Wound covering sheet and wound covering method

Abstract

To provide a sheet for wound coating which has preferable operation ability and which can be stably adhered to a wound part.SOLUTION: The invention relates to a sheet for coating a wound by being transplanted to a wound part, the sheet for wound coating comprises: a serosa; and a cell sheet laminated on a transplantation surface side of the serosa. The sheet for wound coating related to the technique is configured so that since the cell sheet and the serosa are laminated, proper thickness and intensity are provided. Therefore, the sheet for wound coating has stabilized operation ability and can be easily transplanted. In addition, the sheet for wound coating does not flow from a wound part and if necessary, the sheet for wound coating can be fixed to the wound part by suture or ostomy or the like; therefore, the sheet for wound coating can be stably adhered to the wound part.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present technology relates to a wound covering sheet, more specifically to a wound covering sheet for use in covering a wound by being transplanted onto the wound, and a wound covering method using the wound covering sheet. [Background technology]

[0002] When the surrounding tissue including the lesion is excised for the treatment of various visceral diseases such as malignant tumors, surgery is required to suture or anastomose the remaining part of the organ after the lesion is excised. In this case, the problem of anastomosis is called suture failure. For example, in advanced cancer of the digestive tract, the malignant tumor infiltrates the deep layer of the tissue or the surrounding tissue, and the lesion spreads widely, so that the surrounding tissue including the lesion needs to be excised. After the excision of the lesion, the remaining part of the digestive tract needs to be sutured or anastomosed. In this case, the problem of anastomosis is called postoperative digestive tract anastomosis.

[0003] Gastrointestinal postoperative anastomosis is a condition in which the anastomosis does not heal after gastrointestinal anastomosis and dehiscences partially or entirely, causing intestinal contents to leak into the thoracic or abdominal cavity, resulting in severe infection and potentially fatal complications after gastrointestinal surgery. When anastomosis occurs, it can have various effects, such as a decrease in QOL (fever, abdominal pain, nutritional disorders and muscle weakness due to prolonged period of starting meals), intestinal obstruction and the development of new complications, medical economic problems due to prolonged hospital stay, and prognosis (postoperative death due to septic shock, risk of recurrence).

[0004] Suture failure occurs when the healing process (repair phase) of the anastomotic tissue is impeded. Factors that impede the healing process during the repair phase include systemic factors such as preoperative malnutrition, administration of drugs such as steroids, and chronic diseases (diabetes, liver and kidney disorders, etc.), as well as local factors such as blood circulation disorders, hypertension, and infections around the anastomotic site.

[0005] In order to prevent suture leakage, many surgeons have made every effort to improve the patient's overall condition before surgery, develop automated anastomosis machines, select appropriate pretreatment and anastomosis methods according to the site and situation, reduce tension at the anastomosis site, ensure proper blood flow, insert drains, and improve techniques and equipment such as decompression. Nevertheless, suture leakage occurs at a relatively high rate of 3-14%. In particular, the lower the cancer is located, the higher the risk of suture leakage. In addition, the number of cases in which preoperative chemoradiotherapy is performed has recently increased, which can be a risk of suture leakage. As a preventive measure against gastrointestinal suture leakage, there are many cases in which temporary colostomy is performed in exchange for a decrease in quality of life.

[0006] As a means of solving this problem, animal experiments using mesenchymal stem cells and adipose-derived stem cells, which have the effect of promoting wound healing of tissues, have been reported, but most of the transplantation methods used were cell injection methods such as intravenous injection and local injection. In general, the wound healing process at the gastrointestinal anastomosis is divided into an inflammatory period up to 3-4 days after surgery and a repair period up to about 7 days after surgery. During the inflammatory period up to 3-4 days after surgery, collagenase derived from inflammatory cells degrades existing collagen to reconstruct the submucosal tissue at the anastomosis, and in the subsequent repair period, fibroblasts proliferate, collagen is produced and increased, and the continuity and physical tensile strength of the tissue are maintained up to about 7 days after surgery. Therefore, in the transplantation method in which cells are injected into the anastomosis site, not only are the cells difficult to retain in place, but the injected cells are exposed to collagenase, a proteolytic enzyme in the inflammatory period, and there is a possibility that the tissue repair ability will be reduced due to cytotoxicity caused by collagenase.

[0007] In response to this, for example, Patent Document 1 discloses a technology for curing or preventing leakage from a wound in a hollow organ by applying a cell sheet composition containing mesenchymal stem cells to the wound in the hollow organ. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] WO2017 / 130802 Summary of the Invention [Problem to be solved by the invention]

[0009] As mentioned above, in recent years, a technology has been developed to heal or prevent leakage from wounds in hollow organs by attaching a cell sheet to the wound, but since cell sheets are soft, thin, and fragile, they are difficult to manipulate during transplantation in clinical practice. In particular, laparoscopic surgery has become common nationwide in elective colon surgery in recent years, making it very difficult to transplant fragile cell sheets using forceps.

