JP6846402B2 - Method for producing a laminate of sheet-shaped cell culture and fibrin gel - Google Patents

Description

The present invention relates to a method for producing a laminate of a sheet-shaped 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, and the like. Regarding.

In recent years, attempts have been made to transplant various cells for repairing damaged tissues and the like. For example, in order to repair myocardial tissue damaged by ischemic heart disease such as angina pectoris and myocardial infarction, attempts have been made to use fetal myoblasts, skeletal myoblasts, mesenchymal stem cells, heart stem cells, ES cells and the like. (Non-Patent Document 1).

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

As described above, the sheet-shaped cell culture has high usefulness in regenerative medicine and the like, but is generally fragile as it is, and is prone to wrinkles and tears during isolation from the culture substrate and subsequent operations. Operations such as transfer, storage, and transplantation are extremely difficult. In order to improve such a problem, the fibrinogen solution and the thrombin solution are simultaneously sprayed on the sheet-shaped cell culture to form a support layer containing fibrin on the sheet-shaped cell culture, and the sheet-shaped cell culture and fibrin are formed. Attempts have been made on a method for producing a laminate with (Patent Document 2).

Special Table 2007-528755 Japanese Unexamined Patent Publication No. 2011-172925

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

In the subsequent research, regarding the laminate of the sheet-shaped cell culture and fibrin described in Patent Document 2, when the laminate was isolated from the culture substrate, the laminate was torn or the isolation was unsuccessful. It was found that there were cases where it was possible, and cases where the support layer was peeled off from the sheet-like cell culture and a complete laminate could not be obtained. It was also found that thickening the support layer to remedy this problem could result in inhibition of cell function. Therefore, the present invention does not have these problems, has excellent operability, and is suitable for transplantation. A laminate of sheet-shaped cell culture, a method for producing the same, a pharmaceutical composition containing the laminate, and the laminate. It is an object of the present invention to provide the treatment method of the disease using.

While conducting diligent research to solve the above problems, the present inventors first drop a liquid containing fibrinogen onto the sheet-shaped cell culture when forming a layer of fibrin gel on the sheet-shaped cell culture. Then, by spraying a liquid containing thrombin, the layer of fibrin gel formed by the reaction of fibrinogen and thrombin adheres firmly to the sheet-like cell culture, and the resulting fibrin gel and sheet-like cells are firmly adhered to the cell culture. The present invention was completed by finding that the layer of fibrin gel does not peel off from the sheet-like cell culture when the laminate with the culture is isolated from the container.

That is, the present invention relates to the following.
(1) A layer of fibringel is formed by a step of dropping a liquid containing fibrinogen on the upper surface of a sheet-shaped cell culture, a step of spraying a liquid containing thrombin on the surface, and a reaction of fibrinogen and thrombin on the surface. A method for producing a laminate of fibrin gel and sheet-like cell culture, which comprises the step of making.
(2) The method according to (1) above, comprising the step of forming a layer of fibrin gel followed by the step of washing the laminate.
(3) The method according to (1) or (2) above, which further comprises a step of forming a layer of fibrin gel followed by a step of trimming excess fibrin gel.
(4) A laminate of fibrin gel and sheet-shaped cell culture produced by the method according to any one of (1) to (3) above.
(5) A pharmaceutical composition containing the laminate according to (4) above.
(6) The pharmaceutical composition according to (5) above, for treating a disease related to tissue abnormality.
(7) A method for treating a disease related to a tissue abnormality in a subject, wherein an effective amount of the laminate according to (4) above or the pharmaceutical composition according to (5) or (6) above is used. Methods involving administration to subjects in need.

The laminate produced by the method of the present invention has high strength, excellent operability, easy application to the affected area, and there is little difference in operation depending on the skill level of the practitioner, so that reliable treatment of the disease can be performed. Not only will it be possible, but it is also expected that the spread of regenerative medicine using the laminate will increase. Further, since the laminate of the present invention is formed of a biocompatible component, it can be directly transplanted to the transplantation site to decompose the fibrin gel in vivo, and it can be re-operated or in the form of a sheet. The convenience of sheet-shaped cell cultures is greatly enhanced, such as eliminating the need for a special jig for transplanting cell cultures.

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

FIG. 7 is a photographic view showing a state in which the laminate of the present invention is placed on an intestinal spatula. FIG. 8 is a photographic view showing a laminated body having a reinforcing portion of the present invention. Black arrows indicate reinforcements. In addition, it can be seen that the suture thread penetrates the center of the reinforcing portion. FIG. 9 is a photographic view showing an operation of suturing a laminated body of the present invention placed on an intestinal spatula. FIG. 10 is a photographic view showing an operation of transferring the laminate of the present invention from the intestinal spatula to the transplanted portion. FIG. 11 is a photographic view showing an operation of transferring the laminate of the present invention from the intestinal spatula to the transplanted portion. FIG. 12 is a photographic view showing a state in which the suture is detached from the laminated body of the present invention transferred to the transplanted portion.

FIG. 13 is a photographic view showing a situation in which the laminate of the present invention is re-sutured to the transplanted portion. FIG. 14 is a photographic view showing an operation of transferring the laminate of the present invention to the intestinal spatula. FIG. 15 is a photographic view showing an operation of hooking a suture on the edge portion of the laminated body of the present invention. FIG. 16 is a photographic view showing an operation of hanging a suture on the edge portion of the laminated body of the present invention. FIG. 17 is a photographic view showing an operation of hooking a suture on the edge portion of the laminated body of the present invention. FIG. 18 is a photographic view showing a state in which the laminate of the present invention has been transferred to the side surface of the bottle.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. All patents, applications and other publications (including information available on the Internet) referenced herein 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-shaped 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. The present invention relates to a method for producing a laminate of fibrin gel and a sheet-like cell culture, which comprises a step of forming a layer of fibrinogen.

