JPWO2011046104A1 - Culture containing oriented cells in a gel - Google Patents

Abstract

A microfiber-shaped gel including a gel formed by gelable polymer chains, in which the gelable polymer chains are oriented in the fiber major axis direction. A culture containing cells oriented in a certain direction, such as muscle cells, in the gel can be easily produced.

Description

  The present invention relates to a culture containing cells such as oriented muscle cells in a gel, and a microgel fiber for use in the preparation of the culture.

  Biomaterials have recently attracted attention as sensors and actuators (Synthetic Metals, 138, pp.391-398, 2003; Science, 288, pp.2335-2338, 2000; Synthetic Metals, 105, pp.61-64, 1999; see Micro Electro Mechanical Systems, pp.454-457, 2004). In particular, the progress of microactuators using muscle cells as a drive source for moving microobjects is remarkable. For example, a contraction type myocardial sheet (Science, 317, pp.1366-1370, 2007) in which a rat myocardium is cultured on a polymer sheet, a pump (Lab on a Chip, 6 , pp.362-368, 2006), and force measurement devices using mouse skeletal muscles and cantilevers (Proc. of MEMS2009, pp.403-406, 2009).

  When manufacturing an actuator using such muscle cells as a driving source, it is important to orient the muscle cells in a certain direction in order to generate a powerful and efficient contractile force. In order to achieve this orientation, a method of orienting muscle cells by lithography patterning is known (Science, 317, pp.1366-1370, 2007; Advanced Materials, 20, pp.1-5, 2008). However, the method of orienting cells by lithography has a problem that a complicated process is required.

  As a technique for orienting cells using a gel, a method of controlling the orientation of myoblasts using highly oriented hydrogels (The 47th Annual Meeting of the Japan Dental Association) , 25, pp.89) and means for culturing cells using a three-dimensionally oriented collagen gel (three-dimensional collagen gel, Atri Co., Ltd.), but the preparation of the oriented gel is complicated. Operation is necessary.

Synthetic Metals, 138, pp.391-398, 2003 Science, 288, pp.2335-2338, 2000 Synthetic Metals, 105, pp.61-64, 1999 Micro Electro Mechanical Systems, pp.454-457, 2004 Science, 317, pp.1366-1370, 2007 Lab on a Chip, 6, pp.362-368, 2006 Proc. Of MEMS2009, pp.403-406, 2009 Advanced Materials, 20, pp.1-5, 2008 Dental Materials and Equipment, 25, pp.89 http://www.a-tree.co.jp/life_sci/index.html (3D Collagen Gel, Atry Co., Ltd.)

An object of the present invention is to provide a culture containing oriented cells in a gel. More specifically, it is an object of the present invention to provide a culture that contains cells oriented in a certain direction, such as muscle cells, in a gel and can be prepared by a simple method.
Moreover, another subject of this invention is providing the gel for using for preparation of said culture.

  As a result of diligent research to solve the above problems, the present inventors have introduced a fibrin monomer into a glass capillary tube and orientated the fibrin monomer in the long axis direction of the capillary tube, and then gelled it. It has been found that gel fibers containing fibrin fibers oriented in the axial direction can be easily prepared. It was also found that when skeletal muscle cells are brought into contact with the gel fiber, the cells enter the fiber and are aligned in the longitudinal direction along the fibrin fiber in which the cells are oriented. The present invention has been completed based on the above findings.

That is, according to the present invention, there is provided a microfiber-shaped gel including a gel formed of gelable polymer chains, wherein the gelable polymer chains are oriented in the fiber major axis direction. .
According to a preferred embodiment of the present invention, the gel in which the gellable polymer chain is a fibrin monomer, alginic acid, or collagen; the gellable polymer chain is a fibrin monomer, and the gellable polymer chain There is provided the above gel wherein the gel formed by is fibrin; and the above gel in sheet or block form.

According to the present invention, there is also provided a method for producing the above gel, comprising the following steps:
(a) introducing a gellable polymer chain into the capillary tube and orienting the gellable polymer chain in the long axis direction of the capillary; and
(b) A method comprising the step of gelling the gellable polymer chain is provided.

