JP5157139B2 - Cell transplant material - Google Patents

Description

  The present invention relates to a cell transplantation member that is used for cell processing, transplantation, and the like and that can be transplanted while maintaining cell activity, and a cell transplantation device that uses the cell transplantation member.

  In recent years, with the rapid development of medicine along with the development of science, it has now reached a level where almost all organs can be transplanted. Although the development of organ transplantation technology plays a very important role in improving human health, there is a chronic shortage of organ donations, technical difficulties in organ transplantation surgery, high costs, and the use of immunosuppressants. There are limits to the benefit of all humanity from problems such as side effects. Under such circumstances, tissue engineering for researching a method for artificially creating a biological tissue or organ has been developed, and as a result of the research, in scaffolds made from various polymer materials, A method for creating necessary tissues and organs by differentiating and proliferating cells derived from living tissues has been proposed.

  As such a material, for example, a material using a polymer gel has been proposed. It was found that a polymer gel was used as the polymer material, and that the polymer gel was formed into a three-dimensional shape, so that the cells could be cultured three-dimensionally, but were cultured inside the three-dimensional polymer gel. It was difficult to culture the cells uniformly and use them for transplantation. In contrast, cells can be cultured in a uniform sheet form on a membrane made of a biocompatible material derived from a living body such as amniotic membrane. However, those using such a membrane need to be attached to a culture vessel in order to maintain the smoothness of the membrane, or when transferred to another culture vessel to process cultured cells, or When taking out from a culture container for transplantation to a living tissue or the like, deformation or rupture of the membrane occurs, making it difficult to use the cultured cells. Therefore, Patent Document 1 proposes a method for facilitating cell culture using the membrane by proposing an apparatus for fixing the membrane.

  According to this method, cells can be used for transplantation of the epidermis, etc. by culturing the cells in a sheet form, but the cells required for transplantation are not only in sheet form such as the epidermis, for example, It was not cultured in a pattern such as a vascular network such as a blood vessel or a neural network, or cells were simply cultured and assembled, but cultured to express the required function at each transplant destination. Things are required. However, no prior literature has been found that provides a transplantation member that can pattern cells and express their functions in this way, and that can be used for transplantation while maintaining their patterns and functions.

JP 2005-348736 A

  Accordingly, the present invention provides a cell transplantation member that enables functionalization of cell pattern culture, expression of functions, etc., and allows functionalized cells to be transplanted without causing a rejection reaction with a recipient. And a cell transplantation device using the cell transplantation member.

  To achieve the above object, the present invention provides a cell culture membrane comprising a cell culture substrate having a cell functionalized layer on at least one surface, and maintains the flatness of the cell culture membrane during cell culture and cell transplantation. A cell transplantation member comprising: a fixing part that is configured to provide a cell transplantation member, wherein the cell culture membrane and the fixing part are made of a biocompatible material.

  According to the present invention, by having the cell functionalization layer, it is possible to culture and transplant cells so as to functionalize them. Further, since the cell culture membrane is fixed by the fixing portion, the cell culture membrane can be taken out without being deformed or broken when taken out from the cell culture container. Furthermore, the cell culture membrane and the fixing part are made of a biocompatible material, thereby causing an excessive inflammatory reaction or undesirable cell death with a transplanted body tissue such as a living tissue or an artificially cultured / manufactured cell tissue. Transplantation can be possible.

  In the said invention, it is preferable that the said cell functionalization layer has a pattern culture function which can culture a cell in a pattern form. On the cell functionalized layer, cells such as vascular networks such as blood vessels and lymph vessels, and neural networks can be cultured in a pattern. For example, vascular cells cultured in a pattern on a cultured cell tissue or the like can be By transplanting a tissue or a transplanted tissue such as an artificially cultured or produced tissue, it is possible to supply nutrients or the like to the transplanted body or discharge wastes. Therefore, the transplanted body can constantly maintain a desired function.

  Moreover, in the said invention, it is preferable that the said cell culture membrane and the said fixing | fixed part consist of bioabsorbable materials. This is because the cell culture membrane and the fixing part are finally unnecessary substances in the process of tissue regeneration after transplantation. Compared to non-absorbable biocompatible materials, the bioabsorbable material decomposes and deteriorates in the transplanted body after it is transplanted into a transplanted body tissue such as living tissue or artificially cultured / manufactured cell tissue. The cell tissue of the transplanted body is less likely to be recognized as a foreign substance excessively, and has the advantage that the proliferation of the cell tissue is hardly inhibited.

  Furthermore, in the above-mentioned invention, it is preferable to have a support part that is detachable from the fixing part and that prevents the cell culture membrane from coming into contact with the inner wall of the culture vessel during cell culture. For example, when it is necessary to co-culture using feeder cells at the time of cell culture, the problem that the feeder cells are contaminated with cells cultured on the cell culture membrane can be prevented. This is because, when the cells cultured on the membrane are transplanted into the transplanted body, the support can be removed to perform transplantation without causing any damage such as contact with the transplanted body and causing damage.

  The present invention provides a cell transplantation kit comprising the cell transplantation member and a culture plate, wherein the cell culture membrane does not contact the inner wall of the culture plate.

  According to the present invention, the cell culture membrane of the cell transplant member of the present invention is not in contact with the inner wall of the culture plate, so that the cells can be placed on the cell culture membrane under stable conditions without vibrations or the like. It can be cultured. In addition, when cells are cultured on the cell culture membrane and the inner wall of the culture plate, the cells cultured on the cell culture membrane are not contaminated with the cells cultured on the inner wall of the culture plate. Cells can be co-cultured on the cell culture membrane and on the inner wall of the culture plate.

  The present invention provides a cell transplantation device, wherein cells adhere to the cell functionalization layer formed on at least one surface of the cell culture membrane of the cell transplantation member of the present invention.

  According to the present invention, since the cell culture membrane is fixed so as to maintain flatness, the cell culture membrane can be stably cultured on the cell culture membrane. Can be transplanted without loss of pattern or function.

  The present invention provides a method for producing a cellular tissue, characterized by using the above-mentioned cell transfer / transplantation device.

  According to the present invention, the above-mentioned cell transplantation device can be transplanted in a state where the cell culture pattern, the expressed function, etc. are maintained, thereby combining the cells cultured in the pattern, the cells maintaining the function, etc. Cell tissue can be easily manufactured.

  The present invention provides a cell transplantation member that enables functionalization such as cell pattern culture and expression of functions, and that allows functionalized cells to be transplanted without causing a rejection reaction with a recipient, and There is an effect that a cell transplantation device using the cell transplantation member is provided.

  The present invention enables functionalization such as cell pattern culture and expression of functions, and allows functionalized cells to be transplanted with a recipient without causing rejection or undesirable excessive inflammatory reaction. The present invention relates to a cell transplantation member, a cell transplantation device and a cell transplantation kit using the cell transplantation member, and a method for producing a cell tissue using the cell transplantation device. Hereinafter, the cell transplantation member, cell transplantation kit, cell transplantation device, and cell tissue production method of the present invention will be described in detail.

A. Cell Transplant Member First, the cell transplant member of the present invention will be described. The member for cell transplantation of the present invention comprises a cell culture membrane comprising a cell culture substrate having a cell functionalized layer on at least one surface, and a fixing portion that maintains the flatness of the cell culture membrane during cell culture and cell transplantation The cell culture membrane and the fixing part are made of a biocompatible material.