In the present invention, the "sheet-shaped cell culture" refers to cells connected to each other to form a sheet. The cells may be linked to each other directly (including those via cell elements such as adhesion molecules) and / or via intervening substances. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediator is preferably derived from cells, particularly from the cells that make up the cell culture. The cells are at least physically (mechanically) connected, but may be more functionally, for example, chemically or electrically connected. The sheet-like cell culture may be composed of one cell layer (single layer) or two or more cell layers (laminated (multilayer), for example, two layers, three layers, four layers. Layers, 5 layers, 6 layers, etc.) may be used.

Sheet cell cultures preferably do not contain scaffolds. Scaffolds may be used in the art to attach cells to and / or inside the surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride. Although a membrane made of PVDF) or the like is known, the sheet-shaped cell culture in the present invention may be one that can maintain its physical integrity even without such a scaffold. In addition, the sheet-shaped cell culture preferably consists only of cell-derived substances constituting the cell culture, and does not contain any other substances.

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

The cells that make up the sheet cell culture can be derived from any organism that can be treated with the sheet cell culture. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. Further, although only one type of cells may be used to constitute the sheet-shaped cell culture, two or more types of cells may be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a sheet-shaped cell culture, the ratio (purity) of the most abundant cells is 60% or more, preferably 70 at the end of production of the sheet-shaped cell culture. % Or more, more preferably 75% or more.

The cells forming the sheet-like cell culture may be heterologous cells or allogeneic cells. Here, "heterologous cell" means a cell derived from an organism of a species different from the recipient when the sheet-shaped cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to heterologous cells. In addition, "homogeneous cell" means a cell derived from an organism of the same species as the recipient. For example, when the recipient is a human, the human cell corresponds to an allogeneic cell. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie, recipient-derived cells and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferable in the present invention because they do not cause rejection even when transplanted. However, it is also possible to utilize heterologous cells and allogeneic non-autologous cells. When using heterologous cells or allogeneic non-autologous cells, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, that is, allogeneic non-self-derived cells and allogeneic non-self-derived cells may be collectively referred to as non-autologous cells. In one aspect of the invention, the cell is an autologous cell or an allogeneic cell. In one aspect of the invention, the cell is an autologous cell. In another aspect of the invention, the cell is an allogeneic cell.

The sheet-shaped cell culture can be produced by any known method (see, for example, Patent Document 1, Patent Document 2, Japanese Patent Application Laid-Open No. 2010-081829, Japanese Patent Application Laid-Open No. 2011-110368, etc.). The method for producing a sheet-shaped cell culture is typically a step of seeding cells on a culture substrate, a step of sheeting the seeded cells, and isolating the formed sheet-shaped cell culture from the culture substrate. Including, but not limited to, the steps to be performed. Prior to the step of seeding the cells on the culture medium, a step of freezing the cells and a step of thawing the cells may be performed. In addition, a step of washing the cells may be performed after the step of thawing the cells. Each of these steps can be performed by any known technique suitable for the production of the production of sheet cell cultures. The production method of the present invention may further include the step of producing a sheet-like cell culture, in which case the step of producing a sheet-like cell culture may include one or more of the above sub-steps.

The liquid containing fibrinogen (hereinafter, also referred to as fibrinogen solution) is not particularly limited as long as it can react with thrombin to form fibrin gel, and fibrinogen is, for example, 1 mg / mL to 500 mg / mL. Examples thereof include liquids contained in concentrations such as 5, 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 for the fibrinogen solution is typically water. In addition to fibrinogen, fibrinogen solution contains components such as factor XIII, aprotinin, serum albumin, glycine, L-arginine hydrochloride, L-isoleucine, sodium L-glutamate, D-mannitol, sodium citrate hydrate, and sodium chloride. May include. Fibrinogen solutions are commercially available or can be prepared according to known techniques. The commercially available fibrinogen solution is not limited, and for example, ^{the content of vial 1 (fibrinogen lyophilized powder) for bolhel (R)} tissue adhesion (manufactured by Teijin Pharma Limited) is used as the content of vial 2 (fibrinogen solution). Examples thereof include those dissolved in Vial 1 (made by CSL Bering Co., Ltd.) in which the contents (fibrinogen powder) of ^{the beliplast (R) combination set for tissue adhesion are dissolved in the contents of the vial 2 (aprotinin solution).}

The dropping of the fibrinogen solution can be carried out by any known method, for example, a syringe or a pipette. Examples of the syringe include a needleless syringe having a capacity of 0.5 mL to 5 mL, a syringe with a needle (for example, a syringe with a needle of 18G to 27G), and a two-component mixture of a preparation device set attached for ^{bolhel (R) tissue adhesion.} A set (with an apply nozzle having an inner diameter of about 1 mm, manufactured by Nipro), a two-component mixing set of a Veriplast preparation device set (with an apply nozzle, manufactured by Nipro), and 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. Non-limiting examples of the amount of the fibrinogen solution added dropwise are 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, and about 300 μL for a sheet-shaped cell culture having a diameter of 45 mm. ~ About 500 μL and the like can be mentioned. The particle size of the droplet when the fibrinogen solution is dropped is not particularly limited, but for example, the diameter of the droplet attached to the sheet-shaped cell culture after dropping is in the range of about 0.2 cm to about 2.0 cm. You can. The weight of the droplet is not limited, and 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, and the like. The particle size and weight of the droplet can be appropriately adjusted depending on whether or not the needle is attached to the syringe, the number of gauges of the attached needle, the shape of the needle tip, and the like.