Furthermore, the above gel for use in cell culture is also provided by the present invention.
As preferred embodiments, a sheet-shaped cell culture gel comprising a plurality of gels bundled in the transverse direction, a block-shaped cell culture gel in which the sheet-shaped cell culture gels are laminated, and the gel formed into a three-dimensional shape A cell culture gel comprising is provided by the present invention. Examples of a preferable three-dimensional shape of the gel include a twisted yarn structure such as a double chain or a triple chain, a woven fabric structure, a cylinder structure, a spiral structure, or a hollow structure.

From another aspect, the present invention provides a culture containing cells oriented in the fiber long axis direction in the gel.
Preferred embodiments of the present invention include the above-mentioned culture wherein the cells are muscle cells such as skeletal muscle cells and cardiomyocytes, epithelial cells such as nerve cells and vascular endothelial cells, or fibroblasts.
The present invention also provides a method for producing the above culture, which includes a step of culturing the above gel and cells.

Moreover, the cell sheet or cell block containing the said culture is provided by this invention.
This cell sheet can be prepared by bundling microfiber-like cultures prepared by culturing so as to be in contact with cells using a microfiber-like gel. Thus, it can prepare by laminating | stacking suitably the cell sheet obtained. Alternatively, a sheet-shaped cell culture gel containing a plurality of the above-mentioned gels bundled in the lateral direction or a block-shaped cell culture gel in which the sheet-shaped cell culture gel is laminated and cultivating the cells in contact with the cells. Cell sheets or cell blocks can be prepared.

From another aspect, the present invention provides a method for storing the gel, wherein the gel is sucked into a silicon tube and the gel is stretched and stored in the longitudinal direction of the tube.
Moreover, the gel filled in the silicon tube and filled in a state of being stretched in the longitudinal direction of the tube is also provided by the present invention.

  By using the gel of the present invention, a culture containing oriented cells can be prepared very easily without going through complicated steps such as lithography. For example, fibrin gel microfibers obtained by orienting fibrin monomer in the fiber longitudinal direction and then gelling are cultured so as to come into contact with skeletal muscle cells, thereby including skeletal muscle cells oriented along fibrin fibers. A gel can be prepared, but when an electric pulse is applied to this culture, the gel can also expand and contract in synchronization with the contraction of the oriented skeletal muscle cells. Therefore, a sheet or block prepared from this culture can be suitably used as a material for artificial muscle. Further, by bundling a plurality of gels, it becomes possible to use the contraction force of skeletal muscle as a driving force.