  Next, the cell transplanting member of the present invention will be described with reference to the drawings. FIG. 1 is a schematic view showing an example of a cell transplanting member of the present invention. As shown in FIG. 1, the cell transplant member 10 of the present invention includes a cell culture membrane 3 comprising a cell culture substrate 1 and a cell functionalized layer 2 formed on the cell culture substrate 1. And a fixing part 4 for fixing the cell culture membrane 3.

  Since the cell transplant member of the present invention has the cell functionalization layer, it can be cultured and transplanted to functionalize cells. In addition, since the cell culture membrane is fixed by the fixing portion, the cell culture membrane can be taken out without being deformed or broken when taken out from the cell culture container. Furthermore, since the cell culture membrane and the fixing part are made of a biocompatible material, transplantation can be performed without causing a rejection reaction or an undesirable excessive inflammatory reaction with a cell or tissue at the transplant destination. Here, the biocompatibility is a property that is difficult to be recognized as a foreign substance when it comes into contact with a biological tissue or cell. Therefore, a cell transplantation member composed of a cell culture membrane and a fixing part made of a biocompatible material may cause a rejection reaction with a transplanted body tissue such as a living tissue or an artificially cultured / manufactured cell tissue. Inflammatory reaction and thrombus formation can be suppressed. The biocompatible material used in the present invention is not particularly limited as long as it has the biocompatibility.

  The cell transplant member of the present invention comprises a cell culture membrane and a fixing part. Hereafter, each structure of the member for cell transplantation of this invention is demonstrated in detail.

1. Cell Culture Membrane First, the cell culture membrane used for the cell transplant member of the present invention will be described. The cell culture membrane used in the present invention is composed of a cell culture substrate having a cell functionalized layer on at least one surface, and is composed of a biocompatible material.

(1) Cell functionalization layer The cell functionalization layer in the cell culture membrane used for this invention is demonstrated. The cell functionalization layer used in the present invention is formed on the cell culture substrate, and can impart a function necessary for transplantation to a living tissue or the like to a cell, and is a biocompatible material. If it consists of, it will not specifically limit.

  Examples of the biocompatible material used for such a cell functionalized layer include collagen, fibrin, gelatin, albumin, starch, hyaluronic acid, chitin, dextran, poly-β-hydroxybutyrate, nucleic acid (DNA, RNA). Natural polymer materials such as, polyglutamic acid and other polypeptides, polyglycolic acid, polylactic acid, polymalic acid, polylactone and other polyesters, poly (oxycarbonyloxyethylene) and other polycarbonates, polyorthoesters, poly-α-cyano Acrylate, polyphosphazene, polyphosphate ester, silicone, polypropylene, polymethyl methacrylate, polytetrafluoroethylene, polyvinyl chloride, polyethylene terephthalate, polysulfone, segmented polyurethane, high A synthetic polymer material such as droxyethyl methacrylate (HEMA) -styrene-HEMA triblock copolymer can be used. A hydrophilic polymer material can also be used. For example, thermosensitive polymers such as poly (N-isopropylacrylamide), polyalkylene glycols such as polyethylene glycol and polyethylene glycol-polypropylene glycol block copolymers and their derivatives, oligoethylene glycol methacrylate-methacrylic acid copolymers, Examples include zwitterionic polymers such as MPC polymer (trade name) having a phospholipid polar group.

  In the present invention, the biocompatible material is preferably made of a bioabsorbable material. Here, the bioabsorbability is a property of being degraded or degraded enzymatically or non-enzymatically in the living body, and the degradation product is dissolved in a body fluid and metabolized or excreted. Therefore, after transplanted into a transplanted body such as a living tissue or an artificially cultured / manufactured cell tissue, it is desirable that the degradation in the transplanted body is degraded and lost, and the body tissue is less likely to be recognized as a foreign substance. This is because it rarely causes excessive inflammation and inhibits proliferation of cell tissues. Among the biocompatible materials described above, those having bioabsorbability include collagen, fibrin, gelatin, albumin, starch, hyaluronic acid, chitin, dextran, poly-β-hydroxybutyrate, nucleic acid (DNA, RNA) and the like. Natural polymer materials, polypeptides such as polyglutamic acid, polyglycolic acid, polylactic acid, polymalic acid, polyesters such as polylactone, polycarbonates such as poly (oxycarbonyloxyethylene), polyorthoesters, poly-α-cyanoacrylates, Examples thereof include synthetic polymer materials such as polyphosphazenes and polyphosphate esters.

  The cell functionalization layer used in the present invention makes it possible to give a cell a function necessary for transplantation into a living tissue or the like. The function imparted here is not particularly limited as long as it is necessary for transplantation. For example, a pattern culture function capable of culturing cells in a pattern, a function of normally proliferating cells, a desired function, etc. Examples thereof include a function for expressing a function and a function for inducing desired differentiation.

In the present invention, it is preferable to have a pattern culture function capable of culturing cells in a pattern. Since the cell functionalization layer has a pattern culture function, cells such as blood vessels and lymphatic vessels such as vascular networks and neural networks can be cultured in a pattern, for example, cultured in a patterned cell tissue or the like. By transplanting the vascular cells into a living tissue or a transplanted tissue such as an artificially cultured / manufactured cell tissue, it is possible to obtain a transplanted body that can supply nutrients, discharge waste, etc. it can. Therefore, it is possible to make the transplanted body capable of constantly maintaining a desired function.
The type of pattern is not particularly limited as long as it is a two-dimensional pattern. For example, the shape of a line, tree (tree), mesh, grid, circle, square pattern, circle, square, or other graphic A pattern in which cells are arranged throughout the interior can be formed.

  As a method for imparting such a pattern culture function, for example, as shown in FIG. 2, the cell functionalization layer 2 has a cell adhesion region 2a having adhesion to cells and cell adhesion inhibition having no cell adhesion. The method which consists of the area | region 2b is mentioned. When the cell functionalized layer has a cell adhesion region and a cell adhesion inhibition region, cells can be adhered and cultured only on the cell adhesion region, and cells can be transplanted in a pattern.

  A method for forming a cell adhesion region and a cell adhesion inhibition region in order to impart a pattern culture function to the cell functionalized layer is not particularly limited. For example, a contact angle for adjusting a contact angle with water Examples include an adjustment method, a hydration ability adjustment method for adjusting hydration ability, a binder application method for giving a binder substance capable of binding to cells in a pattern, and a shape adjustment method for adjusting the surface according to an uneven shape. .

  As the contact angle adjusting method, the pattern culture function is imparted by setting the contact angles of the cell adhesion region and the cell adhesion inhibition region with water to be different. Here, the cell adhesion region used for culturing and transplanting cells is appropriately selected depending on the type of cells to be transplanted, etc., but the contact angle with water on the cell functionalized layer surface is usually normal. It is preferable that the region be in the range of about 10 ° to 60 °, and more preferably in the range of 15 ° to 55 °. This is because by having such a contact angle with water, the cells can be adhered and cultured on the cell functionalization layer and transplanted to the transplanted body while being active. In addition, when the contact angle with water is larger than the above range, it is possible to adhere the cells on the cell functionalized layer, but because of the high adhesion with the cell functionalized layer, after transplantation, When the cell transplant member of the present invention is composed of a bioabsorbable material, it takes a long time to disassemble the cell transplant member or the like, and it takes time to fuse and integrate the transplanted tissue with the cell tissue. There is a risk of causing such harmful effects. Further, when the contact angle with water is smaller than the above range, it is difficult to adhere the cells on the cell functionalized layer. In addition, when the width | variety of the pattern which adheres a cell is a line or a dot of 1 mm or less, especially the contact angle with the water of the said cell adhesion area | region surface is in the range of 10 degrees-45 degrees, especially 15 degrees- It is preferable to be within a range of 35 °. This is because, within such a range, cells can be transplanted in a high-definition pattern.