The liquid containing thrombin (hereinafter, also referred to as thrombin liquid) is not particularly limited as long as it can react with fibrinogen to form fibrin gel, and thrombin is, for example, 1 unit / mL to 10000 units. / 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 contained in concentrations such as / mL. The solvent for 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 prepared according to known methods. The commercially available thrombin solution is not limited, and for example, ^{the content of the vial 3 (thrombin lyophilized powder) for bolhel (R)} tissue adhesion (manufactured by Teijin Pharma Limited) is used as the content of the vial 4 (thrombin solution). Examples thereof include those dissolved in ^{Vial 3 (thrombin powder) for vilplast (R)} combiset tissue adhesion (manufactured by CSL Bering Co., Ltd.) and dissolved in the contents of vial 4 (calcium chloride solution).

Spraying the thrombin solution can be performed using any known method, such as spraying. As a non-limiting example of spray, a spray tip was attached to the two-component mixing set attached to the ^{Volheel (R)} spray set (manufactured by Sumitomo Akita Bake) and the Veriplast ^{(R) combination set for tissue adhesion (manufactured by CSL Behring).} Things and so on. The amount of the thrombin solution 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 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 amount of spray of thrombin solution, the estimated amount of adhesion to a sheet-like cell culture having a diameter of 45 mm is about 50 μL to about 1000 μL, about 80 μL to about 800 μL, about 100 μL to about 700 μL, and about 200 μL to about 600 μL. , About 300 μL to about 500 μL and the like. The estimated amount of adhesion to the sheet-shaped cell culture is within a predetermined range (for example, 45 mm in diameter) when a predetermined amount of thrombin solution is actually sprayed at a spray, height, spray pressure, and spray angle. The weight of the liquid adhering to the circle) is measured, and this is ^{divided by the density of the thrombin liquid (0.999973 g / cm 3} ) to calculate. Those skilled in the art will be able to determine the spray conditions that result in the desired estimated adherence of the thrombin solution based on the description herein without the need for undue experimentation. For example, in Example 1 below, it is described that the estimated adhesion amounts for the spray amounts of 300 μL, 500 μL, 600 μL, and 900 μL are 100 μL, 180 μL, 300 μL, and 450 μL, respectively. An approximate curve of (μL) = spray amount (μL) × 0.6-88 (μL) is obtained, and based on this, the spray amount that brings about a desired estimated adhesion amount can be obtained. The spraying of the thrombin solution is not particularly limited as long as the thrombin solution can adhere evenly to the upper surface of the sheet-shaped cell culture, preferably the entire upper surface, and is, for example, from a height of about 2 cm to about 15 cm to about 15 ° to about. It can be carried out at a spray angle of 150 ° and a pressure of about 0.005 MPa to about 0.1 MPa.

The ratio of the fibrinogen solution to the thrombin solution applied to the sheet-shaped cell culture is not particularly limited as long as it does not excessively interfere with the transplantation operation of the obtained laminate. For example, the amount of the fibrinogen solution dropped and the thrombin solution are added. As a volume ratio with 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., particularly about 1: 1, and fibrinogen and thrombin adhering to the laminate The ratio (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, etc., and in particular about 8:25.

By adjusting the concentration and amount of the fibrinogen solution and the thrombin solution, the thickness and properties (flexibility, adhesiveness, etc.) of the obtained laminate can be changed. For example, the thickness of the laminate can be increased by increasing the amount of fibrinogen solution, and the volume ratio of the dropping amount of fibrinogen solution (80 mg / mL) to the estimated adhesion amount of thrombin solution (250 units / mL). The closer the ratio is to 1: 1 and the closer the ratio (mg: unit) of fibrinogen to thrombin attached to the laminate is to 8:25, the more flexible and sticky the laminate is, and the better the operability is. ..

The step of forming the layer of fibrin gel is not limited, and can be performed, for example, by spraying the thrombin solution and then allowing the sheet-shaped cell culture to stand for a certain period of time. The standing period is not limited, and more specifically, 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, and the like. 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., especially about 5 minutes. You can.

After the step of forming the layer of fibrin gel, the step of washing the laminate may be performed. Washing is performed in a predetermined amount of cleaning solution, such as water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, etc.) without limitation. , MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc. can be added to the container containing the laminate and discarded. it can. The washing step can be performed once or more than once. Further, even if the cleaning liquid added to the container is immediately discarded, it takes a predetermined time (for example, about 1 minute to about 60 minutes, about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, etc.). It may be discarded after being allowed to stand for about 15 minutes in particular.

After the step of forming the layer of fibrin gel (after the washing step if the step of washing the laminate is included), if necessary, a step of trimming the excess fibrin gel may be performed. Trimming can be done with any instrument that can be used to cut the gel-like material. Such instruments include, but are not limited to, scalpels, scissors, scalpels, and the like. The fibrin gel may be trimmed to the contour of the sheet-like cell culture, or the fibrin gel may protrude around the sheet-like cell culture, that is, the edges of the laminate shall consist solely of the fibrin gel. It may be trimmed so as to.
The step of dropping the liquid containing fibrinogen, the step of spraying the liquid containing trombin, the step of forming a layer of fibrin gel, the step of washing the laminate, and / or the step of trimming the excess fibrin gel are cultured. It may be performed on a sheet-shaped cell culture that remains adhered to the substrate, or may be performed on a sheet-shaped cell culture isolated from the culture substrate.

In one aspect, the manufacturing method of the present invention is performed in vitro in all steps. In another aspect, the production method of the present invention comprises a step performed in vivo, including, but not limited to, the step of collecting, for example, a cell or a tissue to which the cell is supplied from a subject. In one aspect, the production method of the present invention is carried out under sterile conditions in all steps. In one aspect, the production method of the present invention is carried out so that the finally obtained laminate is substantially sterile. In one aspect, the production method of the present invention is carried out so that the finally obtained laminate is sterile.

