A three-layer laminate consisting of an extracellular matrix, a sheet-like cell culture, and a biodegradable gel.

Abstract

The present invention addresses the problem of providing a laminate that is easier to manipulate, in which when a thrombin solution is sprayed, damage to a sheet-shaped cell culture due to the spraying pressure is reduced, and a fibrin gel is formed on one surface of the sheet-shaped cell culture. This problem is solved by: in the production of a sheet-shaped cell culture in which cells are cultured on a temperature-responsive cell culture substrate, fibrinogen is instilled on a sheet-shaped cell culture having an extracellular matrix on the bottom surface, and furthermore, a thrombin solution is sprayed onto the sheet-shaped cell culture from an oblique direction; and by providing a laminate having a fibrin gel layer only on one surface of the sheet-shaped cell culture, and having an extracellular matrix layer on the other surface.

Description

[Technical field]

[0001] The present invention relates to a three-layer laminate composed of, in that order, an extracellular matrix, a sheet-shaped cell culture and a biodegradable gel, a method for producing said laminate, and an apparatus used in said production method. [Background technology]

[0002] In recent years, attempts have been made to transplant various cells to repair damaged tissues, etc. For example, attempts have been made to use fetal cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. to repair myocardial tissue damaged by ischemic heart disease such as angina pectoris and myocardial infarction (Non-Patent Document 1).

[0003] As one example of such an attempt, a sheet-shaped cell culture in which cells are formed into a sheet has been developed. For the application of such cell cultures, a method has been developed in which cells are detached while retaining adhesive proteins secreted by the cells, such as extracellular matrix, by using a temperature-responsive cell culture substrate (Patent Document 1). Furthermore, such sheet-shaped cell cultures are generally fragile, and in order to improve this problem, a method of producing a laminate with fibrin gel has been attempted (Patent Document 2). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 4422540 [Patent Document 2] Patent No. 6495603 [Non-patent literature]

[0005] [Non-Patent Document 1] Haraguchi et al., Stem Cells Trans1 Med. 2012 Feb;1(2):136-41 Summary of the Invention [Problem to be solved by the invention]

[0006] However, with regard to the laminate of sheet-shaped cell culture and fibrin gel described in Patent Document 2, it was found that when a liquid containing thrombin was sprayed in creating the laminate, the sheet-shaped cell culture was damaged by the spray, and fibrin gel was sometimes produced on both sides of the sheet-shaped cell culture, resulting in a laminate with reduced operability. Therefore, an object of the present invention is to provide a laminate with higher operability that does not have these problems. [Means for solving the problem]

[0007] Through extensive research to solve the above problems, the inventors have discovered that in a laminate of a biodegradable gel and a sheet-shaped cell culture, a sheet-shaped cell culture is produced by culturing cells on a cell culture substrate having a stimuli-responsive material, and further, when the biodegradable gel is a fibrin gel, when spraying a liquid containing thrombin, rather than spraying perpendicularly towards the sheet-shaped cell culture, by spraying at a certain angle to the sheet-shaped cell culture, the risk of damage to the sheet-shaped cell culture due to spray pressure is reduced, and further, such spraying weakens the force pushing out thrombin and / or fibrinogen on the sheet-shaped cell culture compared to when spraying perpendicularly, thereby providing a laminate having a fibrin gel layer on only one side of the sheet-shaped cell culture and an extracellular matrix layer on the opposite side.

[0008] That is, the present invention relates to the following. [1] A three-layer laminate consisting of an extracellular matrix, a sheet-like cell culture, and a biodegradable gel. [2] A laminate of [1], wherein the biodegradable gel is fibrin gel. [3] A method for producing a laminate, comprising the steps of: (i) culturing cells on a cell culture substrate having a stimuli-responsive material to produce a sheet-shaped cell culture having an extracellular matrix layer formed on the lower surface; (ii) dripping a liquid containing fibrinogen onto the upper surface of the sheet-shaped cell culture; (iii) spraying a liquid containing thrombin onto the sheet-shaped cell culture from an oblique direction; (iv) forming a fibrin gel layer on the upper surface of the sheet-shaped cell culture by a reaction between the fibrinogen and the thrombin; The method for producing a three-layer laminate consisting of an extracellular matrix, a sheet-like cell culture, and a fibrin gel in that order, comprises the steps of: [4] The method according to [3], further comprising rotating the sheet-shaped cell culture in step (iii).

[0009] [5] A spray nozzle for spraying a liquid containing thrombin; and A stand for supporting the spray nozzle and adjusting the angle of the spray nozzle; An apparatus for use in the method according to [3] or [4], comprising: [6] The apparatus of [5], further comprising a rotating platform. [7] A stand for use with the device described in [5] or [6], comprising a support and a holder. Effect of the Invention

[0010] The laminate of the present invention reduces the risk of damage to the sheet-shaped cell culture due to the spray pressure during thrombin spraying, and because fibrin gel is present on only one side of the sheet-shaped cell culture and a layer of extracellular matrix is ​​present on the opposite side, the sheet-shaped cell culture has improved adhesion to the transplant site when used for transplantation. In other words, according to the present invention, a sheet-shaped cell culture of higher quality and with excellent operability can be provided to the clinical field. [Brief description of the drawings]

