Device capable of simultaneously measuring myocardial-specific contractile force and activity potential

Abstract

The present invention addresses the problem of providing a device for simultaneously measuring an electric signal and a contractile force by using a highly mature myocardial tissue having a 3D / oriented structure close to myocardial structure in a living body. As a solution, provided is a device for measuring the contractile force of a myocardial tissue, the device comprising a contractile unit which includes a rod-like artificial myocardial tissue having a three-dimensional structure and in which cardiomyocytes are oriented in the longitudinal direction, wherein the device is used for measuring the contractile force of the contractile unit.

Description

A device capable of simultaneously measuring cardiac muscle-specific contractile force and action potential

[0001] The present invention relates to artificial myocardial tissue, its uses, and methods for its production.

[0002] Traditionally, drug toxicity assessment and drug discovery and development have relied on animal-derived primary cells and animals, but these methods pose various challenges, including species and lot-by-lot differences compared to human tissue. Furthermore, current guidelines require nonclinical studies of cardiotoxicity (electrocardiogram QT prolongation) for all pharmaceuticals. Drugs with potential for lethal arrhythmias due to QT prolongation are often withdrawn from the market, delayed in approval, or abandoned. The successful generation of human iPS cells has paved the way for industrial applications, including toxicity and pharmacokinetic evaluations using human iPS cell-derived cardiomyocytes. While the issues of differentiation induction efficiency and purity have been largely resolved, remaining challenges include low maturity of the induced cardiomyocytes, poor cell orientation and weak contractile force, frequent arrhythmias in response to QT prolongation during drug toxicity assessment, and significant lot-by-lot variation. Therefore, there is a need for methods to evaluate the safety and efficacy of drugs using highly mature myocardial tissue with a structure similar to that of in vivo cardiac tissue.

[0003] On the other hand, conventional methods for assessing cardiomyocyte function typically involve measuring action potentials using multi-electrodes or measuring contractile force using cell motion or electrical resistance. Multi-electrode arrays used to measure action potentials have the following drawbacks: (1) poor adhesion between the multi-electrode chip and cells, resulting in uneven cell aggregation and easy detachment from the electrode chip. This results in inconsistent propagation of electrical signal excitation during pacing, leading to frequent arrhythmias when drugs are added. Furthermore, cells cannot be cultured long-term on multi-electrodes. (2) Cardiomyocytes on multi-electrode chips have a random structure and lack the inherent myocardial orientation. (3) While two-dimensional (2D) cardiomyocytes can be measured, thick myocardial tissues cannot be measured. Cell motion imaging and electrical resistance, the most commonly used methods for measuring cardiomyocyte contractile force, are suitable for evaluating myocardial contractile force by analyzing the pulsating behavior of 2D cardiomyocytes, but are inapplicable to three-dimensional (3D) myocardial tissues with their thick, oriented structure.

[0004] T. Hayakawa et. al., Journal of Molecular and Cellular Caridology, 77, 178-191, 2014F. Qian et. al., Lab on a Chip, 17, 1732-1739, 2017T. Ohys et. al., Lab on a Chip, 21, 3899-3909, 2021J.U. Lind et. al., Lab on a Chip, 17 3692-3703Yimu Zhao et. al., Cell 176, 913-927I. Mannhardt, et. al., Stem Cell Reports, 15, 983-998, 2020

[0005] While current methods for assessing cardiomyocyte function can measure myocardial-specific action potentials and contractile force separately, the inventors have sought to develop devices and methods that can measure both functions simultaneously under the same environment. While several methods for simultaneously measuring action potentials and contractile force have been reported in recent years, all of these methods evaluate cardiomyocytes at a 2D level. Therefore, the objective of the present invention is to provide a device that can simultaneously measure electrical signals and contractile force using highly mature myocardial tissue with a 3D, oriented structure similar to that of in vivo myocardium.