Another aspect of the present invention relates to a laminate of sheet cell culture and fibrin gel produced by the production method of the present invention. The laminate of the present invention has, for example, compared to the laminate produced by simultaneously spraying a fibrinogen solution and a thrombin solution onto a sheet cell culture described in Patent Document 2, and has a fibrin gel layer in a sheet cell culture. The fibrinogen layer does not come off from the sheet-like cell culture during the operation. Although not bound by any particular theory, in the laminates of the invention, fibrinogen penetrates into sheet cell cultures (eg, cell interstitial spaces) before reacting with thrombin to form fibrin. However, it is considered that the fibrin gel is sterically bound to the sheet-shaped cell culture and anchored, and the contact area between the fibrin gel and the sheet-shaped cell culture is increased, so that the adhesion between the two is strengthened. On the other hand, in the laminate described in Patent Document 2, fibrinogen and thrombin come into contact with the sheet-shaped cell culture at the same time and immediately react to form fibrin gel, so that the fibrin gel is different from the sheet-shaped cell culture. It is considered that it is difficult to form such a three-dimensional bond. Therefore, it is considered that the laminate of the present invention is qualitatively and structurally different from the laminate described in Patent Document 2.

The strength of the laminate is not limited, for example, when measured by the method described in Example 2, 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. It may be .030N or more, about 0.035N or more, about 0.040N or more, about 0.045N or more, and also about 0.010N to about 0.200N, about 0.015N to about 0.100N, about. It may be in the range of 0.020N to about 0.50N 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 the same sheet-like cell culture contained in the laminate except that the fibrin gel is not laminated. It may be 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, and about 1.5 times to about 20 times. , 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 fibrin gel and sheet-like cell culture, may be composed of only fibrin gel, or may be composed of only 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 fibrin gel (hereinafter, may be referred to as a reinforced laminate).
The reinforced laminate of the present invention has at least one reinforcing portion formed by partially further laminating the fibrin gel on the fibrin gel layer of the laminate of the sheet-like cell culture and the fibrin gel. By forming the reinforcing portion, the strength of the required portion can be increased without increasing the thickness of the entire laminated body. The reinforcing portion can be used, for example, as a suture insertion portion or a grip portion by a screwdriver or the like. The reinforcing portion is formed by dropping the fibrinogen solution and the thrombin solution onto the fibrin gel layer, and can be made into various thicknesses and shapes by adjusting the dropping amount, dropping position, dropping pattern, and the like. .. For example, when 50 μL each of fibrinogen solution (80 mg / mL) and thrombin solution (250 units / mL) is dropped, a ^{reinforcing portion having a bottom area of about 1 cm 2} × height of about 1 mm to 2 mm is obtained, and 100 μL of each is dropped. , A reinforcing portion having a bottom area of about 1 cm ² x a height of about 2 mm to 3 mm can be obtained.

The ratio of the fibrinogen solution to be dropped to the thrombin solution is not particularly limited as long as a reinforcing portion having a desired strength can be obtained. For example, the volume ratio of the fibrinogen solution to be dropped to the thrombin solution is about 5: 1 to 1. 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 It may be 1.5: 1 to about 1: 1 etc., particularly about 1: 1 and the ratio (mg: unit) of fibrinogen to thrombin contained in the reinforcing portion 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 etc., especially about 8:25.

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

Another aspect of the present invention is a step of dropping a liquid containing fibrinogen on the upper surface of a sheet-shaped cell culture, a step of spraying a liquid containing fibrinogen on the surface, and fibrin by the reaction of fibrinogen and trombin on the surface. A step of forming a layer of gel, a step of dropping a liquid containing thrombin and a liquid containing fibrinogen onto the layer of fibrinogen so that both liquids are mixed on the layer of fibrinogen, and a step of dropping fibrinogen. A method for producing a laminate of a fibrin gel and a sheet-like cell culture having a reinforcing portion formed of fibrin gel, which comprises a step of forming a reinforcing portion of fibrin gel on a fibrin gel layer by a reaction between the fibrin gel and the fibrin gel. Regarding.

A step of dropping a liquid containing fibrinogen on the upper surface of a sheet-shaped cell culture, a step of spraying a liquid containing thrombin on the surface, a step of forming a layer of fibringel by the reaction of fibrinogen and thrombin on the surface, and a step of forming a layer of fibrin gel on the surface. Regarding the liquid containing thrombin and the liquid containing fibrin, the method for producing a laminate of a sheet-shaped cell culture and fibrin gel is as described above. The method for producing a reinforced laminate includes a step of forming a laminate of a sheet-like cell culture and fibrin gel by the method for producing a laminate of a sheet-like cell culture and fibrin gel, and thrombin. A liquid and a liquid containing fibrinogen are dropped onto the fibringel layer so that both liquids are mixed on the fibringel layer, and the reaction between fibrinogen and thrombin causes the fibrinogen to be placed on the fibringel layer. It may include a step of forming a reinforcing portion of fibrin gel. Therefore, the above method for producing a reinforced laminate includes a step of cleaning the laminate after the step of forming the layer of fibrin gel and a step of cleaning the laminate after the step of forming the layer of fibrin gel (when the step of cleaning the laminate is included). May include various steps describing the method for producing the above-mentioned laminate, such as a step of trimming the excess fibrin gel and a step of producing a sheet-like cell culture (after the washing step). ..

The thrombin solution and the fibrinogen solution may be dropped onto the fibrin gel layer either first or at the same time. For example, the step of dropping the thrombin solution and the fibrinogen solution onto the layer of the fibrin gel is not limited to (1) dropping the thrombin solution onto the layer of the fibrin gel, and then dropping the thrombin solution onto the dropped thrombin solution. , (2) Dropping the fibrinogen solution onto the layer of fibrinogen, and then dropping the thrombin solution onto the dropped fibrinogen solution, and (3) Dropping the thrombin solution and fibrinogen onto the layer of fibrinogen. It includes a step of dropping a liquid at the same time.

