Stem cell adhesion sheet

Abstract

The present invention addresses the problem of achieving the purpose of providing a stem cell adhesion sheet capable of stably holding a stem cell.　This stem cell adhesion sheet comprises a non-woven fabric. The non-woven fabric has a laminated structure including a first layer formed of fibers containing a biocompatible polymer as a main component, and a second layer formed of fibers containing a biocompatible polymer as a main component and having a fiber diameter greater than the fibers forming the first layer. The biocompatible polymers forming the first layer and the second layer contain the same type of polymer material.

Description

Stem cell attachment sheet

[0001] The disclosure in this application relates to a sheet for attaching stem cells.

[0002] In recent years, in the field of regenerative medicine, progress has been made in the development of tissue regeneration promoters that can retain cells inside and promote tissue regeneration by being implanted in the body. For example, Patent Document 1 discloses a sheet-shaped tissue regeneration promoter having at least one of a group consisting of white blood cells and platelets present on its surface. Furthermore, Patent Document 2 discloses a liquid tissue regeneration promoter obtained by mixing a powder of a polymer material with cells.

[0003] Japanese Patent No. 4847129 Japanese Patent Application Laid-Open No. 2015-112262

[0004] However, the tissue regeneration-promoting materials disclosed in Patent Documents 1 and 2 use powders of porous bodies or polymeric materials, which makes it difficult to stably preserve mesenchymal stem cells, which have a high tissue regeneration capacity among cells. This problem is not limited to the case where mesenchymal stem cells are preserved using tissue regeneration-promoting materials, but also exists when other stem cells are preserved.

[0005] The disclosure of the present application has been made against this background, and aims to provide a sheet for stem cell attachment that is capable of stably retaining stem cells.

[0006] The present application discloses the following stem cell attachment sheets: (1) A stem cell attachment sheet comprising a nonwoven fabric, wherein the nonwoven fabric has a laminated structure including: a first layer composed of fibers primarily composed of a biocompatible polymer; and a second layer composed of fibers primarily composed of a biocompatible polymer and having a larger fiber diameter than the fibers comprising the first layer, wherein the biocompatible polymers comprising the first layer and the second layer comprise the same type of polymer material. (2) The stem cell attachment sheet according to (1) above, wherein the polymer material is at least one selected from the group consisting of polylactic acid, polycaprolactone, chitin, chitosan, polyglycolic acid, and copolymers thereof. (3) The stem cell attachment sheet according to (2) above, wherein the polymer material is at least one selected from the group consisting of polylactic acid, polycaprolactone, and copolymers of polylactic acid and polycaprolactone. (4) The stem cell attachment sheet according to (1) above, wherein the biocompatible polymer materials constituting the first layer and the second layer contain at least 25% or more of the same type of polymer material. (5) The stem cell attachment sheet according to (2) above, wherein the biocompatible polymer materials constituting the first layer and the second layer contain at least 25% or more of the same type of polymer material. (6) The stem cell attachment sheet according to (3) above, wherein the biocompatible polymer materials constituting the first layer and the second layer contain at least 25% or more of the same type of polymer material. (7) The stem cell attachment sheet according to any one of (4) to (6) above, wherein the biocompatible polymer materials constituting the first layer and the second layer are 100% the same type of material. (8) The stem cell attachment sheet according to any one of (1) to (7) above, wherein the fiber diameter of the fibers constituting the first layer is in the range of 200 nm or more and 5 μm or less, and the fiber diameter of the fibers constituting the second layer is greater than the fiber diameter of the fibers constituting the first layer and 30 μm or less. (9) The basis weight of the first layer is 0.2 g / m ２ Above, 4g / m ２ Hereinafter, the basis weight of the second layer is 2 g / m ２ Above, 30g / m２ The stem cell adhesion sheet according to any one of (1) to (8) above, wherein the fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding cases where the fiber diameter is 4.0 μm or less and 10 μm or more). (10) The stem cell adhesion sheet according to (3) above, wherein the biocompatible polymer does not contain siloxane, and the fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding cases where the fiber diameter is 4.0 μm or less and 10 μm or more). (11) The stem cell adhesion sheet according to (1) above, wherein the biocompatible polymer does not contain siloxane, and the fiber diameter of the fibers constituting the first layer is 200 nm or more and 5 μm or less (excluding cases where the fiber diameter is 1.5 μm or more), and the fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding cases where the fiber diameter is 4.0 μm or less and 10 μm or more). (12) The biocompatible polymer does not contain siloxane, and the basis weight of the first layer is 0.2 g / m ２ Above, 4g / m ２ The fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding the case where the fiber diameter is 4.0 μm or less and 10 μm or more). The stem cell adhesion sheet described in (1) above.