[0010] Furthermore, when a cell sheet is transplanted into a wound, the cell sheet may flow away from the wound after a few days, which may reduce the effect of healing or preventing leakage of hollow organs from the wound.

[0011] Therefore, the main object of the present technology is to provide a sheet for covering wounds that is easy to handle and can be stably attached to the wound. [Means for solving the problem]

[0012] In order to solve the above problems, the inventors of the present application conducted extensive research into the structure of a sheet for covering wounds, and as a result, by combining it with a specific biological membrane, they succeeded in obtaining a sheet for covering wounds that can be stably attached to the wound, thereby completing the present technology.

[0013] That is, in the present technology, first, a sheet is used for covering a wound by transplanting it to the wound site, The serous membrane, A cell sheet laminated on the transplantation surface side of the serous membrane; The present invention provides a wound covering sheet comprising: As the serous membrane used in the wound covering sheet according to the present technology, a peritoneum can be used. The wound covering sheet according to the present technology can be used on sutured or anastomosed wounds. Furthermore, the wound covering sheet according to the present technology can be used on wounds in hollow organs. In this case, the wound covering sheet according to the present technology can be transplanted onto the outer wall of the hollow organ.

[0014] In this technique, the serous membrane and A cell sheet laminated on the transplantation surface side of the serous membrane; The present invention provides a method for covering a wound by transplanting a wound treatment sheet comprising the steps of: In the wound covering method according to the present technology, the wound treatment sheet can be transplanted to the wound by suturing it. As the serous membrane used in the wound covering method according to the present technology, a peritoneum can be used. The wound covering method according to the present technology can be used to cover sutured or anastomosed wounds. Furthermore, by using the wound covering method according to the present technology, wounds in hollow organs can be covered. In this case, the wound covering sheet can be transplanted onto the outer wall of the hollow organ. Effect of the Invention

[0015] The wound covering sheet according to the present technology has a suitable thickness and strength because the cell sheet and the serous membrane are laminated. Therefore, the sheet is stable in operability and easy to transplant. In addition, the sheet does not flow out of the wound site, and if necessary, the sheet can be fixed to the wound site by suturing or anastomosis, so that the sheet can be stably attached to the wound site. [Brief description of the drawings]

[0016] [Figure 1] Photographs of the back muscle immediately after transplantation of a wound covering sheet, a cell sheet alone, and a peritoneum alone. [Diagram 2] Photographs of the back muscle 3 days after transplantation of the wound covering sheet, the cell sheet alone, and the peritoneum alone. [Diagram 3] Photographs of the back muscle 7 days after transplantation of the wound covering sheet, the cell sheet alone, and the peritoneum alone. [Figure 4] These are micrographs showing the results of hematoxylin-eosin staining of tissue sections removed 3 and 7 days after surgery. [Diagram 5] These are micrographs showing the results of green fluorescent protein (GFP) staining of tissue sections removed 3 and 7 days after surgery. [Figure 6] These are micrographs showing the results of calretinin staining of tissue sections excised on days 3 and 7 after surgery. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] A preferred embodiment for carrying out the present technology will be described below. Note that the embodiment described below shows an example of a typical embodiment of the present technology, and the scope of the present technology is not to be interpreted narrowly by this.

[0018] The wound covering sheet according to the present technology is a sheet used for covering a wound by transplanting it to the wound, and is composed of (1) a serous membrane and (2) a cell sheet laminated on the transplantation side of the serous membrane. The wound covering sheet according to the present technology has an appropriate thickness and strength because the cell sheet and the serous membrane are laminated. Therefore, the sheet is stable in operability and easy to transplant. In addition, the sheet does not flow out of the wound site, and if necessary, it can be fixed to the wound site by suturing or anastomosis, so that the sheet can be stably attached to the wound site.

[0019] As a result, for example, when the wound covering sheet according to the present technology is used on a sutured or anastomosed wound, the effect of preventing suture failure can be improved compared to when a cell sheet is transplanted alone to the wound. Also, for example, when the wound covering sheet according to the present technology is used on a wound in a hollow organ, the effect of preventing or treating leakage from the wound in the hollow organ can be improved compared to when a cell sheet is transplanted alone to the hollow organ.

