Method for culturing adipose-derived stem cells and culture substrate for adipose-derived stem cells

Abstract

To provide a method for culturing adipose-derived stem cells that enables efficient stem cell culturing using adipose tissues containing stem cells, and a culture substrate for adipose-derived stem cells used for culturing adipose-derived stem cells.SOLUTION: A method for culturing adipose-derived stem cells comprises a step of culturing adipose tissues containing stem cells on a nonwoven fabric composed of fibers having an average fiber diameter of 0.1-9 μm, and a step of collecting the stem cells proliferated within the nonwoven fabric.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for culturing adipose stem cells and a substrate for culturing adipose stem cells. [Background technology]

[0002] Adipose tissue is distributed near the surface of the body, so it can be harvested in large quantities relatively safely. Therefore, this cellular tissue is cultured to isolate adipose stem cells, which are then applied to regenerative medicine to form bone, cartilage, cardiac muscle cells, and blood vessels. Because adipose tissue contains only small amounts of stem cells, tissue cells containing stem cells are cultured using nonwoven fabric, and the stem cells that migrate to the nonwoven fabric and proliferate are isolated by trypsin treatment.

[0003] For example, Patent Document 1 discloses a nonwoven fabric treated with hydroxyapatite, but this has the problem that adipose tissue adheres to the nonwoven fabric and cannot be easily separated. Patent Document 2 discloses a nonwoven fabric made of a norbornene-based polymer, but this also has the problem that adipose tissue adheres to the nonwoven fabric and cannot be easily separated. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2018 / 097198 [Patent Document 2] International Publication No. 2018 / 066512 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention aims to provide a method for culturing adipose stem cells that uses adipose tissue containing stem cells to efficiently culture stem cells, and an adipose stem cell culture substrate to be used for culturing adipose stem cells. [Means for solving the problem]

[0006] The present inventors have investigated the adhesion of adipose tissue to nonwoven fabrics and found that the average fiber diameter of the fibers making up the nonwoven fabric has a significant effect, and that if the average fiber diameter is 0.1 to 9 μm, the adipose tissue does not strongly adhere to the nonwoven fabric, allowing it to be easily separated, and stem cells also migrate and proliferate within the nonwoven fabric, thereby completing the present invention.Furthermore, they have found that the effect is even greater if the nonwoven fabric has pores with an average pore diameter of 1 to 100 μm.

[0007] That is, the present invention (1) is a method for culturing adipose stem cells, which comprises the steps of culturing adipose tissue containing stem cells on a nonwoven fabric composed of fibers with an average fiber diameter of 0.1 to 9 μm, and recovering the stem cells proliferated in the nonwoven fabric.

[0008] The present invention (2) is the method for culturing adipose stem cells according to the present invention (1), wherein the fibers in the nonwoven fabric are fibers made of a thermoplastic resin.

[0009] The present invention (3) is the method for culturing adipose stem cells according to the present invention (2), wherein the thermoplastic resin is polylactic acid or a copolymer thereof.

[0010] The present invention (4) is the method for culturing adipose stem cells according to any one of the present inventions (1) to (3), wherein the nonwoven fabric has pores with an average pore size of 1 to 100 μm.

[0011] The present invention (5) is the method for culturing adipose stem cells according to any one of the present inventions (1) to (4), wherein the nonwoven fabric is a nonwoven fabric produced by laser melt electrospinning.

[0012] The present invention (6) is an adipose stem cell culture substrate comprising a nonwoven fabric made of fibers with an average fiber diameter of 0.1 to 9 μm.

[0013] The present invention (7) is the adipose stem cell culture substrate according to the present invention (6), which comprises a nonwoven fabric having pores with an average pore size of 1 to 100 μm.

[0014] The present invention (8) is the adipose stem cell culture substrate according to the present invention (6) or (7), wherein the nonwoven fabric is a nonwoven fabric produced by laser melt electrospinning.

[0015] The present invention (9) is an adipose stem cell culture vessel comprising the adipose stem cell culture substrate according to any one of the present inventions (6) to (8). [Effects of the Invention]

