JP2016077229A - Fiber-like substrate, 3-dimensional cell structure and production method for the same, as well as culture method for 3-dimensional cell structure - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a 3-dimensional cell structure capable of supplying a culture broth to every corner of a tissue, and the production method for the same, as well as a culture method for the 3-dimensional cell structure.SOLUTION: There is provided a 3-dimensional cell structure comprising a fiber-like substrate and a cell layer coating a surface of the substrate. The substrate is a core-shell type fiber-like substrate having a first dissociative hydrogel for forming a core part and a second dissociative hydrogel for forming a shell part. There is also provided a production method for the 3-dimensional cell structure, comprising a step of coating a cell on the core-shell type fiber-like substrate. There is further provided a culture method for the 3-dimensional cell structure, comprising the steps of: coating a cell on the core-shell type fiber-like substrate including the first dissociative hydrogel for forming the core part and the second dissociative hydrogel for forming the shell part; removing the first dissociative hydrogel and hollowing the inside of the substrate; and supplying a culture medium into the inside of the substrate.SELECTED DRAWING: None

Description

  The present invention relates to a three-dimensional cell structure, a method for producing the same, and a method for culturing the three-dimensional cell structure.

  In a plastic cell culture dish that is widely used for cell culture, a culture in which the cultured cells proliferate while extending in two dimensions so as to stick to the dish surface is obtained. Although these cultures are suitable for research purposes such as elucidating the functions of individual cells and elucidating the growth process, the cells are grown and maintained in a living body by forming a tissue that is a three-dimensional structure. Therefore, there is a need for a technique that enables cell culture in a state closer to the in vivo environment.

  In recent years, methods for forming a desired tissue from stem cells such as iPS cells have been developed, and a three-dimensional cell structure is also used to construct a desired three-dimensional tissue using cells differentiated from stem cells. The culture technique for producing the is useful.

  Under such a background, for example, Patent Document 1 describes a method for forming a capillary-like tissue that forms a three-dimensional network, and a blood vessel / spheroid fusion comprising a spheroid having the capillary-like tissue. Yes.

  Patent Document 2 describes a sheet-like three-dimensional structure that is applied to a heart disease composed of myoblasts derived from a portion other than the myocardial tissue constituting an adult heart without using a scaffold. Yes.

JP 2009-213716 A JP 2010-29680 A

  However, in the conventional method, as the thickness of the prepared tissue increases, it becomes difficult to supply the culture solution to every corner of the tissue, and the cell may be necrotized at the center of the tissue.

  Then, an object of this invention is to provide the three-dimensional cell structure which can supply a culture solution to every corner of a structure | tissue. Another object of the present invention is to provide a method for producing and culturing the above three-dimensional cell structure.

The present invention is as follows.
(1) A fiber-shaped base material and a cell layer coated on the surface of the base material, wherein the base material forms a first dissociable hydrogel that forms a core part, and a shell part. A three-dimensional cell structure, which is a core-shell fiber-like substrate having 2 dissociable hydrogels.
(2) The three-dimensional cell structure according to (1), wherein the first and second dissociative hydrogels are dissociated under different dissociation conditions.
(3) The first and second dissociative hydrogels are gelled in the presence of metal ions, enzyme-soluble hydrogels, temperature-responsive hydrogels, pH-responsive hydrogels, and photo-responsive hydrogels. The three-dimensional cell structure according to (2), which is selected from the group consisting of a gel and a magnetic field responsive hydrogel.
(4) The three-dimensional cell structure according to any one of (1) to (3), wherein the cell layer includes a plurality of types of stacked cell layers.
(5) A three-dimensional cell structure comprising a fibrous base material and a cell layer coated on the surface of the base material, wherein the base material is hollow.
(6) The three-dimensional cell structure according to (5), wherein the cell layer includes a plurality of stacked cell layers.
(7) A step of coating cells on a core-shell type fibrous substrate having a first dissociative hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part, A method for producing a three-dimensional cell structure.
(8) The manufacturing method according to (7), further comprising a step of removing the first dissociative hydrogel and making the inside of the base material hollow.
(9) The production method according to (8), further comprising a step of removing the second dissociative hydrogel.
(10) The production method according to any one of (7) to (9), wherein the step of coating the cells is performed by winding a fiber-shaped cell mass on the base material.
(11) A step of coating cells on a core-shell type fiber-shaped substrate having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part, A method for culturing a three-dimensional cell structure, comprising: removing the first dissociative hydrogel to make the inside of the base material hollow; and supplying a medium to the inside of the base material.