[0011] [Figure 1]Figures 1A to 1C are schematic diagrams of a stand 1 according to the present invention. Figure 1A shows a side view of the stand 1. Figure 1B shows a front view of the stand 1. Figure 1C shows a plan view of the stand 1 with a holder 7 and a support pole 3 removed. [Diagram 2] 2A to 2D show enlarged views of the holder 7 portion of the stand 1 according to the present invention. Fig. 2A shows a rear view of the holder 7. Fig. 2B shows a plan view of the holder 7. Fig. 2C shows a side view of the holder 7. Fig. 2D shows a front view of the holder 7. [Diagram 3] FIG. 3 shows a schematic diagram of the stand 1 in combination with a rotating table 9 . [Figure 4] FIG. 4 shows a cross-sectional view of a laminate produced by the method of the present invention. [Diagram 5] FIG. 5 shows a cross-sectional view of a laminate produced by dropping a thrombin solution and then a fibrinogen solution, for comparison. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention will be described in detail below. One aspect of the present invention relates to a three-layer laminate composed in this order of an extracellular matrix, a sheet-shaped cell culture, and a biodegradable gel. In general, the "extracellular matrix" is a component secreted by cells and composed of water, proteins, polysaccharides, etc., including but not limited to collagen, fibronectin, proteoglycan, laminin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, etc. The extracellular matrix serves as a physical scaffold for cells and also plays an important role in tissue morphogenesis, differentiation, and homeostasis.

[0013] In the present invention, the extracellular matrix functions as a physical scaffold, and the extracellular matrix layer of the laminate contributes to adhesion of the sheet-shaped cell culture to the transplantation site. This effect simplifies the step of adhering the sheet-shaped cell culture to the transplantation site, such as suturing, when transplanting the sheet-shaped cell culture, improving operability in clinical application. Since the extracellular matrix is ​​a cell-derived component, there is no problem in transplanting it together with the laminate of the present invention. In the present invention, the thickness of the extracellular matrix layer is 1 nm to 100 nm, and preferably 1 nm to 50 nm.

[0014] In the present invention, the term "sheet-shaped cell culture" refers to cells that are linked to each other to form a sheet. The cells may be directly linked to each other via a cell adhesion factor or indirectly linked to each other via an extracellular matrix. The sheet-shaped cell culture may be a single layer, or may be multi-layered, such as two, three, four or more layers, or may have a three-dimensional structure without a layer structure.

[0015] The cells constituting the sheet-shaped cell culture are not particularly limited as long as they can form a sheet-shaped cell culture, and include, for example, adhesive cells (adherent cells). Adherent cells include, but are not limited to, adhesive somatic cells (e.g., cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin cells, synovial cells, chondrocytes, etc.) and stem cells (e.g., myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), mesenchymal stem cells), etc. The somatic cells may be differentiated from stem cells, particularly iPS cells.

[0016] The cells constituting the sheet-shaped cell culture can be derived from any organism to be treated with the sheet-shaped cell culture, including, but not limited to, humans, primates, dogs, cats, pigs, horses, goats, sheep, rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Furthermore, the sheet-shaped cell culture may be made of only one type of cell, but two or more types of cells can also be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming the sheet-shaped cell culture, the most preferred ratio (purity) at the end of the production of the sheet-shaped cell culture is 60% or more, preferably 70% or more, and more preferably 75% or more. In the present invention, the sheet-shaped cell culture can be produced by any method known to those skilled in the art. For example, it can be prepared by a method including the steps of seeding cells on a culture substrate, forming the seeded cells into a sheet, and isolating the formed sheet-shaped cell culture from the culture substrate. Preferably, the culture substrate is a temperature-responsive cell culture substrate, and more preferably, in order to form an extracellular matrix, the cells are cultured on the temperature-responsive cell culture substrate for one hour or more from seeding to isolation of the sheet-shaped cell culture.

[0017] In the present invention, the term "biodegradable gel" refers to a gel that decomposes in the living body, is absorbed into the body, metabolized, and excreted. Biodegradable gels include, but are not limited to, fibrin gel, gels formed by Adspray (registered trademark) (manufactured by Terumo Corporation), and the like. Biodegradable gels preferably refer to gels that become viscous by mixing two types of liquid. For example, fibrin gel is a gel with high strength that is formed by mixing a liquid containing fibrinogen (hereinafter referred to as fibrinogen liquid) with a liquid containing thrombin (hereinafter referred to as thrombin liquid) and allowing thrombin to act on fibrinogen. In the present invention, the biodegradable gel layer is firmly attached to the sheet-shaped cell culture, and is useful for preventing damage to the sheet-shaped cell culture when the sheet-shaped cell culture is peeled off from the culture vessel and when the sheet-shaped cell culture is moved for transplantation or the like. In addition, since the biodegradable gel can be degraded in vivo, the laminate according to the present invention can be transplanted together with the biodegradable gel layer. In the present invention, the thickness of the biodegradable gel layer is 5 μm to 1300 μm, and preferably 50 μm to 300 μm.

[0018] In one embodiment, the biodegradable gel has a uniform thickness. Here, uniform means that the difference in thickness between the thickest and thinnest parts is 20% or less. Uniform means that the difference in thickness is preferably 10% or less, more preferably 5% or less. In one embodiment, the biodegradable gel is not hard. Preferably, the biodegradable gel is soft and viscous. In one embodiment, the laminate is formed by laminating an extracellular matrix layer, a sheet-shaped cell culture, and a biodegradable gel layer in this order from the bottom.