[0016] In the culture method of the present invention, adipose tissue is cultured on a nonwoven fabric composed of fibers with an average fiber diameter of 0.1 to 9 μm. This means that the adipose tissue does not adhere strongly to the nonwoven fabric, allowing it to be easily separated. Stem cells also migrate to the nonwoven fabric and proliferate, allowing for efficient culture of adipose stem cells. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram showing an example of a method for spinning a nonwoven fabric used in the adipose stem cell culture substrate of the present invention. [Figure 2] 10 is a photograph of a Taylor cone formed in a heat-melted portion. [Figure 3] Regarding the cultured tissue cultured in Example 1, the left photograph is a photograph before the cell tissue was removed, and the right photograph is a photograph after PBS was sprayed. [Figure 4] Regarding the cultured tissue cultured in Example 2, the left photograph is a photograph before the cell tissue was removed, and the right photograph is a photograph after PBS was sprayed. [Figure 5] Regarding the cultured tissue cultured in Comparative Example 1, the upper left photograph is a photograph before cell tissue removal, the upper right photograph is a photograph after PBS spraying, and the lower photograph is a photograph after manual separation. [Figure 6] 1 is a micrograph of a nonwoven fabric after removal of adipose tissue in Example 1 (scale bar length: 400 μm). [Figure 7] 1 is a micrograph of a nonwoven fabric after removal of adipose tissue in Comparative Example 1 (scale bar length: 400 μm). DETAILED DESCRIPTION OF THE INVENTION

[0018] The method for culturing adipose stem cells of the present invention is characterized by comprising the steps of culturing adipose tissue containing stem cells on a nonwoven fabric composed of fibers with an average fiber diameter of 0.1 to 9 μm, and recovering the stem cells proliferated in the nonwoven fabric.

[0019] Adipose tissue is widely distributed over the body surface and can be harvested in large quantities relatively safely, making autologous tissue-based regenerative therapy possible. Adipose tissue contains not only adipose stem cells, but also adipocytes, fat, collagen, vascular endothelial cells, vascular wall cells, leukocytes, hematopoietic stem cells, and fibroblasts. However, adipose stem cells are present in extremely small amounts and must be cultured for proliferation. When adipose cells are cultured on nonwoven fabric, the stem cells migrate to the fabric and proliferate within it. However, if the adipose tissue strongly binds to the fabric, removal of the adipose tissue becomes difficult and the stem cells contain many impurities. Therefore, multiple purification steps are required for use in regenerative medicine. When cultured on nonwoven fabric composed of fibers with an average fiber diameter of 0.1 to 9 μm, the adipose tissue does not strongly bind to the fabric and can be easily separated, resulting in the production of impurity-free stem cells.

[0020] Examples of the medium include KBM ADSC-1 (containing FBS: Kohjin Bio Co., Ltd.) etc. The medium may also contain additional inducers such as proteins, vitamin components, minerals, etc.

[0021] The culture temperature is preferably 15 to 45° C., more preferably 30 to 40° C. The culture atmosphere has a carbon dioxide concentration of preferably 1 to 15%, more preferably 3 to 10%. The culture period is preferably 7 to 21 days, more preferably 10 to 14 days.

[0022] After culturing, the stem cells that have proliferated in the nonwoven fabric are harvested by removing and washing the adipose tissue with, for example, phosphate buffered saline (PBS), and then harvesting the stem cells from the nonwoven fabric using trypsin. The resulting adipose stem cells can differentiate into cells that form bone, cartilage, cardiomyocytes, blood vessels, etc., and can be used for knee osteoarthritis, bedsore treatment, breast reconstruction, urethral sphincter regeneration, anal sphincter regeneration, liver treatment, kidney treatment, and nasolabial fold treatment.

[0023] The nonwoven fabric used in the present invention is composed of fibers with an average fiber diameter of 0.1 to 9 μm. The average fiber diameter of the nonwoven fabric is preferably 1 to 8 μm, and more preferably 2 to 7 μm. If the diameter is less than 0.1 μm, stem cells cannot migrate into the nonwoven fabric, and if the diameter exceeds 9 μm, adipose tissue will penetrate the nonwoven fabric and become strongly bonded to the adipose tissue.

[0024] The thickness of the nonwoven fabric is not particularly limited, but is preferably 0.01 to 10 mm, more preferably 0.03 to 5 mm, even more preferably 0.05 to 1 mm, and most preferably 0.05 to 0.5 mm. The basis weight of the nonwoven fabric is also not particularly limited, but is preferably 0.1 to 100 g / m 2 is preferable, and 0.5 to 50 g / m 2 More preferably, 1 to 30 g / m 2 is more preferable.

[0025] The pore size distribution of the nonwoven fabric is preferably 1 to 100 μm, more preferably 5 to 75 μm, and even more preferably 10 to 50 μm. If the pore size is less than 1 μm, stem cells cannot migrate into the nonwoven fabric, and if it exceeds 100 μm, adipose tissue will enter the nonwoven fabric and become strongly bonded to the nonwoven fabric.

[0026] The nonwoven fabric may be used by stacking multiple sheets, or may be integrated with other nonwoven fabrics (for example, spunbond nonwoven fabrics), woven or knitted fabrics, films, boards, electrode substrates, etc.