  As a method of forming the cell adhesion region and the cell adhesion inhibition region in the cell functionalized layer, for example, after forming a layer having adhesion inhibition properties with cells, only the portion to be the cell adhesion region is made of water. Examples thereof include a method of changing the contact angle, and a method of changing the contact angle with water only in a portion to be a cell adhesion inhibition region after forming a cell adhesive layer. For example, after the cell functionalization layer is formed using the above-described material, only the part to be the cell adhesion region or the part to be the cell adhesion inhibition region is reduced in the contact angle with water. For example, a method of increasing the contact angle with water only for a portion to be a cell adhesion region or only a portion to be a cell adhesion inhibition region after forming a cell functionalization layer using a material.

  Examples of the method for increasing the contact angle of the cell functionalized layer with water as described above include, for example, a method of forming fine irregularities on the surface of the cell functionalized layer. For example, the document Japanese Journal of The method disclosed in Applied Physics, Part 2, 32, L614-L615, 1993 Ogawa et al. Can be used.

  In addition, as a method of reducing the contact angle of the cell functionalized layer with water, for example, a method of decomposing or modifying a group having liquid repellency using the action of a photocatalyst accompanying energy irradiation, heat irradiation, ultraviolet irradiation Examples thereof include a method of decomposing or modifying a group having liquid repellency by light irradiation, electron beam irradiation, or the like. As described above, in the case where a photocatalyst is contained in the cell functionalization layer, the contact angle between the cell functionalization layer and water is reduced using the action of the photocatalyst in the cell functionalization layer. be able to. When the cell functionalization layer does not contain a photocatalyst, for example, a patterning method using a photocatalyst described in JP-A-2001-249219 or JP-A-2003-222626 is described. The contact angle of the cell functionalized layer with water can be reduced using a patterning method using a photocatalyst. Further, patterning by heat irradiation, ultraviolet light irradiation, electron beam irradiation, or the like can be performed in the same manner as a general method of patterning using these energies, and detailed description thereof is omitted here.

As the hydration ability adjustment method, a pattern culture function is imparted by setting the hydration ability of the cell adhesion region and the cell adhesion inhibition region to be different from each other. Here, the cell adhesion region is appropriately selected depending on the type of cells to be transferred, etc., but is formed from a material having a low hydration ability, and the cell adhesion inhibition region is formed from a material having a high hydration ability. The By using a material having high hydration ability, a hydrated layer in which water molecules gather around the material is formed. Such a substance with high hydration ability has a higher affinity with water molecules than with cells, so cells cannot adhere to the above-mentioned material with high hydration ability, and cell adhesion inhibition region It becomes. Here, the hydration ability means the property of hydrating with water molecules, and the high hydration ability means that it easily hydrates with water molecules.
In addition, as a method of reducing the hydration ability of the portion to be the cell adhesion region of the cell functionalized layer, for example, a method using the action of a photocatalyst similar to that used for adjusting the contact angle, Examples thereof include a method of decomposing or modifying a group having hydration ability by ultraviolet light irradiation, electron beam irradiation, or the like.

As the binder application method, usually, a region to which a binder substance is applied functions as a cell adhesion region, and a region to which no binder substance is applied functions as a cell adhesion inhibition region. As such a patterning method, for example, after forming a cell adhesion-inhibiting region on the entire surface of the cell functionalization layer, a binder substance may be applied in a desired pattern, or a binder substance may be applied to the entire surface of the cell functionalization layer. After application, a method using the action of a photocatalyst similar to that used for adjusting the contact angle, a method of decomposing or modifying the binder material by heat irradiation, ultraviolet light irradiation, electron beam irradiation, etc., a binder material by an ink jet method And the like.
Here, the binder substance is not particularly limited as long as it can bind to cells, and examples thereof include sugar chains and proteins that can bind to receptors expressed on the cell surface. In the present invention, it is preferable to use a binder substance that can specifically bind to a receptor expressed in a cell-specific manner. By patterning the binder substance that performs such cell-specific binding on the cell functionalized layer, for example, it is possible to perform patterning on the same cell functionalized layer without mixing two or more different cells. Because.

  As the shape adjustment method, as described later, a concavo-convex shape is imparted to the surface of the cell culture substrate on which the cell functionalization layer is formed, and for example, the cell functionalization layer is formed only on the convex portion or only on the concave portion. The cells can be formed and cultured in a high-definition pattern and transferred.

  In the present invention, the pattern culture function may be formed by one type of method described above, or may be a combination of two or more types.

Moreover, the cell functionalization layer used in the present invention may have a cell regulatory function such as a function of normally proliferating cells, a function of expressing a desired function, a function of inducing a desired differentiation. The method for imparting such a cell regulatory function is not particularly limited as long as it does not affect the transplanted body side when transplanted to the transplanted body. A method of imparting a physiologically active substance to the surface is mentioned. Here, the physiologically active substance is a substance having some biological function, for example, not only an antibody, an adhesion molecule, a cytokine, a growth factor, an enzyme inhibitor or a receptor, but an amino acid, an oligopeptide, a polypeptide, Various substances from low molecular weight substances to high molecular weight substances such as proteins, nucleotides, oligonucleotides, polynucleotides, glycoproteins, monosaccharides, polysaccharides, and vitamins can be used. In the present invention, any of the above physiologically active substances may be used, and can be appropriately selected according to the required function. In the present invention, one type of the above physiologically active substance may be used, or two or more types may be combined.
In addition, these physiologically active substances are all bioabsorbable, and after being transplanted to the transplanted body, they are degraded and deteriorated in the transplanted body and then disappear, and foreign substances are present in the cell tissue of the transplanted body. It is less likely to be recognized as, is less likely to cause undesirable excessive inflammation, and is less likely to inhibit cell tissue growth. Moreover, since it has bioabsorbability, the said physiologically active substance naturally has biocompatibility.

Furthermore, in the present invention, when the cell functionalization layer transplants cells, the cell functionalization layer may have a cell transplantation assisting function that promotes the adhesion and integration of the cells with the cell tissue of the transplanted body. This is because by having such a cell transplantation assisting function, it is possible to more efficiently integrate the transplanted cells with the cell tissue on the transplanted body side. Examples of a method for imparting such a cell transplantation assisting function include a method of imparting a substance that promotes adhesion of the transplanted cell tissue to the cell tissue to the cell functionalized layer. As a substance having such a cell transplantation assisting function, a substance that does not affect the recipient side is selected, for example, a polypeptide such as collagen, a polysaccharide such as hyaluronic acid, polylactic acid, polyglycolic acid, alginic acid, Examples thereof include polyvinyl alcohol and polylysine. The cell functionalization layer may contain metal ions such as calcium ions and magnesium ions.
Such substances having cell transplantation assisting functions are all bioabsorbable, and after being transplanted into the transplanted body, they degrade and disappear in the transplanted body. The tissue is not recognized as a foreign substance, and there is no fear of inhibiting the growth of cellular tissue. Moreover, since it has bioabsorbability, the substance having the cell transplantation assisting function naturally has biocompatibility.

  The cell functionalization layer used in the present invention may have one type of function, or a combination of two or more types.

  The method for forming the cell functionalized layer is appropriately selected according to the type of the cell functionalized layer, the shape of the cell transplanting member, and the like. Usually, the roll coating method, the spin coating method, and the die coating method are used. A wet coating method such as a bead coating method, an adsorption method, an alternate adsorption method, a soft lithography method, or the like can be used.

