WO2020262458A1 - Cell sheet, method for producing same, and fabrication kit - Google Patents

Cell sheet, method for producing same, and fabrication kit Download PDF

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Publication number
WO2020262458A1
WO2020262458A1 PCT/JP2020/024822 JP2020024822W WO2020262458A1 WO 2020262458 A1 WO2020262458 A1 WO 2020262458A1 JP 2020024822 W JP2020024822 W JP 2020024822W WO 2020262458 A1 WO2020262458 A1 WO 2020262458A1
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WIPO (PCT)
Prior art keywords
gelatin
cell sheet
short fibers
cells
average fiber
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PCT/JP2020/024822
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French (fr)
Japanese (ja)
Inventor
田畑泰彦
島田直樹
中村耕一郎
延谷公昭
Original Assignee
日本毛織株式会社
田畑泰彦
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Application filed by 日本毛織株式会社, 田畑泰彦 filed Critical 日本毛織株式会社
Priority to JP2020556991A priority Critical patent/JP7362652B2/en
Publication of WO2020262458A1 publication Critical patent/WO2020262458A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C12N5/0075—General culture methods using substrates using microcarriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2537/00—Supports and/or coatings for cell culture characterised by physical or chemical treatment
    • C12N2537/10—Cross-linking

Definitions

  • the present invention relates to a cell sheet containing gelatin short fibers, a method for producing the same, and a production kit.
  • the present invention provides a cell sheet in which shrinkage after exfoliation from a culture substrate is suppressed and also has high cell activity, a method for producing the same, and a preparation kit.
  • gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet, and the gelatin short fibers have an average fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less. It relates to a cell sheet characterized by being present.
  • cells and gelatin short fibers containing gelatin as a main component are cultured in a medium to form a cell sheet in which the gelatin short fibers are dispersed, and the gelatin short fibers are averaged.
  • the present invention relates to a method for producing a cell sheet, which comprises a fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less, and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less.
  • the present invention also includes cells, gelatin short fibers and a medium, wherein the gelatin short fibers have an average fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less, and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less, and gelatin in a cell sheet.
  • the present invention relates to a cell sheet preparation kit, which is used for forming a cell sheet in which short fibers are dispersed.
  • a cell sheet in which shrinkage after exfoliation from the culture substrate is suppressed and cell activity is high According to the production method of the present invention, a cell sheet having high cell activity while suppressing shrinkage after exfoliation from the culture substrate can be efficiently and easily produced. According to the production kit of the present invention, a cell sheet having high cell activity while suppressing shrinkage after exfoliation from the culture substrate can be efficiently and easily produced.
  • FIG. 1 (a) is an image (4 times) of the cell sheet obtained in one embodiment of the present invention observed with an inverted microscope
  • FIG. 1 (b) shows a cross section of the cell sheet using a microscope. It is an image (10 times) observed in the field of view.
  • FIG. 2 is a photograph of the peeled cell sheet
  • FIG. 2 (a) is Example 5
  • FIG. 2 (b) is Example 7
  • FIG. 2 (c) is Comparative Example 1
  • FIG. 2 (d) is Comparative Example. 2 is shown.
  • FIG. 3 is an image of the peeled cell sheet observed with an inverted microscope.
  • FIG. 3 (a) is Example 4
  • FIG. 3 (b) is Example 6
  • FIG. 3 (c) is Comparative Example 1 and FIG. d) shows Comparative Example 2.
  • FIG. 3 is an image of the peeled cell sheet observed with an inverted microscope.
  • FIG. 3 (a) is Example 4
  • FIG. 3 (b) is Example 6
  • FIG. 3 (c)
  • FIG. 4 is an image (10 times) of the aqueous dispersion of gelatin short fibers used in one example of the present invention observed with an inverted microscope.
  • FIG. 5 is a schematic explanatory view of the nonwoven fabric manufacturing apparatus used in one embodiment of the present invention.
  • 6 (a) and 6 (b) are schematic explanatory views of the filament manufacturing apparatus used in one or more embodiments of the present invention.
  • gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet.
  • the main component means that gelatin is contained in an amount of 90% by mass or more.
  • the gelatin short fiber of the present invention may contain gelatin in an amount of 90% by mass or more and other components in an amount of 10% by mass or less, gelatin in an amount of 95% by mass or more and another component in an amount of 5% by mass or less, substantially 100%. It may be composed of% by mass gelatin. Since gelatin short fibers containing gelatin as the main component with high hydrophilicity are used, it is easy to disperse in the medium and has high affinity with cells. By co-culturing with cells, gelatin short fibers are dispersed.
  • gelatin short fibers containing gelatin as a main component which is highly safe and has excellent bioabsorbability, are used, cell sheets formed using the gelatin short fibers can be transplanted into a living body for regenerative treatment and cell research. It can be suitably used as a three-dimensional cell tissue, an organelle, etc. required for drug discovery research.
  • biocompatible polymers may be other biocompatible polymers, cross-linking agents, drugs, other additives, etc., if necessary.
  • the other biocompatible polymer is not particularly limited, but a biocompatible polymer having adhesiveness to a living body such as a cell can be preferably used.
  • the biocompatible polymer is not particularly limited, but a natural polymer or a synthetic polymer can be used. Examples of natural polymers include proteins and polysaccharides. Examples of the protein include collagen, fibronectin, fibrinogen, laminin, fibrin and the like.
  • polysaccharide for example, natural polymers such as chitosan, calcium alginate, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and daiyutan gum may be used, and natural heights such as carboxymethyl cellulose may be used. Derivatives of the molecule may be used. Examples of the synthetic polymer include polyethylene glycol and the like. As the other biocompatible polymers described above, one type may be used, or two or more types may be used.
  • the type and site of the animal from which collagen, which is the raw material of the gelatin, is derived are not particularly limited.
  • Collagen may be derived from, for example, vertebrates or fish.
  • collagen derived from various organs and tissues such as dermis, ligaments, tendons, bones and cartilage can be appropriately used.
  • the method for preparing gelatin from collagen is not particularly limited, and examples thereof include acid treatment, alkali treatment, and enzyme treatment.
  • the molecular weight of the gelatin is not particularly limited, and those having various molecular weights can be appropriately selected and used.
  • the gelatin short fiber may be composed of one kind of gelatin or may contain two or more kinds of gelatin.
  • the gelatin preferably has a jelly strength of 100 g or more and 400 g or less, and more preferably 150 g or more and 360 g or less.
  • the jelly strength is measured according to JIS K 6503.
  • the gelatin may be a commercially available product.
  • the gelatin short fibers have an average fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less, and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less.
  • the gelatin short fibers have cell adhesion and are mainly composed of gelatin which can be a hydrogel, and the average fiber diameter and the average fiber length are within the above-mentioned ranges, so that the gelatin short fibers are dispersed in the cell sheet. Suppresses shrinkage after exfoliation from the culture substrate and increases cell activity.
  • the gelatin short fibers When the average fiber diameter of the gelatin short fibers is 1 ⁇ m or more, the gelatin short fibers are easily dispersed in the cell sheet, the shrinkage after peeling the cell sheet from the culture substrate is suppressed, and the cell activity is suppressed. Becomes good. In addition, the cell sheet can be easily peeled off from the culture substrate.
  • the gelatin short fibers preferably have an average fiber diameter of 5 ⁇ m or more, more preferably 10 ⁇ m or more, and further preferably 20 ⁇ m or more. Further, since the gelatin short fibers have an average fiber diameter of 400 ⁇ m or less, cells can easily adhere to the gelatin short fibers and gelatin short fibers can be easily arranged between the cells. Therefore, the cell sheet was peeled off from the culture substrate.
  • the gelatin short fibers preferably have an average fiber diameter of 200 ⁇ m or less, and more preferably 55 ⁇ m or less. In the gelatin short fibers, the coefficient of variation of the fiber diameter may be 0.6 or less.
  • the gelatin short fibers have an average fiber length of 10 ⁇ m or more, the gelatin short fibers are easily dispersed in the cell sheet, and a cell sheet in which the gelatin short fibers are dispersed is easily obtained, and the cell sheet is peeled from the culture substrate. The contraction is suppressed after the culturing. In addition, the cell sheet can be easily peeled off from the culture substrate.
  • the gelatin short fibers preferably have an average fiber length of 100 ⁇ m or more, and more preferably 200 ⁇ m or more.
  • the gelatin short fibers have an average fiber length of 2000 ⁇ m or less, the gelatin short fibers do not get entangled with each other, and a cell sheet in which the gelatin short fibers are dispersed can be easily obtained, and after the cell sheet is peeled from the culture substrate. The contraction is suppressed and the cell activity is improved.
  • the gelatin short fibers preferably have an average fiber length of 1500 ⁇ m or less, more preferably 1000 ⁇ m or less, and even more preferably 500 ⁇ m or less. In the gelatin short fiber, the coefficient of variation of the fiber length may be 0.6 or less.
  • the average fiber diameter and average fiber length of gelatin short fibers are the average fiber diameter and average fiber length of gelatin short fibers in a wet cell sheet, or the average fiber diameter and average fibers of swollen gelatin short fibers. Means long.
  • the suspension obtained by dissociating the wet cell sheet with an enzyme such as trypsin was observed with an inverted microscope and photographed, and the fiber diameter and fiber length of 400 fibers arbitrarily selected from the photographed photographs were measured.
  • the average fiber diameter and the average fiber length can be measured by calculating the average of the fiber diameter and the fiber length of 400 fibers, respectively.
  • the fiber diameters and fiber lengths of all the fibers in the suspension may be measured, and the average of the fiber diameters and fiber lengths may be calculated respectively. ..
  • the cell sheet is dry, add distilled water so that the volume of distilled water is 20 mL with respect to 1 g of the mass of the cell sheet, and leave it for 3 minutes or more to obtain a wet cell sheet. Can be done. Further, the average fiber diameter and the average fiber length of the gelatin short fibers used as the raw material for preparing the cell sheet in a swollen state may be measured.
  • gelatin short fibers were dispersed in 100 mL of distilled water, and an aqueous dispersion (suspension) left for 3 minutes or more was observed with an inverted microscope and photographed, and fibers 400 arbitrarily selected from the photographed photographs were taken.
  • the fiber diameter and fiber length of the book are measured, and the average of the fiber diameter and fiber length of 400 fibers is calculated, respectively, and used as the average fiber diameter and the average fiber length.
  • the average fiber diameter and the average fiber length can be measured in the same manner as above, except that the distilled water is 20 mL per 1 g of the gelatin short fibers. ..
  • the fiber diameters and fiber lengths of all the fibers in the aqueous dispersion are measured, and the average of the fiber diameters and fiber lengths is calculated respectively. do it.
  • it means that various operations are performed at room temperature (15 to 25 ° C.).
  • the gelatin short fibers are not particularly limited, but the aspect ratio represented by the average fiber length / average fiber diameter is preferably 3 or more, more preferably 4 or more, and further preferably 5 or more. .. When the aspect ratio is within the above-mentioned range, the effect of suppressing the contraction of the cell sheet by the gelatin short fibers can be improved, and the cell activity can be further enhanced.
  • the upper limit of the aspect ratio is not particularly limited, and may be, for example, 1000 or less, or 500 or less.
  • the aspect ratio is not particularly limited, and specifically, it may be within the range of any combination of the above-mentioned lower limit value and upper limit value, and is preferably 3 or more and 1000 or less, and 4 or more and 500 or less. It is more preferable that the ratio is 5 or more and 500 or less.
  • the gelatin short fibers are preferably crosslinked from the viewpoint of enhancing water resistance, easily maintaining the morphology in co-culture with cells in a medium, and enhancing the handleability of cell sheets.
  • the cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, and from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable.
  • the cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as ⁇ -ray, ultraviolet cross-linking, and the like.
  • it is a thermal dehydration crosslink.
  • the thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example.
  • the gelatin short fiber is not particularly limited, but can be obtained by cutting a gelatin long fiber non-woven fabric.
  • the gelatin long fiber non-woven fabric extrudes a spinning fluid containing gelatin from the nozzle discharge port, is located behind the nozzle discharge port, and is not different from the nozzle discharge port.
  • a pressure fluid is injected forward from the fluid injection port in a contact state, and the extruded spinning liquid is accompanied by the pressure fluid to be directly fiberized by a dry method, and the obtained gelatin filaments are accumulated into a non-woven fabric. It is preferable to prepare the fiber. Since the pressure fluid injection port is arranged rearward independently of the nozzle discharge port and in a non-contact state, the spinning liquid is not mixed. Therefore, it is possible to prevent contamination from being mixed into the product.
  • FIG. 5 is a schematic explanatory view of the nonwoven fabric manufacturing apparatus used in one embodiment of the present invention.
  • the spinning liquid 2 containing gelatin contained in the heating tank 1 is pushed out into the air from the nozzle discharge port 3.
  • a predetermined pressure is applied to the heating tank 1 by the compressor 4.
  • Reference numeral 12 is a heat insulating container.
  • the pressure fluid 7 is injected forward from the fluid injection port 5 which is located behind the nozzle discharge port 3 and is not in contact with the nozzle discharge port 3.
  • a pressure fluid (for example, compressed air) is supplied to the fluid injection port 5 from the compressor 6.
  • the distance between the fluid injection port 5 and the nozzle discharge port 3 is preferably 5 to 30 mm.
  • the extruded spinning liquid is accompanied by the pressure fluid 7 to become gelatin long fibers 8, and is deposited as gelatin long fiber non-woven fabric 9 on the take-up roll 11.
  • the deposited long fibers contain water or are not completely solidified, the fibers in contact with each other at at least a part of the fiber intersections are welded to each other.
  • long fibers may be collected and deposited with a net or the like to form a non-woven fabric.
  • gelatin alone or, if necessary, gelatin and other biocompatible polymers that can be used as the above-mentioned other components are dissolved in water to prepare a spinning solution.
  • the dissolution temperature water temperature
  • the dissolution temperature is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower.
  • gelatin may be dissolved in water and then filtered to remove foreign substances and dust. Further, if necessary, the dissolved air may be removed by vacuum defoaming or vacuum defoaming thereafter. From the viewpoint of efficiently removing gas (air bubbles), the degree of vacuum during decompression defoaming is preferably 5 kPa or more and 30 kPa or less.
  • gelatin Since gelatin is water-soluble, it can be spun in an aqueous solution as a spinning solution, and the safety to the living body is improved.
  • water for example, pure water, distilled water, ultrapure water or the like can be appropriately used.
  • a spinning solution can be prepared by dissolving it in water at the same time as gelatin.
  • the temperature of the spinning solution containing gelatin is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. Within the above range, gelatin can maintain a stable sol state.
  • the gelatin concentration of the gelatin aqueous solution is preferably 30% by mass or more and 55% by mass or less when the gelatin aqueous solution is 100% by mass. A more preferable concentration is 35% by mass or more and 50% by mass or less. At the above concentration, a stable sol state can be maintained.
  • the viscosity of the gelatin aqueous solution is preferably 500 mPa ⁇ s or more and 3000 mPa ⁇ s or less. If the viscosity of the gelatin aqueous solution is within the above range, stable spinning can be achieved.
  • the spinning liquid containing gelatin is discharged from the nozzle of the spinning machine, a pressure fluid is supplied from the periphery of the nozzle, and the discharged gelatin aqueous solution is associated with the pressure fluid to form fibers, and the obtained gelatin filaments are accumulated.
  • a pressure fluid is supplied from the periphery of the nozzle, and the discharged gelatin aqueous solution is associated with the pressure fluid to form fibers, and the obtained gelatin filaments are accumulated.
  • the temperature of the pressure fluid is preferably 20 ° C. or higher and 120 ° C. or lower, and more preferably 80 ° C. or higher and 120 ° C. or lower. Although it depends on the flow velocity of the pressure fluid and the temperature of the ambient atmosphere, stable spinning can be achieved within the above temperature range.
  • Air is preferably used as the pressure fluid, and the pressure is preferably 0.1 MPa or more and 1 MPa or less. Within the above range, the spinning liquid extruded into the air from the nozzle discharge port can be blown off to form fibers.
  • the fibers In the gelatin long fiber non-woven fabric, when the fibers are accumulated (deposited) after spinning, the fibers are laminated in a state of containing water, so that they are welded or entwined with each other and integrated.
  • the density of the non-woven fabric can be easily changed by changing the collection distance when the fibers are deposited.
  • the fiber length of the gelatin long fiber may be several meters to several thousand meters.
  • the average fiber diameter of the gelatin long fibers when swollen with water is preferably 1 ⁇ m or more and 400 ⁇ m or less, more preferably 10 ⁇ m or more and 200 ⁇ m or less, and 20 ⁇ m or more and 55 ⁇ m or less. Is more preferable.
  • the gelatin long-fiber non-woven fabric having a desired average fiber diameter can be obtained by appropriately adjusting the nozzle diameter (inner diameter) and the like.
  • the long fiber non-woven fabric is preferably crosslinked.
  • the cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable.
  • Examples of cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as ⁇ -rays, and ultraviolet cross-linking.
  • thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable.
  • the thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example.
  • the gelatin long fiber non-woven fabric may be dried before being crosslinked. The drying may be air drying at room temperature or vacuum freeze drying.
  • the gelatin long fiber non-woven fabric is cut to obtain gelatin short fibers. From the viewpoint of simple operation, it is preferable to cut in water. Further, from the viewpoint of improving the cutting efficiency, the gelatin long fiber non-woven fabric is cut into a predetermined size such as 5 to 10 mm square, and then the cut gelatin long fiber non-woven fabric is cut (crushed) in water using a mixer. By doing so, it is preferable to obtain gelatin short fibers.
  • the obtained gelatin short fibers may contain branched gelatin short fibers having two or more fibers at intersections, but it is preferable that the individual fibers have a shape separated from each other.
  • As the water for example, pure water, distilled water, ultrapure water or the like can be used.
  • the mixer is not particularly limited as long as it does not damage the fibers and can shorten the length of the fibers.
  • the pulverization is not particularly limited, but in the case of an ultra-high-speed rotary mixer such as a P-11 Messer mill manufactured by Fritsch, for example, the pulverization can be performed for 5 seconds or more and 10 minutes or less under the condition of a rotation speed of 2000 rpm or more and 14000 rpm or less.