[0006] The present inventors have conducted extensive research to solve the above-mentioned problems and have found that by designing a device having a contractile portion containing a rod-shaped artificial myocardial tissue with a three-dimensional structure and cardiomyocytes oriented in the longitudinal direction so as to be able to measure the contractile force of the contractile portion, it is possible to provide a device for measuring the contractile force of highly mature myocardial tissue having a structure similar to that of myocardium in a living body. Such a device can be easily designed so that electrical signals can be measured simultaneously. The present invention was completed through further research based on this finding and includes the following aspects.

[0007] Item 1. A device for measuring the contractile force of myocardial tissue, comprising a contractile section comprising rod-shaped artificial myocardial tissue having a three-dimensional structure and in which cardiomyocytes are oriented in the longitudinal direction, said device being used to measure the contractile force of said contractile section. Item 2. A device for measuring the contractile force of myocardial tissue according to Item 1, wherein said artificial myocardial tissue is obtained by culturing cardiomyocytes on a surface having a contact angle with water of 95° or more. Item 3. A device for measuring the contractile force of myocardial tissue according to Item 1 or 2, further comprising a marker section comprising non-contractile myocardial tissue, said marker section being bound to said marker section so that the contractile force of said contractile section can be measured according to the mobility of said marker section. Item 4. A device for measuring the contractile force of myocardial tissue according to any one of Items 1 to 3, further comprising an action potential measurement section comprising artificial myocardial tissue having a three-dimensional structure and in which cardiomyocytes are oriented, said action potential measurement section being used for measuring action potentials using multiple electrodes. Item 5. Item 6. A method for producing a rod-shaped artificial myocardial tissue having a three-dimensional structure and in which cardiomyocytes are oriented in the longitudinal direction, comprising the step of culturing cardiomyocytes on a surface having a water contact angle of 95° or more to obtain the artificial myocardial tissue. Item 6. The method according to Item 5, wherein in the step, a region (A) having a surface having a water contact angle of 95° or more is bonded to two regions (B) having surfaces having a water contact angle of 85° or less, and the rod-shaped artificial myocardial tissue is formed on the line connecting the two regions (B) by culturing cardiomyocytes in regions (A) and (B).

[0008] According to the present invention, a device for measuring the contractile force of mature myocardial tissue having a structure similar to that of an in vivo myocardial tissue can be provided, and such a device can be easily designed to simultaneously measure electrical signals.

[0009] 1 is a schematic diagram of an example of a contractile force measuring device; FIG. 2 is a photograph of an example of a contractile force measuring device; FIG. 3 is a schematic diagram illustrating the measurement principle of a contractile force measuring device; FIG. 4 is an example of a measurement result using a contractile force measuring device; FIG. 5 is an example of a measurement result using a contractile force measuring device (effect of addition of E4031); FIG. 6 is an example of a measurement result using a contractile force measuring device (effect of addition of verapamil); FIG. 7 is an example of a measurement result using a contractile force measuring device (drug response evaluation using normal / disease iPS cell-derived cardiomyocytes); FIG. 8 is a schematic diagram illustrating an example of an application example of a contractile force measuring device; FIG. 9 is a photograph illustrating the measurement results of the contact angles of various materials with water; and FIG. 10 is a graph illustrating the measurement results of the contact angles of various materials with water.

[0010] 1. Device for measuring contractile force of myocardial tissue The device for measuring contractile force of myocardial tissue of the present invention has a contractile portion having a three-dimensional structure and including rod-shaped artificial myocardial tissue in which cardiomyocytes are oriented in the longitudinal direction, and is used to measure the contractile force of the contractile portion.

[0011] The device for measuring the contractile force of myocardial tissue of the present invention includes a rod-shaped artificial myocardial tissue in which the contractile portion has a three-dimensional structure and the cardiomyocytes are oriented in the longitudinal direction, and can measure the contractile force of this contractile portion. This makes it possible to measure the contractile force of highly mature myocardial tissue that has a structure similar to that of myocardium in vivo. Furthermore, as described below, such a device can be easily designed to simultaneously measure electrical signals.

[0012] The artificial myocardial tissue can be obtained by culturing cardiomyocytes on a surface having a contact angle with water of 95° or more. Preferably, in the culturing step, a region (A) having a surface having a contact angle with water of 95° or more is bonded to two regions (B) that allow non-oriented culture, and by culturing cardiomyocytes in the regions (A) and (B), the rod-shaped artificial myocardial tissue is formed on the line connecting the two regions (B).

