JP2010094114A - Implement for transferring cultured cell, and method for utilizing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To efficiently separate a cultured cell within any range on a cell-culturing substrate and then attach the separated the cultured cell again. <P>SOLUTION: An implement for transferring a cultured cell is provided, which has such a cell-binding part with a convex form under non-loaded state in such a way as to begin to contact from its convex chip, and finally, enables a whole surface of a cell-culturing substrate to be contacted. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for moving cultured cells in the fields of biology, medicine and the like, and a method for using the method.

  Today, animal cell culture technology has made significant progress, and research and development on animal cells has been extended to various fields. The target animal cells are used not only to commercialize the original cells and products of their products, but also to design useful drugs by analyzing the cells and their surface proteins. In some cases, the patient's own cells are proliferated outside the living body, or the function of the patient is increased and then returned to the living body for treatment. Currently, techniques for culturing animal cells and techniques for evaluation, analysis, and utilization are one field that researchers are paying attention to. By the way, many animal cells including human cells are adhesion-dependent. That is, when culturing animal cells in vitro, it is necessary to attach them to the substrate surface once. And it is necessary to peel the cultured cells while keeping the cultured form on the surface of the base material without peeling them apart.

  In particular, with regard to a technique for regenerating a patient's own cells in vitro, organ transplants that attempt to replace difficult-to-treat organs with other people's organs have become common. The target organs are very diverse, such as skin, cornea, kidney, liver, heart, etc., and the progress after surgery has improved remarkably, and has already been established as a medical technology. Taking corneal transplantation as an example, an eye bank was established in Japan about 50 years ago and transplantation activities began. However, the number of donors is still small, and there are about 20,000 patients who need corneal transplants in Japan alone, whereas it is said that there are only about 1/10 of the 2000 patients who can actually perform transplantation treatment. Yes. Despite the almost established technology of corneal transplantation, the current situation is that the next medical technology is required due to the shortage of donors. Under such circumstances, a technique for culturing and transplanting normal cells of a patient to a desired size has been developed.

  On the other hand, for example, Japanese Patent Application Laid-Open No. 05-192138 discloses an upper limit of skin cells on a cell culture support in which a substrate surface is coated with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. A skin cell culture method characterized by culturing at or below the critical dissolution temperature or above the lower critical dissolution temperature and then peeling the cultured skin cells above the upper critical solution temperature or below the lower critical solution temperature is described. Yes. In this method, cells are detached from the culture substrate coated with the temperature-responsive polymer by temperature. However, this method has poor releasability, and the obtained cell sheet has many structural defects. Therefore, it has been difficult to apply the method described in JP-A No. 05-192138 to the construction of myocardial tissue in vitro.

  Furthermore, in International Publication No. WO02 / 08387, myocardial tissue cells are cultured on a cell culture support whose surface is coated with a temperature-responsive polymer to obtain a myocardial cell sheet. By bringing the cultured multi-layered cell sheet into close contact with the polymer membrane and peeling it with the polymer membrane as it is, and making it three-dimensionally structured by a predetermined method. It has been found that a cell sheet having several functions and a three-dimensional structure are constructed as a few in vitro myocardial tissue. However, even with this method, lamination of myocardial cell sheets is not possible with a simple operation, and a technique that can be more simply and accurately laminated has been strongly desired.

  In order to develop the problems as described above, Japanese Patent Application Laid-Open No. 2005-176812 discloses a technique relating to a cultured cell transfer jig having a cell adhesion portion. The cultured cells can be detached, and then the detached cultured cells can be attached again. However, the cell adhesion part shown here is only in a planar form, and it cannot necessarily contact the cultured cells on the surface of a wide range of cell culture substrate uniformly, and all the cells to be moved are moved. It was difficult to do.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel cultured cell transfer jig based on a completely different idea from the prior art. Moreover, an object of this invention is to provide the utilization method.

  In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, it has been found that by making the cell adhesion part of the cultured cell transfer jig convex, it is possible to uniformly contact the entire arbitrary surface on the cell culture substrate. Moreover, it discovered that the peeled cultured cell was made to adhere again to a specific place by weakening the adhesive force of the cell adhesion part and cultured cell of the cultured cell movement jig | tool. The present invention has been completed based on such findings.

