JP2015037381A - Muscle cell output device and muscle cell output evaluation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an output device suitable for the output of the contractile force of a muscle cell.SOLUTION: A muscle cell output device 2 includes a complex 4 which has a support 10 mainly composed of collagen comprising one or plural long part 12 having a predetermined width, and a muscle cell 20 held on the long part 12.

Description

  The present invention relates to a muscle cell output device and a muscle cell output evaluation method.

  There are attempts to evaluate the contractility of muscle cells or their structures. There is also an attempt to use such contractility as a power source. For example, to use muscle cells as a power source or to evaluate the force (contraction force) generated in muscle cells for the treatment of diseases related to muscle cells such as heart disease and screening for drugs therefor. Has been tried.

  In general, adherent cells such as muscle cells are cultured using a plastic dish. However, in such a culture state, since the cells adhere to the culture surface, it is impossible to evaluate the output of myocytes obtained by culturing. Therefore, various evaluation methods have been tried. For example, a method for producing a skeletal muscle-like tissue using the self-assembly ability of muscle cells and measuring the force generated in the tissue is disclosed (Non-Patent Documents 1 and 2). Also disclosed is a method of holding myotube cells with a glass microneedle and measuring the generated force by the deflection of the glass microneedle (Non-patent Document 3). Furthermore, an apparatus for producing a three-dimensional annular structure in which myoblasts and collagen gel are combined in a predetermined shape and measuring the contraction force of the structure is also disclosed (Patent Document 1). Furthermore, an apparatus for measuring the tension of primary cultured cardiomyocytes is also disclosed (Patent Document 2).

JP 2004-500093 A JP 2002-228655 A

Dennis RG et. Al., Am. J. Physiol. Cell. 280 (2): C288-95 Herman Vandengurgh et.al., Muscle & Nerave (2008), 37, p438-447 Macmahon DK et.al., Am. J. Physiol.Cell.266 (6 pt 1): C1795-802

  However, until now, no studies have been conducted on cell structures suitable for evaluating the output of cultured cell structures. The inability to uniformly produce the sample to be evaluated for output greatly reduces the reliability and quantitativeness of the evaluation results.

  That is, and the evaluation method thereof The methods of Non-Patent Documents 1 and 2 described above use cell detachment and accumulation, so the reproducibility is low and the experiment is inefficient. In addition, since the size of the microstructure that can be produced cannot be made uniform, the quantitative property is poor. Furthermore, since the evaluation is performed with the cells folded, the internal structure cannot be observed with the cells alive. Furthermore, since the strength as a cell structure is weak, long-term observation was impossible.

  The method of Non-Patent Document 3 requires a special device and does not hold as a general-purpose evaluation method. Furthermore, in the method described in Patent Document 1, since a single cell is an object to be evaluated, the quantitative property is poor and long-term observation is impossible. Furthermore, in the method of Patent Document 2, it is very complicated to produce an evaluation object, and it is difficult to orient the cells. For this reason, it was difficult to produce a stable cultured cell body as an evaluation target. Moreover, the difficulty in producing the evaluation object means a loss of quantitativeness at the same time.

  Therefore, an object of the present invention is to provide an output device suitable for outputting muscle contraction force of muscle cells. Another object of the present invention is to provide uses such as a cell output evaluation method related to muscle contraction using the output device.

  In order to solve the above-mentioned problems, the present inventors focused on the combination of muscle cells and a support, and used a support having a long portion with a predetermined width as a support for muscle cells. We obtained knowledge that cell contractile force can be output properly. The present inventors have completed the present invention based on these findings. According to the present invention, the following means are provided.

  According to the present invention, an output device for muscle cells, comprising a support mainly composed of collagen having one or more elongated portions having a predetermined width, and held on the elongated portions of the support. An output device is provided comprising a complex having a muscle cell that has been formed. The muscle cells may be one or more selected from skeletal muscle cells, cardiomyocytes, smooth muscle cells, and myotube cells.

  In the output device of the present invention, the predetermined width of the long portion is preferably 250 μm or more and 650 μm or less, and more preferably 400 μm or more and 500 μm or less. Moreover, it is preferable that the length of the said elongate part is 2 mm or more and 50 mm or less.

  In the output device of the present invention, it is preferable that the muscle cells are adhered to the surface of the long portion. In the output device of the present invention, it is also preferable that two or more of the complexes are integrated.

According to the present invention, a myocyte output evaluation method comprising the following steps:
(A) applying one or more stimuli that may promote or inhibit muscle contraction to the muscle cells of the myocyte output device of the present invention;
(B) detecting a contraction force generated in the output device;
A method is provided comprising: The step (a) can be a step of applying at least electrical stimulation to the cultured cells.