[0007] The stem cell attachment sheet disclosed in the present application allows for stable retention of stem cells.

[0008] FIG. 1 is a perspective view showing the configuration of an example of a stem cell adhesion sheet according to an embodiment. FIG. 2 is a perspective view showing the configuration of an example of a stem cell adhesion sheet according to an embodiment. FIG. 3 is a perspective view showing the configuration of an example of a stem cell adhesion sheet according to an embodiment. FIG. 4 is a front view showing an outline of producing a tissue regeneration-promoting sheet using a stem cell adhesion sheet according to an embodiment. FIG. 6 is a graph showing the difference in expression level of vascular endothelial growth factor (VEGF) produced by human adipose-derived mesenchymal stem cells when the tissue regeneration-promoting sheet produced in Example 2 was used and when the tissue regeneration-promoting sheet was not used. FIG. 7 is a graph showing the difference in expression level of vascular endothelial growth factor (VEGF) produced by human adipose-derived mesenchymal stem cells when the tissue regeneration-promoting sheet produced in Example 3 was used and when the tissue regeneration-promoting sheet was not used.

[0009] The stem cell adhesion sheet disclosed in this application will be described below with reference to the drawings. Note that the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc., in order to facilitate understanding. Therefore, the disclosure of this application is not necessarily limited to the position, size, range, etc., disclosed in the drawings.

[0010] Furthermore, in this specification, (1) a numerical range expressed using "~" means a range that includes the numerical values ​​written before and after "~" as the lower and upper limits, (2) numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors that are generally accepted in the technical field, and (3) when "approximately XX" is written, it is interpreted as including not only the exact XX, but also a shape that can be understood to be approximately XX.

[0011] (Embodiment of stem cell adhesion sheet) An outline of a stem cell adhesion sheet 10 according to an embodiment will be described with reference to Figures 1 to 4. Figures 1 to 4 are perspective views showing the configuration of an example of a stem cell adhesion sheet according to an embodiment.

[0012] A stem cell attachment sheet according to an embodiment is used for attaching stem cells. After attaching stem cells, the sheet (hereinafter sometimes referred to as a "tissue regeneration-promoting sheet") is embedded in an injured area within the body, thereby promoting tissue regeneration at the injured area. A stem cell attachment sheet according to an embodiment includes a nonwoven fabric composed of fine fibers, such as microfibers, to which stem cells can attach. A nonwoven fabric is a sheet in which fine fibers are intertwined without being woven. Because nonwoven fabrics can be freely deformed, they are suitable for embedding or attaching to the shape of an injured area. The tissue regeneration-promoting sheet can be attached or embedded in any organ or tissue, such as the heart, blood vessels, lungs, trachea, esophagus, stomach, small intestine, large intestine, liver, kidneys, bladder, bone, muscle, or skin. The tissue regeneration-promoting sheet can function as a scaffold that promotes the proliferation of specific cells within the body.

[0013] 1, the stem cell attachment sheet 10 includes a first layer 12 made of fibers whose main component is a biocompatible polymer, and a second layer 14 made of fibers whose main component is a biocompatible polymer. The second layer 14 is made of fibers whose diameter is thicker than the fibers that make up the first layer 12.