[0019] Another aspect of the present invention relates to a method for producing a three-layer laminate composed of, in that order, an extracellular matrix, a sheet-shaped cell culture, and a fibrin gel, the method including the steps of culturing cells on a cell culture substrate having a stimuli-responsive material to produce a sheet-shaped cell culture having an extracellular matrix layer formed on the lower surface thereof, dripping a fibrinogen liquid on the upper surface of the sheet-shaped cell culture, spraying a thrombin liquid onto the sheet-shaped cell culture from an oblique direction, and forming a fibrin gel layer on the upper surface of the sheet-shaped cell culture by a reaction between the fibrinogen and the thrombin.

[0020] In the present invention, the term "cell culture substrate having a stimuli-responsive material" refers to a substrate whose physical properties change in response to stimuli such as temperature, pH, and light, and is suitable for cell culture. Preferably, the cell culture substrate having a stimuli-responsive material refers to a temperature-responsive cell culture substrate. More preferably, the temperature-responsive cell culture substrate refers to a substrate that exhibits cell adhesion at the temperature at which cells are cultured, and exhibits cell non-adhesion at another temperature. Conventionally, when a sheet-shaped cell culture is detached from a culture vessel, a trypsin treatment or the like is required, and such a treatment damages the extracellular matrix. However, when cells are cultured on a cell culture substrate having a stimuli-responsive material including a temperature-responsive cell culture substrate to produce a sheet-shaped cell culture, such a treatment becomes unnecessary when detaching, and it becomes possible to detach the sheet-shaped cell culture while keeping the extracellular matrix. Any known culture vessel can be used as the culture vessel equipped with the cell culture substrate having a stimuli-responsive material. For example, commercially available Upcell (registered trademark) from CellSeed Co., Ltd. or Cepallet (registered trademark) from DIC Corporation can be used as the temperature-responsive cell culture substrate.

[0021] The fibrinogen solution is not particularly limited as long as it can react with thrombin to form a fibrin gel, and examples of the fibrinogen solution include liquids containing fibrinogen at concentrations of, for example, 1 mg / mL to 500 mg / mL, 5 mg / mL to 400 mg / mL, 10 mg / mL to 250 mg / mL, 20 mg / mL to 150 mg / mL, 40 mg / mL to 100 mg / mL, and 50 mg / mL to 90 mg / mL. The solvent of the fibrinogen solution is typically water. The fibrinogen solution may contain components such as factor XIII, aprotinin, serum albumin, glycine, L-arginine hydrochloride, L-isoleucine, L-sodium glutamate, D-mannitol, sodium citrate hydrate, and sodium chloride in addition to fibrinogen. The fibrinogen solution is commercially available or can be produced based on a known method. Examples of commercially available fibrinogen solutions include, but are not limited to, the solution obtained by dissolving the contents of vial 1 (lyophilized fibrinogen powder) of Bolheal (registered trademark) tissue adhesive (manufactured by Teijin Pharma) in the contents of vial 2 (fibrinogen dissolving solution) and the solution obtained by dissolving the contents of vial 1 (fibrinogen powder) of Beriplast P Combi Set (registered trademark) tissue adhesive (manufactured by CSL Behring) in the contents of vial 2 (aprotinin solution).

[0022] The fibrinogen liquid can be dripped using any known method, for example, a syringe or a pipette. Examples of the syringe that can be used include a needleless syringe with a capacity of 0.5 mL to 5 mL, a syringe with a needle (for example, a syringe with a needle of 18 G to 27 G), a two-liquid mixing set (with an application nozzle with an inner diameter of about 1 mm, manufactured by Nipro) of a preparation set attached to Bolheal (registered trademark) tissue adhesion, and a two-liquid mixing set (with an application nozzle, manufactured by Nipro) of a Veriplast preparation set. The amount of fibrinogen liquid to be dripped is not particularly limited as long as it can coat the upper surface of the sheet-shaped cell culture, and may be, for example, about 6 μL / cm. 2 ~Approx. 70μL / cm 2 , about 9μL / cm 2 ~about 50μL / cm 2 , about 12μL / cm 2 ~Approx. 45μL / cm2 , about 15μL / cm 2 ~about 40μL / cm 2 , about 18μL / cm 2 ~Approx. 32μL / cm 2 The amount of fibrinogen liquid to be dropped is not limited to, but may be about 100 μL to about 1000 μL, about 150 μL to about 800 μL, about 200 μL to about 700 μL, about 250 μL to about 600 μL, about 300 μL to about 500 μL, etc., for a sheet-shaped cell culture with a diameter of 45 mm. The particle size of the droplets when dropping the fibrinogen liquid is not particularly limited, but may be, for example, in a range in which the diameter of the droplets attached to the sheet-shaped cell culture after dropping is about 0.2 cm to about 2.0 cm. The weight of the droplets is not limited to, but may be, for example, in a range of about 10 mg to about 100 mg, about 15 mg to about 50 mg, about 20 mg to about 30 mg, etc. The particle size and weight of the droplets can be appropriately adjusted depending on whether a needle is attached to the syringe, the gauge number of the attached needle, and the shape of the needle tip.