That is, the present invention provides a cell adhesion in a convex shape under non-loading for detaching cells cultured on the surface of the cell culture substrate and then attaching the detached cultured cells to other places again. A cell transfer jig having a portion, and when contacting the cell culture substrate surface, the cell adhesion portion begins to contact from the tip of the convex portion of the cell adhesion portion, the convex shape of the cell adhesion portion is deformed by the load, and finally In particular, a cultured cell transfer jig capable of contacting an entire surface of a cell culture substrate is provided.
In addition, the present invention allows the cultured cells to be detached from the cell culture substrate by attaching the cultured cells on the cell culture substrate to the cell adhesion part provided in the cultured cell transfer jig, and then the cultured cell transfer Provided is a method for moving cultured cells, characterized in that the peeled cultured cells are attached again to a specific location by weakening the adhesion between the cell adhesion part of the jig and the cultured cells.
Furthermore, the present invention provides a treatment method characterized by transplanting a sheet-like cultured cell into a living tissue for an affected part in which a part or all of the tissue is damaged or missing.

  By using the cultured cell moving jig having a convex cell adhesion part described in the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells are removed. It comes to adhere easily again. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately.

  The present invention provides a cultured cell moving jig having a convex cell adhesion part for peeling cultured cells on a cell culture substrate and then attaching the cells again. If the cell adhesion part is convex, when the cell adhesion part of the moving jig is brought closer to the cultured cells on the surface of the cell culture substrate, first, the most convex part of the cell adhesion part can contact. When the cell adhesion part is further brought closer, the cell adhesion part spreads and contacts the entire cell culture surface to be peeled around the most convex part. By contacting the cell adhesion part and the cultured cell in such a state, air bubbles do not enter between the cell adhesion part and the cultured cell, and the cell adhesion part and the cultured cell are brought into close contact with each other. The cultured cells can be detached. At that time, the shape and curvature of the convex part of the cell adhesion part are not particularly limited, and any part of the cell adhesion part may be convex as compared with other parts. In order to peel the cultured cells in a wide range, it is better that the most convex part of the cell adhesion part is at the center of the cell adhesion part. Here, the center of the cell adhesion part means the center part of the cell adhesion part if the most convex part is a dot or a surface having the center as the center, and the most convex part is linear or it. If it is a planar shape centering on, it means the center line part including the center of the cell adhesion part.

  In the present invention, the convex cell adhesion part is brought into contact with the cultured cell surface from the convex part. In that case, you may utilize all the convex parts, or only a part. In any case, the cell adhesion portion of the convex portion first comes into contact with the cultured cell surface, and further the cell adhesion portion approaches the cultured cell surface, so that the cell adhesion portion changes into a flat shape and comes into contact. In that case, the dimension of the convex part is preferably in the range of 0.5 mm to 5 mm, preferably in the range of 0.8 mm to 3 mm, more preferably as the height of the most convex part in the range used for cell adhesion. The range of 1.0 mm to 2.5 mm is good, and the range of 1.2 mm to 2.0 mm is most preferred. When the height of the convex part is 0.5 mm or less, it is not preferable because the cell adhesion part is not flat, and it is not always preferable to peel off all the cells to be moved, and when the height of the convex part is 5 mm or more. The strain when the convex cell adhesion portion finally changes to a flat surface, or the pressure for making the convex cell adhesion portion flat according to circumstances, is unfavorable as a load on the cultured cells.

  In the present invention, the ratio of the convex area to the whole cell adhesion part is not particularly limited, but the ratio of the area of the convex part is preferably in the range of 40% to 100%, preferably Is preferably in the range of 50% to 100%, more preferably in the range of 70% to 100%, and most preferably in the range of 80% to 100%. As a result of various investigations, when the ratio of the area of the convex portion is 40% or less, there are many cases where air bubbles enter between the cell adhesion portion and the cultured cell, which is not preferable as the present invention.

  Furthermore, the shape of the convex part in the present invention is not particularly limited to the shape when observed from the cell adhesion part side and the shape when observed perpendicular to the cell adhesion part side, and when it is vertical For example, the entire cell adhesion part to be used may be convex continuously or stepwise.

