JP4967520B2 - Cell transfer material - Google Patents

Description

  The present invention relates to a cell transfer member that is used for cell processing, transplantation, and the like, and that enables cells to be transferred while being active, and a cell transfer tool using the cell transfer member.

  In recent years, a technique of transplanting a cultured and organized cell as it is attracting attention. This is because artificial skin and artificial blood vessels using synthetic polymers may cause rejection after transplantation and are difficult to use. This is because there is no concern about rejection and can be preferably used for transplantation.

  However, the above cultured cell tissue or the like cultured on a cell culture substrate has a problem that it is difficult to transplant it to a living tissue, for example, or to transfer it to another culture substrate for processing. This is because, for example, when recovering the cultured cell tissue cultured in a pattern on the cell culture substrate, it is difficult to peel only the cultured cell tissue from the cell culture substrate.

  Thus, for example, a method has been proposed in which the cultured cell tissue is recovered by peeling off from the cell culture substrate using, for example, a proteolytic enzyme or a chemical. However, in this method, the cells may be denatured or damaged by the above chemicals or enzymes, and the original function of the cells may be impaired. In addition, there are problems such as complicated processing steps and possible contamination.

Patent Document 1 also discloses a method in which a cell culture support in which a pattern made of a thermosensitive polymer is formed on a base material, cells are cultured on the cell culture support, and then the cell tissue is detached. Proposed. According to this method, by changing the temperature of the temperature-sensitive polymer while the cells cultured on the temperature-sensitive polymer are in close contact with, for example, a polymer membrane, a sheet-like cell (cell Sheet) is peeled from the thermosensitive polymer, and the cell sheet can be brought into close contact with the polymer membrane by the action of the surface tension of the culture solution. After that, the cell sheet held on the polymer membrane side is brought into close contact with the culture substrate, biological tissue, etc., so that the cell sheet can be transferred to another culture substrate or transplanted onto the biological tissue. is there. However, in this method, when the size of the cell sheet is very small, or when the cells are cultured in a sparse state or in a state where small colonies are formed, the effect of surface tension with the polymer membrane is exerted. There was a problem that it was not sufficient, and cells could not be brought into close contact with the polymer membrane side, for example, by sliding on the polymer membrane. In addition, when cells cultured in a pattern, such as vascular networks and nerve networks such as blood vessels and lymphatic vessels, are closely attached to the polymer membrane, the cells in the pattern flow through the polymer membrane. There was a problem that these patterns could not be maintained.
JP 2003-38170 A

  Accordingly, the present invention provides a cell transfer member capable of transferring a pattern-cultured cell while maintaining the pattern, and a cell transfer device using the cell transfer member. Is the main purpose.

  In order to achieve the above object, the present invention provides a cell transfer membrane comprising a cell transfer substrate having a cell transfer layer on one side, a fixing portion for maintaining flatness of the cell transfer membrane during cell culture, There is provided a cell transfer member characterized by comprising a gripping part connected to a fixing part and disposed on a surface side of the cell transfer film where the cell transfer layer is not formed.

  According to the present invention, since the cell transfer membrane is fixed by the fixing portion, the cell transfer membrane can be taken out without being deformed or broken when taken out from the cell culture container.

  Moreover, by having the said holding part, the cell cultured on the said cell transcription | transfer layer was culture | cultivated on the said member for cell transfer by tweezers etc. by holding the said holding part and transferring. When cells are transferred, it is possible to prevent the transfer target such as a living tissue from being damaged by the tip of the tweezers that holds the cell transfer member.

  In the said invention, it is preferable that the said cell transcription | transfer layer has a pattern culture function which can culture a cell in pattern shape. Capable of culturing cells such as vascular networks and nerve networks such as blood vessels and lymphatic vessels in a pattern, for example, by transferring vascular cells cultured in a pattern to cultured cell tissues, etc. This is because it becomes possible to supply waste materials, discharge waste products, etc., and to produce cell tissues and the like having their functions. Moreover, it is because it becomes possible to apply a cultured cell to an artificial organ, a biosensor, a bioreactor, etc. by carrying out pattern culture | cultivation so that a cell may adhere | attach only on a micro part and may be arranged.

  Moreover, in the said invention, it is preferable that the said cell transcription | transfer layer has a bioactive substance on the surface of the said cell transcription | transfer layer. Since the cell transfer layer has the physiologically active substance on the surface of the cell transfer layer, it can regulate the growth, differentiation, function, etc. of cells cultured on the cell transfer layer. This is because the cells given can be cultured and used for transcription.

  Furthermore, in the said invention, it is preferable that the said cell transfer base material is a fine porous structure. Since the cell transfer substrate has a fine porous structure, cells adhered to the cell transfer layer can obtain a desired function from the side of the cell transfer film where the cell transfer layer is not formed. You can receive necessary factors. This is because, for example, epithelial cells such as cells having different functions can be cultured on the front and back of the cells.

  Still further, in the above invention, it is preferable that the gripping part is detachable from the fixing part. Since the gripping part is detachable from the fixing part, for example, when cells cultured in the cell transfer layer are transferred to a transfer target such as a living tissue, gauze or the like is sterilized until the transfer is completed. This is because, in such a case, by making the gripping part detachable, the covering with the protective material is not hindered.

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

  According to the present invention, the cell transfer film of the cell transfer member of the present invention is not in contact with the inner wall of the culture plate, whereby the cells are adhered to the inner wall on the cell transfer layer of the cell transfer film. And can be cultured under stable conditions. In the case of culturing cells on the cell transfer layer and the inner wall of the culture plate, cells cultured on the cell transfer layer are not contaminated with cells cultured on the inner wall of the culture plate. Cells can be co-cultured on the cell transfer layer and on the inner wall of the culture plate.

  The present invention provides a method for culturing cells, characterized by using the cell transfer member of the present invention.

  According to the present invention, by using the cell transfer member of the present invention, the surface of the cell transfer membrane on which the cell transfer layer is not formed does not come into contact with the inner wall of the cell culture container. Cells can be cultured above. Thus, the cells can be cultured from the side of the cell transfer membrane where the cell transfer layer is not formed while supplying factors necessary for the cells to obtain a desired function. Therefore, it is possible to culture cells having different functions on the front and back of the cell, such as epithelial cells. In addition, by using the above-described cell transfer tool, it is possible to easily transfer cultured cells to a transfer target such as a living tissue.

  The present invention provides a cell transfer tool, wherein cells are adhered on the cell transfer layer of the cell transfer member of the present invention.

  According to the present invention, since the cell transfer layer is fixed so as to maintain flatness, the cell transfer layer can be stably cultured on the cell transfer layer. It can be transferred without impairing the pattern or function.

  The present invention provides a method for producing a cell tissue, characterized by using the above-described cell transfer device of the present invention.

  According to the present invention, the cell transfer tool can transfer a cell culture pattern and a function in a maintained state, thereby facilitating a cell tissue in which a pattern cultured cell and a cell having a function maintained are combined. Can be manufactured.

  INDUSTRIAL APPLICABILITY The present invention has an effect of providing a cell transfer member that enables transfer while maintaining a cell culture pattern and function, and a cell transfer tool using the cell transfer member.