[0020] The wound covering sheet according to the present technology may be formed by laminating (1) a serous membrane and (2) a cell sheet, and the lamination method is not particularly limited as long as it does not impair the effect of the present technology. For example, the lamination method may include a method of laminating (1) a serous membrane and (2) a cell sheet as they are, a method of laminating (1) a serous membrane and (2) a cell sheet with an adhesive substance between them, and a method of culturing cells of interest on (1) a serous membrane to prepare a cell sheet on (1) a serous membrane.

[0021] In addition, the wound covering sheet according to the present technology can be formed by laminating a serosal membrane collected in advance and a cell sheet, but it is also possible to collect a serosal membrane from a subject during surgery and use an autologous serosal membrane. For example, when the wound covering sheet according to the present technology is used on a wound in a hollow organ, it is also possible to laminate a serosal membrane such as a peritoneum collected during surgery and a cell sheet prepared in advance to form a wound covering sheet on the spot, and then transplant the wound into the wound during surgery.

[0022] The site to which the wound covering sheet according to the present technology is transplanted may be a wound, and is preferably a sutured or anastomosed wound. If the site is sutured or anastomosed, tissues inflamed by the wound are in close contact with each other, and transplanting the wound covering sheet according to the present technology therein enhances the healing effect. By transplanting the wound covering sheet according to the present technology, the production ability of collagen and the like in the wound is enhanced, remodeling around the wound is promoted, and wound healing is promoted.

[0023] In the present technology, the term "wound" broadly refers to a state in which tissue or organ is injured, and includes, for example, burns, bedsores, contusions, incisions, abrasions, ulcers, surgical wounds, gunshot wounds, blast wounds, stab wounds, impalement wounds, bite wounds, and the like. In the present technology, the term "wound" preferably refers to an incision, surgical wound, stab wound, impalement wound, or bite wound of an organ, and more preferably refers to a surgical wound. In the present technology, the term "surgical wound" refers to a wound formed as a result of a surgical operation, and is, for example, a wound formed using a scalpel, scissors, medical laser, forceps, snare, or the like, but is not limited as long as it is a wound formed by an instrument commonly used in a surgical operation. In the present technology, the term "wound area" refers to the surrounding tissue including the wound area in a tissue or organ, and is, for example, within a radius of 10 cm, 8 cm, 5 cm, 3 cm, or 2 cm from the wound area. The range and shape of the wound area are not limited because they vary depending on the shape of the wound.

[0024] The wound dressing sheet according to the present technology is suitable for use on wounds in hollow organs. By using the wound dressing sheet according to the present technology on wounds in hollow organs, a wound healing effect can be obtained that is strong enough to withstand an increase in pressure inside the lumen. As a result, an effect of preventing or treating leakage from wounds in hollow organs can be obtained.

[0025] The site to which the wound covering sheet according to the present technology is transplanted may be the inner and / or outer wall of a hollow organ, or from the standpoint of ease of transplantation during surgery, it may be only the outer wall.

[0026] In the present technology, the term "hollow organ" refers to an organ having a luminal structure, such as the digestive tract, vascular system, bladder, vagina, etc., and is preferably the digestive tract. In the present invention, the digestive tract refers to an organ that runs from the oral cavity to the anus, such as the esophagus, stomach, small intestine, and large intestine.

[0027] In this technology, "leakage" from a wound in a hollow organ refers to the leakage of the contents of the hollow organ to the outside of the hollow organ. For example, when the hollow organ is the digestive tract, it refers to the leakage of solid matter such as food and digested products, liquids such as gastric juice and saliva, and gases such as air from the wound into the thoracic or abdominal cavity.

[0028] In particular, when cancer that has developed in a hollow organ, such as the digestive tract, has infiltrated deep into the digestive tract, it is necessary to resect a part of the digestive tract. In this case, it is necessary to suture or anastomose the remaining digestive tract. As described above, the sutured or anastomosed part of the digestive tract may suffer from suture failure, which causes leakage of contents, due to various reasons. In this technology, the wound covering sheet according to the present technology is transplanted to the sutured or anastomosed wound in a hollow organ, particularly the digestive tract, to promote healing of the wound and prevent or heal suture failure.

[0029] Furthermore, the use of the wound dressing sheet of this technology shows significantly higher pressure resistance than when a cell sheet is transplanted into a luminal organ alone, and maximizes tissue repair ability, bringing about groundbreaking effects in preventing or curing gastrointestinal anastomotic leakage caused by various factors that impede the healing process (repair phase) of anastomotic tissue. This can contribute to reducing the significant decline in patient QOL caused by additional treatments such as artificial anus and enterostomy caused by anastomotic leakage, as well as social losses such as the investment of a large amount of medical resources.