  Here, the thickness of the cell functionalization layer as described above is appropriately selected depending on the type of the cell functionalization layer and the like, but is usually in the range of 1 nm to 1000 nm, preferably 1.5 nm to A range of 750 nm is preferable, and a range of 20 nm to 500 nm is particularly preferable. This is because, by setting the thickness of the cell functionalized layer within the above range, cells can be stably cultured and transplanted.

(2) Cell culture substrate The cell culture substrate in the cell culture membrane used in the present invention will be described. The cell culture substrate used in the present invention is particularly suitable if it can form the above-mentioned cell functionalized layer uniformly and can be sterilized by a known method, and further comprises a biocompatible material. It is not limited. As a known sterilization method, for example, a method of sterilizing by irradiating γ rays, a method of sterilizing by irradiating ultraviolet light, a method of sterilizing by irradiating an electron beam, a method of sterilizing by an autoclave, a method of sterilizing by alcohol, The method etc. which sterilize with ethylene oxide gas are mentioned.

  In the present invention, a biocompatible material is used as the material used for the cell culture substrate, and a bioabsorbable material is particularly preferable. In the transplanted body, it degrades and deteriorates, is rarely recognized as a foreign substance in living tissue, rarely causes unwanted excessive inflammation, and hardly inhibits proliferation of cellular tissue. is there. In the present invention, as the biocompatible material and the bioabsorbable material used as the material for the cell culture substrate, the same materials as described in “(1) Cell functionalized layer” can be used.

  Here, although the thickness of the cell culture substrate described above varies depending on the material to be selected, the range of 1 μm to 250 μm is preferable, and the range of 5 μm to 200 μm is particularly preferable. When the thickness of the cell culture substrate is thinner than the above range, the flexibility is so high that handling becomes very difficult, the strength is insufficient, and it may be easily torn or torn. is there. When the thickness is larger than the above range, a very long time is required for decomposition even if the tissue regeneration after transplantation is inhibited or the cell culture substrate is made of a bioabsorbable material. It is necessary.

The cell culture substrate used in the present invention may have a fine porous structure. The cells that adhere to the cell culture membrane because the cell culture substrate has a fine porous structure are factors necessary for obtaining a desired function from the cell-adhered surface side of the cell culture membrane. Etc. can be supplied. This is because, for example, epithelial cells such as cells having different functions can be cultured on the front and back of the cells.
In addition, there is an effect that the culture solution and oxygen are uniformly distributed to all cells during the cell transplantation operation.

  When the cell culture substrate used in the present invention has a porous structure, the average size of the pores constituting the porous structure depends on the cell type, but is usually 0.1 μm to 3.5 μm. It is preferable that the thickness is 0.2 μm to 1.5 μm. This is because when the size of the pores constituting the porous structure is less than 0.1 μm, the supply efficiency of nutrients and liquid factors to cultured cells is deteriorated. In addition, when the size of the pores constituting the porous structure exceeds 3.5 μm, the proportion of cultured cells adhering to the inside of the pores and the back surface of the cell culture substrate increases, and different cells are cultured on both sides of the cell culture membrane. Or culture in a desired pattern.

  In the present invention, the cell culture substrate may have such a fine porous structure as, for example, a non-woven fabric obtained by processing the above-described biocompatible material or bioabsorbable material into fibers. And a method of providing fine through holes in the film.

  In addition, as shown in FIG. 3, for example, the cell culture substrate 1 used in the present invention is a cell culture substrate 1 in which concave and convex portions are formed in a pattern in which the cell functionalized layer 2 is formed. There may be. In this case, cells can be cultured in a high-definition pattern by forming a cell functionalization layer only on the convex part or only on the concave part. The width of such recesses and projections is usually about 10 μm to 500 μm, and in particular, about 20 μm to 200 μm. In addition, the height of the concave portion or the convex portion can be about 0.1 μm to 100 μm, and more preferably about 0.2 μm to 20 μm. In the present invention, the unevenness of the cell culture substrate is defined in the above range because, if it is larger than the above range, the thickness of the concave portion of the cell culture substrate becomes too thin, so that the strength is insufficient and can be easily torn or torn. It is because there is a case where it is.

(3) Cell culture membrane The cell culture membrane used in the present invention comprises the cell culture substrate and a cell functionalized layer formed on at least one surface of the cell culture substrate, and is biocompatible. It consists of materials.

  The cell culture membrane used in the present invention comprises the cell culture substrate and a cell functionalized layer formed on at least one surface of the cell culture substrate, and is made of a biocompatible material. For example, the structure is not particularly limited, and the cell functionalization layer may be provided on both sides. In this way, cells can be cultured and transplanted in a pattern on both sides of the cell culture membrane, or one side can be used as a cell cultured in a pattern, and one side can be used as a cell with a desired function. it can. Such a configuration is appropriately selected depending on the use of the cell transplant member.

In the present invention, the shape and size of the cell culture membrane are not particularly limited, and can be selected according to the use of the cell transplant member of the present invention.
In addition, in order to efficiently perform cell culture (adhesion) on the cell culture membrane, a ring-shaped ring having an appropriate height is in close contact with the cell functionalization layer until the cell adheres to the cell functionalization layer surface. You may enclose it. The shape of the enclosure is not particularly limited, but an O-ring shape is preferable. The material of such an enclosure is not particularly limited as long as it can be sterilized by a known method. For example, it is preferably made of a silicone resin. This is because the silicone resin has good adhesion to the cell functionalization layer. Moreover, although the height of the said enclosure can be selected according to the quantity of the culture medium to be used, about 2 mm-20 mm are preferable.

2. Fixed part Next, the fixed part used for the member for cell transplantation of the present invention is explained. The fixing unit used in the present invention fixes the cell culture membrane, maintains the flatness of the cell culture membrane during cell culture, and removes the cell culture membrane when removing it from the cell culture container. The material is not particularly limited as long as it can be taken out without causing deformation or breakage and is made of a biocompatible material.

As such a fixing portion, as shown in FIG. 4, a ring-shaped one connected to either side of the cell culture membrane, as shown in FIG. 5, on either side of the cell culture membrane. As shown in FIG. 6, the fixed portion 4 is a separable member made up of an upper fixed portion 4a and a lower fixed portion 4b, and the upper fixed portion 4a and the lower fixed portion 4b. The cell culture membrane 3 is disposed between the upper and lower fixing portions 4a and 4b, and the cell culture membrane 3 is fixed. In addition, as the separable fixing portion composed of the upper fixing portion and the lower fixing portion, as shown in FIG. 7, the cell culture is provided in the gap between the ring-shaped upper fixing portion 4a and the lower fixing portion 4b. It is good also as what fixes the film | membrane 3 so that it may be wound.
In the present invention, a ring-shaped member connected to any surface of the cell culture membrane as shown in FIG. 4 is particularly preferable. This is because the proportion of the fixing portion in the cell transplanting member of the present invention can be reduced, so that the proliferation of cells in the transplanted body is not inhibited. In addition, when a cell functionalized layer is formed on one side of the cell culture membrane, the cell cultured on the cell functionalized layer is applied to the cell culture membrane from the side where the cell functionalized layer is not formed. In the case where it is easy to supply factors necessary for cells or the cell culture membrane has flexibility, the cell culture membrane can be fixed without impairing the flexibility. This is because there is an advantage.