  ADVANTAGE OF THE INVENTION According to this invention, the three-dimensional cell structure which can supply a culture solution to every corner of a structure | tissue can be provided. Moreover, the manufacturing method and culture | cultivation method of said three-dimensional cell structure can be provided.

FIG. 5 is a schematic diagram for explaining an embodiment of a manufacturing process of a cell fiber 300. It is a schematic diagram explaining one Embodiment of the manufacturing process of the core-shell type fiber-like base material 400. FIG. (A), (b) is the microscope picture of the three-dimensional cell structure immediately after winding a cell fiber. (C) is a photomicrograph of the three-dimensional cell structure after culturing for 2 days after winding the cell fiber. (A) is a photograph showing a process of coating a second cell on a three-dimensional cell structure coated with a first cell. (B) is a fluorescence micrograph showing the result of observing a cryostat section of a three-dimensional cell structure. (A) is a photograph which shows the state before the core part of a fiber-like base material is sol-ized. FIG. 5B is a photograph showing a state in which blue ink flows through the core portion of the fiber-shaped substrate.

[Three-dimensional cell structure]
In one embodiment, the present invention comprises a fibrous base material, and a cell layer coated on the surface of the base material, wherein the base material forms a first dissociating hydrogel, Provided is a three-dimensional cell structure, which is a core-shell fiber-like substrate having a second dissociable hydrogel that forms a shell portion.

  It is said that the thickness of the cell layer of the three-dimensional cell structure that can be maintained in the absence of blood vessels is about 200 μm at the maximum. Therefore, as the cell layer of the three-dimensional cell structure becomes thicker, it is necessary to start supplying the culture solution at an early stage after the formation of the three-dimensional cell structure to supply nutrients, oxygen, and the like to the cells. As a method for supplying the culture solution, for example, it is conceivable to supply the culture solution through a hollow portion formed by removing the substrate after coating the cell layer on the surface of the substrate. However, when the base material is not a core-shell type base material, the base material cannot be removed until the cells coated on the surface of the base material are cultured for a certain time or more and the cells are in close contact with each other. Otherwise, the three-dimensional structure of the three-dimensional cell structure will be broken.

  On the other hand, according to the three-dimensional cell structure of this embodiment, the inside of the fibrous substrate can be made hollow by removing the first dissociative hydrogel. And a culture solution can be supplied to every corner of a structure | tissue by sending a culture solution to this hollow part. Moreover, even if the supply of the culture solution to the hollow portion is started without waiting for the cells to be closely bonded to each other, the shell portion of the fiber-like substrate can maintain the mechanical strength. The three-dimensional structure of the three-dimensional cell structure is not destroyed.

  For this reason, according to the three-dimensional cell structure of this embodiment, the supply of the culture solution can be started at an early stage after the formation of the three-dimensional cell structure. Thus, a three-dimensional cell structure having a large cell layer thickness can be provided without necrotizing cells at the center of the tissue. That is, a tissue having a high cell density can be easily obtained by the three-dimensional cell structure of the present embodiment.

  As a method of forming a three-dimensional cell structure having a large cell layer thickness, it is also conceivable to previously coat the cell layer on the surface of a fiber-like substrate having a hollow interior. However, the inventors do not have sufficient mechanical strength, and the substrate tends to be crushed or broken at the stage of coating the cell layer. I found out.

  It is also conceivable to use a hollow fiber membrane as a base material and coat the cell layer on the surface. Although the hollow fiber membrane has sufficient mechanical strength, the hollow fiber membrane cannot be removed after the three-dimensional cell structure is formed. For this reason, application of a three-dimensional cell structure may be limited.