  • the gelatin short fibers extrude a spinning liquid containing gelatin into the air from a nozzle discharge port, pass through a heated spinning cylinder for dry spinning, and cut the obtained gelatin filament yarn. You can also get it.
  • a spinning solution containing gelatin As the spinning liquid, the same one as that used for producing the gelatin long fiber non-woven fabric can be used. Simply put, for example, gelatin alone or, if desired, gelatin and other biocompatible polymers that can be used as the other components described above are dissolved in water and then defoamed under reduced pressure to remove the dissolved air. This makes it possible to obtain a spinning solution. After being dissolved, foreign matter and dust may be removed by filtration.
  • the melting temperature is preferably 40 ° C. or higher and 90 ° C. or lower.
  • the degree of vacuum at the time of defoaming under reduced pressure is preferably 5 kPa or more and 30 kPa or less.
  • the spinning liquid containing gelatin is extruded into the air from the nozzle discharge port of the spinning machine, passed through a heated spinning cylinder, and dry-spun. It is preferable that the temperature of the heated spinning cylinder is maintained at 120 ° C. or higher and 180 ° C. or lower, and the residence time of the extruded product in the heated spinning cylinder is 5 seconds or longer. As a result, water is rapidly removed from the extruded material, and threads are formed.
  • the heated spinning cylinder is not particularly limited, but for example, a spinning cylinder having a length of 2 m in which a heater is wound around a stainless steel pipe having an inner diameter of 200 mm can be used.
  • the heat spinning cylinder 24 may be divided into a plurality of compartments, and temperature control may be performed in each compartment.
  • the heated spinning cylinder is preferably oriented in the vertical direction. Then, at the position where the heated spinning cylinder is exited, the gelatin hollow fiber is a hollow fiber, and by cutting here, the obtained gelatin filament becomes a hollow fiber.
  • the reason why it becomes hollow at the position where it comes out of the heat spinning cylinder is considered to be that water is rapidly removed in the heating spinning cylinder. By winding the yarn without cutting it, it is possible to obtain a flat yarn having a flat hollow and a flat cross section.
  • the average fiber diameter when swollen with water is preferably 1 ⁇ m or more and 400 ⁇ m or less, more preferably 10 ⁇ m or more and 200 ⁇ m or less, and more preferably 20 ⁇ m or more and 55 ⁇ m or less.
  • a gelatin filament having a desired average fiber diameter can be obtained by appropriately adjusting the nozzle diameter (inner diameter) and the like.
  • the fiber diameter is an average value of a major axis and a minor axis in each fiber.
  • the gelatin filament is preferably crosslinked.
  • Crosslinking can be performed in the same manner as described for the method for producing a gelatin long fiber non-woven fabric. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable.
  • the thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of, for example, 10 kPa or less.
  • the gelatin filament may be dried before cross-linking. The drying may be air drying at room temperature or vacuum freeze drying.
  • the gelatin filament is cut to obtain gelatin short fibers. From the viewpoint of simple operation, it is preferable to cut in water. Further, from the viewpoint of increasing the cutting efficiency, the gelatin filament is cut so that the fiber length is, for example, 5 to 10 mm, and then further cut (crushed) in water using a mixer to obtain gelatin short fibers. It is preferable to obtain it.
  • the water for example, pure water, distilled water, ultrapure water or the like can be used.
  • the mixer is not particularly limited as long as it does not damage the fibers and can shorten the length of the fibers.
  • the pulverization is not particularly limited, but in the case of an ultra-high-speed rotary mixer such as a P-11 Messer mill manufactured by Fritsch, for example, the pulverization can be performed for 5 seconds or more and 10 minutes or less under the condition of a rotation speed of 2000 rpm or more and 14000 rpm or less.
  • FIG. 6 (a) and 6 (b) are schematic explanatory views of the filament manufacturing apparatus 20 used in one or more embodiments of the present invention.
  • the gelatin-containing spinning solution 22 contained in the syringe 21 is pushed out into the air from the nozzle 23.
  • 0.1 MPa of pressurized air can be sent from the end of the syringe 21 to push out the spinning liquid 22 from the nosol 23.
  • the nozzle 23 may have a normal round cross section.
  • a heat spinning cylinder 24 is directly connected under the nozzle 23.
  • the heat spinning cylinder 24 is composed of four compartments of 24a-24d, and the temperature can be controlled in each compartment. As shown in FIG.
  • the gelatin filament 25 passes through the guide roll 26 and is wound around the winding machine 27 to obtain a flat yarn.
  • a hollow fiber having a predetermined length can be obtained by cutting the gelatin filament 25 that drops by its own weight at the lower part of the heated spinning cylinder 24.
  • the gelatin short fibers may be sterilized by ethylene oxide gas sterilization, steam sterilization, electron beam irradiation, or irradiation with ⁇ -rays or the like.
  • the gelatin short fibers and cells By co-culturing (adhesive culture) the gelatin short fibers and cells in a medium, a cell sheet in which gelatin short fibers are dispersed can be formed.
  • the main component is gelatin, which has cell adhesion and can be a hydrogel, and gelatin short fibers having an average fiber diameter and an average fiber length within the above-mentioned ranges are co-cultured with cells in a medium. Therefore, cells can easily adhere to the surface of gelatin short fibers that are uniformly dispersed and swollen in the medium, and gelatin short fibers are arranged between the cells to obtain a cell sheet in which gelatin short fibers are dispersed. Can be done.
  • the cell may be an animal cell, and its origin is not particularly limited.
  • the animal may be a human or a non-human animal. Examples of animals other than humans include primates such as monkeys and chimpanzees, rodents such as mice, rats and hamsters, and ungulates such as cows, sheep, goats and pigs.
  • the cells include individual cells, cell lines, cells obtained by culturing such as primary culture, and the like. The cells are not particularly limited, and examples thereof include somatic cells, stem cells, progenitor cells, germ cells and the like.
  • Somatic cells include somatic cells that make up the living body and cancer cells derived from somatic cells.
  • the somatic cells constituting the living body are not particularly limited, and are, for example, fibroblasts, muscle cells, endothelial cells, osteoblasts, endothelial cells, bladder cells, lung cells, osteoblasts, nerve cells, hepatocytes, and chondrocytes. , Epithelial cells, mesenteric cells and the like.
  • the cancer cells are not particularly limited, and examples thereof include breast cancer cells, renal cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, cervical cancer cells, esophageal epithelial cancer, pancreatic cancer, colon cancer, and bladder cancer.
  • Stem cells are cells that have the potential to differentiate into various specialized cell types.
  • the stem cells are not particularly limited, and are, for example, embryonic stem cells (ES cells), embryonic cancer tumor cells (EC), embryonic germ stem cells (EG), artificial pluripotent stem cells (iPS cells), adult stem cells, and scutellum.
  • ES cells embryonic stem cells
  • EC embryonic cancer tumor cells
  • EG embryonic germ stem cells
  • iPS cells artificial pluripotent stem cells
  • adult stem cells and scutellum.
  • Spore-derived stem cells reproductive ridge-derived stem cells, malformation-derived stem cells, oncostatin-independent stem cells (OISC), bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, sheep water-derived mesenchymal stem cells, skin-derived mesenchymal stem cells Examples thereof include stem cells and bone membrane-derived mesenchymal stem cells.
  • Progenitor cells are cells that can develop from the stem cells and differentiate into the final differentiated cells that make up the living body.
  • germ cells examples include sperm, sperm cells, eggs, and egg cells.
  • one type may be used alone, or two or more types may be used in combination depending on the purpose and the like.
  • the medium is not particularly limited, and a medium containing components necessary for cell survival and proliferation can be appropriately used depending on the type of cells.
  • the medium may contain serum, antibiotics, growth factors and the like.
  • serum for example, bovine serum, fetal bovine serum, horse serum, human serum and the like can be appropriately used.
  • antibiotic penicillin, streptomycin, gentamicin, amphotericin, ampicillin, minocycline, kanamycin and the like can be appropriately used.
  • As the growth factor a cell proliferation factor, a differentiation inducing factor, a cell adhesion factor and the like can be appropriately used.
  • Culturing can be performed under adhesive culture conditions that can form cell sheets.
  • the culture substrate those that can be used for adhesive culture can be appropriately used.
  • cell culture vessels having a flat portion (also referred to as a culture surface) to which cells can adhere can be used, such as dishes, plates, flasks and the like.
  • the seeding surface density of the cells is not particularly limited, and can be appropriately determined based on the cell type, the thickness of the cell sheet, and the like. For example, it is preferably 5,000 cells / cm 2 or more and 100,000 cells / cm 2 or less, more preferably 20,000 cells / cm 2 or more and 80,000 cells / cm 2 or less, and 40,000 cells / cm 2. It is more preferably cells / cm 2 or more and 60,000 cells / cm 2 or less. When the seeding surface density of the cells is within the above-mentioned range, the cells are not biased and easily adhere to the culture surface.
  • the addition density of the gelatin short fibers is not particularly limited, but is preferably, for example, 0.05 mg / cm 2 or more and 1 mg / cm 2 or less, and 0.1 mg / cm / cm. It is more preferably cm 2 or more and 0.9 mg / cm 2 or less, and further preferably 0.2 mg / cm 2 or more and 0.6 mg / cm 2 or less.
  • the addition density of the gelatin short fibers is within the above-mentioned range, it is easy to uniformly disperse on the culture surface.
  • the seeding volume density of the cells is not particularly limited, but is preferably 0.1x10 5 cells / mL or more and 3x10 5 cells / mL or less, preferably 0.3x10 5 cells. More preferably / mL or 2x10 is 5 cells / mL or less, still more preferably not more than 0.5 ⁇ 10 5 cells / mL or more 1.5 ⁇ 10 5 cells / mL.
  • the seeding volume density of the cells is within the above range, nutrients and oxygen are easily supplied to the cells, and by co-culturing with gelatin short fibers, it becomes easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed. ..
  • the concentration of the gelatin short fibers is not particularly limited, but is preferably 0.01 mg / mL or more and 2 mg / mL or less, and 0.02 mg / mL or more and 1. It is more preferably 5 mg / mL or less, and further preferably 0.04 mg / mL or more and 1 mg / mL or less.
  • concentration of gelatin short fibers is within the above range, the dispersibility in the medium is high, and co-culturing with cells makes it easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed.
  • the ratio of the seeded amount of the cells to the added amount of gelatin short fibers is not particularly limited, but is, for example, 0.1x10 5 cells / mg or more and 7x10 5 cells / mg or less. it is preferably, 0.5 ⁇ 10 more preferably 5 cells / mg or 5x10 5 cells / mg or less, and more preferably not more than 3x10 5 cells / mg 1x10 5 cells / mg or more.
  • the ratio of the seeded amount of cells to the amount of gelatin short fibers added is within the above range, the distribution of cells is not biased, it becomes easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed, and the obtained cells are obtained.
  • the cell activity of the sheet can be increased.
  • the co-culture of cells and gelatin short fibers may be carried out, for example, at 27 ° C. or higher and 40 ° C. or lower, or 31 ° C. or higher and 37 ° C. or lower.
  • Carbon dioxide may be in the range of 2% or more and 10% or less.
  • the culturing time may be appropriately determined according to the cell type, the number of cells, etc. so as to form a cell sheet.
  • the culturing may be continued for 2 to 8 days, or continuously for 3 to 7 days. It may be continued for 4 to 6 days.
  • the medium may be changed every 2-3 days.
  • DS2 / DS1 is preferably 0.56 or more and 1 or less. It is more preferably 0.60 or more and 0.95 or less, and further preferably 0.64 or more and 0.9 or less.
  • the thickness of the cell sheet may be appropriately determined depending on the cell type, use, etc., but from the viewpoint of handleability and cell activity, it is preferably, for example, 30 ⁇ m or more and 150 ⁇ m or less, and more preferably 40 ⁇ m or more and 140 ⁇ m or less. More preferably, it is 50 ⁇ m or more and 130 ⁇ m or less.
  • the thickness of the cell sheet is measured by microscopic observation of a cross-sectional image of a frozen section.
  • the ratio of the mass of cell number and gelatin short fibers is, 1x10 5 cells / mg or more 25x10 It is preferably 5 cells / mg or less, more preferably 2x10 5 cells / mg or more and 20x10 5 cells / mg or less, and further preferably 3x10 5 cells / mg or more and 15x10 5 cells / mg or less.
  • the above-mentioned kit containing gelatin short fibers, cells and a medium can be used as a cell sheet preparation kit, and specifically, it is used to form a cell sheet in which gelatin short fibers are dispersed in the cell sheet. be able to.
  • the kit may include a culture medium.
  • the cell sheet can be used for cell therapy such as growth, repair or regeneration of human or non-human animal tissues, for example.
  • the tissue is not particularly limited, and examples thereof include connective tissues such as bone, cartilage, tendon, half moon, muscle and fat.
  • the cell sheet By containing gelatin short fibers, the cell sheet has high strength, and therefore, has good handleability in cell therapy applications.
  • peeled from the culture substrate it can easily return to its original shape by grasping it with tweezers and immersing it in a liquid such as water.
  • the cell sheet By containing gelatin short fibers, the cell sheet can prevent cell damage due to freezing, and specifically, can suppress a decrease in cell activity due to freezing.
  • the present invention is not particularly limited, but preferably includes the following aspects.
  • Gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet.
  • the gelatin short fiber is a cell sheet having an average fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less.
  • the cell sheet according to [1] wherein the gelatin short fibers are crosslinked.
  • the ratio of the number of cells to the mass of gelatin short fibers is 1x10 5 cells / mg or more and 2.5x10 6 cells / mg or less [1]. Or the cell sheet according to [2].
  • DS2 / DS1 is 0.56 or more and 1 or less.
  • the ratio of the seeded amount of the cells to the added amount of gelatin short fibers is not particularly limited, but is, for example, 0.1x10 5 cells / mg or more 7x10 5 cells.
  • the gelatin short fibers extrude a spinning liquid containing gelatin into the air from a nozzle discharge port, are located behind the nozzle discharge port, and forward from a fluid injection port in a state of non-contact with the nozzle discharge port.
  • a pressure fluid is jetted toward the surface, the extruded spinning liquid is associated with the pressure fluid to form fibers, and the fiber-formed fibers are accumulated to form a gelatin long-fiber non-woven fabric, and the gelatin long-fiber non-woven fabric is cut.
  • the method for producing a cell sheet according to any one of [5] to [7].
  • the gelatin short fibers are obtained by extruding a spinning liquid containing gelatin into the air from a nozzle discharge port, passing it through a heated spinning cylinder, dry spinning, and cutting the obtained gelatin filament yarn.
  • the gelatin short fibers have an average fiber diameter of 1 ⁇ m or more and 400 ⁇ m or less, and an average fiber length of 10 ⁇ m or more and 2000 ⁇ m or less.
  • a kit for producing a cell sheet which is used for forming a cell sheet in which gelatin short fibers are dispersed in the cell sheet.
  • the cell sheet preparation kit according to [12] wherein the gelatin short fibers are crosslinked.
  • the measurement method is as follows. ⁇ Average fiber diameter and average fiber length> An aqueous dispersion containing 5 g of gelatin short fibers and 100 mL of water was observed and photographed with an inverted microscope (Olympus, CKX53, 4x) equipped with a digital microscope (Zeiss, product name "AxioCAM ERc5"). Next, using computer software (PhotoRuler, The Genus Incybe), the fiber diameter and fiber length of 400 fibers arbitrarily selected from the photographed photographs were measured, and the average of the fiber diameters and fiber lengths of the 400 fibers was measured. Was calculated and used as the average fiber diameter and the average fiber length.
  • the temperature of the spinning liquid is 60 ° C
  • the nozzle diameter (inner diameter) is 250 ⁇ m
  • the discharge pressure is 0.2 MPa
  • the nozzle height is 5 mm
  • the air pressure is 0.375 MPa
  • the air temperature is 100 ° C
  • the distance between the fluid injection port 5 and the nozzle discharge port 3 is.
  • the gelatin long fiber non-woven fabric 9 was wound up by the winding roller 11 with a collection distance of 5 mm and a collection distance of 50 cm. Then, the gelatin long fiber non-woven fabric was air-dried at room temperature (20 ° C.) overnight, and then heat-crosslinked under vacuum (1 kPa) at 140 ° C. for 48 hours.
  • the obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used.
  • the gelatin long fiber non-woven fabric was cut by stirring at 2000 rpm for 3 minutes to obtain an aqueous dispersion of gelatin short fibers.
  • the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 ⁇ m (coefficient of variation 0.13).
  • FIG. 4 shows a photograph taken by observing an aqueous dispersion of gelatin short fibers with an inverted microscope (Olympus "CKX53", 10x) equipped with a digital microscope (Zeiss, product name "AxioCAM ERc5"). Indicated. Next, the fiber suspension was frozen in a freezer at ⁇ 30 ° C. and then vacuum freeze-dried to obtain dried gelatin short fibers.
  • a gelatin long-fiber non-woven fabric was produced in the same manner as in Production Example 1.
  • the obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used. By stirring at 2000 rpm for 1 minute, an aqueous dispersion of gelatin short fibers was obtained.
  • the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 ⁇ m (coefficient of variation 0.13). ), The average fiber length was 828 ⁇ m (coefficient of variation 0.58), and the aspect ratio was 20.7.
  • the fiber suspension was frozen in a freezer at ⁇ 30 ° C. and then freeze-dried to a degree of vacuum to obtain dried gelatin short fibers.
  • a gelatin long-fiber non-woven fabric was produced in the same manner as in Production Example 1.
  • the obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used. By stirring at 2000 rpm for 10 seconds, an aqueous dispersion of gelatin short fibers was obtained.
  • the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 ⁇ m (coefficient of variation 0.13). ), The average fiber length was 1469 ⁇ m (coefficient of variation 0.47), and the aspect ratio was 36.7.
  • the fiber suspension was frozen in a freezer at ⁇ 30 ° C. and then freeze-dried to a degree of vacuum to obtain dried gelatin short fibers.