[0014] As shown in Figure 3 of JP 2023-47494 A, stem cells are captured in the stem cell adhesion sheet 10 by adhering to the fibers rather than being trapped in the gaps between the fibers that make up the nonwoven fabric. The procedure for attaching stem cells to the stem cell adhesion sheet 10 will be described later. Stem cells can adhere to both the first layer 12 and the second layer 14. Furthermore, the second layer 14 is composed of fibers with a diameter thicker than the fibers that make up the first layer 12, so it not only adheres stem cells but also functions as a reinforcing sheet that reinforces the first layer 12. A stem cell adhesion sheet formed solely from the first layer 12, which has a narrow fiber diameter, tends to curl when exposed to a cell suspension. The stem cell adhesion sheet 10 according to the embodiment has improved handleability by laminating the first layer 12 and the second layer 14. The first layer 12 and the second layer 14 need only be laminated so as not to easily separate. Without being limited thereto, the fibers may be intertwined during the manufacture of the nonwoven fabric, or the first layer 12 and the second layer 14 may be fabricated separately and bonded to each other using an adhesive or heat fusion.

[0015] Figure 1 shows an example of a stem cell adhesion sheet 10 in which one first layer 12 and one second layer 14 are laminated, but there is no particular limit to the number of first layers 12 and second layers 14 as long as the number of first layers 12 and second layers 14 is within a range that allows the cell suspension containing stem cells to be filtered and the stem cells to adhere to the stem cell adhesion sheet 10.

[0016] For example, as shown in Fig. 2, a first layer 12 may be sandwiched between two upper and lower second layers 14. Alternatively, as shown in Fig. 3, a plurality of first layers 12 (two layers in the example shown in Fig. 3) may be stacked between two upper and lower second layers 14. Alternatively, as shown in Fig. 4, first layers 12 and second layers 14 may be stacked alternately.

[0017] The first layer 12 and the second layer 14 are made of fibers whose main component is a biocompatible polymer. In this specification, the term "biocompatible polymer" refers to a polymer that has affinity with biological tissues and organs and does not cause foreign body reactions or rejection reactions when applied to a living body.

[0018] The polymer material used to prepare the biocompatible polymer is not particularly limited as long as it is highly biocompatible. Examples include, but are not limited to, polylactic acid (PLA), polycaprolactone (PCL), chitin, chitosan, polyglycolic acid (PGA), and copolymers thereof. However, siloxane may be omitted from the polymer material used to prepare the biocompatible polymer. In other words, the biocompatible polymer may be a polymer that does not contain siloxane.

[0019] Among the polymer materials exemplified above, polycaprolactone is a semi-crystalline, biodegradable thermoplastic polyester that can be suitably used in the production of nonwoven fabrics. The polycaprolactone may be, for example, poly-ε-caprolactone. The molecular weight of the polycaprolactone may be a molecular weight suitable for injection molding, for example, in the range of 70,000 to 200,000. Furthermore, the molecular weight of polylactic acid may be 50,000 to 200,000, and the molecular weight of polyglycolic acid may be 50,000 to 500,000.

[0020] The fibers constituting the first layer 12 and the second layer 14 may be composed solely of biocompatible polymers formed from the above-mentioned polymer materials, or may contain other substances. Examples of other substances include drugs that promote tissue regeneration and other biodegradable polymers. Drugs that promote tissue regeneration include hydroxyapatite (calcium hydroxide phosphate) and tricalcium phosphate. Examples of other biodegradable polymers include polydioxanone. The mass ratio of the biocompatible polymer in the fibers may be 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 100%, etc.

[0021] Although not approved in Japan, nonwoven fabrics made from 100% polylactic acid (PLA) have been approved in the United States. Furthermore, in Japan, a copolymer of 50% polycaprolactone (PCL) and 50% polylactic acid (PLA) has been approved as a synthetic artificial dura mater (product name: Seamdura, manufactured by Gunze), and a copolymer of 25% polycaprolactone (PCL) and 75% polylactic acid (PLA) has been approved as an absorbable suture (product name: P(LA / CL) suture, manufactured by Gunze). In other words, polylactic acid and polycaprolactone can be considered polymer materials whose safety in humans has been confirmed. Therefore, from the perspective of reducing the burden of pharmaceutical approval applications, including safety testing when applied to living organisms, polylactic acid, polycaprolactone, and copolymers of polylactic acid and polycaprolactone are more preferred polymer materials.