[0030] As described above, the wound covering sheet according to the present technology does not flow from the wound site, and if necessary, it can be fixed to the wound site by suturing or anastomosis, so that it can be stably attached to the wound site. When the wound covering sheet according to the present technology is suturing or anastomosis to the wound site, the suturing or anastomosis method is not particularly limited as long as it is a method used in conventional surgical operations. The suture used when suturing or anastomosis of the wound covering sheet according to the present technology to the wound site may be either soluble or non-soluble, but from the viewpoint of invasiveness, soluble sutures are preferred. The thickness of the suture may be appropriately selected depending on the size and site of the wound.

[0031] Below, (1) the serous membrane and (2) the cell sheet will be described in detail.

[0032] (1) Serosa The serosal membrane used in the wound covering sheet according to the present technology can be freely selected from serosal membranes that can be harvested from a living body depending on the location of the wound, etc., as long as the effect of the present technology is not impaired. For example, serosal membranes such as peritoneum, pleura, and pericardium can be used in the wound covering sheet according to the present technology.

[0033] In this technology, the origin of the animal species from which the serosal membrane is collected may be, for example, mammals such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or immunodeficient animals thereof, birds, reptiles, amphibians, fish, insects, etc. When the wound covering sheet according to this technology is used for treating humans, it is preferable to use a serosal membrane derived from humans, when it is used for treating pigs, when it is used for treating monkeys, it is preferable to use a serosal membrane derived from chimpanzees. In addition, when the treatment is performed on humans, the serosal membrane may be collected from the patient himself (autotransplantation), or may be collected from another person (allotransplantation).

[0034] (2) Cell Sheets The cell sheet used in the wound covering sheet according to the present technology can be any general cell sheet that is freely selected depending on the location of the wound, etc., as long as it does not impair the effect of the present technology. More specifically, a sheet consisting of one or more layers of sheet-like cell groups obtained by culturing on a cell culture substrate and peeling off from the cell culture substrate can be used.

[0035] Examples of methods for obtaining a cell sheet include a method in which cells are cultured on a stimuli-responsive culture substrate coated with a polymer whose molecular structure changes in response to stimuli such as temperature, pH, and light, and the surface of the stimuli-responsive culture substrate is changed by changing the conditions of stimuli such as temperature, pH, and light to change the surface of the stimuli-responsive culture substrate, thereby peeling the cells from the stimuli-responsive culture substrate in a sheet form while maintaining the adhesion state between the cells, and a method in which cells are cultured on an arbitrary culture substrate and physically peeled from the edge of the cell culture substrate using tweezers or the like. A preferred embodiment includes a method in which a temperature-responsive culture substrate coated with a polymer whose hydration force changes in a temperature range of 0 to 80 ° C. is used as the stimuli-responsive culture substrate. The method is a method in which cells are cultured on a temperature-responsive culture substrate in a temperature range in which the hydration force of the polymer is weak, and then the culture solution is changed to a temperature in which the hydration force of the polymer is strong, thereby culturing the cells, and the cells are peeled off in a sheet form and collected. At that time, the cells are cultured on a cell culture substrate coated with a polymer whose hydration force changes in a temperature range of 0 to 80 ° C., in a temperature range in which the hydration force of the polymer is weak. The temperature range is usually the temperature at which cells are cultured, for example, preferably 33° C. to 40° C. The temperature-responsive polymer used in the present technology may be either a homopolymer or a copolymer.

[0036] An example (temperature-responsive culture dish) will be described using poly(N-isopropylacrylamide) as a stimuli-responsive polymer, particularly a temperature-responsive polymer. Poly(N-isopropylacrylamide) is known as a polymer with a lower critical dissolution temperature of 31°C. In a free state, it dehydrates in water at temperatures of 31°C or higher, causing the polymer chains to aggregate and become cloudy. Conversely, at temperatures below 31°C, the polymer chains become hydrated and dissolved in water. In this technology, this polymer is coated and fixed on the surface of a substrate such as a petri dish. Therefore, at temperatures above 31°C, the polymer on the surface of the culture substrate also dehydrates in the same way, but since the polymer chains are fixed to the surface of the culture substrate, the surface of the culture substrate becomes hydrophobic. Conversely, at temperatures below 31°C, the polymer on the surface of the culture substrate becomes hydrated, but since the polymer chains are coated on the surface of the culture substrate, the surface of the culture substrate becomes hydrophilic. The hydrophobic surface at this time is a moderate surface to which cells can attach and grow, and the hydrophilic surface is a surface to which cells cannot attach. Therefore, when the substrate is cooled to 31° C. or lower, the cells are detached from the substrate surface. If the cells are cultured to confluence over the entire culture surface, a cell sheet can be collected by cooling the substrate to 31° C. or lower. The temperature-responsive culture dish is not limited as long as it has the same effect, and examples thereof include UpCell (registered trademark) available from CellSeed Inc.