  The present invention relates to a cell transfer member that enables transfer while maintaining a cell culture pattern and function, a cell transfer tool using the cell transfer member, a cell transfer kit, and a cell culture method And a method for producing a cellular tissue using the cell transfer tool. Hereinafter, the cell transfer member, cell transfer kit, cell culture method, cell transfer tool, and cell tissue manufacturing method of the present invention will be described in detail.

A. Cell Transfer Member First, the cell transfer member of the present invention will be described. The cell transfer member of the present invention includes a cell transfer film having a cell transfer layer on one side, a fixing part that maintains the flatness of the cell transfer film during cell culture, the cell transfer film connected to the fixing part, and the cell And a gripping part disposed on the side of the transfer film on which the cell transfer layer is not formed.

  Next, the cell transfer member of the present invention will be described with reference to the drawings. FIG. 1 is a schematic cross-sectional view showing an example of the cell transfer member of the present invention. As shown in FIG. 1, the cell transfer member 10 of the present invention includes a cell transfer film 3 comprising a cell transfer substrate 1 and a cell transfer layer 2 formed on the cell transfer substrate 1, and the above A fixing part 4 for fixing the cell transfer film 3; and a grip part 5 connected to the fixing part 4 and disposed on the surface of the cell transfer film 3 where the cell transfer layer 2 is not formed. Is.

  In the cell transfer member of the present invention, since a cell transfer layer having cell transferability is formed, cells cultured on the cell transfer layer are directly transferred to a transfer medium such as a cell culture substrate or a living tissue. Can be transferred. Further, since the cell transfer membrane is fixed by the fixing portion, the cell transfer membrane can be taken out without causing deformation or breakage when taken out from the cell culture container.

  In addition, since the cell transfer member of the present invention has the grip portion, in the transfer operation of the cells cultured on the cell transfer member, by holding the grip portion, for example, tweezers When the cells cultured on the cell transfer member are transferred by, for example, it is possible to prevent the transfer target body such as a biological tissue from being damaged by the tip of the tweezers that grips the cell transfer member.

  The cell transfer member of the present invention comprises a cell transfer film, a fixing part, and a grip part. Hereafter, each structure of the member for cell transfer of this invention is demonstrated in detail.

1. Cell Transfer Membrane First, the cell transfer membrane used for the cell transfer member of the present invention will be described. The cell transfer membrane used in the present invention is composed of a cell transfer substrate having a cell transfer layer on one side.

(1) Cell transfer layer The cell transfer layer in the cell transfer film used in the present invention will be described. The cell transfer layer used in the present invention is formed on the cell transfer substrate, and can be cultured with the cells adhered to the surface, and the cultured cells can be cultured in the culture pattern. In addition, transfer is performed on a transferred body such as another cell culture substrate or living tissue without impairing the function.

  In the present invention, cell transferability means that cells cultured and adhered on a cell transfer layer are brought into contact with the transfer target while being adhered on the cell transfer layer, and after a predetermined time, A property that can be transferred to a transfer target. In the present invention, at least 48% of the cells adhered on the cell transfer layer are transferred to the transferred body within 48 hours at the latest after the cells cultured on the cell transfer layer are brought into contact with the transferred body. When done, the cell transfer layer shall have cell transferability.

  The cell transferability is evaluated by bringing the cells adhered on the cell transfer layer into contact with the transferred body for a predetermined time, peeling off the cell transfer layer, and contacting the cell transfer layer and the transferred body through the cells. The number of cells present on the surface of the cell transfer layer and the transfer target in the predetermined region can be evaluated by observing with a commercially available microscope. When the transferred body is a living tissue or the like, it may be difficult to observe the transferred body under a microscope.In this case, the number of cells in a predetermined region of the cell transfer layer before the transfer operation is counted in advance, After cell transfer, evaluation can be performed by counting the number of cells remaining in the region of the cell transfer layer.

  Further, the cell transfer layer used in the present invention preferably has a time required to transfer 80% or more of the cells cultured on the cell transfer layer within 24 hours, preferably within 6 hours. Is more preferable.

  The cell transfer layer is not particularly limited as long as it is made of the material having the cell transfer property. Examples of such a material include a material containing fluorine and oxygen, and specific examples include ionic fluoropolymers such as Nafion (trade name), fluorine-based silane coupling agents, fluoroalkyl chains and hydroxyl groups. And a gel containing ethylene glycol-based silane coupling agent.

  Moreover, a hydrophilic polymer material can also be used as the cell transfer layer. Specifically, thermosensitive polymers such as poly (N-isopropylacrylamide), polyalkylene glycols such as polyethylene glycol and polyethylene glycol-polypropylene glycol block copolymers and their derivatives, MPC polymers having phospholipid polar groups ( And zwitterionic polymers such as trade name).

  Furthermore, the cell transfer layer may contain a photocatalyst. Specifically, the cell transfer layer is a layer made of a mixture of the cell transferable material and photocatalyst fine particles, or the cell transfer layer is composed of a photocatalyst-containing layer containing at least a photocatalyst and the cell transferability. A layer composed of two layers, such as a layer composed of a material comprising In this case, the photocatalyst used can be the same as that described in, for example, JP-A-2001-249219.

The cell transfer layer used in the present invention is not particularly limited as long as it has cell transferability, and may have other functions. In the present invention, it is preferable to have a pattern culture function capable of culturing cells in a pattern. Capable of culturing cells such as vascular networks and nerve networks such as blood vessels and lymph vessels in a pattern, for example, transferring vascular cells cultured in a pattern to cultured cell tissues, thereby providing nutrients to cell tissues, etc. This is because it becomes possible to supply waste materials, discharge waste products, and the like, and it becomes possible to produce cell tissues and the like that retain their functions. In addition, by culturing a pattern so that cells are adhered and arranged only at minute portions, the cultured cells can be applied to artificial organs, biosensors, bioreactors, and the like.
As a method for imparting such a pattern culture function, for example, as shown in FIG. 2, the cell transfer layer 2 has a cell adhesion region 6 having adhesion to cells and a cell adhesion inhibition region having no cell adhesion. And a method consisting of 7. When the cell transfer 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 the cells can be transferred in a pattern. The cell adhesion region formed in the cell transfer layer may be one place or a plurality of places. The shape is not particularly limited, and various modes such as a circle, a polygon, a line, a line having a branch, a network, a ring, and a combination of these may be considered.

  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 transfer layer is not particularly limited. For example, contact angle adjustment for adjusting the contact angle with water Examples thereof include a method for adjusting the hydration ability, a method for adjusting the hydration ability, a binder application method for applying a binder substance capable of binding to cells in a pattern, and a shape adjustment method for adjusting the surface according to the concavo-convex 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 transferring the cells is appropriately selected depending on the type of cells to be transferred and the like. Usually, the contact angle with water on the surface of the cell transfer layer is 10. It is preferable that the region is in the range of about 60 ° 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 to the cell transfer layer, cultured, and transferred to the transfer target while being active. When the contact angle with water is larger than the above range, it is possible to adhere the cells on the cell transfer layer, but it becomes difficult to transfer, and when the contact angle with water is smaller than the above range. This is because it becomes difficult to adhere cells on the cell transfer layer. Note that the contact angle with water on the surface of the cell adhesion region is in the range of 10 ° to 45 °, particularly 15 ° to 45 °, particularly when the width of the pattern for transferring cells is a line or dot having a width of 1 mm or less. It is preferable to be within a range of 35 °. This is because, within such a range, it becomes possible to transfer cells in a highly fine pattern.

