriers, 1 × 10 ７ It has been reported that cells can be fixed at a rate of cells / gram. Furthermore, Non-Patent Literature 2 states that if HUVEC cells, which are vascular endothelial cells, are not fixed, 4-5 × 10⁶ cells can be fixed in 6 days. ８ It has been reported that it produces exosomes at a rate of 1 / mL.

[0008] Therefore, in order to improve the amount of cells that can be immobilized (retention rate) and made available for use in various regenerative medicine applications, there is a need for a scaffold material that can easily and quickly immobilize supplied cells at high density without loss.

[0009] Japanese Patent Publication No. 2004-208692, Japanese Patent Publication No. 2006-254722, Japanese Patent Publication No. 2007-325543

[0010] Ferrari C et al., Biotechnology Progress, Vol. 28, pp. 780-787 (2012). Davidson et al., Scientific Reports, Vol. 8, Article No. 15885 (2018).

[0011] The purpose of this disclosure is to provide a cell-immobilizing microfiber material that allows cells to be immobilized deep into the microfiber material in a short time without loss simply by dropping them onto the microfiber material, while simultaneously enabling high-density cell production.

[0012] The present inventors investigated a microfiber immobilization method and found that a microfiber material uniformly containing a mixture of a water-insoluble polymer (component A) that swells after microfiberization and a cell-adhesion polymer (component B) allows (i) cell suspensions to be spontaneously absorbed (water-retained) into the interfiber spaces and taken up deep into the cell, and (ii) the taken-up cells adhere efficiently to component B and subsequently proliferate at high density. Furthermore, the present inventors found that the microfiber material for cell immobilization described herein maintains oxygen and nutrient supply through appropriate fiber diameter, void structure, and water retention, resulting in good cell activity and exosome production.

[0013] In other words, the present disclosure is as follows: Item 1. A microfiber material for cell immobilization, comprising a water-insoluble polymer material that swells when microfiberized and a cell-adhering polymer material. Item 2. The microfiber material for cell immobilization according to Item 1, wherein the water-insoluble polymer material that swells when microfiberized is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 3. The microfiber material for cell immobilization according to Item 1, wherein the water-insoluble polymer material that swells when microfiberized is at least one compound selected from the group consisting of cellulose acetate material, polyketone material, and polyethylene vinyl alcohol material. Item 4. A cell-containing microfiber material comprising the microfiber material for cell immobilization according to any one of Items 1 to 3, and further comprising cells. Item 5. A cell sheet comprising the cell-containing microfiber material according to Item 4. Item 6. A method for producing a microfiber material for cell immobilization, comprising: Step (I): A mixture production step of mixing a water-insoluble polymer material that swells when microfiberized with a cell-adhering polymer material to obtain a mixture; and Step (II): A microfiberization step of obtaining microfibers from the mixture obtained in the mixture production step. Item 7. The method for producing a microfiber material for cell immobilization according to Item 6, wherein the water-insoluble polymer material that swells when microfiberized is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 8. The method for producing a microfiber material for cell immobilization according to Item 6, wherein the water-insoluble polymer material that swells when microfiberized is at least one compound selected from the group consisting of cellulose acetate material, polyketone material, and polyethylene vinyl alcohol material. Item 9. The method for producing a microfiber material for cell immobilization according to Item 6, wherein the mixture production step of Step (I) is carried out in the presence of a solvent.Item 10. A method for producing a cell-containing microfiber material, comprising: Step (I): A mixture production step of mixing a water-insoluble polymer material that swells when microfiberized with a cell-adhering polymer material to obtain a mixture; Step (II): A microfiberization step of obtaining microfibers from the mixture obtained in the mixture production step; and Step (III): A step of contacting cells with the microfibers obtained in the microfiberization step. Item 11. A cell-immobilization microfiber material obtained from a mixture containing a water-insoluble polymer material that swells when microfiberized with a cell-adhering polymer material. Item 12. A cell-containing microfiber material obtained from a mixture containing the cell-immobilization microfiber material described in any one of Items 1 to 3 and cells. Item 13. A method for using a microfiber material obtained from a mixture containing a water-insoluble polymer material that swells when microfiberized with a cell-adhering polymer material for cell immobilization. Item 14. A method for immobilizing cells on a microfiber material, comprising the step of bringing the cells into contact with the cell-immobilizing microfiber material described in any one of items 1 to 3. Item 15. Use for manufacturing cell sheets using a microfiber material containing a water-insoluble polymer material that swells when microfiberized and a cell-adhering polymer material.

[0014] Item 1A. A microfiber material for cell immobilization, comprising microfibers containing (A) a water-insoluble polymer material that swells when microfiberized, and (B) a cell-adhering polymer material. Item 2A. The microfiber material for cell immobilization according to Item 1A, wherein the water-insoluble polymer material that swells when microfiberized (A) is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 3A. The microfiber material for cell immobilization according to Item 1A or Item 2A, wherein the water-insoluble polymer material that swells when microfiberized (A) is at least one compound selected from the group consisting of cellulose acetate materials, polyketone materials, and polyethylene vinyl alcohol materials. Item 4A. The cell-immobilizing microfiber material according to any one of claims 1A to 3A, wherein the (B) cell-adhering polymer material is at least one polymer material selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin. Claim 5A. The cell-immobilizing microfiber material according to claim 4A, wherein the average diameter of the microfibers in the cell-immobilizing microfiber material is 0.3 μm to 20 μm. Claim 6A. The cell-immobilizing microfiber material according to any one of claims 1A to 5A, wherein the (A) water-insoluble polymer material that swells when microfiberized has a water retention rate of 0.01 g or more per gram of microfiber. Claim 7A. A cell-immobilizing microfiber material according to any one of items 1A to 6A, wherein the mass ratio of (A) a water-insoluble polymer material that swells when microfiberized to (B) a cell-adhering polymer material is 0.1 to 2.5:1. Item 8A. A cell-containing microfiber material comprising the cell-immobilizing microfiber material according to any one of items 1A to 7A, and further comprising cells. Item 9A. A cell sheet comprising the cell-containing microfiber material according to item 8A.Item 10A. A method for producing a microfiber material for cell immobilization according to any one of items 1A to 7A, comprising: step (I): a mixture production step of mixing (A) a water-insoluble polymer material having swelling properties when microfibrillated and (B) a cell-adhering polymer material to obtain a mixture; and step (II): a microfibrillation step of obtaining microfibers from the mixture obtained in the mixture production step. Item 11A. A method for producing a microfiber material for cell immobilization according to item 10A, wherein the water-insoluble polymer material having swelling properties when microfibrillated is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 12A. A method for producing a microfiber material for cell immobilization according to item 10A or item 11A, wherein the water-insoluble polymer material having swelling properties when microfibrillated is at least one compound selected from the group consisting of cellulose acetate material, polyketone material, and polyethylene vinyl alcohol material. Item 13A. A method for producing a cell-immobilizing microfiber material according to any one of claims 10A to 12A, wherein the (B) cell-adhering polymer material is at least one polymer material selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin. Claim 14A. A method for producing a cell-immobilizing microfiber material according to any one of claims 10A to 13A, wherein the mixture production step of step (I) is carried out in the presence of a solvent. Claim 15A. A method for producing a cell-containing microfiber material according to claim 8A, comprising: step (I): a mixture production step of mixing a water-insoluble polymer material that swells when microfibrillated with a cell-adhering polymer material to obtain a mixture; step (II): a microfibrillation step of obtaining microfibers from the mixture obtained in the mixture production step; and step (III): a step of contacting cells with the microfibers obtained in the microfibrillation step. Claim 16A. A cell-immobilizing microfiber material according to any one of items 1A to 7A, wherein the average diameter of the microfibers is 0.3 to 20 μm. Item 17A. A cell-immobilizing microfiber material according to any one of items 1A to 7A, wherein the cell uptake rate after 16 hours is 90 to 100%.Item 18A. A cell-immobilizing microfiber material according to any one of items 1A to 7A, wherein the number of immobilized cells per 4 mg is 100,000 or more on day 2 of culture and 400,000 or more on day 15 of culture.

[0015] Item 1B. A microfiber material for cell immobilization, comprising microfibers containing (A) a water-insoluble polymer material that swells when microfiberized, and (B) a cell-adhering polymer material. Item 2B. The microfiber material for cell immobilization according to Item 1B, wherein the water-insoluble polymer material that swells when microfiberized (A) is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 3B. The microfiber material for cell immobilization according to Item 2B, wherein the water-insoluble polymer material that swells when microfiberized (A) is at least one compound selected from the group consisting of cellulose acetate materials, polyketone materials, and polyethylene vinyl alcohol materials. Item 4B. The cell-immobilizing microfiber material according to item 1B, wherein the (B) cell-adhering polymer material is at least one polymer material selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin. Item 5B. The cell-immobilizing microfiber material according to item 4B, wherein the average diameter of the microfibers in the cell-immobilizing microfiber material is 0.3 μm to 20 μm. Item 6B. The cell-immobilizing microfiber material according to item 1B, wherein the (A) water-insoluble polymer material that swells when microfiberized has a water retention rate of 0.01 g or more per gram of microfiber. Item 7B. The cell-immobilizing microfiber material according to item 6B, wherein the water retention rate per gram of microfiber is 0.1 g or more and 17 g or less. Item 8B. The cell-immobilizing microfiber material according to item 1B, wherein the mass ratio of (A) a water-insoluble polymer material that swells when microfiberized to (B) a cell-adhering polymer material is 0.1 to 2.5:1. Item 9B. The cell-immobilizing microfiber material according to item 1B, wherein the average diameter of the microfibers is 0.3 μm to 20 μm. Item 10B. The cell-immobilizing microfiber material according to item 1B, wherein the cell uptake rate after 16 hours in contact with a cell suspension is 90% or more. Item 11B. The cell-immobilizing microfiber material according to item 10B, wherein the number of immobilized cells per 4 mg is 100,000 or more on day 2 of culture and 400,000 or more on day 15 of culture.Item 12B. A cell-containing microfiber material comprising the cell-immobilizing microfiber material described in Item 1B, and further comprising cells. Item 13B. The cell-containing microfiber material according to Item 12B, wherein the cells are animal cells. Item 14B. The cell-containing microfiber material according to Item 13B, wherein the animal cells are at least one selected from the group consisting of mesenchymal stem cells, neural stem cells, iPS cells, fibroblasts, vascular endothelial cells, nerve cells, β-cells, and hepatocytes. Item 15B. A cell sheet comprising the cell-containing microfiber material described in Item 12B. Item 16B. The cell sheet according to Item 15B, for cartilage regeneration, skin regeneration, or bone regeneration. Item 17B. A method for producing a microfiber material for cell immobilization, comprising: step (I): a mixture production step of mixing (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adherent polymer material to obtain a mixture; and step (II): a microfibrillation step of obtaining microfibers from the mixture obtained in the mixture production step. Item 18B. The method according to item 17B, wherein the mixture production step of step (I) is carried out in the presence of a solvent. Item 19B. A method for producing a cell-containing microfiber material, comprising: step (I): a mixture production step of mixing (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adherent polymer material to obtain a mixture; step (II): a microfibrillation step of obtaining microfibers from the mixture obtained in the mixture production step; and step (III): a step of contacting a cell suspension containing animal cells with the microfibers obtained in the microfibrillation step to immobilize the animal cells on the microfibers. Item 20B. Use of the cell-immobilizing microfiber material described in Item 1B in the manufacture of cell sheets for regenerative medicine.