It is a figure which shows an example of the output device and support body of this invention. It is a figure which shows another example of the support body used for the output device of this invention. It is a figure which shows an example of the method of evaluating the output of a muscle cell using the output device of this invention. It is a figure which shows the evaluation system of the myocyte used in the Example. It is a figure which shows an output evaluation result when the width | variety of a long part shall be 250 micrometers-650 micrometers. It is a figure which shows the output evaluation result about an output device provided with a 2 type | mold support body. It is a figure which shows the output evaluation result about an output device each provided with the support body from which thickness differs (20 micrometers, 35 micrometers).

  The present invention relates to a myocyte output device, a manufacturing method thereof, and an application thereof. According to the output device of the present invention, the muscle cells are combined with the long portion of the support, and when the muscle contraction stimulus is applied to the muscle cells, the support is included by the contraction force of the muscle cells. The composite can be effectively shrunk, and as a result, it can be output as a tension or the like. For this reason, the output device of the present invention is suitable for obtaining the output of myocytes, and can easily evaluate the contraction force of myocytes. Moreover, since the composite body in such an output device has a simple structure and is easy to handle, it can be produced more uniformly as an evaluation object than in the past. Therefore, it is excellent in accuracy and reproducibility as compared with the prior art, and it is possible to evaluate the shrinkage force with more quantitativeness.

  Further, according to such an output device, when a stimulus that may induce muscle contraction is applied on the support, the contraction ability of the muscle cell can be detected, so that the function, responsiveness, and differentiation of the muscle cell itself are detected. In addition to evaluating the degree, drug screening and the like are also possible.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. FIG. 1 is a view showing an example of the output device and the support thereof according to the present invention, FIG. 2 is a view showing another example of the support used in the output device of the present invention, and FIG. 3 is a view showing the present invention. It is a figure which shows an example of the method of evaluating the output of a muscle cell using this output device.

(Muscle cell output device)
As shown in FIG. 1, the output device 2 of the present invention includes a complex 4 including a support 10 having a long portion 12 and a muscle cell 20 as its constituent elements.

  The muscle cell 20 applied to the output device 2 of the present invention includes a cell having a contractility that is the same as that of a mature cell of the same type as that of a mature cell, in addition to a cell matured with respect to the muscle contractility. It is out. For example, when the mature cell is a skeletal muscle cell, it is sufficient if the mature cell has a tendency to contract in the same quality as the skeletal muscle cell, and the degree thereof is not limited. Accordingly, the myocytes include immature cells such as cells undergoing differentiation into mature myocytes and immature cells of mature myocytes. The origin of the muscle cell is not particularly limited. Examples include cells derived from humans or non-human animals. Muscle cells may be derived from iPS cells or ES cells. Furthermore, gene introduction or gene disruption may be performed.

  Cells matured for muscle contraction ability can be selected from skeletal muscle cells, cardiomyocytes and smooth muscle cells. Any of these may be used, but the output device 2 of the present invention is preferably applied to skeletal muscles that require cell orientation for expression of contractility.

  Examples of immature cells of skeletal muscle cells include myotube cells, myoblasts, skeletal muscle satellite cells, and mesenchymal stem cells. Examples of immature cells of cardiomyocytes include immature cells of myocardial cells or cells in the middle of differentiation, such as myocardial progenitor cells, myocardial stem cells, mesenchymal stem cells, and the like. Examples of immature cells of smooth muscle cells include immature cells of smooth muscle cells or cells in the middle of differentiation, such as smooth muscle progenitor cells and mesenchymal stem cells.

  The muscle cell is preferably a cultured cell. In the present specification, the cultured cell means a cell cultured in vitro. Therefore, it may be a cell cultured for a certain period after being collected from a living body, and includes primary cultured cells.

  The muscle cell 20 may be provided directly on the long portion 12 or may be provided via a heterogeneous cultured cell layer, but is directly attached to the long portion 12. It is preferable. Especially, it is preferable that it is the cell seed | inoculated on the surface of the elongate part 12, and was cultured. More specifically, it is preferable that the muscle cell 20 is cultured to a confluent state following the elongated planar form of the elongated portion 12. In other words, it is preferable to form a cultured cell layer according to the shape of the long portion 12. In such a state, contractility is most likely to be exhibited, which is preferable for output extraction and evaluation. Further, as long as the muscle cells 20 are provided in at least a single layer state, the muscle cells 20 may be further layered, or cultured cells of heterogeneous cells such as endothelial cells may be layered.