[0013] For example, if a strip (a) having a surface with a water contact angle of 95° or more is prepared, and both ends of the strip are bonded to substrates (b) having a surface with a water contact angle of 85° or less, and cardiomyocytes are cultured in suspension on the strips using a liquid medium, the cardiomyocytes will initially tend to gather on the substrates (b) at both ends, forming a cardiomyocyte cell population there. On the other hand, the inventors have found that, although cardiomyocytes are less likely to accumulate on the strip (a) having a surface with a water contact angle of 95° or more due to weak adhesion, they gradually accumulate in the central portion of the strip (a) along its longitudinal axis, bridging the cardiomyocyte populations at both ends, forming a rod-shaped artificial myocardial tissue oriented in the longitudinal direction. The inventors have confirmed that this rod-shaped artificial myocardial tissue has a three-dimensional structure, in which cardiomyocytes are oriented in the longitudinal direction, resulting in a highly mature myocardial tissue with a structure similar to that in vivo. By utilizing this principle, it is possible to obtain an artificial myocardial tissue of a desired shape, regardless of the shape of the surface having a water contact angle of 95° or more (even if it is a long, thin piece (strip)). Furthermore, by utilizing this principle, it is possible to obtain an artificial myocardial tissue with or without a base material (b).

[0014] In addition, in the early stage of the culture, it is preferable to plasma-treat the surface of the elongated piece (strip) (a) to facilitate the accumulation of cardiomyocytes on the surface. In this case, the contact angle of the surface with water temporarily decreases to 85° or less, and therefore cardiomyocytes tend to accumulate on the entire surface of the elongated piece (strip) (a) immediately after the start of the culture. However, as the culture continues, the effect of the plasma treatment weakens and the contact angle of the surface with water gradually returns to the value before the plasma treatment, so that cardiomyocytes gradually accumulate in the central part of the elongated piece (strip) (a), as described above.

[0015] For a surface having a water contact angle of 95° or more, a water contact angle of 100° or more is preferred, and a water contact angle of 105° or more is even more preferred, from the viewpoint of efficiently obtaining an artificial myocardial tissue. Note that for this surface, a water contact angle of 120° or less is preferred, a water contact angle of 115° or less is even more preferred, and a water contact angle of 110° or less is even more preferred, from the viewpoint of efficiently obtaining an artificial myocardial tissue.

[0016] The surface having a contact angle with water of 95° or more may be made of any material as long as the contact angle with water is within the above range, and examples thereof include silicone (silicon resin), with PDMS (polydimethylsiloxane), silicone rubber, etc. being particularly preferred.

[0017] The substrate (b) having a surface with a water contact angle of 85° or less is preferably a water contact angle of 80° or less, more preferably a water contact angle of 75° or less, and even more preferably a water contact angle of 70° or less, from the viewpoint of efficiently obtaining an artificial myocardial tissue. Note that the substrate (b) is preferably a water contact angle of 50° or more, and more preferably a water contact angle of 60° or more, from the viewpoint of efficiently obtaining an artificial myocardial tissue.

[0018] The substrate (b) can be selected from a wide range of materials known to have the above-mentioned water contact angle and be suitable for cell culture. Examples include polyimine, polyethylene terephthalate, polyethyleneimine, polystyrene, and glass. Alternatively, oriented fibers, as described below, may be used as the substrate (b). In this case, the artificial myocardial tissue formed in the region can be oriented.

[0019] In the present invention, the contact angle of water is determined by irradiating a droplet on the surface to be measured with light, capturing an image of the droplet with a camera from the opposite side, and performing calculations through image analysis.

[0020] The rod-shaped artificial myocardial tissue highly expresses β-MHC, which is a maturation marker. In the present invention, whether or not the myocardial tissue highly expresses β-MHC can be confirmed by immunohistochemical staining.