  The cell adhesion part in the present invention starts to contact the cultured cell from the convex part of the cell adhesion part, and the cell adhesion part becomes flat over the entire range of the cultured cell to be finally peeled off. In the present invention, the action mechanism of the change, the material of the cell adhesion part, etc. are not particularly limited as long as the form of the cell adhesion part can be changed as described above at the time of culture cell detachment. And a cell adhesion portion made of a flexible material having a convex shape. If it is a cell adhesion part of such a material, it will deform | transform without requiring a big load and can finally contact with the whole arbitrary surface of a cell culture substratum. The material of such equipment is not particularly limited. For example, silicone rubber, acrylic rubber, acrylonitrile butadiene rubber, isoprene rubber, urethane rubber, ethylene propylene rubber, epichlorohydrin rubber, chloroprene rubber, styrene butadiene rubber, butadiene rubber, Rubbers such as fluoro rubber, polyisobutylene, gum arabic and guar gum, polyurethanes, urethane foams, polyacrylamide, polydimethylacrylamide, polyacrylic acid and salts thereof, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, Polyvinylpyrrolidone, cellulose, carboxymethyl cellulose, fibrin gel, collagen, water-containing gel such as gelatin, or more Mixtures thereof, and the like. Moreover, the film | membrane of such a material may be a bag shape, and the thing filled with gas and liquid may be sufficient as it.

The surface of the cell adhesion part in the present invention may be further coated with, for example, one or more of cell adhesion protein, cell adhesion peptide, or temperature responsive polymer. Among them, examples of the cell adhesion protein include one or more of fibrin gel, fibronectin, laminin, collagen, gelatin and the like. Examples of the cell adhesion peptide include one or more of RGD peptide, RGDS peptide, GRGD peptide, GRGDS peptide and the like. The method for immobilizing the cell adhesion protein and the cell adhesion peptide in the cell adhesion part is not particularly limited, and physical adsorption or the like may be performed by applying a cell adhesion protein or a cell adhesion peptide aqueous solution known as a conventional method. The amount of the cell adhesion protein and cell adhesion peptide immobilized in the cell adhesion part is not particularly limited as long as it is sufficient to immobilize the cells to be moved. the amount is 0.005 / cm ² or more, preferably 0.01 [mu] g / cm ² or more, more preferably 0.02 .mu.g / cm ² or more. Measurement of the amount of immobilized cell adhesion protein and cell adhesion peptide may be in accordance with a conventional method. For example, a method of directly measuring a cell adhesion part using FT-IR-ATR, a cell adhesion protein labeled in advance, a cell Examples include a method of inferring from the amount of the cell-adhesive peptide, a labeled cell-adhesive protein immobilized on the cell-adhesive portion by immobilizing the adhesive peptide by the same method, and the method of inferring from the amount of the cell-adhesive peptide.

The surface of the cell adhesion part in the present invention may be coated with a temperature-responsive polymer. The temperature-responsive polymer may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. The method for coating the surface of the base material with various polymers is not particularly limited. For example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption. The amount of the hydrophilic polymer immobilized in the cell adhesion part is not particularly limited as long as the amount sufficient to attach the cells to be moved is immobilized, but the amount immobilized is 0.5 μg / cm ² or more, preferably 1.0 μg / cm ² or more, more preferably 1.5 μg / cm ² or more. The amount of the temperature-responsive polymer immobilized may be measured according to a conventional method. For example, the cell adhesion part is directly measured using FT-IR-ATR, and the temperature-responsive polymer previously labeled is immobilized by the same method. A method inferred from the amount of the labeling temperature-responsive polymer immobilized on the cell adhesion part may be mentioned, and any method may be used.

  The cell adhesion part in the cultured cell moving jig used in the present invention is not limited at all as long as it is determined at any time according to the size of the cultured cell, cultured cell mass, and cultured cell sheet to be moved. Moreover, what is necessary is just to determine at any time according to the magnitude | size of a cell adhesion part also in the culture cell movement jig | tool which has a cell adhesion part. Furthermore, the shape of the cultured cell moving jig is not particularly limited, and a mechanism that can join a grip and other devices necessary for moving the jig may be provided.

  Examples of cells used in the present invention include corneal epithelial cells, corneal endothelial cells, retinal pigment cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatocytes, Examples include skeletal muscle myoblasts, mesenchymal stem cells, alveolar epithelial cells, mesothelial cells, chondrocytes, synoviocytes, bone cells, periodontal ligament cells, other stem cells, or a mixture of two or more. However, the type is not restricted at all. The origin of the cells is not particularly limited, and examples include humans, dogs, cats, rabbits, rats, pigs, sheep, and the like. When the cultured cells of the present invention are used for human therapy, humans are used. It is desirable to use cells derived from them.