[0016] Item 1C. A microfiber material for cell immobilization, comprising microfibers containing (A) a water-insoluble polymer material that swells when microfiberized, and (B) a cell-adhering polymer material, wherein the components (A) and (B) are mixed within the microfibers, and the water retention rate per gram of the microfibers is 0.01 g or more. Item 2C. A microfiber material for cell immobilization according to Item 1C, wherein the water-insoluble polymer material that swells when microfiberized (A) is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups. Item 3C. A microfiber material for cell immobilization according to item 1C or item 2C, wherein the (A) water-insoluble polymer material that swells when microfibrillated is at least one compound selected from the group consisting of cellulose acetate, polyketone, and polyethylene vinyl alcohol. Item 4C. A microfiber material for cell immobilization according to any one of items 1C to 3C, wherein the (B) cell-adhering polymer material is at least one compound selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin. Item 5C. A microfiber material for cell immobilization according to any one of items 1C to 4C, wherein the mass ratio of the (A) water-insoluble polymer material that swells when microfibrillated to the (B) cell-adhering polymer material is 0.1 to 2.5:1. Item 6C. A cell-immobilizing microfiber material according to any one of items 1C to 5C, wherein the average diameter of the microfibers is 0.3 μm to 20 μm. Item 7C. A cell-containing microfiber material comprising a cell-immobilizing microfiber material according to any one of items 1C to 6C and animal cells immobilized on the microfiber material. Item 8C. A cell-containing microfiber material according to item 7C, wherein the animal cells are at least one selected from the group consisting of mesenchymal stem cells, neural stem cells, iPS cells, fibroblasts, vascular endothelial cells, nerve cells, β cells, and hepatocytes.Item 9C. A cell-containing microfiber material as described in Item 7C or Item 8C, wherein the number of immobilized cells per 4 mg is 100,000 or more on day 2 of culture and 400,000 or more on day 15 of culture. Item 10C. A cell sheet for regenerative medicine, comprising a cell-containing microfiber material as described in any one of Items 7C to 9C. Item 11C. A method for producing a cell-immobilizing microfiber material, comprising: (a) a step of mixing (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adhering polymer material in a solvent to obtain a mixed solution; and (b) a step of microfibrillating the mixed solution to obtain microfibers in which component (A) and component (B) are mixed. Item 12C. A method for producing a cell-containing microfiber material, wherein step (b) is performed by electrospinning, the applied voltage is 5 to 35 kV, and the discharge rate is 0.01 to 2 mL / min. 13C. A method for producing a cell-containing microfiber material, comprising: (a) a step of obtaining microfibers by the method described in 11C or 12C; and (c) a step of contacting the microfibers with a cell suspension containing animal cells, allowing the microfibers to absorb the cell suspension by swelling, and immobilizing the animal cells on the microfibers. 14C. Use of a cell-containing microfiber material according to any one of 7C to 9C in cartilage regeneration, skin regeneration, bone regeneration, blood vessel repair, or nerve repair. 15C. A cell-containing microfiber material according to any one of 7C to 9C for use in the treatment of osteoarthritis.

[0017] Furthermore, the inventions of the products defined in this disclosure, specifically those related to the manufacturing process, are described using product-by-process claims because it is currently impossible or impractical to specify all of their components or their structure.

[0018] According to this disclosure, it is possible to provide a cell-immobilizing microfiber material that allows cells to be immobilized deep into the microfiber material in a short time without loss simply by dropping them onto the microfiber material, while simultaneously enabling high-density cell growth. Furthermore, it has been shown that the microfiber material of this disclosure maintains the supply of oxygen and nutrients due to appropriate voids between the microfibers, thereby preventing cell death due to overcrowding, maintaining good cell activity, and enabling cell proliferation and exosome production.

[0019] Figure 1 is a schematic diagram showing an example of the process of immobilizing cells by contacting them with a microfiber material for cell immobilization. Figure 2 is a scanning electron microscope (SEM) image of the microfiber materials of Comparative Examples 1 to 6. Figure 3 is the Fourier transform infrared spectroscopy (FT-IR) spectrum of the microfiber materials of Example 1 and Reference Examples 1 to 3. Figure 4 is a scanning electron microscope (SEM) image of the microfiber material of Example 1. Figure 5 is a scanning electron microscope (SEM) image of the cell-containing microfiber material of Example 1 with cells immobilized on it after 15 days of culture. Figure 6 is a scanning electron microscope (SEM) image of the microfiber materials of Comparative Examples 7 and 8. Figure 7(a-1) is a graph showing the diameter distribution of the microfiber material of Example 13 (EVG50), and (a-2) is a scanning electron microscope (SEM) image of the microfiber material of Example 13 (EVG50). Figure 8(a) is a photograph of the microfiber material (20 weeks) of Example 13 (EVG50) in which human mesenchymal stem cells were immobilized; (b) is a photograph of cartilage tissue (3 weeks) in which human mesenchymal stem cells were immobilized on EVG50, stained with Alisian blue (cartilage tissue is stained blue); and (c) is a scanning microscope image of the surface (c-1) and interior (c-2) of cartilage tissue (3 weeks) in which human mesenchymal stem cells were immobilized on EVG50.

[0020] This disclosure relates to a microfiber material for cell immobilization, comprising (A) a water-insoluble polymer material that swells when microfibrillated (hereinafter sometimes referred to as "component (A)") and (B) a cell-adherent polymer material (hereinafter sometimes referred to as "component (B)"). In particular, the microfiber material for cell immobilization contains microfibers in which (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adherent polymer material coexist. In this specification, "microfiber" refers to a fiber with a diameter on the order of nanometers to micrometers. Therefore, the microfiber includes nanofibers with a diameter on the order of nanometers (1 nm or more and less than 1 μm) and microfibers with a diameter on the order of micrometers (1 μm or more and less than 1 mm). The diameter of the microfiber is preferably 0.1 μm (100 nm) to 50 μm, and more preferably 0.3 μm (300 nm) to 20 μm.

[0021] Measurement of Fiber Diameter Here, the diameter of the microfibers can be measured, for example, by the method described below. Microfibers stacked on a collector are imaged using a scanning electron microscope (SEM) at a predetermined magnification (for example, a magnification between 1000 and 3000 times). Then, a specific number of microfibers (for example, 100) are selected from the image, and the diameter of the selected microfibers is measured using software such as ImageJ. The arithmetic mean of the measured diameters of the specific number of microfibers is then taken as the average diameter. Note that the diameter is the diameter of the cross-section perpendicular to the longitudinal direction of the microfiber. If the cross-section of the microfiber is not circular, the maximum width of the cross-section can be considered as the diameter and measured. Also, microfibers that are entangled with each other are excluded from the measurement.

[0022] (A) Component: A water-insoluble polymer material that swells when microfibrillated. (A) A water-insoluble polymer material that swells when microfibrillated means that the polymer material used as the raw material is hardly soluble in water (slightly soluble in water) in particulate or film form, but after microfibrillation, when the microfibrillations swell, it takes in water between the microfibrillations (retains moisture). As the (A) water-insoluble polymer material that swells when microfibrillated, a compound containing at least one group (structure) selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups can be used. An ester group is a group represented as -(C=O)O-. A ketone group is a group represented as -(C=O)-. A hydroxyl group is a group represented as -OH. An ethylene group is -CH ２ CH ２ - refers to a group or ethylene unit represented by -. Examples of the water-insoluble polymer material having swelling properties when microfibrillated include (A1) cellulose acetate material, (A2) polyketone material, and (A3) polyethylene vinyl alcohol material. The water-insoluble polymer material having swelling properties when microfibrillated may be used alone or two or more may be used in appropriate mixtures.

[0023] (A1) Cellulose acetate material is a biomass material obtained by chemically modifying (esterifying) the hydroxyl groups in cellulose, which is derived from non-edible plants such as wood fibers and cotton, with acetic acid, and is a compound having ester groups that is highly safe for living organisms. Therefore, cellulose acetate has not only ester groups but also chemical structures derived from cellulose. (A1) There are no particular limitations on the cellulose acetate material, and examples include diacetylcellulose, triacetylcellulose, and co-esters containing cellulose acetate (cellulose acetate-cellulose propionate (CAP), cellulose acetate-cellulose butyrate (CAB), etc.), with diacetylcellulose being preferred. Here, cellulose acetate is also called cellulose acetate. (A1) Cellulose acetate material may be used alone or two or more types may be mixed as appropriate.

[0024] (A2) The polyketone material is a new polymer material with excellent environmental properties synthesized from carbon monoxide and olefins (ethylene, propylene, etc.). There is no particular limitation on this polyketone material. For example, polyketone compounds having a ketone group and an ethylene group, such as those represented by the following general formula (1), can be mentioned. Specifically, examples of this polyketone include a linear polymer in which a divalent organic group derived from a carbonyl group (C=O) and an ethylenically unsaturated compound or a divalent organic group formed by linking two or more of such organic groups are alternately bonded, and it is usually represented by the general formula (1).

[0025]

[0026] In the formula (1), A is a divalent organic group derived from an ethylenically unsaturated compound. m is an integer of 1 to 6. n is an integer of 2 or more, preferably an integer of 2 to 6000. Such polyketones are known and can usually be obtained by polymerizing carbon monoxide and an ethylenically unsaturated compound.

[0027] There is no particular limitation on the ethylenically unsaturated compound. For example, α-olefins having 2 to 12 carbon atoms such as ethylene, propylene, 1-butene, isobutylene, 1-pentene; dienes such as butadiene, isoprene, 2-chlorobutadiene-1,3 or their halides; vinylidene compounds such as vinylidene chloride or their halides; vinyl esters such as vinyl acetate, vinyl chloroacetate, vinyl dimethylacetate, vinyl trimethylacetate or their halides; vinyl halides such as tetrafluoroethylene, chloroethylene; vinyl acetals such as ketene methyl (vinyl) acetal; vinyl ketones such as vinyl methyl ketone, vinyl ethyl ketone; styrene or its derivatives such as styrene, chlorostyrene, α-methylstyrene; acrylic acid and methacrylic acid, and their esterified products, amidated products, nitrided products and acid halides; vinyl esters of unsaturated carboxylic acids such as vinyl hexenoate, vinyl crotonate can be mentioned.