[0021] The rod-shaped artificial myocardial tissue preferably has a width in the direction of the culture surface of 0.03 to 30 mm, more preferably 0.1 to 10 mm, and even more preferably 0.3 to 3 mm, from the viewpoint of convenience in measuring contractile force. Similarly, the rod-shaped artificial myocardial tissue preferably has a width in the direction perpendicular to the culture surface of 0.003 to 30 mm, more preferably 0.03 to 3 mm, and even more preferably 0.1 to 1 mm.

[0022] The type of cardiomyocytes can be selected appropriately depending on the purpose of the measurement. For example, when used to evaluate the cardiotoxicity of drugs, etc., cardiomyocytes derived from normal human iPS cells can be used. Furthermore, when used for drug discovery and development, cardiomyocytes derived from iPS cells for cardiac disease can also be used as needed.

[0023] As a specific example of the configuration of the device for measuring the contractile force of myocardial tissue of the present invention, there is a configuration in which a base material having a hollow area around the contractile portion is provided at the bottom, as shown in Figure 3. This configuration has the advantage that the contractile portion is likely to be in a floating state, making it easier to measure the contractile force. Furthermore, if necessary, a configuration in which the periphery is surrounded by a partition member can be adopted, as shown in Figure 3.

[0024] In the above case, the material of the member having a cavity is not particularly limited, but examples thereof include polyethylene terephthalate, polyethyleneimine, and polystyrene.

[0025] In the above case, the purpose of the partition member is to prevent leakage of the liquid culture medium to the outside, and as long as this purpose is achieved, the shape is not particularly limited, but it can be, for example, a ring-shaped member. The material of the partition member is not particularly limited, but examples thereof include PDMS (polydimethylsiloxane), polystyrene, silicone rubber, and glass.

[0026] The device for measuring the contractile force of myocardial tissue of the present invention may further include a marker portion including non-contractile myocardial tissue, and the contractile portion may be coupled to the marker portion so that the contractile force of the contractile portion can be measured depending on the mobility of the marker portion.

[0027] Specifically, the contraction portion and marker portion are suspended in a liquid medium, and when the myocardium beats, the movement distance of the floating marker portion is detected using image recognition technology, the magnitude of the movement vector and its change over time are recorded, and the contraction force can be measured by converting it into force and recalculating it.

[0028] More specifically, measuring contractile force based on the distance traveled by the marker can be performed, for example, as follows. As shown in Figure 3, a device called "MicroTester" (Cell Scale) is used to contract myocardial tissue. The sensor in contact with the tissue moves with the myocardial pulsation, so the distance traveled by the sensor is recorded as a video. The "MUSCLEMOTION" plugin in the free software "ImageJ" is then used to obtain displacement curves of the sensor over different times. Finally, this information is used to convert the intracellular forces exerted by the sensor at different times.

[0029] In the above example, such a marker portion may include myocardial tissue formed in region (B) having a surface with a contact angle to water of 85° or less.

[0030] In the present invention, "non-contractile myocardial tissue" means that the contractility is lower than that of a rod-shaped artificial myocardial tissue at the contractile portion, and does not necessarily mean that the tissue has no contractility at all. This is because the orientation of cardiomyocytes is lower than that of a rod-shaped artificial myocardial tissue at the contractile portion.

[0031] The device for measuring contractile force of myocardial tissue of the present invention further includes an action potential measurement unit having a three-dimensional structure and including artificial myocardial tissue in which cardiomyocytes are oriented, and the action potential measurement unit may be used for action potential measurement using multiple electrodes. This makes it possible to provide a device that can simultaneously measure electrical signals and contractile force.

[0032] Specifically, an existing multi-electrode system for measuring electrical signals can be applied, and a part of the device can be attached to the multi-electrode chip to measure action potentials.

[0033] The method for obtaining an artificial myocardial tissue having a three-dimensional structure of the action potential measurement section and oriented cardiomyocytes is not particularly limited. For example, as previously reported by the present inventors, it can be obtained by culturing cardiomyocytes using oriented fibers as a cell scaffold (J. Li et al., 2017 Stem Cell Reports, 9, 1-14, 2017). Oriented fibers can be obtained, for example, by electrospinning polymers as raw materials. Polymers can be selected from a wide range of polymers as long as they do not adversely affect the proliferation and physiological activity of cardiomyocytes, depending on the intended use. Examples include lactic acid-glycolic acid copolymer (PLGA), polylactic acid (PLA), polystyrene (PS), polyethylene terephthalate (PET), gelatin, collagen, etc.