  • Example 1 ⁇ Preparation of cell sheet> Medium (MEM ⁇ , 10% fetal bovine serum, 1% penicillin /) in which ascorbic acid phosphate was added to each well of a 24-well plate (Corning, flat bottom, culture surface area 1.9 cm 2 ) so as to be 10 ⁇ M. After adding 1 mL of streptomycin) and the gelatin short fibers obtained in Production Example 1 in the blending amounts shown in Table 1 below, the number of mouse fibroblasts MC3T3-E1 cells per well was 1.0 ⁇ 10 5 The cells were seeded to form cells. The 24-well plate was then incubated at 37 ° C. with 5% CO 2 . The medium was changed once every two days. The cells were cultured for 5 days to prepare cell sheets.
  • MEM ⁇ 10% fetal bovine serum, 1% penicillin /
  • Example 5 Using the gelatin short fibers obtained in Production Example 2 as the gelatin short fibers, a cell sheet was prepared in the same manner as in Example 1 except that the blending amount was 1 mg.
  • Example 6 The gelatin short fibers obtained in Production Example 3 were used as the gelatin short fibers, and a cell sheet was prepared in the same manner as in Example 1 except that the blending amount was 1 mg.
  • Example 7 The well plate was changed to a 12-well plate (flat bottom, culture surface area 3.8 cm 2 ) of a temperature-sensitive culture dish (UpCell, CellSeed), and the amount of gelatin short fibers obtained in Production Example 3 was changed to 0.
  • a cell sheet was prepared in the same manner as in Example 6 except that the dose was 4 mg.
  • Example 1 A cell sheet was prepared in the same manner as in Example 1 except that gelatin short fibers were not added.
  • Example 3 A cell sheet was prepared in the same manner as in Example 7 except that gelatin short fibers were not added.
  • the detached cell sheet was transferred with medium to a new 24-well plate. The medium was then removed and incubated in the absence of medium at 37 ° C. and 5% CO 2 for 30 minutes to allow the cell sheets to adhere to the well plate. 2 mL of a medium (MEM ⁇ , 10% fetal bovine serum, 1% penicillin / streptomycin) to which ascorbic acid phosphate was added to 10 ⁇ M was added, and the cells were cultured at 37 ° C. and 5% CO 2 .
  • a medium MEM ⁇ , 10% fetal bovine serum, 1% penicillin / streptomycin
  • the culture supernatant was collected at the 24th hour, and the glucose concentration in the collected culture supernatant was quantified using a glucose measurement kit (glucose CII-Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.), and the glucose concentration in the unused medium. Glucose consumption was calculated by comparison with. The results are shown in Table 2 below.
  • Example 7 (Measurement of cell sheet peeling time)
  • the temperature-sensitive culture dish (UpCell, CellSeed) formed by the cell sheet was placed on a shaker (ThermoMixerC, Eppendorf) and shaken at 25 ° C. and 300 rpm.
  • the time until the cell sheet was peeled off from the culture dish and floated in the medium was measured as the peeling time.
  • the results are shown in Table 3 below.
  • FIG. 1 shows a micrograph of the cell sheet of Example 1.
  • FIG. 1A is an image obtained by observing the cell sheet of Example 1 before peeling from the culture substrate with an inverted microscope (manufactured by Olympus, CKX53, 4x).
  • FIG. 1B is an image (10 times) of the cell sheet of Example 1 after being exfoliated from the culture substrate, observed in a bright field using a microscope (manufactured by KEYENCE, BX-X710).
  • FIG. 2 shows a photograph of the peeled cell sheet spread on the bottom surface of the well plate and taken with a digital camera (“VH-515” manufactured by Olympus Corporation).
  • VH-515 manufactured by Olympus Corporation.
  • FIG. 2 (a) to (d) are images of the peeled cell sheets of Example 5, Example 7, Comparative Example 1, and Comparative Example 2, respectively.
  • FIG. 3 shows an image of the peeled cell sheet observed with an inverted microscope (manufactured by Olympus, CKX53, 4x).
  • (a) to (d) are images of the peeled cell sheets of Example 4, Example 6, Comparative Example 1, and Comparative Example 2, respectively.
  • gelatin short fibers are dispersed in the cell sheet, and the cells are adhered to the surface of the gelatin short fibers.
  • the cell sheet of the example in which gelatin short fibers are dispersed in the cell sheet is compared with the cell sheet of Comparative Example 1 containing no gelatin short fibers and Comparative Example 2 using PET fibers. Therefore, the amount of glucose consumed per cell is high, and the cell activity is improved.
  • the cell sheet of the present invention can be used for the growth, repair or regeneration of human or non-human animal tissues.

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Abstract

The present invention relates to a cell sheet in which gelatin short fibers made mainly of gelatin are dispersed in the cell sheet, and the gelatin short fibers have an average fiber diameter of from 1 μm to 400 μm and an average fiber length of from 10 μm to 2000 μm. The present invention also relates to a method for producing a cell sheet in which a cell sheet in which gelatin short fibers are dispersed is formed by dispersing cells and gelatin short fibers made mainly of gelatin in culture medium and culturing, and the gelatin short fibers have an average fiber diameter of from 1 μm to 400 μm and an average fiber length of from 10 μm to 2000 μm. A cell sheet having high cell activity in which shrinkage after detachment from the culture substrate is suppressed, a method for producing the same, and a fabrication kit are provided thereby.

Description

細胞シート、その製造方法及び作製キットCell sheet, its manufacturing method and manufacturing kit
　本発明は、ゼラチン短繊維を含む細胞シート、その製造方法及び作製キットに関する。 The present invention relates to a cell sheet containing gelatin short fibers, a method for producing the same, and a production kit.
　細胞移植において、単に細胞を移植しても細胞が組織に着床しにくいことから、近年、胚性幹細胞（ＥＳ細胞）や人工多能性幹細胞（ｉＰＳ）等の幹細胞、体細胞等の細胞を、シート状に培養して用いることが行われている。この場合、培養後の細胞シートを培養基材から剥離して回収する必要があるが、特許文献１では、温度応答性細胞培養基材を用いることで、培養後の細胞シートを回収しやすくすることが提案されている。 In cell transplantation, it is difficult for cells to implant into tissues even if cells are simply transplanted. Therefore, in recent years, stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS), and cells such as somatic cells have been introduced. , It is used by culturing it in the form of a sheet. In this case, it is necessary to peel off the cultured cell sheet from the culture substrate and collect it. However, in Patent Document 1, the cell sheet after culturing can be easily collected by using the temperature-responsive cell culture substrate. Has been proposed.
特開平０５－１９２１３８号公報Japanese Unexamined Patent Publication No. 05-192138
　しかし、細胞シートは、培養基材から剥離した後に収縮しやすいという問題があった。 However, there is a problem that the cell sheet tends to shrink after being peeled from the culture substrate.
　本発明は、前記従来の問題を解決するため、培養基材から剥離した後の収縮が抑制されるとともに、細胞活性も高い細胞シート、その製造方法及び作製キットを提供する。 In order to solve the above-mentioned conventional problems, the present invention provides a cell sheet in which shrinkage after exfoliation from a culture substrate is suppressed and also has high cell activity, a method for producing the same, and a preparation kit.
　本発明は、ゼラチンを主成分とするゼラチン短繊維が細胞シート中に分散しており、前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シートに関する。 In the present invention, gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet, and the gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less and an average fiber length of 10 μm or more and 2000 μm or less. It relates to a cell sheet characterized by being present.
　本発明は、また、細胞及びゼラチンを主成分とするゼラチン短繊維を培地中で培養することで、前記ゼラチン短繊維が分散している細胞シートを形成しており、前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シートの製造方法に関する。 In the present invention, cells and gelatin short fibers containing gelatin as a main component are cultured in a medium to form a cell sheet in which the gelatin short fibers are dispersed, and the gelatin short fibers are averaged. The present invention relates to a method for producing a cell sheet, which comprises a fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less.
　本発明は、また、細胞、ゼラチン短繊維及び培地を含み、前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であり、細胞シート中にゼラチン短繊維が分散している細胞シートを形成するのに用いることを特徴とする細胞シートの作製キットに関する。 The present invention also includes cells, gelatin short fibers and a medium, wherein the gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less, and gelatin in a cell sheet. The present invention relates to a cell sheet preparation kit, which is used for forming a cell sheet in which short fibers are dispersed.
　本発明によれば、培養基材から剥離した後の収縮が抑制されるとともに、細胞活性も高い細胞シートを提供することができる。
　本発明の製造方法によれば、培養基材から剥離した後の収縮が抑制されるとともに、細胞活性も高い細胞シートを効率よく簡便に作製することができる。
　本発明の作製キットによれば、培養基材から剥離した後の収縮が抑制されるとともに、細胞活性も高い細胞シートを効率よく簡便に作製することができる。 According to the present invention, it is possible to provide a cell sheet in which shrinkage after exfoliation from the culture substrate is suppressed and cell activity is high.
According to the production method of the present invention, a cell sheet having high cell activity while suppressing shrinkage after exfoliation from the culture substrate can be efficiently and easily produced.
According to the production kit of the present invention, a cell sheet having high cell activity while suppressing shrinkage after exfoliation from the culture substrate can be efficiently and easily produced.
図１（ａ）は、本発明の一実施例で得られた細胞シートを倒立顕微鏡で観察した画像（４倍）であり、図１（ｂ）は同細胞シートの断面を顕微鏡を用いて明視野で観察した画像（１０倍）である。FIG. 1 (a) is an image (4 times) of the cell sheet obtained in one embodiment of the present invention observed with an inverted microscope, and FIG. 1 (b) shows a cross section of the cell sheet using a microscope. It is an image (10 times) observed in the field of view. 図２は剥離した細胞シートの写真であり、図２（ａ）は実施例５、図２（ｂ）は実施例７、図２（ｃ）は比較例１、図２（ｄ）は比較例２を示す。FIG. 2 is a photograph of the peeled cell sheet, FIG. 2 (a) is Example 5, FIG. 2 (b) is Example 7, FIG. 2 (c) is Comparative Example 1, and FIG. 2 (d) is Comparative Example. 2 is shown. 図３は剥離した細胞シートを倒立顕微鏡で観察した画像であり、図３（ａ）は実施例４、図３（ｂ）は実施例６、図３（ｃ）は比較例１、図３（ｄ）は比較例２を示す。FIG. 3 is an image of the peeled cell sheet observed with an inverted microscope. FIG. 3 (a) is Example 4, FIG. 3 (b) is Example 6, and FIG. 3 (c) is Comparative Example 1 and FIG. d) shows Comparative Example 2. 図４は本発明の一実施例で用いたゼラチン短繊維の水分散液を倒立顕微鏡で観察した画像（１０倍）である。FIG. 4 is an image (10 times) of the aqueous dispersion of gelatin short fibers used in one example of the present invention observed with an inverted microscope. 図５は本発明の一実施例で使用する不織布製造装置の模式的説明図である。FIG. 5 is a schematic explanatory view of the nonwoven fabric manufacturing apparatus used in one embodiment of the present invention. 図６（ａ）及び（ｂ）は本発明の１以上の実施形態で用いるフィラメント製造装置の模式的説明図である。6 (a) and 6 (b) are schematic explanatory views of the filament manufacturing apparatus used in one or more embodiments of the present invention.
　本発明の１以上の実施形態において、細胞シート中にゼラチンを主成分とするゼラチン短繊維が分散している。本発明において、主成分とは、ゼラチンを９０質量％以上含むことを意味する。本発明のゼラチン短繊維は、ゼラチンを９０質量％以上、他の成分を１０質量％以下含んでもよく、ゼラチンを９５質量％以上、他の成分を５質量％以下含んでもよく、実質的に１００質量％のゼラチンで構成されてもよい。親水性が高いゼラチンを主成分とするゼラチン短繊維を用いるため、培地中に分散しやすい上、細胞との親和性が高く、細胞と共培養することで、ゼラチン短繊維が分散している細胞シートを得ることができる。また、安全性が高く、生体吸収性に優れるゼラチンを主成分とするゼラチン短繊維を用いることから、該ゼラチン短繊維を用いて形成した細胞シートは、生体に移植して再生治療用、細胞研究および創薬研究に必要となる3次元細胞組織体、オルガネラ等として好適に用いることができる。 In one or more embodiments of the present invention, gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet. In the present invention, the main component means that gelatin is contained in an amount of 90% by mass or more. The gelatin short fiber of the present invention may contain gelatin in an amount of 90% by mass or more and other components in an amount of 10% by mass or less, gelatin in an amount of 95% by mass or more and another component in an amount of 5% by mass or less, substantially 100%. It may be composed of% by mass gelatin. Since gelatin short fibers containing gelatin as the main component with high hydrophilicity are used, it is easy to disperse in the medium and has high affinity with cells. By co-culturing with cells, gelatin short fibers are dispersed. You can get a sheet. In addition, since gelatin short fibers containing gelatin as a main component, which is highly safe and has excellent bioabsorbability, are used, cell sheets formed using the gelatin short fibers can be transplanted into a living body for regenerative treatment and cell research. It can be suitably used as a three-dimensional cell tissue, an organelle, etc. required for drug discovery research.
　他の成分は、必要に応じて、他の生体適合ポリマー、架橋剤、薬剤、他の添加剤等であってもよい。他の生体適合ポリマーは、特に限定されないが、細胞等の生体と接着性を有する生体適合性ポリマーを好適に用いることができる。このような生体適合性ポリマーとしては、特に限定されないが、天然高分子や合成高分子を用いることができる。天然高分子としては、例えばタンパク質や多糖類等が挙げられる。タンパク質としては、例えばコラーゲン、フィブロネクチン、フィブリノーゲン、ラミニン、フィブリン等が挙げられる。多糖類としては、例えばキトサン、アルギン酸カルシウム、ジェランガム、アガロース、グァーガム、キサンタンガム、カラギーナン、ペクチン、ローカストビーンガム、タマリンドガム、ダイユータンガム等の天然高分子を用いてもよく、カルボキシメチルセルロース等の天然高分子の誘導体を用いてもよい。合成高分子としては、例えば、ポリエチレングリコール等が挙げられる。上述した他の生体適合ポリマーは、１種を用いてもよく、２種以上を用いてもよい。 Other components may be other biocompatible polymers, cross-linking agents, drugs, other additives, etc., if necessary. The other biocompatible polymer is not particularly limited, but a biocompatible polymer having adhesiveness to a living body such as a cell can be preferably used. The biocompatible polymer is not particularly limited, but a natural polymer or a synthetic polymer can be used. Examples of natural polymers include proteins and polysaccharides. Examples of the protein include collagen, fibronectin, fibrinogen, laminin, fibrin and the like. As the polysaccharide, for example, natural polymers such as chitosan, calcium alginate, gellan gum, agarose, guar gum, xanthan gum, carrageenan, pectin, locust bean gum, tamarind gum, and daiyutan gum may be used, and natural heights such as carboxymethyl cellulose may be used. Derivatives of the molecule may be used. Examples of the synthetic polymer include polyethylene glycol and the like. As the other biocompatible polymers described above, one type may be used, or two or more types may be used.
　前記ゼラチンの原材料となるコラーゲンが由来する動物の種類や部位は特に限定されない。コラーゲンは、例えば脊髄動物由来でもよく、魚由来でもよい。また、真皮、靭帯、腱、骨、軟骨等の様々な器官や組織由来のコラーゲンを適宜用いることができる。また、コラーゲンからゼラチンを調製する方法も特に限定されず、例えば酸処理、アルカリ処理、及び酵素処理等が挙げられる。前記ゼラチンの分子量も特に限定されず、様々な分子量のものを適宜選択して用いることができる。また、ゼラチン短繊維は、１種のゼラチンで構成されてもよく、２種以上のゼラチンを含んでもよい。 The type and site of the animal from which collagen, which is the raw material of the gelatin, is derived are not particularly limited. Collagen may be derived from, for example, vertebrates or fish. In addition, collagen derived from various organs and tissues such as dermis, ligaments, tendons, bones and cartilage can be appropriately used. Further, the method for preparing gelatin from collagen is not particularly limited, and examples thereof include acid treatment, alkali treatment, and enzyme treatment. The molecular weight of the gelatin is not particularly limited, and those having various molecular weights can be appropriately selected and used. Further, the gelatin short fiber may be composed of one kind of gelatin or may contain two or more kinds of gelatin.
　前記ゼラチンは、適度な柔軟性及び硬さを有する観点から、ゼリー強度が１００ｇ以上４００ｇ以下であることが好ましく、より好ましくは１５０ｇ以上３６０ｇ以下である。本発明において、ゼリー強度は、ＪＩＳ　Ｋ　６５０３に準じて測定する。前記ゼラチンは、市販品であってもよい。 From the viewpoint of having appropriate flexibility and hardness, the gelatin preferably has a jelly strength of 100 g or more and 400 g or less, and more preferably 150 g or more and 360 g or less. In the present invention, the jelly strength is measured according to JIS K 6503. The gelatin may be a commercially available product.
　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、平均繊維長が１０μｍ以上２０００μｍ以下である。前記ゼラチン短繊維が細胞接着性を有し、ハイドロゲルとなり得るゼラチンを主成分とするとともに、平均繊維径と平均繊維長が上述した範囲内であることで、該ゼラチン短繊維が分散した細胞シートは、培養基材から剥離した後の収縮が抑制されるとともに、細胞活性も高くなる。 The gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less. The gelatin short fibers have cell adhesion and are mainly composed of gelatin which can be a hydrogel, and the average fiber diameter and the average fiber length are within the above-mentioned ranges, so that the gelatin short fibers are dispersed in the cell sheet. Suppresses shrinkage after exfoliation from the culture substrate and increases cell activity.