  In addition, when the cell transfer layer has a cell adhesion inhibition region, the cell adhesion inhibition region is about 10 ° to 40 ° from the contact angle of the cell adhesion region with water, particularly about 15 ° to 35 ° with water. It is preferable that the contact angle is small. This is because in such a region, the adhesiveness with cells can be poor. In addition, a region having a large contact angle with water of about 50 ° to 160 °, particularly about 60 ° to 150 °, than the contact angle with water of the cell adhesion region, can also have poor adhesion to cells, This region can be a cell adhesion inhibition region.

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

  Examples of the method for increasing the contact angle of the cell transfer layer with water as described above include, for example, a method of forming fine irregularities on the surface of the cell transfer layer. For example, the document Japanese Journal of Applied Use the method disclosed in Physics, Part 2, 32, L614-L615, 1993 Ogawa et al., And the material disclosed in Langmuir, 19: 10624-10627, 2003 Teshima et al. Can do.

  In addition, as a method of reducing the contact angle of the cell transfer 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 light Examples include a method of decomposing or modifying a group having liquid repellency by irradiation, electron beam irradiation, or the like. As described above, when the photocatalyst is contained in the cell transfer layer, the contact angle between the cell transfer layer and water can be reduced by utilizing the action of the photocatalyst in the cell transfer layer. . When the cell transfer 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. By using a patterning method using a photocatalyst, the contact angle of the cell transfer layer with water can be reduced. 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 for reducing the hydration ability of the portion to be used as the cell adhesion region of the cell transfer layer, for example, a method using the action of a photocatalyst similar to that used for adjusting the contact angle, heat irradiation, ultraviolet irradiation, Examples thereof include a method of decomposing or modifying a group having hydration ability by 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 inhibition region on the entire surface of the cell transfer layer, a binder substance may be applied in a desired pattern, or a binder substance is applied on the entire surface of the cell transfer layer. Thereafter, a method using the action of a photocatalyst similar to that used for the adjustment of the contact angle, a method of decomposing or modifying the binder substance by heat irradiation, ultraviolet light irradiation, electron beam irradiation, etc. can be mentioned.
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 transfer layer, for example, it is possible to perform patterning on the same cell transfer layer without mixing two or more different cells. is there.

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

  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.

  The cell transfer layer used in the present invention preferably has a physiologically active substance on the surface of the cell transfer layer. The physiologically active substance used in the present invention is a substance having some biological function. For example, it is not limited to antibodies, adhesion molecules, cytokines, growth factors, enzyme inhibitors and receptors, but amino acids, oligopeptides and polypeptides. 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. Since the cell transfer layer has such a physiologically active substance, it is possible to regulate the growth, differentiation, function, etc. of cells cultured on the cell transfer layer. This is because it can be cultured and used for transcription.

  The method for forming the cell transfer layer used in the present invention is appropriately selected according to the type of the cell transfer layer, the shape of the cell transfer member, and the like. Usually, the roll coating method, the die coating method, Wet coating methods such as bead coating and spin coating, thermal CVD, silane coupling, adsorption, and alternate adsorption can be used.

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

(2) Cell transfer substrate The cell transfer substrate used in the present invention will be described. The cell transfer substrate used in the present invention is capable of uniformly forming the cell transfer layer, and the type of cell transfer substrate is particularly limited as long as it can be sterilized by a known method. It is not a thing. 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.

  As such a cell transfer substrate, a substrate that can be used as a substrate of a general cell culture substrate can be used. Specifically, inorganic materials such as glass, metal, and silicon, and organic materials typified by plastics can be used. This is appropriately selected depending on the culture conditions and the like. In the present invention, among these, those made of organic materials are preferable. Since the organic material is excellent in moldability and easy to adjust flexibility and the like, for example, by making the cell transfer substrate highly flexible, cells comprising the cell transfer substrate can be obtained. The transfer film is also flexible so that it can be transferred to a body to be transferred such as a living tissue having a curved surface while maintaining the pattern and function, and used for various purposes. It is because it is easy.

  Such an organic material is appropriately selected based on functions required for the cell transfer membrane using the cell transfer substrate such as flexibility and transparency. For example, polypropylene, polymethylpentene, polypropylene copolymer Fluorine resin such as coalescence, Teflon (registered trademark) and ethylene-tetrafluoroethylene copolymer, polycarbonate, acetal resin, polysulfone, polyvinyl chloride, silicone resin, polystyrene, styrene-acrylonitrile copolymer, polymethyl methacrylate, etc. A bioabsorbable polymer such as acrylic resin, polyurethane, polyester, polyimide, polyglycolic acid, polylactic acid, collagen, and elastin can be used. Moreover, a nonwoven fabric, a woven fabric, etc. can also be used.

  In the present invention, when a polymer substrate such as polyester is used as the organic material, the cell transfer substrate may be provided with an intermediate layer in order to improve adhesion with the cell transfer layer. The intermediate layer is not particularly limited as long as it can adhere to the cell transfer substrate and the cell transfer layer and can uniformly provide the cell transfer layer, but is preferably a polar polymer or a metal oxide. Examples of the polar polymer include polyvinyl alcohol, poly 2-hydroxyethyl methacrylate, polyvinyl pyrrolidone, polyacrylic acid, and polylysine. Examples of the metal oxide include silicon-based oxide, titanium-based oxide, aluminum-based oxide, and phosphorus oxide. These metal oxide layers may contain an organic substance or a fluoride.

  As such an intermediate layer deposition method, a wet coating method such as roll coating, die coating, or bead coating, or a dry coating method such as sputtering, PVD (physical vapor deposition), CVD (chemical vapor deposition), or plasma is preferably used. be able to.

  The cell transfer substrate used in the present invention preferably has a fine porous structure. Cells that adhere to the cell transfer layer because the cell transfer substrate has a fine porous structure can obtain a desired function from the side of the cell transfer membrane where the cell transfer layer is not formed. You can receive necessary factors. This is because, for example, epithelial cells such as cells having different functions can be cultured on the front and back of the cells.

  The cell transfer substrate having such a fine porous structure is particularly useful when it is necessary to co-culture using feeder cells before transferring the cells cultured on the cell transfer layer. In this case, there is also an effect that the culture solution and oxygen are uniformly distributed to all cells during the cell transfer operation. In this case, although the average size of the pores depends on the type of cell, it is usually preferably 0.1 μm to 5 μm, and more preferably 0.2 μm to 1.5 μm. This is because if the size of the pores is less than 0.1 μm, the supply efficiency of nutrients and humoral factors to the cultured cells is deteriorated. Further, if the size of the pores exceeds 5 μm, the proportion of the cultured cells adhering to the inside of the pores or the back surface of the cell transfer substrate increases, and the cultured cells cannot be transferred efficiently.