[0022] The biocompatible polymers constituting the first layer 12 and the second layer 14 preferably contain the same type of polymer material. When the tissue regeneration-promoting sheet is implanted at an injured site in a living body, the biocompatible polymer is degraded within the body. If the polymer materials of the first layer 12 and the second layer 14 are different, differences in the environment at the implantation site, such as the degradation rate, degradation products, and pH, may occur. If the biocompatible polymers constituting the first layer 12 and the second layer 14 contain the same type of polymer material, it is expected that the differences in the environmental changes at the implantation site after degradation will be smaller.

[0023] It is expected that the greater the proportion of the same type of polymer material, the greater the effect that can be obtained. Examples of the proportion of the same type of biocompatible polymer in the polymer material constituting the first layer 12 and the second layer include, but are not limited to, 5% or more, 10%, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45%, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100%, etc.

[0024] In this specification, the proportion of the same type of polymer material refers to the mass of the component of the same type when the mass of the polymer material of the first layer 12 is taken as 100 and the mass of the component of the polymer material of the second layer 14 is taken as 100. Furthermore, when the polymer material is a copolymer, the mass of the component of the same type contained in the copolymer is compared, and the mass of the component with the smaller amount is taken as the proportion of the same type of polymer material. The same type may be one type or two or more types. When two or more types of the same type of polymer material (component) are contained, the proportions can be added together. More specific examples of polymer materials X, Y, and Z are shown below. The term "X + Y" refers to a copolymer of X and Y.

[0025] There are no particular limitations on the fiber diameters of the fibers constituting the first layer 12 and the second layer 14, as long as the fiber diameter of the fibers constituting the first layer 12 is smaller than the fiber diameter of the fibers constituting the second layer 14, stem cells can be attached, and the stem cell attachment sheet is easy to handle. Examples of lower limits for the fiber diameter of the fibers constituting the first layer 12 include, but are not limited to, 200 nm or more, 300 nm or more, 400 nm or more, 500 nm or more, 600 nm or more, 700 nm or more, 800 nm or more, 900 nm or more, and 1 μm or more. Examples of upper limits include 5 μm or less, 4.75 μm or less, 4.5 μm or less, 4.25 μm or less, and 4 μm or less. The lower limit of the fiber diameter of the fibers constituting the second layer 14 may be larger than the fiber diameter of the fibers constituting the first layer 12, and examples of upper limits include 30 μm or less, 27.5 μm or less, 25 μm or less, 22.5 μm or less, 20 μm or less, 17.5 μm or less, and 15 μm or less. Furthermore, assuming that the fiber diameter of the fibers constituting the first layer 12 is 1, the fiber diameter of the fibers constituting the second layer 14 is not limited, and examples thereof include 1.2 times or more, 1.4 times or more, 1.6 times or more, 1.8 times or more, 2 times or more, 3 times or more, 4 times or more, 5 times or more, 6 times or more, 7 times or more, 8 times or more, 9 times or more, and 10 times or more. The above-mentioned fiber diameters are merely examples, and the range between the lower limit and the upper limit may be expressed using any numerical value not exemplified. The fiber diameter of the fibers constituting the first layer 12 may be expressed as, for example, 200 nm or more and less than 1.5 μm, so long as it is in the range of 200 nm or more and 5 μm or less. The fiber diameter of the fibers constituting the second layer 14 may be expressed as, for example, 4.0 μm or more and less than 10 μm, so long as it is in the range of 200 nm or more and 30 μm or less.