　前記ゼラチン短繊維は、平均繊維径が１μｍ以上であることで、ゼラチン短繊維が細胞シート中に分散しやすく、細胞シートを培養基材から剥離した後に収縮することが抑制されるとともに、細胞活性が良好になる。また、培養基材から細胞シートを剥離しやすくなる。前記ゼラチン短繊維は、平均繊維径が５μｍ以上であることが好ましく、より好ましくは１０μｍ以上であり、さらに好ましくは２０μｍ以上である。また、前記ゼラチン短繊維は、平均繊維径が４００μｍ以下であることで、ゼラチン短繊維に細胞が接着しやすく、細胞間にゼラチン短繊維が配置されやすいため、細胞シートを培養基材から剥離した後に収縮することが抑制されるとともに、細胞活性が良好になる。前記ゼラチン短繊維は、平均繊維径が２００μｍ以下であることが好ましく、５５μｍ以下であることがより好ましい。前記ゼラチン短繊維において、繊維径の変動係数は０．６以下であってもよい。 When the average fiber diameter of the gelatin short fibers is 1 μm or more, the gelatin short fibers are easily dispersed in the cell sheet, the shrinkage after peeling the cell sheet from the culture substrate is suppressed, and the cell activity is suppressed. Becomes good. In addition, the cell sheet can be easily peeled off from the culture substrate. The gelatin short fibers preferably have an average fiber diameter of 5 μm or more, more preferably 10 μm or more, and further preferably 20 μm or more. Further, since the gelatin short fibers have an average fiber diameter of 400 μm or less, cells can easily adhere to the gelatin short fibers and gelatin short fibers can be easily arranged between the cells. Therefore, the cell sheet was peeled off from the culture substrate. Later contraction is suppressed and cell activity is improved. The gelatin short fibers preferably have an average fiber diameter of 200 μm or less, and more preferably 55 μm or less. In the gelatin short fibers, the coefficient of variation of the fiber diameter may be 0.6 or less.
　前記ゼラチン短繊維は、平均繊維長が１０μｍ以上であることで、ゼラチン短繊維が細胞シート中に分散しやすく、ゼラチン短繊維が分散した細胞シートが得られやすく、細胞シートを培養基材から剥離した後に収縮することが抑制される。また、培養基材から細胞シートを剥離しやすくなる。前記ゼラチン短繊維は、平均繊維長が１００μｍ以上であることが好ましく、２００μｍ以上であることがより好ましい。また、前記ゼラチン短繊維は、平均繊維長が２０００μｍ以下であることで、ゼラチン短繊維が互いに絡まず、ゼラチン短繊維が分散した細胞シートが得られやすく、細胞シートを培養基材から剥離した後に収縮することが抑制されるとともに、細胞活性が良好になる。前記ゼラチン短繊維は、平均繊維長が１５００μｍ以下であることが好ましく、１０００μｍ以下であることがより好ましく、さらに好ましくは５００μｍ以下である。前記ゼラチン短繊維において、繊維長の変動係数は０．６以下であってもよい。 Since the gelatin short fibers have an average fiber length of 10 μm or more, the gelatin short fibers are easily dispersed in the cell sheet, and a cell sheet in which the gelatin short fibers are dispersed is easily obtained, and the cell sheet is peeled from the culture substrate. The contraction is suppressed after the culturing. In addition, the cell sheet can be easily peeled off from the culture substrate. The gelatin short fibers preferably have an average fiber length of 100 μm or more, and more preferably 200 μm or more. Further, since the gelatin short fibers have an average fiber length of 2000 μm or less, the gelatin short fibers do not get entangled with each other, and a cell sheet in which the gelatin short fibers are dispersed can be easily obtained, and after the cell sheet is peeled from the culture substrate. The contraction is suppressed and the cell activity is improved. The gelatin short fibers preferably have an average fiber length of 1500 μm or less, more preferably 1000 μm or less, and even more preferably 500 μm or less. In the gelatin short fiber, the coefficient of variation of the fiber length may be 0.6 or less.
　本発明において、ゼラチン短繊維の平均繊維径及び平均繊維長は、湿潤状態の細胞シートにおけるゼラチン短繊維の平均繊維径及び平均繊維長、或いは、膨潤状態のゼラチン短繊維の平均繊維径及び平均繊維長を意味する。湿潤状態の細胞シートをトリプシン等の酵素で解離して得られた懸濁液を倒立顕微鏡で観察して撮影し、撮影した写真から任意に選択した繊維４００本の繊維径と繊維長を計測し、４００本の繊維の繊維径と繊維長の平均をそれぞれ算出することで、平均繊維径及び平均繊維長を測定することができる。前記懸濁液中の繊維の本数が４００本未満の場合は、懸濁液中の全ての繊維の繊維径と繊維長を計測し、それらの繊維径と繊維長の平均をそれぞれ算出すればよい。細胞シートが乾燥している場合は、細胞シートの質量１ｇに対して蒸留水の体積が２０ｍＬになるように、蒸留水を添加して３分間以上放置することで湿潤状態の細胞シートを得ることができる。また、細胞シートの作製に用いた原料のゼラチン短繊維を膨潤状態の平均繊維径及び平均繊維長を測定してもよい。具体的には、ゼラチン短繊維５ｇを蒸留水１００ｍＬに分散させ、３分間以上放置した水分散液（懸濁液）を倒立顕微鏡で観察して撮影し、撮影した写真から任意に選択した繊維４００本の繊維径と繊維長を計測し、４００本の繊維の繊維径と繊維長の平均をそれぞれ算出し、平均繊維径及び平均繊維長とする。ゼラチン短繊維の質量が５ｇ未満の場合は、ゼラチン短繊維の質量１ｇあたりに蒸留水が２０ｍＬになるようにした以外は、上記と同様にして平均繊維径及び平均繊維長を測定することができる。ゼラチン短繊維の水分散液中の繊維の本数が４００本未満の場合は、水分散液中の全ての繊維の繊維径と繊維長を計測し、それらの繊維径と繊維長の平均をそれぞれ算出すればよい。なお、本発明において、特に指摘がない場合、各種操作は、室温（１５～２５℃）で行うことを意味する。 In the present invention, the average fiber diameter and average fiber length of gelatin short fibers are the average fiber diameter and average fiber length of gelatin short fibers in a wet cell sheet, or the average fiber diameter and average fibers of swollen gelatin short fibers. Means long. The suspension obtained by dissociating the wet cell sheet with an enzyme such as trypsin was observed with an inverted microscope and photographed, and the fiber diameter and fiber length of 400 fibers arbitrarily selected from the photographed photographs were measured. , The average fiber diameter and the average fiber length can be measured by calculating the average of the fiber diameter and the fiber length of 400 fibers, respectively. When the number of fibers in the suspension is less than 400, the fiber diameters and fiber lengths of all the fibers in the suspension may be measured, and the average of the fiber diameters and fiber lengths may be calculated respectively. .. When the cell sheet is dry, add distilled water so that the volume of distilled water is 20 mL with respect to 1 g of the mass of the cell sheet, and leave it for 3 minutes or more to obtain a wet cell sheet. Can be done. Further, the average fiber diameter and the average fiber length of the gelatin short fibers used as the raw material for preparing the cell sheet in a swollen state may be measured. Specifically, 5 g of gelatin short fibers were dispersed in 100 mL of distilled water, and an aqueous dispersion (suspension) left for 3 minutes or more was observed with an inverted microscope and photographed, and fibers 400 arbitrarily selected from the photographed photographs were taken. The fiber diameter and fiber length of the book are measured, and the average of the fiber diameter and fiber length of 400 fibers is calculated, respectively, and used as the average fiber diameter and the average fiber length. When the mass of the gelatin short fibers is less than 5 g, the average fiber diameter and the average fiber length can be measured in the same manner as above, except that the distilled water is 20 mL per 1 g of the gelatin short fibers. .. When the number of fibers in the aqueous dispersion of gelatin short fibers is less than 400, the fiber diameters and fiber lengths of all the fibers in the aqueous dispersion are measured, and the average of the fiber diameters and fiber lengths is calculated respectively. do it. In the present invention, unless otherwise specified, it means that various operations are performed at room temperature (15 to 25 ° C.).
　前記ゼラチン短繊維は、特に限定されないが、平均繊維長/平均繊維径で表されるアスペクト比が３以上であることが好ましく、４以上であることがより好ましく、５以上であることがさらに好ましい。アスペクト比が上述した範囲内であると、ゼラチン短繊維による細胞シートの収縮抑制効果を向上させるとともに、細胞活性をより高めることができる。アスペクト比の上限は、特に限定されず、例えば、１０００以下であってもよく、５００以下であってもよい。アスペクト比は、特に限定されず、具体的には、上述した下限値及び上限値の任意の組み合わせの範囲内であってもよく、例えば、３以上１０００以下であることが好ましく、４以上５００以下であることがより好ましく、５以上５００以下であることが特に好ましい。 The gelatin short fibers are not particularly limited, but the aspect ratio represented by the average fiber length / average fiber diameter is preferably 3 or more, more preferably 4 or more, and further preferably 5 or more. .. When the aspect ratio is within the above-mentioned range, the effect of suppressing the contraction of the cell sheet by the gelatin short fibers can be improved, and the cell activity can be further enhanced. The upper limit of the aspect ratio is not particularly limited, and may be, for example, 1000 or less, or 500 or less. The aspect ratio is not particularly limited, and specifically, it may be within the range of any combination of the above-mentioned lower limit value and upper limit value, and is preferably 3 or more and 1000 or less, and 4 or more and 500 or less. It is more preferable that the ratio is 5 or more and 500 or less.
　前記ゼラチン短繊維は、耐水性を高め、培地中での細胞との共培養において形態を維持しやすく、細胞シートのハンドリング性を高める観点から、架橋されていることが好ましい。架橋は、架橋剤等の化合物を用いた化学架橋であってもよく、生体安全性の観点から、生体安全性を有する架橋剤を用いる架橋、架橋剤を用いない架橋であることが好ましい。架橋剤を用いない架橋としては、例えば、熱架橋、電子線架橋、γ線等の放射線架橋、紫外線架橋等が挙げられ、簡便に所望の架橋効果を得やすい観点から、熱架橋であることが好ましく、熱脱水架橋であることがより好ましい。熱脱水架橋は、例えば、１００℃以上１６０℃以下で、２４時間以上９６時間以下行ってもよい。また、熱脱水架橋は、例えば、１ｋＰａ以下の真空下で行ってもよい。 The gelatin short fibers are preferably crosslinked from the viewpoint of enhancing water resistance, easily maintaining the morphology in co-culture with cells in a medium, and enhancing the handleability of cell sheets. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, and from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable. Examples of the cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as γ-ray, ultraviolet cross-linking, and the like. Preferably, it is a thermal dehydration crosslink. The thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example.
　前記ゼラチン短繊維は、特に限定されないが、ゼラチン長繊維不織布を切断することで得ることができる。夾雑物の発生を抑制し、製品汚染を防ぐ観点から、前記ゼラチン長繊維不織布は、ゼラチンを含む紡糸液をノズル吐出口から押し出し、ノズル吐出口の後方に位置し、前記ノズル吐出口とは非接触状態の流体噴射口から前方に向けて圧力流体を噴射し、前記押し出された紡糸液を前記圧力流体に随伴させて乾式でダイレクトに繊維化し、得られたゼラチン長繊維を集積させて不織布にすることで作製することが好ましい。圧力流体噴射口は、ノズル吐出口とは独立にかつ非接触状態で後方に配置されているため、紡糸液が混入することはない。このため、製品にコンタミが混入することを防止できる。 The gelatin short fiber is not particularly limited, but can be obtained by cutting a gelatin long fiber non-woven fabric. From the viewpoint of suppressing the generation of impurities and preventing product contamination, the gelatin long fiber non-woven fabric extrudes a spinning fluid containing gelatin from the nozzle discharge port, is located behind the nozzle discharge port, and is not different from the nozzle discharge port. A pressure fluid is injected forward from the fluid injection port in a contact state, and the extruded spinning liquid is accompanied by the pressure fluid to be directly fiberized by a dry method, and the obtained gelatin filaments are accumulated into a non-woven fabric. It is preferable to prepare the fiber. Since the pressure fluid injection port is arranged rearward independently of the nozzle discharge port and in a non-contact state, the spinning liquid is not mixed. Therefore, it is possible to prevent contamination from being mixed into the product.
　図５は本発明の一実施例で使用する不織布製造装置の模式的説明図である。不織布製造装置１０において、加温槽１に入れたゼラチンを含む紡糸液２をノズル吐出口３から空気中に押し出す。加温槽１にはコンプレッサー４により、所定の圧力をかけておく。１２は保温容器である。
　また、ノズル吐出口３の後方に位置し、ノズル吐出口３とは非接触状態の流体噴射口５から前方に向けて圧力流体７を噴射させる。流体噴射口５にはコンプレッサー６から圧力流体（例えば圧空）が供給される。流体噴射口５とノズル吐出口３との距離は５～３０ｍｍが好ましい。
　押し出された紡糸液は圧力流体７に随伴されてゼラチン長繊維８となり、巻き取りロール１１上でゼラチン長繊維不織布９となって堆積される。この時、堆積された長繊維は水分を含んでいたり、完全には固化していないので、繊維交点の少なくとも一部において接している繊維が互いに溶着する。なお、巻き取りロール以外でもネット等で長繊維を捕集し堆積して不織布にしてもよい。 FIG. 5 is a schematic explanatory view of the nonwoven fabric manufacturing apparatus used in one embodiment of the present invention. In the non-woven fabric manufacturing apparatus 10, the spinning liquid 2 containing gelatin contained in the heating tank 1 is pushed out into the air from the nozzle discharge port 3. A predetermined pressure is applied to the heating tank 1 by the compressor 4. Reference numeral 12 is a heat insulating container.
Further, the pressure fluid 7 is injected forward from the fluid injection port 5 which is located behind the nozzle discharge port 3 and is not in contact with the nozzle discharge port 3. A pressure fluid (for example, compressed air) is supplied to the fluid injection port 5 from the compressor 6. The distance between the fluid injection port 5 and the nozzle discharge port 3 is preferably 5 to 30 mm.
The extruded spinning liquid is accompanied by the pressure fluid 7 to become gelatin long fibers 8, and is deposited as gelatin long fiber non-woven fabric 9 on the take-up roll 11. At this time, since the deposited long fibers contain water or are not completely solidified, the fibers in contact with each other at at least a part of the fiber intersections are welded to each other. In addition to the take-up roll, long fibers may be collected and deposited with a net or the like to form a non-woven fabric.
　まず、ゼラチン単独、或いは、必要に応じてゼラチンと上述した他の成分として用いることができる他の生体適合ポリマーを水に溶解して紡糸液を調製する。溶解温度（水の温度）は２０℃以上９０℃以下が好ましく、４０℃以上９０℃以下であることがより好ましい。必要に応じて、ゼラチンを水に溶解した後、フィルトレーションして異物やごみなどを除去してもよい。また、必要に応じて、その後、減圧脱泡又は真空脱泡して溶解空気を除去してもよい。効率よく気体（気泡）を除去する観点から、減圧脱泡時の真空度は５ｋＰａ以上３０ｋＰａ以下であることが好ましい。ゼラチンが水溶性であることで、紡糸液として水溶液の状態で紡糸でき、生体に対する安全性が高くなる。水としては、例えば、純水、蒸留水、超純水等を適宜用いることができる。なお、他の成分として、他の生体適合性水溶性高分子を用いる場合、ゼラチンと同時に水に溶解することで、紡糸液を調製することができる。 First, gelatin alone or, if necessary, gelatin and other biocompatible polymers that can be used as the above-mentioned other components are dissolved in water to prepare a spinning solution. The dissolution temperature (water temperature) is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. If necessary, gelatin may be dissolved in water and then filtered to remove foreign substances and dust. Further, if necessary, the dissolved air may be removed by vacuum defoaming or vacuum defoaming thereafter. From the viewpoint of efficiently removing gas (air bubbles), the degree of vacuum during decompression defoaming is preferably 5 kPa or more and 30 kPa or less. Since gelatin is water-soluble, it can be spun in an aqueous solution as a spinning solution, and the safety to the living body is improved. As the water, for example, pure water, distilled water, ultrapure water or the like can be appropriately used. When another biocompatible water-soluble polymer is used as another component, a spinning solution can be prepared by dissolving it in water at the same time as gelatin.
　前記ゼラチンを含む紡糸液（ゼラチン水溶液）の温度は２０℃以上９０℃以下であることが好ましく、４０℃以上９０℃以下であることがより好ましい。前記の範囲であればゼラチンは安定したゾル状態を維持できる。また、前記ゼラチン水溶液のゼラチン濃度は、ゼラチン水溶液を１００質量％とした時、３０質量％以上５５質量％以下であることが好ましい。さらに好ましい濃度は３５質量％以上５０質量％以下である。前記の濃度であれば安定したゾル状態を維持できる。前記ゼラチン水溶液（紡糸液）の粘度は５００ｍＰａ・ｓ以上３０００ｍＰａ・ｓ以下が好ましい。ゼラチン水溶液の粘度が前記の範囲であれば安定した紡糸ができる。 The temperature of the spinning solution containing gelatin (gelatin aqueous solution) is preferably 20 ° C. or higher and 90 ° C. or lower, and more preferably 40 ° C. or higher and 90 ° C. or lower. Within the above range, gelatin can maintain a stable sol state. The gelatin concentration of the gelatin aqueous solution is preferably 30% by mass or more and 55% by mass or less when the gelatin aqueous solution is 100% by mass. A more preferable concentration is 35% by mass or more and 50% by mass or less. At the above concentration, a stable sol state can be maintained. The viscosity of the gelatin aqueous solution (spinning solution) is preferably 500 mPa · s or more and 3000 mPa · s or less. If the viscosity of the gelatin aqueous solution is within the above range, stable spinning can be achieved.
　前記ゼラチンを含む紡糸液を紡糸機のノズルから吐出し、前記ノズル周囲から圧力流体を供給し、前記吐出したゼラチン水溶液を前記圧力流体に随伴させて繊維形成させ、得られたゼラチン長繊維を集積してゼラチン長繊維不織布とする。 The spinning liquid containing gelatin is discharged from the nozzle of the spinning machine, a pressure fluid is supplied from the periphery of the nozzle, and the discharged gelatin aqueous solution is associated with the pressure fluid to form fibers, and the obtained gelatin filaments are accumulated. To make a gelatin long fiber non-woven fabric.