  The culture medium for cell culture in the present invention is not particularly limited as long as it is a commonly used medium for cells to be cultured. However, when the obtained cultured cells are used for human therapy, the components of the medium to be used are Those with a clear origin or those recognized as pharmaceuticals are desirable.

  The shape of the culture substrate in the present invention is not particularly limited, and examples thereof include dishes, multiplates, flasks, cell inserts, and flat membranes.

  In the present invention, the cultured cells are detached from the cell culture substrate by attaching the cultured cells on the cell culture substrate to the cell adhesion part provided in the cultured cell transfer jig, and then the cultured cell transfer jig Provided is a method for moving cultured cells, characterized in that the peeled cultured cells are attached again to a specific location by weakening the adhesion between the cell adhesion part and the cultured cells. According to this method, the cultured cells on the cell culture substrate can be easily detached, and the detached cultured cells are simply attached again. For this reason, it has been found that the cultured cells can be easily moved to a desired location and can be moved accurately. Furthermore, by either automating either or both of the steps of detaching cultured cells from the cell culture substrate and reattaching the detached cultured cells to a specific location, the cultured cells can be moved further. It has been found that it can be carried out easily and accurately.

  In order to move the cultured cells in the present invention, it is necessary to first attach the cultured cells on the cell culture substrate to the cell adhesion portion provided in the cultured cell moving jig. The attachment method is not limited in any way, but the cultured cell transfer jig in the present invention is provided with a cell adhesion part, so that the part is placed on the cultured cell to be moved and left to stand. good. When the above-mentioned hydrogel is used for the cell adhesion part of the present invention, the cultured cells can be sucked up and supplemented using its own water absorption in addition to physical adsorption. In the latter case, it is convenient to change the supplemental force by changing the amount of water absorbed by the hydrogel in advance. When the cell adhesion protein or cell adhesion peptide is coated on the surface of the cell adhesion part of the present invention, the cultured cells adhere to the cultured cell migration jig via the cell adhesion protein and cell adhesion peptide. Further, when the surface of the cell adhesion part is coated with a temperature-responsive polymer, the cultured cells physically adhere to the hydrophilicity / hydrophobicity of the cell adhesion part polymer layer surface. At that time, for the purpose of promoting the adhesion, a load may be applied to the extent that the cultured cells are not burdened, or a sufficient time may be taken until the cells adhere. Furthermore, an operation for promoting adhesion of cultured cells, such as increasing or decreasing the amount of medium and changing the culture temperature, may be used in combination. Moreover, you may perform automatically the adhesion operation using the Z-stage which can operate | move up and down.

  The cultured cells attached to the cultured cell moving jig by the above-described method can be freely moved to a desired place by moving together with the cultured cell moving jig. At that time, it is better that the cultured cells are moved aseptically in order to prevent the cultured cells from being contaminated. Further, the moving operation may be performed under humidification so that the attached cells are not dried. Further, the attaching operation may be automatically performed using a stage that can be operated in the vertical and horizontal directions.

  In the present invention, the cultured cells that have been moved by the above-described method are placed on a place where they are to be reattached and reattached. The reattachment method is not particularly limited, but usually, after the cultured cells that have migrated are attached to the place where they want to be reattached, the adhesion between the cell adhesion part of the cultured cell transfer jig and the cultured cells is performed. The operation is completed by weakening and separating the cultured cell moving jig from the cultured cells. At that time, if there is no particular problem even if the cell adhesion portion of the cultured cell migration jig remains, a method of peeling the cell adhesion portion and the cultured cell together at the cell adhesion portion of the culture cell migration jig may be used. In the case of the method of peeling the cell adhesion part and the cultured cell together, it is preferable that the cell adhesion part dissolves with time and the lysate does not affect the cell, and as such, collagen or gelatin is convenient. The present invention is not limited to these. Alternatively, as described above, when a water-containing gel is used for the cell adhesion part, if the water-containing gel has sufficiently absorbed water, the adhesion force of the cells adhering to the cell adhesion part is weak, and the cells can be easily removed from the cell adhesion part. Can be peeled off. Similarly, even when cultured cells are adsorbed by the water-absorbing power of the water-containing gel, the adhesive force of the cells attached to the cell adhesion portion is weakened by sufficiently absorbing the water-containing gel. In order to weaken the adhesion between the surface of the cell adhesion part of the cultured cell migration jig and the cultured cell, for example, when the surface of the cell adhesion part is coated with a cell adhesion protein or a cell adhesion peptide, Examples thereof include a method of adding an amino acid, a peptide, a protein, or the like that adheres more strongly than an adhesive property, and a method of sufficiently adding a medium. Further, when the temperature-responsive polymer is coated on the surface of the cell adhesion part of the cultured cell migration jig, the cultured cell can be peeled off by sufficiently changing the surface of the cell adhesion part polymer layer. On the other hand, in order to promote the attachment to the place where it wants to reattach, it is used in combination with applying a load to the extent that the cultured cells are not burdened, allowing sufficient time to attach, and changing the culture temperature. You may do it. Moreover, you may perform automatically the adhesion operation using the Z-stage which can operate | move up and down.