[0034] The diameter of the oriented fibers is not particularly limited, but may be, for example, 0.1 to 10 μm, preferably 1 to 8 μm, and more preferably 3 to 5 μm.

[0035] When applying a multi-electrode system and attaching a portion of artificial myocardial tissue to a multi-electrode chip to measure action potentials, the oriented fibers must be formed into a sheet having the required area to obtain artificial myocardial tissue of that size.

[0036] The number of fibers (density) per mm of width in the minor axis direction (the direction perpendicular to the orientation direction) of the oriented fiber can vary depending on the diameter of the fiber used. The density is 10 fibers / mm or more, preferably 30 to 15,000 fibers / mm, and more preferably 50 to 13,000 fibers / mm.

[0037] For example, a device having one set each of the contraction portion and the action potential measurement portion can be sized to fit into one well of a multi-well microplate (for example, 24-well, 48-well, or 96-well devices are commonly used), and by using this device as a single unit and connecting multiple unit devices so that each is accommodated in a different well of the multi-well microplate, simultaneous measurement of multiple unit devices can be performed using a commonly used multi-well microplate.

[0038] 2. Method for Producing Rod-Shaped Artificial Myocardial Tissue The method for producing rod-shaped artificial myocardial tissue of the present invention, which has a three-dimensional structure and in which cardiomyocytes are oriented in the longitudinal direction, comprises the step of culturing cardiomyocytes on a surface having a contact angle with water of 95° or more to obtain the artificial myocardial tissue.

[0039] In the above step, the culture method is not particularly limited and can be appropriately selected from known methods for culturing cardiomyocytes. The culture may be performed in a static state or with shaking.

[0040] In the above step, a region (A) having a surface with a contact angle to water of 95° or more may be bonded to two regions (B) having surfaces with a contact angle to water of 85° or less, and the rod-shaped artificial myocardial tissue may be formed on a line connecting the two regions (B) by culturing cardiomyocytes in the regions (A) and (B). By this step, the above-mentioned device for measuring the contractile force of myocardial tissue having a marker portion including non-contractile myocardial tissue can be produced.

[0041] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0042] (1) Fiber Sheet Processing: PLGA (75 / 25; Sigma-Aldrich, USA) was mixed with hexafluoro-2-propanol (HFIP, Wako Pure Chemical Industries, Tokyo, Japan) in a centrifuge tube (1.2 g:3 mL, w / v) and then synthesized using an automated fiber fabrication system (NF-103, MECC, Fukuoka, Japan). Specifically, the mixed solution was loaded into a 3 mL syringe and connected to the anode of a high-voltage power supply (10 kV) connected to a 0.6 mm inner diameter needle. An aluminum foil sheet was placed on the surface of a drum. The drum rotated at 1000 rpm, and PLGA fibers ejected from the needle were collected on the aluminum foil sheet. The distance between the needle tip and the drum was kept 15 cm, and the spinning process was set for 60 min. The fiber sheet collected on the aluminum foil was then transferred to the device. Fiber quality was evaluated by scanning electron microscopy.

[0043] (2) Device Fabrication. PDMS (SYLGARD 184; Dow Corning, Midland, MI, USA) was cast into a culture plate (Nunc Cell Culture Plate, Thermo Fisher) and cured overnight at 80°C to create a 1 mm-thick PDMS sheet. PDMS was cast onto a silicon wafer spun at 1500 rpm using a spin coater and cured overnight at 80°C to create a 100 μm-thick PDMS sheet. The PDMS sheet was cut into strips (3 mm x 1 mm) using a cutter (SV-8, Roland DG). PDMS rings (outer diameter = 8 mm, inner diameter = 6 mm) were created using a biopsy punch (Kai Medical, Japan). PET sheet (SFL-A4, AS ONE) was cut into disks using an 8 mm-diameter biopsy punch, and two 2 mm holes were drilled in the disk to attach the PDMS strip and fiber sheet. The PDMS ring and PET disk were then attached. The fiber sheet was transferred to a PET disk using an adhesive transfer tap (3M®). The PDMS strip was then attached to the PET disk using tweezers. A polyimide (PI) sheet (3-1966-06, AS ONE) was punched with a 1 mm biopsy punch to create a disk. This disk was then attached to the PDMS strip to create a force sensor (device for measuring contractile force) (Figure 1). Before seeding the cells, the device was treated with plasma for 2 minutes.