[0028] The ethylenically unsaturated compound is preferably an α-olefin having 2 to 12 carbon atoms, more preferably a linear α-olefin having 2 to 6 carbon atoms, and even more preferably ethylene alone, or containing ethylene and propylene. Therefore, as the polyketone, ethylene-carbon monoxide copolymer and ethylene-propylene-carbon monoxide copolymer are preferred.

[0029] The ethylenically unsaturated compound may be used alone or in combination of two or more. When two or more ethylenically unsaturated compounds are used, it is preferable to use ethylene in combination with a linear α-olefin having 3 to 6 carbon atoms, particularly propylene. When ethylene and a linear α-olefin having 3 to 6 carbon atoms are used, the molar ratio of ethylene to the linear α-olefin having 3 to 6 carbon atoms is preferably greater than 1, and more preferably between 2 and 30.

[0030] The melting point of polyketones is typically 175 to 300°C, preferably 210 to 270°C. The intrinsic viscosity number (IV) of polyketones measured in m-cresol using a standard capillary viscometer is typically 0.5 to 10 dl / g, preferably 0.8 to 4 dl / g.

[0031] Specific examples of polyketones include, for instance, a linear alternating polyketone terpolymer of carbon monoxide, ethylene, and other ethylenically unsaturated compounds (e.g., propylene) as described in U.S. Patent No. 4,880,903. There are no particular limitations on the method for producing polyketones; for example, they can be produced by polymerizing carbon monoxide and the aforementioned ethylenically unsaturated compounds using known methods. Details of the production method are described, for example, in Japanese Patent Publication No. 47-32100, Japanese Patent Publication No. 5-87527, and Japanese Patent Publication No. 6-13608.

[0032] Commercially available polyketones can be used. For example, AKRO-PLASTIC's product name AKROTEK:PK-HM and Shell Chemical's product name Carilon can be used.

[0033] (A2) The polyketone material may be used alone or may be appropriately mixed and used in two or more kinds.

[0034] (A3) The polyethylene vinyl alcohol material is a synthetic resin also called EVOH (Eval), ethylene vinyl alcohol copolymer, etc., and is a compound having a hydroxyl group and an ethylene group. EVOH has excellent gas barrier properties and is used in various applications such as food packaging and fuel tanks. This polyethylene vinyl alcohol (EVOH) is a copolymer of ethylene and vinyl alcohol. Polyethylene vinyl alcohol can also be referred to as an ethylene-vinyl alcohol copolymer or an ethylene-vinyl alcohol copolymer. The chemical structure of EVOH has the following repeating units, and these units are repeatedly arranged alternately, for example, it can be represented as (C ２ H ４ )-[CH ２ CH(OH)])-. - Ethylene unit: -CH ２ -CH ２ - - Vinyl alcohol unit: -CH ２ -CH(OH)- As the arrangement of ethylene and vinyl alcohol in the copolymer, for example, random copolymerization, block copolymerization, alternating copolymerization, graft copolymerization, etc. can be mentioned. Polyethylene vinyl alcohol can be produced, for example, by saponifying an ethylene-vinyl acetate copolymer resin obtained from a vinyl acetate monomer and ethylene. Commercially available products of polyethylene vinyl alcohol include the "Soarnol (registered trademark)" series manufactured by Mitsubishi Chemical Corporation, the "Eval (registered trademark)" series manufactured by Kuraray Co., Ltd., etc. (A3) The polyethylene vinyl alcohol material may be used alone or may be appropriately mixed and used in two or more kinds.

[0035] As the (A) water-insoluble polymer material having swelling properties when microfibrillated, cellulose acetate, polyketone, and polyethylene vinyl alcohol are preferable because of their biocompatibility.

[0036] The fine fibers obtained by microfibling the water-insoluble polymer material that has swelling properties when microfibrillated as described above (A) can incorporate water between the fine fibers. For example, the water retention rate per gram of fine fiber is usually 0.01 g or more, with no particular upper limit, preferably 0.1 to 30 g, more preferably 1 to 25 g, and particularly preferably 3 to 20 g. The water retention rate can be rephrased as water retention capacity (g - water / g - fiber).

[0037] Water retention (g / g): For example, a sample (4 mg) weighed in a dry state is immersed in 1 mL of deionized water for 1 hour, then removed and allowed to stand for 30 minutes. The water retention (g - water / g - fiber) is calculated by subtracting the dry mass from the wet mass of the sample after 30 minutes of standing, and dividing by the dry mass. The measurement is performed at least three times, and the average value is calculated.

[0038] Furthermore, when the non-water-soluble polymer material that swells when microfibrillated (A) is subjected to cell uptake treatment on the microfibrillations after microfibrillation, the cell uptake rate after 16 hours is usually 5% or more, preferably 80-100%, and more preferably 95-100%. Cell uptake rate (%) [16-hour value] For example, the initial seeding cell count (N1) is dropped onto the microfibrillations, and after 16 hours, the number of unuptaken cells in the tube (N2) is measured with a cell counter, and the cell uptake rate is calculated using the following formula: Cell uptake rate (%) = {(N1-N2) / N1} × 100 (n≧3)

[0039] (B) Component: Cell-adhering polymer material (B) The cell-adhering polymer material is not particularly limited and includes, for example, naturally derived protein-based materials such as gelatin and collagen; synthetic polypeptide-based materials such as polyglutamic acid, polylysine, polyarginine, and polyornithine; and extracellular matrix (ECM) protein-based materials such as fibronectin and laminin.

[0040] Gelatin is extracted by applying heat to collagen, the main component of connective tissue such as animal skin, bones, and tendons. Gelatin is mainly composed of linear polymers (proteins) of amino acids. Bovine gelatin is preferably used. Collagen is one of the proteins that mainly make up the dermis, ligaments, tendons, bones, and cartilage of vertebrates. Collagen is the main component of the extracellular matrix of multicellular animals. Human recombinant collagen can also be used as collagen. Polyglutamic acid is a type of polypeptide that uses glutamic acid as the polymerization unit. Polyglutamic acid is a high-molecular-weight polymer made by linearly linking glutamic acid, a type of amino acid. Polylysine (ε-poly-L-lysine, EPL) is a high-molecular-weight natural homopolymer of L-lysine, one of the essential amino acids, and is produced by bacterial fermentation. Polyarginine is a polypeptide in which multiple arginine molecules are linked together by peptide bonds. The number of arginine molecules linked together is not particularly limited. Polyornithine is a compound composed of many ornithine molecules linked together. Ornithine is an amino acid that plays a particularly important role in the liver, where it detoxifies ammonia as part of the urea cycle. Fibronectin is a cell adhesion glycoprotein found in blood, cell surfaces, and the extracellular matrix of tissues. Laminin is a large protein that constitutes the basement membrane of the extracellular matrix.

[0041] As described above, gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin are proteins or peptides and all have peptide bonds (peptide groups). Therefore, the cell-adhesive polymer material (B) can be rephrased as a peptide group-containing polymer material. Gelatin, collagen, polylysine, polyglutamic acid, and fibronectin are preferred as the cell-adhesive polymer material (B), and gelatin and polyglutamic acid are more preferred. The cell-adhesive polymer material (B) may be used alone or by mixing two or more as appropriate.

[0042] In particular, the cell immobilization microfiber material of the present disclosure preferably comprises (A) cellulose acetate, polyketone, or polyethylene vinyl alcohol, and (B) gelatin or polyglutamic acid; more preferably, (A) cellulose acetate and polyketone, or polyethylene vinyl alcohol, and (B) gelatin or polyglutamic acid; and even more preferably, (A) cellulose acetate and polyketone, and (B) gelatin or polyglutamic acid; or (A) polyethylene vinyl alcohol, and (B) gelatin.

[0043] The mixing ratio of component (A) and component (B) is not particularly limited. For example, component (A) and component (B) can be mixed in a mass ratio of typically 0.01 to 10:1, preferably 0.05 to 5:1, and more preferably 0.1 to 2.5:1. When two or more compounds are used as component (A), the total amount of each compound can be adjusted as appropriate so that it matches the above mixing ratio. For example, when (A1) cellulose acetate and (A2) polyketone are used as component (A), cellulose acetate and polyketone can be mixed in a mass ratio of typically 0.2 to 8:1, preferably 0.5 to 5:1, and more preferably 1.5 to 1.8:1.

[0044] Method for manufacturing microfiber material for cell immobilization A microfiber material for cell immobilization can be manufactured by the following steps: Step (I): A mixture manufacturing step in which (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adherent polymer material are mixed to obtain a mixture; and Step (II): A microfibrillation step in which microfibers are obtained from the mixture obtained in the mixture manufacturing step. Steps (I) and (II) may be performed with step (II) following step (I), or steps (I) and (II) may be performed simultaneously.

[0045] Step (I) (Mixture Manufacturing Step) The first step is to obtain a mixture by mixing (A) a water-insoluble polymer material that swells when microfibrillated and (B) a cell-adhesive polymer material. Preferably, the water-insoluble polymer material (A) that swells when microfibrillated is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups, and more preferably at least one compound selected from the group consisting of (A1) cellulose acetate material, (A2) polyketone material, and (A3) polyethylene vinyl alcohol material. Preferably, the water-insoluble polymer material (A) that swells when microfibrillated can be at least one compound selected from the group consisting of cellulose acetate, polyketone, and polyethylene vinyl alcohol.

[0046] The mixture manufacturing step (I) described above is preferably carried out in the presence of a solvent.

[0047] The solvent is not particularly limited as long as it dissolves components (A) and (B) and evaporates during the microfiberization stage to form fibers. Examples of the solvent include water; acetonitrile; alcohols such as methanol, ethanol, n-propanol, isopropanol, 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), benzyl alcohol, and phenol; carboxylic acids such as formic acid and acetic acid; esters such as methyl formate and methyl propionate; ketones such as acetone, methyl ethyl ketone, hexafluoroacetone, and cyclohexanone; ethers such as diethyl ether and 1,2-dimethoxyethane; chlorocarbons such as carbon tetrachloride, chloroform, dichloromethane (methylene chloride), and trichloroethane; and carbonized water such as cyclohexane, benzene, and toluene. Examples of solvents include: amides such as N,N-dimethylformamide and N,N-dimethylacetamide; sulfur-containing compounds such as dimethyl sulfoxide and sulfolane; carbonates such as ethylene carbonate, propylene carbonate, butylene carbonate, dimethyl carbonate, diethyl carbonate, and ethyl methyl carbonate; and heterocyclic compounds such as pyridine, 1-methyl-2-pyrrolidone, N-methylmorpholine-N-oxide, 1,3-dimethyl-2-imidazolidinone, 3-methyloxazolidine-2-one, tetrahydrofuran, 2-methyltetrahydrofuran, 1,3-dioxolane, 1,4-dioxane, and 1,4-butyrolactone. The solvent may be used alone or as a mixture of multiple solvents. When a solvent is added, the amount of solvent is typically 10 to 1000 parts by mass, preferably 20 to 700 parts by mass, and more preferably 30 to 500 parts by mass, per 1 part by mass of component (A).