  The place to be reattached in the present invention is not particularly limited. For example, the surface of the culture substrate, the surface of the in vivo tissue, the surface of the in vitro tissue, another cultured cell, or another cultured cell sheet described later. It ’s fine. Examples of the in vivo tissue surface and in vitro tissue surface herein include, but are not limited to, humans, dogs, cats, rabbits, rats, pigs, and sheep. Another cultured cell includes corneal epithelial cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatic parenchymal cells, or a mixture of two or more. However, the type is not limited at all, but when the cultured cells of the present invention are used for human therapy, it is desirable to use human-derived cells.

  If the temperature-responsive polymer is coated on the surface of the cell culture substrate, the cultured cells can be peeled off from the surface of the cell culture substrate just by changing the culture temperature as shown in International Application Publication No. WO02 / 08387. By using the technique of the present invention, the peeling operation, moving operation, and reattachment operation can be performed easily and accurately. In that case, the temperature-responsive polymer coated on the substrate surface has an upper critical solution temperature or a lower critical solution temperature of 0 ° C. to 80 ° C., more preferably 20 ° C. to 50 ° C. in an aqueous solution. If the upper critical lysis temperature or the lower critical lysis temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, if the upper critical lysis temperature or the lower critical lysis temperature is lower than 0 ° C., the cell growth rate is generally extremely reduced or cells are killed, which is also not preferable.

  The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer.

A method for coating the surface of the base material with the temperature-responsive polymer is not particularly limited. For example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption. The coating amount of the temperature-responsive polymer is preferably in the range of 0.4 to 4.5 μg / cm ² , preferably 0.7 to 3.5 μg / cm ² , more preferably 0.9 to 3.0 μg / cm ^2. cm ² . When the coating amount is less than 0.2 μg / cm ² , the cells on the polymer are difficult to peel off even when a stimulus is applied, and the working efficiency is remarkably deteriorated. On the other hand, if it is 4.5 μg / cm ² or more, it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells.

  In the present invention, in order to exfoliate cultured cells in a sheet form and stack the cultured cell sheets using a cultured cell moving jig, or to transplant the cultured cell sheets to in vivo tissues or in vitro tissues. The cells must be cultured on a cell culture substrate coated with a temperature-responsive polymer, and the cultured cells must be peeled into a sheet. At that time, the temperature of the medium is not more than that temperature when the polymer coated on the surface of the culture substrate has an upper critical solution temperature, and particularly if it is more than that temperature if the polymer has a lower critical solution temperature. Not limited. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. The culture conditions other than the temperature may be in accordance with conventional methods and are not particularly limited. For example, the medium to be used may be a medium to which serum such as known fetal calf serum (FCS) is added, or a serum-free medium to which such serum is not added.

  In the method of the present invention, in order to peel and recover the cultured cell sheet from the cell culture substrate coated with the temperature-responsive polymer, the cultured cell sheet is attached to the cultured cell moving jig, and the temperature of the culture substrate surface is adjusted. Separation can be achieved by setting the temperature to the upper critical solution temperature or higher or the lower critical solution temperature or lower. It should be noted that peeling the cultured cell sheet can be performed in a culture solution in which cells are cultured or in another isotonic solution, and can be selected according to the purpose.