(Support)
The support 10 has a long portion 12 having a predetermined width. The long portion 12 is formed mainly of collagen. Collagen is preferable in that it can have good cell adhesion and moderate flexibility and can maintain transparency under culture conditions. Collagen can be classified into insoluble collagen and soluble collagen. As the collagen used in the raw material of the present invention, soluble or solubilized collagen is used. Specifically, enzyme-solubilized collagen (atelocollagen), alkali-solubilized collagen, acid-soluble collagen, salt-soluble collagen and the like can be used, and atelocollagen is particularly desirable. Since the solubility of collagen depends on the degree of cross-linking of collagen, and the degree of cross-linking becomes higher as the degree of cross-linking increases, the degree of cross-linking of collagen used in the present invention is preferably, for example, a trimer or less, more preferably 2 amounts. Below the body. For example, the molecular weight of collagen is preferably about 300,000 to about 900,000, more preferably about 300,000 to about 600,000.

  Collagen may be derived from living tissue or recombinant collagen. When the tissue is derived from a living tissue, the tissue to be used is not particularly limited. For example, the dermis layer is easily collected and easily dissolved. When derived from a living tissue, there is no particular limitation on the animal species, and there is no problem as long as the collagen has a denaturation temperature at which the collagen does not undergo thermal denaturation during culture. Specifically, it can be derived from mammals such as cattle and pigs, derived from birds such as chickens, warm currents such as tuna and sea bream, derived from fish living in tropical regions, and the like. Chemically modified products of the side chains of the constituent amino acids of collagen, specifically, acylation such as acetylation, succinylation and phthalation, esterification such as methylation and ethylation, and the like can be used.

  In addition, the elongate part 12 can also mix other biocompatible materials besides collagen. Examples of biocompatible materials include gelatin, fibrin, albumin, hyaluronic acid, heparin, chondroitin sulfate, chitin, chitosan, alginic acid, pectin, agarose, hydroxyapatite, polypropylene, polyethylene, polydimethylsiloxane, or glycolic acid, lactic acid or amino acid. Or a copolymer thereof, or a mixture of two or more of these biocompatible materials.

  The long portion 12 of the support 10 is preferably in the form of a film having a substantially constant thickness. The long portion 12 preferably has a rectangular shape having approximately the same width throughout the long portion 12. The width of the long portion 12 is set so that the complex 4 having the myocytes 20 on the long portion 12 is developed, and when the myocytes 20 are placed in a constant isometric contraction state, a greater contractility is expressed. It is preferable that This is because the contractility of muscle cells can be detected with high sensitivity.

  In general, the contractility of skeletal muscle cells and the like increases as the orientation of the cells increases, and the generated tension when the cells are in an isometric contraction state is also large. In addition, in order to obtain the cell orientation, it is said that the width of the cell layer (direction perpendicular to the contraction direction) needs to be 100 μm or less. However, according to the present inventors, when a muscle cell or the like is cultured on the surface of the long portion 12 in the shape of a long shape following the planar shape and a contracted state is formed along the long axis direction, It has been found that the generated tension becomes maximum at a width exceeding 100 μm. The width of the long portion 12 can be determined by a muscle cell output evaluation method described later. This will be described later.

  The long portion 12 preferably has a width of 250 μm or more and 650 μm or less. If it is less than 250 μm, the strength is insufficient and handling becomes difficult, and evaluation in an isometric contraction state is also difficult. On the other hand, if it exceeds 650 μm, the orientation of the skeletal muscle cells or their precursor cells is too low to be suitable for taking out and evaluating the contractile force. More preferably, it is 400 μm or more. This is because if it is 400 μm or more, the contraction force per unit cross-sectional area of the long portion 12 tends to be stabilized. More preferably, it is 440 micrometers or more. The upper limit is more preferably 500 μm or less. The range of the width of the long portion 12 is more preferably 400 μm or more and 500 μm or less, and most preferably 440 μm or more and 460 μm or less.

  The thickness of the long portion 12 is not particularly limited, but is preferably 25 μm or more and 100 μm or less in consideration of handling property and economic efficiency in addition to taking out and evaluating muscle contraction force. This is because if the thickness is less than 25 μm, handling is difficult due to fragility, and the collagen membrane of the long portion 12 may be deformed by the force generated by the cells during cell culture. On the other hand, when the thickness exceeds 100 μm, it is difficult to produce a collagen film having such a film thickness or high cost, and the transparency is lowered and cell observation becomes difficult. More preferably, it is 30 μm or more and 60 μm or less.

