JP2019047819A - Production method for laminated body of sheet-like cell culture and fibrin gel - Google Patents

Abstract

To provide a laminated body of sheet-like cell culture which is excellent in operativity and suitable for transplantation.SOLUTION: The invention provides a method for producing a laminated body of a fibrin gel and sheet-like cell culture comprising dropping a liquid containing fibrinogen on the upper face of the sheet-like cell culture, spraying a liquid containing a thrombin to the face, and forming a layer of a fibrin gel on the face by the reaction of the fibrinogen and the thrombin; and a laminated body of the fibrin gel and the sheet-like cell culture produced by the method.SELECTED DRAWING: None

Description

  The present invention provides a method for producing a laminate of a sheet-like cell culture and a fibrin gel, a laminate produced by the production method, a pharmaceutical composition containing the laminate, a method for treating a disease using the laminate, etc. About.

  In recent years, attempts have been made to implant various cells for the repair of damaged tissues and the like. For example, use of fetal cardiac muscle cells, skeletal myoblasts, mesenchymal stem cells, cardiac stem cells, ES cells, etc. is attempted to repair myocardial tissue damaged by ischemic heart disease such as angina pectoris or myocardial infarction. (Non-Patent Document 1).

As part of such an attempt, a cell structure formed using a scaffold and a sheet-like cell culture in which cells are formed into a sheet have been developed (Patent Document 1).
For therapeutic application of sheet-like cell cultures, use of cultured epidermal sheet for skin damage caused by burns, use of corneal epithelial sheet-like cell culture for corneal damage, oral mucosal sheet for endoscopic resection of esophageal cancer Studies are focused on regenerative medicine, such as the use of cell cultures.

  Thus, although sheet-like cell cultures have high utility in regenerative medicine and the like, they are generally fragile as they are, and they are prone to wrinkles and breaks during isolation from culture substrates and during subsequent operations. Operations such as transfer, storage and transplantation are extremely difficult. In order to ameliorate such problems, the sheet-like cell culture is simultaneously sprayed with a fibrinogen solution and a thrombin solution to form a support layer containing fibrin on the sheet-like cell culture, and the sheet-like cell culture and fibrin are formed. The method of manufacturing the laminated body with is tried (patent document 2).

Japanese Patent Application Publication No. 2007-528755 JP 2011-172925 A

Haraguchi et al., Stem Cells Trans. Med. 2012 Feb; 1 (2): 136-41

  In the subsequent research, regarding the laminate of the sheet-like cell culture described in Patent Document 2 and fibrin, when the laminate is isolated from the culture substrate, the laminate may be broken, or the isolation may not be completed. It was found that there are cases that are possible, cases in which the support layer is detached from the sheet-like cell culture, and cases in which a complete laminate can not be obtained. It has also been found that thickening the support layer to ameliorate such problems can result in the inhibition of cell function. Therefore, the present invention is a laminate of a sheet-like cell culture which is excellent in operability and suitable for transplantation, does not have these problems, a method for producing the same, a pharmaceutical composition comprising the laminate, and the laminate To provide a method of treating a disease using

  The inventors of the present invention undertook intensive research to solve the above problems, and first, when forming a layer of fibrin gel on a sheet-like cell culture, first drop a liquid containing fibrinogen on the sheet-like cell culture. Then, by spraying a liquid containing thrombin, the layer of fibrin gel formed by the reaction of fibrinogen and thrombin firmly adheres to the sheet-like cell culture, and thus the fibrin gel and sheet-like cells thus obtained In isolation of the laminate with the culture from the container, it was found that the layer of fibrin gel did not peel off from the sheet-like cell culture, and completed the present invention.

That is, the present invention relates to the following.
(1) A step of dropping a liquid containing fibrinogen on the upper surface of a sheet-like cell culture, a step of spraying a liquid containing thrombin on the surface, and forming a fibrin gel layer on the surface by the reaction of fibrinogen and thrombin And producing a laminate of fibrin gel and sheet-like cell culture.
(2) The method according to (1) above, which further comprises the step of washing the laminate after the step of forming a layer of fibrin gel.
(3) The method according to (1) or (2) above, which further comprises the step of trimming excess fibrin gel after the step of forming a layer of fibrin gel.
(4) A laminate of a fibrin gel and a sheet-like cell culture produced by the method according to any one of the above (1) to (3).
(5) Pharmaceutical composition containing the laminated body as described in said (4).
(6) The pharmaceutical composition according to (5) above, for treating a disease associated with a tissue abnormality.
(7) A method of treating a disease associated with tissue abnormality in a subject, which comprises an effective amount of the laminate according to (4) above or the pharmaceutical composition according to (5) or (6) above, Administering to a subject in need thereof.

  The laminate manufactured by the method of the present invention is high in strength, excellent in operability, easy to apply to the affected area, and small in operational difference depending on the skill level of the practitioner. Not only is this possible, but the spread and spread of regenerative medicine and the like using the laminate can be expected. In addition, since the laminate of the present invention is formed of a biocompatible component, it can be implanted as it is into a graft site and the fibrin gel can be decomposed in vivo, so that re-operation or sheet-like The convenience of the sheet-like cell culture is greatly enhanced, as the use of a dedicated jig for transplantation of the cell culture becomes unnecessary.

FIG. 1 is a photograph showing a sheet-like cell culture in a culture dish. FIG. 2 is a photographic view showing a dropping operation of a fibrinogen solution onto a sheet-like cell culture. FIG. 3 is a photographic view showing the operation of spraying a thrombin solution onto a sheet-like cell culture. FIG. 4 is a photograph showing a trimming operation of a fibrin gel coagulated outside of a sheet-like cell culture. FIG. 5 is a photograph showing a sheet-like cell culture after simultaneous spraying of a fibrinogen solution and a thrombin solution. FIG. 6 is a photographic view showing a situation in which isolation of a laminate of a sheet-like cell culture and a fibrin gel obtained by simultaneous spraying of a fibrinogen solution and a thrombin solution has failed.

FIG. 7 is a photographic view showing the laminated body of the present invention placed on the intestinal folds. FIG. 8 is a photographic view showing a laminate having the reinforcing portion of the present invention. Black arrows indicate reinforcements. It can also be seen that the suture penetrates the center of the reinforcement. FIG. 9 is a photographic view showing the operation of applying a suture to the laminate of the present invention placed on the intestinal folds. FIG. 10 is a photographic view showing the operation of transferring the laminate of the present invention from the intestine to the transplant site. FIG. 11 is a photographic view showing the operation of transferring the laminate of the present invention from the enterella to the transplant site. FIG. 12 is a photographic view showing a state in which the suture is detached from the laminate of the present invention transferred to the implantation site.