[0048] Component (A) and component (B) may be dissolved in the same solvent or in different solvents. If steps (I) and (II) are performed simultaneously, component (A) and component (B) may be dissolved in different solvents. However, if step (II) is performed after step (I), it is preferable to dissolve component (A) and component (B) in the same solvent.

[0049] When step (II) is performed after step (I), the mixture obtained in step (I) is a polymer solution obtained by dissolving component (A) and component (B) in a solvent. In addition to component (A) and component (B) described above, the polymer solution may contain any other component as long as it does not hinder the formation of microfibers. Examples of such optional components include surfactants, adhesives, electrolytes, etc.

[0050] While there are no particular limitations on the surfactant, surfactants used in the field of biochemistry are preferred. Examples of such surfactants include nonionic surfactants. Examples of nonionic surfactants include polyethylene oxide and octylphenol ethoxylate (poly(oxyethylene)octylphenol ether). A commercially available example of octylphenol ethoxylate is the "Tritoon® X" series from Dow Chemical. When adding a surfactant, it is preferable to add it in an amount of 0.5 to 10% by mass relative to the total amount of component (A) and component (B) in the polymer solution.

[0051] The adhesive is not particularly limited as long as it can bond the manufactured fine fibers together and is soluble in the solvent of the polymer solution. Examples of adhesives include hot-melt resin adhesives, elastomer adhesives, acrylic adhesives, epoxy adhesives, and vinyl adhesives. Examples of elastomer adhesives include polychloroprene rubber, styrene-butadiene rubber, butyl rubber, acrylonitrile-butadiene rubber, ethylene-propylene rubber, chlorosulfonated polyethylene rubber, and epichlorohydrin rubber. When an adhesive is added, the manufactured fine fibers are bonded together at the contact points, so when obtaining the fine fibers in the form of a nonwoven fabric, a strong, flexible nonwoven fabric with less fuzzing due to friction can be obtained. When an adhesive is added, it is preferable to add 0.5 to 10% by mass relative to the total amount of component (A) and component (B) in the polymer solution.

[0052] The electrolyte is not particularly limited as long as it is soluble in the polymer solution and ionizes in the polymer solution. Examples of electrolytes include sodium chloride, calcium chloride, magnesium chloride, sodium carbonate, sodium bicarbonate, sodium dihydrogen carbonate, and magnesium carbonate. By adding an electrolyte, the charge density on the surface of the polymer solution can be increased, and as a result, the spinnability can be improved. When adding an electrolyte, it is desirable to add it in an amount that does not cause salting out of components (A) and (B) in the polymer solution, and it is preferable to add 0.5 to 10% by mass relative to the total amount of components (A) and (B) in the polymer solution.

[0053] The polymer solution may be prepared by dissolving component (A) and component (B) separately in a solvent and mixing the two solutions, or by adding and dissolving the other component to a solution in which either component is dissolved in a solvent.

[0054] When steps (I) and (II) are performed simultaneously, instead of dissolving components (A) and (B) separately in solvents and mixing the resulting solutions, the steps of preparing solutions for component (A) and component (B) separately and mixing them to obtain a mixture are performed simultaneously with the microfiberization step. The solvents used to dissolve components (A) and (B) may be the same solvent or different solvents. The temperature of the solvent during mixing is not particularly limited, for example, usually 0 to 40°C, preferably 10 to 35°C, and more preferably 15 to 30°C. The time for dissolving components (A) and (B) in the solvent, or for adding one of the components to a solution in which the other is dissolved and dissolving it, is not particularly limited, for example, usually 10 hours or more, preferably 20 hours or more. Furthermore, during preparation, heating, stirring, etc. may be performed as appropriate, as long as components (A) and (B) are not denatured. When stirring, the stirring speed is not particularly limited, but is typically 5 to 3000 rpm, preferably 10 to 1000 rpm, and more preferably 50 to 500 rpm. The stirring speed may be constant, or it may be changed in stages. For example, the stirring speed may be gradually increased. The concentration of component (A) in the polymer solution is typically 0.1 to 10% by mass, preferably 0.15 to 5% by mass, and more preferably 0.2 to 3% by mass. The concentration of component (B) in the polymer solution is typically 0.1 to 10% by mass, preferably 0.15 to 5% by mass, and more preferably 1 to 2% by mass. The mixing ratio of component (A) and component (B) in the polymer solution is typically 0.01 to 10:1 by mass, preferably 0.05 to 5:1, and more preferably 0.1 to 2.5:1.

[0055] Step (II): Microfiberization Step (II) is a microfiberization step in which microfibers are obtained from the mixture obtained in the mixture manufacturing step. The microfiberization method is not particularly limited as long as it is a method that can produce microfibers. Examples of microfiberization methods include electrospinning and methods using microfluidic channels, with electrospinning being preferred. When step (II) is performed after step (I), by performing electrospinning using the polymer solution, fibers (nanofibers or microfibers) having a fine and uniform diameter on the nanoscale or microscale can be produced. Using electrospinning yields a fiber material with a large specific surface area, and it is also easy to obtain it as a nonwoven fabric, making it suitable for application as a high-performance and easy-to-handle material for cell immobilization.

[0056] Electrospinning is a technique that involves applying a high voltage to a polymer solution and using the resulting electric field to extract and spin the polymer solution. Electrospinning can be performed by well-known means. Specifically, a voltage is applied between a nozzle filled with the polymer solution (spinning solution) and a collector (substrate), and the polymer solution is discharged from the nozzle to collect a nonwoven fabric-like fine fiber film (sheet) on the collector. The conditions for performing electrospinning are not particularly limited and can be appropriately adjusted according to the type of polymer solution, the intended use of the resulting fine fibers, etc. A commercially available general-purpose electrospinning apparatus can be used as the device. The electrospinning conditions in the method disclosed herein can be general conditions. For example, the applied voltage is usually 5 to 35 kV, preferably 10 to 25 kV, and more preferably 15 to 20 kV. The discharge rate (syringe rate) is usually 0.01 to 2 mL / min, preferably 0.1 to 1 mL / min, and 0.05 to 0.2 mL / min. The polymer solution prepared in step (I) can be dispensed using a syringe or the like at the applied voltage and dispensing speed within the aforementioned range. The spinning environment is preferably a relative humidity of 30 to 70% and a temperature of 10 to 40°C, but it is not necessary to control these conditions strictly. When steps (I) and (II) are performed simultaneously using the electrospinning method, an electrospinning apparatus with two nozzles can be used. The solution of component (A) is filled into one nozzle and the solution of component (B) into the other nozzle, and by dispensing them, mixing and microfiber formation can be performed simultaneously. When components (A) and (B) are dissolved in different solvents, in step (II), a double nozzle can be used, for example, by dispensing the solution of component (A) from the inside and the solution of component (B) from the outside. When performing steps (I) and (II) simultaneously using a microfluidic channel, the solution of component (A) and the solution of component (B) are set in each channel of a microfluidic channel having two channels, and mixing and microfibrillation can be performed simultaneously by discharging from each channel.

[0057] Step (II) can yield fibers (fine fibers) with diameters ranging from nanometers to micrometers. When microfiber formation is performed using the electrospinning method, a nonwoven fine fiber film (sheet) is obtained. The average diameter of the fine fibers is typically 0.1 μm to 50 μm, preferably 0.3 μm to 20 μm. If a solvent is used in step (I), some solvent may remain in the obtained fine fiber material.

[0058] The cell-immobilization microfiber material is a material containing microfibers obtained by the manufacturing method described above, and can be in any form as long as it contains microfibers. Examples of forms include fibers, woven fabrics, nonwoven fabrics (sheets, membranes, etc.), and particles. When the cell-immobilization microfiber material is in the form of particles, the average particle diameter is usually 1 mm to 10 cm, preferably 2 mm to 5 cm. The microfibers obtained by microfibulating the cell-immobilization microfiber material have a water retention rate of usually 0.01 g or more per gram of microfiber, with no particular upper limit, preferably 0.1 to 30 g, more preferably 1 to 25 g, and particularly preferably 3 to 20 g. When the microfibers obtained by microfibulating the cell-immobilization microfiber material are subjected to cell uptake treatment, the cell uptake rate after 16 hours is usually 90% or more, preferably 90 to 100%, and more preferably 95 to 100%. When cells are immobilized on the microfibers obtained by microfiberizing the aforementioned cell immobilization microfiber material, the number of immobilized cells per 4 mg of microfiber is 100,000 or more on day 2 of culture, 400,000 or more on day 5 of culture, 400,000 or more on day 15 of culture, and 400,000 or more on day 20 of culture.

[0059] Cell-containing microfiber material: The cell-containing microfiber material contains the cell-immobilization microfiber material described above, as well as cells. The cell-containing microfiber material can be rephrased as "a microfiber composite material in which cells are immobilized." If the cell-immobilization microfiber material is manufactured in the presence of a solvent, the cell-containing microfiber material may contain the solvent.

[0060] Method for producing cell-containing microfiber material A method for producing cell-containing microfiber material comprises: Step (I): A mixture production step of mixing (A) a water-insoluble polymer material that swells when microfiberized and (B) a cell-adhering polymer material to obtain a mixture; Step (II): A microfiberization step of obtaining microfibers from the mixture obtained in the mixture production step; and Step (III): A step of bringing cells into contact with the microfibers obtained in the microfiberization step.

[0061] Steps (I) and (II) of the method for producing cell-containing microfiber material are the same as steps (I) and (II) of the method for producing cell-immobilizing microfiber material described above. Therefore, the explanation is omitted here.

[0062] Step (III): Cell Contact Step Step (III) is a step in which cells are brought into contact with the microfibers obtained in the microfiber formation step. By bringing cells into contact with the microfibers obtained in step (II), the cells are immobilized on the microfibers, and a cell-containing microfiber material is obtained. Therefore, step (III) can also be called the cell immobilization step.

[0063] The cells used in step (III) are animal cells. The animal cells may be either adherent or non-adherent cells. Examples of animal cells include stem cells such as mesenchymal stem cells, neural stem cells, and iPS cells, as well as somatic cells such as fibroblasts, vascular endothelial cells, nerve cells, β-cells, and hepatocytes. Furthermore, the cells used may be from any species; for example, cells from humans, mice, rats, or cattle can be used.