  In the present invention, the fixing method of the cell culture membrane of the fixing portion as shown in FIG. 4 is a method of fixing the cell culture membrane and the fixing portion by thermocompression bonding or fixing via an adhesive. The method of doing is mentioned. In the present invention, it is appropriately selected according to the type of material constituting the cell culture membrane, but when using an adhesive, an adhesive that has been confirmed to be low enough that cytotoxicity does not become a problem. It is necessary to select.

  In the present invention, the material used for the fixing portion has biocompatibility, and among them, a material having bioabsorbability is preferable. As such materials, materials similar to those described in the section “(1) Cell functionalized layer” of “1. Cell culture membrane” can be used, and thus description thereof is omitted here. To do.

3. Cell transplant member The cell transplant member of the present invention comprises a cell culture membrane comprising a cell culture substrate having a cell functionalized layer on at least one surface, and a fixing portion for maintaining the flatness of the cell culture membrane during cell culture. And the cell culture membrane and the fixing part are not particularly limited as long as they are made of a biocompatible material.

The member for cell transplantation of the present invention preferably has a support part that can be detached from the fixing part and that prevents the cell culture membrane from coming into contact with the inner wall of the culture vessel during cell culture. In the present invention, by having the support portion, for example, when co-cultured with feeder cells, the feeder cells on the inner wall of the cell culture container and the cells cultured in the cell transplanting member of the present invention do not come into contact with each other. This is because co-culture can be performed.
As such a support part, as shown in FIG. 8A, a truncated cone-like support part 5 arranged on the entire circumference of the fixed part is formed with the cell functionalized layer 2 of the cell culture membrane 3. Examples include a shape projecting to the opposite side of the surface, and a shape projecting to the surface side where the cell functionalized layer 2 of the cell culture membrane 3 is formed, as shown in FIG. it can. Other shapes include those having spaced columnar support portions. As the separated columnar support portions, those shown in FIG. 9 can be cited. As shown in FIG. 9A, the separated columnar support portions 5 are connected to the fixing portion 4 and extend in a substantially vertical direction of the cell culture membrane 3. FIG. 9B is a schematic view showing a cross section taken along the line AA ′ of FIG. 9A, in which the cell culture membrane is circular and the fixing portion 4 is ring-shaped, and is separated from the space. This shows a case where the columnar support portions 5 are cylindrical and four are arranged. For the stability during cell culture using the cell transplanting member of the present invention, it is preferable that the spaced columnar support portions are composed of three or more.

  In the present invention, the arrangement of the fixing part and the support part can be appropriately selected according to culture conditions and the like. In the present invention, it is preferable that the support portions are arranged in a truncated cone shape as shown in FIG. This is because when the cell transplanting member of the present invention is placed on a culture vessel and the cells are cultured, the stability of the cell transplanting member is improved. In this case, when the plane parallel to the cell culture membrane surface of the fixing section is the fixing section plane 20, the angle 21 formed by the support section 5 and the fixing section plane 20 is preferably in the range of 45 ° to 135 °. The range of 65 ° to 115 ° is particularly preferable. In the present invention, the angle formed by the support part and the fixing part surface of the fixing part is within the above range. If the angle is larger than the above range, the stability of the cell transplanting member at the time of cell culture decreases, This is because if the cell size is smaller than the range, the cell culture membrane becomes too small particularly in the case of a cell transplantation kit described later.

  Further, the support portion used in the present invention has a frustum-like shape arranged on the entire periphery of the fixed portion as shown in FIG. Also good. This is because when cells are cultured on both surfaces of the cell culture membrane of the transplanting member, factors necessary for cell culture are equally supplied to both cells. In addition, when feeder cells are cultured on the bottom surface of the culture container for the purpose of producing factors necessary for cell culture, it is easy to supply nutrients and replace the medium to the feeder cells cultured on the bottom surface of the culture container. This is because the factors produced by the feeder cells cultured on the bottom surface of the culture vessel are supplied to the cells on the cell culture membrane surface. Further, the presence / absence of the hole in the support part, the size of the hole and the like are not particularly limited, and can be selected according to the use and the like of the member for cell transplantation of the present invention.

  In the case where the support part used in the present invention is detachable from the fixing part, a method of making it detachable includes a method of applying an adhesive to the connection part of the fixing part and the support part, or the fixing There is a fitting type method in which one end of the support part is inserted into a hole arranged in the part and fixed. In the present invention, considering that it is a member for culturing cells for cell transplantation, it is preferable to make it detachable by adopting a fitting type in which an adhesive component or the like is not mixed.

  Moreover, the support part used for this invention may be arrange | positioned only on the single side | surface side of the said cell culture membrane surface, and good also as what was arrange | positioned on both surfaces. In the present invention, the one arranged only on one side of the cell culture membrane surface is preferable. This is selected according to the use of the cell transplanting member of the present invention.

  In the present invention, as a material used for the support portion, a material used as a material for a general cell culture instrument can be used. As such a material, specifically, an inorganic material such as glass, metal, silicon, and an organic material typified by plastic can be used. In the present invention, an organic material typified by plastic is preferable. This is because the organic material is easy to process. Moreover, since the said support part is removed and used at the time of a transplant, as a material used for the said support part, it is not necessary to consist of what has biocompatibility and bioabsorbability.

B. Cell transplant kit Next, the cell transplant kit of the present invention will be described. The cell transplantation kit of the present invention comprises the above-described cell transplantation member and a culture plate, and is characterized in that the cell culture membrane does not contact the inner wall of the culture plate. .

  According to the present invention, the cell culture membrane of the cell transplant member and the inner wall of the culture plate are not in contact with each other, so that the cells are cultured on the cell culture membrane under stable conditions free from vibration or the like. be able to. In addition, when cells are cultured on the cell culture membrane and the inner wall of the culture plate, the cells cultured on the cell culture membrane are not contaminated with the cells cultured on the inner wall of the culture plate. Cells can be co-cultured on the cell culture membrane and on the inner wall of the culture plate.

  The cell transplantation kit of the present invention comprises a cell transplantation member and a culture plate. Hereinafter, each component of the cell transplantation kit of the present invention will be described in detail.

1. Cell Transplant Member First, the cell transplant member used in the present invention will be described. The cell transplant member used in the present invention has the same contents as those described in the above section “A. Cell Transplant Member”, and therefore the description thereof is omitted here.

2. Culture plate Next, the culture plate used in the present invention will be described. The culture plate used for this invention will not be specifically limited if the said cell culture membrane and the said culture plate inner wall do not contact. Examples of such a culture plate include a plate that can fix the cell transplant member in a state where the cell culture membrane and the inner wall of the culture plate are not in contact with each other.

  In the present invention, the method for fixing the cell transplantation member to the culture plate is not particularly limited, and examples thereof include a method using an adhesive and a fitting method. In the present invention, in consideration of the fact that cells used for cell transplantation are cultured, a method such as a fitting type is preferred. As such a culture plate, when the cell transplant member has a support portion, for example, as shown in FIG. 10, the support portion of the cell transplant member 10 is shown. An example of the culture plate 31 includes a bottom recess 32 that can be fixed by inserting one end of the plate 5.

  Further, in the present invention, when the culture plate is capable of fixing the cell transplantation member so that the cell culture membrane and the inner wall of the culture plate are not in contact with each other, Cells can be cultured on the cell culture membrane without giving unnecessary vibrations. Further, when cells are cultured both on the cell transplant member and on the bottom of the culture plate, the cell transplant Since the member does not move on the bottom surface of the culture plate, the cells can be co-cultured on the cell transplanting member and on the bottom surface of the culture plate without damaging the cells on the bottom surface of the culture plate.