  On the other hand, the three-dimensional cell structure of the present embodiment has sufficient strength to cover the cell layer, and if necessary, the inside of the fiber-like substrate is hollowed, and the culture solution is used. Can be supplied. Furthermore, if necessary, the dissociative hydrogel is again introduced into the inside of the fiber-shaped base material that has been once hollowed and then cured, whereby a treatment such as coating of cells can be performed again.

  For example, a larger and more complicated three-dimensional cell structure can be produced by bundling a plurality of three-dimensional cell structures produced once. Thereby, theoretically, a large three-dimensional cell structure can be produced without limiting the size while maintaining the distance of the cells from the culture solution within 200 μm.

  In addition, by placing vascular endothelial cells in advance in the cell fiber to be wound, a capillary network can be produced inside the cell layer while culturing. And a culture solution can be supplied through the capillary network produced in this way. Therefore, by constructing a capillary network inside the wound cell layer and then winding a new cell layer, theoretically, the distance from the culture solution is maintained within 200 μm without any size limitation. A large three-dimensional cell structure can be produced. In this case, the thickness of the cell layer may be, for example, 400 μm or more, for example, 1 mm or more, for example, 2 mm or more.

  In addition, by coating the surface of the three-dimensional cell structure with a cell layer of a different type from the cells coated first, a three-dimensional cell structure having a plurality of types of stacked cell layers can be obtained.

  In the three-dimensional cell structure of this embodiment, the first and second dissociative hydrogels are preferably dissociated under different dissociation conditions. Examples of such dissociable hydrogels include hydrogels that gel in the presence of metal ions, enzyme-soluble hydrogels, temperature-responsive hydrogels, pH-responsive hydrogels, photo-responsive hydrogels, and magnetic-field-responsive hydrogels. A gel etc. are mentioned.

  A hydrogel that gels in the presence of metal ions can be dissociated by removing the metal ions. Examples of the metal ion include calcium ion, magnesium ion, strontium ion, barium ion, sodium ion, potassium ion and the like. Moreover, the enzyme-soluble hydrogel can decompose and dissolve molecules constituting the gel by the action of an enzyme. Further, the temperature-responsive hydrogel can change the state of solification and gelation by changing the temperature beyond the phase transition temperature. The pH-responsive hydrogel can be dissociated by changing the pH. The photoresponsive hydrogel can be dissociated by, for example, irradiating light with a certain wavelength. The magnetic field responsive hydrogel can be dissociated by changing the magnetic field, for example.

  When the first and second dissociative hydrogels are such, each hydrogel can be selectively dissociated and removed. Hydrogels that gel in the presence of metal ions include alginate gels that gel in the presence of divalent or trivalent metal ions, carrageenan gels that gel in the presence of calcium ions and potassium ions, and sodium ions. An acrylic acid-based synthetic gel that gels in the presence of the resin may be used. Examples of the enzyme-soluble hydrogel include alginic acid gel, chitosan gel, cellulose gel, collagen gel, and fibrin gel. As temperature-responsive hydrogel, poly (N-isopropylacrylamide) cross-linked with polyethylene glycol, temperature-responsive hydrogel (commercial name: meviol gel), methylcellulose, hydroxypropylcellulose, polylactic acid and polyethylene glycol copolymer, polyethylene And triblock copolymers of glycol and polypropylene oxide (commercial names: Pluronic, Poloxamer). Examples of the pH-responsive hydrogel include alginic acid gel, chitosan gel, carboxymethylcellulose gel, and acrylic acid-based synthetic gel. Photoresponsive hydrogels include synthetic gels combining azobenzene and cyclodextrin in the skeleton, gels composed of supramolecules using fumaric acid amide as a spacer, gels crosslinked or bonded via nitrobenzyl groups, etc. Can be mentioned. Examples of the magnetic field responsive hydrogel include a gel made of crosslinked poly (N-isopropylacrylamide) containing magnetic particles.