It is a schematic diagram of a microfiber-like gel containing oriented skeletal muscle cells. It shows that a fibrin monomer solution is oriented in a microchannel by aspiration and gelled, then cultured with skeletal muscle cells, and cells are taken up into the fiber to obtain an oriented culture. It is the figure which showed that a myotube cell was formed with the skeletal muscle cell cultured using the dish and the fibrin hydrogel layer. (a) shows the culture results on the dish and (b) shows the culture results on the fibrin hydrogel. It is the figure which showed the result which the skeletal muscle cell cultured using the dish and the fibrin gel layer contracts by electrical stimulation. (a) shows the culture results on the dish and (b) shows the culture results on the fibrin hydrogel. It is the figure which showed the preparation methods of the fibrin gel fiber containing the oriented skeletal muscle cell. (a) The inner wall of the glass capillary is coated with Pluronic 127, (b) the fibrin monomer solution is sucked into the glass capillary, and (c) the microfiber-like fibrin gel is discharged onto the PDMS coated dish to float the cells. The solution is added, (d) fibrin gel is fixed to the PDMS layer, and (e) skeletal muscle cells are grown in the microfiber. FIG. 3 is a view showing a state in which skeletal muscle cells (C2C12 cells) are attached to fibrin gel fibers (diameter: about 100 μm). It is the figure which showed the result of having observed the cell cultured on the dish and the cell cultured in the fibrin gel fiber by the fluorescence immuno-staining method. (a) shows cells grown randomly on the dish, and (b) shows cells grown by orientation in fibrin gel fibers. It is the figure which showed the result of having observed the formation of the skeletal muscle on a dish, and the formation of the skeletal muscle in a fibrin gel fiber by the fluorescent immunostaining method. (a) shows skeletal muscle formation on a dish, (b) shows skeletal muscle formation in a fibrin gel fiber, and (c) shows skeletal muscle cells oriented in the fiber long axis direction in the fibrin gel fiber. Show the state. It is the figure which showed the conceptual diagram of the method of preparing a woven fabric structure using microfiber-like gel, and the prepared woven fabric structure. (A) shows a conceptual diagram of a loom (left) and a woven fabric preparation method (right), and (B) shows a specific example of a method for preparing a woven fabric using a microfiber gel in water. (C) shows the prepared woven fabric structure gel, (D) shows a fluorescent image of the woven fabric, (E) shows an enlarged view of the image of (D), and (F) shows a cross-sectional view of the sheet. Indicates. In the figure, Wrap gel wire indicates a microfiber gel that is a warp, and Weft gel wire indicates a microfiber gel that is a weft. (A) shows the microfibrous gel sucked into the silicon tube, and (B) shows an enlarged view. It is a figure showing the result of fluorescent immunostaining after culturing a core-shell type fiber having fibrin gel (core part) and alginate gel (shell part) encapsulating oriented skeletal muscle cells C2C12 or cardiomyocytes P19LC6 . Production of α-actinin was observed after staining, confirming that skeletal muscle cells and cardiomyocytes were differentiated. It is the figure which showed the result that the primary cardiomyocyte P19LC6 included in the core part repeats contraction spontaneously after culture | cultivation, and the whole fiber contracts synchronizing with the motion of these cells. (a) shows contraction movement of primary cardiomyocytes, and (b) shows fiber contraction.

The gel of the present invention is a microfiber-shaped gel including a gel formed by a gelable polymer chain, and the gelable polymer chain is oriented in the fiber major axis direction. Yes.
The microfiber shape means, for example, a fiber shape having an outer diameter of about 10 μm to 1 mm, but the outer diameter is not particularly limited to the above range. The cross-sectional shape may be various shapes such as a circle, an elliptical system, or a polygon such as a quadrangle or a pentagon. The length is not particularly limited, but is about several mm to several tens of cm, and preferably about several cm.

  The gel includes a gel formed by a gelable polymer chain. In the present specification, “gelatinizable polymer chain” means a gellable polymer chain capable of forming a gel, preferably a hydrophilic polymer chain, in other words, a gel in a state before gelation. Means a polymer chain which can be converted, preferably a hydrophilic polymer chain. For example, when the gel formed by the gelable polymer chain is fibrin, the fibrin monomer before forming an aggregate by gelation corresponds to the gelable polymer chain. Examples of the gelable polymer chain include natural polymer chains such as polypeptide chains, sugar-modified polypeptides, or polysaccharides, and non-natural polymer chains such as polyethylene glycol chains and polyacrylic acid chains. However, it is not limited to these. Preferred polymer chains capable of gelation in the present invention are polymer chains having a property of gelling in the presence of metal ions such as fibrin monomer and alginic acid, or collagen that gels by heating. Although the molecular weight of the gelable polymer chain is not particularly limited, it is, for example, about 10 to 600 kilodaltons.

  The gelable polymer chain that is particularly preferably used in the present invention is a fibrin monomer. By fibrin monomer being gelled in the presence of thrombin, fibrin is a gel formed by the gelable polymer chain. Can be prepared. It is also preferred to use alginic acid or collagen as a gelable polymer chain. In the case of using a non-natural gelable polymer chain, an appropriate gelling agent can be used for gelation, but it goes without saying that such a gelling agent can be appropriately selected by those skilled in the art. Yes.