　前記圧力流体の温度は、２０℃以上１２０℃以下であることが好ましく、８０℃以上１２０℃以下であることがより好ましい。圧力流体の流速及び周囲雰囲気の温度にもよるが、前記の温度範囲であれば安定した紡糸ができる。圧力流体は空気を使用することが好ましく、圧力は０.１ＭＰａ以上１ＭＰａ以下であることが好ましい。前記の範囲であれば、ノズル吐出口から空気中に押し出された紡糸液を吹き飛ばして繊維化できる。 The temperature of the pressure fluid is preferably 20 ° C. or higher and 120 ° C. or lower, and more preferably 80 ° C. or higher and 120 ° C. or lower. Although it depends on the flow velocity of the pressure fluid and the temperature of the ambient atmosphere, stable spinning can be achieved within the above temperature range. Air is preferably used as the pressure fluid, and the pressure is preferably 0.1 MPa or more and 1 MPa or less. Within the above range, the spinning liquid extruded into the air from the nozzle discharge port can be blown off to form fibers.
　前記ゼラチン長繊維不織布は紡糸後に繊維を集積（堆積）させる時に繊維同士が、水分を含んだ状態で積層されるため、溶着したり互いに絡んで一体化されている。繊維を堆積させる際の捕集距離を変えることで、容易に不織布密度を変えることができる。ゼラチン長繊維不織布において、ゼラチン長繊維の繊維長は数メートル～数千メートルであってもよい。 In the gelatin long fiber non-woven fabric, when the fibers are accumulated (deposited) after spinning, the fibers are laminated in a state of containing water, so that they are welded or entwined with each other and integrated. The density of the non-woven fabric can be easily changed by changing the collection distance when the fibers are deposited. In the gelatin long fiber non-woven fabric, the fiber length of the gelatin long fiber may be several meters to several thousand meters.
　前記ゼラチン長繊維不織布において、水で膨潤した際のゼラチン長繊維の平均繊維径は、１μｍ以上４００μｍ以下であることが好ましく、１０μｍ以上２００μｍ以下であることがより好ましく、２０μｍ以上５５μｍ以下であることがより好ましい。ノズル径（内径）等適宜を調整することで、所望の平均繊維径を有する前記ゼラチン長繊維不織布を得ることができる。 In the gelatin long fiber non-woven fabric, the average fiber diameter of the gelatin long fibers when swollen with water is preferably 1 μm or more and 400 μm or less, more preferably 10 μm or more and 200 μm or less, and 20 μm or more and 55 μm or less. Is more preferable. The gelatin long-fiber non-woven fabric having a desired average fiber diameter can be obtained by appropriately adjusting the nozzle diameter (inner diameter) and the like.
　前記長繊維不織布は、架橋することが好ましい。これにより形態安定性及び耐水性を高めることができ、ゼラチン長繊維不織布を切断してゼラチン短繊維を得る工程の作業性が良好になる。架橋は、架橋剤等の化合物を用いた化学架橋であってもよいが、生体安全性の観点から、生体安全性を有する架橋剤を用いる架橋、架橋剤を用いない架橋であることが好ましい。架橋剤を用いない架橋としては、例えば、熱架橋、電子線架橋、γ線等の放射線架橋、紫外線架橋等が挙げられる。電子線照射、γ線等の放射線照射の場合は、滅菌と架橋を同時にすることもできる。簡便に所望の架橋効果を得やすい観点から、熱架橋であることが好ましく、熱脱水架橋であることがより好ましい。熱脱水架橋は、例えば、１００℃以上１６０℃以下で、２４時間以上９６時間以下行ってもよい。また、熱脱水架橋は、例えば、１ｋＰａ以下の真空下で行ってもよい。前記ゼラチン長繊維不織布は、架橋する前に乾燥してもよい。乾燥は、室温における風乾でもよく、真空凍結乾燥でもよい。 The long fiber non-woven fabric is preferably crosslinked. As a result, morphological stability and water resistance can be improved, and workability in the process of cutting the gelatin long fiber non-woven fabric to obtain gelatin short fibers is improved. The cross-linking may be a chemical cross-linking using a compound such as a cross-linking agent, but from the viewpoint of biosafety, a cross-linking using a cross-linking agent having biosafety or a cross-linking without using a cross-linking agent is preferable. Examples of cross-linking without using a cross-linking agent include thermal cross-linking, electron beam cross-linking, radiation cross-linking such as γ-rays, and ultraviolet cross-linking. In the case of electron beam irradiation or radiation irradiation such as γ-rays, sterilization and cross-linking can be performed at the same time. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. The thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of 1 kPa or less, for example. The gelatin long fiber non-woven fabric may be dried before being crosslinked. The drying may be air drying at room temperature or vacuum freeze drying.
　次に、前記ゼラチン長繊維不織布を切断してゼラチン短繊維を得る。操作が簡便である観点から、水中で切断することが好ましい。また、切断効率を高める観点から、前記ゼラチン長繊維不織布を、例えば、５～１０ｍｍ角等の所定のサイズに切断してから、切断したゼラチン長繊維不織布を水中でミキサーを用いて切断（粉砕）することで、ゼラチン短繊維を得ることが好ましい。得られたゼラチン短繊維は、２本以上の繊維が交点を有する枝分かれ形状のゼラチン短繊維を含んでもよいが、個々の繊維は互いに分離した形状であることが好ましい。水としては、例えば、純水、蒸留水、超純水等を用いることができる。ミキサーとしては、繊維にダメージを与えず、繊維の長さを短くすることができるものであればよく、特に限定されない。粉砕は、特に限定されないが、例えば、フリッチュ社製のＰ－１１メッサーミル等の超高速回転型ミキサーの場合、回転数２０００ｒｐｍ以上１４０００ｒｐｍ以下の条件下で５秒以上１０分以下行うことができる。 Next, the gelatin long fiber non-woven fabric is cut to obtain gelatin short fibers. From the viewpoint of simple operation, it is preferable to cut in water. Further, from the viewpoint of improving the cutting efficiency, the gelatin long fiber non-woven fabric is cut into a predetermined size such as 5 to 10 mm square, and then the cut gelatin long fiber non-woven fabric is cut (crushed) in water using a mixer. By doing so, it is preferable to obtain gelatin short fibers. The obtained gelatin short fibers may contain branched gelatin short fibers having two or more fibers at intersections, but it is preferable that the individual fibers have a shape separated from each other. As the water, for example, pure water, distilled water, ultrapure water or the like can be used. The mixer is not particularly limited as long as it does not damage the fibers and can shorten the length of the fibers. The pulverization is not particularly limited, but in the case of an ultra-high-speed rotary mixer such as a P-11 Messer mill manufactured by Fritsch, for example, the pulverization can be performed for 5 seconds or more and 10 minutes or less under the condition of a rotation speed of 2000 rpm or more and 14000 rpm or less.
　本発明の１以上の実施形態において、ゼラチン短繊維は、ゼラチンを含む紡糸液をノズル吐出口から空気中に押し出し、加熱紡糸筒を通過させて乾式紡糸し、得られたゼラチンフィラメント糸を切断することでも得ることができる。 In one or more embodiments of the present invention, the gelatin short fibers extrude a spinning liquid containing gelatin into the air from a nozzle discharge port, pass through a heated spinning cylinder for dry spinning, and cut the obtained gelatin filament yarn. You can also get it.
　まず、ゼラチンを含む紡糸液を調製する。紡糸液は、ゼラチン長繊維不織布の製造に用いるものと同様のものを用いることができる。簡単に言うと、例えば、ゼラチン単独、或いは、必要に応じてゼラチンと上述した他の成分として用いることができる他の生体適合ポリマーを水に溶解した後、減圧脱泡して溶解空気を除去することで紡糸液を得ることができる。溶解した後、フィルトレーションして異物やごみなどを除去してもよい。溶解温度は４０℃以上９０℃以下が好ましい。減圧脱泡時の真空度は５ｋＰａ以上３０ｋＰａ以下であることが好ましい。 First, prepare a spinning solution containing gelatin. As the spinning liquid, the same one as that used for producing the gelatin long fiber non-woven fabric can be used. Simply put, for example, gelatin alone or, if desired, gelatin and other biocompatible polymers that can be used as the other components described above are dissolved in water and then defoamed under reduced pressure to remove the dissolved air. This makes it possible to obtain a spinning solution. After being dissolved, foreign matter and dust may be removed by filtration. The melting temperature is preferably 40 ° C. or higher and 90 ° C. or lower. The degree of vacuum at the time of defoaming under reduced pressure is preferably 5 kPa or more and 30 kPa or less.
　次に、前記ゼラチンを含む紡糸液を紡糸機のノズル吐出口から空気中に押し出し、加熱紡糸筒を通過させて乾式紡糸する。前記加熱紡糸筒は、温度が１２０℃以上１８０℃以下に保持されており、かつ押し出し物の加熱紡糸筒中における滞留時間は５秒以上とするのが好ましい。これにより、押し出し物から急激に水分が除去され、糸条が形成される。 Next, the spinning liquid containing gelatin is extruded into the air from the nozzle discharge port of the spinning machine, passed through a heated spinning cylinder, and dry-spun. It is preferable that the temperature of the heated spinning cylinder is maintained at 120 ° C. or higher and 180 ° C. or lower, and the residence time of the extruded product in the heated spinning cylinder is 5 seconds or longer. As a result, water is rapidly removed from the extruded material, and threads are formed.
　前記加熱紡糸筒は、特に限定されないが、例えば内径が２００ｍｍステンレス管にヒーターを巻きつけた長さ２ｍの紡糸筒を用いることができる。前記加熱紡糸筒２４は、複数の区画に区分けられてもよく、それぞれの区画において温度制御を行ってもよい。前記加熱紡糸筒は鉛直方向に向いていることが好ましい。そして、加熱紡糸筒を出た位置では、ゼラチン中空糸は中空状体であり、ここでカットすることで、得られるゼラチンフィラメントは中空糸となる。加熱紡糸筒を出た位置で中空になる理由は、加熱紡糸筒内で急激に水分が除去されるためと思われる。カットせずに、巻き取ることで、中空がつぶれて断面が扁平になった扁平糸を得ることができる。 The heated spinning cylinder is not particularly limited, but for example, a spinning cylinder having a length of 2 m in which a heater is wound around a stainless steel pipe having an inner diameter of 200 mm can be used. The heat spinning cylinder 24 may be divided into a plurality of compartments, and temperature control may be performed in each compartment. The heated spinning cylinder is preferably oriented in the vertical direction. Then, at the position where the heated spinning cylinder is exited, the gelatin hollow fiber is a hollow fiber, and by cutting here, the obtained gelatin filament becomes a hollow fiber. The reason why it becomes hollow at the position where it comes out of the heat spinning cylinder is considered to be that water is rapidly removed in the heating spinning cylinder. By winding the yarn without cutting it, it is possible to obtain a flat yarn having a flat hollow and a flat cross section.
　前記ゼラチンフィラメントにおいて、水で膨潤した際の平均繊維径は、１μｍ以上４００μｍ以下であることが好ましく、１０μｍ以上２００μｍ以下であることがより好ましく、２０μｍ以上５５μｍ以下であることがより好ましい。ノズル径（内径）等適宜を調整することで、所望の平均繊維径を有するゼラチンフィラメントを得ることができる。なお、前記ゼラチンフィラメントが扁平糸の場合、個々の繊維において、繊維径は、長径と短径の平均値である。 In the gelatin filament, the average fiber diameter when swollen with water is preferably 1 μm or more and 400 μm or less, more preferably 10 μm or more and 200 μm or less, and more preferably 20 μm or more and 55 μm or less. A gelatin filament having a desired average fiber diameter can be obtained by appropriately adjusting the nozzle diameter (inner diameter) and the like. When the gelatin filament is a flat yarn, the fiber diameter is an average value of a major axis and a minor axis in each fiber.
　前記ゼラチンフィラメントは、架橋することが好ましい。これにより形態安定性及び耐水性を高めることができ、前記ゼラチンフィラメントを切断してゼラチン短繊維を得る工程の作業性が良好になる。架橋は、ゼラチン長繊維不織布の製造方法について説明したのと同様に行うことができる。簡便に所望の架橋効果を得やすい観点から、熱架橋であることが好ましく、熱脱水架橋であることがより好ましい。熱脱水架橋は、例えば、１００℃以上１６０℃以下で、２４時間以上９６時間以下行ってもよい。また、熱脱水架橋は、例えば、１０ｋＰａ以下の真空下で行ってもよい。前記ゼラチンフィラメントは、架橋する前に乾燥してもよい。乾燥は、室温における風乾でもよく、真空凍結乾燥でもよい。 The gelatin filament is preferably crosslinked. As a result, morphological stability and water resistance can be improved, and workability in the step of cutting the gelatin filament to obtain gelatin short fibers is improved. Crosslinking can be performed in the same manner as described for the method for producing a gelatin long fiber non-woven fabric. From the viewpoint of easily obtaining the desired crosslinking effect, thermal crosslinking is preferable, and thermal dehydration crosslinking is more preferable. The thermal dehydration crosslinking may be carried out, for example, at 100 ° C. or higher and 160 ° C. or lower for 24 hours or longer and 96 hours or shorter. Further, the thermal dehydration crosslinking may be performed under a vacuum of, for example, 10 kPa or less. The gelatin filament may be dried before cross-linking. The drying may be air drying at room temperature or vacuum freeze drying.
　次に、前記ゼラチンフィラメントを切断してゼラチン短繊維を得る。操作が簡便である観点から、水中で切断することが好ましい。また、切断効率を高める観点から、前記ゼラチンフィラメントを、例えば、繊維長が５～１０ｍｍになるように切断し、その後、水中でミキサーを用いてさらに切断（粉砕）することで、ゼラチン短繊維を得ることが好ましい。水としては、例えば、純水、蒸留水、超純水等を用いることができる。ミキサーとしては、繊維にダメージを与えず、繊維の長さを短くすることができるものであればよく、特に限定されない。粉砕は、特に限定されないが、例えば、フリッチュ社製のＰ－１１メッサーミル等の超高速回転型ミキサーの場合、回転数２０００ｒｐｍ以上１４０００ｒｐｍ以下の条件下で５秒以上１０分以下行うことができる。 Next, the gelatin filament is cut to obtain gelatin short fibers. From the viewpoint of simple operation, it is preferable to cut in water. Further, from the viewpoint of increasing the cutting efficiency, the gelatin filament is cut so that the fiber length is, for example, 5 to 10 mm, and then further cut (crushed) in water using a mixer to obtain gelatin short fibers. It is preferable to obtain it. As the water, for example, pure water, distilled water, ultrapure water or the like can be used. The mixer is not particularly limited as long as it does not damage the fibers and can shorten the length of the fibers. The pulverization is not particularly limited, but in the case of an ultra-high-speed rotary mixer such as a P-11 Messer mill manufactured by Fritsch, for example, the pulverization can be performed for 5 seconds or more and 10 minutes or less under the condition of a rotation speed of 2000 rpm or more and 14000 rpm or less.
　図６（ａ）及び（ｂ）は、本発明の１以上の実施形態で用いるフィラメント製造装置２０の模式的説明図である。シリンジ２１に入れたゼラチンを含む紡糸液２２をノズル２３から空気中に押し出す。具体的には、シリンジ２１の末端より０．１ＭＰａの加圧空気を送ってノゾル２３から紡糸液２２を押し出すことができる。ノズル２３は通常の丸断面でよい。ノズル２３の下には加熱紡糸筒２４が直結している。加熱紡糸筒２４は２４ａ-２４ｄの４区画からなり、それぞれの区画で温度制御が可能となっている。
　図６（ａ）に示すように、ゼラチンフィラメント２５を、ガイドロール２６を通過して巻き取り機２７に巻き取ることで、扁平糸が得られる。
　図６（ｂ）に示すように、加熱紡糸筒２４を出た下部で自重落下するゼラチンフィラメント２５をカットすることで、所定長さの中空糸が得られる。 6 (a) and 6 (b) are schematic explanatory views of the filament manufacturing apparatus 20 used in one or more embodiments of the present invention. The gelatin-containing spinning solution 22 contained in the syringe 21 is pushed out into the air from the nozzle 23. Specifically, 0.1 MPa of pressurized air can be sent from the end of the syringe 21 to push out the spinning liquid 22 from the nosol 23. The nozzle 23 may have a normal round cross section. A heat spinning cylinder 24 is directly connected under the nozzle 23. The heat spinning cylinder 24 is composed of four compartments of 24a-24d, and the temperature can be controlled in each compartment.
As shown in FIG. 6A, the gelatin filament 25 passes through the guide roll 26 and is wound around the winding machine 27 to obtain a flat yarn.
As shown in FIG. 6B, a hollow fiber having a predetermined length can be obtained by cutting the gelatin filament 25 that drops by its own weight at the lower part of the heated spinning cylinder 24.
　前記ゼラチン短繊維は、エチレンオキサイドガス滅菌、蒸気滅菌、電子線照射やγ線等の放射線照射により滅菌してもよい。 The gelatin short fibers may be sterilized by ethylene oxide gas sterilization, steam sterilization, electron beam irradiation, or irradiation with γ-rays or the like.
　前記ゼラチン短繊維及び細胞を培地中で共培養（接着培養）することで、細胞シート中にゼラチン短繊維が分散している細胞シートを形成することができる。上述したとおり、細胞接着性を有し、ハイドロゲルとなり得るゼラチンを主成分とするとともに、平均繊維径と平均繊維長が上述した範囲内であるゼラチン短繊維を細胞と培地中で共培養することで、培地中で均一に分散して膨潤したゼラチン短繊維の表面に細胞が接着しやすく、細胞間にゼラチン短繊維が配置されることで、ゼラチン短繊維が分散している細胞シートを得ることができる。 By co-culturing (adhesive culture) the gelatin short fibers and cells in a medium, a cell sheet in which gelatin short fibers are dispersed can be formed. As described above, the main component is gelatin, which has cell adhesion and can be a hydrogel, and gelatin short fibers having an average fiber diameter and an average fiber length within the above-mentioned ranges are co-cultured with cells in a medium. Therefore, cells can easily adhere to the surface of gelatin short fibers that are uniformly dispersed and swollen in the medium, and gelatin short fibers are arranged between the cells to obtain a cell sheet in which gelatin short fibers are dispersed. Can be done.