FIG. 13 is a photographic view showing the situation in which the laminate of the present invention is re-stitched to the implantation site. FIG. 14 is a photographic view showing the operation of transferring the laminate of the present invention to the intestine. FIG. 15 is a photographic view showing an operation of applying a suture to the peripheral portion of the laminate of the present invention. FIG. 16 is a photographic view showing an operation of applying a suture to the peripheral portion of the laminate of the present invention. FIG. 17 is a photographic view showing an operation of applying a suture to the peripheral portion of the laminate of the present invention. FIG. 18 is a photograph showing a state in which the laminate of the present invention has been transferred to the side of the bottle.

  Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications referenced herein, including the information available from the Internet, are hereby incorporated by reference in their entirety.

  One aspect of the present invention is a step of dropping a liquid containing fibrinogen on the upper surface of a sheet-like cell culture, a step of spraying a liquid containing thrombin on the surface, and a fibrin gel by the reaction of fibrinogen and thrombin on the surface. Forming a layer of fibrin gel and sheet-like cell culture.

  In the present invention, “sheet-like cell culture” refers to cells in which cells are linked to each other to form a sheet. The cells may be linked to each other directly (including via cell components such as adhesion molecules) and / or via an intermediary substance. The mediator is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples include extracellular matrix and the like. The mediator is preferably of cell origin, in particular of the cells constituting the cell culture. The cells are at least physically (mechanically) linked, but may be further functionally linked, for example, chemically or electrically. The sheet-like cell culture is composed of one cell layer (monolayer), but is composed of two or more cell layers (laminate (multilayer), for example, two layers, three layers, 4 Layers, five layers, six layers, etc.).

  The sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within their surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride (eg, Membranes and the like made of PVDF) are known, but the sheet-like cell culture in the present invention may be able to maintain its physical integrity even without such scaffolds. In addition, the sheet-like cell culture preferably comprises only the substance derived from cells constituting the cell culture, and does not contain any other substance.

  The cells constituting the sheet-like cell culture are not particularly limited as long as they can form a sheet-like cell culture, and include, for example, adherent cells (adherent cells). The adherent cells are, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, hepatocytes, pancreatic cells, pancreatic cells, renal cells, adrenal cells, periodontal ligament cells, gingival cells, periosteal cells, skin) Cells, synoviocytes, chondrocytes etc. and stem cells (eg myoblasts, tissue stem cells such as cardiac stem cells, embryonic stem cells, pluripotent stem cells such as induced pluripotent stem cells, mesenchymal stem cells etc) And so on. Somatic cells may be differentiated from stem cells, in particular iPS cells. Non-limiting examples of cells constituting the sheet-like cell culture include, for example, myoblasts (eg, skeletal myoblasts etc.), mesenchymal stem cells (eg, bone marrow, adipose tissue, peripheral blood, skin, hair root, Muscle tissue, endometrium, placenta, umbilical cord blood, etc.), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synoviocytes, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells) , Retinal pigment epithelial cells, nasal mucosal epithelial cells, etc., endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, hepatic parenchymal cells), pancreatic cells (eg, pancreatic islet cells), renal cells, adrenal cells Periodontal ligament cells, gingival cells, periosteum cells, skin cells and the like.

  The cells that make up the sheet-like cell culture can be derived from any organism that can be treated by the sheet-like cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. Moreover, although the cell which comprises a sheet-like cell culture thing may be only one type, 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 a sheet-like cell culture, the ratio (purity) of the largest number of cells is 60% or more, preferably 70% at the end of production of sheet-like cell culture. % Or more, more preferably 75% or more.

  The cells forming the sheet-like cell culture may be xenogeneic cells or allogeneic cells. Here, "heterologous cell" means a cell derived from an organism of a species different from that of the recipient when a sheet-like cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. Also, "allogeneic derived cells" mean cells derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie cells derived from a recipient and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present invention because no rejection occurs upon transplantation. However, it is also possible to use heterologous cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, ie, xenogeneic cells and allogeneic nonautologous cells may be collectively referred to as nonautologous cells. In one aspect of the invention, the cells are autologous cells or allogeneic cells. In one aspect of the invention, the cells are autologous cells. In another aspect of the invention, the cells are allogeneic cells.

  The sheet-like cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, JP 2010-081829, JP 2011-110368, etc.). The method for producing a sheet-like cell culture typically comprises: seeding cells on a culture substrate; sheeting the seeded cells; and isolating the formed sheet-like cell culture from the culture substrate Including but not limited to. Prior to seeding the cells onto the culture substrate, the steps of freezing the cells and thawing the cells may be performed. Furthermore, the step of washing the cells may be performed after the step of thawing the cells. Each of these steps can be performed in any known manner suitable for the production of sheet cell cultures. The manufacturing method of the present invention may further include the step of manufacturing a sheet-like cell culture, in which case the step of manufacturing the sheet-like cell culture may include one or more of the above-described substeps.

The liquid containing fibrinogen (hereinafter sometimes referred to as fibrinogen solution) is not particularly limited as long as it can react with thrombin to form a fibrin gel, and fibrinogen, 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, 50 mg / mL to 90 mg / mL and the like. The solvent of the fibrinogen solution is typically water. Fibrinogen solution is a component such as factor XIII, aprotinin, serum albumin, glycine, L-arginine hydrochloride, L-isoleucine, L-glutamate sodium, D-mannitol, sodium citrate hydrate, sodium chloride, etc. in addition to fibrinogen May be included. Fibrinogen solutions are commercially available or can be prepared according to known procedures. Examples of commercially available fibrinogen solutions include, but are not limited to, for example, the contents of vial 1 of Borheal ^{(R) for} tissue adhesion (manufactured by Teijin Pharma) (Fibrinogen freeze-dried powder) and the contents of vial 2 (Fibrinogen solution) And those obtained by dissolving the contents (fibrinogen powder) of the vial 1 for Beriplast ^(R) Combiset tissue adhesion (manufactured by CSL Behring Co., Ltd.) with the contents of the vial 2 (aprotinin solution).