  In the present invention, in preparing the gel, it is necessary to introduce a gelable polymer chain into the capillary tube so that the gelable polymer chain is oriented in the long axis direction of the capillary tube. The material of the capillary tube is not particularly limited, and for example, a glass capillary tube, a silicon capillary tube, a plastic capillary tube, or the like can be used. The inner diameter of the capillary may be appropriately selected according to the desired gel outer diameter. For example, it is preferable to use a capillary having an inner diameter of about 10 μm to 1 mm. It is also preferable to appropriately coat the inner wall of the capillary in order to facilitate the discharge of the gel after gel formation. For example, when a glass capillary is used, it is preferable to coat the inner wall of the capillary with a copolymer surfactant such as polypropylene glycol ethylene oxide adduct (pluronic nonionic surfactant). The method for introducing the gelable polymer chain into the capillary tube is not particularly limited, and for example, a method such as suction or injection can be employed.

  By introducing an aqueous solution of a polymer chain that can be gelled into the tube using a capillary tube with such an inner diameter, the flow rate of the aqueous solution that passes through the capillary tube is greatly different between the inner wall and the central portion. Very fast flow rate near the part. By utilizing this phenomenon, it is possible to efficiently orient the gelable polymer chain in the long axis direction of the capillary, that is, in the direction of the solution flow. The speed at which the solution containing the gelable polymer chain is introduced into the capillary is not particularly limited, but for example, the moving speed of the tip of the solution sucked into the capillary is 1 so that the above phenomenon occurs efficiently. It is preferably about ˜5 cm / sec.

  The concentration of the aqueous solution containing a gellable polymer chain is not particularly limited, and can be appropriately selected from the viewpoint of the viscosity of the solution and the gel hardness after gelation. For example, when a fibrin monomer is used, 1 to The concentration can be about 10 mg / ml. In addition to appropriate buffers, medium components for cell culture, biological substances such as arbitrary peptides, proteins, saccharides, lipids, and nucleic acids may be added to the aqueous solution containing gelable polymer chains. However, the components that can be added are not limited to those described above. In addition, from the viewpoint of enhancing the orientation, an appropriate microfiber, for example, a natural microfiber such as a polysaccharide chain, a polypeptide chain, or a polynucleotide chain, or a non-natural microfiber such as a carbon nanofiber may be added. Is possible. But the component which can be added is not limited to these.

  The method of gelling the gelable polymer chain introduced into the capillary and oriented is not particularly limited, and an appropriate gelation method may be selected according to the type of the gelable polymer chain. For example, when a fibrin monomer is used as the gelable polymer chain, it can be gelled with thrombin, and when alginic acid is used, it can be gelled using calcium ions. When collagen is used, it can be gelled by heating. It is also preferable to heat appropriately when gelling. For example, gelation may be promoted by heating at a temperature of about 30 to 40 ° C., preferably about 37 ° C.

  After gelation, the obtained gel can be discharged from a capillary tube to prepare a microfiber gel. This gel is a microfibrous gel in which the gelable polymer chains oriented in the long axis direction of the capillary in the capillary tube form a three-dimensional structure, and the gel can be oriented in the gel. Conserved polymer chains. The hardness of the gel is not particularly limited, but it is preferable that the gel has a hardness comparable to that of a biological material such as a normal collagen gel, alginic acid gel, or agarose gel. The hardness of the gel may be appropriately selected according to the type of cells used for culture. An appropriate gel strength can be selected so that cells can be easily penetrated into the gel during cell culture, and the culture obtained after the culture has sufficient mechanical strength.

  The discharged microfiber-like gel can be appropriately cut, but can preferably be used by cutting in a direction perpendicular to the major axis. For example, a flake obtained by cutting in a direction perpendicular to the long axis to form a thin section contains short polymer chains aligned in a direction perpendicular to the cross-section (ie, the long axis direction of the capillary tube). It goes without saying that such a section aspect is also included in the scope of the present invention.

  The microfiber gel thus obtained can be sucked into a silicon tube, and the gel can be stretched and stored in the longitudinal direction of the tube. It is generally difficult to keep the gel in a straight line when the gel is discharged from the capillary tube into water or buffer after gelation, but after discharging the microfibrous gel into water or buffer, By dipping the tip of a silicon tube having an inner diameter of about 100 μm to several mm in an aqueous medium and sucking the silicon tube, the microfibrous gel was sucked into the silicon tube from the tip and stretched in the longitudinal direction of the tube. It is sucked into the silicon tube in a straight state. This state is shown in FIG. In this state, the gel can be stored, and when used, it is possible to prepare a gel having a desired length by cutting a silicon tube containing a microfibrous gel into an appropriate length. In storage, appropriate agents such as preservatives, pH adjusters and buffering agents can be added to the tube as necessary.