[0026] The larger the basis weight of the first layer 12, the more stem cells can be attached, but filtration of the cell suspension becomes increasingly difficult. The basis weight of the first layer 12 can be determined taking into consideration the amount of attached stem cells and ease of manufacturing. Although not limited, the lower limit of the basis weight of the first layer 12 is 0.2 g / m ２ Above, 0.4g / m ２ Above, 0.6g / m ２ Above, 0.8g / m ２ Above, 1g / m ２The upper limit is 4 g / m ２ Below, 3g / m ２ Below, 2.8g / m ２ Below, 2.6g / m ２ Below, 2.4g / m ２ Below, 2.2g / m ２ Below, 2.0g / m ２ The following may be mentioned:

[0027] On the other hand, as described above, the second layer 14 adheres stem cells and also functions as a reinforcing sheet that reinforces the first layer 12. Therefore, the basis weight of the second layer 14 can be determined appropriately by taking into consideration the attachment of stem cells and ease of handling. Although not limited thereto, the lower limit is 2 g / m ２ Above, 2.5g / m ２ Above, 3 / m ２ Above, 3.5g / m ２ Above, 4g / m ２ Above, 4.5g / m ２ Above, 5 / m ２ Above, 5.5g / m ２ Above, 6g / m ２ Above, 6.5g / m ２ Above, 7g / m ２ Above, 7.5 / m ２ Above, 8g / m ２ Above, 8.5g / m ２ Above, 9g / m ２ Above, 9.5 / m ２ Above, 10 / m ２ The upper limit is 30 g / m ２ Below, 29g / m ２ Below, 28g / m ２ Below, 27g / m ２ Below, 26g / m ２ Below, 25g / m ２ Below, 24g / m ２ Below, 23g / m ２ Below, 22g / m ２ Below, 21g / m ２ Below, 20g / m ２The following can be mentioned: With regard to the fiber diameter, the fiber diameter of the fibers constituting the second layer 14 needs to be thicker than the fiber diameter of the fibers constituting the first layer 12. On the other hand, with regard to the basis weight, the basis weight of the first layer 12 may be smaller than, the same as, or larger than the basis weight of the second layer 14, as long as it is easy to handle and does not interfere with the filtration of stem cells.

[0028] The thickness of the laminated structure formed by stacking the first layer 12 and the second layer 14 may be determined appropriately while taking into consideration ease of handling with tweezers. Examples of thicknesses of the laminated structure include, but are not limited to, 200 μm or more, 300 μm or more, 400 μm or more, 500 μm or more, 600 μm or more, and 700 μm or more. On the other hand, the upper limit of the thickness of the laminated structure may be 1700 μm or less, 1600 μm or less, 1500 μm or less, or 1400 μm or less. Furthermore, because the second layer 14 functions as a reinforcing sheet, there are no problems with handling even if the first layer 12 is thin. The thickness of the first layer 12 in the laminate structure may be 10 μm or more, 20 μm or more, 40 μm or more, 60 μm or more, 80 μm or more, 100 μm or more, and the upper limit may be less than 200 μm, 180 μm or less, 160 μm or less, 140 μm or less, etc.

[0029] Examples of stem cells to be attached to the stem cell attachment sheet 10 include mesenchymal stem cells (MSCs) or cells differentiated from mesenchymal stem cells, embryonic stem (ES) cells, and induced pluripotent stem (iPS) cells. Mesenchymal stem cells are somatic stem cells that have the ability to differentiate into cells belonging to the mesenchymal system, and may be derived from adipose tissue, periosteum, synovium, cancellous bone, bone marrow, amniotic membrane, umbilical cord blood, or placenta. Stem cells attached to the nonwoven fabric may be of one type or a combination of two or more types.

[0030] The stem cells to be attached to the stem cell attachment sheet 10 may be collected from a patient or may be an established cell line. To culture stem cells in vitro, for example, a medium containing fetal bovine serum (FBS) or human serum, preferably human serum derived from a patient, or a serum-free medium may be used. Stem cells cultured in the medium may be attached to the nonwoven fabric as is, or the stem cells may be differentiated to express specific cells, such as osteoblasts, before being attached to the nonwoven fabric. To express specific cells, for example, a differentiation-inducing factor may be added to the stem cell medium.