The dripping of the fibrinogen solution can be performed using any known method, for example, a syringe or a pipette. As a syringe, for example, a syringe without a needle having a volume 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 mixture of a preparation set attached for Borheal ^(R) tissue adhesion A set (with an application nozzle with an inner diameter of about 1 mm, manufactured by Nipro), a two-liquid mixing set with a beriplast preparation set (with an application nozzle, manufactured by Nipro), or the like can be used. Dropping amount of fibrinogen solution is not particularly limited as long covering the upper surface of the sheet-like cell culture, for example, about 6 [mu] L / cm ² ~ about 70 [mu] L / cm ^2, about 9 [mu] L / cm ² ~ about 50 [mu] L / cm ^2, about 12μL / cm ² ~ about 45 [mu] L / cm ^2, from about 15 [mu] L / cm ² ~ about 40 [mu] L / cm ^2, may be about 18 [mu] L / cm ² ~ about 32 [mu] L / cm ² and the like. As a non-limiting example of the dripping amount of the fibrinogen solution, 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, for a sheet cell culture of 45 mm in diameter -About 500 microliters etc. are mentioned. The particle size of the droplets when the fibrinogen solution is dropped is not particularly limited. For example, the diameter of the droplets attached to the sheet-like cell culture after dropping is in the range of about 0.2 cm to about 2.0 cm. You may Also, the weight of the droplets may be, for example, in the range of about 10 mg to about 100 mg, about 15 mg to about 50 mg, about 20 mg to about 30 mg, etc. without limitation. The particle size and weight of the droplet can be appropriately adjusted by the presence or absence of the attachment of the needle to the syringe, the number of gauges of the attached needle, the selection of the shape of the needle tip, and the like.

The liquid containing thrombin (hereinafter sometimes referred to as thrombin liquid) is not particularly limited as long as it can react with fibrinogen to form a fibrin gel, for example, 1 unit / mL to 10000 units of thrombin. / ML, 10 units / mL to 5000 units / mL, 25 units / mL to 2500 units / mL, 50 units / mL to 1000 units / mL, 100 units / mL to 500 units / mL, 250 units / mL to 300 units Examples include liquids containing concentrations such as / 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. Thrombin solutions are commercially available or can be prepared according to known procedures. Examples of commercially available thrombin solutions include, but are not limited to, for example, contents of vial 3 (Thrombin lyophilized powder) for Borheal ^(R) tissue adhesion (manufactured by Teijin Pharma Co., Ltd.) and contents of vial 4 (thrombin solution) And those obtained by dissolving the contents (thrombin powder) of the vial 3 for Beriplast ^(R) Combiset tissue adhesion (manufactured by CSL Behring Co., Ltd.) with the contents of the vial 4 (calcium chloride solution).

The spraying of the thrombin solution can be performed using any known method, such as a spray. As a non-limiting example of spray, a spray tip was attached in a two-liquid mixing set attached to Borheal ^(R) spray set (made by Akita Sumitomo Bake Co., Ltd.) and Beriplast ^(R) combination set for tissue adhesion (made by CSL Behring) The thing is mentioned. The spray amount of the thrombin solution is not particularly limited as long as the top surface of the sheet-like cell culture can be coated, and the estimated adhesion amount to the sheet-like cell culture is, for example, about 3 μL / cm ² to about 70 μL / cm ² , about 5 μL / cm ² ~ about 50 [mu] L / cm ^2, about 6 [mu] L / cm ² ~ about 45 [mu] L / cm ^2, from about 12 [mu] L / cm ² ~ about 40 [mu] L / cm ^2, may be about 18 [mu] L / cm ² ~ about 32 [mu] L / cm ^2, etc. . As a non-limiting example of the spray amount of the thrombin solution, 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 as estimated adhesion to a 45-mm diameter sheet-like cell culture And about 300 μL to about 500 μL and the like. The estimated amount of adhesion to the sheet-like cell culture is within a predetermined range (for example, 45 mm in diameter) when a predetermined amount of thrombin solution is sprayed at an actual spray, height, spray pressure and spray angle. The weight of the liquid adhering to the circle) is measured and divided by the density of the thrombin liquid (0.999973 g / cm ³ ) to calculate. Based on the description herein, one of ordinary skill in the art can determine spray conditions that result in the desired estimated amount of adhesion of the thrombin solution without undue experimentation. For example, since the estimated adhesion amount with respect to the spray amount of 300 μL, 500 μL, 600 μL and 900 μL is described in Example 1 below as 100 μL, 180 μL, 300 μL and 450 μL respectively, the estimated adhesion amount by the least squares method An approximate curve of (μL) = spray amount (μL) × 0.6-88 (μL) is obtained, and based on this, it is possible to determine the spray amount that yields the desired estimated adhesion amount. The spray of the thrombin solution is not particularly limited as long as the thrombin solution can be uniformly attached to the upper surface, preferably the entire upper surface, of the sheet-like cell culture, for example, about 15 ° to about 15 from a height of about 2 cm to about 15 cm. It can be carried out at a pressure of about 0.005 MPa to about 0.1 MPa at a spray angle of 150 °.

  The ratio of the fibrinogen solution to the thrombin solution applied to the sheet-like cell culture is not particularly limited as long as it does not excessively inhibit the grafting operation of the resulting laminate, and for example, the amount of the fibrinogen solution dropped and the thrombin solution As a volume ratio to the estimated adhesion amount of 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: 1 to about It may be 1: 1, about 2: 1 to about 1: 1, about 1.5: 1 to about 1: 1, etc., in particular about 1: 1, and it is also possible to attach fibrinogen and thrombin to the laminate. As a ratio (mg: unit), 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, etc., particularly about 8:25.

  By adjusting the concentrations and amounts of the fibrinogen solution and the thrombin solution, it is possible to change the thickness and properties (flexibility, adhesiveness, etc.) of the resulting laminate. For example, the thickness of the laminate can be increased by increasing the amount of fibrinogen solution, and the volume ratio of the dripping amount of fibrinogen solution (80 mg / mL) to the estimated adhesion amount of thrombin solution (250 units / mL) The closer to 1: 1 the ratio, and the closer the ratio of fibrinogen to thrombin attached to the laminate (mg: unit) to 8:25, the more the laminate becomes more flexible and sticky, and the operability improves. .

  The step of forming a layer of fibrin gel can be performed without limitation, for example, by spraying a thrombin solution and then allowing the sheet-like cell culture to stand for a certain period of time. The standing period is not limited, 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., particularly about 5 minutes. You may

  After the step of forming the layer of fibrin gel, the step of washing the laminate may be performed. The wash may be a predetermined amount of wash solution, such as, but not limited to, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640). , MCDB (MCDB 102, 104, 107, 120, 131, 153, 199 etc.), L15, SkBM, RITC 80-7, DMEM / F 12 etc. etc. to the container that holds the stack, and discarding it can. The washing step can be performed once or twice or more. Also, even if the cleaning liquid added to the container is immediately discarded, it may be discarded for a predetermined period of time (for example, without limitation, about 1 minute to about 60 minutes, about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, etc. In particular, it may be discarded after standing for about 15 minutes.