The dropping of the thrombin solution and the fibrinogen solution onto the layer of fibrin gel can be performed by any known method, for example, a syringe or a pipette. Examples of the syringe include a needleless syringe having a capacity of 0.5 mL to 5 mL, a syringe with a needle (for example, a syringe with a needle of 18G to 27G), and a two-component mixture of a preparation device set attached for ^{bolhel (R) tissue adhesion.} A set (with an apply nozzle having an inner diameter of about 1 mm, manufactured by Nipro), a two-component mixing set of a Veriplast preparation device set (with an apply nozzle, manufactured by Nipro), and the like can be used. The particle size of the droplets when the thrombin solution or the fibrinogen solution is dropped is not particularly limited, but for example, the diameter of the droplets attached to the sheet-shaped cell culture after dropping is about 0.2 cm to about 2.0 cm. It may be a range. The weight of the droplet when dropping the thrombin solution or the fibrinogen solution is not limited, and 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, and the like. The particle size and weight of the droplet can be appropriately adjusted depending on whether or not the needle is attached to the syringe, the number of gauges of the attached needle, the shape of the needle tip, and the like. The dropping amount 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 reinforced laminate.

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

After the step of forming the reinforcing portion of the fibrin gel, the step of cleaning the reinforcing laminate can be performed. Washing is performed in a predetermined amount of cleaning solution, such as water, saline, various buffers (eg, PBS, HBSS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI1640, etc.) without limitation. , MCDB (MCDB102, 104, 107, 120, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.), etc. are added to the container containing the reinforced laminate and discarded. Can be done. The washing step can be performed once or more than once. Further, even if the cleaning liquid added to the container is immediately discarded, it takes a predetermined time (for example, about 1 minute to about 60 minutes, about 5 minutes to about 30 minutes, about 10 minutes to about 20 minutes, etc.). It may be discarded after being allowed to stand for about 15 minutes in particular.

The laminates of the present invention (including reinforced laminates; the same shall apply hereinafter) are useful in treating various diseases associated with tissue abnormalities. Therefore, in one aspect, the laminate of the present invention is intended 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 the sheet-shaped cell culture, and the other side of the sheet-shaped cell culture is intact. Since fibrin gel is eventually decomposed and disappears in vivo, it can be applied to tissues and diseases that can be treated with sheet-like cell culture. Tissues to be treated include, but are not limited to, myocardium, cornea, retina, esophagus, skin, articular cartilage, liver, pancreas, gingiva, kidney, thyroid gland, skeletal muscle, middle ear and the like. The diseases to be treated include, for example, heart diseases (for example, myocardial injuries (myocardial infarction, cardiac trauma, etc.)) and myocardial diseases (ischemic myocardial disease, dilated myocardial disease, dilated phase fertilizer). (Major myocardial disease, etc.), Corneal disease (eg, corneal epithelial stem cell exhaustion, corneal injury (heat / chemical corrosion), corneal ulcer, corneal opacity, corneal perforation, corneal scar, Stevens Johnson syndrome, ocular herbitis ), Retinal diseases (eg, retinal pigment degeneration, age-related yellow spot degeneration, etc.), esophageal diseases (eg, prevention of esophageal inflammation / stenosis after esophageal surgery (removal of esophageal cancer)), skin diseases (eg, prevention of esophageal inflammation / narrowing) Skin damage (trauma, burns, etc.), joint disease (eg, osteoarthritis), cartilage disease (eg, cartilage damage), liver disease (eg, chronic liver disease), pancreatic disease (eg, diabetes, etc.) ), Dental disease (eg periodontal disease), kidney disease (eg renal failure, renal anemia, renal osteodystrophy, etc.), thyroid disease (eg, hypothyroidism), muscle disease (eg, thyroid dysfunction) , Muscle injury, myitis, etc.), middle ear diseases (eg, middle ear inflammation, etc.).

The usefulness of sheet cell cultures for the above 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., 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.

The laminate of the present invention can be applied to the tissue to be treated and used for repairing and regenerating the tissue, but as a source of a physiologically active substance such as a hormone, other than the tissue to be treated. It can also be transplanted to a site (eg, subcutaneous tissue) (eg, 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. In addition, the laminate of the present invention can also be used as a graft.

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 cell culture contained in the laminate of the present invention is not genetically engineered. In another embodiment, the sheet cell culture contained in the laminate of the present invention is genetically engineered. Genetic manipulation includes, but is not limited to, the introduction of, for example, genes that enhance the viability, engraftment, function, etc. of sheet cell cultures and / or genes that are useful in the treatment of disease. The gene to be introduced is not limited, and examples thereof include cytokine genes such as HGF gene and VEGF gene.

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 may contain various additional ingredients such as a pharmaceutically acceptable carrier, viability, engraftment and / or function of a sheet cell culture. It may contain an ingredient that enhances the temperature, other active ingredients that are useful for treating the target disease, and the like. Any known additional ingredient can be used, and those skilled in the art are familiar with these additional ingredients. In addition, the pharmaceutical composition of the present invention can be used in combination with an ingredient that enhances the viability, engraftment and / or function of a sheet-shaped cell culture, and other active ingredients that are useful for treating a target disease. .. In one aspect, the pharmaceutical composition of the present invention is for use in the treatment of diseases associated with tissue abnormalities. 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 a tissue abnormality in a subject, wherein an effective amount of the laminate or pharmaceutical composition of the present invention is administered to the subject in need thereof. Regarding how to include. The tissues and diseases that are the targets of 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, a component that enhances the viability, engraftment and / or function of the sheet-shaped cell culture, other active ingredients useful for treating the target disease, and the like are laminated with the present invention. It can be used in combination with the body or pharmaceutical composition.