[0031] Furthermore, it is not necessary to attach stem cells in advance to the stem cell attachment sheet 10 disclosed in the present application. After placing the stem cell attachment sheet 10 at an injured site in the body, stem cells may be attached to the stem cell attachment sheet in the body by administering a cell suspension containing stem cells into the vascular system.

[0032] Next, we will explain the specific procedure for attaching stem cells to the stem cell attachment sheet 10 according to the embodiment. Stem cells can be attached to the stem cell attachment sheet 10 by contacting it with a cell suspension containing stem cells for a while, but it is preferable to follow the procedure below to prevent a decrease in growth factor production ability during stem cell culture.

[0033] First, as shown in Figure 5, the stem cell adhesion sheet 10 according to the embodiment is set between a pair of holders 20 that make up the filtration device. Each holder 20 is configured to allow the cell suspension to flow in and out, and in the example shown in Figure 5, the cell suspension comes into contact with the first layer 12 when flowing from one holder 20 to the other holder 20.

[0034] Next, a cell suspension containing stem cells is slowly dripped dropwise from the top, and the cell suspension is filtered through the first layer 12. The filtration rate can be, for example, a rate of dripping 1 ml of cell suspension over 2 to 3 minutes. The stem cells contained in the dripped cell suspension then adhere to the fibers of the first layer 12. While the exact mechanism is unknown, filtering the cell suspension containing stem cells little by little through the stem cell attachment sheet stably adheres to each fiber, and the attached stem cells are activated, promoting the production of growth factors. Stem cells that pass through the first layer 12 without adhering may adhere to the second layer 14, but adjusting the conditions may allow all stem cells to adhere to the first layer 12. In other words, although the second layer 14 has the function of adhering stem cells, attachment of stem cells to the second layer 14 is not essential when used as a tissue regeneration-promoting sheet.

[0035] The above-described embodiments are merely examples, and the present invention is not limited to these. Various embodiments are possible within the scope of the invention as set forth in the claims. The components described in the embodiments can be freely combined. Furthermore, inventions equivalent to the inventions set forth in the claims are also included in the present invention.

[0036] For example, although not shown, the stem cell adhesion sheet 10 shown in Figure 5 may be the stem cell adhesion sheet 10 shown in Figures 2 to 4. The cell suspension may be applied from the second layer 14 side. The examples shown in Figures 2 to 4 show a laminated structure of three or more layers, but the layers are of two types: a first layer 12 and a second layer 14. Alternatively, a third layer may be laminated that differs from the first layer 12 and the second layer 14 in terms of the polymer material, fiber diameter, etc. that make it up. Other layers may be included as long as the first layer 12 and the second layer 14 disclosed in the present application are included.

[0037] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the technical scope of the disclosure in the present application.

[0038] Example 1 Preparation of Various Nonwoven Fabrics A 50:50 copolymer of polycaprolactone (PCL) and polylactic acid (PLA), which are already used clinically as artificial dura mater, was used as the polymer material for the first layer 12 and the second layer 14. Using a commercially available nonwoven fabric preparation device, nonwoven fabrics with the fiber diameters, basis weights, and thicknesses shown in Table 1 below were prepared.

[0039] [Stem Cell Adsorption (Capture) to the Prepared Nonwoven Fabric] Next, the prepared nonwoven fabric was used as a stem cell attachment sheet. The stem cell attachment sheet was sandwiched between a filtration sterilization holder, and a cell suspension containing human adipose-derived mesenchymal stem cells (ADSCs, Promocell, product code C-12977) was passed through to attach human mesenchymal stem cells to the stem cell attachment sheet, and the attachment rate was evaluated. A Swinex filter holder (Merck Millipore) was used as the filtration sterilization holder. The cell suspension was added drop by drop. Experiments were performed on three samples for each experiment.

[0040] The results of the adhesion (capture) experiment are also shown in Table 1. The capture rate was calculated by counting the number of cells before and after filtration using a hemocytometer and calculating (number of cells before filtration - number of cells after filtration) / number of cells before filtration x 100. As is clear from the results shown in Table 1, it was confirmed that stem cells could be attached with extremely high efficiency by using a stem cell adhesion sheet comprising the nonwoven fabric produced in Example 1.