  The above microfibrous gel can be used for cell culture. By culturing the cells using the gel of the present invention, it is possible to prepare a culture in which the cells enter the inside of the gel and are oriented along the gelable polymer chain. Any cell can be used as long as it has orientation. For example, muscle cells such as skeletal muscle cells and cardiomyocytes, neuronal cells, epithelial cells such as vascular endothelial cells, or fibroblasts can be used, but are not limited thereto. The method for culturing cells using the microfibrous gel of the present invention is not particularly limited, and those skilled in the art appropriately select appropriate culture conditions such as medium, culture temperature, carbon dioxide concentration, etc. according to the type of cells used. it can. The appearance of cells entering and aligning in the gel can be easily observed under a normal optical microscope.

  In this specification, the “orientation” of cells means that non-circular cells of the same type are aligned in the same direction. Typically, substantially elliptical or vertically long cells are aligned along the major axis direction. Although it means an aligned state, the term “orientation” should not be interpreted in a limited way in any sense, and this term should be interpreted in the broadest sense. For example, a skeletal muscle cell has a vertically long outer shape, and a culture in which the major axis direction of the cell is aligned so as to substantially coincide with the fiber major axis direction of the microfiber can be prepared. A conceptual diagram of a culture having such an orientation is shown in FIG. In addition to the cells, the gel can appropriately contain biological substances such as proteins, nucleic acids, polysaccharides, antibodies, lipids, and non-biological substances such as inorganic materials and organic substances as necessary.

  A microfiber gel for use in culture may be used independently, but a plurality of gels may be used in a bundle. As an example of using a microfiber gel independently, a conceptual diagram is shown in FIG. A microfiber gel gelled in a capillary tube connected to a cylinder is discharged on a dish so that the gel is almost linear, and is stretched with a needle etc. In FIG. 4, skeletal muscle cells) can be added and cultured.

  As an example of a case where a plurality of microfiber gels are used in a bundle, for example, a plurality of gels are bundled in the lateral direction to form a sheet gel composed of a single row of gels, and then cell culture is performed. A method for preparing (referred to herein as a “cell sheet”) can be exemplified. Further, a block-shaped cell culture (referred to as “cell block” in this specification) can be prepared by stacking a plurality of sheet-shaped gels to form a block-shaped gel and performing cell culture. When stacking a plurality of sheet-like gels, it can be stacked so that the fiber major axis directions of the microfibers constituting each sheet coincide with each other, but the fiber major axis direction of each sheet has an appropriate angle difference. Sheets may be stacked.

  In addition, the cell sheet is prepared by using a microfiber gel in an independent state, cultivating the microfiber culture by contacting the cells, and bundling the obtained culture horizontally. It can also be prepared. The cell block may be prepared by appropriately laminating the cell sheets obtained as described above.

  Moreover, the microfibrous gel for use in culture may be molded to have an arbitrary three-dimensional shape. The three-dimensional shape can be formed by a single gel or a plurality of gels. Examples of the three-dimensional shape that can be formed by a single gel include a spiral shape and a cylinder structure. Examples of the three-dimensional shape that can be formed by a plurality of gels include a twisted yarn structure such as a double chain or a triple chain, and a woven fabric structure. The three-dimensional shape may be a hollow structure. However, the three-dimensional shape is not limited to the above shape.

  For example, in order to prepare a gel having a woven fabric structure, a micro loom having a warp spacing of about 1 to 5 mm is used, the above-mentioned microfiber gel is used as a warp, and an arbitrary height as a weft A woven fabric structure gel may be prepared using microfibers made of molecular gel. An example of this conceptual diagram and a gel having a woven fabric structure is shown in FIG. In the woven fabric structure shown in FIG. 8 (C), by using the microfiber gel of the present invention as the warp and using, for example, an alginate microfiber gel or the like as the weft, the gel of the woven fabric structure having excellent mechanical strength. Can be prepared.