The treatment method of the present invention may further include the step of producing a laminate according to the production method of the present invention. In the treatment method of the present invention, before the step of producing a laminate, the step of producing a sheet-shaped cell culture, or collecting cells or a tissue that is a source of cells for producing a sheet-shaped cell culture from a subject. Further steps may be included. In one aspect, the subject from which the cell or tissue that is the source of the cell is harvested is the same individual that is treated with the laminate or pharmaceutical composition. In another embodiment, the subject from which the cell or tissue from which the cell is sourced is harvested is a separate entity of the same species as the subject to whom the laminate or pharmaceutical composition is administered. In another embodiment, the subject from which the cell or tissue that is the source of the cell is harvested is an individual that is heterologous to the subject to whom the laminate or pharmaceutical composition is administered.

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

Also, the term "treatment" shall include all types of medically acceptable prophylactic and / or therapeutic interventions aimed at curing, transient remission or prevention of disease. For example, the term "treatment" is medically acceptable for a variety of purposes, including delaying or stopping the progression of a disease associated with a tissue abnormality, regressing or eliminating a lesion, preventing the onset or recurrence of the disease, and the like. Including interventions to be performed.

In the present invention, the effective amount is, for example, an amount capable of suppressing the onset or recurrence of the disease, reducing the symptoms, or delaying or stopping the progression (for example, the size and weight of the laminate), and is preferable. An amount that prevents the onset and recurrence of the disease or cures the disease. In addition, an amount that does not cause an adverse effect exceeding the benefit of administration is preferable. Such an amount can be appropriately determined by, for example, a test in an experimental animal such as a mouse, a rat, a dog or a pig, or a disease model animal, and such a test method is well known to those skilled in the art. In addition, the size of the tissue lesion to be treated can be an important index for determining the effective amount.

The method of administration typically includes direct application to tissues. The frequency of administration is typically once per treatment, but multiple doses can be administered if the desired effect is not obtained. When applied to a tissue, the laminate or pharmaceutical composition of the present invention may be fixed to the target tissue by a locking means such as a suture or a staple. When a reinforcing laminate is used, the locking means can be applied to the reinforcing portion.

The present invention will be described in more detail with reference to the following examples, but these show specific specific examples of the present invention, and the present invention is not limited thereto.

Example 1 Production of a laminate of fibrin gel and sheet-shaped cell culture Skeletal myoblasts (CD56 positive) cryopreserved in a cell freezing preservation solution (MCDB medium containing 10% DMSO) were thawed at 37 ° C. and 0. Washed twice with physiological buffer containing 5.5% serum albumin. The washed cells 6.0 × 10 ⁷ were suspended in DMEM medium (10 mL) containing 20% human serum ^{and seeded in a cell culture dish (UpCell (R)} 10 cm dish, CS3005, manufactured by Cellseed) having a diameter of 10 cm. .. After seeding, the cells were cultured for 20 hours in an incubator (BNA-121D, manufactured by ESPEC) set at 37 ° C. and 5% CO _2. After culturing, the culture dish was taken out from the incubator, it was confirmed that the sheet-shaped cell culture was adhered so as to cover the entire bottom surface of the culture dish, and the medium was discarded. Then, the sheet-shaped cell culture was peeled from the culture dish by temperature treatment (standing at room temperature (20 to 25 ° C.) for 5 to 30 minutes) and pipetting. The obtained sheet-shaped cell culture had a size of 47 mm × 47 mm.

After removing the culture solution in the culture dish and shaping the sheet-shaped cell culture as necessary (Fig. 1), the fibrinogen solution (for boulder ^(R) tissue adhesion (manufactured by Teijin Pharma Limited)) was placed on the sheet-shaped cell culture. ) Vial 1 contents (fibrinogen lyophilized powder) dissolved in vial 2 contents (fibrinogen solution), fibrinogen concentration 80 mg / mL, the same applies hereinafter) 500 μL, attached for ^{Bolheel (R) tissue adhesion} The cells were added dropwise using a two-component mixing set (with an apply nozzle having a length of about 6 cm and an inner diameter of about 1 mm, manufactured by Nipro Co., Ltd.). Next, the contents (thrombin lyophilized powder) of the vial 3 of the thrombin solution (for bolhel ^(R) tissue adhesion (manufactured by Teijin Pharma Limited)) dissolved in the contents of the vial 4 (thrombin solution), the thrombin concentration was 250 units. Using 800 μL of / mL (the same applies hereinafter) and a Volheel ^(R) spray set (manufactured by Sumitomo Akita Bake Co., Ltd.), the spray nozzle was sprayed at a pressure of 0.03 MPa at a distance of about 7 cm from the cell sheet (Fig. 3). Since the sheet-shaped cell culture shrinks by peeling from the culture dish and becomes smaller than the bottom surface of the culture dish (Fig. 1), about 500 μL of the sprayed 800 μL thrombin solution adheres to the sheet-shaped cell culture. It is presumed that it was done. The estimated amount of adhering to the thrombin solution is as follows: a predetermined amount of thrombin solution (800 μL in this case), a predetermined spray (Bolheel ^(R) spray set in this example), and a spray pressure (0 in this example). 03MPa), height (about 7 cm in this example), spray angle (45 ° in this example), and actually sprayed within a predetermined range (in this example, within a circle with a diameter of about 45 mm). The weight of the thrombin solution adhering to the water was measured with 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 allowing to stand for about 5 minutes, add 24 mL of Hanks balanced salt solution (HBSS (+), Cat No. 14025, manufactured by Life Technologies, the same applies hereinafter) to the culture dish and immediately remove the sheet-like cell culture. The containing culture dish was washed. Thereby, the unreacted fibrinogen solution and the thrombin solution can be removed. Next, 24 mL of Hanks balanced salt solution was added to the culture dish again and allowed to stand for about 15 minutes, the solution in the culture dish was removed, and the fibrin gel coagulated except on the sheet-like cell culture was trimmed with a scalpel (Fig.). 4), a laminate of fibring gel and sheet-like culture was isolated (laminate 1). The isolated laminate was stored in a culture dish with 24 mL of Hanks balanced salt solution added until use.