[0041] Example 2 A nonwoven fabric was produced in the same manner as in Example 1, except that the nonwoven fabric was produced using 100% polycaprolactone instead of the polymer material used in Example 1. The sizes of the first and second layers of the produced nonwoven fabric were as follows. The cell capture rate was 100%. First layer: fiber diameter 0.5 μm, basis weight 2 g / m ２ , thickness 100 μm. Second layer: fiber diameter 5 μm, basis weight 10 g / m ２ , thickness 500 μm.

[0042] [Confirmation of growth factor production ability] Next, using the nonwoven fabric prepared in Example 2, stem cells were attached to the nonwoven fabric using the same procedure as in Example 1 to prepare a tissue regeneration-promoting sheet. The prepared tissue regeneration-promoting sheet was transferred to a petri dish containing a culture medium and cultured for 4 weeks. As a control, stem cells (ADSCs) were cultured directly in the petri dish for 4 weeks. After 4 weeks of culture, the expression level of vascular endothelial growth factor (VEGF) was measured using ELISA. The results are shown in Figure 6.

[0043] Example 3: Growth factor production ability was confirmed using a nonwoven fabric prepared in the same manner as in Example 2, except that the polymer material of Example 1 was used. The sizes of the first and second layers of the prepared nonwoven fabric were as follows. The cell capture rate was 99.7%. The results are shown in Figure 7. First layer: fiber diameter 0.5 μm, basis weight 2 g / m ２ , thickness 100 μm. Second layer: fiber diameter 4 μm, basis weight 10 g / m ２ , thickness 500 μm.

[0044] As is clear from Figures 6 and 7, it was confirmed that when stem cells were attached to the stem cell attachment sheet disclosed in the present application and then cultured, the stem cells were activated compared to when the stem cells were cultured directly in medium (ADSCs in Figures 6 and 7). Furthermore, because the stem cell attachment sheet disclosed in the present application has a laminated structure consisting of a first layer and a second layer with a fiber diameter thicker than that of the first layer, a series of experiments confirmed that there were no particular problems with the handleability of the stem cell attachment sheet.

[0045] Example 4 A nonwoven fabric was produced and stem cell adsorption (capture) was performed in the same manner as in Example 1, except that a copolymer of polycaprolactone (PCL) and polylactic acid (PLA) in a ratio of 25:75, which is already used clinically as an absorbable suture, was used as the polymer material for the first layer 12 and the second layer 14 instead of the polymer material used in Example 1. Table 2 shows the fiber diameter, basis weight, and thickness of the first and second layers, as well as the number of layers and stem cell capture rate.

[0046]

[0047] As is clear from the results shown in Table 2, it was confirmed that stem cells could be attached with extremely high efficiency by using the stem cell attachment sheet comprising the nonwoven fabric prepared in Example 4.

[0048] Comparative Example 1: A nonwoven fabric having only a first layer was produced using only polycaprolactone as the polymer material. The size of the produced nonwoven fabric was as follows: First layer: fiber diameter 0.5 μm, basis weight 2 g / m ２ , thickness 100 μm.

[0049] An attempt was made to set the produced nonwoven fabric in a holder, but it was difficult to handle and could not be set in the holder.

[0050] Comparative Example 2 A nonwoven fabric was produced using the polymer material described in Example 4 in the same manner as in Example 1, except that the fiber diameter, basis weight, thickness, and number of layers shown in Table 3 below were set to 1, and stem cell adsorption (capture) was performed. The capture rate is also shown in Table 3 below.