  The length of the long portion 12 is not particularly limited, but in consideration of output extraction and evaluation, it is preferably 2 mm or more and 50 mm or less, more preferably 5 mm or more and 50 mm or less, more preferably 10 mm or more. 25 mm or less.

  Although the support body 10 may be provided with one long portion 12, for example, two or more long portions 12 may be provided in parallel so that the long axis direction is aligned. By providing the plurality of long portions 12, it is possible to extract and evaluate a large output. In the form in which the support body 10 includes a plurality of long portions 12, the contraction that occurs in two or more long portions 12 may be arranged so as not to inhibit the contraction in the adjacent long portions 12. preferable. An example of the support 10 having two or more long portions 12 is shown in FIG. In this case, the long portions 12 can be appropriately spaced so as not to interfere with each other's contraction.

  The support 10 may include an element for connecting to another member (an output destination or an evaluation unit) for extracting an output of muscle contraction and / or evaluating the output. These elements may use both ends or one of the long portions 12 along the long axis direction, or may be provided as connecting portions 14 at both ends or one end along the long axis direction of the long portions 12. . The connecting portion 14 may be formed integrally with the long portion 12. That is, it may be formed as a single film mainly composed of collagen and continuous with the long portion 12. In this case, as illustrated in FIG. 1, the connecting portion 14 may have a form that can be clearly distinguished from the long portion 12, but may be a form in which the long portion 12 is formed to be extended. The connection part 14 can be provided with a preferable form for extracting or evaluating the output of muscle contraction. For example, as shown in FIG. 1, it may have a size, thickness, and / or shape that can ensure a greater strength than the long portion 12 in favor of fixing or the like. Moreover, the process of providing the through-hole part 16 for fixation etc. may be made | formed. The connecting portion 14 does not need to have the muscle cells 20 on the surface, but when the connecting portion 14 is formed of a cell adhesive material like the long portion 12, the muscle cells are adhered and held on the surface. May be.

  The complex 4 including the film-like support 10 and the muscle cells 20 on the long portion 12 is a laminate of the support 10 and the muscle cells 20. That is, the composite 2 has a substantially sheet-like form having at least a part of a planar form corresponding to the long portion 12. For this reason, the muscle cell 20 is supported and reinforced by the support body 10, and has excellent handleability.

  The composite 10 may constitute the entire output device 2 of the present invention as it is, or two or more composites 4 may be integrated to form the output device 2 of the present invention. For example, the composites 4 may be integrated so that the composites 4 are arranged in parallel in the same plane, or the composites 4 may have different heights (directions along the thickness of the long portion 12). You may accumulate. Furthermore, these may be combined and accumulated in the planar direction and the height direction. When the composites 4 are arranged in parallel, the composites 4 may be brought into close contact with each other or may be arranged in parallel with a gap. In the case of stacking in the height direction, the composites 4 may be directly stacked or stacked with a gap. When accumulating in the height direction, the support 10 may be held by an appropriate holding and fixing means so that an appropriate space is formed between the composites 4. In any of these integrated forms, in order to effectively extract or evaluate the output, it is preferable that the long axis direction of the long portion 12 of each composite 4 is oriented in the same direction or substantially the same direction.

  As described above, the output device 2 of the present invention contracts the muscle cell 20 by applying a stimulus that induces muscle contraction to the muscle cell 20, and outputs the contraction force as tension or the like to the outside. can do. In the complex 4 that is a basic component included in the output device 2 of the present invention, the muscle cells 20 are cultured in a substantially sheet shape on the long portion of the support. That is, it is not a complicated structure as in the prior art, but has a simple structure. For this reason, it has a structure suitable for outputting as tension generated based on the contractility of muscle cells. Therefore, it is suitable to use or evaluate such an output as a power source. In addition, since it has a simple structure, it is easy to manufacture and the accuracy and reproducibility of manufacture are improved, so that stable output can be taken out, and it has excellent accuracy and reproducibility, and highly quantitative evaluation. Is possible.

  Moreover, since the output device of the present invention has muscle cells in the long part of the support, it also has good handling properties. Since handling is easy, it is possible to simplify output extraction and evaluation. As a result, a stable output can be taken out, and the evaluation is excellent in accuracy and reproducibility, and highly quantitative.

  Furthermore, since the support which is a basic component of the output device of the present invention has a simple structure, a tension generation system based on shrinkage can be easily optimized. In particular, the width of the long portion greatly affects the generated tension. By optimizing the length, the output capability can be easily optimized. As a result, according to the output device of the present invention, a larger output can be easily taken out from a smaller elongated portion. At the same time, it is possible to increase the detection sensitivity when detecting the contractility as the generated tension.