  The cultured cell sheet peeled from the cell culture substrate coated with the temperature-responsive polymer in the present invention and obtained using a cultured cell transfer jig is a proteolytic enzyme represented by dispase, trypsin, etc. The cell-substrate basement membrane-like protein formed during culture is not damaged by the enzyme, and the cell-cell desmosome structure is retained, resulting in structural defects. There is little and high strength. Furthermore, by using the cultured cell moving jig, it is possible to accurately stack the cultured cell sheets or move them accurately to the affected tissue. For these reasons, for example, at the time of transplantation of a cultured cell sheet, it can be adhered to the affected tissue well and accurately, and an efficient treatment can be performed.

  The method for fixing the cultured cell sheet and the living tissue shown in the present invention is not particularly limited, and the cultured cell sheet and the living tissue may be sutured, or the cultured cell sheet shown in the present invention is a living body. The cultured cell sheet attached to the affected part does not need to be sutured to the living body side so as to be engrafted with the tissue quickly.

  By using the cultured cell migration jig having the convex cell adhesion part of the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells can be simply and easily removed again. It becomes possible to adhere. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

Using a mold in which silicone sheets are overlapped as shown in FIG. 1, a T-shaped base with a handle (part A) according to the present invention and a flexible material (part B) having a water-absorbing shape that is formed in a downward convex shape. A cultured cell transfer jig (FIG. 2) was prepared. As the material of each part, polycarbonate was selected for the base part and gelatin gel was selected for the flexible material. The appearance of the cell adhesion part at that time is shown in FIG. By combining these parts, a cultured cell transfer jig was obtained. Human skeletal muscle myoblasts were grown until the culture surface of the temperature-responsive culture dish coated with poly-N-isopropylacrylamide, which can control the adhesion and non-adhesion of cells depending on the temperature, became full. The culture method followed a conventional method (cell seeding number 1 × 10 ⁵ cells / cm ² , 37 ° C., 5% CO ₂ , culture period 4 days). The coating amount of the poly-N-isopropylacrylamide was 1.9 μg / cm ² . Next, the cultured cell moving jig was gently brought into contact with the cultured cell surface, and the cultured cell moving jig was placed on the cultured cell surface. When the culture temperature was further lowered and the surface of the culture dish was made non-adhered to supplement the cells with the cultured cell migration jig, the cells adhered to the gelatin gel which is the cell adhesion part of the culture cell migration jig. FIG. 4 shows a photograph of the culture dish in which only the portion where the gelatin gel, which is the cell adhesion portion, is adhered to the skeletal muscle myoblast is peeled off. The cells left in the culture dish in FIG. 4 are confirmed by visualization by staining. Three culture dishes were prepared, and sheet-shaped skeletal myoblasts laminated in three layers were obtained on a gelatin gel by continuously attaching a culture cell migration jig. The cultured cell transfer jig to which the sheet-shaped skeletal myoblasts are attached is temporarily brought into close contact with another culture dish, and the cultured cell transfer jig is released while holding the periphery of the sheet-shaped laminated cells with tweezers. Only skeletal muscle myoblasts could be detached (FIG. 5).

In the same manner as in Example 1, a cultured cell moving jig (T-shaped handle base (part A) according to the present invention and a flexible material (part B) having water absorption formed in a downward convex shape) FIG. 2) was produced. As the material of each part, polycarbonate was selected for the base portion, collagen gel was selected for the flexible material, and the surface of the collagen gel was covered with a non-woven fabric made of polyglycolic acid. As in Example 1, canine periodontal ligament cells were allowed to grow until the culture surface of the temperature-responsive culture dish coated with poly-N-isopropylacrylamide, which can control the adhesion and non-adhesion of cells depending on the temperature, became full. It was. The culture method followed a conventional method (cell seeding number 1 × 10 ⁵ cells / cm ² , 37 ° C., 5% CO ₂ , culture period 4 days). The coating amount of the poly-N-isopropylacrylamide was 2.0 μg / cm ² . Next, the cultured cell moving jig was gently brought into contact with the cultured cell surface, and the cultured cell moving jig was placed on the cultured cell surface. Furthermore, when the culture temperature was lowered and the culture dish surface was made non-adhered to supplement the cells with the cultured cell migration jig, the cells were placed on the polyglycolic acid nonwoven fabric that is the surface of the cell adhesion part of the cultured cell migration jig. Attached. Three culture dishes were prepared, and sheet-like periodontal ligament cells laminated in three layers were obtained on a non-woven fabric made of polyglycolic acid by continuously attaching a culture cell migration jig. The cultured cell transfer jig to which the sheet-like periodontal ligament cells have adhered is temporarily brought into close contact with another culture dish, and the culture cell transfer jig is released while holding the periphery of the non-woven fabric made of polyglycolic acid with tweezers. Can be peeled off together with the polyglycolic acid nonwoven fabric.