  In the present invention, the culture plate may be capable of inserting two or more cell transplant members. In this way, the culture plate can be inserted with two or more cell transplanting members when, for example, it is necessary to co-culture two or more cell types without contamination with each other. Useful.

  In the present invention, as a material used for the culture plate, a material used as a base material for a general cell culture instrument can be used. Specifically, inorganic materials such as glass, metal, silicon, and organic materials typified by plastics can be used. If necessary, the bottom surface of the culture plate may be subjected to surface treatment. Examples of such a surface treatment include those that impart functions having the same contents as those of the cell functionalization layer described in “A. Cell transplantation member” described above.

3. Cell Transplant Kit The cell transplant kit of the present invention is not particularly limited as long as it comprises the above-described cell transplant member and culture plate. As such a cell transplantation kit, for example, as illustrated in FIG. 10, the cell transplantation kit 30 of the present invention can fix the cell transplantation member 10 and the cell transplantation member 10 to the bottom surface. And a culture plate 31 having a recess 32. In the case where the support portion of the cell transplant member has a shape protruding to the cell functionalization layer side of the cell culture membrane, as shown in FIG. 11, the cell transplant member 10 and the cell transplant It may consist of a culture plate 31 to which the support part 5 of the member 10 can be fixed. As such a culture plate 31, it is good also as what provided the wall surface recessed part 33 which can be fitted with the said support part 5. FIG. This is because the stability during culture can be increased. Furthermore, in the case where the cell transplant member does not have the support part, as shown in FIG. 12, the cell transplant kit 30 is arranged with the fixing part recess 6 in the fixing part 4. The cell transplant member 10 and a culture plate 31 having a transplant member support 7 that can be fitted into the fixing portion recess 6 on the inner wall may be used. In the case where the transplanting member support part is provided on the inner wall of the culture plate, the transplanting member support part may be the bottom surface of the inner wall of the culture plate as shown in FIG. There may be.

C. Next, the cell transplantation tool of the present invention will be described. The cell transplantation device of the present invention is characterized in that cells adhere to the cell functionalized layer formed on at least one surface of the cell culture membrane of the cell transplantation member of the present invention.

  The cell transplantation device of the present invention will be described with reference to the drawings. FIG. 13 is a schematic view showing an example of the cell transplantation device of the present invention. As illustrated in FIG. 13, the cell transplantation device 50 of the present invention is cultured on the cell transplantation member 10 of the present invention and the cell functionalized layer 2 formed on at least one surface of the cell culture membrane 3. And the cell 40 that has been formed.

  According to the present invention, since the cell culture membrane is fixed so as to maintain flatness, the cell culture membrane can be stably cultured on the cell culture membrane. Can be transplanted without loss of pattern or function. Therefore, it can be set as the cell transplantation tool which can be transplanted in the state which maintained the culture pattern and function of the cell.

  The cell transplantation device of the present invention comprises the above-mentioned cell transplantation member of the present invention and cells. Hereinafter, each configuration of the cell transplantation device of the present invention will be described in detail.

1. Cell Transplant Member The cell transplant member used in the present invention has the same contents as those described in the above section “A. Cell Transplant Member”, and therefore the description thereof is omitted here.

2. Cells In the present invention, the cells to be seeded, adhered and cultured on the cell culture membrane may be floating cells such as blood cells and lymphoid cells, and may be adhesive cells. In the present invention, a cell having adhesiveness is particularly preferable. Examples of such adhesive cells include liver cells that are liver parenchymal cells, vascular endothelial cells, fibroblasts, epidermal cells such as epidermal keratinocytes, tracheal epithelial cells, gastrointestinal epithelial cells, cervical cervix, etc. Epithelial cells, epithelial cells such as lens epithelial cells, mammary gland cells, muscle cells such as smooth muscle cells and cardiomyocytes, kidney cells, pancreatic Langerhans islet cells, nerve cells such as peripheral and optic nerve cells, chondrocytes, bone cells Etc. In addition, these cells may be primary cells directly collected from tissues or organs, or may be obtained by substituting them for several generations. Moreover, the established cell may be sufficient. Furthermore, these cells may be any of ES cells that are undifferentiated cells, pluripotent stem cells having multipotency, unipotent stem cells having unipotency, and cells that have been differentiated. In addition, the cells may be cultured as a single species or as a coculture of two or more cells.

3. Cell transplantation device The cell transplantation device of the present invention is not particularly limited as long as it has cells on at least the cell functionalized layer of the cell transplantation member of the present invention and the cell culture membrane of the cell transplantation member. It is not something. In the present invention, cells may be adhered even on the surface of the cell culture membrane on which the cell functionalization layer is not formed, and the cell functionalization layer is formed on both sides of the cell culture membrane. A cell may be adhered on the cell functionalized layer. The cell transplantation device of the present invention is used for transplanting cells cultured on the cell culture membrane of the above-mentioned cell transplantation member to a transplanted body such as a living tissue or a cell tissue artificially cultured and produced. It becomes.

D. Cell tissue manufacturing method The cell tissue manufacturing method of the present invention is characterized by using the cell transplantation device of the present invention. According to the present invention, by using the above-described cell transplantation device, it is possible to easily produce a cell tissue in which a pattern cultured cell, a cell expressing a desired function, or the like is combined. Therefore, for example, in order to maintain the function as a cell tissue, it is necessary to stretch blood vessels and supply necessary nutrients and discharge waste products. By transplanting cultured cells and constructing a cell tissue that stretches around the vascular network, it can be a cell tissue that can maintain its function constantly even though it is an artificially constructed cell tissue. .

  The method for producing a cell tissue of the present invention is not particularly limited as long as it is a method for producing a cell tissue using the cell transplantation device of the present invention. Examples of such a method include a method comprising a transplantation step of producing a cell tissue by transplanting the above-mentioned cell transplantation device into a transplanted body. Hereinafter, the transplanting process will be described.

1. Transplantation step The transplantation step in the present invention is a step of producing a cell tissue by transplanting the above-mentioned cell transplantation device into a transplanted body such as a living tissue or an artificially cultured and produced cell tissue. In this step, the cell transplantation tool is not particularly limited as long as it can be transplanted into a transplanted body. As such a process, for example, in the case where the transplanted body is a massive cell tissue, and transplanting the inside thereof, as shown in FIG. 14 and an incision location 62 necessary for inserting the cell transplant device 50 into the transplant location 61 are determined (FIG. 14A), and then the incision is made using a sharp knife such as a scalpel. After the incision is made in the location 62 to create the insertion location 63, the cell transplantation device 50 is inserted into the transplantation location 61 (FIG. 14B), and the incision is sutured to form a cellular tissue by a known suturing method ( FIG. 14 (c)) can be mentioned.

  When the transplant is a cell cultured on a cell culture substrate, a cell culture substrate 71 and a cultured cell 72 cultured on at least one side of the cell culture substrate 71 as shown in FIG. The cell culture member 70 and the cell transplant device 50 are arranged so that the cultured cells 72 and the cells 40 on the cell transplant device face each other (FIG. 15 (a)), and then The cell culture member 70 and the cell transplantation device 50 are laminated so that the cultured cells 72 and the cells 40 are in contact with each other (FIG. 15B), and a process of forming a cell tissue can be exemplified. In the case shown in FIG. 15, the number of the cell culture member and the cell transplantation device stacked and the material of the cell culture substrate are appropriately selected depending on the conditions of the transplanted body. For example, the cell culture member and the transplantation device may be alternately laminated in one layer or two or more layers, and the cell culture member may be composed of the cell transplantation device of the present invention.