[0027] The material for the fibers of the nonwoven fabric is not particularly limited as long as it is a thermoplastic resin, but thermoplastic resins with biocompatibility and low cytotoxicity are preferred, including polyglycolic acid, polylactic acid (poly-L-lactic acid, poly-D-lactic acid, poly-DL-lactic acid), polylactic acid-polyglycolic acid copolymers, polycaprolactone, polydioxanone, polytrimethylene carbonate, polybutylene succinate, polyethylene succinate, and their copolymers, as well as homopolymers, copolymers, and mixtures of these polymers such as N-methylpyrrolidone, trimethylene carbonate, paradioxanone, and 1,5-dioxepan-2-one. Among these, aliphatic polyesters are preferred, with polyglycolic acid, polylactic acid, and polycaprolactone being more preferred. Polylactic acid or its copolymers are even more preferred due to their excellent biocompatibility and low cytotoxicity, and poly-DL-lactic acid (PDLLA) and its copolymers being particularly preferred.

[0028] The orientation direction of the fibers in the nonwoven fabric is not particularly limited, and examples thereof include uniaxial orientation, biaxial orientation, random orientation, unevenness with coarse and fine fibers, etc. The nonwoven fabric may also be provided with an outer frame.

[0029] The method for producing the nonwoven fabric is not particularly limited, but laser melt electrospinning is preferred because it does not cause contamination that would be an impurity for stem cells. Examples of laser melt electrospinning include the methods described in JP 2015-158026 A, JP 2012-112067 A, and JP 2010-275661 A.

[0030] The nonwoven fabric sheet used in the present invention can be produced, for example, by irradiating a sheet-like material made of a thermoplastic resin with laser light to heat and melt the edge of the sheet-like material, and then creating a potential difference between the heat-melted portion of the sheet-like material and a collecting member, causing the fibers to fly in the direction of the collecting member and be layered on the collecting member. In this case, it is important to use a collecting member having a plurality of through holes as the collecting member, and by using such a collecting member, it is possible to form a nonwoven fabric sheet having a large number of through holes at the same time as molding the nonwoven fabric sheet.

[0031] Nonwoven fabric sheets produced by this method can be obtained without using a solvent, unlike solvent-based electrostatic spinning methods.Furthermore, unlike melt-based electrostatic spinning methods that use a heat source other than laser light, there is no thermal degradation of the thermoplastic resin during spinning, and it is easy to create a clean manufacturing environment free of impurities, resulting in high-quality nonwoven fabric sheets.

[0032] The method for producing the nonwoven fabric sheet will now be described in more detail. Fig. 1(a) is a schematic diagram showing a method for manufacturing the nonwoven fabric sheet. When manufacturing the nonwoven fabric sheet, as shown in Fig. 1(a), a laser beam 12 emitted from a laser source 11 is irradiated via a laser beam scanning means 15 to scan an edge 17a of a sheet-like material 17 made of a thermoplastic resin and held by a holding member 18, and a voltage is applied from a power source 20 to generate a potential difference between the edge 17a and a collecting member 19 arranged opposite the edge 17a of the sheet-like material 17. As a result, as shown in Fig. 1(b), the edge 17a of the sheet-like material 17 is heated and melted by the irradiation of the laser beam 12, and an electric charge is imparted to the heated and melted portion. Then, at the heated and melted portion to which an electric charge has been applied, charges gather on its surface and repel each other, gradually forming a plurality of needle-like protrusions (hereinafter also referred to as Taylor cones) 117, and when the repulsive force of the charges exceeds the surface tension, the molten thermoplastic resin is ejected as fibers from the tip of Taylor cone 117 toward collection member 19 by electrostatic attraction, and flies in the direction of collection member 19. As a result, the elongated fibers are deposited on collection member 19 and collected.

[0033] The collection member 19 is electrically conductive. Specific examples of such collection members include a metal drum, a metal mesh, and a punched metal. A metal drum is a metal roll whose rotation speed can be controlled, and the metal roll may be capable of oscillating in a direction perpendicular to the direction of flight of the fibers. This is because controlling the location where the flying fibers are collected makes it possible to produce a nonwoven fabric sheet with a uniform basis weight.

[0034] In the case of a metal mesh, the metal wires are woven together, resulting in unevenness (undulations) on the collection surface. However, in the case of a metal drum or perforated metal, the collection surface is flat, making it possible to produce a nonwoven fabric sheet with a uniform thickness and opening diameter. Examples of materials for the metal drum, metal mesh, and perforated metal include iron, stainless steel, brass, aluminum, copper, titanium, and the like, as well as plated, surface-treated, or painted metal plates and alloys of the above metals. A nonconductive material may be embedded in the through-holes of the perforated metal. This is because flying fibers avoid the nonconductive material and are collected on the surface of the conductive perforated metal, making it more suitable for producing a nonwoven fabric sheet with a uniform thickness and opening diameter.