  Furthermore, according to the output device of the present invention, the muscle cells are formed in a sheet shape (layer shape) on the long portion. Further, the long part is mainly made of collagen and has high transparency. For this reason, it is excellent in the visibility (including visual observation and under a microscope) of various states including the differentiation state and orientation state of myocytes, and can be easily observed. Visibility is important in muscle research and use. Therefore, the output device of the present invention is an output device suitable for observation and research.

  According to the output device of the present invention, it is possible to simplify, improve the efficiency, and improve the accuracy of research use, screening of new drugs, evaluation of cultured cells, etc. by applying to the output evaluation method of muscle cells described later. it can.

  The output device 2 of the present invention can be used for an actuator that uses functions such as skeletal muscle in vitro. Further, the output device 2 of the present invention is a disease related to contraction of muscle cells or the like (heart muscle related diseases such as heart diseases, skeletal muscle related diseases such as muscular dystrophy, smooth muscle related diseases such as hypertension, asthma and digestive dysfunction). Available for treatment. For example, it can be used for regenerative medical materials for the treatment of such diseases in humans or the like or for evaluation thereof. It can also be used for drug screening for the treatment of such diseases or for evaluating the efficacy of therapeutic agents.

  According to the present invention, a method for acquiring output of muscle cells or the like using the output device 2 of the present invention is also provided. That is, the output can be effectively taken out by giving a stimulus for inducing muscle contraction to the output device 2 of the present invention. The contractile force of the myocyte 20 of the output device 2 can be taken out as tension or the like.

  In addition, according to this invention, the support body for output devices is also provided. The support of the present invention can have various forms of the support described above. Typically, the support can take the form of a collagen film having a long portion with a width of 250 μm or more and 650 μm or less, and more preferably a collagen film having a thickness of 25 μm or less and 100 μm or less.

(Production method of myocyte output device)
The output device 2 of the present invention can be manufactured, for example, by the following method. Typically, the above-described support 10 is prepared, and the myocytes 20 are obtained by seeding and culturing the myocytes 20 or their precursor cells on the long portion 12 of the support 10.

  The support 10 can be produced using various collagens by a known method, for example. In order to obtain the long portion 12 having a predetermined shape and size, appropriate molding conditions may be given. Collagen films as cell culture carriers and methods for their production are well known to those skilled in the art. Moreover, you may obtain a sheet-like film commercially and process it into a predetermined shape as needed. In addition to the long portion 12, a connecting portion 14 or the like may be separately formed on the support 10 as necessary.

  When the support 10 includes a plurality of long portions 12, for example, as illustrated in FIG. 2, when the plurality of long portions 12 are provided in the same plane, the support 10 having a shape as illustrated in FIG. Can be produced.

  Next, the muscle cell 20 is obtained on the long portion 12 of the support 10. When culturing myocytes directly on the long portion 12, the long portion 12 is seeded with myocytes or precursor cells thereof prior to the culture. The seeded cells may be muscle cell precursor cells. In the culturing process on the long portion 12, the myocytes 20 can be finally obtained by differentiating the myocytes by a known method. The cells to be seeded include, for example, skeletal muscle cells, smooth muscle cells, cardiomyocytes and myotube cells, skeletal muscle-derived stem cells, cardiac muscle-derived stem cells, smooth muscle-derived stem cells, etc. Examples include blast cells, myoblasts, myotube cells, vascular endothelial cells, mesenchymal stem cells, ES cells, iPS cells, osteoblasts, bone cells, chondrocytes, fat cells, nerve cells and the like. In addition, the seed | inoculated cell contains the cell of the state which was extract | collected from the living body, and comprised the structure | tissue, and the cell artificially formed in the sheet form besides the individual cell. Therefore, the cells to be seeded do not necessarily take a form such as a cell suspension, and may be a tissue such as a muscle tissue or a part thereof (tissue piece) or a cell sheet.

  The culture conditions are not particularly limited, and are appropriately set according to the type of seeded cells. Moreover, when it is necessary to differentiate, a required culture medium and a compound are used suitably. For example, when seeding myoblasts, it can be differentiated into myotube cells by supplying a differentiation medium containing 2% bovine serum.