  The transplant used in this step is not particularly limited, and may be a cell cultured in a sheet shape, a cell cultured in a three-dimensionally arranged state in a polymer gel, or a massive shape. It may be a cell. The transplant is appropriately selected according to the target cell tissue.

  As the cells used in this step, the same cells as those described in the above section “C. Cell transplantation device” can be used, and the description thereof is omitted here.

  In addition, as a cell used for this process, it is not limited to one type, You may be used combining several types of cells.

2. Method for Producing Cell Tissue The method for producing cell tissue of the present invention is not particularly limited as long as it produces cell tissue using the cell transplantation device of the present invention. In the present invention, the above-described cell transplantation device is transplanted into a transplanted body to form a cell tissue, and the operation of transplanting the cells of the above-mentioned cell transplantation device into the transplanted body composed of the cell tissue is performed a plurality of times. Thus, a cell tissue having a higher function can be obtained. In the present invention, by using the above-described cell transplantation device, cells can be transplanted in a pattern or in a state in which the function is maintained, so that a vascular network in which a desired pattern is formed inside the massive cell can be constructed or desired. It becomes possible to produce a cell tissue in which cells having these functions are combined. In addition, after transplantation, it is not necessary to remove the cell transplantation member constituting the cell transplantation device, so that the cell tissue can be produced without inhibiting the growth of the cell tissue or damaging the cell tissue. can do.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  The following examples illustrate the present invention more specifically.

[Example 1]
1. Preparation of Cell Transplant Material A 100-μm-thick gelatin film (25 mmφ) was prepared as a cell culture substrate, and oligoethylene glycol methacrylate (OEGMA, Scientific Polymer Products) and methacryl as a cell functionalization layer on the cell culture substrate. A cell culture membrane having a line pattern of a copolymer with acid (MA, Scientific Polymer Products) was prepared. Hereinafter, a method for producing a cell transplant member will be described.

(1) Preparation of cell functionalized layer Preparation of oligoethylene glycol-methacrylate and methacrylic acid to a mass ratio of 4: 1, and 2,2′-azobis (2-amidinopropane) dihydrochloride as an initiator (Wako Pure Chemical Industries, Ltd.) was used and reacted in methanol at 60 ° C. for 16 hours to obtain a copolymer (poly (OEGMA-co-MA)). A polydimethylsiloxane (PDMS) replica was produced using a silicon master having a groove of 60 μm and a ridge of 20 μm. 10% poly (OEGMA-co-MA) was placed as an ink on the convex part of the PDMS replica and air-dried.

(2) Preparation of cell culture substrate An 8% gelatin solution containing 0.2 M acetic acid is cast in a glass petri dish, dried, chemically crosslinked with 0.5% glutaraldehyde, washed with deionized water, and dried. As a result, a gelatin film having a thickness of 100 μm was prepared and used as a cell culture substrate.

(3) Production of Cell Culture Membrane The PDMS replica poly (OEGMA-co-MA) was contacted with the gelatin film surface, which is the cell substrate produced by the above method, for 3 seconds, and then the PDMS replica was removed.
In this way, a cell culture membrane was obtained in which a poly (OEGMA-co-MA) pattern having a line and space of 20 μm / 60 μm was present as a cell functionalization layer on a gelatin film having a thickness of 100 μm as a cell culture substrate.

(4) Production of cell transplantation member The cell culture membrane produced by the above method was sterilized with 70% ethanol, and the fixing part made of Teflon (registered trademark) was subjected to autoclave sterilization treatment. A one-component RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used for adhesion between the cell culture substrate and the fixing part, and the same configuration as in FIG. In this way, a cell transplant member was produced.
Next, the fixing part and the support part were bonded to the cell transplantation member produced by the above method using a one-component RTV rubber deoxime type. The shape of the support portion is a ring-shaped member that adheres to the lower surface of the fixed portion, and the angle formed by the support portion and the fixed portion is 85 °, and has the same configuration as FIG.

2. Cell culture After placing the transplanting member produced by the above method in a culture plate and surrounding the cell culture membrane with an O-ring, the O-ring touches the medium with HuMedia-EG2 (2% FBS & supplement) manufactured by Kurabo Industries. Until normal human umbilical vein endothelial cells (HUVEC) suspended in the same medium are seeded on the cell functionalized layer in the O-ring so as to be 4.0 × 10 ⁵ cells / cm ² , The cells were cultured for 16 hours with 5% CO ₂ . It was confirmed with a phase contrast microscope that HUVEC was adhered to the cell functionalization layer in a line pattern shape having a width of 20 μm. When the culture was further continued at 37 ° C. and 5% CO ₂ , HUVEC formed a lumen after 5 days. In this way, a cell transplantation device was produced.

3. Transplantation operation The abdomen of a nude mouse (age 10 months, female) was incised with a scalpel, and the support was removed from the cell transplantation device produced by the method described above, and transplanted to the subcutaneous site of the nude mouse and sutured.

4). Evaluation (confirmation of inflammatory reaction at transplant site)
When an inflammatory reaction occurs at the transplanted site, tissue fibrosis is observed. Therefore, as an evaluation method, the tissue at the transplantation site was taken out, a section was prepared, hematoxylidine-eosin staining was performed, and the presence or absence of tissue fibrosis around the transplantation site was observed.
On the 8th day after the transplantation, the tissue at the transplantation site was taken out, a section was prepared, and hematoxylidine-eosin staining was observed. As a result, almost no tissue fibrosis around the transplantation site was observed.

[Example 2]
1. Preparation of Cell Transplant Material Using a 100 μm-thick poly (glycerol-sebacate) as a cell culture substrate, a copolymer of oligoethylene glycol methacrylate and methacrylic acid as a cell functionalization layer is formed on the cell culture substrate. A cell culture membrane having a line pattern was prepared. Hereinafter, a method for producing a cell transplant member will be described.

(1) Preparation of cell functionalization layer preparation Preparation of a cell functionalization layer was prepared in the same manner as in Example 1.

(2) Preparation of cell culture substrate Glycerin (Wako Pure Chemical Industries) and sebacic acid (Tokyo Kasei) were prepared at a molar ratio of 2: 3, and reacted at 120 ° C. for 24 hours in a nitrogen gas atmosphere. The acid was dissolved in glycerin. The prepolymer obtained by the above method is dissolved in 10% tetrahydrofuran, cast into a Teflon (registered trademark) petri dish, and subjected to a dehydration condensation reaction at 120 ° C. for 72 hours while reducing the pressure at 3 mmHg using an oil pump. A cell culture substrate made of 100 μm poly (glycerol-sebacate) was prepared.

(3) Preparation of cell culture membrane After contacting PDMS replica poly (OEGMA-co-MA) for 3 seconds on the surface of the cell culture substrate made of poly (glycerol-sebacate) prepared by the above method, the PDMS replica was Removed.
Thus, a cell culture membrane in which a poly (glycerol-sebacate) with a thickness of 100 μm as a cell culture substrate has a poly (OEGMA-co-MA) pattern with a line and space of 20 μm / 60 μm as a cell functionalized layer. Got.

(4) Production of Cell Transplant Member The cell culture membrane and Teflon (registered trademark) produced by the above method were subjected to autoclave sterilization treatment. A one-component RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used for adhesion between the cell culture substrate and the fixing part, and the same configuration as in FIG. In this way, a cell transplant member was produced.
Next, the fixing part and the support part were bonded to the cell transplantation member produced by the above method using a one-component RTV rubber deoxime type. The shape of the support portion is a ring-shaped member that adheres to the lower surface of the fixed portion, and the angle formed by the support portion and the fixed portion is 85 °, and has the same configuration as FIG.