[0023] The thrombin solution is not particularly limited as long as it can react with fibrinogen to form a fibrin gel, and examples of the thrombin solution include a liquid containing thrombin at a concentration of, for example, 1 unit / mL to 10,000 units / mL, 10 units / mL to 5,000 units / mL, 25 units / mL to 2,500 units / mL, 50 units / mL to 1,000 units / mL, 100 units / mL to 500 units / mL, or 250 units / mL to 300 units / mL. The solvent of the thrombin solution is typically water. The thrombin solution may contain components such as sodium citrate hydrate and sodium chloride in addition to thrombin. The thrombin solution is commercially available or can be produced based on a known method. Examples of commercially available thrombin solutions include, but are not limited to, a solution obtained by dissolving the contents of vial 3 (lyophilized thrombin powder) of Bolheal (registered trademark) tissue adhesive (manufactured by Teijin Pharma) in the contents of vial 4 (thrombin dissolving solution) and a solution obtained by dissolving the contents of vial 3 (thrombin powder) of Beriplast P Combiset (registered trademark) tissue adhesive (manufactured by CSL Behring) in the contents of vial 4 (calcium chloride solution).

[0024] The thrombin solution can be sprayed by any known method, for example, a spray. Examples of sprays include, but are not limited to, the Bolheal (registered trademark) spray set (manufactured by Akita Sumitomo Bake Co., Ltd.) and the two-liquid mixing set attached with a spray tip included in the Veriplast P Combi Set (registered trademark) for tissue adhesion (manufactured by CSL Behring). The amount of thrombin solution to be sprayed is not particularly limited as long as it can cover the upper surface of the sheet-shaped cell culture, and the estimated amount of adhesion to the sheet-shaped cell culture is, for example, about 3 μL / cm. 2 ~Approx. 70μL / cm 2 , about 5μL / cm 2 ~about 50μL / cm 2 , about 6μL / cm 2 ~Approx. 45μL / cm 2 , about 12μL / cm 2 ~about 40μL / cm 2 , about 18μL / cm 2 ~Approx. 32μL / cm 2 and the like. Non-limiting examples of the amount of thrombin liquid sprayed include, as the estimated amount of adhesion to a sheet-shaped cell culture with a diameter of 45 mm, about 50 μL to about 1000 μL, about 80 μL to about 800 μL, about 100 μL to about 700 μL, about 200 μL to about 600 μL, about 300 μL to about 500 μL, and the like. The estimated amount of adhesion to a sheet-shaped cell culture is determined by measuring the weight of the liquid adhered within a predetermined range (for example, within a circle with a diameter of 45 mm) when a predetermined amount of thrombin liquid is sprayed with the spray, height, spray pressure, and spray angle actually used, and calculating this based on the density of the thrombin liquid (0.999973 g / cm 3 ) to calculate. Based on the description in this specification, a person skilled in the art can determine the spray conditions that will result in the desired estimated adhesion amount of thrombin solution without undue experimentation. For example, since it has been confirmed that the estimated adhesion amounts for spray amounts of 300 μL, 500 μL, 600 μL, and 900 μL are 100 μL, 180 μL, 300 μL, and 450 μL, respectively, an approximation curve of estimated adhesion amount (μL) = spray amount (μL) × 0.6-88 (μL) can be obtained by the least squares method, and the spray amount that will result in the desired estimated adhesion amount can be determined based on this.

[0025] In the present invention, the thrombin solution is sprayed onto the fibrinogen solution that has been evenly dropped onto the sheet-shaped cell culture. The thrombin solution must be sprayed so that the sprayed thrombin solution and / or the dropped fibrinogen solution do not get around to the underside of the sheet-shaped cell culture and do not damage the sheet-shaped cell culture. If the sprayed thrombin solution and / or the fibrinogen solution get around to the underside of the sheet-shaped cell culture, a fibrin gel layer is also formed on the underside of the sheet-shaped cell culture. If this happens, at the time of transplantation, the fibrin gel layer will be between the sheet-shaped cell culture and the transplantation site, preventing the sheet-shaped cell culture from coming into contact with the transplantation site and hindering the exchange of substances secreted by cells, including the extracellular matrix involved in cell adhesion, thereby reducing operability.

[0026] In general, when a liquid is sprayed using a spray nozzle, it is sprayed in a straight line perpendicular to the target. However, when spraying a sheet-shaped cell culture in this manner, all spray pressure is applied perpendicularly to the sheet-shaped cell culture, increasing the risk of damaging the sheet-shaped cell culture. In addition, the fibrinogen liquid already dripped and / or the sprayed thrombin liquid may be pushed strongly and far away due to the high spray pressure and the reaction when it hits the sheet-shaped culture. If the spray pressure is reduced by moving away from the sheet-shaped cell culture, the thrombin liquid sprayed from the nozzle will spread out more radially, so that the thrombin liquid will follow the edge of the culture vessel and wrap around the underside of the sheet-shaped cell culture. In addition, the thrombin liquid will be sprayed around the periphery of the sheet-shaped cell culture, requiring extra thrombin liquid to be sprayed.