  In one embodiment, the present invention provides a three-dimensional cell structure comprising a fibrous substrate and a cell layer coated on the surface of the substrate, wherein the interior of the substrate is hollow.

  The three-dimensional cell structure of this embodiment corresponds to the above-described three-dimensional cell structure in which the first dissociative hydrogel is removed to make the inside of the fiber-shaped substrate hollow. According to the three-dimensional cell structure of the present embodiment, the culture solution can be supplied to every corner of the tissue by feeding the culture solution to the hollow portion inside the fiber-like substrate.

  In addition, a three-dimensional cell structure having a hollow interior lacks mechanical strength, and it may be difficult to coat cells. In such a case, the dissociable hydrogel may be introduced again into the inside of the fiber-shaped substrate once hollowed and cured. Thereby, the mechanical strength of the fiber-shaped substrate is increased, and it becomes possible to perform a process such as coating the cells again. Here, a three-dimensional cell structure including a plurality of types of stacked cell layers can be obtained by coating a cell layer of a different type from the cells coated first.

[Method for producing three-dimensional cell structure]
In one embodiment, the present invention provides a cell on a core-shell type fiber-shaped substrate having a first dissociative hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part. Provided is a method for producing a three-dimensional cell structure comprising a coating step.

  In the method for producing a three-dimensional cell structure according to this embodiment, the step of coating cells may be performed by winding a fiber-shaped cell mass around a core-shell type fiber-shaped substrate. According to this method, a tissue having a high cell density can be easily produced in a short time. In addition, a three-dimensional cell structure having a complicated function and structure can be produced by coating a plurality of types of cells.

(Manufacture of cell fibers)
First, a method for producing a fiber-like cell mass (hereinafter sometimes referred to as “cell fiber”) will be described. The method for producing the cell fiber is not particularly limited. For example, the cell fiber can be easily produced by using a double coaxial microfluidic device 100 as shown in FIG. For example, Wonje Jeong, et al., Hydrodynamic microfabrication via "on the fly" photopolymerization of microscale fibers and Wonje Jeong, et al. tubes, Lab Chip, 2004, 4, 576-580, FIG. 1 is also specifically described.

  FIG. 1 is a schematic diagram for explaining one embodiment of a manufacturing process of the cell fiber 300. As an example, a case where a collagen solution is used as the material of the core part and a sodium alginate solution before crosslinking is used as the material of the shell part will be described.

  First, a collagen solution containing cells 115 is introduced and ejected from the inlet 110 of the microfluidic device 100. Further, a sodium alginate solution before cross-linking is introduced and injected from the inlet 120 of the microfluidic device 100. In addition, a calcium chloride solution is introduced and injected from the inlet 130 of the microfluidic device 100. Then, the sodium alginate solution in the shell part is gelled, and the cell fiber 200 in which the shell part 220 is an alginate gel can be manufactured. Moreover, the collagen solution containing the cells 115 of the core part 210 can be gelled by heating the cell fiber 200 at about 37 ° C. for about several minutes to about 1 hour.

  The injection speed of the solution at the inlets 110 and 120 is not particularly limited, but may be about 10 to 500 μL / min when the diameter of the microfluidic device 100 is about 50 μm to 2 mm. By adjusting the injection speed of the solution at the inlets 110 and 120, the diameter of the core part and the coating thickness of the shell part can be adjusted as appropriate. The injection speed of the solution at the inlet 130 is not particularly limited, but may be, for example, about 1 to 10 mL / min.

  The outer diameter of the cell fiber 200 is not particularly limited, and may be, for example, about 10 μm to 2 mm, for example, about 200 μm to 2 mm, for example, about 50 μm to 1 mm. The length of the cell fiber 200 is not particularly limited, and may be about several mm to several m. Examples of the cross-sectional shape of the cell fiber 200 include a circle, an elliptical system, a polygon such as a quadrangle and a pentagon.

  Cells can be grown by culturing the cell fiber 200 in a culture solution. The cell fiber 200 can be cultured for several months by appropriately replacing the culture medium.