After the step of forming the layer of fibrin gel (if the step of washing the laminate is included after the washing step), a step of trimming extra fibrin gel may be performed, if necessary. The trimming can be performed with any device that can be used to cut gelled material. As such a device, for example, scalpel, scissors, scalpel and the like can be mentioned without limitation. The fibrin gel may be trimmed to the contours of the sheet-like cell culture, or the fibrin gel may protrude around the sheet-like cell culture, ie the edges of the laminate consist only of the fibrin gel It may be trimmed to
The steps of dripping the liquid containing fibrinogen, spraying the liquid containing thrombin, forming a layer of fibrin gel, washing the laminate, and / or trimming excess fibrin gel It may be performed on sheet-like cell cultures that remain adhered to the substrate or on sheet-like cell cultures isolated from the culture substrate.

  In one aspect, the manufacturing method of the present invention performs all the steps in vitro. In another embodiment, the production method of the present invention comprises the steps performed in vivo, including, but not limited to, harvesting the cells or tissue from which the cells are sourced. In one aspect, the manufacturing method of the present invention is carried out under sterile conditions for all the steps. In one aspect, the production method of the present invention is performed such that the finally obtained laminate is substantially sterile. In one aspect, the production method of the present invention is performed such that the finally obtained laminate is sterile.

  Another aspect of the present invention relates to a laminate of sheet-like cell culture and fibrin gel produced by the production method of the present invention. The laminate of the present invention is, for example, compared to a laminate prepared by simultaneously spraying a fibrinogen solution and a thrombin solution onto a sheet-like cell culture described in Patent Document 2, to a sheet-like cell culture of a fibrin gel layer. It has a strong adhesive strength, and the fibrin gel layer does not peel off from the sheet-like cell culture during operation. While not wishing to be bound by any particular theory, in the laminates of the invention, fibrinogen penetrates into the sheet-like cell culture (e.g., intercellular space) before reacting with thrombin to form fibrin. Since the fibrin gel is sterically coupled to the sheet-like cell culture and anchored, and the contact area between the fibrin gel and the sheet-like cell culture becomes large, it is thought that the adhesion between the two becomes strong. On the other hand, in the laminate described in Patent Document 2, the fibrinogen gel and the thrombin simultaneously contact the sheet-like cell culture and react immediately to form a fibrin gel. It is considered that it is difficult to form such a steric bond. Therefore, the laminate of the present invention is considered to be qualitatively and structurally different from the laminate described in Patent Document 2.

The strength of the laminate is not limited, and is, for example, about 0.010 N or more, about 0.015 N or more, about 0.020 N or more, about 0.025 N or more, about 0 as measured by the method described in Example 2. .030 N or more, about 0.035 N or more, about 0.040 N or more, about 0.045 N or more, etc., and also about 0.010 N to about 0.200 N, about 0.015 N to about 0.100 N, about It may be in the range of 0.020 N to about 0.50 N or the like. In addition, the strength of the laminate is about 1.5 times or more, about 2 times or more, about 3 times or more, about 3 times or more that of the same sheet-like cell culture as that included in the laminate except that the fibrin gel is not laminated. 4 times or more, about 5 times or more, about 6 times or more, about 7 times or more, about 8 times or more, about 9 times or more, about 10 times or more, etc., and also about 1.5 times to about 20 times And may be in the range of about 2 times to about 15 times, about 2.5 times to about 10 times, and the like.
Further, the edge of the laminate may be composed of a laminate of a fibrin gel and a sheet-like cell culture, may be composed of only a fibrin gel, or may be composed of only a sheet-like cell culture.

Another aspect of the present invention relates to a laminate of a sheet-like cell culture having a reinforcing portion formed of fibrin gel and a fibrin gel (hereinafter sometimes referred to as a reinforcement laminate).
The reinforced laminate of the present invention has at least one reinforcing portion formed by partially laminating a fibrin gel on the fibrin gel layer of a laminate of a sheet-like cell culture and a fibrin gel. By forming the reinforcing portion, the strength of the necessary portion can be increased without increasing the thickness of the entire laminate. The reinforcing portion can be used, for example, as a suture thread insertion portion or a gripping portion by forceps or the like. The reinforcing portion is formed by dropping the fibrinogen solution and the thrombin solution on the fibrin gel layer, but various thicknesses and shapes can be obtained by adjusting the dropping amount, the dropping position, the dropping pattern, and the like. . For example, if 50 μL each of fibrinogen solution (80 mg / mL) and thrombin solution (250 units / mL) are dropped, reinforcements with a bottom area of about 1 cm ² × height of about 1 mm to ² mm are obtained. A reinforced portion with a base area of about 1 cm ² × height of about ² mm to ³ mm is obtained.

  The ratio between the fibrinogen solution and the thrombin solution to be dropped is not particularly limited as long as a reinforcing portion having a desired strength can be obtained, and for example, about 5: 1 to 5 as a volume ratio between the fibrinogen solution and the thrombin solution to be dropped. About 1: 3, about 4: 1 to about 1: 2, about 3: 1 to about 1: 1.5, about 2.5: 1 to about 1: 1, about 2: 1 to about 1: 1, about The ratio may be 1.5: 1 to about 1: 1, in particular about 1: 1, and the ratio of fibrinogen to thrombin (mg: unit) contained in the reinforcing part may be 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 , Etc., and in particular about 8:25.

  The strength of the reinforcing portion is not limited, and is, for example, about 0.04 N or more, about 0.05 N or more, about 0.06 N or more, about 0.07 N or more, or about 0 as measured by the method described in Example 2. .08 N or more, about 0.09 N or more, about 0.10 N or more, about 0.12 N or more, about 0.15 N or more, etc., and also about 0.04 N to about 0.50 N, about 0.05 N to about It may be in the range of about 0.40 N, about 0.06 N to about 0.30 N, about 0.07 N to about 0.25 N, and so on. In addition, the strength of the reinforced portion is about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 6 times, about 7 times, about 8 times or more, about 9 times or more, about 10 times or more, etc., and also about 1.5 times to about 25 times, about 2 times to about 20 times, about 3 times to about 15 times, about 4 times It may be in the range of, for example, about 10 times.

  Another aspect of the present invention is the steps of dropping a liquid containing fibrinogen onto the top surface of a sheet-like cell culture, spraying a liquid containing thrombin onto the surface, and reacting the surface with fibrinogen on the surface by means of the reaction between fibrinogen and thrombin. Forming a layer of gel; dripping a liquid containing thrombin and a liquid containing fibrinogen onto the layer of fibrin gel such that both liquids are mixed on the layer of fibrin gel; fibrinogen And a step of forming a reinforcement of fibrin gel on a fibrin gel layer by reaction with thrombin, a method of producing a laminate of a sheet-like cell culture having a reinforcement formed of fibrin gel and a fibrin gel About.