  Here, although the thickness of the cell transfer substrate described above varies depending on the material to be selected, a range of 1 μm to 250 μm is preferable, and a range of 5 μm to 200 μm is particularly preferable. When the thickness of the cell transfer substrate is thinner than the above range, the flexibility is very high, and the handling becomes very difficult because the strength is insufficient and it may be easily torn or torn. is there. In addition, when the thickness is larger than the above range, the flexibility is insufficient, and therefore, when the cells cultured on the cell transfer member are transferred, the adhesion with the transfer target may be deteriorated. .

  For example, as shown in FIG. 3, the cell transfer substrate 1 may have a concave or convex portion formed in a pattern in which the cell transfer layer 2 is formed. In this case, it is possible to culture and transfer cells in a high-definition pattern by forming a cell transfer layer only on the convex portion or only on the concave portion. 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 transfer 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 transfer substrate becomes too thin, so that the strength is insufficient and it can be easily torn or torn. This is because there are cases in which the

(3) Cell transfer membrane The cell transfer membrane used in the present invention comprises the cell transfer substrate and a cell transfer layer formed on one side of the cell transfer substrate.

  The cell transfer membrane is not particularly limited as long as it has the cell transfer substrate and the cell transfer layer, and may have other layers as needed, for example, the cell A cell transfer auxiliary layer or the like may be formed on the transfer layer. Hereinafter, the cell transfer auxiliary layer used in the present invention will be described.

(Cell transfer auxiliary layer)
The cell transfer auxiliary layer is formed on the cell transfer layer, and the cells are adhered on the cell transfer auxiliary layer. In such a cell transfer auxiliary layer, when cells are cultured on the cell transfer auxiliary layer and then transferred, at least a part of the cell transfer auxiliary layer is transferred together with the cells to the transfer target side. In the present invention, by forming such a cell transfer auxiliary layer, it becomes possible to transfer cells onto the transfer target more efficiently. As such a cell transfer auxiliary layer, a layer that does not affect the transferred body side is selected, for example, a polypeptide such as collagen, a polysaccharide such as hyaluronic acid, polylactic acid, polyglycolic acid, alginic acid, polyvinyl alcohol, Polylysine etc. are mentioned. The cell transfer auxiliary layer may contain metal ions such as calcium ions and magnesium ions.

  In the present invention, the shape and size of the cell transfer membrane are not particularly limited, and can be selected according to the use of the cell transfer member of the present invention.

2. Next, the fixing part used for the cell transfer member of the present invention will be described. The fixing part used in the present invention fixes the cell transfer membrane and maintains the flatness of the cell transfer membrane during cell culture. When the cell transfer membrane is removed from the cell culture container, If it can take out without raise | generating deformation | transformation and a fracture | rupture, it will not specifically limit.

  As such a fixing portion, as shown in FIG. 4, a ring-shaped one connected to the surface side of the cell transfer film where the cell transfer layer is not formed, or as shown in FIG. As shown in FIG. 6, a sheet-like member bonded to the surface side on which the cell transfer layer is not formed, the fixing portion 4 is a splittable member composed of an upper fixing portion 8 and a lower fixing portion 9, The cell transfer membrane 3 is disposed between the upper fixing portion 8 and the lower fixing portion 9, and the upper fixing portion 8 and the lower fixing portion 9 are fitted together to fix the cell transfer membrane 3. And what to do. In addition, as a separable fixing part composed of the upper fixing part and the lower fixing part, as shown in FIG. 7, the cell transfer is performed in the gap between the ring-shaped upper fixing part 8 and the lower fixing part 9. 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 the surface side of the cell transfer membrane as shown in FIG. 4 where the cell transfer layer is not formed is preferable. This is because, during cell transfer, transfer can be performed without damaging the transfer target, and the cell transfer layer of the cell transfer film is not formed on the cells cultured on the cell transfer layer. When the cell transfer membrane has flexibility, it is easy to supply factors necessary for cells from the surface side, and the cell transfer membrane is fixed without impairing the flexibility. This is because there is an advantage of being able to.

  In the present invention, the fixing method of the cell transfer membrane of the fixing part as shown in FIG. 4 is a method of fixing the cell transfer film and the fixing part by thermocompression bonding or fixing via an adhesive. The method of doing is mentioned. In the present invention, in consideration of being a member for culturing cells, a method of fixing by thermocompression bonding is preferable. Moreover, when using an adhesive agent, it is preferable to consist of a component which has no influence on a cell.

  As a material used for such a fixing portion, since the same material as the cell transfer substrate described in the above section “1. Cell transfer membrane” can be used, description thereof is omitted here.

3. Next, the gripping part used for the cell transfer member of the present invention will be described. The gripping part used in the present invention is connected to a fixing part for fixing the cell transfer membrane, and is disposed on the surface side of the cell transfer film where the cell transfer layer is not formed. The cells cultured on the layer are not particularly limited as long as the cells can be transferred by holding the holding part.

As such a gripping portion, the cell transfer layer of the fixed membrane 20 formed by fixing the cell transfer membrane by the fixing portion as shown in FIG. 8 is not formed at the connection position with the fixing portion. Examples thereof include those that are the fixed portion surface 21 that is the surface side and those that are the fixed portion side surface 22 that is a surface perpendicular to the fixed portion surface of the fixed portion as shown in FIG. 9.
Moreover, as shown in FIG. 8, the arrangement of the gripping portion 5 includes a truncated cone-shaped one disposed around the entire periphery of the fixed portion, and one using a separated columnar gripping portion. FIG. 10 is a schematic diagram illustrating an example of the separated columnar gripping portions. As shown in FIG. 10A, the separated columnar gripping portions are connected to the fixing portion and arranged on the surface side of the cell transfer membrane where the cell transfer layer is not formed. FIG. 10B is a schematic cross-sectional view showing a cross-section taken in the direction of the arrow in FIG. 10A, in which the cell transfer membrane and the fixing portion are circular, and the four separated columnar gripping portions are arranged. Shows the case. For the stability during cell culture using the cell transfer member of the present invention, it is preferable that the separated columnar gripping portions are composed of three or more. Moreover, the said holding part may be connected to the convergence part 30, as shown in FIG. 11, when the said holding part is the said spaced apart columnar shape. This is because, depending on the size of the cell transfer member of the present invention, the separated columnar gripping portion is connected to the converging portion at the time of transfer, so that it can be gripped more stably. The number of the converging parts is not particularly limited as long as the number is less than the number of the columnar gripping parts spaced apart. In the present invention, the number of the converging portions is preferably 1 or 2 from the viewpoint of handleability.

  In the present invention, the connection position of the gripping part and the fixing part, and the arrangement of the gripping part can be used in appropriate combination according to the culture conditions and the like. In the present invention, it is preferable that the connection position between the grip portion and the fixing portion is the fixing portion surface of the fixing portion. This is because the possibility of damaging the transfer target during transfer can be reduced. Moreover, in this invention, it is preferable that the arrangement | positioning of the said holding | grip part shall be a truncated cone shape arrange | positioned at the perimeter of the said fixing | fixed part as shown in FIG. This is because when the cell transfer member of the present invention is placed on a culture vessel and the cells are cultured, the stability of the cell transfer member is improved. In this case, the angle 23 formed by the grip portion 5 and the fixing portion surface 21 of the fixing portion is preferably in the range of 45 ° to 135 °, particularly preferably in the range of 65 ° to 115 °. In the present invention, the angle formed by the grip portion and the fixing portion surface of the fixing portion is within the above range. If the angle is smaller than the above range, the stability of the cell transfer member during cell culture is reduced, This is because if it is larger than the range, it is difficult to grip the grip portion during transfer.