  The core part 210 includes various growth factors suitable for maintenance, proliferation or functional expression of the cells 115, such as epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor (TGF), insulin-like Growth factor (IGF), fibroblast growth factor (FGF), nerve growth factor (NGF) and the like may be contained. When a growth factor is contained, an appropriate concentration can be selected according to the type of growth factor.

  By selectively dissociating the second dissociable hydrogel forming the shell part 220 of the cell fiber 200, the shell part 220 can be removed, and a fiber-like cell mass (cell fiber 300) can be obtained.

  In the case where the shell part 220 is an alginate gel, only the shell part can be dissociated by causing a chelating agent such as EDTA to act at an appropriate concentration to remove calcium ions. Examples of the method for dissociating the alginate gel include a method in which a weak acid such as citric acid and an enzyme such as alginate lyase are used in addition to the chelating agent.

  As a density | concentration when using alginate lyase, 0.01-1 mg / mL, for example, about 0.1 mg / mL is mentioned, for example. For example, only the shell portion can be dissociated by immersing the cell fiber 300 in an alginate lyase solution and treating the cell fiber 300 at 37 ° C. for about 10 minutes to several hours while shaking with a shaker.

  The cell fiber 300 thus obtained can be easily operated using tweezers or the like. The cell can be coated by winding the cell fiber 300 around a core-shell type fiber-like substrate.

(Core-shell type fiber base material)
Next, a method for producing a core-shell type fiber-like substrate will be described. The method for manufacturing the core-shell type fiber-shaped substrate is not particularly limited, but for example, it can be easily manufactured by using the microfluidic device 100 described above.

  FIG. 2 is a schematic view for explaining one embodiment of a manufacturing process of the core-shell type fiber-like substrate 400. As an example, a temperature-responsive hydrogel (commercial name: meviol gel) obtained by crosslinking poly (N-isopropylacrylamide) with polyethylene glycol was used as the core solution, and a sodium alginate solution before crosslinking was used as the shell solution. The case will be described.

  Meviol gel is a temperature-responsive hydrogel that has a phase transition temperature of 22 to 25 ° C. and is in a sol (liquid) state on the low temperature side and in a gel state on the high temperature side. The phase transition is reversible and proceeds rapidly.

  First, meviol gel is introduced and injected from the inlet 110 of the microfluidic device 100. Further, a sodium alginate solution before cross-linking is introduced and injected from the inlet 120 of the microfluidic device 100. In addition, a calcium chloride solution is introduced and injected from the inlet 130 of the microfluidic device 100. The injection speed of the solution at the inlets 110, 120 and 130 is the same as described above. As a result, the sodium alginate solution in the shell is gelled, and the fiber-shaped substrate 400 in which the shell 420 is the alginate gel can be manufactured.

  Moreover, the mebiol gel of the core part 410 can be rapidly gelatinized by putting the fiber-like base material 400 in a temperature environment equal to or higher than a phase transition temperature such as an incubator set at 37 ° C., for example. In the state where the core portion 410 is gelled, the fiber-shaped substrate 400 exhibits sufficient mechanical strength. For this reason, the cell can be coated on the fibrous substrate 400 by a method of winding the cell fiber 300 or the like.

  The outer diameter of the fiber-shaped substrate 400 is not particularly limited, and may be, for example, about 10 μm to 2 mm, for example, 200 μm to 2 mm, for example, about 50 μm to 1 mm. The length of the fiber-shaped substrate 400 is not particularly limited, and may be about several mm to several m. Examples of the cross-sectional shape of the fiber-shaped substrate 400 include a circle, an elliptical system, a polygon such as a quadrangle and a pentagon.

(Removal of the core part of the fiber base material of the three-dimensional cell structure)
The manufacturing method of the three-dimensional cell structure according to the present embodiment further includes a step of removing the dissociable hydrogel constituting the core portion of the fiber-shaped substrate 400, so that the inside of the fiber-shaped substrate 400 is hollow. A three-dimensional cell structure can be produced.