[0027] In the present invention, spraying is performed from an oblique direction. By spraying from an oblique direction, the spray pressure is dispersed in a direction perpendicular to and parallel to the sheet-shaped cell culture, and the pressure applied to the sheet-shaped cell culture is smaller than that applied from a perpendicular direction, which is thought to reduce the risk of damaging the sheet-shaped cell culture. In addition, the thrombin solution is evenly distributed due to the pressure dispersed in a direction parallel to the sheet-shaped cell culture. The pressure dispersed in a horizontal direction to the sheet-shaped cell culture at this time is weaker than the force that strongly pushes the fibrinogen solution and / or the sprayed thrombin solution farther when sprayed from a perpendicular direction, and in addition, it prevents the fibrinogen that has already reacted with thrombin from being pushed away further due to its viscosity, pushing the fibrinogen solution and / or the thrombin solution from the edge of the sheet-shaped cell culture, reducing the risk of fibrin gel being formed on the underside of the sheet-shaped cell culture, and furthermore, since the pressure applied in a perpendicular direction to the sheet-shaped cell culture is also reduced, it is not necessary to move the solution away from the sheet-shaped cell culture as compared to when spraying from a perpendicular direction, and it is thought that the solution will not run along the edge of the culture vessel.

[0028] Such spraying from an oblique direction is determined by the height and angle at which the thrombin solution is sprayed onto the sheet-shaped cell culture. The effects of the present invention can be achieved by spraying from a spray nozzle at a height of about 2 cm to about 15 cm from the sheet-shaped cell culture, from a direction inclined at about 15° to about 150° from the sheet-shaped cell culture, and at a pressure of about 0.005 MPa to about 0.1 MPa. Preferably, the spray nozzle is sprayed at a height of 7 cm from the sheet-shaped cell culture, and spraying is performed from a direction inclined at 45° from the sheet-shaped cell culture.

[0029] In a preferred embodiment, the thrombin solution is sprayed while rotating the sheet-shaped cell culture. Here, "rotation" refers to rotating the sheet-shaped cell culture around an axis that passes through the center of the sheet-shaped cell culture and is perpendicular to the surface containing the sheet-shaped cell culture. By rotating the sheet-shaped cell culture, the thickness of the fibrin gel layer can be made more uniform. This makes it possible to reduce unevenness in the thickness of the fibrin gel in the laminate, and prevents damage to the thin parts when peeling off from the culture vessel or moving to the transplant site. The rotation may be performed manually by an operator or using an instrument such as a turntable. The rotation may also be a combination of vertical and / or horizontal rocking, for example, when the sheet-shaped cell culture is curled up. Preferably, the rotation is performed continuously during spraying.

[0030] In one aspect, the step of preparing a fibrin gel in the present invention can be carried out before detaching the sheet-shaped cell culture from the temperature-responsive cell culture substrate. In such an aspect, the surface of the sheet-shaped cell culture on which the extracellular matrix is ​​formed is adhered to the temperature-responsive cell culture substrate and liquids from each do not penetrate, thereby reducing the risk of fibrin gel being formed on the side of the sheet-shaped cell culture containing the extracellular matrix. In one aspect, all steps of the method according to the present invention are carried out under aseptic conditions.

[0031] The ratio of fibrinogen solution to thrombin solution applied to the sheet-shaped cell culture is not particularly limited as long as it does not excessively inhibit the transplantation operation of the resulting laminate, and for example, the volume ratio of the amount of fibrinogen solution dropped to the estimated amount of thrombin solution attached is about 5:1 to about 1:3, about 4:1 to about 1:2, about 3:1 to about 1:1.5, about 2.5 to about 1:1, about 2:1 to about 1:1, about 1.5 to about 1:1, and preferably about 1:1. The ratio of fibrinogen to thrombin attached to the laminate (mg: unit) is about 8:5 to about 8:75, about 32:25 to about 4:25, about 24:25 to about 16:75, about 4:5 to about 8:25, about 16:25 to about 8:25, about 12:25 to about 8:25, and preferably about 8:25.

[0032] By adjusting the concentrations and amounts of the fibrinogen solution and the thrombin solution, the thickness and properties (flexibility, adhesiveness, etc.) of the resulting laminate can be changed. For example, the thickness of the laminate can be increased by increasing the amount of fibrinogen solution, and the closer the volume ratio of the dropped amount of fibrinogen solution (80 mg / mL) to the estimated attached amount of thrombin solution (250 units / mL) is to 1:1, and the closer the ratio (mg:unit) of fibrinogen to thrombin attached to the laminate is to 8:25, the greater the flexibility and adhesiveness of the laminate and the improved operability.

[0033] The step of forming a fibrin gel layer is not limited, and can be performed, for example, by spraying a thrombin solution and then leaving the sheet-shaped cell culture for a certain period of time. The leaving period is not limited to, but is, for example, about 1 minute to about 60 minutes, about 2 minutes to about 30 minutes, about 3 minutes to about 20 minutes, about 4 minutes to about 10 minutes, etc., more specifically, for example, about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, etc., and is preferably about 5 minutes.

[0034] Another aspect of the present invention relates to an apparatus for spraying a liquid containing thrombin from an oblique direction, comprising a spray nozzle and a stand. In the present invention, the "spray nozzle" may be any known one, and examples that can be used include, but are not limited to, the Bolheal (registered trademark) Spray Set (manufactured by Akita Sumitomo Bake Co., Ltd.) and the two-liquid mixing set attached with a spray tip included in the Beriplast P Combi Set (registered trademark) for tissue adhesion (manufactured by CSL Behring).