The stack 2 was subjected to the same procedure except that the amount of the fibrinogen solution dropped was 300 μL and the amount of the thrombin solution sprayed was about 600 μL (estimated adhesion amount of about 300 μL). The procedure was the same except that 900 μL (estimated adhesion amount of about 450 μL) was used, and the amount of fibrinogen solution dropped was 300 μL and the amount of thrombin solution sprayed was about 300 μL (estimated adhesion amount of about 100 μL). The laminate 5 was produced by the same procedure except that the amount of the fibrinogen solution dropped was 500 μL and the amount of the thrombin solution sprayed was 500 μL (estimated adhesion amount of about 180 μL).

For comparison, we attempted to produce a laminate of fibrin gel and sheet-like cell culture by spraying fibrinogen solution and thrombin solution at the same time.
A sheet-shaped cell culture was formed in the same manner as in the laminated body 1, and was peeled off from the culture dish. After removing the culture solution in the culture dish and shaping the sheet-shaped cell culture as necessary, 800 μL of fibrinogen solution and 800 μL of trombine solution and Volheel ^(R) spray set (Sumitomo Akita) on the sheet-shaped cell culture. Using Bake), the spray nozzles were simultaneously sprayed at a pressure of 0.03 MPa at a distance of about 7 cm from the cell sheet (estimated amount of adhesion to the sheet-like cell culture was about 500 μL, respectively). 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. Then, 24 mL of Hanks balanced salt solution was added to the culture dish again, allowed to stand for about 15 minutes, and then the solution in the culture dish was removed (FIG. 5). An attempt was made to isolate a laminate of fibrin gel and sheet cell culture by trimming the fibrin gel that had coagulated other than on the sheet cell culture with a scalpel, but the fibrin gel peeled off from the sheet cell culture. No laminate could be obtained (Fig. 6).

Example 2 Evaluation of a laminate of fibrin gel and sheet-shaped cell culture The size, weight, strength, thickness, operability and properties of the laminates 1 to 5 obtained in Example 1 were evaluated. The size was measured with a ruler, the weight was measured with an electronic non-automatic scale (AT201, manufactured by Mettler-Toledo), and the thickness was measured with a dial thickness gauge (SM-124, manufactured by Teclock). The intensity was measured as follows. First, the laminate was scooped up with a stainless steel intestinal spatula (width 45 mm, the same applies hereinafter) in a state of being stretched in the liquid, and the laminate was placed outside the liquid in a state of being attached to the surface of the intestinal spatula. A suture with a needle (6-0 proline) was inserted between the laminate and the intestinal spatula and penetrated from the lower surface to the upper surface of the laminate. Both ends of the thread were tied together to form a ring, which was connected to a gauge (general-purpose digital force gauge, FGC-1B, manufactured by Nidec Symposium). The thread locked to the laminate was pulled horizontally through a gauge, and the maximum load (tensile fracture load) until the laminate broke was measured. The measurement was performed at three different locations of the laminated body (n = 3). For operability, the laminate in the liquid is placed on the intestinal spatula (Fig. 7), and it is easy to operate in the operation of transferring it to the side surface of the bottle imitating the heart (easy to put on the intestinal spatula, intestine when moving). Comprehensive evaluation based on the difficulty of falling from the spatula, the ease of transfer from the intestinal spatula to the bottle, etc., and the condition of the laminate (whether it is wrinkled or torn during operation), etc. It was expressed in 5 stages of 5 to 1. As for the properties, the state of the laminated body observed during the evaluation of operability was qualitatively evaluated. Further, for comparison, the same evaluation was performed on the sheet-shaped cell culture (produced under the same conditions as the laminated body 1) on which the fibrin gel was not laminated. The results are shown in Table 1.

表１の結果から、いずれの積層体も、フィブリンゲルが積層されていないシート状細胞培養物よりはるかに高い強度を示すこと、フィブリノゲン液の量が多い方が積層体の重量、強度および厚みが増大することが分かる。また、いずれの積層体も模擬移植操作が可能であったが、フィブリノゲン液とシート状細胞培養物に付着したトロンビン液の体積比が１：１に近いほど操作性が向上する傾向が見られた。

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

Laminates 6 to 9 having different numbers of cells, number of laminated sheet-like cell cultures, and the like were prepared by the same method as that of laminate 1, and the same evaluation as above was performed. The results are shown in Table 2. The sheet-shaped cell culture was formed in a cell culture dish having a diameter of 3.5 cm (UpCell ^(R) 3.5 cm dish, manufactured by CellSeed). The dry weight of the laminate was obtained by measuring the freeze-dried laminate with an electronic balance. The moisture content was calculated by the following formula: Moisture content = (wet weight of laminate-dry weight of laminate) ÷ wet weight of laminate x 100 (%). In addition, the sheet-shaped cell cultures were laminated as follows. First, a support (CellShifter ^TM , manufactured by Cellseed) is placed on the sheet-shaped cell culture without bubbles, and the support to which the sheet-shaped cell culture is attached is turned over from the end to form the sheet-shaped cell culture. The entire support was collected and layered on another sheet cell culture after removing the medium. After allowing to stand at 37 ° C. for about 30 minutes, it was confirmed that the sheet-shaped cell cultures adhered to each other, and then the support was removed from the laminated sheet-shaped cell cultures. If necessary, the same operation was repeated to perform further lamination.