　本発明の１以上の実施形態において、細胞は、動物細胞であればよく、その由来は特に限定されない。動物としては、ヒトでもよく、ヒト以外の動物でもよい。ヒト以外の動物としては、例えば、サル、チンパンジー等の霊長類、マウス、ラット、ハムスター等の齧歯類、ウシ、ヒツジ、ヤギ、ブタ等の有蹄類等が挙げられる。また、本発明の１以上の実施形態において、細胞は、個々の細胞、細胞株、初代培養等培養で得られる細胞等を含む。前記細胞としては、特に限定されないが、例えば、体細胞、幹細胞、前駆細胞、生殖細胞等が挙げられる。 In one or more embodiments of the present invention, the cell may be an animal cell, and its origin is not particularly limited. The animal may be a human or a non-human animal. Examples of animals other than humans include primates such as monkeys and chimpanzees, rodents such as mice, rats and hamsters, and ungulates such as cows, sheep, goats and pigs. In addition, in one or more embodiments of the present invention, the cells include individual cells, cell lines, cells obtained by culturing such as primary culture, and the like. The cells are not particularly limited, and examples thereof include somatic cells, stem cells, progenitor cells, germ cells and the like.
　体細胞は、生体を構成する体細胞や体細胞から派生した癌細胞を含む。生体を構成する体細胞としては、特に限定されず、例えば、線維芽細胞、筋細胞、内皮細胞、骨芽細胞、内皮細胞、膀胱細胞、肺細胞、骨細胞、神経細胞、肝細胞、軟骨細胞、上皮細胞、中皮細胞等が挙げられる。癌細胞としては、特に限定されず、例えば、乳癌細胞、腎癌細胞、前立腺癌細胞、肺癌細胞、肝癌細胞、子宮頸癌細胞、食道上皮癌、膵癌、大腸癌、膀胱癌等が挙げられる。 Somatic cells include somatic cells that make up the living body and cancer cells derived from somatic cells. The somatic cells constituting the living body are not particularly limited, and are, for example, fibroblasts, muscle cells, endothelial cells, osteoblasts, endothelial cells, bladder cells, lung cells, osteoblasts, nerve cells, hepatocytes, and chondrocytes. , Epithelial cells, mesenteric cells and the like. The cancer cells are not particularly limited, and examples thereof include breast cancer cells, renal cancer cells, prostate cancer cells, lung cancer cells, liver cancer cells, cervical cancer cells, esophageal epithelial cancer, pancreatic cancer, colon cancer, and bladder cancer.
　幹細胞は、様々な特殊化した細胞型へ分化する可能性がある細胞である。幹細胞としては、特に限定されず、例えば、胚性幹細胞（ＥＳ細胞）、胚性癌腫細胞（ＥＣ）、胚性生殖幹細胞（ＥＧ）、人工多能性幹細胞（ｉＰＳ細胞）、成体幹細胞、胚盤胞由来幹細胞、生殖***由来幹細胞、奇形腫由来幹細胞、オンコスタチン非依存性幹細胞（ＯＩＳＣ）、骨髄由来間葉系幹細胞、脂肪由来間葉系幹細胞、羊水由来間葉系幹細胞、皮膚由来間葉系幹細胞、骨膜由来間葉系幹細胞等が挙げられる。 Stem cells are cells that have the potential to differentiate into various specialized cell types. The stem cells are not particularly limited, and are, for example, embryonic stem cells (ES cells), embryonic cancer tumor cells (EC), embryonic germ stem cells (EG), artificial pluripotent stem cells (iPS cells), adult stem cells, and scutellum. Spore-derived stem cells, reproductive ridge-derived stem cells, malformation-derived stem cells, oncostatin-independent stem cells (OISC), bone marrow-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, sheep water-derived mesenchymal stem cells, skin-derived mesenchymal stem cells Examples thereof include stem cells and bone membrane-derived mesenchymal stem cells.
　前駆細胞は、前記幹細胞から発生し生体を構成する最終分化細胞へ分化することができる細胞である。 Progenitor cells are cells that can develop from the stem cells and differentiate into the final differentiated cells that make up the living body.
　生殖細胞としては、***、精細胞、卵子、卵細胞等が挙げられる。 Examples of germ cells include sperm, sperm cells, eggs, and egg cells.
　上述した細胞は、１種を単独で用いてもよく、目的等に応じて２種以上を併用してもよい。 As the above-mentioned cells, one type may be used alone, or two or more types may be used in combination depending on the purpose and the like.
　前記培地（液体培地）としては、特に限定されず、細胞の種類に応じて、細胞の生存増殖に必要な成分を含むものを適宜用いることができる。前記培地は、血清、抗生物質及び成長因子等を含んでもよい。血清は、例えば、ウシ血清、ウシ胎児血清、ウマ血清、ヒト血清等を適宜用いることができる。抗生物質は、ペニシリン、ストレプトマイシン、ゲンタマイシン、アンフォテリシン、アンピシリン、ミノマイシン、カナマイシン等を適宜用いることができる。成長因子は、細胞増殖因子、分化誘導因子、細胞接着因子等を適宜用いることができる。 The medium (liquid medium) is not particularly limited, and a medium containing components necessary for cell survival and proliferation can be appropriately used depending on the type of cells. The medium may contain serum, antibiotics, growth factors and the like. As the serum, for example, bovine serum, fetal bovine serum, horse serum, human serum and the like can be appropriately used. As the antibiotic, penicillin, streptomycin, gentamicin, amphotericin, ampicillin, minocycline, kanamycin and the like can be appropriately used. As the growth factor, a cell proliferation factor, a differentiation inducing factor, a cell adhesion factor and the like can be appropriately used.
　培養は、細胞シートを形成することができる接着培養条件で行うことができる。培養基材としては、接着培養に用いることができるものを適宜用いることができる。例えば、細胞が接着し得る平坦な部分（培養面とも称される。）を有する細胞培養容器、例えば、ディッシュ、プレート、及びフラスコ等を用いることができる。 Culturing can be performed under adhesive culture conditions that can form cell sheets. As the culture substrate, those that can be used for adhesive culture can be appropriately used. For example, cell culture vessels having a flat portion (also referred to as a culture surface) to which cells can adhere can be used, such as dishes, plates, flasks and the like.
　前記細胞の播種面密度（細胞の播種量/培養面の表面積）は、特に限定されず、細胞種類や細胞シートの厚み等に基づいて適宜決めることができる。例えば、５，０００細胞/ｃｍ2以上１００，０００細胞/ｃｍ2以下であることが好ましく、２０，０００細胞/ｃｍ2以上８０，０００細胞/ｃｍ2以下であることがより好ましく、４０，０００細胞/ｃｍ2以上６０，０００細胞/ｃｍ2以下であることがさらに好ましい。細胞の播種面密度が上述した範囲内であると、細胞が偏ることがなく、培養面に接着しやすくなる。 The seeding surface density of the cells (seed amount of cells / surface area of the culture surface) is not particularly limited, and can be appropriately determined based on the cell type, the thickness of the cell sheet, and the like. For example, it is preferably 5,000 cells / cm 2 or more and 100,000 cells / cm 2 or less, more preferably 20,000 cells / cm 2 or more and 80,000 cells / cm 2 or less, and 40,000 cells / cm 2. It is more preferably cells / cm 2 or more and 60,000 cells / cm 2 or less. When the seeding surface density of the cells is within the above-mentioned range, the cells are not biased and easily adhere to the culture surface.
　前記ゼラチン短繊維の添加密度（ゼラチン短繊維の質量/培養面の表面積）は、特に限定されないが、例えば、０．０５ｍｇ／ｃｍ2以上１ｍｇ／ｃｍ2以下であることが好ましく、０．１ｍｇ／ｃｍ2以上０．９ｍｇ／ｃｍ2以下であることがより好ましく、０．２ｍｇ／ｃｍ2以上０．６ｍｇ／ｃｍ2以下であることがさらに好ましい。ゼラチン短繊維の添加密度が上述した範囲内であると、培養面に均一に分散しやすくなる。 The addition density of the gelatin short fibers (mass of gelatin short fibers / surface area of the culture surface) is not particularly limited, but is preferably, for example, 0.05 mg / cm 2 or more and 1 mg / cm 2 or less, and 0.1 mg / cm / cm. It is more preferably cm 2 or more and 0.9 mg / cm 2 or less, and further preferably 0.2 mg / cm 2 or more and 0.6 mg / cm 2 or less. When the addition density of the gelatin short fibers is within the above-mentioned range, it is easy to uniformly disperse on the culture surface.
　前記細胞の播種体積密度（細胞の播種量/培地の体積）は、特に限定されないが、例えば、０．１ｘ１０5細胞／ｍＬ以上３ｘ１０5細胞／ｍＬ以下であることが好ましく、０．３ｘ１０5細胞／ｍＬ以上２ｘ１０5細胞／ｍＬ以下であることがより好ましく、０．５ｘ１０5細胞／ｍＬ以上１．５ｘ１０5細胞／ｍＬ以下であることがさらに好ましい。細胞の播種体積密度が上述した範囲内であると、細胞に栄養や酸素が供給されやすく、ゼラチン短繊維と共培養することで、ゼラチン短繊維が均一に分散している細胞シートを得やすくなる。 The seeding volume density of the cells (seed amount of cells / volume of medium) is not particularly limited, but is preferably 0.1x10 5 cells / mL or more and 3x10 5 cells / mL or less, preferably 0.3x10 5 cells. more preferably / mL or 2x10 is 5 cells / mL or less, still more preferably not more than 0.5 × 10 5 cells / mL or more 1.5 × 10 5 cells / mL. When the seeding volume density of the cells is within the above range, nutrients and oxygen are easily supplied to the cells, and by co-culturing with gelatin short fibers, it becomes easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed. ..
　前記ゼラチン短繊維の濃度（ゼラチン短繊維の質量/培地の体積）は、特に限定されないが、例えば、０．０１ｍｇ／ｍＬ以上２ｍｇ／ｍＬ以下であることが好ましく、０．０２ｍｇ／ｍＬ以上１．５ｍｇ／ｍＬ以下であることがより好ましく、０．０４ｍｇ／ｍＬ以上１ｍｇ／ｍＬ以下であることがさらに好ましい。ゼラチン短繊維の濃度が上述した範囲内であると、培地中の分散性が高く、細胞と共培養することで、ゼラチン短繊維が均一に分散している細胞シートを得やすくなる。 The concentration of the gelatin short fibers (mass of gelatin short fibers / volume of medium) is not particularly limited, but is preferably 0.01 mg / mL or more and 2 mg / mL or less, and 0.02 mg / mL or more and 1. It is more preferably 5 mg / mL or less, and further preferably 0.04 mg / mL or more and 1 mg / mL or less. When the concentration of gelatin short fibers is within the above range, the dispersibility in the medium is high, and co-culturing with cells makes it easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed.
　前記細胞の播種量とゼラチン短繊維の添加量の比（細胞の播種量/ゼラチン短繊維の添加量）は、特に限定されないが、例えば、０．１ｘ１０5細胞/ｍｇ以上７ｘ１０5細胞/ｍｇ以下であることが好ましく、０．５ｘ１０5細胞/ｍｇ以上５ｘ１０5細胞/ｍｇ以下であることがより好ましく、１ｘ１０5細胞/ｍｇ以上３ｘ１０5細胞/ｍｇ以下であることがさらに好ましい。細胞の播種量とゼラチン短繊維の添加量の比が上述した範囲であると、細胞の分布が偏らず、ゼラチン短繊維が均一に分散している細胞シートを得やすくなる上、得られた細胞シートの細胞活性を高めることができる。 The ratio of the seeded amount of the cells to the added amount of gelatin short fibers (cell seeded amount / added amount of gelatin short fibers) is not particularly limited, but is, for example, 0.1x10 5 cells / mg or more and 7x10 5 cells / mg or less. it is preferably, 0.5 × 10 more preferably 5 cells / mg or 5x10 5 cells / mg or less, and more preferably not more than 3x10 5 cells / mg 1x10 5 cells / mg or more. When the ratio of the seeded amount of cells to the amount of gelatin short fibers added is within the above range, the distribution of cells is not biased, it becomes easy to obtain a cell sheet in which gelatin short fibers are uniformly dispersed, and the obtained cells are obtained. The cell activity of the sheet can be increased.
　細胞とゼラチン短繊維の共培養は、例えば、２７℃以上４０℃以下で行ってもよく、３１℃以上３７℃以下であってもよい。二酸化炭素は、２％以上１０％以下の範囲であってもよい。 The co-culture of cells and gelatin short fibers may be carried out, for example, at 27 ° C. or higher and 40 ° C. or lower, or 31 ° C. or higher and 37 ° C. or lower. Carbon dioxide may be in the range of 2% or more and 10% or less.
　培養時間は、細胞シートを形成するよう、細胞種類、細胞数等に応じて適宜決めればよいが、例えば、２～８日継続して培養してもよく、３～７日継続して行ってもよく、４～６日継続して行ってもよい。培地は、２～３日毎に交換してもよい。 The culturing time may be appropriately determined according to the cell type, the number of cells, etc. so as to form a cell sheet. For example, the culturing may be continued for 2 to 8 days, or continuously for 3 to 7 days. It may be continued for 4 to 6 days. The medium may be changed every 2-3 days.
　前記細胞シートは、ゼラチン短繊維を含むことにより、培養基材から剥離した後の収縮が抑制されている。培養基材から剥離する前の細胞シートの直径をＤＳ１とし、培養基材から剥離した後の細胞シートの直径をＤＳ２とした場合、ＤＳ２／ＤＳ１は０．５６以上１以下であることが好ましく、０．６０以上０．９５以下であることがより好ましく、０．６４以上０．９以下であることがさらに好ましい。 Since the cell sheet contains gelatin short fibers, shrinkage after peeling from the culture substrate is suppressed. When the diameter of the cell sheet before peeling from the culture substrate is DS1 and the diameter of the cell sheet after peeling from the culture substrate is DS2, DS2 / DS1 is preferably 0.56 or more and 1 or less. It is more preferably 0.60 or more and 0.95 or less, and further preferably 0.64 or more and 0.9 or less.
　前記細胞シートの厚みは、細胞種類や用途等により適宜決めればよいが、ハンドリング性及び細胞活性の観点から、例えば、３０μｍ以上１５０μｍ以下であることが好ましく、より好ましくは４０μｍ以上１４０μｍ以下であり、さらに好ましくは５０μｍ以上１３０μｍ以下である。本発明において、細胞シートの厚みは、凍結切片の断面像の顕微鏡観察で測定する。 The thickness of the cell sheet may be appropriately determined depending on the cell type, use, etc., but from the viewpoint of handleability and cell activity, it is preferably, for example, 30 μm or more and 150 μm or less, and more preferably 40 μm or more and 140 μm or less. More preferably, it is 50 μm or more and 130 μm or less. In the present invention, the thickness of the cell sheet is measured by microscopic observation of a cross-sectional image of a frozen section.
　前記細胞シートにおいて、特に限定されないが、例えば、ハンドリング性及び細胞活性の観点から、細胞数とゼラチン短繊維の質量の比（細胞数/ゼラチン短繊維の質量）は、１ｘ１０5細胞/ｍｇ以上２５ｘ１０5細胞/ｍｇ以下であることが好ましく、より好ましくは２ｘ１０5細胞/ｍｇ以上２０ｘ１０5細胞/ｍｇ以下であり、さらに好ましくは３ｘ１０5細胞/ｍｇ以上１５ｘ１０5細胞/ｍｇ以下である。 In the cell sheet is not particularly limited, for example, from the viewpoints of handling properties and cellular activity, the ratio of the mass of cell number and gelatin short fibers (cells / gelatin short fiber mass) is, 1x10 5 cells / mg or more 25x10 It is preferably 5 cells / mg or less, more preferably 2x10 5 cells / mg or more and 20x10 5 cells / mg or less, and further preferably 3x10 5 cells / mg or more and 15x10 5 cells / mg or less.
　上述したゼラチン短繊維、細胞及び培地を含むキットは、細胞シートの作製キットとして用いることができ、具体的には、細胞シート中にゼラチン短繊維が分散している細胞シートを形成するのに用いることができる。前記キットは、培養基材を含んでもよい。 The above-mentioned kit containing gelatin short fibers, cells and a medium can be used as a cell sheet preparation kit, and specifically, it is used to form a cell sheet in which gelatin short fibers are dispersed in the cell sheet. be able to. The kit may include a culture medium.
　前記細胞シートは、例えば、ヒト又はヒト以外の動物の組織の増大、修復又は再生等の細胞治療に用いることができる。組織としては、特に限定されず、例えば、骨、軟骨、腱、半月、筋肉及び脂肪等の結合組織等が挙げられる。 The cell sheet can be used for cell therapy such as growth, repair or regeneration of human or non-human animal tissues, for example. The tissue is not particularly limited, and examples thereof include connective tissues such as bone, cartilage, tendon, half moon, muscle and fat.
　前記細胞シートは、ゼラチン短繊維を含むことにより、強度が高くなり、それゆえ、細胞治療用途の際のハンドリング性が良好になる。培養基材から剥離した際にピンセットで把持して水等の液体に浸すと元の形状に戻りやすい。 By containing gelatin short fibers, the cell sheet has high strength, and therefore, has good handleability in cell therapy applications. When peeled from the culture substrate, it can easily return to its original shape by grasping it with tweezers and immersing it in a liquid such as water.
　前記細胞シートは、ゼラチン短繊維を含むことにより、凍結による細胞損傷を防ぐことができ、具体的には、凍結による細胞活性の低減を抑制することができる。 By containing gelatin short fibers, the cell sheet can prevent cell damage due to freezing, and specifically, can suppress a decrease in cell activity due to freezing.