2. Cell culture After placing the cell transplantation member prepared by the above method in a culture plate and surrounding the cell culture membrane with an O-ring, add a predetermined amount of MEM medium containing 5% serum and suspend it in the same medium. Bovine carotid artery-derived vascular endothelial cells (bAEC) were seeded at 4.0 × 10 ⁵ cells / cm ² and cultured at 37 ° C. and 5% CO ₂ for 16 hours. It was confirmed with a phase contrast microscope that bAEC adhered to the cell transfer layer in a line pattern having a width of 20 μm. When the culture was further continued at 37 ° C. and 5% CO ₂ , bAEC formed a lumen after 5 days. In this way, a cell transplantation device was produced.

3. Transplantation operation The nude mouse (age 10 months, male) was cut open with a scalpel, and the support was removed from the cell transplantation device prepared by the above method, and transplanted to the subcutaneous site of the nude mouse and sutured.

4). Evaluation (confirmation of inflammatory reaction at transplant site)
In the same manner as in Example 1, the presence or absence of fibrosis of the tissue at the transplant site was confirmed.
On the 8th day after the transplantation, the tissue at the transplantation site was taken out, a section was prepared, and hematoxylidine-eosin staining was observed. As a result, almost no tissue fibrosis around the transplantation site was observed.

(Evaluation of bioabsorbability of cell transplant materials)
In addition, as an evaluation of the bioabsorbability of the cell transplantation member, the transplantation tool is taken out after a predetermined number of days after transplantation, washed thoroughly with a phosphate buffer, dried, and then observed for shape using a scanning electron microscope The weight of the transplantation device before and after drying was measured, and the moisture content of the transplantation device was calculated and evaluated. The water content was calculated by the following formula.
Water content = (Wwet−Wdry) / Wdry × 100
Wwet is the weight before the transplant tool is dried, and Wdry is the weight after the transplant tool is dried. Here, it shows that absorption and decomposition | disassembly are progressing, so that a moisture content is low.
In order to evaluate the bioabsorbability of the cell transplantation member, the transplantation device was taken out on the 7, 14, 21, 28, and 35 days after transplantation, washed thoroughly with a phosphate buffer, and dried. When the obtained dried product was observed with a scanning electron microscope, it was confirmed that the size of the transplantation tool was gradually reduced as the number of days elapsed after transplantation increased. The water content decreased linearly as the number of days elapsed after transplantation increased, and was about 20% of the weight immediately after transplantation 35 days after transplantation.

[Example 3]
1. Preparation of Cell Transplantation Member A polyurethane nonwoven fabric was prepared as a cell culture substrate. The fixed part consisting of the cell culture substrate and Teflon (registered trademark) was autoclaved. For adhesion between the cell culture substrate and the fixed part, a one-component RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used. Thereafter, a thin layer of porcine skin-derived type I collagen was provided as a cell functionalization layer on the cell culture substrate to obtain a cell transplantation member. Next, the fixing part and the support part were bonded to the cell transplantation member produced by the above method using a one-component RTV rubber deoxime type. The shape of the support portion is a ring-shaped member that adheres to the lower surface of the fixed portion, and the angle formed by the support portion and the fixed portion is 85 °, and has the same configuration as FIG.

2. Cell culture After placing the cell transplantation member prepared by the above method in a culture plate and surrounding the cell culture membrane with an O-ring, add a predetermined amount of MEM medium containing 5% serum and suspend it in the same medium. Bovine vascular endothelial cells were seeded at 1.3 × 10 ⁶ cells / cm ² and cultured at 37 ° C. and 5% CO ₂ for 16 hours.

3. Transplantation operation The abdomen of a nude mouse (age 1 year, 2 months, male) was incised with a scalpel and the support removed from the cell transplantation device prepared by the above method was transplanted to the subcutaneous site of the nude mouse and sutured. .

4). Evaluation (confirmation of inflammatory reaction at transplant site)
On the 14th day after transplantation, the tissue at the transplant site was taken out and a section was prepared. Subsequently, in order to confirm the presence or absence of fibrosis, observation was performed with Azan staining, and almost no tissue fibrosis was observed around the transplant site.

It is the schematic which shows an example of the member for cell transplantation of this invention. It is the schematic which shows an example of the cell culture membrane used for this invention. It is the schematic which shows the other example of the cell culture membrane used for this invention. It is the schematic which shows an example of the fixing | fixed part used for this invention. It is the schematic which shows the other example of the fixing | fixed part used for this invention. It is the schematic which shows the other example of the fixing | fixed part used for this invention. It is the schematic which shows the other example of the fixing | fixed part used for this invention. It is the schematic which shows an example of the support part used for this invention. It is the schematic which shows the other example of the support part used for this invention. It is the schematic which shows an example of the kit for cell transplantation of this invention. It is the schematic which shows the other example of the kit for cell transplantation of this invention. It is the schematic which shows the other example of the kit for cell transplantation of this invention. It is the schematic which shows an example of the cell transplantation tool of this invention. It is the schematic which shows an example of the manufacturing method of the cell tissue of this invention. It is the schematic which shows the other example of the manufacturing method of the cell tissue of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Cell culture base material 2 ... Cell functionalization layer 2a ... Cell adhesion area | region 2b ... Cell non-adhesion area | region 3 ... Cell culture membrane 4 ... Fixed part 4a ... Upper side fixing | fixed part 4b ... Lower side fixing | fixed part 5 ... Supporting part 6 ... Fixed Portion concave part 7 ... Transplanting member support 10 ... Cell transplantation member 20 ... Supporting part fixing part surface 21 ... Angle formed by supporting part and fixing part surface 30 ... Cell transplantation kit 31 ... Culture plate 32 ... Bottom concave part 33 ... Wall surface Recess 40 ... Cell 50 ... Cell transplantation tool 60 ... Cell tissue 61 ... Transplantation site 62 ... Incision site 63 ... Insertion site 70 ... Cell culture member 71 ... Cell culture substrate 72 ... Cell

Claims (10)

A cell transplantation member comprising a cell culture membrane comprising a cell culture substrate having a cell functionalized layer on at least one surface, and a fixing part for maintaining flatness of the cell culture membrane during cell culture and cell transplantation. there are, the cell culture membrane and the fixing unit, Ri Do from a biocompatible material,
A member for cell transplantation , wherein the biocompatible material is a bioabsorbable material . The cell transplantation member according to claim 1, wherein the cell functionalization layer has a pattern culture function capable of culturing cells in a pattern. The support part according to claim 1 or 2 , further comprising a support part that is detachable from the fixing part and that prevents the cell culture membrane from coming into contact with an inner wall of a culture vessel during cell culture. Cell transplant member. The cell transplantation member according to claim 3 , wherein the fixing part and the support part are detachable by being fitted. The cell fixing member according to any one of claims 1 to 4 , wherein the fixing portion has a ring shape. The cell transplant member according to any one of claims 1 to 5 , wherein the fixing portion is formed on any surface side of the cell culture membrane. The cell transplantation member according to any one of claims 1 to 6, wherein the cell culture membrane has flexibility. A cell transplantation kit comprising the cell transplantation member according to any one of claims 1 to 7 and a culture plate, wherein the cell culture membrane does not contact the inner wall of the culture plate. Cell transplant kit. A cell is adhered to the cell functionalization layer formed on at least one surface of the cell culture membrane of the cell transplantation member according to any one of claims 1 to 7. Cell transplantation tool. A method for producing a cell tissue, wherein the cell transplantation device according to claim 9 is used.

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