Comparative Example 1

  In order to collect the cultured cells, an acrylic cultured cell transfer jig as shown in FIG. 6 was prepared. In FIG. 6, the disk part below the right part is a cell adhesion part. This was used by inserting the shaft of the right part of FIG. 7 into the cylindrical part of the left part of FIG. As the cell culture method and the cell culture substrate coated with the temperature-responsive polymer used, the same method and substrate as in Example 1 were used. After 4 days of culture, after confirming that the skeletal myoblasts on the culture substrate had become confluent, the cultured cell transfer jig was gently placed on the cultured cells. After the cell culture substrate was treated at 20 ° C. for 15 minutes, the cultured cell transfer jig was gently separated from the culture substrate. As a result, only about 20% of cultured cells were detached. This is probably because the cell adhesion part of the cultured cell transfer jig was flat and could not uniformly contact the entire part to which the cultured cells adhered.

  By using the cultured cell moving jig having a convex cell adhesion part described in the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells are removed. It comes to adhere easily again. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately. Furthermore, if a cell culture substrate surface coated with a temperature-responsive polymer is used, a cultured cell sheet with extremely high engraftment on living tissue can be obtained. The cultured cell sheet obtained by this method is strongly expected to have clinical applications such as corneal transplantation, skin transplantation, corneal disease treatment, ischemic heart disease treatment and the like. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.

  The manufacturing method of the cultured cell movement jig | tool shown in Example 1 is shown.   The cultured cell movement jig | tool shown in Example 1 is shown.   2 is a photograph showing a cell adhesion part of the cultured cell transfer jig shown in Example 1. FIG.   It is a figure which shows the appearance of the peeled cultured cell shown in Example 1. FIG. Cells that have not been detached are stained.   It is a figure which shows the state of the transplanted cell sheet shown in Example 1. FIG. The transplanted cell sheet is stained.   The part of the cultured cell movement jig which shape | molded the acrylic board shown in the comparative example 1 is shown.   The place of the cultured cell movement jig | tool which shape | molded the acrylic board shown in the comparative example 1 is shown being assembled and utilized on a cell culture substrate.

Claims (8)

Cultured cell migration with convex cell adhesion under non-load to peel the cultured cells on the cell culture substrate surface, and then attach the detached cultured cells to other places again When it comes into contact with the surface of the cell culture substrate, the jig starts to contact from the tip of the convex portion of the cell adhesion portion, and the convex shape of the cell adhesion portion is deformed by the load, and finally the cell culture substrate A cell migration jig that can contact the entire surface of any cell. The cultured cell migration jig according to claim 1, wherein the cell adhesion part is a hydrous gel. The cultured cell migration according to any one of claims 1 and 2, wherein any one of cell adhesion protein, cell adhesion peptide, or temperature-responsive polymer, or two or more thereof are attached to the cell adhesion part surface. jig. The cultured cell transfer jig according to claim 3, wherein the cell adhesion protein is composed of one or more of fibrin gel, fibronectin, laminin, and collagen. The cultured cells are detached from the cell culture substrate by attaching the cultured cells on the cell culture substrate to the cell adhesion portion provided in the cultured cell transfer jig according to claim 1, and then the cultured cell migration is performed. A method for moving cultured cells, characterized by reattaching the detached cultured cells to a specific location by weakening the adhesion between the cell adhesion part of the jig and the cultured cells. The cultured cell transfer method according to claim 5, wherein the surface of the cell culture substrate is coated with a temperature-responsive polymer. The cultured cell transfer method according to any one of claims 5 and 6, wherein the sheet-shaped cultured cells are transplanted into a living tissue. The cultured cell transfer method according to any one of claims 5 to 7, wherein a sheet-like cultured cell is transplanted to an affected part in which a part or all of the tissue is damaged or missing.

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