[0037] In the present technology, the origin of the animal species of the cells used in the cell sheet can be, for example, mammals such as humans, rats, mice, guinea pigs, marmosets, rabbits, dogs, cats, sheep, pigs, goats, monkeys, chimpanzees, or immunodeficient animals thereof, birds, reptiles, amphibians, fish, insects, etc. When the wound covering sheet according to the present technology is used for treating humans, it is preferable to use cells derived from humans, when it is used for treating pigs, when it is used for treating monkeys, it is preferable to use cells derived from chimpanzees. In addition, when the treatment is performed on humans, the cells may be taken from the patient himself (autologous cells), may be taken from the cells of another person (allogeneic cells), or may be a commercially available cell line.

[0038] In this technology, cells used for cell sheets include, for example, germ cells (sperm, eggs, etc.), somatic cells that make up a living organism, stem cells, progenitor cells, cells (cell lines) that have been isolated from a living organism and acquired immortalization ability and stably maintained outside the body, cells that have been isolated from a living organism and artificially genetically modified, and cells that have been isolated from a living organism and whose nuclei have been artificially exchanged.

[0039] In the present technology, it is preferable to use mesenchymal stem cells. Mesenchymal stem cells are cells isolated from tissues in vivo, such as bone marrow, adipose tissue, umbilical cord blood, dental pulp, synovium, and placenta, and can be isolated by known methods.

[0040] For example, bone marrow-derived mesenchymal stem cells can be isolated as adhesive cells by separating blood cells from bone marrow fluid taken from the bone marrow using density gradient centrifugation, seeding them on plastic culture dishes, and culturing them in an environment of 37°C and 5% CO2.

[0041] Adipose tissue-derived mesenchymal stem cells are prepared by mincing collected adipose tissue, digesting the tissue with collagenase type II at 37°C for 1 hour, adding medium and centrifuging. The precipitated cells are then washed with basal medium, filtered through a mesh such as a cell strainer, seeded on a plastic culture dish, and cultured at 37°C in a 5% CO2 environment, allowing the cells to be isolated as adhesive cells. Methods for isolating mesenchymal stem cells derived from other tissues may be any known method and are not limited thereto.

[0042] The mesenchymal stem cells may be mesenchymal stem cells obtained by inducing differentiation from pluripotent stem cells. In the present technology, pluripotent stem cells are cells that have self-renewal and multi-differentiation abilities, and are capable of forming all the cells that make up the body (pluripotency). Self-renewal ability refers to the ability to create two undifferentiated cells identical to itself from one cell. The pluripotent stem cells used in the present technology include embryonic stem cells (ES cells), embryonic carcinoma cells (EC cells), trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), embryonic germ cells (EG cells), multipotent germline stem cells (mGS cells), induced pluripotent stem cells (iPS cells), and the like.

[0043] The origin of the mesenchymal stem cells used in the present technology is not limited, but bone marrow or adipose tissue is preferred, as collection and isolation methods are widely established. Furthermore, adipose tissue is preferred because a larger number of mesenchymal stem cells can be collected compared to bone marrow.

[0044] In the present technology, when a cell sheet using mesenchymal stem cells is used, it may contain cells other than mesenchymal stem cells. For example, vascular endothelial cells, vascular endothelial precursor cells, fibroblasts, epithelial cells, interstitial cells, etc. can be appropriately selected depending on the transplantation site and purpose and used in combination with mesenchymal stem cells. In addition, it may contain cells derived from the tissue from which the mesenchymal stem cells were collected.

[0045] In the present technology, the number of cells contained in the cell sheet can be changed depending on the size and degree of the wound to be covered. The percentage of cells contained in the cell sheet is not limited, and can be, for example, 30% or more, 40% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 93% or more, 95% or more, 97% or more, 98% or more, or 99% or more. The higher the percentage of cells contained in the cell sheet, the higher the effect of covering the wound. For example, when the wound covering sheet according to the present technology is used on a sutured or anastomosed wound, the effect of preventing suture failure can be improved. For example, when the wound covering sheet according to the present technology is used on a wound of a hollow organ, the effect of preventing or treating leakage from the wound of the hollow organ can be improved.