  Further, the gripping part used in the present invention has a hole on the side surface when the gripping part is in the shape of a truncated cone arranged on the entire periphery of the fixed part as shown in FIG. Also good. In the case where feeder cells are cultured on the bottom surface of the culture vessel for the purpose of causing the cells cultured on the cell transfer layer of the transfer member to produce factors necessary for obtaining a desired function, the bottom surface of the culture vessel To the cells to be cultured on the above-mentioned cell transfer layer, it is possible to easily supply nutrients to the feeder cells cultured at It is because it can supply not only from the surface side which adhere | attached with the said cell transfer layer of a cell but from the surface side which is not adhere | attached on the said cell transfer layer. Further, the presence / absence of a hole in the gripping part, the size of the hole, and the like are not particularly limited, and can be selected according to the use of the cell transfer member of the present invention.

  The gripping part used in the present invention is preferably detachable from the fixing part. Since the gripping part is detachable from the fixing part, for example, when the cells cultured in the cell transfer layer are transferred to a transferred body such as a living tissue, gauze or other sterilization is performed until the transfer is completed. The protective material may be covered, but in such a case, the gripping portion is made removable so that the covering with the protective material is not hindered.

  Such a detachable method includes applying a pressure-sensitive adhesive to the connecting portion between the fixed portion and the grip portion, or inserting one end of the grip portion into a hole disposed in the fixed portion. There is a method of fixing and fitting. In this invention, since it is a member which culture | cultivates the cell for cell transcription | transfer, it is preferable to make it detachable by using a fitting type. Moreover, when using an adhesive, it is preferable to consist of a component which has no influence on a cell.

  As the material of the gripping part used in the present invention, since the same material as the cell transfer substrate described in the section “1. Cell transfer membrane” can be used, the description thereof is omitted here.

4). Cell transfer member The cell transfer member of the present invention comprises a cell transfer membrane comprising a cell transfer substrate having a cell transfer layer on one side, and a fixing portion for maintaining the flatness of the cell transfer membrane during cell culture, If it has the holding part connected to the said fixing | fixed part and arrange | positioned in the surface side in which the said cell transfer layer of the said cell transfer film is not formed, it will not specifically limit.

  The cell transfer member of the present invention is to transfer cells cultured on the cell transfer layer onto another culture substrate for processing, or to transfer the cells to a transferred body such as a living tissue. Will be used.

B. Cell Transcription Kit Next, the cell transcription kit of the present invention will be described. The cell transfer kit of the present invention is a cell transfer kit comprising the cell transfer member of the present invention and a culture plate, wherein the cell transfer membrane does not contact the inner wall of the culture plate. To do.

  According to the present invention, the cell transfer film of the cell transfer member of the present invention is not in contact with the inner wall of the culture plate, so that the cells are not vibrated on the cell transfer layer of the cell transfer film. It can be cultured under stable conditions. In the case of culturing cells on the cell transfer layer and the inner wall of the culture plate, the cells cultured on the cell transfer layer are not contaminated with the cells cultured on the inner wall of the culture plate. Cells can be co-cultured on the cell transfer layer and on the inner wall of the culture plate.

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

1. Cell Transfer Member First, the cell transfer member used in the present invention will be described. The cell transfer member used in the present invention has the same contents as those described in the above section “A. Cell Transfer 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 in the present invention is not particularly limited as long as the cell transfer membrane and the culture plate inner wall do not contact each other. Examples of such a culture plate include a plate that can fix the cell transfer member in a state where the cell transfer 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 transfer member to the culture plate is not particularly limited, but a method using an adhesive or the cell transfer member in a hole arranged in the culture plate. There is a fitting type method of fixing by inserting one end of the gripping part. In the present invention, in consideration of the fact that cells used for cell transcription are cultured, a method such as a fitting type is preferred. As such a culture plate, for example, as shown in FIG. 12, a culture plate 41 having a bottom recess 42 that can be fixed by inserting one end of the grip portion 5 of the cell transfer member 10 is mentioned. Can do.

  Further, in the present invention, when the culture plate can fix the cell transfer member so that the cell transfer membrane and the inner wall of the culture plate do not contact, the cell transfer member Cells can be cultured on the cell transfer layer without giving unnecessary vibrations. Further, when cells are cultured both on the cell transfer member and on the bottom of the culture plate, the cell transfer layer Since the member does not move on the bottom surface of the culture plate, cells can be co-cultured on the cell transfer 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 transfer members. In this way, the culture plate can be inserted with two or more cell transfer members when, for example, it is necessary to co-cultivate two or more cell types without contaminating 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 substrate 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 forming a layer having cell transferability and a layer having a physiologically active substance described in the above-mentioned section “A. Cell Transfer Member”.

3. Cell Transfer Kit The cell transfer kit of the present invention is not particularly limited as long as it comprises a cell transfer member and a culture plate. As such a cell transcription kit, for example, as shown in FIG. 12, the cell transcription kit 40 of the present invention fixes the cell transcription member 10 of the present invention and the cell transcription member 10 described above. And a culture plate 41 having a possible bottom recess 42. Moreover, the cell transfer kit of the present invention may be one in which the cell transfer layer of the cell transfer member is fixed so as to face the bottom surface of the culture plate. As such a cell transfer kit, as shown in FIG. 13, the holding portion 5 of the cell transfer member 10 and a wall surface recess 43 provided on the inner wall of the culture plate 41 are fixed by fitting. Things. In the present invention, whether the cell transfer layer and the bottom surface of the culture plate are opposed or not is appropriately selected depending on the intended cell culture conditions and the like, and is particularly limited. Not what you want.

C. Cell Culture Method Next, the cell culture method of the present invention will be described. The present invention is characterized by using the cell transfer member of the present invention. According to the present invention, by using the cell transfer member of the present invention, the surface of the cell transfer membrane on which the cell transfer layer is not formed does not come into contact with the inner wall of the cell culture container. Cells can be cultured above. Thus, the cells can be cultured from the side of the cell transfer membrane where the cell transfer layer is not formed while supplying factors necessary for the cells to obtain a desired function. Therefore, it is possible to culture cells having different functions on the front and back of the cell, such as epithelial cells. In addition, by using the above-described cell transfer tool, it is possible to easily transfer cultured cells to a transfer target such as a living tissue.

  The method for culturing cells of the present invention is not particularly limited as long as the cells are cultured using the cell transfer member of the present invention. As such a cell culture method, a cell transfer member is inserted into a cell culture plate, and a cell culture member preparation step as a cell culture member, and cells are seeded on the cell transfer layer of the cell culture member. And a cell culturing step for culturing the cells. Hereinafter, each of these steps will be described.

1. Cell Culture Member Preparation Step First, the cell culture member preparation step in the present invention will be described. This step is a step of inserting a cell transfer member into a cell culture plate to form a cell culture member. As the cell transfer member used in this step, the cell transfer member of the present invention is used. The cell transfer member of the present invention has the same contents as those described in the above-mentioned section “A. Cell Transfer Member”, and thus the description thereof is omitted here.