  Applying a liquid containing fibrinogen onto the top surface of the sheet-like cell culture, spraying a liquid containing thrombin onto the surface, forming a layer of fibrin gel on the surface by reacting fibrinogen and thrombin, and The liquid containing thrombin and the liquid containing fibrin are as described above for the method for producing a laminate of sheet-like cell culture and fibrin gel. The method for producing a reinforcing laminate described above comprises the step of forming a laminate of a sheet-like cell culture and a fibrin gel by the method of producing a laminate of a sheet-like cell culture and a fibrin gel, and thrombin. Dropping a liquid and a liquid containing fibrinogen onto the layer of the fibrin gel so that both liquids are mixed on the layer of the fibrin gel, and reacting the fibrinogen with thrombin on the fibrin gel layer Forming a reinforcement of the fibrin gel. Therefore, the method of manufacturing a reinforcing laminate as described above includes the steps of washing the laminate after the step of forming the layer of fibrin gel, and the steps of forming the layer of fibrin gel (if the step of cleaning the laminate is performed After the washing step) may include trimming the excess fibrin gel, as well as the steps of producing the sheet-like cell culture, and the various steps described for the method of producing a laminate described above .

  The dropping of the thrombin solution and the fibrinogen solution onto the layer of the fibrin gel may be either first or simultaneously. For example, the step of dropping the thrombin solution and the fibrinogen solution onto the layer of the fibrin gel is not limited, but (1) the thrombin solution is dropped onto the layer of the fibrin gel, and then the fibrinogen solution is dropped onto the dropped thrombin solution. (2) dripping fibrinogen solution on the layer of fibrin gel, then dropping thrombin solution on the dropped fibrinogen solution, and (3) thrombin solution and fibrinogen on layer of fibrin gel Including a step of simultaneously dropping the solution and the like.

The dropping of the thrombin solution and the fibrinogen solution onto the layer of fibrin gel can be performed using any known method such as a syringe or a pipette. As a syringe, for example, a syringe without a needle having a volume 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 mixture of a preparation set attached for Borheal ^(R) tissue adhesion A set (with an application nozzle with an inner diameter of about 1 mm, manufactured by Nipro), a two-liquid mixing set with a beriplast preparation set (with an application nozzle, manufactured by Nipro), or the like can be used. The particle size of the droplets when the thrombin solution or fibrinogen solution is dropped is not particularly limited. For example, after dropping, the diameter of the droplets attached to the sheet-like cell culture becomes about 0.2 cm to about 2.0 cm. It may be a range. Also, the weight of the droplets upon dropping the thrombin solution or fibrinogen solution may be, for example, in the range of about 10 mg to about 100 mg, about 15 mg to about 50 mg, about 20 mg to about 30 mg, etc. without limitation. The particle size and weight of the droplet can be appropriately adjusted by the presence or absence of the attachment of the needle to the syringe, the number of gauges of the attached needle, the selection of the shape of the needle tip, and the like. The dripping amounts of the thrombin solution and the fibrinogen solution, and the ratio of the thrombin solution and the fibrinogen solution to be dropped are as described above for the reinforcing laminate.

  The step of forming the reinforcing portion of fibrin gel on the fibrin gel layer is not limited, and can be performed, for example, by allowing the laminate to stand for a certain period of time after dropping a thrombin solution and a fibrinogen solution. The standing period is not limited, 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., particularly about 5 minutes. You may

  After the step of forming the fibrin gel reinforcement, a step of washing the reinforcement laminate can be performed. The wash may be a predetermined amount of wash solution, such as, but not limited to, water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640). , MCDB (MCDB 102, 104, 107, 120, 131, 153, 199 etc.), L15, SkBM, RITC 80-7, DMEM / F 12 etc. etc. to the container containing the reinforced laminate, and discarding. Can. The washing step can be performed once or twice or more. Also, even if the cleaning liquid added to the container is immediately discarded, it may be discarded for a predetermined period of time (for example, without limitation, about 1 minute to about 60 minutes, about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, etc. In particular, it may be discarded after standing for about 15 minutes.

  The laminates of the present invention (including reinforced laminates, and the same hereinafter) are useful for treating various diseases associated with tissue abnormalities. Thus, in one aspect, the laminate of the present invention is for use in the treatment of diseases associated with tissue abnormalities. The laminate of the present invention has a structure in which a layer of biocompatible fibrin gel is laminated on one side of a sheet-like cell culture, while the other side of the sheet-like cell culture is intact. Since the fibrin gel is degraded and eventually disappears in vivo, it can be applied to tissues and diseases that can be treated with a sheet-like cell culture. Tissues to be treated include, but are not limited to, for example, cardiac muscle, cornea, retina, esophagus, skin, joints, cartilage, liver, pancreas, gingiva, kidney, thyroid, skeletal muscle, middle ear and the like. In addition, the disease to be treated is not limited, for example, heart disease (for example, myocardial injury (myocardial infarction, cardiac trauma, etc.), cardiomyopathy (ischemic cardiomyopathy, dilated cardiomyopathy, diastolic phase fertilization) Etc.), corneal disease (eg, corneal epithelial stem cell exhaustion, corneal injury (heat and chemical corrosion), corneal ulcer, corneal opacification, corneal perforation, corneal scarring, Stevens-Johnson syndrome, ocular pemphigus) Etc.), retinal diseases (eg, retinitis pigmentosa, age-related macular degeneration, etc.), esophagus diseases (eg, prevention of inflammation / stenosis of the esophagus after esophagus surgery (esophageal cancer removal), etc.), skin diseases (eg, Skin damage (traumatic injury, burn injury etc.), joint disease (eg, osteoarthritis etc.), cartilage disease (eg, cartilage damage etc.), liver disease (eg, chronic liver disease etc.), pancreatic disease (eg, diabetes etc.) ), Dental diseases (eg Periodontal disease etc.), kidney disease (eg renal failure, renal anemia, renal osteodystrophy etc) thyroid disease (eg hypothyroidism etc) muscle disease (eg muscle damage, myositis etc) , Middle ear disease (eg, otitis media etc.).