表２の結果から、フィブリンゲルによる積層体の重量および強度への寄与は、シート状細胞培養物の積層によるものより大きいことが分かる。

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

Example 3 Manufacture of a laminate having a reinforcing portion 50 μL of thrombin solution was added dropwise to one end of the laminate 2, 50 μL of fibrinogen solution was added dropwise, and the mixture was allowed to stand for about 5 minutes, and then 24 mL of Hanks balanced salt was placed in a culture dish. The solution was added to remove unreacted fibrinogen and thrombin. In this way, the reinforcing portion was formed in the laminated body 2. The thrombin solution and the fibrinogen solution used were the same as those used for forming the fibrin gel in the non-reinforcing portion. Further, 100 μL of the thrombin solution was dropped onto one end of the laminated body 3, and then 100 μL of the fibrinogen solution was dropped to form a reinforcing portion in the same manner (FIG. 8). The strength of the laminated bodies 2 and 3 with the reinforcing portion at the reinforcing portion was evaluated in the same manner as in Example 2. The results are shown in Table 3.

A laminate 10 having a reinforcing portion was prepared using a cell culture dish having a diameter of 3.5 cm by the same method as described above, 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 reinforcing portion can be imparted with about 4.5 to 8.5 times the strength of the non-reinforced portion. Further, it can be seen that the strength of the reinforcing portion correlates with the amount of the thrombin solution and the fibrinogen solution dropped for forming the reinforcing portion.

Example 4 A laminate manufactured under the same conditions as the laminate 3 (300 μL of fibrinogen solution + about 100 μL of thrombin solution (estimated adhesion amount) spray) in order to evaluate the operability of the laminated laminate when transplanting the fixed laminate with suture. The following tests were performed using the body (n = 2). The laminate in liquid is placed on the intestinal spatula, sutures (7-0 proline) are hung on the ends of the laminate (Fig. 9), and the laminate is transferred from the intestinal spatula to the surgically exposed pig heart (Fig. 9). 10-11), fixed with suture. After transplanting the laminate, the chest was closed and the condition of the animal was observed thereafter. Five laminates were transplanted per pig. In each test, in one of the five transplanted laminates, after the laminate was sutured to the surface of the heart, the laminate could not withstand the tension of the suture and broke from the penetration, thereby causing the suture. Although it came off (Fig. 12) and the suture was redone (Fig. 13), all the laminates were finally able to be fixed to the surface of the heart.

Further, the following test was carried out using the laminate produced under the same conditions as the laminate 1 (fibrinogen solution 500 μL dropping + thrombin solution about 500 μL (estimated adhesion amount) spray). An operation in which the laminate in the liquid is placed on the intestinal spatula (Fig. 14), sutures (7-0 proline) are hung on the margin (Figs. 15 to 17), and transferred to the side surface of the bottle imitating the heart (Fig. 18). Was done. The laminate had moderate flexibility and stickiness, was easy to put on the intestinal spatula, moved smoothly from the intestinal spatula to the bottle, and fit well on the curved surface of the bottle. In addition, the suture remained firmly hooked on the laminate during the series of operations, and the laminate was not damaged and the suture did not come off.

Example 5 Treatment with Laminates Laminates were used to treat human patients suffering from severe cardiomyopathy (ischemic cardiomyopathy, dilated cardiomyopathy, etc.). A laminate was produced under the same conditions as the laminate 1 (fibrinogen solution 500 μL dropping + thrombin solution about 500 μL (estimated adhesion amount) spray), placed on an intestinal spatula, and transferred onto the heart of a patient exposed under thoracotomy. After fixing with suture, the chest was closed and the patient's condition was observed. Improvements in cardiac function, exercise tolerance, quality of life, morbidity and prognosis were seen in all patients.

The various features of the invention described herein can be combined in various ways, and all aspects obtained by such combinations, including combinations not specifically described herein, are of the present invention. It is within the range. Those skilled in the art also understand that a number of various modifications are possible that do not deviate from the spirit of the present invention. Therefore, it should be understood that the embodiments described herein are merely exemplary and are not intended to limit the scope of the invention.

Claims (8)

A laminate consisting of fibrin and a sheet-like cell culture, in which the fibrin gel is laminated on one side of the sheet-like cell culture, and the sheet-like cell culture consists of cells and substances derived from the cells. , The strength of the laminate is 0 . The laminate having a value of 010 N or more. The laminate according to claim 1, wherein the sheet-shaped cell culture is ^{formed by skeletal myoblasts seeded at a density of 1.0 × 10 6} cells / cm ^{2 or more.} The laminate according to claim 1 or 2, which is for transplantation. The laminate according to any one of claims 1 to 3, which is housed in a container. A step of dropping a liquid containing fibrinogen on one side of a sheet-shaped cell culture, a step of spraying a liquid containing thrombin on the surface on which the liquid was dropped, and a layer of fibrin gel by the reaction of fibrinogen and thrombin on the surface. A method for producing a laminate in which a sheet-shaped cell culture comprises a cell and a fibrin composed of a substance derived from the cell and a sheet-shaped cell culture. The method according to claim 5, further comprising a step of exfoliating the sheet-shaped cell culture from the culture vessel before the step of dropping the liquid containing fibrinogen on one side of the sheet-shaped cell culture. The method of claim 5 or 6, further comprising the step of cleaning the laminate. The method of any one of claims 5-7, further comprising the step of accommodating the manufactured laminate in a container.

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