  The muscle cells are adherent cells, and are preferably cultured to a confluent state by adhering to the surface of the long portion 12 in a desired differentiation state following the planar shape. In order to obtain the myocytes 20 in the form of the long portions 12 by suppressing the adhesion of cells to other than the long portions 12, and to facilitate the peeling of the support 10 from the culture substrate, It is preferable to seed and adhere the cells with the support 10 placed on the adhesive surface. For example, it is preferable to culture after seeding cells with the support 10 placed on the surface of a substrate such as polydimethylsiloxane (PDMS), allowing them to adhere, and removing unadhered cells by washing or the like. Note that PDMS also has an advantage that it has the hardness and strength necessary to fix a pin or the like for fixing the support 10. The fixing of the support 10 is important in order to avoid the possibility of damaging the muscle cells 20 being cultured on the support 10 by mistake in the medium exchange operation. In addition, it is preferable to continue culture | cultivation in the state which arrange | positioned the support body 10 on the cell non-adhesive surface as it is after adhesion | attachment.

  After culturing as a myocyte until a desired differentiation state and a confluent state, the culturing process can be terminated. The differentiation state of the myocyte 20 can also be confirmed as a specific protein or the like as required. Further, the orientation state of the myocyte 20 can also be confirmed by cell staining or the like.

  In addition, when obtaining the layered form of the myocytes 20, the myocytes 20 obtained on the long portion 12 may be layered with the myocytes 20 separately formed in a sheet shape, or on the long portion 12. The cultured myocytes 20 may be further seeded with myocytes and cultured. In addition, when taking a layered form of cultured muscle cells 20 and a cultured cell layer of heterologous cells, the heterogeneous cells are seeded and cultured in the long part 12 in advance to be confluent, and then the myocytes are seeded and cultured. Or the sheet | seat of the myocyte 20 may be laminated | stacked.

  As described above, the composite 4 of the output device 2 of the present invention can be obtained. When the composite 4 is further integrated, the composites 4 are arranged and / or stacked according to a desired integration form. In order to collect the composites 4, suitable holding means for clamping or holding the support 10 or its connecting portion 14 may be used to collect a plurality of composites 4 as necessary.

  According to the output device manufacturing method of the present invention, the output device 2 can be manufactured by a simple operation. That is, in the complex 4 that is a basic component of the output device 2, the muscle cells 20 are substantially cultured in a sheet shape, and it is not necessary to produce a complicated structure as in the conventional case. For this reason, it is possible to easily obtain the output device 2 in a homogeneous state as compared with the conventional case.

(Method for evaluating myocyte output)
The output evaluation method for muscle cells of the present invention uses the output device 2 of the present invention. The evaluation method of the present invention includes a step of applying a stimulus (stimulation step) that may induce muscle contraction to the muscle cells 20 of the output device 2 of the present invention, and a contraction force generated in the output device 2. And a step of detecting (detection step). Hereinafter, the output evaluation method of the present invention will be described with reference to FIG. FIG. 3 is a diagram showing an example of the output evaluation method of the present invention.

  In evaluating the output of myocytes, an output device 2 including the myocytes 20 to be evaluated is prepared. For example, muscle cells derived from human or non-human animals are used for output evaluation as a power source or the like, without any particular limitation. In addition, for evaluation as a regenerative medical material, muscle cells derived from a heterologous animal or from the same species (autologous or exogenous) suitable for a transplant destination organism are used. Furthermore, for screening for a drug that promotes or inhibits muscle cell contraction, a target animal, that is, a muscle cell derived from a human or non-human animal is used. Furthermore, muscle cells derived from cells collected from a specific individual are used for the evaluation of drug efficacy for tailor-made treatment. These various myocytes may be obtained by differentiating from each progenitor cell, and the cultured myocytes may be derived from iPS cells. Furthermore, gene introduction or gene disruption may be performed.

(Stimulation process)
In the stimulus applying step, a stimulus that may promote or inhibit muscle contraction is given to the muscle cells 20 of the output device 2. In giving such a stimulus, the output device 2 of the present invention may be based on either an evaluation form based on isometric contraction or an evaluation form based on isotonic contraction, but as shown in FIG. It is preferable that it is based on the evaluation form by scale contraction. That is, it is preferable that the length of the long portion 12 in the output device 2 can be held constant, and the muscle cells 20 in this state are preferably stimulated and evaluated. This is because the contraction ability of the myocyte 20 can be easily evaluated by measuring the tension generated by the contraction of the myocyte 20 at the isometric contraction.

  It is sufficient that the stimulus to be applied has the possibility of promoting or inhibiting muscle contraction, and it does not matter whether the muscle contraction is promoted or inhibited as a result. Such stimuli include physical stimuli and / or chemical stimuli. When evaluating the basic ability of muscle cells, that is, the presence or absence of muscle contraction ability (tension generation ability), it is preferable to use electrical stimulation that causes muscle contraction. This is because it is simple and reproducibility can be secured easily.