  The usefulness of the sheet-like cell culture for the above-mentioned diseases is described, for example, in Patent Document 1, Non-patent Document 1, Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Ito et al. al., Tissue Eng. 2005 Mar-Apr; 11 (3-4): 489-96, Yaji et al., Biomaterials. 2009 Feb; 30 (5): 797-803, Yaguchi et al., Acta Otolaryngol. 2007 Oct; 127 (10): 1038-44, Watanabe et al., Transplantation. 2011 Apr 15; 91 (7): 700-6, Shimizu et al., Biomaterials. 2009 Oct; 30 (30): 5943-9, Ebihara et al., Biomaterials. 2012 May; 33 (15): 3846-51, Takagi et al., World J Gastroenterol. 2012 Oct 7; 18 (37): 5145-50, and the like.

  The laminate of the present invention can be applied to the tissue to be treated and used to repair or regenerate it, but as a source of physiologically active substances such as hormones, it can be used for other than the tissue to be treated. It can also be transplanted to a site (e.g., subcutaneous tissue) (e.g., Arauchi et al., Tissue Eng Part A. 2009 Dec; 15 (12): 3943-9, Shimizu et al., Biomaterials. 2009 Oct; 30. (30): 5943-9, etc.). Since the laminate of the present invention can be used for regenerative medicine, it can also be used as a product for regenerative medicine and the like. The laminate of the present invention can also be used as an implant.

  In one aspect, the laminate of the present invention is substantially sterile. In one aspect, the laminate of the present invention is sterile. In one aspect, the sheet-like cell culture contained in the laminate of the present invention is not genetically engineered. In another embodiment, the sheet-like cell culture contained in the laminate of the present invention is genetically engineered. Genetic engineering includes, but is not limited to, for example, introduction of genes that enhance the viability, engraftment ability, function, etc. of sheet-like cell cultures, and / or genes useful for treating diseases. As a gene to be introduced, for example, cytokine genes such as HGF gene and VEGF gene can be mentioned without limitation.

Another aspect of the present invention relates to a pharmaceutical composition comprising the laminate of the present invention.
In addition to the laminate of the present invention, the pharmaceutical composition of the present invention includes various additional components, such as pharmaceutically acceptable carriers, viability, engraftment and / or function of sheet-like cell cultures, etc. Ingredients to enhance the E. coli, other active ingredients useful for treating the target disease, and the like. As such additional components, any known ones can be used, and those skilled in the art are familiar with these additional components. In addition, the pharmaceutical composition of the present invention can be used in combination with a component that enhances the viability, engraftment and / or function of the sheet-like cell culture, and other active ingredients useful for the treatment of a target disease. . In one aspect, the pharmaceutical composition of the present invention is for use in the treatment of a disease associated with abnormal tissue. The tissues and diseases to be treated are as described above for the laminate of the present invention.

  Another aspect of the present invention is a method of treating a disease associated with abnormal tissue in a subject, which comprises administering an effective amount of the laminate or the pharmaceutical composition of the present invention to a subject in need thereof. On the method involved. The tissues and diseases targeted by the treatment method of the present invention are as described above for the laminate of the present invention. Further, in the treatment method of the present invention, the component of the present invention for enhancing the viability, engraftment and / or function of the sheet-like cell culture, other active components useful for the treatment of the target disease, etc. It can be used in combination with the body or the pharmaceutical composition.

  The treatment method of the present invention may further include the step of producing a laminate in accordance with the production method of the present invention. According to the treatment method of the present invention, the step of producing a sheet-like cell culture or the step of producing a sheet-like cell culture from a subject prior to the step of producing a laminate is performed. The method may further include the step of In one aspect, the subject from which the cell or tissue from which the cell is derived is harvested is the same individual as the subject undergoing treatment with the laminate or pharmaceutical composition. In another embodiment, the subject from which the cell or tissue from which the cell is harvested is harvested is a separate entity that is allogeneic to the subject being treated for administration of the laminate or pharmaceutical composition. In another embodiment, the subject from which the cell or tissue from which the cell is harvested is harvested is an individual that is xenogeneic to the subject being treated for administration of the laminate or pharmaceutical composition.

  In the present invention, the term "subject" means any living individual, preferably an animal, more preferably a mammal, more preferably a human. In the present invention, the subject may be healthy or may be suffering from any disease, but when treatment of a disease associated with tissue abnormality is intended, typically the disease is Means a subject who is suffering from or at risk of suffering from.

  Also, the term "treatment" is intended to encompass all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, temporary remission or prevention of disease. For example, the term "treatment" refers to medically acceptable for a variety of purposes, including delaying or halting the progression of a disease associated with tissue abnormalities, regression or disappearance of a lesion, prevention of the onset of the disease, or prevention of recurrence. Interventions are included.

  In the present invention, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of a disease, reducing the symptoms, or delaying or stopping the progression (for example, the size or weight of the laminate), preferably An amount that prevents the onset and recurrence of the disease or cures the disease. Also preferred is an amount that does not adversely affect the benefits of administration. Such amount can be determined as appropriate, for example, by tests in experimental animals such as mice, rats, dogs or pigs, or in disease model animals, and such test methods are well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important indicator for determining the effective dose.

  Methods of administration typically include direct application to tissues. Although the frequency of administration is typically once per treatment, multiple administrations can also be performed if the desired effect can not be obtained. When applied to tissue, the laminate or pharmaceutical composition of the present invention may be fixed to the tissue of interest by a locking means such as sutures or staples. When using a reinforcement laminate, the locking means can be applied to the reinforcement.

  The invention will be described in more detail with reference to the following examples, which illustrate specific embodiments of the invention and the invention is not limited thereto.

Example 1 Production of Laminate of Fibrin Gel and Sheet-Like Cell Culture Skeletal myoblasts (CD56-positive) cryopreserved in cell preservation medium (MCDB medium containing 10% DMSO) are thawed at 37 ° C. . Washed twice with physiological buffer containing 5% serum albumin. Washed cells 6.0 × 10 ⁷ cells are suspended in human serum containing 20% DMEM media (10 mL), were seeded in a cell culture dish with a diameter of 10cm ^(UpCell (R) 10cm dish, CS3005, manufactured by Cellseed Co.) . After seeding, the cells were cultured for 20 hours in an incubator (BNA-121D, manufactured by Espec Corp.) set at 37 ° C. and 5% CO ₂ . After the culture, the culture dish was removed from the incubator, and it was confirmed that the sheet-like cell culture was adhered so as to cover the entire bottom of the culture dish, and the culture medium was discarded. Thereafter, the sheet-like cell culture was detached from the culture dish by temperature treatment (standing at room temperature (20 to 25 ° C.) for 5 to 30 minutes) and pipetting. The resulting sheet-like cell culture was 47 mm × 47 mm in size.