[0035] The collection member (or metal member) is preferably grounded, as this improves the handleability of the produced nonwoven fabric sheet.

[0036] The laser beam scanning means 15 is an assembly of optical components for irradiating the edge 17a of the sheet-like material 17 with laser beams to scan the edge 17a, and is composed of a reflecting mirror 13 and a polygon mirror 14. By introducing the laser beam emitted from the laser source 11 via the reflecting mirror 13 into the polygon mirror 14, which rotates at high speed, the laser beam can be uniformly irradiated via the polygon mirror 14 to scan the edge 17a of the sheet-like material 17.

[0037] 1(a), the entire edge of the sheet-like material is irradiated with laser light via polygon mirror 14, but the edge of the sheet-like material may be irradiated with laser light by other methods as long as the entire edge of the sheet-like material can be irradiated with laser light, for example, a galvanometer mirror may be used to irradiate the laser light instead of polygon mirror 14. In the example shown in FIG. 1(a), holding member 18 that holds sheet-like material 17 also functions as an electrode, and when a voltage is applied to holding member 18 by a high-voltage generator that is power source 20, an electric charge is imparted to edge 17a of sheet-like material 17.

[0038] Furthermore, the holding member 18 continuously delivers the sheet-like material 17 toward the collecting member 19 as fibers are discharged from the end 17a of the sheet-like material 17. When the sheet-like material is continuously delivered, the feed speed is not particularly limited, but is typically 0.01 to 150.0 mm / min, preferably 0.05 to 100.0 mm / min, and more preferably 0.1 to 60.0 mm / min. Increasing the speed increases productivity, but if it is too fast, the thermoplastic resin near the laser light irradiation area does not melt sufficiently, making it difficult to spin fibers. On the other hand, if the speed is too slow, the thermoplastic resin may decompose, resulting in low productivity.

[0039] The holding member 18 also has multiple N2 gas outlets 18b arranged in parallel on the side facing the collection member 19, and an N2 gas supply port 18a arranged on the side perpendicular to the surface with the N2 gas outlets 18b. The N2 gas outlets 18b and the N2 gas supply port 18a are connected via internal piping (not shown). During production, N2 gas is introduced through the N2 gas supply port 18a and supplied to the edge of the sheet-like material, thereby preventing oxidative decomposition of the thermoplastic resin. The gas that prevents oxidative decomposition is not limited to N2 gas; it can be any inert gas such as helium or argon, or a non-oxidizing gas such as carbon dioxide. Note that N2 gas does not necessarily need to be supplied when producing the nonwoven fabric sheet.

[0040] 1(a) uses a heated air supply device 22 for supplying heated air to the fiber flight space. By supplying heated air to the fiber flight space in this way, the fiber diameter of the flying fibers can be reduced. In other words, by heating the spinning space, a rapid temperature drop in the fibers being formed can be suppressed, thereby facilitating the elongation or stretching of the fibers and producing a nonwoven fabric sheet made of finer fibers. It should be noted that, when producing the nonwoven fabric sheet, dry air does not necessarily have to be supplied to the fiber flight space.

[0041] The temperature of the flight space depends on the type of sheet-like material and is not particularly limited, but is preferably 20 to 300°C. If the temperature of the space into which the fibers fly is high, the molten thermoplastic resin can be electrospun while remaining in a molten state within the flight space, making it easier to achieve the desired fiber diameter. If the temperature is below 20°C, the fibers flying from the sheet-like material may solidify before they become thinner due to elongation or stretching caused by electrostatic attraction. Conversely, if the temperature exceeds 300°C, the fibers flying from the sheet-like material may thermally decompose and reach the collection member in a molten state, forming a film and not becoming a porous nonwoven fabric sheet.

[0042] Examples of the heating means in the heated air supply device include a heater (such as a halogen heater), etc. The temperature of the heated air can be selected depending on the melting point of the thermoplastic resin, for example, within a range from 50°C or higher to below the ignition point of the thermoplastic resin, but from the viewpoint of spinnability, a temperature below the melting point of the thermoplastic resin is preferred.

[0043] The humidity of the above-mentioned flight space is not particularly limited, but is preferably 50% or higher, more preferably 60% or higher, and particularly preferably 70% or higher. As humidity increases, water molecules adsorb to the fiber surface, forming a thin water layer on the surface, rapidly reducing surface resistance due to the creeping ion conduction effect. The creeping ion conduction effect facilitates electrospinning even with thermoplastic resins that inherently have high surface resistance, and reduces the surface resistance of the fiber surface captured in the collection section, facilitating the formation of a nonwoven fabric sheet with numerous fine openings and multiple through-holes that reflect the shape of the collection member. The concept of the creeping ion conduction effect is described in detail in "Static Electricity Engineering Series 1: Basics of Static Electricity, p. 139-2.2: Creeping Ion Conduction," published by Asakura Shoten Co., Ltd. On the other hand, at humidity levels below 50%, the creeping ion conduction effect is so small that electrospinning is difficult for thermoplastic resins with high surface resistance. Furthermore, the surface resistance of the collection section surface increases due to the fibers captured in the collection section, making it impossible to form through-holes that reflect the shape of the collection member.