  Physical stimulation includes electrical stimulation and mechanical stimulation known to contract muscles. Examples of the electrical stimulation include a pulse current that generates a single contraction. Moreover, pulling by a motor etc. is mentioned as mechanical stimulation. In the myocardium and the like, the output evaluation at the time of muscle stretching is useful. Insect flying muscle also has a phenomenon of muscle contraction due to pulling. Moreover, administration of various compounds etc. is mentioned as chemical irritation | stimulation. The chemical stimulation includes a test compound at the time of screening for a drug that promotes or inhibits muscle cell contraction. Moreover, the drug etc. at the time of evaluation of the reactivity (medicine effect) with respect to the drug which accelerates | stimulates or inhibits muscle contraction in custom-made treatment etc. are mentioned. Such a test compound or drug can be supplied to a liquid medium or the like filled in the culture container of the output evaluation system shown in FIG. It may be applied alone to evaluate whether it causes or inhibits muscle contraction by itself, but may also be applied in combination with other stimuli as described below.

  Two or more of these stimuli can be combined. For example, applying physical stimuli (stimuli known to cause muscle contraction) such as electrical pulses in the presence of chemical stimuli such as test compounds or drugs that may promote or inhibit muscle contraction be able to. By doing so, the presence or absence and the extent of the effect of these test compounds and drugs on the contraction ability of muscle cells can be evaluated from the contrast with the contraction ability of muscle cells in the absence of chemical stimulation.

(Detection process)
In the detection step, the contraction force is detected when the output device 2 contracts due to the stimulus. In order to detect such contraction force, for example, it can be detected as tension in an evaluation system based on isometric contraction. In the isometric contraction system, as shown in FIG. 3, if the length of the myocytes 20 of the output device 2 (the length of the long portion 12) is fixed to be kept constant, Tension acts on the strain gauge by the generated contraction force, and displacement (strain) occurs in the strain gauge. Due to this displacement, the resistance value of the strain gauge changes, so that the contraction force generated in the output device 2 can be detected and measured as the tension acting on the strain gauge by this change in resistance value. In addition, the electrical signal (resistance value) obtained in the strain gauge is output to a computer or the like equipped with a CPU that operates a program that can calculate tension or strain based on the electrical signal, such as a PC, and is processed in the computer. Output as tension. The configuration of the evaluation system for detecting the contractile force is not particularly limited. In addition to the known isometric shrinkage evaluation system shown in FIG. 3, an isotonic shrinkage evaluation system can be applied. In the isometric contraction evaluation system, the contraction force (contraction amount) generated in the muscle cell 20 is very small, and unlike the normal isometric contraction evaluation system, it is pulled with a motor or the like to be in an isometric state. It is not always necessary to maintain, and the long portion 12 may be simply fixed to the connecting portion 14 or the like through a certain length. A conventionally known strain gauge can be used as the strain gauge.

  According to the evaluation method of the present invention, the contractile force (output) of myocytes can be evaluated with an evaluation system using a strain gauge or the like. Conventionally, the output of the muscle has been measured only by a special device or an expensive device. However, according to the evaluation method of the present invention, it can be applied to a simple evaluation system by using the output device 2 of the present invention. As a result, it is possible to perform output evaluation with excellent accuracy and reproducibility and as a result, quantitative output. In addition, muscle output evaluation experiments can be performed without using experimental animals. Furthermore, it is possible to easily realize tests and research such as output evaluation, drug screening, evaluation of materials for regenerative treatment, effect evaluation in tailor-made treatment of muscle-related diseases such as muscular dystrophy related to contraction force of muscle cells. It becomes like this.

  Hereinafter, specific examples of the present invention will be described with reference to examples. The following examples do not limit the present invention.

(Production of support)
A collagen film (manufactured by Koken Co., Ltd., CLF01, thickness 35 μm) is formed with a strip-shaped long portion having a length of 18 mm and a width of 250 μm to 1200 μm, and a substantially hexagonal connecting portion having a diameter of 1 mm at both ends thereof A through hole was formed in the center to form a support. Moreover, the two-type support body was produced similarly to the support body produced previously except the point provided with two long parts with a width of 450 micrometers-750 micrometers using the same collagen film. Moreover, the support body provided with the elongate part whose width | variety of a elongate part is 450 micrometers was also produced separately using the same collagen film except thickness being 20 micrometers.

(Production of output device)
Each of the prepared supports having long portions having different widths was fixed on a 35 mm plastic dish coated with PDMS, and placed under sterilization under ultraviolet irradiation for 5 minutes. Thereafter, 2 ml of a growth medium (DMEM medium containing 10% fetal bovine serum) containing 0.5 × 10 ⁵ cells / ml of C2C12 myoblasts was dropped and seeded with C2C12 myoblasts.