[0051]

[0052] As shown in Table 3, the fiber diameter of the fibers constituting the nonwoven fabric produced in Comparative Example 2 was quite close to the fiber diameter of the second layer in Examples 1 to 4, and therefore, even when the basis weight was approximately the same as in Comparative Example 1, there were no particular problems with handleability. Furthermore, although the stem cell capture rate in Comparative Example 2 was lower than in Examples 1 to 4, a predetermined amount of stem cells was captured. Therefore, it was confirmed that the second layer of the stem cell attachment sheet disclosed in the present application functions to attach (capture) stem cells and to reinforce the first layer. Furthermore, the results shown in Table 3 confirmed that, if a low stem cell capture rate is not a problem, by setting the fiber diameter to a predetermined thickness or greater, the nonwoven fabric can function as a stem cell attachment sheet even in a single layer.

[0053] From the above results, it was confirmed that, rather than producing a stem cell attachment sheet using only a first layer with a thin fiber diameter, by forming a laminated structure with a second layer composed of fibers with a fiber diameter thicker than that of the first layer, handling properties and stem cell capture efficiency when attaching stem cells to the stem cell attachment sheet are improved.

[0054] 10 Stem cell attachment sheet 12 First layer 14 Second layer 20 Holder

Claims

1. A sheet for stem cell attachment comprising a nonwoven fabric, the nonwoven fabric having a laminated structure including: a first layer composed of fibers whose main component is a biocompatible polymer; and a second layer composed of fibers whose main component is a biocompatible polymer and whose fiber diameter is thicker than the fibers constituting the first layer, the biocompatible polymers constituting the first layer and the second layer containing the same type of polymer material.

2. The sheet for attaching stem cells according to claim 1, wherein the polymer material is at least one selected from the group consisting of polylactic acid, polycaprolactone, chitin, chitosan, polyglycolic acid, and copolymers thereof.

3. The sheet for attaching stem cells according to claim 2, wherein the polymer material is at least one selected from the group consisting of polylactic acid, polycaprolactone, and copolymers of polylactic acid and polycaprolactone.

4. The sheet for attachment of stem cells according to claim 1, wherein the biocompatible polymer materials constituting the first layer and the second layer contain the same type of polymer material in a proportion of at least 25% or more.

5. The sheet for attachment of stem cells according to claim 2, wherein the biocompatible polymer materials constituting the first layer and the second layer contain the same type of polymer material at a ratio of at least 25% or more.

6. The sheet for attachment of stem cells according to claim 3, wherein the biocompatible polymer materials constituting the first layer and the second layer contain the same type of polymer material in a proportion of at least 25% or more.

7. A sheet for stem cell attachment according to any one of claims 4 to 6, wherein the polymer materials of the biocompatible polymers constituting the first layer and the second layer are 100% the same type of material.

8. A sheet for stem cell attachment according to any one of claims 1 to 6, wherein the fiber diameter of the fibers constituting the first layer is in the range of 200 nm or more and 5 μm or less, and the fiber diameter of the fibers constituting the second layer is larger than the fiber diameter of the fibers constituting the first layer and is in the range of 30 μm or less.

9. The basis weight of the first layer is 0.2 g / m ２ Above, 4g / m ２ Hereinafter, the basis weight of the second layer is 2 g / m ２ Above, 30g / m ２ The sheet for stem cell attachment according to any one of claims 1 to 6, wherein the thickness is in the range of:

10. The biocompatible polymer does not include siloxane, and the fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding the fiber diameter being 4.0 μm or less and 10 μm or more). ） 　 The sheet for stem cell attachment according to claim 3 .

11. The biocompatible polymer does not include siloxane, the fiber diameter of the fibers constituting the first layer is 200 nm or more and 5 μm or less (excluding the case where the fiber diameter is 1.5 μm or more), and the fiber diameter of the fibers constituting the second layer is 3.2 μm to 15 μm (excluding the case where the fiber diameter is 4.0 μm or less and 10 μm or more). ） 　 The sheet for attaching stem cells according to claim 1 .

12. The biocompatible polymer does not include siloxane, and the basis weight of the first layer is 0.2 g / m ２ Above, 4g / m ２ The fiber diameter of the fibers constituting the second layer is in the range of 3.2 μm to 15 μm (excluding the case where the fiber diameter is 4.0 μm or less and 10 μm or more). The sheet for attaching stem cells according to claim 1 .

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