[0044] When nonwoven fabric sheets produced by the above-described method are continuously produced, the collection member may be moved relative to the edge of the sheet-like material over time. In this case, the collection member may be moved relative to the edge of the sheet-like material, or the position of the edge of the sheet-like material may be moved relative to the collection member, or both may be moved simultaneously. The movement of the collection member or the edge of the sheet-like material may be continuous or intermittent. Another example of a method for moving the collection member relative to the edge of the sheet-like material is to apply a mechanical, magnetic, or electrical force to the fibers flying from the Taylor cone toward the collection member to move the collection position, such as by blowing air at the flying fibers or by suctioning them toward the collection section. Of course, these methods for moving the collection member relative to the edge of the sheet-like material may be used in combination.

[0045] The moving speed of the collection member etc. is not particularly limited and may be determined appropriately taking into consideration the basis weight of the fiber sheet to be produced. For example, 2 When the feeding speed of the sheet material is 0.5 mm / min, the moving speed of the collection member is set to about 100 mm / min, and the basis weight is 0.5 g / m 2 This allows continuous production of nonwoven fabric sheets of this size.

[0046] Next, we will explain in more detail the process of forming a Taylor cone in the heated and melted portion of the charged sheet-like material and discharging the fibers. Figure 1(b) is a photograph of the Taylor cone formed in the heated and melted portion of Figure 1(a). As shown in Figures 1(a) and 1(b), when a laser beam 12 is irradiated so as to scan the edge 17a of the sheet-like material 17 while a voltage is applied, a linear heated and melted portion is formed at the edge 17a of the sheet-like material 17. Furthermore, a wavy perturbation (meniscus instability phenomenon) occurs at the tip of the heated and melted portion. This perturbation (meniscus) develops to form a Taylor cone 117. When the repulsive force of the electric charge exceeds the surface tension, fibers are discharged from the Taylor cone 117 toward the collection member 19 (right side in Figure 2).

[0047] The thickness of the sheet material used in the nonwoven fabric sheet is not particularly limited, but is usually 0.01 to 10 mm, preferably 0.03 to 5.0 mm. The number of Taylor cones (the spacing between Taylor cones (W in FIG. 1(b))) can be adjusted by appropriately adjusting the thickness of the sheet material. Specifically, the thicker the sheet material, the fewer the Taylor cones (the greater the spacing between Taylor cones). While the reason for this is unclear, it is thought that as the thickness of the sheet material increases, the volume of the molten material at the edge of the sheet material increases, resulting in the Taylor cones developing well and increasing the electrostatic repulsion between the Taylor cones, which in turn increases the spacing between the Taylor cones. The development of Taylor cones means an increase in the height of the Taylor cones (H in FIG. 1(b)).

[0048] The sheet-like material is made of a thermoplastic resin and may be a fiber assembly (nonwoven fabric, woven fabric, knitted fabric, etc.) made of thermoplastic resin fibers, or may be a sheet produced by kneading a thermoplastic resin in advance and then molding it. The shape of the sheet-like material is not particularly limited, and examples thereof include a film, plate, board, etc.

[0049] Among the above thermoplastic resins, low-viscosity thermoplastic resins are preferred because they are easy to form ultrafine fibers such as nanofibers. Furthermore, polar thermoplastic resins are preferred because they are easy to generate electric traction forces due to electric charges and to form Taylor cones. Furthermore, when used as a cell culture scaffold for regenerative medicine, polylactic acid (PLA), particularly poly(DL-lactic acid) (PDLLA), is preferred.

[0050] The sheet-like material is not limited to those made solely of thermoplastic resins, but may also be made of a thermoplastic resin composition containing various additives commonly used in fibers, such as stabilizers (antioxidants, UV absorbers, heat stabilizers, etc.), antistatic agents, fillers, lubricants, humectants, plasticizers, thickeners, dispersants, foaming agents, surfactants, etc. Proteins effective for cell culture, tissue regeneration, etc., may also be used as additives. Examples include cytokines, fibronectin, laminin, collagen, glycosaminoglycans (heparan sulfate, hyaluronic acid, etc.), heparin, chitin, collagen, polylysine, polyarginine, sericin, cellulose, dextran, and pullulan. These additives may be used alone or in combination. Furthermore, in the present invention, in addition to the thermoplastic resin, drugs such as growth factors for bones and biological tissues, antibacterial substances such as tetracyclines such as minocycline and doxycycline, macrolides such as clarithromycin and azithromycin, new quinolones such as levofloxacin, and ketolides such as telithromycin, anti-inflammatory drugs such as non-steroids such as flurbiprofen and steroids such as dexamethasone, naturally occurring substances such as azulene, and bone resorption inhibitors such as bisphosphonates can be appropriately blended.