After incubating at 37 ° C. under 5% CO ₂ for 24 hours and confirming that C2C12 cells adhered to the collagen film, unadhered cells were washed and removed with a growth medium. Thereafter, the adhered cells are cultured at 37 ° C. under 5% CO ₂ using a growth medium until confluent, and the medium is replaced with a differentiation medium (DMEM medium containing 2% fetal calf serum). Differentiation was induced. Differentiated myotubes were obtained by changing the differentiation medium every 24 hours and culturing for one week. When the cells were stained with a fluorescent dye and observed with a fluorescence microscope, myotube cells oriented in the long axis direction of the long part of the support were confirmed. These were used as output devices.

(Output evaluation)
Each output device obtained was applied to the evaluation system shown in FIG. That is, the culture container filled with the differentiation medium was placed on a temperature control hot plate, and the medium inside was kept at 28 ° C. In the container, the output device was fixed to be isometrically contractible by a pin so as not to loosen through the through hole of the connecting portion of the output device. Moreover, one connection part was connected with the strain gauge so that the contraction force of an output device might be transmitted. The electric signal from the strain gauge can be output to the PC. Carbon electrodes were arranged on each surface along the long axis direction of the long part of the output device, and connected to an amplifier so that an electric pulse necessary for contraction of myotube cells could be applied.

  An electrical pulse (1 V / mm, 10 ms plus duration) is applied to the myotube cells on the long part of each output device, and the tension based on the contractile force generated in the myotube cells is measured using a strain gauge. did. The results are shown in FIG.

  As shown in FIG. 5, the contractile force of the muscle cells on the long part could be detected as the tension in the range of 250 μm to 650 μm. In particular, the tension generated almost corresponding to the increase in the width of the long portion up to about 500 μm was also increased. From around 500 μm, the tension became constant regardless of the thickness of the support. From this, it was found that when the width of the long portion exceeds 500 μm, effective muscle contraction does not occur and tension according to the width does not occur. Further, the width of the support is preferably 250 μm to about 500 μm in that a tension corresponding to the width is generated. However, in order to extract or evaluate the tension with the most effective muscle contraction with respect to the stimulus, 400 μm It was found that the thickness was 500 μm or less, more preferably around 450 μm (440 μm or more and 460 μm or less).

  Also, when the generated tension was evaluated in the same manner for the output device manufactured using the two-type support, as shown in FIG. 6, the output power was larger than that of the single-type output device having the same width. It was found that can be taken out. From this result, it was found that a larger output corresponding to the number can be obtained by providing a plurality of long portions having appropriate widths.

  Furthermore, the generated tension was tested under the same conditions for the output device using the support (the width of the long portion was 450 μm) prepared using collagen films having different thicknesses (35 μm and 20 μm). The results are shown in FIG. As shown in FIG. 7, when the thickness was 20 μm, the generated tension was halved compared to the case where the thickness was 35 μm. From this, it was found that when the thickness of the long portion is about 20 μm, it is difficult to extract the contraction force of the muscle cells, and the detection sensitivity of the contraction force of the muscle cells tends to decrease. From the above results, it has been found that the thickness of the long portion or the support is preferably 25 μm or more in consideration of output extraction and evaluation based on the contractile force of muscle cells.

2 Output device 4 Composite body 10 Support body 12 Long part 14 Connection part 16 Through-hole part 20 Myocyte

Claims (9)

An output device for muscle cells,
A complex comprising a support mainly composed of collagen having one or more long portions having a predetermined width, and myocytes held on the long portions of the support;
An output device comprising:   The output device according to claim 1, wherein the predetermined width of the long portion is not less than 250 μm and not more than 650 μm.   The output device according to claim 2, wherein the predetermined width of the long portion is not less than 400 μm and not more than 500 μm.   The length of the said elongate part is an output device in any one of Claims 1-3 which are 2 mm or more and 50 mm or less.   The output device according to claim 1, wherein the muscle cells are adhered to the surface of the long portion.   The output device according to claim 1, wherein two or more of the complexes are integrated.   The output device according to any one of claims 1 to 6, wherein the muscle cells are one or more selected from skeletal muscle cells, cardiomyocytes, smooth muscle cells, and myotube cells. A method for evaluating myocyte output,
The following steps:
(A) applying one or more stimuli that may promote or inhibit muscle contraction to the muscle cells of the muscle cell output device according to any one of claims 1 to 7;
(B) detecting a contraction force generated in the output device;
A method comprising:   The method according to claim 8, wherein the step (a) is a step of applying at least electrical stimulation to the cultured cells.