  For example, when the core part 410 is the above-mentioned meviol gel, the core part 410 can be made into a sol by placing the three-dimensional cell structure in a constant temperature environment below the phase transition temperature for several seconds. After the core part 410 is made into a sol, the culture solution or the like is fed from one end of the fiber-like substrate 400, whereby the core part 410 can be washed away and replaced with the culture solution or the like.

  In this specification, the inside of the fiber base material being hollow means that the core portion of the fiber base material is not gelled. In this state, for example, if air is introduced into the fiber-shaped substrate, the core portion of the fiber-shaped substrate can be made hollow. Moreover, if a fluid such as a culture solution is introduced, the core portion of the fiber base material can be replaced with the fluid.

(Removal of shell part of fiber base material of three-dimensional cell structure)
The method for producing a three-dimensional cellular structure according to the present embodiment further includes a step of removing the dissociable hydrogel that constitutes the shell portion 420 of the fibrous substrate 400 to thereby remove the fibrous substrate from the three-dimensional cellular structure. A three-dimensional cell structure from which 400 is removed can be produced.

  According to the manufacturing method of the present embodiment, for example, after a desired three-dimensional cell structure is manufactured, the fibrous base material can be completely removed to obtain a tissue formed only from cells. Such a tissue is considered useful, for example, when transplanted into a living body.

[Culture method of three-dimensional cell structure]
In one embodiment, the present invention provides a cell on a core-shell type fiber-shaped substrate having a first dissociative hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part. A three-dimensional cell structure comprising: a coating step; a step of removing the first dissociative hydrogel to make the inside of the base material hollow; and a step of supplying a medium to the inside of the base material. A culture method is provided.

  According to the culture method of the present embodiment, after the cells are coated on the fiber-shaped substrate to form a three-dimensional cell structure, the cells are cultured in the hollow portion without waiting for the cells to adhere and bond to each other. The liquid supply can be started. Even if the cells are not closely bonded to each other, the shell portion of the fiber-like base material can maintain the mechanical strength, so that the culture solution can be obtained without destroying the three-dimensional structure of the three-dimensional cell structure. Supply can be made.

  As a result, a three-dimensional cell structure having a large cell layer thickness can be provided without necrotizing cells at the center of the tissue. That is, a tissue having a high cell density can be easily obtained by the culture method of the present embodiment.

  Examples of the method for supplying the culture solution include a method in which a silicon tube is connected to the end of the fiber-like substrate and the culture solution in the tube is circulated using a peristaltic pump or the like. As a method of connecting the tube made of silicon to the end of the fiber base material, for example, by using a bioadhesive such as photocrosslinkable gelatin at the end of the fiber base material, Examples include a method of bonding one end and connecting the other end to a silicon tube.

  Hereinafter, although an example of an experiment explains the present invention, the present invention is not limited to the following example of an experiment.

[Experiment 1]
(Manufacture of cell fibers)
Cell fibers were produced according to the production process schematically shown in FIG. As the cells, mouse fibroblast 3T3, rat skeletal muscle cell L6 and bovine vascular endothelial cell HH were used. A collagen solution (concentration 2 mg / mL) containing cells with a cell density of 1 × 10 ⁸ cells / mL is introduced from the introduction port 110 of the microfluidic device 100 at a flow rate of 25 μL / min. By introducing v% sodium alginate at a flow rate of 75 μL / min and injecting and injecting a 3% sucrose solution containing 100 mM calcium chloride from the inlet 130 at a flow rate of 3600 μL / min, the core diameter is about 100 μm and the outer diameter is about 200 μm. Cell fiber was obtained.

  After culturing each obtained cell fiber in a culture solution for 1 day, it was immersed in Dulbecco's modified Eagle medium (DMEM) containing 50 mM EDTA and treated at 37 ° C. for 10 seconds, whereby the alginate gel in the shell portion was treated. The removed cell fiber was produced. This cell fiber maintained a fiber-like shape, and had sufficient mechanical strength to be handled with tweezers in the culture solution.