[0046] In this technology, the number of cells to be seeded to create a cell sheet varies depending on the animal species and cell type. For example, 0.3 × 10 4 ~10×10 6 pieces / cm 2 may be 0.5 x 10 4 ~8×10 6 pieces / cm 2 may be 0.7 x 10 4 ~5×10 6 pieces / cm 2In the present technology, in order to detach and recover a cell sheet from a temperature-responsive culture substrate, the temperature of the culture substrate to which cells have attached and become confluent or subconfluent is set to be equal to or higher than the upper critical dissolution temperature or lower than the lower critical dissolution temperature of the coating polymer, thereby obtaining a cell sheet. In this case, the cell sheet can be prepared in a culture solution or in another isotonic solution, and can be selected according to the purpose. In order to detach and recover a cell sheet more quickly and efficiently, a method of tapping or shaking the culture substrate, a method of stirring the medium using a pipette, a method of using tweezers, and the like may be used alone or in combination. Culture conditions other than temperature may be in accordance with conventional methods. For example, the medium used may be a medium containing a known serum such as fetal bovine serum (FBS), or a serum-free medium.

[0047] In the present technology, the shape of the cell culture substrate when preparing a cell sheet can be, for example, a dish, a multi-plate, a flask, a flat membrane, etc. Materials for the cell culture substrate include compounds such as glass, modified glass, polystyrene, polymethyl methacrylate, and polycarbonate that are generally used in cell culture, and substances that can generally be shaped, such as polymer compounds other than those mentioned above, and ceramics.

[0048] In the present technology, the cell culture substrate used in preparing the cell sheet may be a cell culture substrate having both a cell-adherent region and a cell-non-adherent region on the same culture surface. For example, by using a cell culture substrate having a plurality of circular cell-adherent regions and other cell-non-adherent regions on the same culture surface, it is possible to prepare a plurality of cell sheets at once. In this case, the shape of the cell-adherent region may be a desired shape such as a circle, a square, a triangle, a rectangle, etc., depending on the purpose, and the size of the region may be appropriately changed. There is no particular limitation on the method of providing the cell-non-adherent region, but examples include a method of coating with a hydrophilic polymer such as poly-N-acryloylmorpholine, polyacrylamide, polydimethylacrylamide, polyethylene glycol, cellulose, etc., which is a cell-non-adherent polymer with low affinity to cells, or a strongly hydrophobic polymer such as a silicone polymer or a fluorine polymer.

[0049] In the present technology, the method of detaching the cells from the cell culture substrate when preparing a cell sheet is not particularly limited as long as it does not impair the effects of the present technology. For example, a general method such as a method of detaching and recovering the cells using an enzyme can be used, but it is preferable to use a method of culturing the cells on a stimuli-responsive substrate and detaching the cells with little damage.

[0050] The cell sheet used in the wound covering sheet according to the present technology may be a layered cell sheet in which multiple cell sheets are layered. In the present technology, when a layered cell sheet is used, the number of cells to be applied is increased, and the effect of covering the wound can be improved. For example, when the wound covering sheet according to the present technology is used on a sutured or anastomosed wound, the effect of preventing suture failure can be improved. In addition, for example, when the wound covering sheet according to the present technology is used on a wound in a hollow organ, the effect of preventing or treating leakage from the wound in the hollow organ can be improved.

[0051] Methods for obtaining a layered cell sheet include a method of sucking up a cell sheet floating in a culture medium together with the culture medium using a pipette or the like, releasing it onto a cell sheet in another culture dish, and layering it by the flow of the liquid medium, and a method of layering it using a cell migration tool. In the present technology, the method of using a cell migration tool is preferable because it allows layering without damaging the cell sheet. The cell migration tool may be any tool that has the function of capturing a cell sheet, and for example, polyvinylidene difluoride (PVDF), silicone resin, polyvinyl alcohol, urethane, cellulose and its derivatives, chitin, chitosan, collagen, gelatin, fibrin gel, etc. may be used as the material. The cell migration tool may be, for example, in the form of a stamp, a membrane, a porous membrane, a nonwoven fabric, or a woven fabric. In an embodiment of the present technology, the cell migration tool may be any tool that has the function of recovering a cell sheet without damaging it and layering it on another cell sheet, and is preferably a cultured cell migration tool having a cell attachment part consisting of one or more of cell adhesive proteins, cell adhesive peptides, or hydrophilic polymers. For example, a stamp-type cultured cell migration tool having a cell attachment portion is preferred. The stamp-type cultured cell migration tool prevents the cell sheet from being damaged by the cell attachment portion, and enables recovery while preventing the cell sheet from shrinking when the cell sheet is peeled off from the culture dish. The cell migration tool allows a cell sheet to be easily moved onto another cell sheet and stacked without shrinking the cell sheets. By stacking the cell sheets without shrinking them, the cell sheets are stacked without gaps, and a stacked cell sheet having a high-density three-dimensional structure can be obtained.