After removing the culture solution in the culture dish and shaping the sheet-like cell culture as needed (FIG. 1), a fibrinogen solution (Borheal ^{(R) for} tissue adhesion on a sheet-like cell culture (manufactured by Teijin Pharma Co., Ltd.) Dissolve the contents of vial 1) (fibrinogen freeze-dried powder) with the contents of vial 2 (fibrinogen solution), fibrinogen concentration 80 mg / mL, same below) 500 μL, attached for Borheal ^(R) tissue adhesion The solution was dropped using a two-liquid mixing set (approximately 6 cm in length, with an apply nozzle with an inner diameter of approximately 1 mm, manufactured by Nipro Corporation) of the preparation set (Fig. 2). Then, the contents of a vial 3 (thrombin freeze-dried powder) of a thrombin solution (Borheal ^{(R) for} tissue adhesion (manufactured by Teijin Pharma Ltd.) dissolved in the contents of a vial 4 (thrombin solution), thrombin concentration 250 units The spray nozzle was sprayed about 7 cm away from the cell sheet at a pressure of 0.03 MPa (FIG. 3), using 800 μL of the following ^(the same applies hereinafter), Borheal ^(R) spray set (manufactured by Akita Sumitomo Bake Co., Ltd.). Since the sheet-like cell culture shrinks by peeling from the culture dish and becomes smaller than the bottom of the culture dish (FIG. 1), about 500 μL of the sprayed 800 μL thrombin solution adheres on the sheet-like cell culture It is estimated that The estimated adhesion amount of the thrombin solution is a predetermined amount of thrombin solution (800 μL in this case), a predetermined spray (in the case of this example, Volheal ^(R) spray set), and a spray pressure (in the case of this example) 0. 03MPa), height (about 7 cm in the case of this example), spray angle (45 ° in the case of this example) actually, within a predetermined range (in the case of this example, within a circle of about 45 mm in diameter) The weight of the thrombin solution attached thereto was measured by an electronic balance and calculated by dividing by the density of the thrombin solution.

  The reaction between the fibrinogen solution and the thrombin solution forms a fibrin gel. After standing for about 5 minutes, add 24 mL of Hanks Balanced Salt Solution (HBSS (+), Cat No. 14025, Life Technologies, same hereafter) to the culture dish and immediately remove the sheet-like cell culture by removing it. The containing culture dishes were washed. Thereby, unreacted fibrinogen solution and thrombin solution can be removed. Next, 24 mL of Hanks Balanced Salt Solution is again added to the culture dish, and after standing for about 15 minutes, the solution in the culture dish is removed, and the fibrin gel coagulated outside the sheet-like cell culture is trimmed with a scalpel (Figure 4) A laminate of fibrin gel and sheet-like culture was isolated (laminate 1). The isolated laminate was stored until use, with 24 mL of Hanks Balanced Salt Solution added to culture dishes.

  Follow the same procedure except that the dripping amount of fibrinogen solution is 300 μL and the spraying amount of thrombin solution is about 600 μL (estimated adhesion amount of about 300 μL), the dripping amount of fibrinogen solution is 300 μL, and the spraying amount of thrombin solution is about Follow the same procedure except using 900 μL (estimated adhesion amount of about 450 μL), the same procedure except using 300 μL of the amount of fibrinogen solution dropped and 300 μL of thrombin solution sprayed (about 100 μL of estimated adhesion amount) in the same procedure. A layered product 5 was manufactured in the same procedure as that of the layered product 4, except that the dropping amount of the fibrinogen solution was 500 μL and the spraying amount of the thrombin solution was 500 μL (the estimated adhesion amount was about 180 μL).

In addition, for comparison, manufacture of a laminate of a fibrin gel and a sheet-like cell culture was attempted by a method of simultaneously spraying a fibrinogen solution and a thrombin solution.
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 a fibrinogen solution and 800 μL of a thrombin solution on the sheet-like cell culture, Borheal ^(R) spray set (Akita Sumitomo The spray nozzle was sprayed about 7 cm away from the cell sheet and simultaneously sprayed at a pressure of 0.03 MPa (estimated adhesion amount to the sheet-like cell culture was about 500 μL each) using Bake Co., Ltd.). After standing for about 5 minutes, the culture dish containing the sheet-like cell culture was washed by adding 24 mL of Hanks Balanced Salt Solution to the culture dish and immediately removing it. Next, 24 mL of Hanks Balanced Salt Solution was again added to the culture dish and allowed to stand for about 15 minutes, after which the solution in the culture dish was removed (FIG. 5). The fibrin gel coagulated outside the sheet-like cell culture was trimmed with a scalpel, and an attempt was made to isolate a laminate of the fibrin gel and the sheet-like cell culture, but the fibrin gel peeled off from the sheet-like cell culture, A laminate could not be obtained (FIG. 6).

Example 2 Evaluation of Laminates of Fibrin Gel and Sheet-Like Cell Culture The size, weight, strength, thickness, operability and properties of laminates 1 to 5 obtained in Example 1 were evaluated. The size was measured by a ruler, and the weight was measured by an electronic non-automatic balance (AT 201, manufactured by Mettler-Toledo), and the thickness by a dial thickness gauge (SM-124, manufactured by Techlock). The strength was measured as follows. First, in a state in which the laminate was stretched in a liquid, it was scooped up with a stainless steel enterella (width 45 mm, the same applies hereinafter), and placed outside the liquid in a state where the laminate adhered to the surface of the intestine. A suture with a needle (6-0 proline) was inserted between the laminate and the intestinal fold and penetrated from the lower surface to the upper surface of the laminate. The two ends of the yarn are connected to form an annulus, which is connected to a gauge (general-purpose digital force gauge, FGC-1B, manufactured by Nidec-Shimpo). The yarn locked to the laminate was pulled horizontally via a gauge, and the maximum load (tensile breaking load) until breakage of the laminate was measured. The measurement was performed at three different places of the laminate (n = 3). For ease of operation, the stack in the liquid is placed on the intestinal folds (Figure 7), and this is transferred to the side of the heart-mocking bottle (easiness to be placed on the intestinal folds, the intestines during movement) Evaluate comprehensively on the basis of difficulty in falling from the tongue, ease of transfer from the intestines to the bottle, etc., and the state of the laminate (whether it is wrinkled or broken at the time of operation), etc. It was expressed in five steps of 5 to 1. The property qualitatively evaluated the state of the laminate observed in the evaluation of the operability. Further, for comparison, the same evaluation was performed on a sheet-like cell culture (made under the same conditions as the laminate 1) in which no fibrin gel was laminated. The results are shown in Table 1.