　本発明は、特に限定されないが、好ましくは以下の態様を含む。
　［１］　ゼラチンを主成分とするゼラチン短繊維が細胞シート中に分散しており、
　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シート。
　［２］　前記ゼラチン短繊維は、架橋されている、［１］に記載の細胞シート。
　［３］　前記細胞シートにおいて、細胞数とゼラチン短繊維の質量の比（細胞数/ゼラチン短繊維の質量）は、１ｘ１０5細胞/ｍｇ以上２．５ｘ１０6細胞/ｍｇ以下である、［１］又は［２］に記載の細胞シート。
　［４］　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である、［１］～［３］のいずれかに記載の細胞シート。
　［５］　細胞及びゼラチンを主成分とするゼラチン短繊維を培地中で培養することで、前記ゼラチン短繊維が分散している細胞シートを形成しており、
　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シートの製造方法。
　［６］　培養基材から剥離する前の細胞シートの直径をＤＳ１とし、培養基材から剥離した後の細胞シートの直径をＤＳ２とした場合、ＤＳ２／ＤＳ１は０．５６以上１以下である、［５］に記載の細胞シートの製造方法。
　［７］　前記細胞の播種量とゼラチン短繊維の添加量の比（細胞の播種量/ゼラチン短繊維の添加量）は、特に限定されないが、例えば、０．１ｘ１０5細胞/ｍｇ以上７ｘ１０5細胞/ｍｇ以下である、［５］又は［６］に記載の細胞シートの製造方法。
　［８］　前記ゼラチン短繊維は、ゼラチンを含む紡糸液をノズル吐出口から空気中に押し出し、前記ノズル吐出口の後方に位置し、前記ノズル吐出口とは非接触状態の流体噴射口から前方に向けて圧力流体を噴射し、前記押し出された紡糸液を前記圧力流体に随伴させて繊維形成させ、前記繊維形成した繊維を集積させてゼラチン長繊維不織布とし、前記ゼラチン長繊維不織布を切断することで得られる、［５］～［７］のいずれかに記載の細胞シートの製造方法。
　［９］　前記ゼラチン短繊維は、ゼラチンを含む紡糸液をノズル吐出口から空気中に押し出し、加熱紡糸筒を通過させて乾式紡糸し、得られたゼラチンフィラメント糸を切断することで得られる、［５］～［７］のいずれかに記載の細胞シートの製造方法。
　［１０］　前記ゼラチン長繊維不織布又はゼラチンフィラメント糸は架橋した後に切断する、［５］～［９］のいずれかに記載の細胞シートの製造方法。
　［１１］　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である、［５］～［１０］のいずれかに記載の細胞シートの製造方法。
　［１２］　細胞、ゼラチン短繊維及び培地を含み、
　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であり、
　細胞シート中にゼラチン短繊維が分散している細胞シートを形成するのに用いることを特徴とする細胞シートの作製キット。
　［１３］　前記ゼラチン短繊維は、架橋されている、［１２］に記載の細胞シートの作製キット。
　［１４］　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である、［１２］又は［１３］に記載の細胞シートの作製キット。 The present invention is not particularly limited, but preferably includes the following aspects.
[1] Gelatin short fibers containing gelatin as a main component are dispersed in the cell sheet.
The gelatin short fiber is a cell sheet having an average fiber diameter of 1 μm or more and 400 μm or less and an average fiber length of 10 μm or more and 2000 μm or less.
[2] The cell sheet according to [1], wherein the gelatin short fibers are crosslinked.
[3] In the cell sheet, the ratio of the number of cells to the mass of gelatin short fibers (number of cells / mass of gelatin short fibers) is 1x10 5 cells / mg or more and 2.5x10 6 cells / mg or less [1]. Or the cell sheet according to [2].
[4] The cell sheet according to any one of [1] to [3], wherein the gelatin short fibers have an aspect ratio represented by an average fiber length / average fiber diameter of 3 or more.
[5] By culturing cells and gelatin short fibers containing gelatin as a main component in a medium, a cell sheet in which the gelatin short fibers are dispersed is formed.
A method for producing a cell sheet, wherein the gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less.
[6] When the diameter of the cell sheet before peeling from the culture substrate is DS1 and the diameter of the cell sheet after peeling from the culture substrate is DS2, DS2 / DS1 is 0.56 or more and 1 or less. The method for producing a cell sheet according to [5].
[7] The ratio of the seeded amount of the cells to the added amount of gelatin short fibers (cell seeded amount / added amount of gelatin short fibers) is not particularly limited, but is, for example, 0.1x10 5 cells / mg or more 7x10 5 cells. The method for producing a cell sheet according to [5] or [6], which is / mg or less.
[8] The gelatin short fibers extrude a spinning liquid containing gelatin into the air from a nozzle discharge port, are located behind the nozzle discharge port, and forward from a fluid injection port in a state of non-contact with the nozzle discharge port. A pressure fluid is jetted toward the surface, the extruded spinning liquid is associated with the pressure fluid to form fibers, and the fiber-formed fibers are accumulated to form a gelatin long-fiber non-woven fabric, and the gelatin long-fiber non-woven fabric is cut. The method for producing a cell sheet according to any one of [5] to [7].
[9] The gelatin short fibers are obtained by extruding a spinning liquid containing gelatin into the air from a nozzle discharge port, passing it through a heated spinning cylinder, dry spinning, and cutting the obtained gelatin filament yarn. 5] The method for producing a cell sheet according to any one of [7].
[10] The method for producing a cell sheet according to any one of [5] to [9], wherein the gelatin long fiber non-woven fabric or gelatin filament yarn is crosslinked and then cut.
[11] The method for producing a cell sheet according to any one of [5] to [10], wherein the gelatin short fibers have an aspect ratio represented by an average fiber length / average fiber diameter of 3 or more.
[12] Containing cells, gelatin short fibers and medium
The gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less.
A kit for producing a cell sheet, which is used for forming a cell sheet in which gelatin short fibers are dispersed in the cell sheet.
[13] The cell sheet preparation kit according to [12], wherein the gelatin short fibers are crosslinked.
[14] The cell sheet preparation kit according to [12] or [13], wherein the gelatin short fibers have an aspect ratio represented by an average fiber length / average fiber diameter of 3 or more.
　以下、実施例を用いてさらに具体的に説明する。なお、本発明は下記の実施例に限定されるものではない。 Hereinafter, a more specific description will be given using examples. The present invention is not limited to the following examples.
　測定方法は下記のとおりである。
　＜平均繊維径及び平均繊維長＞
　ゼラチン短繊維５ｇと水１００ｍＬを含む水分散液をデジタルマイクロスコープ（Ｚｅｉｓｓ社製、品名「ＡｘｉｏＣＡＭ　ＥＲｃ５」）を搭載した倒立顕微鏡（Ｏｌｙｍｐｕｓ社製、ＣＫＸ５３、４倍）で観察して撮影した。次に、コンピュータソフトウェア（ＰｈｏｔｏＲｕｌｅｒ，Ｔｈｅ　Ｇｅｎｕｓ　Ｉｎｃｙｂｅ）を使用して、撮影した写真から任意に選択した繊維４００本の繊維径と繊維長を計測し、４００本の繊維の繊維径と繊維長の平均をそれぞれ算出し、平均繊維径及び平均繊維長とした。 The measurement method is as follows.
<Average fiber diameter and average fiber length>
An aqueous dispersion containing 5 g of gelatin short fibers and 100 mL of water was observed and photographed with an inverted microscope (Olympus, CKX53, 4x) equipped with a digital microscope (Zeiss, product name "AxioCAM ERc5"). Next, using computer software (PhotoRuler, The Genus Incybe), the fiber diameter and fiber length of 400 fibers arbitrarily selected from the photographed photographs were measured, and the average of the fiber diameters and fiber lengths of the 400 fibers was measured. Was calculated and used as the average fiber diameter and the average fiber length.
　（製造例１）
　＜ゼラチン長繊維不織布の作製＞
　ゼラチンとして新田ゼラチン社製（ゼリー強度２６２ｇ、原料：アルカリ処理牛骨）を使用し、ゼラチン：水＝３：５の質量比（ゼラチン濃度３７．５質量％）とし、温度６０℃で溶解した。ゼラチン水溶液の６０℃における粘度は９６０～９７０ｍＰａ・ｓであった。このゼラチン水溶液を紡糸液とし、図５に示す不織布製造装置を使用してゼラチン長繊維不織布を製造した。紡糸液の温度は６０℃、ノズル直径（内径）２５０μｍ、吐出圧０．２ＭＰａ、ノズル高さ５ｍｍ、エアー圧力０．３７５ＭＰａ、エアー温度１００℃、流体噴射口５とノズル吐出口３との距離は５ｍｍ、捕集距離５０ｃｍとし、巻き取りローラ１１でゼラチン長繊維不織布９を巻き取った。
　次いで、ゼラチン長繊維不織布を室温（２０℃）で一晩風乾し、その後、真空下（１ｋＰａ）、１４０℃で４８時間熱架橋させた。 (Manufacturing Example 1)
<Making gelatin long fiber non-woven fabric>
Nitta Gelatin Co., Ltd. (jelly strength 262 g, raw material: alkali-treated beef bone) was used as gelatin, and gelatin: water = 3: 5 mass ratio (gelatin concentration 37.5 mass%) and dissolved at a temperature of 60 ° C. .. The viscosity of the aqueous gelatin solution at 60 ° C. was 960 to 970 mPa · s. This gelatin aqueous solution was used as a spinning solution, and a gelatin long fiber non-woven fabric was manufactured using the non-woven fabric manufacturing apparatus shown in FIG. The temperature of the spinning liquid is 60 ° C, the nozzle diameter (inner diameter) is 250 μm, the discharge pressure is 0.2 MPa, the nozzle height is 5 mm, the air pressure is 0.375 MPa, the air temperature is 100 ° C, and the distance between the fluid injection port 5 and the nozzle discharge port 3 is. The gelatin long fiber non-woven fabric 9 was wound up by the winding roller 11 with a collection distance of 5 mm and a collection distance of 50 cm.
Then, the gelatin long fiber non-woven fabric was air-dried at room temperature (20 ° C.) overnight, and then heat-crosslinked under vacuum (1 kPa) at 140 ° C. for 48 hours.
　＜ゼラチン短繊維の作製＞
　得られたゼラチン長繊維不織布を５～１０ｍｍ角に切断し、切断したゼラチン長繊維不織布５ｇに蒸留水を１００ｍＬ加えて、超高速回転型ミキサー（フリッチュ社製の「Ｐ－１１メッサーミル」）を用いて２０００ｒｐｍで３分間撹拌することで、ゼラチン長繊維不織布を切断してゼラチン短繊維の水分散液を得た。得られたゼラチン短繊維の水分散液（懸濁液）を用いて、上述したとおり、ゼラチン短繊維の平均繊維径と平均繊維長を測定したところ、平均繊維径が４０μｍ（変動係数０．１３）であり、平均繊維長が２３０μｍ（変動係数０．５２）であり、アスペクト比は５．７５であった。図４に、ゼラチン短繊維の水分散液をデジタルマイクロスコープ（Ｚｅｉｓｓ社製、品名「ＡｘｉｏＣＡＭ　ＥＲｃ５」）を搭載した倒立顕微鏡（Ｏｌｙｍｐｕｓ社製「ＣＫＸ５３」、１０倍）で観察して撮影した写真を示した。
　次に、繊維懸濁液を－３０℃の冷凍庫で凍結させた後、真空凍結乾燥を行い、乾燥したゼラチン短繊維を得た。 <Making gelatin short fibers>
The obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used. The gelatin long fiber non-woven fabric was cut by stirring at 2000 rpm for 3 minutes to obtain an aqueous dispersion of gelatin short fibers. As described above, the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 μm (coefficient of variation 0.13). ), The average fiber length was 230 μm (coefficient of variation 0.52), and the aspect ratio was 5.75. FIG. 4 shows a photograph taken by observing an aqueous dispersion of gelatin short fibers with an inverted microscope (Olympus "CKX53", 10x) equipped with a digital microscope (Zeiss, product name "AxioCAM ERc5"). Indicated.
Next, the fiber suspension was frozen in a freezer at −30 ° C. and then vacuum freeze-dried to obtain dried gelatin short fibers.
　（製造例２）
　製造例１と同様にして、ゼラチン長繊維不織布を作製した。
　得られたゼラチン長繊維不織布を、５～１０ｍｍ角に切断し、切断したゼラチン長繊維不織布５ｇに蒸留水を１００ｍＬ加えて、超高速回転型ミキサー（フリッチュ社製の「Ｐ－１１メッサーミル」）を用いて２０００ｒｐｍで１分間撹拌することで、ゼラチン短繊維の水分散液を得た。得られたゼラチン短繊維の水分散液（懸濁液）を用いて、上述したとおり、ゼラチン短繊維の平均繊維径と平均繊維長を測定したところ、平均繊維径が４０μｍ（変動係数０．１３）であり、平均繊維長が８２８μｍ（変動係数０．５８）であり、アスペクト比は２０．７であった。
　次に、繊維懸濁液を－３０℃の冷凍庫で凍結させた後、真空度凍結乾燥を行い、乾燥したゼラチン短繊維を得た。 (Manufacturing Example 2)
A gelatin long-fiber non-woven fabric was produced in the same manner as in Production Example 1.
The obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used. By stirring at 2000 rpm for 1 minute, an aqueous dispersion of gelatin short fibers was obtained. As described above, the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 μm (coefficient of variation 0.13). ), The average fiber length was 828 μm (coefficient of variation 0.58), and the aspect ratio was 20.7.
Next, the fiber suspension was frozen in a freezer at −30 ° C. and then freeze-dried to a degree of vacuum to obtain dried gelatin short fibers.
　（製造例３）
　製造例１と同様にして、ゼラチン長繊維不織布を作製した。
　得られたゼラチン長繊維不織布を、５～１０ｍｍ角に切断し、切断したゼラチン長繊維不織布５ｇに蒸留水を１００ｍＬ加えて、超高速回転型ミキサー（フリッチュ社製の「Ｐ－１１メッサーミル」）を用いて２０００ｒｐｍで１０秒間撹拌することで、ゼラチン短繊維の水分散液を得た。得られたゼラチン短繊維の水分散液（懸濁液）を用いて、上述したとおり、ゼラチン短繊維の平均繊維径と平均繊維長を測定したところ、平均繊維径が４０μｍ（変動係数０．１３）であり、平均繊維長が１４６９μｍ（変動係数０．４７）であり、アスペクト比は３６．７であった。
　次に、繊維懸濁液を－３０℃の冷凍庫で凍結させた後、真空度凍結乾燥を行い、乾燥したゼラチン短繊維を得た。 (Manufacturing Example 3)
A gelatin long-fiber non-woven fabric was produced in the same manner as in Production Example 1.
The obtained gelatin long-fiber non-woven fabric is cut into 5 to 10 mm squares, 100 mL of distilled water is added to 5 g of the cut gelatin long-fiber non-woven fabric, and an ultra-high-speed rotary mixer (“P-11 Messer Mill” manufactured by Fritsch) is used. By stirring at 2000 rpm for 10 seconds, an aqueous dispersion of gelatin short fibers was obtained. As described above, the average fiber diameter and the average fiber length of the gelatin short fibers were measured using the obtained aqueous dispersion (suspension) of the gelatin short fibers, and the average fiber diameter was 40 μm (coefficient of variation 0.13). ), The average fiber length was 1469 μm (coefficient of variation 0.47), and the aspect ratio was 36.7.
Next, the fiber suspension was frozen in a freezer at −30 ° C. and then freeze-dried to a degree of vacuum to obtain dried gelatin short fibers.
　（実施例１～４）
　＜細胞シートの作製＞
　２４ウェルプレート（コーニング社製、平底、培養面の面積１．９ｃｍ2）の各ウェルにアスコルビン酸リン酸塩を１０μＭとなるように添加した培地（ＭＥＭα、１０％牛胎児血清、１％ペニシリン／ストレプトマイシン）１ｍＬと、製造例１で得られたゼラチン短繊維を下記表１に示す配合量で添加した後、マウス線維芽細胞ＭＣ３Ｔ３－Ｅ１細胞を１ウェル当たりの細胞数が１．０×１０5細胞となるように播種した。その後、２４ウェルプレートを３７℃、５％のＣＯ2の条件でインキュベートした。培地の交換は２日に一回行った。５日間培養し細胞シートを作製した。 (Examples 1 to 4)
<Preparation of cell sheet>
Medium (MEMα, 10% fetal bovine serum, 1% penicillin /) in which ascorbic acid phosphate was added to each well of a 24-well plate (Corning, flat bottom, culture surface area 1.9 cm 2 ) so as to be 10 μM. After adding 1 mL of streptomycin) and the gelatin short fibers obtained in Production Example 1 in the blending amounts shown in Table 1 below, the number of mouse fibroblasts MC3T3-E1 cells per well was 1.0 × 10 5 The cells were seeded to form cells. The 24-well plate was then incubated at 37 ° C. with 5% CO 2 . The medium was changed once every two days. The cells were cultured for 5 days to prepare cell sheets.
　（実施例５）
　ゼラチン短繊維として製造例２で得られたゼラチン短繊維を用い、配合量を１ｍｇにした以外は、実施例１と同様にして細胞シートを作製した。 (Example 5)
Using the gelatin short fibers obtained in Production Example 2 as the gelatin short fibers, a cell sheet was prepared in the same manner as in Example 1 except that the blending amount was 1 mg.
　（実施例６）
　ゼラチン短繊維として製造例３で得られたゼラチン短繊維を用い、配合量を１ｍｇにした以外は、実施例１と同様にして細胞シートを作製した。 (Example 6)
The gelatin short fibers obtained in Production Example 3 were used as the gelatin short fibers, and a cell sheet was prepared in the same manner as in Example 1 except that the blending amount was 1 mg.
　（実施例７）
　ウェルプレートを温度感応性培養皿（ＵｐＣｅｌｌ、ＣｅｌｌＳｅｅｄ社）の１２ウェルプレート（平底、培養面の面積３．８ｃｍ2）に変更し、製造例３で得られたゼラチン短繊維の配合量を０．４ｍｇとした以外は、実施例６と同様にして細胞シートを作製した。 (Example 7)
The well plate was changed to a 12-well plate (flat bottom, culture surface area 3.8 cm 2 ) of a temperature-sensitive culture dish (UpCell, CellSeed), and the amount of gelatin short fibers obtained in Production Example 3 was changed to 0. A cell sheet was prepared in the same manner as in Example 6 except that the dose was 4 mg.
　（比較例１）
　ゼラチン短繊維を添加していない以外は、実施例１と同様にして細胞シートを作製した。 (Comparative Example 1)
A cell sheet was prepared in the same manner as in Example 1 except that gelatin short fibers were not added.