[0035] In the present invention, the "stand" is used to support the spray nozzle for spraying thrombin, to determine the position of the spray nozzle, i.e., the spray start position of the tip of the spray nozzle and the angle of the spray nozzle, and to spray at a constant angle from that position onto the sheet-shaped cell culture. Here, the angle of the spray nozzle refers to the inclination of the spray nozzle with respect to the target to be sprayed, i.e., the plane including the sheet-shaped cell culture portion. In one embodiment, the stand is used after sterilization. The present invention will be described in detail below based on preferred embodiments with reference to the drawings. Note that the numerical values ​​in the drawings are for the preferred embodiments and should not be construed as being limited thereto.

[0036] FIG. 1 shows three views of the stand according to the present invention. In FIG. 1, the stand 1 is composed of a shaft holder 2, a support 3, a collar 4, a clamp lever 5, a plate 6, and a holder 7. FIG. 1A shows a side view of the stand 1 equipped with a spray nozzle 8. The height of the stand 1 can be changed depending on the size of the support 3, and the spray position of the spray nozzle can be adjusted to a preferred height in the manufacturing method of the laminate. The support 3 is fixed by the shaft holder 2, and is stably installed on the work table by the plate 6. A holder 7 is attached to the upper end of the support 3, and the holder fixes the spray nozzle 8. The angle of the spray nozzle 8 can be changed depending on the mounting angle of the holder 7 to the support 3, and the spray angle can be adjusted to a preferred angle in the manufacturing method of the laminate. FIG. 1B shows a front view of the stand 1. FIG. 1C shows a plan view of the stand 1 with the holder 7 and the support 3 removed. In FIG. 1C, a metal collar 4 and a clamp lever 5 are shown. In one embodiment, by manipulating the clamp lever 5, the shaft holder 2 can be loosened or tightened, thereby allowing the post 3 to be attached or detached.

[0037] FIG. 2 shows an enlarged four-sided view of the holder 7 in the stand 1 shown in FIG. 1. As shown in FIG. 2A and FIG. 2B, the holder 7 has a suitable shape for fixing the spray nozzle, and in an embodiment using, for example, the spray tip of Bolheal (registered trademark) Spray Set (manufactured by Akita Sumitomo Bake Co., Ltd.) or Beriplast P Combi Set (registered trademark) for tissue adhesion (manufactured by CSL Behring Co., Ltd.), the shape is such that the opening size becomes smaller as it advances to the tip. In addition, the slit 71 in the plan view of FIG. 2B allows an airline for supplying gas to the spray nozzle to pass when the above-mentioned spray nozzle is used. FIG. 2C and FIG. 2D show a side view and a front view of the holder 7. In each figure, an opening 72 for connecting the support 3 is shown, and the angle of the holder, i.e., the angle of the spray nozzle fixed to the holder, can be adjusted by the angle of the opening relative to the holder body.

[0038] When using the stand 1, the support 3 and holder 7 are adjusted in advance so that they are at a height and angle suitable for use in the laminate manufacturing method, and in this state the spray nozzle 8 is fixed to the holder 7 so that the airline fits into the slit section 71 of the holder 7. By using the stand of the present invention, the operator can continue spraying under constant conditions such as spray height and angle, reducing the need for skill to perform spraying work. In addition, by fixing the spray nozzle, one hand is freed, making it possible for a single operator to spray the thrombin solution while rotating the sheet-shaped cell culture. The material constituting the stand 1 is not particularly limited, but is preferably a sterilizable material. Examples of the sterilizable material include, but are not limited to, stainless steel, polypropylene, polyvinylidene fluoride, tetrafluoroethylene-ethylene copolymer resin, etc. Preferably, the sterilizable material is stainless steel.

[0039] In one embodiment, the apparatus further comprises a turntable. In the present invention, the "turntable" refers to an apparatus having a top plate on which a culture vessel containing a sheet-shaped cell culture can be placed and capable of rotating the top plate at a constant angular velocity. In one embodiment, the turntable is an electrically powered one that continues to rotate automatically once settings are input. Fig. 3 is a schematic diagram of an apparatus including a rotating table. For ease of explanation, the size and positional relationship of each member in Fig. 3 are appropriately emphasized, and the size and positional relationship of each member shown in the figure do not represent the actual size and positional relationship.

[0040] As shown in Fig. 3, in this device, liquid is sprayed from a spray nozzle 8 fixed to a stand 1 onto a sample rotating on a turntable 9. In this embodiment, the rotation of the sheet-shaped cell culture and support of the spray nozzle are covered when spraying the thrombin liquid in the above-mentioned laminate manufacturing method, so that an operator can carry out the spraying step simply by pushing in the syringe of thrombin liquid. The positional relationship between the stand 1 and the turntable 9 is preferably such that the center of the sample on the turntable 9 is located at the extension of the tip of the spray nozzle 8 supported by the stand 1. The stand 1 and the turntable 9 may be physically connected, or may be separate. EXAMPLES

[0041] Example 1. Manufacturing of laminates Skeletal myoblasts (CD56 positive cells) cryopreserved in a cell freezing storage solution (MCDB medium containing 10% DMSO) were thawed at 37°C and washed twice with physiological buffer containing 0.5% serum albumin. The washed cells (6.0 × 10 7 The cells were suspended in 20% human serum-containing DMEM medium (10 mL) and seeded on a 10 cm diameter cell culture dish (UpCell (registered trademark) 10 cm dish, CS3005, manufactured by CellSeed). After seeding, the cells were incubated at 37°C, 5% CO 2 The cells were cultured for 20 hours in an incubator (BNA-121D, Espec Corp.) set at 4°C. After culturing, the culture dish was removed from the incubator, and it was confirmed that the sheet-like cell culture had adhered to and covered the entire bottom surface of the culture dish, and the medium was discarded. The sheet-like cell culture was then detached from the culture dish by temperature treatment (standing at room temperature (20-25°C) for 5-30 minutes) and pipetting. The obtained sheet-like cell culture measured 47 mm x 47 mm in size.