[0064] Figure 1 shows a schematic diagram illustrating an example of the process of fixing cells by contacting them with the cell-fixing microfiber material in step (III). The process of fixing cells by contacting them with the cell-fixing microfiber material will be described below in accordance with Figure 1, but the procedure for fixing cells by contacting them is not limited to this. (A) Prepare the microfiber material to be used for cell fixation. The photograph showing the state of this step (A) is the top left photograph in Figure 1. The microfiber material may be the microfiber sheet obtained in steps (I) and (II) above, or it may be cut to an appropriate size before use. Suspend the cells in a culture medium to prepare a cell suspension. The concentration of the cell suspension is not particularly limited. The concentration of the cell suspension can be any, for example, 10 ５ ~10 ９ Examples include pieces / mL.

[0065] (B) A cell suspension equivalent to or less than the swollen mass of the microfibers is dropped onto the microfiber material. The swollen mass of the microfibers is the amount of water contained per unit mass of microfibers after water has been absorbed into the microfibers and then removed. The photograph showing the state at this stage (B) is the second photograph from the left in the top row of Figure 1. (C) The microfiber material swells, and all of the cell suspension is incorporated into the microfiber material or the interior (deep part) of the microfiber material. The photograph showing the state at this stage (C) is the third photograph from the left in the top row of Figure 1. (D) After being left for 4 hours or more, the cells incorporated into the microfiber material adhere to the microfiber material. The photograph showing the state at this stage (D) is the fourth photograph from the left in the top row of Figure 1. (E) By culturing while changing the culture medium, the cells proliferate and become dense. The photograph showing the state at this stage (E) is the first photograph from the right in the top row of Figure 1.

[0066] Thus, in the method for producing cell-containing microfiber material of this disclosure, suspended cells are added to the microfibers. As a result, the cell suspension is spontaneously incorporated by the swelling properties of the microfibers, and by allowing it to stand, the cells can be spontaneously and uniformly adhered to the microfibers. In conventional methods using hollow fibers, in order to immobilize cells on the hollow fibers, it was necessary to add the cell solution, let it stand for more than 8 hours to allow the cells to adhere to the upper part of the hollow fiber, then rotate it slightly and let it stand for more than 8 hours to allow the cells to adhere to a different part of the hollow fiber, and repeat this process many times. In order to immobilize cells on microcarriers, it was necessary to put the cell solution into a container containing microcarriers, stir it gently for a short time, let it stand for more than 8 hours to allow the cells to adhere, stir it again briefly, let it stand for more than 8 hours to allow the cells to bind to a different surface, and repeat this process many times. Similarly, in conventional methods using nanofibers, it was necessary to repeatedly stir the cell solution gently for a short time and let it stand for several hours to allow the cells to adhere in order to incorporate the cells. In contrast, the method for producing cell-containing microfiber material according to this disclosure does not require operations such as rotation or stirring to immobilize the cells, as all of the dropped cell solution is incorporated into the microfibers, thus eliminating the need to repeat the same process. However, there is no particular problem even if operations such as rotation or stirring are performed when immobilizing the cells.

[0067] Therefore, by using the method for producing cell-containing microfiber materials described above, cells can be immobilized onto the microfiber material inexpensively and efficiently. Furthermore, cell productivity is increased by performing high-density culture, making the cell-immobilization microfiber material disclosed herein highly valuable for industrial use. Moreover, using the cell-immobilization microfiber material disclosed herein, cell-containing microfiber materials can be produced simply by dropping a cell suspension and leaving it for several hours. Since no other method has yet been found that can produce cell-containing microfiber materials such as cell sheets in such a short time, this method is considered to have high industrial value.

[0068] The cell-immobilizing microfiber material and the cell-containing microfiber material can be used in regenerative medicine. The cell-immobilizing microfiber material can be used, for example, as a material that contains cells. The cell-immobilizing microfiber material can be used, for example, as a hydrogel (a material that encloses and protects cells while providing a suitable environment), a porous scaffold (for example, a material with a structure that allows cells to easily penetrate, used for bone or cartilage regeneration), or microfibers (for example, microfibers for supporting cells, used for blood vessel or nerve regeneration). Furthermore, the cell-immobilizing microfiber material of this disclosure can be crushed or finely cut into particles, or cells immobilized on such material can be injected by means of injection or the like and used as a bone graft material aimed at bone regeneration. In addition, the cell-containing microfiber material obtained by the above-described method can be used as a cell-containing material used in regenerative medicine. Examples of cell-containing materials used in regenerative medicine include cell sheets. This disclosure includes cell sheets containing the cell-containing microfiber material. The cell sheet containing the aforementioned cell-containing microfiber material can be used in many medical fields as a therapeutic cell sheet, such as a cell sheet to accelerate the repair of burns or dermatitis, a cell sheet to repair bone or cartilage, a cell sheet with immobilized mesenchymal stem cells (stromal stem cells) to treat allergic diseases such as allergies and atopic dermatitis; a cell sheet with immobilized pancreatic cells to treat type 1 diabetes, a cell sheet with immobilized vascular cells to repair and treat damaged blood vessels, a cell sheet with immobilized nerve cells or glial cells to repair or treat neurological diseases such as dementia or nerve damage, and a cell sheet with immobilized pluripotent stem cells differentiated into organoids to treat various diseases.

[0069] The immobilized cells contained in the cell-containing microfiber material may be used directly for the production of useful substances, or the cells may be propagated by changing the culture medium. When culturing cells, the cell-containing microfiber material may or may not be stirred. Oxygen may also be supplied if necessary. The cell-containing microfiber material includes the entire microfiber material containing immobilized cells, substances secreted from the immobilized cells (e.g., exosomes), microfiber material containing cells that have been propagated from the immobilized cells, substances secreted from the propagated cells, etc. The microfibers after microfibulation of the cell-immobilizing microfiber material can produce exosomes. In this case, the amount of exosomes produced is not particularly limited, for example, 0.1 to 100 × 10 per 8 mg of microfibers. ９ The particles are of a certain size, preferably 1 to 80 × 10 ９ The particles are of a certain size, and more preferably 3 to 80 × 10 ９ It is at the particle level.

[0070] The present disclosure will be described in more detail below with reference to examples, but the technical scope of the present disclosure is not limited to these examples.

[0071] Materials (A) Non-water-soluble polymer materials that swell when microfiberized: Polystyrene (Sigma-Aldrich, 182427-25G), Polyethylene vinyl alcohol (Sigma-Aldrich, 414093-100G), Polyketone (Asahi Kasei Corporation, product name: #360; Mitsui Chemicals Fine, Inc., M330A), Polylactic acid (Nature3D), Cellulose acetate (Dicellulose acetate, Daicel Corporation, L44), Polyethylene vinyl acetate (Sigma-Aldrich, 340502-250G)

[0072] (B) Cell-adhering polymer materials: Gelatin (derived from bovine bone, manufactured by Fujifilm Wako Pure Chemical Corporation, 071-06291), Sodium alginate (manufactured by Fujifilm Wako Pure Chemical Corporation, 193-13321), Polyglutamic acid (manufactured by Fujifilm Wako Pure Chemical Corporation)

[0073] Solvents: Dimethylacetamide (DMAC) (Sigma-Aldrich), Acetone (Sigma-Aldrich), Chloroform (Sigma-Aldrich), N,N-Dimethylformamide (Sigma-Aldrich), 1,1,1,3,3,3-Hexafluoroisopropanol (Sigma-Aldrich) Surfactants: Polyethylene oxide (Merck, 182029-250G), Octylphenol ethoxylate (Nacalai Tesque, product name: Triton® X-100)

[0074] Preliminary Test Example 1 (Investigation of Component (A)) Comparative Example 1 A polymer solution was prepared by dissolving polystyrene in N,N-dimethylformamide to a concentration of 20% by mass. 20 mL of the obtained polymer solution was placed in a 20 mL syringe (product name: Terumo Syringe 20 mL SS-20ESZ, manufactured by Terumo Corporation), set in an electrospinning device (product name: NEU, manufactured by Kato Tech Co., Ltd.), and dispensed at a syringe speed of 0.05 to 0.2 mL / min under a voltage of 15 to 20 kV to produce fine fibers. This operation was repeated twice, and comparative fine fiber material 1 was produced from a total of 40 mL of spinning solution.

[0075] Comparative Example 2 A comparative fine fiber material 2 was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with polyethylene vinyl alcohol and dissolved in 1,1,1,3,3,3-hexafluoroisopropanol to a concentration of 6% by mass to prepare a polymer solution.

[0076] Comparative Example 3 A comparative fine fiber material 3 was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with polyketone (PK) and dissolved in 1,1,1,3,3,3-hexafluoroisopropanol to prepare a polymer solution at a concentration of 5% by mass.

[0077] Comparative Example 4 A comparative fine fiber material 4 was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with polylactic acid and dissolved in chloroform to a polymer solution of 6% by mass.

[0078] Comparative Example 5 A comparative fine fiber material 5 was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with cellulose acetate and dissolved in a solvent prepared by mixing dimethylacetamide (DMAC) and acetone in a 1:3 ratio to make a polymer solution of 16% by mass.

[0079] Comparative Example 6 A comparative fine fiber material 6 was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with polyethylene vinyl acetate and dissolved in chloroform to prepare a polymer solution at a concentration of 16 wt%.

[0080] The film thickness of the comparative microfiber materials prepared in Comparative Examples 1 to 6 was measured using a film thickness gauge (Peacock Model H). Scanning electron microscope (SEM) images were taken. Each comparative microfiber material was coated with platinum using a platinum coater (JEOL JFC-1600) and observed using a scanning electron microscope (Thermo Fisher Scientific Phenom ProX G6). The obtained SEM images are shown in Figure 2. Diameter measurement The diameter was measured from the SEM images using ImageJ software (n=50). The average diameter of the microfibers is shown in Table 1.

[0081]

[0082] As shown in Table 1 above, the average diameter of the comparative microfibers in Comparative Examples 1 to 5 was 0.8 to 3.2 μm. The average diameter of the microfiber material in Comparative Example 6 (polyethylene vinyl acetate) was 10 μm.

[0083] Test Example 1 (Measurement of Moisture Retention Rate) Each comparative microfiber material prepared was cut with scissors into 4 mg pieces (approximately 5 to 12 mm x approximately 5 to 12 mm, film thickness approximately 0.2 to 0.7 mm). Hereinafter, these will be referred to as microfiber material fragments or microfiber fragments. 1 mL of deionized water was added to a 1.5 mL microtube containing 4 mg of microfiber material fragments, and after standing for 1 hour, the microfiber fragments were removed and left for 30 minutes. Subsequently, the moisture retention amount (water content) was calculated by measuring the mass of deionized water retained within the microfiber fragments. The water content per microfiber (g - water / g - microfiber) is shown in Table 2.

[0084]

[0085] Table 2 of the results shows that the microfiber materials of Comparative Example 2 (polyethylene vinyl alcohol), Comparative Example 3 (polyketone), and Comparative Example 5 (cellulose acetate) had high moisture retention rates. From this, it was found that polyethylene vinyl alcohol, polyketone, and cellulose acetate do not retain any moisture in the powder form used in the experiment, but when microfiberized, they swell and change to properties that allow them to retain moisture.