[0052] In this technology, when preparing the cell sheet, the cells may be further cultured in a medium supplemented with ascorbic acid (Reference: Kato Y, et al. Allogeneic Transplantation of an Adipose-Derived Stem Cell Sheet Combined With Artificial Skin Accelerates Wound Healing in a Rat Wound Model of Type 2 Diabetes and Obesity. Diabetes. 2015 Aug; 64(8): 2723-34). Cell sheets obtained by culturing in a medium supplemented with ascorbic acid are stronger and less likely to tear than cell sheets obtained by culturing without the addition of ascorbic acid. This results in a cell sheet that is more suitable for transplantation.

[0053] The cell sheet used in the wound covering sheet according to the present technology may further contain angiogenesis-inducing factors. Examples of angiogenesis-inducing factors include vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), angiopoietin, platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β), matrix metalloproteinase (MMP), VE-cadherin, ephrin, plasminogen activator, inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), placenta growth factor (PlGF), etc. By including these in the cell sheet, angiogenesis at the transplant site is also promoted. EXAMPLES

[0054] The present technology will be described in more detail below based on examples. Note that the examples described below are representative examples of the present technology, and the scope of the present technology is not to be interpreted narrowly by them. Note that the experimental protocol in this example was approved by the Ethics Committee for Animal Experiments at Tokyo Women's Medical University, and was carried out in accordance with the "Guide for the Care and Use of Laboratory Animals" (revised version, 1996) published by the National Institutes of Health (NIH).

[0055] <Animals, reagents, and kits used> ·GFP transgenic rats (Sankyo Lab Services) SD rats (Sankyo Lab Services) Penicillin and streptomycin (Fujifilm Wako Pure Chemical Industries, #168-23191) Fetal bovine serum (FBS; Life Technologies, #10270-106) Trypsin-EDTA(X1) (Fujifilm Wako Pure Chemical Industries, #208-17251) L-Ascorbic acid phosphate magnesium salt n-hydrate (Wako Pure Chemical Industries, #013-19641) Povidone-iodine (Isodine®, Meiji Seika Kaisha, #50400) Collagenase (SERVA, #17465 NB 4G Proved Grade) Distilled water (Otsuka Pharmaceutical) RNeasy® Fibrous Tissue Mini Kit (QIAGEN, #74704) Mitomycin C (Wako Pure Chemical Industries, #134-07911) ·Hepatocyte Growth Factor (Heapapoietin A, Scatter Factor) (HGF) ELISA Kit (antibodies, online.Com, #ABIN367412) ·FGF basic Pig ELISA Kit (Abcam, #ab156467) D-MEM (high glucose) (Fujifilm Wako Pure Chemical Industries, #043-30085)

[0056] <Equipment used> 75cm 2 Flask (BD Falcon, #353810) 3.5cm (35mm) temperature-responsive culture dish (UpCell®) (CellSeed, #CS3007)

[0057] 1. Isolation and culture of adipose-derived stem cells and production of cell sheets (1) Isolation of adipose-derived stem cells Adipose-derived stem cells were isolated according to the method described in the paper by Watanabe N. et al. (Watanabe N., et al. Genetically Modified Adipose Tissue-Derived Stem / Stromal Cells, Using Simian Immunodeficiency Virus-Based Lentiviral Vectors, in the Treatment of Hemophilia B. Hum Gene Ther. 2013 Mar;24(3):283-294.).

[0058] 20g of subcutaneous fat tissue was collected from the inguinal region of a GFP transgenic rat under local anesthesia, with care taken to avoid the inclusion of blood cells as much as possible. The collected adipose tissue was then sterilized with povidone-iodine and washed twice with antibiotic medium (D-MEM containing 1% penicillin-streptomycin). After washing, the tissue was finely divided using scissors on a dish. 4g of each tissue was placed in a 50mL tube, 35mL of antibiotic medium was added, and 1mL of collagenase at a concentration of 0.27 pzu / mL was added to each tube. The tube was shaken at 37℃ and 130 rpm for 1 hour. The tube was then centrifuged at 4℃ and 2000 rpm for 5 minutes. The tube was shaken manually for 30 seconds. The tube was then centrifuged again at 4℃ and 300G for 5 minutes. Large tissue pieces floating on the surface of the tube were removed and passed through a 100μm cell strainer (BD Japan Becton Dickinson, #352360). The cells were then passed through a 40 μm cell strainer (BD Japan Becton Dickinson, #352340) and centrifuged at 1500 rpm at 4°C for 5 minutes. The supernatant was removed and the pellet was suspended in a medium containing 10% FBS and then filtered through a 75 cm 2 The cells were seeded into five flasks and cultured in an incubator at 37°C.