[0051] Among these additives, it is preferable to use a surfactant, because when a high voltage is applied to a sheet-like material to inject an electric charge, a sheet-like material made of a thermoplastic resin has high electrical insulation, making it difficult to inject an electric charge to the heat-fused portion where the electrical resistance is low, whereas a fiber aggregate made of thermoplastic resin fibers has a lower electrical resistance when a surfactant or the like is applied to the surface of the fibers, which have high electrical insulation, and therefore the electric resistance of the sheet-like material can be sufficiently injected to the heat-fused portion.

[0052] These additives can be used in a proportion of 50 parts by mass or less, preferably 0.01 to 30 parts by mass, and more preferably 0.1 to 5 parts by mass, per 100 parts by mass of the thermoplastic resin.

[0053] Examples of the laser light source include a YAG laser, a carbon dioxide (CO2) laser, an argon laser, an excimer laser, and a helium-cadmium laser. Among these, a carbon dioxide laser is preferred because of its high power efficiency and high melting ability of thermoplastic resins. The wavelength of the laser light is, for example, about 200 nm to 20 μm, preferably about 500 nm to 18 μm, and more preferably about 5 to 15 μm.

[0054] The output power of the laser beam may be controlled so that the temperature of the heat-melting zone is equal to or higher than the melting point of the thermoplastic resin and equal to or lower than the ignition point of the thermoplastic resin; however, a higher output power is preferable from the viewpoint of reducing the diameter of the extruded fibers. The specific output power of the laser beam can be appropriately selected depending on the physical properties (melting point, LOI (limiting oxygen index)) and shape of the thermoplastic resin used, the feed rate of the thermoplastic resin, and the like. The temperature of the heat-melting zone is not particularly limited as long as it is equal to or higher than the melting point of the thermoplastic resin and equal to or lower than the ignition point, but is usually about 100 to 600°C, and preferably 200 to 400°C.

[0055] Furthermore, the scanning speed of the laser beam is preferably 30 m / s or more. If the scanning speed is less than 30 m / s, it may not be possible to heat and melt the entire edge of the sheet-like material at the same time.

[0056] In such a method for producing a nonwoven fabric sheet, the potential difference generated between the edge of the sheet-like material and the collection member is preferably a high voltage within a range that does not cause discharge. This can be selected appropriately depending on the required fiber diameter, the distance between the electrode and the collection member, the amount of laser light irradiation, etc., but is usually about 0.1 to 30 kV / cm, preferably 0.5 to 20 kV / cm, and more preferably 1 to 10 kV / cm.

[0057] The voltage application method for the molten thermoplastic resin may be a direct application method in which the laser beam irradiation portion (the heated and melted portion of the thermoplastic resin) is aligned with an electrode portion for imparting a charge. However, an indirect application method (particularly a method in which the laser beam irradiation portion is located downstream in the direction of thermoplastic resin supply) is preferred, due to the following advantages: the device can be easily fabricated, the laser beam can be effectively converted into thermal energy, the direction of laser beam reflection can be easily controlled, and safety is high. In particular, in the nonwoven fabric sheet manufacturing method described above, the thermoplastic resin is irradiated with laser beam downstream of the electrode portion, and the distance between the electrode portion and the laser beam irradiation portion is preferably adjusted to a specific range (e.g., approximately 10 mm or less). This distance can be selected depending on the electrical conductivity, thermal conductivity, glass transition point, and laser beam dose of the thermoplastic resin, and is, for example, approximately 0.5 to 10 mm, preferably 1 to 8 mm, more preferably 1.5 to 7 mm, and particularly preferably 2 to 5 mm. When the distance between them is within this range, the molecular mobility of the thermoplastic resin in the vicinity of the laser beam irradiation portion is increased, and a sufficient charge can be imparted to the molten thermoplastic resin, thereby improving productivity.

[0058] Furthermore, the distance between the edge of the sheet-like material and the collection member is not particularly limited and is usually 5 mm or more, but in order to efficiently produce nonwoven fabric sheets, it is preferably 10 to 300 mm, more preferably 15 to 250 mm, even more preferably 50 to 220 mm, and particularly preferably about 80 to 200 mm.