  The cell culture plate used in this step can be a general cell culture plate and is not particularly limited. In the present invention, among them, the culture plate described in the above-mentioned section “B. Cell transcription kit” can be most preferably used. Since the cell transfer member inserted into the cell culture plate is the cell transfer member of the present invention, the cell transfer member used in the cell transfer kit of the present invention can be fixed as the cell culture plate. By using a culture plate, a cell culture member capable of culturing cells on the cell culture layer without contacting the cell transfer membrane of the cell transfer member with the inner wall of the cell culture plate. Because it can.

  Further, the cell culture plate used in this step may be subjected to surface treatment on the bottom surface of the cell culture plate. Examples of such a surface treatment include those forming a layer having cell transferability and a layer having a physiologically active substance described in the above-mentioned section “A. Cell Transfer Member”.

  In this step, the method for inserting the cell transfer member into the cell culture plate is not particularly limited as long as the cell transfer member and the cell culture plate are sterilized by a known method and then inserted. Is not to be done. Known sterilization methods include, for example, a method of sterilizing by γ-ray irradiation, a method of sterilizing by irradiating ultraviolet light, a method of sterilizing by irradiating an electron beam, a method of sterilizing by an autoclave, and sterilizing with ethylene oxide gas. And a method of sterilizing with alcohol. In the present invention, the cell transfer layer of the cell transfer member may or may not be disposed so as to face the bottom surface of the cell culture plate. The insertion method in this step is appropriately selected according to the cell culture conditions used in the present invention.

2. Cell Culture Process Next, the cell culture process in the present invention will be described. This step is a step of seeding cells on the cell transfer layer of the cell culture member, adhering the cells, and culturing.

As such a cell seeding method, for example, a medium in which cells can be cultured is added to the cell culture member prepared in “1. Cell culture member preparation step” described above, and then cells are seeded on the medium. Is mentioned.

  In this step, the cells to be seeded, adhered and cultured on the cell transfer layer may be floating cells such as blood cells and lymphoid cells, or may be adherent cells. In this step, 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.

  In this step, feeder cells may be seeded on the bottom surface of the cell culture plate of the cell culture member and cultured. This is because, by culturing feeder cells on the bottom surface of the cell culture plate, factors necessary for cells to be cultured on the cell transfer layer of the cell culture member can be produced.

  In this step, in order to efficiently culture cells on the cell transfer layer, the cell transfer layer is in close contact with the cell transfer layer until the cells adhere to the surface of the cell transfer layer, and has a suitable height. You may enclose with. 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 transfer layer. Moreover, although the height of the said enclosure can be selected according to the quantity of the culture medium to be used, 2 mm-20 mm are preferable.

3. Cell Culture Method The cell culture method of the present invention is not particularly limited as long as the cells are cultured using the cell transfer member of the present invention. In the present invention, when feeder cells are cultured on the bottom surface of the cell culture plate for the purpose of supplying factors necessary for cells cultured on the cell transfer layer, the cell transfer member is used as the cell transfer plate. Before insertion into the feeder, feeder cells may be seeded, adhered, and cultured on the bottom surface of the cell culture plate. By seeding and adhering the feeder cells first, it is possible to prevent the contamination of the feeder cells to the cells to be cultured on the cell transfer layer of the cell culture member in the cell culture step described later. It is.

  In the present invention, when the cell transfer member and the cell culture plate are inserted so that the cell transfer layer and the bottom surface of the cell culture plate are opposed to each other, After seeding and adhering cells on the cell transfer layer, the cell transfer member may be inserted into the cell culture plate in the cell culture member preparation step. This is because it is difficult to seed, adhere and culture desired cells on the cell transfer layer when the cell transfer layer is inserted so that the bottom surface of the cell culture plate faces.

  In the present invention, in order to efficiently culture cells on the cell transfer layer, the cell transfer layer is in close contact with the cell transfer layer until the cells adhere to the surface of the cell transfer layer and has a suitable height. You may enclose with. 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 transfer layer. Moreover, although the height of the said enclosure can be selected according to the quantity of the culture medium to be used, 2 mm-20 mm are preferable.

D. Cell Transfer Tool Next, the cell transfer tool of the present invention will be described. The cell transfer tool of the present invention is characterized in that cells adhere to the cell transfer layer of the cell culture member of the present invention.

  The cell transfer tool of the present invention will be described with reference to the drawings. FIG. 14 is a schematic cross-sectional view showing an example of the cell transfer tool of the present invention. As shown in FIG. 14, the cell transfer device 60 of the present invention includes the cell transfer member 10 of the present invention and cells 50 cultured on the cell transfer layer 2 of the cell transfer member 10. Is.

  According to the present invention, since the cell transfer layer is fixed so as to maintain flatness, the cell transfer layer can be stably cultured on the cell transfer layer. It can be transferred without impairing the pattern or function. Therefore, it can be set as the cell transcription | transfer tool which can transcribe | transfer in the state which maintained the culture pattern and function of the cell.

  The cell transfer tool of the present invention comprises the cell transfer member of the present invention and cells. Hereafter, each structure of the cell transfer tool of this invention is demonstrated in detail.

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

2. Cells As the cells used in the present invention, the same cells as those described in the above section “C. Cell Culturing Method” can be used, and the description thereof is omitted here.

3. Cell Transfer Tool The cell transfer tool of the present invention is not particularly limited as long as it has cells on the cell transfer member of the present invention and the cell transfer layer of the cell transfer member. As the method for producing the cell transfer tool of the present invention, the same method as described in the above “C. Cell culture method” can be used, and the description thereof is omitted here.

  The cell transfer tool of the present invention can transfer cells cultured on the cell transfer layer of the cell transfer member onto another culture substrate for processing, or transfer the cells to a body to be transferred such as a living tissue. It will be used when transferring.

E. Cell tissue manufacturing method The cell tissue manufacturing method of the present invention is characterized by using the above-described cell transfer tool of the present invention. According to the present invention, by using the above-mentioned cell transfer tool, it is possible to perform transfer while maintaining the cell culture pattern and function, and therefore it is possible to produce a cell tissue in which cells having various functions are combined. Can do. Therefore, for example, in order to maintain the function as a cellular tissue, it is necessary to supply necessary nutrients and discharge waste products by blood vessels, etc. By constructing a cell tissue in which a cell is stretched, it is possible to obtain a cell tissue that can maintain its function constantly while being an artificially constructed cell tissue.

  The present invention is not particularly limited as long as it is a method for producing a cell tissue using the cell transfer device of the present invention. Examples of such a method include a cell transfer tool manufacturing step for manufacturing the cell transfer tool, and a transfer step for transferring cells cultured on the cell transfer tool to a transfer target. Hereinafter, each of these steps will be described.

1. Cell transfer tool manufacturing process The cell transfer tool manufacturing process of the present invention is for manufacturing the cell transfer tool of the present invention. Such a method has the same contents as those described in the above section “D. Cell Transfer Tool”, and therefore the description thereof is omitted here.