  The microfibrous gel used as the weft and warp is preferably placed in the loom while being stored in the silicon tube as described above so that the microfibrous gel is supplied from within the silicon tube. FIG. 8 (A) is a conceptual diagram showing that warp yarn is supplied from within the silicon tube. When skeletal muscle cells are cultured using the woven fabric structure gel thus obtained, a culture in which skeletal muscle cells are oriented along the axial direction of the warp can be prepared. Can be used as a cell block constituting an artificial muscle. For example, Proc. Of MEMS2009, pp.403-406, 2009 and Proc. Of Transducers, pp.124-127, 2009, etc., have been reported on methods for using skeletal muscle as a driving source, including skeletal muscle cells. The culture can also be used as a driving source according to the methods described in these publications.

EXAMPLES Hereinafter, although an Example demonstrates this invention further more concretely, the scope of the present invention is not limited to the following Example.
Example 1
The mouse myoblast cell line C2C12 cell established as one of the cell lines was used as the skeletal muscle cell. Examples of the medium include a growth medium for growing myoblasts and a differentiation medium for promoting myoblast differentiation and fusion to form myotube cells. Growth medium consists of DMEM (Dulbecco's Modified Eagle Medium) + FBS (Fetal Bovine Serum) 10% + AB (antibiotic-antimycotic) 1%, differentiation medium consists of DMEM + HS (Horse Serum) 5% + AB 1% Is done. The seeding concentration at the time of passage was about 3000 cells / cm ² . Passaging was performed twice a week. Moreover, the differentiation medium was added when it became confluent 3 days after passage, and the differentiation medium was changed once every two days.

A fibrin gel was prepared by mixing 400 μl of fibrinogen solution, 100 μl of PBS (phosphate buffered saline), and 100 μl of thrombin solution. The fibrinogen solution was prepared by adding 7.5 ml of HBSS (Hanks' Balanced Salt Solution) to 1.5 ml of fibrinogen, and the thrombin solution was prepared by adding CaCl ₂ and 100 μl thrombin to 2 ml of PBS.

  It was confirmed that myotube cells were formed on the fibrin gel as in the dish (FIG. 2). This result shows that myotube cells may be formed even in fibrin gel fibers. In addition, it was confirmed that myotube cells on the fibrin gel layer contracted by an external electric pulse in the same manner as myotube cells on the dish (FIG. 3). This result shows that when skeletal muscle cells are cultured in fibrin gel fibers and the skeletal muscle cells form myotube cells, the myotube cells can contract within the fibrin gel fiber. At that time, it was also confirmed that the fibrin gel layer under the myotube cells contracted in synchronism with the contraction of the myotube cells (FIG. 3). This result indicates that when myotube cells contract in the fibrin gel fiber, the entire gel fiber may contract. In this experiment, an electric pulse having a voltage of 5 V and a frequency of 50 Hz was applied, and the distance between the electrodes was about 45 mm. The contraction rate graph in FIG. 3 shows the contraction rate based on the distance between the two points on the myotube and the two points on the fibrin gel before applying the electric pulse.

FIG. 4 shows a method for producing a fibrin gel fiber enclosing oriented skeletal muscle cells. In order to easily peel the gel fiber from the capillary, the inner wall of the glass capillary was coated with pluronic 127 (FIG. 4 (a)). The fibrin monomer was sucked into a glass capillary tube so that the fibrin monomer was micro-oriented (FIG. 4 (b)). After the fibrin monomer was gelled, the obtained fibrin gel microfiber was extruded onto a dish coated with PDMS (polydimethylsiloxane), and a cell suspension was added (FIG. 4 (c)). The fibrin gel microfibers were fixed with a needle in a layer of PDMS (FIG. 4 (d)), and cultured so as to contact the skeletal muscle cells and fibrin gels microfibers at 37 ° C. under conditions of 5% CO ₂ (FIG. 4 (e)). As a result, as shown in FIG. 5, a culture in which skeletal muscle cells C2C12 invaded into the fibrin gel microfiber having a diameter of about 100 μm and proliferated was obtained.