[Experiment 2]
(Fabrication of core-shell type fiber base material)
In accordance with the manufacturing process schematically shown in FIG. 2, a core-shell type fiber-shaped base material in which the core part is a temperature-responsive hydrogel and the shell part is an alginate gel was produced. From the introduction port 110 of the microfluidic device 100, a temperature-responsive hydrogel obtained by crosslinking poly (N-isopropylacrylamide) with polyethylene glycol (commercial name: meviol gel, concentration 10 w / v%) is introduced at a flow rate of 100 μL / min. By introducing 1.5 w / v% sodium alginate from the port 120 at a flow rate of 200 μL / min, and introducing and injecting a 3% sucrose solution containing 100 mM calcium chloride from the inlet 130 at a flow rate of 1000 μL / min, A core-shell fiber-shaped substrate having a diameter of about 250 μm and an outer diameter of about 450 μm was obtained.

[Experiment 3]
(Production of three-dimensional cell structure 1)
In the culture solution, the 3T3 cell fiber prepared in Experimental Example 1 was wrapped around the fiber-shaped substrate prepared in Experimental Example 2 using tweezers, and a three-dimensional cell structure was prepared. 3 (a) and 3 (b) are photomicrographs of the three-dimensional cell structure immediately after winding the cell fiber. A thick tissue having a cell density of 1 × 10 ⁸ cells / cm ³ could be formed in one day. Subsequently, the obtained three-dimensional cell structure was cultured in a culture solution. FIG. 3C is a photomicrograph of a three-dimensional cell structure after culturing for 2 days after winding the cell fiber. FIG. 3 (c) shows that cells are firmly bonded to each other and an integral tissue is formed.

[Experimental Example 4]
(Production of three-dimensional cell structure 2)
A part of the 3T3 cell fiber prepared in Experimental Example 1 was stained with an orange fluorescent dye using Cell Tracker Orange (Invitrogen). Further, the remaining part of the cell fiber prepared in Experimental Example 1 was stained with a green fluorescent dye using Cell Tracker Green (Invitrogen). Subsequently, each of the two fiber-like base materials prepared in Experimental Example 2 was covered with the cell fiber dyed in orange, and a three-dimensional cell structure was prepared (covering with the first cell layer). ). Subsequently, two of these three-dimensional cell structures were bundled, and the above-described green-stained cell fiber was wrapped and covered to prepare a three-dimensional cell structure (covering with the second cell layer). FIG. 4 (a) is a photograph showing the process of coating the second cells.

  Subsequently, the three-dimensional cell structure was cultured for 2 days and then fixed to prepare a cryostat section, which was observed with a fluorescence microscope. FIG. 4B is a fluorescence micrograph showing the result of observing a cryostat section of a three-dimensional cell structure. As a result, it was shown that the first cell layer and the second cell layer maintain a hierarchical structure. It was shown that a three-dimensional cell structure having a high cell density and a complicated hierarchical structure can be rapidly produced by the method of this experimental example.

[Experimental Example 5]
(Dissociation of the core part of the core-shell type fiber base material)
The core part of the fiber base material produced in Experimental Example 2 was selectively dissociated to form a hollow part inside the fiber base material.

  Fig.5 (a) is a photograph which shows the state before the core part of a fiber-like base material is sol-ized. Subsequently, the fibrous substrate was left in an environment of 4 ° C. for 30 seconds. Thereby, the meviol gel of the core part of a fiber-like base material was sol-ized. As a result of connecting a silicon tube to the fiber base material and feeding blue ink, it was observed that the blue ink flowed into the core portion of the fiber base material. FIG. 5B is a photograph showing a state in which blue ink flows through the core portion of the fiber-shaped substrate. From this result, it was shown that the core part of the fiber base material can be selectively dissociated and removed.

  ADVANTAGE OF THE INVENTION According to this invention, the three-dimensional cell structure which can supply a culture solution to every corner of a structure | tissue can be provided. Moreover, the manufacturing method and culture | cultivation method of said three-dimensional cell structure can be provided.