2. Transfer step The transfer step in the present invention is a step of transferring the cells on the cell transfer tool to the transfer destination cells of the transfer target. This step is not particularly limited as long as the cells on the cell transfer tool can be transferred to the transfer destination cells of the transfer target. As such a process, for example, as shown in FIG. 15A, the cell transfer tool 60 manufactured in the cell transfer tool manufacturing process, the transfer destination cell culture substrate 71, and the transfer destination cell After the transfer body 70 composed of transfer destination cells 72 cultured on the culture substrate 71 is arranged so that the cells 50 of the cell transfer tool 60 and the transfer destination cells 72 face each other, FIG. As shown to b), the said cell 50 and the said transcription | transfer destination cell 72 are made to contact, the culture medium which can culture both is added, and it cocultivates. Next, as shown in FIG. 15 (c), the cell transfer member 10 has a cell tissue body 73 in which the cells 50 adhere to the transfer destination cells 72 by peeling off the cell transfer member 10. A process for forming the transfer object 80 can be given. In this step, the co-culture conditions are appropriately selected depending on the conditions of the cells of the cell transfer tool, the transfer destination cells of the transfer target, and the like.

  The transfer target used in this step is not particularly limited, and may be a cell cultured on a general culture vessel or a cell cultured on the above-described cell transfer member of the present invention. Further, it may be a living tissue or an organ. The transfer target is appropriately selected according to the target cell tissue. Further, the transfer destination cell of the transfer target is not limited to a planar one, but may be a cell mass having a three-dimensional shape.

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

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

3. Cell tissue production method The present invention is not particularly limited as long as it is a method for producing a cell tissue using the above-described cell transfer tool of the present invention. In the present invention, the cells on the cell transfer tool are transferred to the transfer destination cells of the transferred body to form a cell tissue, or the cells of the cell transfer tool are transferred to the transferred body having the cell tissue. It is possible to obtain a cell tissue having a higher level of function by performing the operation of transferring a plurality of times.

  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 Transfer Member (1) Formation of Intermediate Layer As the cell transfer substrate, a porous polyester film having a thickness of 10 μm was used. The film size was 25 mmφ, the pore size was 0.4 μm, the pore density was 1.6 × 10 ⁶ pieces / cm ² , and the water contact angle was 65 °. This was placed in a CVD chamber, and predetermined amounts of hexamethyldisiloxane, oxygen gas, and helium gas were charged. The degree of vacuum at that time was controlled to 30 MPa. As a result, a silicon oxide thin film (intermediate layer) was formed with a thickness of about 100 nm by plasma vapor deposition (CVD).

(2) Formation of cell transfer layer Next, the CVD-treated film is placed in a Teflon (registered trademark) container containing 0.2 cc of pentafluorophenylpropyltrimethoxysilane, and the container is sealed and kept in an oven at 100 ° C. for 5 hours. did. The water contact angle of the cell transfer layer obtained by the above method was 120 °.

(3) Exposure processing of cell transfer layer A photocatalyst containing layer of a 5-inch square quartz plate coated with a titanium oxide photocatalyst on the entire surface and the cell transfer layer are opposed to each other, and 3.5 J from the quartz plate side by a mercury lamp. Irradiation with ultraviolet rays was performed at a dose of / cm ² . As a result, a cell transfer film in which pentafluorophenylpropyltrimethoxysilane constituting the cell transfer layer was subjected to oxidative decomposition treatment (exposure treatment) was obtained. The water contact angle of the cell transfer layer of the cell transfer film after the exposure treatment was 15 °.

(4) Formation of cell transfer member The fixing part and the grip part attached to the cell transfer film were made of polystyrene. The cell transfer membrane prepared by the above method was sterilized by autoclave, and the fixed part and the grip part were sterilized by 70% ethanol. A one-component RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used for adhesion between the cell transfer membrane, the fixing portion and the gripping portion, and the same configuration as in FIG.

2. Cell Culture After the cell transfer layer was surrounded by a silicone resin O-ring, a cell transfer member was placed in the culture plate so that the cell transfer layer was on the top surface. MEM medium containing 5% serum was then added to the culture plate to such an extent that the O-ring touched the medium. Next, bovine vascular endothelial cells (bEC) suspended in the MEM medium in advance were seeded on the cell transfer layer in the O-ring so as to be 5.3 × 10 ⁵ cells / cm ² , at 37 ° C., 5%. Incubate for 16 hours in CO ₂ . It was confirmed with a phase contrast microscope that cells were confluently adhered on the cell transfer layer. Thus, a cell transfer tool was obtained in which cells adhered to the cell transfer layer of the cell transfer member.

3. Cell transfer A mouse-derived fibroblast sheet that has been cultured to be confluent in a commercially available cell culture dish is brought into contact with the cells (bEC) on the cell transfer member, and 5% FBS (bovine Fetal serum) -containing DMEM medium was added and incubated at 37 ° C., 5% CO ₂ . After removing the cell transfer member after 24 hours and observing with a phase contrast microscope, it was confirmed that all the cells adhering to the cell transfer layer were transferred onto the fibroblast sheet. . In addition, the cell transfer membrane on which the cell transfer layer was formed did not deform or break in the cell culture operation and the cell transfer operation described above.

[Example 2]
1. Preparation of Cell Transfer Member (1) Formation of Intermediate Layer As the cell transfer substrate, a porous polyester film having a thickness of 20 μm was used. The film size was 25 mmφ, the pore size was 1.0 μm, the pore density was 1.6 × 10 ⁶ pieces / cm ² , and the water contact angle was 70 °. This was placed in a CVD chamber, and predetermined amounts of hexamethyldisiloxane, oxygen gas, and helium gas were charged. The degree of vacuum at that time was controlled to 30 MPa. As a result, a silicon oxide thin film with a thickness of about 100 nm was formed by plasma vapor deposition (CVD).

(2) Formation of cell transfer layer Next, the CVD-treated film was placed in a Teflon (registered trademark) container containing 0.2 cc of pentafluorophenylpropyltrichlorosilane, and the container was sealed and kept in an oven at 100 ° C. for 5 hours. . The water contact angle of the cell transfer layer obtained by the above method was 115 °.

(3) Exposure processing of cell transfer layer A photomask having a grid pattern in which openings of 100 μm square are uniformly arranged at a pitch of 100 μm, and an opening having a width of about 1.5 cm for measuring a contact angle, A 5-inch square photomask for exposure in which a titanium oxide photocatalyst was applied to the entire surface was prepared. Next, the photocatalyst containing layer of the photomask for exposure treatment and the cell transfer layer were opposed to each other, and ultraviolet irradiation was performed from the photomask side with a mercury lamp at ^an irradiation amount of 4.0 J / cm ² . Thereby, the pentafluorophenylpropyltrimethoxysilane constituting the cell transfer layer is subjected to oxidative decomposition treatment (exposure treatment), and the cell transfer having a pattern-like cell adhesion region in which 100 μm square cell adhesion regions are uniformly arranged at a pitch of 100 μm. A cell transfer membrane having a layer formed thereon was obtained. Further, when the surface water contact angle was measured at an opening portion having a width of 1.5 cm formed around the mask for contact angle measurement, the surface water contact angle was 15 °.