  FIG. 6 shows the results of observation of the cells cultured on the dish and the cells that have invaded and proliferated into the fibrin gel microfiber by fluorescent immunostaining. The fluorescent immunostaining was performed according to the following procedure. (1) Fix the sample with paraformaldehyde; (2) Perform cell membrane permeabilization with TritonX-100 in PBS; (3) Block with goat serum in PBS; (4) Add Alexa 488-phalloidin 2 μl to goat serum Add 1 ml in PBS and let stand for 2 hours at room temperature; (5) Add 1 μl Hoechst 33342 and let stand for 5 minutes at room temperature; (6) Roll the parafilm at 4 ° C to prevent the sample from drying Stored in the dark. In FIG. 6, the cell nucleus is stained blue and F-actin (cytoskeleton) is stained green. On the dish, the cells were cultured in an unspecified direction, whereas in the fibrin gel microfiber, the cells were oriented in the long axis direction of the fiber.

  The presence or absence of skeletal muscle formation in skeletal muscle cells was similarly confirmed by fluorescent immunostaining. The following processes were performed between the procedures (3) and (4) of the fluorescent immunostaining described above. (3-1) Add 20 μl of mouse anti-α-actinin (Sarcomeric) monoclonal antibody to 1 ml of goat serum in PBS and let stand for 2 hours at room temperature; (3-2) 5 μl of Alexa 568-anti-mouse antibody in PBS It was left at room temperature for 2 hours after adding 1 ml of middle goat serum. In FIG. 7, α-actinin is stained in red, and the portion stained in yellow indicates a region of skeletal muscle in which green (F-actin) and red (α-actinin) are mixed. It was confirmed that skeletal muscle was formed in the fibrin gel microfiber in FIG. 7 (b) as well as skeletal muscle was formed on the dish in FIG. 7 (a). On the dish, skeletal muscles are formed in an unspecified direction, whereas in fibrin gel microfibers, skeletal muscles are oriented in the long axis direction of the fiber (Fig. 7 (c)). It was shown that when the oriented skeletal muscle contracts in response to an electric pulse, a larger contraction force can be obtained as a whole than a skeletal muscle formed in an unspecified direction.

Example 2
A core-shell type fiber having a fibrin gel containing skeletal muscle cells C2C12 or cardiomyocytes P19LC6 oriented as a core part and an alginate gel as a shell part (Lab. Chip, 4, pp. 576, 2004, Fig. 1) by the method of Onoe et al. (MEMS, pp. 248-251, 2010). The fiber diameter was about 150 μm, and the cell density in the core after culture was 1.0 × 10 ⁸ cells / ml. When fluorescent immunostaining was performed after the culture, α-actinin production was observed, confirming that skeletal muscle cells and cardiomyocytes were differentiated (FIG. 10). Primary cardiomyocytes P19LC6 encapsulated in the core spontaneously repeated contraction after culture, and contraction of the entire fiber was also observed in synchronization with the movement of these cells (FIG. 11).

Claims (10)

A microfiber-shaped gel including a gel formed by gelable polymer chains, wherein the gelable polymer chains are oriented in the fiber major axis direction. The gel according to claim 1, wherein the gelable polymer chain is a fibrin monomer, alginic acid, or collagen. The gel according to claim 1 or 2, which has a sheet or block shape. It is a manufacturing method of the gel of any one of Claim 1 thru | or 3, Comprising: The following processes:
(a) introducing a gellable polymer chain into the capillary tube and orienting the gellable polymer chain in the long axis direction of the capillary; and
(b) A method comprising a step of gelling the gelable polymer chain. The gel according to any one of claims 1 to 3, for use in cell culture. The culture containing the cell orientated in the fiber major axis direction in the gel of any one of Claim 1 thru | or 3. The culture according to claim 6, wherein the cells are muscle cells. A cell sheet or a cell block comprising the culture according to claim 6 or 7. The method for storing a gel according to any one of claims 1 to 3, wherein the gel is sucked into a silicon tube and the gel is stretched in the longitudinal direction of the tube and stored. The gel according to any one of claims 1 to 3, wherein the gel is filled in a silicon tube and is stretched in the longitudinal direction of the tube.