[0086] Test Example 2 (Cell Immobilization Test) Microfiber material fragments of Comparative Examples 1 and 3-6 were placed in 1.5 mL microtubes and sterilized by autoclaving (120°C, 15 minutes). Microfiber material fragments of Comparative Example 2 (polyethylene vinyl alcohol) were sterilized with 70% ethanol and then aseptically dried in a 65°C dryer. For the immobilization experiment, TKD2 vascular endothelial cells (JCRB Cell Bank, IFO50374) were used as cells. The culture medium used was a mixture of Dulbecco's modified Eagle medium containing 10% bovine serum (MP Biomedics) and 10% bovine serum (Gibco, 26140-079) in a 9:1 ratio, to which 1 / 100th volume of penicillin-streptomycin solution (Fujifilm Wako Pure Chemical Industries, Ltd., 168231-9) was added (hereinafter referred to as DMEM medium). 1 × 10 ５ 100 μL of a suspension of TKD2 cells in DMEM medium was pipetteed into a microtube containing microfiber material fragments and left for 16 hours. The number of cells that remained unincorporated was then measured using a cell counter (WC2-100, Waken B-Tech Co., Ltd.), and the cell incorporation rate (%) into the microfibers was calculated. The results are shown in Table 3.

[0087] Furthermore, the microfiber fragments that had been treated to incorporate these cells were transferred to a 48-well plate (manufactured by Iwaki Corporation, 1830-048) to which cells could not adhere, 1 mL of DMEM medium was added, and CO2 was added at 37°C. ２Cells were cultured in an incubator for two days. Then, on the second day, the number of cells immobilized within the microfiber fragments was analyzed using a Cell Container Kit (Fujifilm Wako Pure Chemical Industries, Ltd., 34107761). A solution prepared by adding 15 μL of WST (Water-Soluble Tetrazolium; a cell counting reagent containing water-soluble tetrazolium salt)-8 solution to 300 μL of DMEM medium was placed in wells containing microfibers and left at 37°C for 20–120 minutes. A 50 μL sample was taken and placed in a 96-well plate (Areaspectraplate, 6052630), and the absorbance at 450 nm was measured using a microplate reader (INFINITE, PLEX). The number of immobilized cells was then calculated from a previously obtained calibration curve of cell count and absorbance. Furthermore, the microfiber fragments to which these cells were bound were cultured for 20 days, changing the DMEM medium every 2-3 days, until the increase in cell number stopped. Then, on days 15 and 20, the number of cells immobilized within the microfiber fragments was analyzed using Cell Counting Kit-8 (manufactured by Dojin Chemical Research Institute Co., Ltd.). The results are shown in Table 3. Here, the number of immobilized cells (cells / 4 mg) can be calculated by measuring the absorbance using WST-8, etc., and applying it to a calibration curve created by pre-measuring the relationship between cell number and absorbance. The number of immobilized cells was measured over time (days 2, 15, and 20).

[0088]

[0089] As shown in Table 3, the microfiber materials of Comparative Example 2 (polyethylene vinyl alcohol (EVOH)), Comparative Example 3 (polyketone (PK)), and Comparative Example 5 (cellulose acetate (CA)) were able to rapidly incorporate cells together with the culture medium into the deep layers of the microfiber material. On the other hand, it was found that the microfiber materials of Comparative Example 1 (polystyrene), Comparative Example 4 (polylactic acid), and Comparative Example 6 (polyethylene vinyl acetate) were almost unable to incorporate the cell suspension into the deep layers. Furthermore, from the results of the number of immobilized cells in Table 3, 1 × 10⁶ ５Despite supplying individual cells to microfiber material fragments, in Comparative Examples 1-6, the cells either did not bind to the microfiber material, or were initially incorporated but then fell off. Nearly 90% of the cells leaked out, with only a small number of cells binding to the microfibers slowly proliferating. The amount of cells immobilized became almost constant across all microfibers after 15 days. These results indicate that when a microfiber sheet is made using only water-insoluble polymer materials that swell when microfibrillated, the cell suspension can be incorporated deep into the microfiber sheet along with the cells. However, it was found that if the microfiber sheet only has swelling properties, the incorporated cells do not adhere sufficiently and leak out.

[0090] In Example 1, a microfiber material was prepared using cellulose acetate and polyketone as component (A) and gelatin as component (B). 20 mL of the obtained polymer solution was placed in a 20 mL syringe (Terumo Corporation, Terumo Syringe 20 mL SS-20ESZ), set in an electrospinning device (Kato Tech Co., Ltd., product name: NEU), and dispensed at a syringe speed of 0.1 mL / min under a voltage of 20 kV to produce the microfiber material of Example 1 (hereinafter sometimes referred to as CPG microfiber material). This operation was repeated twice to produce a total of 40 mL of microfiber material from the polymer solution. The FT-IR spectrum of the microfiber material of Example 1 was measured using a Fourier transform infrared spectrophotometer (Thermo Fisher Scientific, product name: Nicolet iS5). The results are shown in Figure 3.

[0091] For comparison, polymer solutions were prepared by dissolving polyketone, cellulose acetate, and gelatin in 1,1,1,3,3,3-hexafluoroisopropanol to concentrations of 1.05% by mass, 1.76% by mass, and 1.29% by mass, respectively. Microfiber materials for Reference Example 1 (polyketone), Reference Example 2 (cellulose acetate), and Reference Example 3 (gelatin) were then prepared in the same manner as in Example 1. The FT-IR spectra of the microfiber materials for Reference Examples 1 to 3 were then measured in the same manner as described above, and the results are shown in Figure 3.

[0092] Furthermore, SEM images of Example 1 were taken in the same manner as in Comparative Examples 1 to 6 above, and the average diameter of the microfibers was calculated from these SEM images. The SEM images are shown in Figure 4, and the average diameters are shown in Table 4. Then, the moisture retention rate was measured using the method described in Test Example 1 above. The results are shown in Table 4.

[0093]

[0094] Reference Example 1 in Result Figure 3 shows 2900 cm －１ There is a specific peak for polyketones, and in reference example 2, at 1250 cm⁻¹ －１ There is a specific peak for cellulose acetate, and in Reference Example 3, at 3200-3600 cm⁻¹, －１ A specific peak for gelatin is present. Since Example 1 in Figure 3 has all of these peaks, it was confirmed that the microfiber material of Example 1 is a mixture of component (A), cellulose acetate and polyketone, and component (B), gelatin. From Table 4 and Figure 4, the microfiber material of Example 1 contained uniform microfibers with an average diameter of 1.3 μm. Furthermore, the microfiber material of Example 1, which contains components (A) and (B), had a high water retention rate. From this, it was found that the microfiber material of Example 1, which contains component (A), cellulose acetate and polyketone, has a high ability to swell and retain water.

[0095] Using the microfiber material of Example 1 (4 mg, film thickness approximately 0.60 mm), Test Example 2 (cell immobilization test) was performed, and the cell uptake rate and the number of immobilized cells were measured. The results are shown in Table 5. Figure 5 shows a photograph of cells immobilized on the microfiber material of Example 1 on day 15 of culture. For comparison, microfiber materials of Comparative Examples 7 to 10 were prepared, and the cell uptake rate and the number of immobilized cells were measured using the method of Test Example 2. The results are shown in Table 5. For the microfiber materials of Comparative Examples 7 and 8, SEM images were taken in the same manner as for Comparative Examples 1 to 6, and the SEM images are shown in Figure 6.

[0096] (B) Comparative Example 7 of a single-component microfiber material A microfiber material was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with gelatin and dissolved in 1,1,1,3,3,3-hexafluoroisopropanol to a mass of 3%, in order to prepare a polymer solution.

[0097] Comparative Example 8 A microfiber material was prepared in the same manner as in Comparative Example 1, except that polystyrene was replaced with sodium alginate, sodium alginate was dissolved in water to a concentration of 13.5% by mass, polyethylene oxide, a surfactant, was dissolved in water to a concentration of 4% by mass, the sodium alginate aqueous solution and the polyethylene oxide aqueous solution were mixed in a ratio of 3:7, and then octylphenol ethoxylate, a surfactant, was added to a concentration of 0.1% by mass to prepare a polymer solution.

[0098] Comparative Example 9 of a microfiber material in which microfibers of component (A) are coated with component (B) 1 mL of 1% gelatin solution (Stemsure, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to a 1.5 mL microtube containing a fragment of the microfiber material of Comparative Example 3 (polyketone), and the mixture was left to stand for 12 hours to coat the microfibers with gelatin. All of the gelatin solution was removed to obtain a microfiber material in which component (A) (polyketone) was coated with component (B) (gelatin).

[0099] Comparative Example 10 A microfiber material was obtained in the same manner as in Comparative Example 9, except that the microfiber material fragment of Comparative Example 3 (polyketone) was replaced with the microfiber material fragment of Comparative Example 5 (cellulose acetate), by coating component (A) (cellulose acetate) with component (B) (gelatin).

[0100]

[0101] As shown in Results Table 5, the microfiber material of Example 1 not only had high water retention capacity but was also able to rapidly incorporate cells into the microfibers along with the culture medium. Furthermore, the microfiber material of Example 1 was 120% immobilized after 2 days, indicating nearly 100% immobilization. As shown in Tables 3 and 5, the amount of immobilized cells remained almost constant for all microfiber materials used after 15 days, and the immobilized cell density was highest for the microfiber material of Example 1. On the other hand, the microfiber material of Comparative Example 7 (microfiber material of gelatin, component (B)) dissolved in 1-2 days, making immobilized culture impossible. The microfiber of Comparative Example 8 (microfiber material of sodium alginate, component (B)) was pretreated with 1 mL of 100 mM sodium chloride solution to undergo a crosslinking reaction. After that, it was sterilized by placing it in 1 mL of 70% ethanol. The removed microfiber material showed dissolution between fibers. Thus, the microfiber material of Comparative Example 7 (gelatin) and the microfiber material of Comparative Example 8 (sodium alginate) could not be used for the immobilization of animal cells. Furthermore, the microfibers of Comparative Examples 9 and 10 (microfiber materials in which microfibers of component (A) alone were coated with component (B)) showed low numbers of immobilized cells on the 2nd day of culture, the 15th day of culture, and the 20th day of culture, similar to the results of the microfiber materials of Comparative Examples 1 to 6. Thus, in Comparative Examples 9 and 10, where microfibers were prepared using only component (A) and then coated with component (B) to improve adhesion, adhesion was improved, but the low swelling rate resulted in the loss of supplied cells. From the above, it was found that microfiber materials having a mixture of component (A) and component (B) have high cell uptake and cell immobilization effects.

[0102] In Example 2, a microfiber material was prepared using cellulose acetate and polyketone as component (A) and polyglutamic acid as component (B). Specifically, the microfiber material was prepared in the same manner as in Example 1, except that polyglutamic acid was used instead of gelatin as component (B), the voltage was changed to 15-20 kV, and the syringe speed was changed to 0.05-0.2 mL / min.