(4) Formation of cell transfer member The fixing part and the grip part to be attached to the cell transfer film subjected to the exposure treatment were made of polystyrene. The cell transfer membrane prepared by the above method was sterilized by autoclave, and the fixed part and the grip part were sterilized by 70% ethanol. A one-pack RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used for adhesion between the cell transfer membrane, the fixing part and the gripping part, and the same configuration as in FIG.

2. Cell Culture After the cell transfer layer was surrounded by a silicone resin O-ring, a cell transfer member was placed in the culture plate so that the cell transfer layer was on the top surface. Medium 106S (2% FBS & supplement) manufactured by Cascade was added to the extent that the O-ring touched the medium, and normal human skin fibroblasts (HDFa) suspended in the same medium were added to 2.0 × 10 ⁵ cells / cm ² . so as seeded on the cell transfer layer of O- in the ring, 37 ° C., in 5% CO _2, and incubated for 16 hours. As a result of observation with a phase contrast microscope, it was confirmed that the cells were adhered only to the portions subjected to the exposure treatment of the cell transfer layer. As a result, a cell transfer tool was obtained in which cells adhered in a pattern on the cell transfer layer of the cell transfer member.

3. Cell transfer After removing the O-ring with tweezers, 200 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) was spread on a 35 mm dish (Falcon) using a cell scraper and allowed to stand at room temperature for about 2 minutes. Cells (HDFa) adhered on the cell transfer layer were contacted for a predetermined time, MEM medium containing 5% serum was added, and incubated at 37 ° C. and 5% CO ₂ . After removing the member for cell transfer after 12 hours and confirming with the phase-contrast microscope, it was confirmed that all the cells adhered on the cell transfer layer were transferred onto the Matrigel while maintaining the pattern. confirmed. In addition, the cell transfer membrane on which the cell transfer layer having the patterned cell adhesion region was formed did not deform or break in the cell culture operation and the cell transfer operation described above.

[Example 3]
1. Preparation of Cell Transfer Member A cell transfer layer obtained by physically adsorbing vascular endothelial cell growth factor (VEGF) on the cell transfer substrate using a pig skin-derived type I collagen sheet having a thickness of 25 μm as a cell transfer substrate. A cell transfer membrane was prepared. Hereinafter, a method for producing a cell transfer member will be described.

(1) Formation of cell transfer member The collagen sheet used as the cell transfer substrate was preliminarily UV-sterilized. Next, the fixed part and the grip part made of polystyrene were sterilized with 70% ethanol. A one-component RTV rubber deoxime type (manufactured by Shin-Etsu Silicone) was used for adhesion between the cell transfer substrate, the fixing part, and the gripping part, and the same configuration as in FIG. The O-ring was brought into close contact with the cell transfer layer forming surface of the member thus formed before the cell transfer layer was formed.

(2) Formation of cell transfer layer Next, a filter-sterilized VEGF phosphate buffer solution having a concentration of 100 ng / mL is spread on the cell transfer substrate surrounded by the O-ring, and the member is placed in a sealed container. And kept at room temperature for 2 hours. As a result, a cell transfer member comprising a cell transfer film having a VEGF adsorption layer formed as a cell transfer layer was obtained.

2. After the cell transfer O-ring is placed in the culture plate and the VEGF solution is removed by suction, the KuMedia's HuMedia-EG2 (2% FBS & supplement) is immediately added to the O-ring. Add to the extent that it touches the medium, and seed normal human umbilical vein endothelial cells (HUVEC) suspended in the medium to 5.3 × 10 ⁵ cells / cm ² on the cell transfer layer in the O-ring, The cells were cultured at 37 ° C. and 5% CO ₂ for 16 hours. It was confirmed with a phase contrast microscope that cells were confluently adhered to the cell transfer layer.

3. Cell transfer After removing the O-ring with tweezers, 200 μl of ice-cooled growth factor-reduced Matrigel (Becton Dickinson) was spread on a 35 mm dish (Falcon) using a cell scraper and allowed to stand at room temperature for about 2 minutes. Cells (HUVEC) adhered on the cell transfer layer were brought into contact, and HuMedia-EG2 (2% FBS & supplement) was added, and incubated at 37 ° C., 5% CO ₂ . After removing the member for cell transfer after 12 hours, as a result of confirmation with a phase contrast microscope, it was confirmed that all the cells adhered on the cell transfer layer were transferred onto the Matrigel. Further, the cell transfer membrane using a collagen sheet as the cell transfer substrate did not deform or break in the cell culture operation and the cell transfer operation described above.

It is the schematic which shows an example of the member for cell transcription | transfer of this invention. It is the schematic which shows an example of the cell transfer film used for this invention. It is the schematic which shows the other example of the cell transfer film 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 a rough disconnection 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 holding part used for this invention. It is the schematic which shows the other example of the holding part used for this invention. It is the schematic which shows the other example of the holding part used for this invention. It is the schematic which shows an example of the convergence part used for this invention. It is the schematic which shows an example of the cell transcription kit of this invention. It is the schematic which shows the other example of the cell transcription kit of this invention. It is the schematic which shows an example of the cell transcription | transfer tool of this invention. It is the schematic which shows an example of the manufacturing method of the cell tissue of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Cell transfer base material 2 ... Cell transfer layer 3 ... Cell transfer film 4 ... Fixing part 5 ... Grasping part 6 ... Cell adhesion area 7 ... Cell adhesion inhibition area 8 ... Upper fixing part 9 ... Lower fixing part 10 ... Cell transfer Member 30 ... Convergence part 40 ... Cell transfer kit 41 ... Culture plate 42 ... Bottom recess 43 ... Wall recess 50 ... Cell 60 ... Cell transfer tool 70, 80 ... Transfer target 71 ... Transfer destination cell culture substrate 72 ... Target Transcription destination cell 73 ... Cell tissue

Claims (9)

A cell transfer membrane comprising a cell transfer substrate having a cell transfer layer on one side; a fixing portion that maintains flatness of the cell transfer membrane during cell culture; and a connection to the fixing portion, and the cell transfer membrane a gripping portion which the cell transfer layer is disposed on the side not formed, Ri Tona,
The cell transfer member, wherein the cell transfer layer has a pattern culture function capable of culturing cells in a pattern . 2. The cell transfer member according to claim 1 , wherein the cell transfer substrate has a recess or a protrusion formed in a pattern in which the cell transfer layer is formed . The cell transfer member according to claim 1 or 2, wherein the cell transfer layer is made of a thermosensitive polymer . The cell transfer member according to any one of claims 1 to 3, wherein the cell transfer substrate has a fine porous structure. The fixing part is a ring-shaped one capable of supplying factors necessary for cells from the surface side where the cell transfer layer is not formed,
5. The cell transfer member according to claim 4 , wherein the fixing portion is a separable member including an upper fixing portion and a lower fixing portion capable of sandwiching the cell transfer membrane . A cell transfer kit comprising the cell transfer member according to any one of claims 1 to 5 and a culture plate, wherein the cell transfer membrane does not contact the inner wall of the culture plate. Characteristic cell transcription kit. A cell culture method using the cell transfer member according to any one of claims 1 to 5. A cell transfer tool, wherein cells are adhered on the cell transfer layer of the cell transfer member according to any one of claims 1 to 5. A method for producing a cell tissue, wherein the cell transfer tool according to claim 8 is used.

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