[0042] The culture medium in the culture dish was removed, and the sheet-like cell culture was shaped as necessary. Then, 500 μL of fibrinogen solution (the content of vial 1 (freeze-dried fibrinogen powder) of Bolheal (registered trademark) for tissue adhesion (manufactured by Teijin Pharma) was dissolved in the content of vial 2 (fibrinogen dissolving solution), fibrinogen concentration 80 mg / mL, same below) was dripped onto the sheet-like cell culture using a two-liquid mixing set (with an apply nozzle approximately 6 cm long and approximately 1 mm in inner diameter, manufactured by Nipro) of the preparation set provided with Bolheal (registered trademark) for tissue adhesion. Next, 800 μL of thrombin solution (contents of vial 3 (lyophilized thrombin powder) of Bolheal (registered trademark) for tissue adhesion (manufactured by Teijin Pharma) dissolved in contents of vial 4 (thrombin solution), thrombin concentration 250 units / mL, same below) was sprayed at a pressure of 0.03 MPa using a Bolheal (registered trademark) spray set (manufactured by Akita Sumitomo Bake Co., Ltd.) with the spray nozzle approximately 7 cm away from the cell sheet. The sheet-like cell culture contracted when detached from the culture dish and became smaller than the bottom surface of the culture dish, so it is estimated that approximately 500 μL of the 800 μL of thrombin solution sprayed adhered to the sheet-like cell culture. The estimated amount of adhered thrombin liquid was calculated by actually spraying a predetermined amount of thrombin liquid (800 μL in this case) using a predetermined spray (Bolheal (registered trademark) spray set in this case), spray pressure (0.03 MPa in this case), height (approximately 7 cm in this case), and spray angle (45° in this case), measuring the weight of the thrombin liquid adhered within a predetermined range (within a circle with a diameter of approximately 45 mm in this case) using an electronic balance, and dividing the weight by the density of the thrombin liquid.

[0043] A fibrin gel is formed by the reaction of dropping the fibrinogen solution and spraying the thrombin solution. After standing for about 5 minutes, 24 mL of Hank's balanced salt solution (HBSS(+), Cat No. 14025, Life Technologies, the same applies below) was added to the culture dish and immediately removed, washing the culture dish containing the sheet-shaped cell culture. This allows the removal of unreacted fibrinogen solution and thrombin solution. Next, 24 mL of Hank's balanced salt solution was added again to the culture dish and left to stand for about 15 minutes, after which the solution in the culture dish was removed, and the fibrin gel that had solidified outside the sheet-shaped cell culture was trimmed with a scalpel to isolate a laminate of the fibrin gel and the sheet-shaped culture (Laminate 1). When the cross section of the isolated laminate was examined, it was confirmed that the fibrin gel was formed only on the upper surface of the sheet-shaped cell culture (upside down in FIG. 4), and that the thickness of the gel was uniform without unevenness (FIG. 4).

[0044] For comparison, an attempt was made to manufacture a laminate of fibrin gel and a sheet-shaped cell culture (comparative laminate 1) by dropping a thrombin solution and then a fibrinogen solution in that order. A sheet-like cell culture was formed in the same manner as in the laminate 1, and peeled off from the culture dish. After removing the culture solution in the culture dish and shaping the sheet-like cell culture as necessary, 800 μL of fibrinogen solution and 800 μL of thrombin solution were dropped onto the sheet-like cell culture in order using a two-liquid mixing set (with an application nozzle of about 6 cm in length and about 1 mm in inner diameter, manufactured by Nipro Corporation) of a preparation set attached to Bolheal (registered trademark) tissue adhesion. After standing for about 5 minutes, 24 mL of Hanks' balanced salt solution was added to the culture dish and immediately removed to wash the culture dish containing the sheet-like cell culture. Next, 24 mL of Hanks' balanced salt solution was added again to the culture dish and left for about 15 minutes, and the solution in the culture dish was removed. When the cross section was observed, it was confirmed that fibrin gel was formed not only on the upper surface of the sheet-like cell culture but also on the lower surface (upside down in FIG. 5). Furthermore, the fibrin gel layer did not have a uniform gel thickness, and operability was reduced (FIG. 5).

[0045] Similarly, for comparison, attempts were made to manufacture a laminate of fibrin gel and sheet-shaped cell culture by dripping fibrinogen solution and then thrombin solution (Comparative Laminate 2), a laminate sprayed with both fibrinogen solution and thrombin (Comparative Laminate 3), and a laminate sprayed with fibrinogen solution after dripping thrombin solution (Comparative Laminate 4). The manufacture was carried out in the same manner as the manufacture of Comparative Laminate 1, except for the dripping and / or spraying of each liquid and the order in which they were applied. Comparative laminate 2 was a thick laminate exceeding 1300 μm and formed a hard gel, which was not suitable for transplantation. Comparative laminate 3 had the fibrin gel peeled off from the sheet-shaped cell culture and did not form a laminate. Comparative laminate 4 had a fibrin gel layer formed in patches, the back side of the layer was not solidified, and did not form a laminate.