[0103] In Example 3, a fine fiber material was prepared using polyethylene vinyl alcohol (EVOH) as component (A) and gelatin as component (B). Specifically, a spinning solution was prepared by dissolving gelatin and polyethylene vinyl alcohol (EVOH) in 1,1,1,3,3,3-hexafluoroisopropanol to concentrations of 1.5% by mass and 0.24% by mass, respectively. The fine fiber material was then prepared in the same manner as in Example 1, except that the voltage was changed to 15-20 kV and the syringe speed to 0.05-0.2 mL / min.

[0104] Test Example 3 (Cell Immobilization Test) 1 x 10 ５ 100 μL of a suspension of 100 TKD2 cells in DMEM medium was pipetted onto 4 mg of the microfiber fragments of Example 2 or 3 and left for 16 hours. Then, the microfiber material fragments of Example 2 or 3 were transferred to a 48-well plate (Iwaki Corporation, 1830-048) that would not allow cells to adhere, and 1 mL of DMEM medium was added and heated at 37°C CO2. ２ The cells were cultured in an incubator. The number of cells immobilized within the microfiber material fragments of Example 2 or 3 after 2 and 5 days was analyzed using the same method as in Test Example 2. The number of immobilized cells is shown in Table 6.

[0105]

[0106] Results: The microfiber materials of Examples 2 and 3, which contained a mixture of component (A) and component (B), were found to have a high cell immobilization effect, similar to the microfiber material of Example 1, as they were able to bind supplied cells to the microfibers without loss.

[0107] Test Example 4 (Cell Densification Test) Using the thin-film microfiber material of Example 1, an experiment was conducted to increase cell density by reducing the amount of microfiber relative to the supplied cells. The microfiber material of Example 1 (4 mg, film thickness approximately 0.60 mm) was divided into two sheets, and one of the sheets (0.3 mg, film thickness 0.25 mm) (hereinafter referred to as the microfiber of Example 4) was sterilized in an autoclave (120°C, 15 minutes). 1 × 10 ５ The TKD2 vascular endothelial cells were suspended in 25 μL of DMEM medium and dropped onto the microfiber material of Example 4 using a pipette, allowing the entire cell solution to be absorbed.２ After culturing in an incubator for 6 hours with 25 μL of DMEM medium added to prevent drying, and then culturing for 8 hours, cells were immobilized on these microfibers using the same procedure as in Test Example 2. 1 × 10⁶ cells were added to the microfiber material of Example 4. ５ 50 μL of a suspension of individual TKD2 vascular endothelial cells in DMEM medium was pipetteed onto a microfiber material to swell it, allowing all cells to be absorbed. The material was then left for 16 hours. Subsequently, the microfiber material was transferred to a 48-well plate for suspension cells (Iwaki Corporation, 1830-048), and the cells were grown while changing 1 mL of DMEM medium. The number of fixed cells was analyzed using Cell Counting Kit-8 (Dojin Chemical Research Institute Co., Ltd.). The number of fixed cells is shown in Table 7.

[0108]

[0109] As a result, the microfiber material of Example 4, which has a thickness of 0.25 mm, was similar to the microfiber material of Example 1, which has a thickness of 1 mm, in that it was supplied with 1 × 10⁻¹⁶ ５ No individual cells leaked out of the microfibers and were lost. Furthermore, the microfiber material of Example 4 had an average of 9 × 10⁶ cells per 0.3 mg. ５ pieces, that is, 3 x 10 ９ It was confirmed that cells were immobilized at an extremely high density of 1 x 10⁻¹⁶ cells / g-fibers. Furthermore, Non-Patent Literature 1 mentioned above states that Cytodex-1 (registered trademark), currently one of the best porous microimmobilization carriers, achieves 1 x 10⁻¹⁶ cells / g-fibers. ７ It has been reported that cells can be immobilized at a rate of cells / gram, and by immobilizing them on the microfibers of Example 4, which contain cellulose acetate, polyketone, and gelatin, it became possible to immobilize approximately 300 times more cells than with the aforementioned Cytodex-1 (registered trademark).

[0110] Test Example 5 (Exosome Productivity Confirmation Test) In Test Example 2, cells were cultured for 20 days and immobilized onto microfibers. The microfiber materials containing cellulose acetate, polyketone, and gelatin (Examples 5 and 6), which had a high cell density, were used. The productivity of exosomes was then investigated using the following materials: a polylactic acid microfiber material with a relatively high cell density (Comparative Example 11), a microfiber material made by coating polyketone microfibers with gelatin (Comparative Example 12), and a microfiber material made by coating cellulose acetate microfibers with gelatin (Comparative Example 13). Examples 5 and 6 used the microfiber materials produced in Example 1. Comparative Example 11 used the microfiber material produced in Comparative Example 4. Comparative Example 12 used the microfiber material produced in Comparative Example 9. Comparative Example 13 used the microfiber material produced in Comparative Example 10. The culture medium used was DMEM medium containing 10% exosome-free FBS (SBI Exo-FBS-50A-1) (hereinafter referred to as exoFBS-DMEM medium).

[0111] For the microfiber materials of Examples 5, Comparative Examples 11, 12, and 13, 0.5 mL of exoFBS-DMEM was added to 4 mg of microfiber material fragments, and the culture medium was changed after incubation for 2 days. This procedure was repeated three times. For microfibers containing cellulose acetate, polyketone, and gelatin, 0.25 mL of exoFBS-DMEM was added to 4 mg of microfiber material, and the culture medium was changed after incubation for 1 day. This procedure was repeated three times (Example 6). After each incubation, the culture medium was filtered through a 0.2 μm sterile filter (Kurabo Industries Ltd., S-1302), and 500 μL of the filtrate was centrifuged at 10,000 × g for 10 minutes using a centrifugal filter (Merck Ltd., UFC501096) that separates molecules with a molecular weight of 10,000. 500 μL of PBS, from which particles were removed by centrifugation at 10,000 × g for 5 minutes using a centrifugal filter capable of separating particles with a molecular weight of 30,000, was added and washed twice. The resulting exosome solution was diluted 20 to 80 times with ultrapure water, and immediately subjected to nanoparticle tracking analysis (NTA) assay using a nanoparticle analyzer (NanoSight NS500-HSBZ-K, manufactured by Quantum Design Japan Co., Ltd.) to measure the number of particles per volume. The number of exosome particles obtained is shown in Table 8.

[0112]

[0113] As a result, the exosome productivity when immobilized on the microfiber material of Example 5, which contains cellulose acetate, polyketone, and gelatin, was far higher than when immobilized on microfiber materials other than those of Example 1 (microfiber materials of Comparative Examples 11, 12, and 13). When 0.25 mL of culture medium was added per microfiber material containing cellulose acetate, polyketone, and gelatin (Example 6), the yield was 40 × 10⁶. ９ We confirmed that a very high productivity of particles / mL / day can be achieved. Furthermore, Non-Patent Literature 2, mentioned above, states that if HUVEC cells, another type of vascular endothelial cell, are not fixed, the productivity over 6 days is 0.4–0.5 × 10⁻⁶. ９ It has been reported that it produces exosomes at a rate of 1 / mL, and by immobilizing it on the microfibers of Example 6, which contain cellulose acetate, polyketone, and gelatin, the exosome productivity was increased by approximately 100 times, and the production rate was increased by approximately 600 times.

[0114] Test Example 6 (Immobilization Test of Cells with Low Cell Adhesion) The immobilization of mesenchymal stem cells with low cell adhesion onto microfiber material was investigated. The cells used were MSCs-OUMS human mesenchymal stem cells differentiated by the inventors from OUMS-36T-2 human embryonic fibroblasts (JCRB1006.2), and KBM ADSC-5 medium (Cosmo Bio Co., Ltd., 16030060) was used as the mesenchymal stem cell retention medium. Example 7 used the microfiber material produced in Example 1 above. Comparative Example 14 used the microfiber material produced in Comparative Example 4 above. Comparative Example 15 used the microfiber material produced in Comparative Example 5 above.

[0115] 0.3 mL each of 0.1% fibronectin solution (Merck, F01895) and 1% gelatin solution (Fujifilm Wako Pure Chemical Industries, STEMsure) were added dropwise to cell culture dishes (Eppendorf, 0030700015) and left for 1 hour. Afterward, these solutions were removed to prepare dishes coated with 0.1% fibronectin solution and dishes coated with 1% gelatin solution. 5 × 10 ４When individual MSCs-OUMS mesenchymal stem cells were seeded in these three types of dishes and cultured for three days, the cells coated with 0.1% fibronectin solution and 1% gelatin solution measured 1.5 × 10⁶. ５ Although the cells were divided into individual cells, when no coating was applied, the cells did not proliferate at all. This confirmed that mesenchymal stem cells also bind to gelatin. Furthermore, the same immobilization procedure as in Test Example 2 was performed using polylactic acid microfiber fragments (Comparative Example 14) and cellulose acetate microfiber fragments (Comparative Example 15), which showed relatively good results in Test Example 2, as well as microfiber fragments containing cellulose acetate, polyketone, and gelatin (Example 7), which showed good results in Test Example 2. 1.0 × 10 ５ A 25 μL suspension of individual MSCs-OUMS mesenchymal stem cells was dropped onto 2 mg of microfiber material fragments in a 48-well plate, and then 25 μL of KBM ADSC-5 medium was added. The mixture was incubated for 6 hours, ensuring it did not dry out. Using tweezers, the microfiber material was transferred to a new 48-well plate, and the mixture was incubated at 37°C CO2, with 1 mL of KBM ADSC-5 medium being replaced. ２ The cells were cultured in an incubator. Table 9 shows the number of fixed cells on day 2, day 15, and day 20 of culture.

[0116]

[0117] The results showed that the microfiber materials of Comparative Examples 14 and 15 did not allow cells to bind and did not provide any immobilization at all. In contrast, it was confirmed that the microfiber material of Example 7 could effectively immobilize even mesenchymal stem cells with low cell adhesion.

[0118] Test Example 7 (Cell Adhesion Test) The adhesion of various cells to a microfiber material containing cellulose acetate, polyketone, and gelatin was investigated. The cells used were 3T3-L1 mouse adipose progenitor cells (ATCC CL-173), J774.1 mouse macrophage cells (JCRB Cell Bank 0018), 3T3-E1 osteoblasts (RIKEN BRC RCB1126), C2C12 myoblasts (ATCC CRL1772), and NMuLi mouse hepatocytes (ATCC CRL-1638). Examples 8 to 12 used the microfiber material produced in Example 1 above.