  DESCRIPTION OF SYMBOLS 100 ... Microfluidic device, 110, 120, 130 ... Inlet, 115 ... Cell, 210, 410 ... Core part, 220, 420 ... Shell part, 200, 300 ... Cell fiber, 400 ... Fiber-like base material.

The present invention is as follows.
(1) A core-shell fiber-shaped substrate having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part.
(2) The fiber-shaped substrate according to (1), wherein the first and second dissociative hydrogels are dissociated under different dissociation conditions.
(3) The first and second dissociative hydrogels are gelled in the presence of metal ions, enzyme-soluble hydrogels, temperature-responsive hydrogels, pH-responsive hydrogels, and photo-responsive hydrogels. The fiber substrate according to (2), which is selected from the group consisting of a gel and a magnetic field responsive hydrogel.
(4) A three-dimensional cell structure comprising the fibrous substrate according to any one of (1) to (3) and a cell layer coated on the surface of the substrate.
( 5 ) The three-dimensional cell structure according to ( 4 ), wherein the cell layer includes a plurality of types of stacked cell layers.
( 6 ) A three-dimensional cell structure comprising a fibrous base material and a cell layer coated on the surface of the base material, wherein the base material is hollow.
( 7 ) The three-dimensional cell structure according to ( 6 ), wherein the cell layer includes a plurality of types of stacked cell layers.
( 8 ) comprising a step of coating cells on a core-shell type fibrous substrate having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part, A method for producing a three-dimensional cell structure.
( 9 ) The manufacturing method according to ( 8 ), further comprising a step of removing the first dissociative hydrogel and making the inside of the base material hollow.
( 10 ) The production method according to ( 9 ), further comprising a step of removing the second dissociative hydrogel.
( 11 ) The method according to any one of ( 8 ) to ( 10 ), wherein the step of coating the cells is performed by winding a fiber-shaped cell mass on the base material.
( 12 ) a step of coating cells on a core-shell type fiber-shaped substrate having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part; A method for culturing a three-dimensional cell structure, comprising: removing the first dissociative hydrogel to make the inside of the base material hollow; and supplying a medium to the inside of the base material.

Claims (11)

A fibrous substrate;
A cell layer coated on the surface of the substrate;
And the base material is a core-shell fiber-like base material having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part. Cell structure.   The three-dimensional cell structure according to claim 1, wherein the first and second dissociative hydrogels are dissociated under different dissociation conditions.   The first and second dissociative hydrogels gel in the presence of metal ions, enzyme-soluble hydrogels, temperature-responsive hydrogels, pH-responsive hydrogels, photo-responsive hydrogels, and magnetic fields The three-dimensional cell structure according to claim 2, which is selected from the group consisting of responsive hydrogels.   The three-dimensional cell structure according to any one of claims 1 to 3, wherein the cell layer includes a plurality of types of stacked cell layers. A fibrous substrate;
A cell layer coated on the surface of the substrate;
A three-dimensional cell structure in which the inside of the substrate is hollow.   The three-dimensional cell structure according to claim 5, wherein the cell layer includes a plurality of types of cell layers stacked.   A three-dimensional cell comprising a step of coating a cell on a core-shell type fibrous substrate having a first dissociable hydrogel that forms a core part and a second dissociative hydrogel that forms a shell part Manufacturing method of structure.   The manufacturing method according to claim 7, further comprising a step of removing the first dissociative hydrogel and making the inside of the base material hollow.   The manufacturing method according to claim 8, further comprising a step of removing the second dissociative hydrogel.   The manufacturing method according to any one of claims 7 to 9, wherein the step of coating the cells is performed by winding a fiber-shaped cell mass on the base material. Coating a cell on a core-shell type fibrous substrate having a first dissociable hydrogel forming a core part and a second dissociative hydrogel forming a shell part;
Removing the first dissociative hydrogel and making the interior of the substrate hollow;
Supplying a medium inside the substrate;
A method for culturing a three-dimensional cell structure.