[0046] Example 2. Evaluation of laminates Using the same procedure as for manufacturing laminate 1, laminate 2 was manufactured with a fibrinogen liquid drip amount of 300 μL and a thrombin liquid spray amount of approximately 600 μL (estimated adhesion amount of approximately 300 μL), laminate 3 with a fibrinogen liquid drip amount of 300 μL and a thrombin liquid spray amount of 900 μL (estimated adhesion amount of approximately 450 μL), laminate 4 with a fibrinogen liquid drip amount of 300 μL and a thrombin liquid spray amount of 300 μL (estimated adhesion amount of approximately 100 μL), and laminate 5 with a fibrinogen liquid drip amount of 500 μL and a thrombin liquid spray amount of 500 μL (estimated adhesion amount of approximately 180 μL).

[0047] The size, weight, strength, thickness and properties of the laminate 1 and laminates 2 to 5 obtained in Example 1 were evaluated. The size was measured using a ruler, the weight was measured using an electronic non-automatic scale (AT201, manufactured by Mettler-Toledo), and the thickness was measured using a dial thickness gauge (SM-124, manufactured by Techlock). The strength was measured by scooping up the laminate in a state where it was stretched in the liquid with a stainless steel intestinal spatula (width 45 mm, the same applies below), placing the laminate outside the liquid with the laminate attached to the surface of the intestinal spatula, penetrating the laminate from the bottom to the top, connecting both ends to a gauge (general-purpose digital force gauge, FGC-1B, manufactured by Nidec-Shimpo), pulling it horizontally, and measuring the maximum load (tensile breaking load) until the laminate broke. The strength was measured at three different points of the laminate (n=3). The properties were qualitatively evaluated based on the state observed when the laminate was transferred to the side of a bottle resembling a heart.

[0048] [Table 1] The results in Table 1 make it clear that while there is not much difference in strength due to the mixing ratio, the mixing ratio does cause differences in properties such as weight, hardness, and viscosity. In addition, based on the ease of transferring the laminate to a bottle modeled after a heart (ease of placing it on the spatula, how easily it does not fall off the spatula when moved, and how easily it can be transferred from the spatula to the bottle) and the state of the laminate during the operation (wrinkling, tearing, etc.), a mixing ratio of 1:1 provided the best operability. [Industrial Applicability]

[0049] Use of the laminate according to the present invention can reduce the risk of damage when the produced sheet-shaped cell culture is peeled off from the culture substrate and when it is moved to the transplant site, etc., and can also simplify the step of adhering the sheet-shaped cell culture at the transplant site. By providing such a sheet-shaped cell culture with high operability, regenerative medicine using sheet-shaped cell cultures can become more widely used. [Explanation of symbols]

[0050] 1 Stand 2 Shaft holder 3 pillars 4 Color 5 Clamp lever 6 Plate 7 Holder 71 Slit 72 Opening 8 Spray Nozzle 9 Rotating table

Claims

1. A three-layer laminate consisting of an extracellular matrix, a sheet-shaped cell culture, and a fibrin gel in this order, comprising: (i) culturing cells on a cell culture substrate having a stimuli-responsive material to produce a sheet-shaped cell culture having an extracellular matrix layer formed on the lower surface; (ii) dripping a liquid containing fibrinogen onto the upper surface of the sheet-shaped cell culture; (iii) spraying the liquid containing thrombin from an oblique direction onto the sheet-shaped cell culture using a spray nozzle for spraying the liquid containing thrombin and a stand for supporting the spray nozzle, wherein the stand includes a holder for fixing the angle of the spray nozzle and a support for determining the height of the holder; (iv) forming a fibrin gel layer on the upper surface of the sheet-shaped cell culture by a reaction between the fibrinogen and the thrombin; The laminate produced by a method comprising the steps of:

2. 1. A method for producing a laminate, comprising: (i) culturing cells on a cell culture substrate having a stimuli-responsive material to produce a sheet-shaped cell culture having an extracellular matrix layer formed on the lower surface; (ii) dripping a liquid containing fibrinogen onto the upper surface of the sheet-shaped cell culture; (iii) spraying the liquid containing thrombin from an oblique direction onto the sheet-shaped cell culture using a spray nozzle for spraying the liquid containing thrombin and a stand for supporting the spray nozzle, wherein the stand includes a holder for fixing the angle of the spray nozzle and a support for determining the height of the holder; (iv) forming a fibrin gel layer on the upper surface of the sheet-shaped cell culture by a reaction between the fibrinogen and the thrombin; The method for producing a three-layer laminate consisting of an extracellular matrix, a sheet-like cell culture, and a fibrin gel in that order, comprises the steps of:

3. The method according to claim 2, wherein the liquid containing thrombin is sprayed at a constant angle onto the sheet-shaped cell culture by fixing the angle of the spray nozzle.

4. The method according to claim 2 or 3, further comprising rotating the sheet-shaped cell culture in step (iii).

5. The method according to claim 4, wherein the sheet-shaped cell culture is rotated using a rotating platform that is physically connected to the stand.

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