[0119] Each 1 x 10 ５ 100 μL of a suspension of individual cells in DMEM medium was pipetted onto a microfiber material fragment containing 4 mg of cellulose acetate, polyketone, and gelatin, and left for 16 hours. The microfiber fragment was then transferred to a 48-well plate (Iwaki Corporation, 1830-048) to prevent cell adhesion, 1 mL of DMEM medium was added, and the plate was heated at 37°C CO2. ２ The cells were cultured in an incubator for two days. The number of cells immobilized within the microfiber material fragments was analyzed using the same method as in Test Example 2. The number of immobilized cells is shown in Table 10.

[0120]

[0121] Results: When using microfiber materials containing components (A) (cellulose acetate and polyketone) and (B) (gelatin), it was confirmed that all cells could bind to the microfibers without any cell loss.

[0122] Test Example 8 (Cell Preservation and Recovery Test) The microfiber material of Example 4 (cellulose acetate + polyketone + gelatin, film thickness 0.25 mm) was investigated to determine whether it could be used for cell preservation and recovery. A fragment of the microfiber material of Example 4 (film thickness 0.25 mm), on which cells had been immobilized for 10 days in Test Example 4, was placed in a sterile tube for freezing, and 500 μL of cryoprotection solution (Takara Bio Inc., Cellbanker 1) was added. The tube was placed in a cell freezing container (Waken B-Tech Co., Ltd., Cool Cell LX) and stored in a -80°C freezer for one week. The sterile tube for freezing was removed and thawed in a 37°C constant temperature bath for one minute. The microfiber fragment was removed with sterile tweezers and placed in a 48-well plate for suspension cells (Iwaki Corporation, 1830-048), and cultured for two days while changing 1 mL of DMEM medium. The number of viable cells was measured using the Cell Counting Kit, as in Test Example 2. As a result, the cell viability was 96% of that before freezing. This confirmed that microfiber materials containing cellulose acetate, polyketone, and gelatin can be cryopreserved while cells are immobilized. Furthermore, in Test Example 4, a microfiber material fragment (film thickness 0.25 mm) from Example 4, in which cells were immobilized for 10 days, was placed in a 1.5 mL microtube, and 500 μL of Trp-LE solution (Thermo Fisher Scientific, 12604021) was added. The solution was gently stirred by pipetting, and the cells were detached by leaving it at room temperature for 10 minutes. After that, the mixture was centrifuged at 1000 × g for 3 minutes, and the nanofiber material and supernatant were removed. The cells were then suspended in PBS solution, and the number of cells was measured using a cell counter (FPI Co., Ltd., OneCellCounter). Furthermore, trypan blue staining solution (Nacalai Tesque Co., Ltd., 20577-34) was added to the cell suspension in a 1:1 ratio with trypan blue solution, and the percentage of viable cells was measured using a cell counter. As a result, the cell recovery rate was 82%, and the percentage of viable cells was 97%. This confirmed that microfiber materials containing cellulose acetate, polyketone, and gelatin can also be used for cell recovery.As described above, it has been found that the microfiber material of this disclosure can immobilize cells deep into the microfiber material in a short time without loss simply by dropping cells onto the microfiber material, and at the same time, it can immobilize cells at a high density.

[0123] In Example 13, a microfiber material was prepared using polyethylene vinyl alcohol (EVOH) as component (A) and gelatin as component (B). Specifically, the microfiber material of Example 13 was prepared in the same manner as in Example 1, except that polyethylene vinyl alcohol (EVOH) and gelatin were dissolved in 1,1,1,3,3,3-hexafluoroisopropanol to concentrations of 2.5% by mass and 2.5% by mass, respectively, to prepare a spinning solution. Hereinafter, the microfiber material of Example 13 may also be referred to as "EVG50". Similar to the scanning electron microscope (SEM) photography in Preliminary Test Example 1, the microfiber material of Example 13 was coated with platinum, observed with a scanning electron microscope (SEM), and photographs were taken. Furthermore, the diameter of the microfiber material of Example 13 was measured according to the diameter measurement in Preliminary Test Example 1. Figure 7(a-1) shows a graph illustrating the diameter distribution of the microfiber material of Example 13 (EVG50), and Figure 7(a-2) shows a scanning electron microscope (SEM) image.

[0124] As shown in Figure 7(a-1), the average particle size of the fine fiber material in Example 13 (EVG50) was 1.16 μm.

[0125] Test Example 9 (Preparation of cartilage tissue using microfiber material immobilized with human mesenchymal stem cells) We investigated whether the microfiber material of this disclosure can be used as a cell sheet for cartilage regeneration. 100,000 HAdpc-25-Bmi-1-TERT human adipose-derived mesenchymal stem cells (stromal stem cells) (JCRB Cell Bank, JCRB1550) were suspended in 20 μL of KBM ADSC-5 medium (Cosmo Bio, 16030060) to prepare a cell culture medium. This cell culture medium was dropped onto a fragment (approximately 5 mm x 5 mm) of the microfiber material of Example 13 (EVG50) to allow the cell suspension to be absorbed into the microfiber material fragment, and then subjected to CO2 at 37°C. ２The material was incubated in an incubator for 2 hours. After that, to prevent the microfibers from drying out, 20 μL of KBM ADSC-5 medium was added to the microfiber material fragments as needed, and the material was incubated at 37°C CO2. ２ The cells were cultured in an incubator for a further 4 hours to immobilize them onto microfiber material fragments. These microfiber material fragments containing the cells were then transferred to a 48-well plate using tweezers, 1 mL of KBM ADSC-5 medium was added, and the plate was heated at 37°C CO2. ２ The cells were cultured in an incubator for 16 hours. Subsequently, the microfiber material fragments containing the cells were cultured for 10 days while changing the KBM ADSC-5 medium. Then, cartilage tissue was prepared by culturing the cells in 1 mL of chondrocyte differentiation medium (Funakoshi, Chondrocyte Differentiation Induction Kit BMK-R010) for 3 weeks while changing the medium. A photograph of the prepared cartilage tissue is shown in Figure 8(a). The obtained cartilage tissue was stained using a cartilage staining kit (Alcian Blue staining kit, Funakoshi, BMK-R011). A photograph of the stained cartilage tissue is shown in Figure 8(b). Furthermore, after fixing and dehydrating the obtained cartilage tissue, the cartilage tissue was coated with platinum in the same manner as the scanning electron microscope (SEM) imaging in preliminary test example 1, and the surface and interior of the fibers were observed using a scanning microscope. A surface SEM image is shown in Figure 8(c-1), and an internal SEM image is shown in Figure 8(c-2).

[0126] As shown in Figure 8(a), the fabricated cartilage tissue maintained a consistent shape due to the microfibers. Furthermore, the photograph shown in Figure 8(b) shows that the tissue was uniformly stained blue with Alcian blue, confirming the presence of cartilage tissue. In addition, Figure 8(c-1) shows that the surface of the microfibers was covered with cartilage tissue, and Figure 8(c-2) shows that the interior of the microfibers had elasticity due to the presence of a sponge-like structure covered with cartilage tissue. From the above, it was found that the microfiber material of this disclosure can be used as a cell sheet for creating three-dimensional cartilage tissue for the treatment of chondrodegenerative arthropathy.

[0127] As described above, the microfiber material of this disclosure not only allows for high-density immobilization of cells, but also maintains the supply of oxygen and nutrients due to the appropriate void structure between the microfibers. This prevents cell death due to overcrowding and maintains good cell activity for a long period of time. As a result, it has been confirmed that cell proliferation and long-term continuous production of exosomes are possible, and that the material can be used as a cell sheet for therapeutic purposes.

[0128] The cell-immobilization microfiber material and cell-containing microfiber material disclosed herein are available for use in the field of regenerative medicine.

[0129] This application is based on a Japanese patent application, Japanese Patent Application No. 2025-004827, filed on January 14, 2025. Japanese Patent Application No. 2025-004827 is incorporated herein by reference.

Claims

1. A microfiber material for cell immobilization, comprising: (A) a water-insoluble polymer material that swells when microfibrillated; and (B) a cell-adhering polymer material; and a microfiber material comprising microfibers in which these materials coexist.

2. The cell-immobilizing microfiber material according to claim 1, wherein the (A) water-insoluble polymer material having swelling properties when microfiberized is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups.

3. The cell-immobilizing microfiber material according to claim 1, wherein the (A) water-insoluble polymer material having swelling properties when microfiberized is at least one compound selected from the group consisting of cellulose acetate material, polyketone material, and polyethylene vinyl alcohol material.

4. The cell-immobilizing microfiber material according to claim 3, wherein the (B) cell-adhering polymer material is at least one polymer material selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin.

5. The cell immobilization microfiber material according to claim 4, wherein the average diameter of the microfibers in the cell immobilization microfiber material is 0.3 μm to 20 μm.

6. The cell-immobilizing microfiber material according to claim 1, wherein the water-insoluble polymer material having swelling properties when microfiberized (A) has a water retention rate of 0.01 g or more per gram of microfiber.

7. The cell-immobilizing microfiber material according to claim 1, wherein the mass ratio of (A) a water-insoluble polymer material having swelling properties when microfiberized to (B) a cell-adhering polymer material is 0.1 to 2.5:

1.

8. A cell-containing microfiber material comprising a cell-immobilizing microfiber material according to any one of claims 1 to 7, and further a cell.

9. A cell sheet comprising the cell-containing microfiber material according to claim 8.

10. A method for producing a microfiber material for cell immobilization according to any one of claims 1 to 7, comprising: step (I): a mixture production step of mixing (A) a water-insoluble polymer material having swelling properties when microfiberized with (B) a cell-adhering polymer material to obtain a mixture; and step (II): a microfiberization step of obtaining microfibers from the mixture obtained in the mixture production step.

11. The method for producing a microfiber material for cell immobilization according to claim 10, wherein the water-insoluble polymer material that swells when microfiberized is a compound containing at least one substituent selected from the group consisting of ester groups, ketone groups, hydroxyl groups, and ethylene groups.

12. The method for producing a microfiber material for cell immobilization according to claim 10, wherein the (A) water-insoluble polymer material having swelling properties when microfiberized is at least one compound selected from the group consisting of cellulose acetate material, polyketone material, and polyethylene vinyl alcohol material.

13. The method for producing a microfiber material for cell immobilization according to claim 10, wherein the (B) cell-adhering polymer material is at least one polymer material selected from the group consisting of gelatin, collagen, polyglutamic acid, polylysine, polyarginine, polyornithine, fibronectin, and laminin.

14. The method for producing a microfiber material for cell immobilization according to claim 10, wherein the mixture production step of step (I) is carried out in the presence of a solvent.

15. A method for producing a cell-containing microfiber material according to claim 8, comprising: step (I): a mixture production step of mixing a water-insoluble polymer material having swelling properties when microfiberized with a cell-adhering polymer material to obtain a mixture; step (II): a microfiberization step of obtaining microfibers from the mixture obtained in the mixture production step; and step (III): a step of bringing cells into contact with the microfibers obtained in the microfiberization step.

Images