From the results in Table 1, it can be seen that all laminates show much higher strength than the sheet-like cell culture in which the fibrin gel is not laminated, and the weight, strength and thickness of the laminate are larger when the amount of fibrinogen solution is larger. It can be seen that it increases. In addition, simulated transfer operation was possible in any of the laminates, but the operability tended to improve as the volume ratio of the fibrinogen solution and the thrombin solution attached to the sheet-like cell culture was closer to 1: 1. .

Laminates 6 to 9 different in the number of cells, the number of laminated sheet-like cell cultures, and the like were prepared in the same manner as in the laminate 1 and were evaluated as described above. The results are shown in Table 2. In addition, the formation of the sheet-like cell culture was performed in a cell culture dish (UpCell ^(R) 3.5 cm dish, Cellseed Co., Ltd.) with a diameter of 3.5 cm. The dry weight of the laminate was obtained by measuring the freeze-dried laminate with an electronic balance. The moisture content was determined by the following equation: moisture content = (wet weight of laminate−dry weight of laminate) ÷ wet weight of laminate × 100 (%). Moreover, lamination of the sheet-like cell culture was performed as follows. First, the support (CellShifter ^TM, manufactured by Cellseed Co.) superimposed on a sheet-like cell cultures were prevented from entering bubbles, a sheet-like cell cultures flipping a support sheet cell cultures deposited from the end The support was collected, which was overlaid on another sheet cell culture after removal of the medium. After standing for about 30 minutes at 37 ° C., after confirming that the respective sheet-like cell cultures were adhered to each other, the support was removed from the laminated sheet-like cell cultures. The same operation was repeated as necessary to perform further lamination.

From the results in Table 2, it can be seen that the contribution of the fibrin gel to the weight and strength of the laminate is greater than that of the sheet cell culture laminate.

Example 3 Production of a Laminate Having a Reinforcing Part 50 μL of a thrombin solution was dropped at one end of the laminate 2, 50 μL of a fibrinogen solution was dropped, and after standing for about 5 minutes, 24 mL of Hanks balanced salt in a culture dish The solution was added to remove unreacted fibrinogen and thrombin. Thus, a reinforcing portion was formed in the laminate 2. The thrombin solution and the fibrinogen solution used were the same as those used for forming the non-reinforcing portion fibrin gel. Further, 100 μL of a thrombin solution was dropped to one end of the laminate 3 and then 100 μL of a fibrinogen solution was dropped to form a reinforced portion in the same manner (FIG. 8). The strengths of the laminates 2 and 3 with the reinforcements were evaluated in the same manner as in Example 2 in the reinforcements. The results are shown in Table 3.

In the same manner as described above, a laminate 10 having a reinforcing portion was produced using a cell culture dish having a diameter of 3.5 cm, and the same evaluation as described above was performed. The results are shown in Table 4.

  From the results shown in Tables 3 to 4, it can be seen that the reinforced portion can be given about 4.5 to 8.5 times the strength of the non-reinforcing portion. Also, it can be seen that the strength of the reinforcing portion correlates with the amount of thrombin solution and fibrinogen solution dropped for forming the reinforcing portion.

Example 4 In order to evaluate the operability at the time of grafting of the fixed laminate with a suture of the laminate, the laminate manufactured under the same conditions as in the laminate 3 (300 μL of fibrinogen solution + about 100 μL of thrombin solution (estimated adhesion amount) sprayed) The following tests were performed using the body (n = 2). Place the laminate in the liquid in the intestinal folds and apply a suture (7-0 proline) to the end of the laminate (Figure 9) and transfer the laminate from the intestinal folds to the surgically exposed pig heart ( Figures 10-11), fixed with sutures. After implanting the laminate, the chest was closed and the condition of the animal thereafter was observed. Five laminates were implanted per pig. In each test, after fixing the laminate on the surface of the heart with a suture in one of the five laminates implanted, the laminate can not withstand the tension of the suture and tears from the penetrating part, thereby the suture Although it was released (FIG. 12) and sutured again (FIG. 13), finally all laminates could be fixed to the surface of the heart.

  Moreover, the following tests were performed using the laminated body manufactured on the same conditions as the laminated body 1 (500 microliters of fibrinogen solution dripping + about 500 microliters of thrombin solutions (estimated adhesion amount) spraying). Place the laminate in the liquid on the intestines (Fig. 14), put a suture (7-0 proline) on the edge (Fig. 15-17), and transfer it to the side of the heart-simulating bottle (Fig. 18) Did. The laminate had adequate flexibility and viscosity, was easy to put on the intestinal folds, was smooth in movement from the intestinal folds to the bottles, and fitted well on the curved surfaces of the bottles. Also, the suture remained firmly attached to the laminate during the series of operations, and the laminate did not break and the suture could not be removed.

Example 5 Treatment with a Laminate The laminate was used to treat a human patient suffering from severe cardiomyopathy (ischemic cardiomyopathy, dilated cardiomyopathy etc). A laminate is produced under the same conditions as in the laminate 1 (500 μL of fibrinogen solution + about 500 μL of thrombin solution (estimated adhesion amount) sprayed), placed on the intestine and transferred onto the exposed patient's heart under open chest After fixing with sutures, chest was closed and the condition of the patient was observed. All patients showed improvement in cardiac function, tolerability, quality of life, morbidity and prognosis.

  The various features of the invention described herein may be combined in various ways, and the embodiments obtained by such combinations are all included in the invention, including combinations not specifically described herein. It is in the range. Those skilled in the art will also appreciate that numerous and various modifications are possible without departing from the spirit of the present invention. Accordingly, it should be understood that the embodiments described herein are merely exemplary and are not intended to limit the scope of the present invention.

Claims (7)

  The steps of dropping a liquid containing fibrinogen onto the top surface of the sheet-like cell culture, spraying a liquid containing thrombin onto the surface, and forming a layer of fibrin gel on the surface by the reaction of fibrinogen and thrombin. A method of producing a laminate of a fibrin gel and a sheet-like cell culture, comprising:   The method according to claim 1, further comprising the step of washing the laminate after the step of forming a layer of fibrin gel.   The method according to claim 1 or 2, further comprising the step of trimming excess fibrin gel after forming the layer of fibrin gel.   A laminate of a fibrin gel and a sheet-like cell culture produced by the method according to any one of claims 1 to 3.   A pharmaceutical composition comprising the laminate according to claim 4.   The pharmaceutical composition according to claim 5, for treating a disease associated with a tissue abnormality.   A method of treating a disease associated with a tissue abnormality in a subject, comprising administering to a subject in need thereof an effective amount of the laminate of claim 4 or the pharmaceutical composition of claim 5 or 6. How to do that.