[0059] (2) Cultivation of adipose-derived stem cells and production of cell sheets The medium was replaced on the third day after cell seeding, and on the fifth day, the cells were detached with 0.25% trypsin and plated on ten 75 cm plates.2 The cells were subcultured in a flask. After 2-3 days, the cells were subcultured again. After 2-3 days, the cells were detached again with 0.25% trypsin, counted, and then 2.3 × 10 6 The cells were suspended in 2 mL of medium, seeded on a 35 mm UpCell (registered trademark), and cultured for 2 days at 37°C. After 2 days, the medium was replaced with a medium containing 16.4 μg / mL ascorbic acid. After another 2 days, the medium was replaced with a medium containing ascorbic acid, and the cells were collected in a sheet form by incubating for 20 to 30 minutes in a 20°C incubator just before transplantation of the cell sheet.

[0060] (3) Preparation of wound covering sheet Peritoneum was collected from SD rats and layered on the cell sheet prepared above to prepare a wound covering sheet.

[0061] 2. Transplantation of wound covering sheets and cell sheets The fascia was removed from the central back of SD rats to expose the muscles. The wound covering sheet prepared above was transplanted to the back muscles by suturing them four times. For comparison, the cell sheet alone and the peritoneum alone were also transplanted by attaching them to the back muscles.

[0062] 3. Macroscopic Evaluation The state of the back muscle after transplantation was observed immediately after surgery, 3 days after surgery, and 7 days after surgery. Figure 1 is a photograph of the back muscle immediately after transplantation of the wound dressing sheet, the cell sheet alone, and the peritoneum alone. Figure 2 is a photograph of the back muscle 3 days after transplantation of the wound dressing sheet, the cell sheet alone, and the peritoneum alone. Figure 3 is a photograph of the back muscle 7 days after transplantation of the wound dressing sheet, the cell sheet alone, and the peritoneum alone.

[0063] As shown in Figure 2, on the third day after surgery, the cell sheet remained in both the area where the wound dressing sheet had been transplanted and the area where only the cell sheet had been transplanted, but it was confirmed that the cell sheet had flowed slightly in the area where only the cell sheet had been transplanted.

[0064] As shown in Figure 3, 7 days after surgery, the cell sheet maintained its shape at the site where the wound-covering sheet made of peritoneum and cell sheet was transplanted, but it was confirmed that the cell sheet had almost completely flowed away at the site where only the cell sheet was transplanted. It was also confirmed that the site where the wound-covering sheet made of peritoneum and cell sheet was transplanted had a more stable engraftment than the site where only the peritoneum was transplanted.

[0065] 4. Histological Evaluation The samples were extracted on the 3rd and 7th days after surgery, embedded in compound, frozen in liquid nitrogen, and tissue sections were prepared. Hematoxylin-eosin staining, green fluorescent protein (GFP) staining, and calretinin staining were performed. Figure 4 is a micrograph showing the results of hematoxylin-eosin staining. Figure 5 is a micrograph showing the results of green fluorescent protein (GFP) staining. Figure 6 is a micrograph showing the results of calretinin staining.

[0066] The cell sheet was positive for green fluorescent protein (GFP) staining and calretinin staining. As shown in Figures 5 and 6, even on the 7th day after surgery, it was confirmed that the cell sheet was stably engrafted at the site where the wound covering sheet consisting of the peritoneum and the cell sheet was transplanted.

Claims

1. A sheet for use in covering a wound by transplanting the sheet onto the wound, The serous membrane, an adipose-derived stem cell sheet laminated on the transplantation surface side of the serous membrane; A wound covering sheet comprising:

2. The wound covering sheet according to claim 1 , wherein the serous membrane is a peritoneum.

3. 3. The wound covering sheet according to claim 1, wherein the wound is a sutured or anastomosed wound.

4. The wound covering sheet according to claim 1 , wherein the wound is a wound in a hollow organ.

5. A wound covering sheet described in any one of claims 1 to 4, which is transplanted onto the outer wall of a tubular organ.

6. The serous membrane, an adipose-derived stem cell sheet laminated on the transplantation surface side of the serous membrane; A method for covering a wound (excluding methods for treating humans), comprising transplanting a wound treatment sheet comprising the steps of:

7. 7. The method for covering a wound according to claim 6, wherein the wound treatment sheet is transplanted to the wound by suturing.

8. The method of claim 6 or 7, wherein the serous membrane is a peritoneum.

9. 9. The method of claim 6, wherein the wound is a sutured or anastomosed wound.

10. The method for covering a wound according to claim 6 , wherein the wound is a wound in a hollow organ.

11. The method for covering a wound according to claim 10, wherein the wound treatment sheet is transplanted onto the outer wall of the hollow organ.

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