[0059] In the nonwoven fabric sheet manufacturing method described above with reference to FIG. 1, the edge of the sheet-like material is irradiated with laser light from only one direction. However, for example, laser light may be irradiated with the edge of the sheet-like material from two directions via a reflecting mirror. Even if the sheet-like material is thick, the edge can be melted more uniformly. Alternatively, multiple sheets of the above-mentioned sheet-like material may be arranged in parallel and placed along the direction of movement of the collection member, and fibers may be ejected simultaneously from the edge of each sheet-like material. In this case, the production speed of the nonwoven fabric sheet can be increased several times.

[0060] In the nonwoven fabric sheet manufacturing method, the space between the edge of the sheet-like material and the collection member (fiber flight space) may be an inert gas atmosphere. By creating an inert gas atmosphere in the flight space, ignition of the fibers can be suppressed, thereby increasing the output of the laser light. Examples of inert gases include nitrogen gas, helium gas, argon gas, and carbon dioxide gas. Of these, nitrogen gas is usually used. These inert gases may be heated. Furthermore, the use of the inert gas can suppress oxidation reactions in the heat-melting zone.

[0061] The nonwoven fabric sheet produced by this method can be made solely from the resin material, free of residual impurities such as solvents. Therefore, it can be suitably used as a cell culture scaffold for use in regenerative medicine, etc. The nonwoven fabric sheet is more porous than a resin film, allowing it to supply sufficient oxygen and nutrients to cells and quickly expel carbon dioxide and waste products. Furthermore, if the nonwoven fabric sheet is made of fibers with an average fiber diameter of 20 μm or less, it has a large specific surface area and high cell adhesion. The cross section of the fiber is irregular, which further increases the specific surface area compared to fibers with a circular cross section, providing a sufficient area for cells to adhere to the fiber surface. The nonwoven fabric sheet of the present invention is suitable as a cell culture scaffold.

[0062] The adipose stem cell culture substrate of the present invention is characterized by including a nonwoven fabric made of fibers with a fiber diameter of 0.1 to 9 μm.

[0063] The average fiber diameter of the nonwoven fabric is preferably 1 to 8 μm, and more preferably 2 to 7 μm. If it is less than 0.1 μm, stem cells cannot migrate into the nonwoven fabric, and if it exceeds 9 μm, adipose tissue will penetrate the nonwoven fabric and become strongly bonded to the nonwoven fabric.

[0064] The nonwoven fabric preferably has pores with an average pore diameter of 1 to 100 μm, preferably 5 to 75 μm, and more preferably 10 to 50 μm.

[0065] The adipose stem cell culture substrate of the present invention may consist of only a nonwoven fabric, or may have a frame to fix the nonwoven fabric in addition to the nonwoven fabric. The shape of the frame is not particularly limited, and examples include ring-shaped, lattice-shaped, cup-shaped, and dish-shaped.

[0066] The adipose stem cell culture vessel of the present invention is characterized by containing the adipose stem cell culture substrate. The vessel may be in the shape of a well plate, flask, tube, dish, cup, or the like. [Example]

[0067] The present invention will be explained in more detail below by way of examples, but the present invention is not limited to these examples.

[0068] Manufacturing Example 1 (Production of nonwoven fabric) PDLLA (poly DL-lactic acid) pellets were hot-pressed at 140°C to produce a PDLLA sheet with a thickness of 0.3 mm and a width of 80 mm. This sheet was used to produce a nonwoven fabric sheet using the manufacturing apparatus shown in Figure 1. The following components were used: Laser light source: CO2 laser (COHERENT, DIAMOND J-3-10.6, wavelength 10.6 μm, output rating 250 W, beam diameter 8.5 mm) Laser beam scanning means: Au-coated Cu mirror Beam expander and polygon mirror Power supply: High-voltage DC power supply (Matsusada Precision, HAR-120R0.83, maximum voltage ±120kV, maximum current 0.83mA) Collection material: Stainless steel drum (diameter 200 mm)

[0069] A laser beam (output: 137 W) with a beam diameter of 1.6 mm was irradiated onto the edge of a sheet-like material held by a holding member (electrode) with a scanning width of 250 mm and a scanning speed of 188 m / s. The distance between the holding member (electrode) and the laser beam irradiation unit was 2 mm, the distance between the edge of the sheet-like material and the capturing surface of the capturing member was 20 cm, and the potential difference between the holding member (electrode) and the capturing member was -3 kV / cm. The sheet-like material was fed at a speed of 3 mm / min. The capturing member was rotated at 0.04 rpm to produce a nonwoven fabric.

[0070] PDLLA fiber with a basis weight of 11.5g / m 2 A nonwoven fabric with a thickness of 0.3 mm was obtained. The average fiber diameter of the PDLLA fibers was 5.9 μm, and the average pore size was 47.1 μm.