　（比較例２）
　ゼラチン短繊維に変えてポリエチレンテレフタレート（ＰＥＴ）短繊維［平均繊維径５４．１μｍ（変動係数０．１０）、平均繊維長１７００μｍ（変動係数０．５１）］を用い、配合量を１ｍｇとした以外は、実施例１と同様にして細胞シートを作製した。 (Comparative Example 2)
Polyethylene terephthalate (PET) short fibers [average fiber diameter 54.1 μm (coefficient of variation 0.10), average fiber length 1700 μm (coefficient of variation 0.51)] were used instead of gelatin short fibers, except that the blending amount was 1 mg. Made a cell sheet in the same manner as in Example 1.
　（比較例３）
　ゼラチン短繊維を添加していない以外は、実施例７と同様にして細胞シートを作製した。 (Comparative Example 3)
A cell sheet was prepared in the same manner as in Example 7 except that gelatin short fibers were not added.
　＜細胞シートの収縮性の評価＞
　まず、細胞シートを剥離する前に、デジタルカメラ（オリンパス社製「ＶＨ－５１５」）で撮影した。得られた画像から、細胞シートの直径（ＤＳ１）を測定した。
　次に、ウェルプレートと細胞シートの界面に、ピペットを用いて培地を吐出した。その液流によりウェルプレートから細胞シートを剥離した。ウェルプレートから培地を除去して、細胞シートをウェルプレート底面に広げ、デジタルカメラ（オリンパス社製「ＶＨ－５１５」）で撮影した。得られた画像から、細胞シートの直径（ＤＳ２）を測定した。
　次に、剥離した後の細胞シートの直径ＤＳ２と剥離する前の細胞シートの直径ＤＳ１の比、ＤＳ２／ＤＳ１を求め、細胞シートの収縮性を評価した。その結果を下記表１に示した。 <Evaluation of cell sheet contractility>
First, before peeling off the cell sheet, an image was taken with a digital camera (“VH-515” manufactured by Olympus Corporation). From the obtained image, the diameter of the cell sheet (DS1) was measured.
Next, the medium was discharged to the interface between the well plate and the cell sheet using a pipette. The cell sheet was peeled from the well plate by the liquid flow. The medium was removed from the well plate, the cell sheet was spread on the bottom surface of the well plate, and photographed with a digital camera (“VH-515” manufactured by Olympus Corporation). From the obtained image, the diameter of the cell sheet (DS2) was measured.
Next, the ratio of the diameter DS2 of the cell sheet after exfoliation to the diameter DS1 of the cell sheet before exfoliation, DS2 / DS1, was determined, and the contractility of the cell sheet was evaluated. The results are shown in Table 1 below.
　（グルコース消費量の測定）
　剥離した細胞シートを培地とともに新しい２４ウェルプレートに移動した。その後、培地を除去し、培地がない状態で３７℃、５％ＣＯ2の条件で、３０分間インキュベートして細胞シートをウェルプレートに接着させた。アスコルビン酸リン酸塩を１０μＭとなるように添加した培地（ＭＥＭα、１０％牛胎児血清、１％ペニシリン／ストレプトマイシン）を２ｍＬ加えて、３７℃、５％ＣＯ2の条件で培養した。２４時間目に培養上清を回収し、回収した培養上清中のグルコース濃度をグルコース測定キット（グルコースＣＩＩ－テストワコー、富士フイルム和光純薬）を用いて定量し、未使用の培地のグルコース濃度と比較することで、グルコース消費量を計算した。その結果を下記表２に示した。 (Measurement of glucose consumption)
The detached cell sheet was transferred with medium to a new 24-well plate. The medium was then removed and incubated in the absence of medium at 37 ° C. and 5% CO 2 for 30 minutes to allow the cell sheets to adhere to the well plate. 2 mL of a medium (MEMα, 10% fetal bovine serum, 1% penicillin / streptomycin) to which ascorbic acid phosphate was added to 10 μM was added, and the cells were cultured at 37 ° C. and 5% CO 2 . The culture supernatant was collected at the 24th hour, and the glucose concentration in the collected culture supernatant was quantified using a glucose measurement kit (glucose CII-Test Wako, Fujifilm Wako Pure Chemical Industries, Ltd.), and the glucose concentration in the unused medium. Glucose consumption was calculated by comparison with. The results are shown in Table 2 below.
　（細胞数の計測）
　グルコース消費量の測定で用いた２４ウェルプレートから培地を除去し、トリプシンＥＤＴＡ溶液を０．６ｍＬ加えて１０分間３７℃でインキュベートした。その後マイクロピペットでピペッティングをして細胞シートを解離し、さらに１０分間３７℃でインキュベートした。０．４ｍＬの培地を加えたあと、トリパンブルーで染色し、血球計算板で細胞数を計測した。 (Measurement of cell number)
Medium was removed from the 24-well plate used to measure glucose consumption, 0.6 mL of trypsin EDTA solution was added and incubated for 10 minutes at 37 ° C. The cell sheet was then dissociated by pipetting with a micropipette and incubated for another 10 minutes at 37 ° C. After adding 0.4 mL of medium, the cells were stained with trypan blue and the number of cells was counted with a hemocytometer.
　（細胞シートの剥離時間の測定）
　実施例７及び比較例３において、細胞シートが形成した温度感応性培養皿（ＵｐＣｅｌｌ、ＣｅｌｌＳｅｅｄ社）を、振盪機（ＴｈｅｒｍｏＭｉｘｅｒＣ、エッペンドルフ社）の上に置き２５℃、３００ｒｐｍで振盪させた。細胞シートが培養皿から剥がれて、培地中で浮遊するまでの時間を剥離時間として計測した。その結果を下記表３に示す。 (Measurement of cell sheet peeling time)
In Example 7 and Comparative Example 3, the temperature-sensitive culture dish (UpCell, CellSeed) formed by the cell sheet was placed on a shaker (ThermoMixerC, Eppendorf) and shaken at 25 ° C. and 300 rpm. The time until the cell sheet was peeled off from the culture dish and floated in the medium was measured as the peeling time. The results are shown in Table 3 below.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　図１には、実施例１の細胞シートの顕微鏡写真を示した。図１（ａ）は、培養基材から剥離する前の実施例１の細胞シートを倒立顕微鏡（Ｏｌｙｍｐｕｓ社製、ＣＫＸ５３、４倍）で観察した画像である。図１（ｂ）は、培養基材から剥離した後の実施例１の細胞シートを顕微鏡（キーエンス製、BX-X710）を用いて明視野で観察した画像（１０倍）である。 FIG. 1 shows a micrograph of the cell sheet of Example 1. FIG. 1A is an image obtained by observing the cell sheet of Example 1 before peeling from the culture substrate with an inverted microscope (manufactured by Olympus, CKX53, 4x). FIG. 1B is an image (10 times) of the cell sheet of Example 1 after being exfoliated from the culture substrate, observed in a bright field using a microscope (manufactured by KEYENCE, BX-X710).
　図２には、剥離した細胞シートをウェルプレート底面に広げ、デジタルカメラ（オリンパス社製「ＶＨ－５１５」）で撮影した写真を示した。図２において、（ａ）～（ｄ）は、それぞれ、実施例５、実施例７、比較例１、及び比較例２の剥離した細胞シートの画像である。 FIG. 2 shows a photograph of the peeled cell sheet spread on the bottom surface of the well plate and taken with a digital camera (“VH-515” manufactured by Olympus Corporation). In FIG. 2, (a) to (d) are images of the peeled cell sheets of Example 5, Example 7, Comparative Example 1, and Comparative Example 2, respectively.
　図３には、剥離した細胞シートを倒立顕微鏡（Ｏｌｙｍｐｕｓ社製、ＣＫＸ５３、４倍）で観察した画像を示した。図３において、（ａ）～（ｄ）は、それぞれ、実施例４、実施例６、比較例１、及び比較例２の剥離した細胞シートの画像である。 FIG. 3 shows an image of the peeled cell sheet observed with an inverted microscope (manufactured by Olympus, CKX53, 4x). In FIG. 3, (a) to (d) are images of the peeled cell sheets of Example 4, Example 6, Comparative Example 1, and Comparative Example 2, respectively.
　図１及び図３から分かるように、実施例の細胞シートにおいて、ゼラチン短繊維が細胞シート中に分散しており、細胞がゼラチン短繊維の表面に接着している。 As can be seen from FIGS. 1 and 3, in the cell sheet of the example, gelatin short fibers are dispersed in the cell sheet, and the cells are adhered to the surface of the gelatin short fibers.
　表１及び図２から分かるように、ゼラチン短繊維が細胞シート中に分散している実施例の細胞シートでは、ゼラチン短繊維を含まない比較例１、及びＰＥＴ繊維を用いた比較例２の細胞シートに比べて、培養基材から剥離した後の収縮が抑制されていた。実施例１～４の対比から分かるように、細胞数に対するゼラチン短繊維の配合量が多いほど、培養基材から剥離した後の細胞シートの収縮を抑制する効果が高い。また、実施例４～６の対比から分かるように、平均繊維径が同じである場合、平均繊維長が８２８μｍ以下である方が、培養基材から剥離した後の細胞シートの収縮を抑制する効果が高い。 As can be seen from Table 1 and FIG. 2, in the cell sheet of the example in which gelatin short fibers are dispersed in the cell sheet, the cells of Comparative Example 1 containing no gelatin short fibers and Comparative Example 2 using PET fibers. Compared with the sheet, the shrinkage after peeling from the culture substrate was suppressed. As can be seen from the comparison of Examples 1 to 4, the larger the amount of gelatin short fibers blended with respect to the number of cells, the higher the effect of suppressing the shrinkage of the cell sheet after exfoliation from the culture substrate. Further, as can be seen from the comparison of Examples 4 to 6, when the average fiber diameter is the same, the one having the average fiber length of 828 μm or less has the effect of suppressing the shrinkage of the cell sheet after peeling from the culture substrate. Is high.
　表２から分かるように、ゼラチン短繊維が細胞シート中に分散している実施例の細胞シートでは、ゼラチン短繊維を含まない比較例１、及びＰＥＴ繊維を用いた比較例２の細胞シートに比べて、細胞当たりのグルコース消費量が高く、細胞活性が向上している。 As can be seen from Table 2, the cell sheet of the example in which gelatin short fibers are dispersed in the cell sheet is compared with the cell sheet of Comparative Example 1 containing no gelatin short fibers and Comparative Example 2 using PET fibers. Therefore, the amount of glucose consumed per cell is high, and the cell activity is improved.
　表３から分かるように、ゼラチン短繊維が細胞シート中に分散している実施例では、ゼラチン短繊維を含まない比較例３に比べて、培養基材から細胞シートを剥離するのに必要な時間が短く、ハンドリング性が良好であった。 As can be seen from Table 3, in the example in which the gelatin short fibers were dispersed in the cell sheet, the time required to peel the cell sheet from the culture substrate was compared with the comparative example 3 in which the gelatin short fibers were not contained. Was short and the handleability was good.
　本発明の細胞シートは、ヒト又はヒト以外の動物の組織の増大、修復又は再生に用いることができる。 The cell sheet of the present invention can be used for the growth, repair or regeneration of human or non-human animal tissues.
１　加温槽
２、２２　紡糸液
３　ノズル吐出口
４，６　コンプレッサー
５　流体噴射口
７　圧力流体
８　ゼラチン長繊維
９　ゼラチン長繊維不織布
１０　不織布製造装置
１１　巻き取りロール
１２　保温容器
２０　フィラメント製造装置
２１　シリンジ
２３　ノズル（吐出口）
２４　加熱紡糸筒
２５　ゼラチンフィラメント
２６　ガイドロール
２７　巻き取り機 1 Heating tanks 2, 22 Spinning liquid 3 Nozzle discharge port 4, 6 Compressor 5 Fluid injection port 7 Pressure fluid 8 Gelatin long fiber 9 Gelatin long fiber non-woven fabric 10 Non-woven fabric manufacturing equipment 11 Winding roll 12 Heat insulation container 20 Filament manufacturing equipment 21 Syringe 23 Nozzle (discharge port)
24 Heat spinning cylinder 25 Gelatin filament 26 Guide roll 27 Winding machine

Claims (14)

  1. 　ゼラチンを主成分とするゼラチン短繊維が細胞シート中に分散しており、
    　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シート。     Gelatin short fibers containing gelatin as the main component are dispersed in the cell sheet.
    The gelatin short fiber is a cell sheet having an average fiber diameter of 1 μm or more and 400 μm or less and an average fiber length of 10 μm or more and 2000 μm or less.
  2. 　前記ゼラチン短繊維は、架橋されている請求項１に記載の細胞シート。 The cell sheet according to claim 1, wherein the gelatin short fibers are crosslinked.
  3. 　前記細胞シートにおいて、細胞数とゼラチン短繊維の質量の比（細胞数/ゼラチン短繊維の質量）は、１ｘ１０5細胞/ｍｇ以上２．５ｘ１０6細胞/ｍｇ以下である請求項１又は２に記載の細胞シート。 In the cell sheet, the ratio of the mass of cell number and gelatin short fibers (cells / gelatin short fiber mass) is claimed in claim 1 or 2 or less 2.5 × 10 6 cells / mg 1x10 5 cells / mg or more Cell sheet.
  4. 　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である請求項１～３のいずれかに記載の細胞シート。 The cell sheet according to any one of claims 1 to 3, wherein the gelatin short fiber has an aspect ratio of 3 or more represented by an average fiber length / average fiber diameter.
  5. 　細胞及びゼラチンを主成分とするゼラチン短繊維を培地中で培養することで、前記ゼラチン短繊維が分散している細胞シートを形成しており、
    　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であることを特徴とする細胞シートの製造方法。     By culturing cells and gelatin short fibers containing gelatin as a main component in a medium, a cell sheet in which the gelatin short fibers are dispersed is formed.
    A method for producing a cell sheet, wherein the gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less.
  6. 　培養基材から剥離する前の細胞シートの直径をＤＳ１とし、培養基材から剥離した後の細胞シートの直径をＤＳ２とした場合、ＤＳ２／ＤＳ１は０．５６以上１以下である請求項５に記載の細胞シートの製造方法。 When the diameter of the cell sheet before peeling from the culture substrate is DS1 and the diameter of the cell sheet after peeling from the culture substrate is DS2, DS2 / DS1 is 0.56 or more and 1 or less. The method for producing a cell sheet according to the above.
  7. 　前記細胞の播種量とゼラチン短繊維の添加量の比（細胞の播種量/ゼラチン短繊維の添加量）は、特に限定されないが、例えば、０．１ｘ１０5細胞/ｍｇ以上７ｘ１０5細胞/ｍｇ以下である請求項５又は６に記載の細胞シートの製造方法。 The ratio of the seeded amount of the cells to the added amount of gelatin short fibers (cell seeded amount / added amount of gelatin short fibers) is not particularly limited, but is, for example, 0.1x10 5 cells / mg or more and 7x10 5 cells / mg or less. The method for producing a cell sheet according to claim 5 or 6.
  8. 　前記ゼラチン短繊維は、ゼラチンを含む紡糸液をノズル吐出口から空気中に押し出し、前記ノズル吐出口の後方に位置し、前記ノズル吐出口とは非接触状態の流体噴射口から前方に向けて圧力流体を噴射し、前記押し出された紡糸液を前記圧力流体に随伴させて繊維形成させ、前記繊維形成した繊維を集積させてゼラチン長繊維不織布とし、前記ゼラチン長繊維不織布を切断することで得られる請求項５～７のいずれかに記載の細胞シートの製造方法。 The gelatin short fibers push a spinning liquid containing gelatin into the air from a nozzle discharge port, and are located behind the nozzle discharge port, and pressure is directed forward from a fluid injection port in a state of non-contact with the nozzle discharge port. It is obtained by injecting a fluid, forming fibers by accompanying the extruded spinning liquid with the pressure fluid, and accumulating the fiber-formed fibers to form a gelatin long fiber non-woven fabric, and cutting the gelatin long-fiber non-woven fabric. The method for producing a cell sheet according to any one of claims 5 to 7.
  9. 　前記ゼラチン短繊維は、ゼラチンを含む紡糸液をノズル吐出口から空気中に押し出し、加熱紡糸筒を通過させて乾式紡糸し、得られたゼラチンフィラメント糸を切断することで得られる請求項５～７のいずれかに記載の細胞シートの製造方法。 The gelatin short fibers are obtained by extruding a spinning liquid containing gelatin into the air from a nozzle discharge port, passing it through a heat spinning cylinder, dry spinning, and cutting the obtained gelatin filament yarns. The method for producing a cell sheet according to any one of.
  10. 　前記ゼラチン長繊維不織布又はゼラチンフィラメント糸は架橋した後に切断する請求項５～９のいずれかに記載の細胞シートの製造方法。 The method for producing a cell sheet according to any one of claims 5 to 9, wherein the gelatin long fiber non-woven fabric or gelatin filament yarn is crosslinked and then cut.
  11. 　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である請求項５～１０のいずれかに記載の細胞シートの製造方法。 The method for producing a cell sheet according to any one of claims 5 to 10, wherein the gelatin short fiber has an aspect ratio represented by an average fiber length / average fiber diameter of 3 or more.
  12. 　細胞、ゼラチン短繊維及び培地を含み、
    　前記ゼラチン短繊維は、平均繊維径が１μｍ以上４００μｍ以下であり、かつ平均繊維長が１０μｍ以上２０００μｍ以下であり、
    　細胞シート中にゼラチン短繊維が分散している細胞シートを形成するのに用いることを特徴とする細胞シートの作製キット。     Contains cells, gelatin short fibers and medium
    The gelatin short fibers have an average fiber diameter of 1 μm or more and 400 μm or less, and an average fiber length of 10 μm or more and 2000 μm or less.
    A kit for producing a cell sheet, which is used for forming a cell sheet in which gelatin short fibers are dispersed in the cell sheet.
  13. 　前記ゼラチン短繊維は、架橋されている請求項１２に記載の細胞シートの作製キット。 The cell sheet preparation kit according to claim 12, wherein the gelatin short fibers are crosslinked.
  14. 　前記ゼラチン短繊維は、平均繊維長/平均繊維径で表されるアスペクト比が３以上である請求項１２又は１３に記載の細胞シートの作製キット。 The cell sheet preparation kit according to claim 12 or 13, wherein the gelatin short fibers have an aspect ratio of 3 or more represented by an average fiber length / average fiber diameter.
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