[0071] (average fiber diameter) The surface of the nonwoven fabric was observed using a scanning electron microscope (SEM), and the surface widths of 100 randomly selected nanofibers in the electron microscope photograph were measured. The average value was taken as the average fiber diameter. The observation magnification was 100x to 1000x. The fiber diameter was measured using image analysis software.

[0072] (Average pore diameter) The surface of the nonwoven fabric was observed under a scanning electron microscope (SEM), and the widths of the openings of 20 randomly selected recesses or through-holes in the electron micrograph were measured, and the average value was taken as the average pore diameter. The observation magnification was 50x to 500x. The opening diameter was measured using image analysis software.

[0073] Manufacturing Example 2 A nonwoven fabric was produced in the same manner as in Production Example 1, except that the rotation speed of the collection member was set to 750 rpm. The basis weight of the nonwoven fabric was 7.0 g / m 2 A nonwoven fabric with a thickness of 0.2 mm was obtained. The average fiber diameter was 4.3 μm and the average pore diameter was 17.7 μm.

[0074] Example 1 The nonwoven fabric (2 cm diameter) prepared in Production Example 1 was hydrophilized by immersion in 70% ethanol, washed with PBS, and then placed in a 6 cm dish containing 5 mL of KBM ADSC-1 medium. Approximately 3-5 mL of adipose tissue collected from human subcutaneous fat was placed on the nonwoven fabric and cultured in a cell culture device at 37°C and 5% carbon dioxide for 10-14 days to culture the adipose tissue. The medium was not changed during this period.

[0075] Example 2 Adipose tissue was cultured in the same manner as in Example 1, except that the nonwoven fabric prepared in Production Example 2 was used.

[0076] Comparative Example 1 Adipose tissue was cultured in the same manner as in Example 1, except that a nonwoven fabric of polyethylene-polypropylene core-sheath structure fiber coated with hydroxyapatite (manufactured by Bio Mirai Kogyo Co., Ltd., average fiber diameter: 22.6 μm, average pore diameter: 161.6 μm) was used.

[0077] In Comparative Example 1, stem cells were isolated from the adipose tissue and could be cultured to proliferate, but the adipose tissue adhered firmly to the nonwoven fabric, and could not be separated even when sprayed with PBS, and manual separation using a pipette tip did not allow complete removal of the adipose tissue. Furthermore, the adipose tissue that had been removed fell into pieces. Figure 7 shows a micrograph of the nonwoven fabric after removal of the cultured tissue cultured in Comparative Example 1, revealing the presence of round droplets of fat adhering to the nonwoven fabric.

[0078] On the other hand, in Examples 1 and 2, stem cells could be isolated from the adipose tissue and proliferated by culture, and when PBS was sprayed onto the nonwoven fabric, the adipose tissue could be easily separated without adhering firmly to the fabric. Figure 6 shows a micrograph of the nonwoven fabric after removal of the cultured tissue cultured in Example 1. Unlike Comparative Example 1, no round droplets of fat adhered to the nonwoven fabric.

[0079] Table 1 shows a summary of the number of stem cells that could be recovered in the Examples and Comparative Examples. [Table 1]

[0080] In both cases, sufficient stem cells could be cultured. [Explanation of symbols]

[0081] 1 nonwoven fabric sheet 2 through holes 11 Laser light source 12 Laser light 13 Reflective mirror 14 Polygon Mirror 15 Laser beam scanning means 17 Sheet-like objects 18 Retaining member 19 Collection member 20 Power supply 22 Heated air supply device 117 Taylor Cone

Claims

1. Culturing adipose tissue containing stem cells on a nonwoven fabric made of fibers with an average fiber diameter of 0.1 to 9 μm; A step of recovering stem cells proliferated in the nonwoven fabric A method for culturing adipose stem cells, comprising:

2. 2. The method for culturing adipose stem cells according to claim 1, wherein the fibers in the nonwoven fabric are made of a thermoplastic resin.

3. 3. The method for culturing adipose stem cells according to claim 2, wherein the thermoplastic resin is polylactic acid or a copolymer thereof.

4. 3. The method for culturing adipose stem cells according to claim 1, wherein the nonwoven fabric has pores with an average pore size of 1 to 100 μm.

5. 3. The method for culturing adipose stem cells according to claim 1 or 2, wherein the nonwoven fabric is a nonwoven fabric produced by laser melt electrospinning.

6. An adipose stem cell culture substrate comprising a nonwoven fabric made of fibers with an average fiber diameter of 0.1 to 9 μm.

7. The adipose stem cell culture substrate according to claim 6, comprising a nonwoven fabric having pores with an average pore size of 1 to 100 μm.

8. The adipose stem cell culture substrate according to claim 6 or 7, wherein the nonwoven fabric is a nonwoven fabric produced by laser melt electrospinning.

9. An adipose stem cell culture vessel comprising the adipose stem cell culture substrate according to claim 8.

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