[0044] This invention utilizes three-dimensional, oriented fiber technology to construct myocardial tissue with a three-dimensional, oriented structure. Furthermore, it applies an existing multi-electrode system for measuring electrical signals, attaching a portion of the device to a multi-electrode chip to measure action potentials. Furthermore, by levitating a portion of the device, image recognition technology is used to detect the distance traveled outside the circular circle of the floating portion as the myocardium pulsates, recording the magnitude and time-dependent changes of the movement vector, which is then converted into force and recalculated (Figures 2-4).

[0045] Although the cells used for measurement are not always the same, as will be shown below, using the device fabricated in the present invention, we were able to simultaneously measure the changes in electrical signals and contractile force due to the addition of drugs using the same cardiomyocytes under the same conditions.

[0046] (3) Measurement results using the device E4031 is an antiarrhythmic drug that inhibits the HERG potassium channel. As the concentration increased, QT prolongation was observed from 100 nM. On the other hand, a gradual weakening of contractile force was observed with increasing concentration (Figure 5).

[0047] Verapamil is an antiarrhythmic drug with calcium channel blocking properties. As the concentration increased, the beating rate gradually increased, and at concentrations up to 10 μM, action potentials ceased. Contractile force gradually weakened, becoming almost unmeasurable at 1 μM (Figure 6).

[0048] Furthermore, we evaluated drug response and efficacy using normal and diseased iPS cell-derived cardiomyocytes. As a result, no differences were observed in the electrical signals and contractile force of heterozygous myocardium for myosin-binding protein C (MYBPC3), a sarcomere-associated factor in hypertrophic cardiomyopathy (HCM), compared with normal cardiomyocytes. In contrast, in the case of homozygous mutant myocardium, no differences were observed in the electrical signals between normal and heterozygous myocardium, but the technology of this invention clearly demonstrated a decrease in contractile force (Figure 7).

[0049] On the other hand, while existing methods require samples to be discarded after one measurement, this system allows for the reuse of samples and evaluation systems, leading to cost reductions. It is easy to operate and handle, can be mass-produced, and can be upgraded to a 24 / 48 / 96-well device to meet the needs of high-throughput drug discovery (Figure 8).

[0050] (4) Measurement of Water Contact Angle of Various Materials In addition to the various materials used in the examples (polyimide, polyethylene terephthalate, PDMS), silicone rubber was also prepared and the water contact angle of each material was measured. The water contact angle after plasma treatment was also measured in the same way. The results are shown in Figures 9 and 10.

Claims

1. A device for measuring the contractile force of myocardial tissue, comprising a contractile section having a three-dimensional structure and including rod-shaped artificial myocardial tissue in which cardiomyocytes are oriented in the longitudinal direction, and used for measuring the contractile force of the contractile section.

2. A device for measuring the contractile force of myocardial tissue according to claim 1, wherein the artificial myocardial tissue is obtained by culturing myocardial cells on a surface having a contact angle with water of 95° or more.

3. A device for measuring the contractile force of myocardial tissue as described in claim 1 or 2, further comprising a marker portion including non-contractile myocardial tissue, the contractile portion being coupled to the marker portion such that the contractile force of the contractile portion can be measured depending on the mobility of the marker portion.

4. A device for measuring the contractile force of myocardial tissue as described in claim 1 or 2, further comprising an action potential measuring unit having a three-dimensional structure and including artificial myocardial tissue in which cardiomyocytes are oriented, the action potential measuring unit being used for measuring action potentials using multiple electrodes.

5. A method for producing rod-shaped artificial myocardial tissue having a three-dimensional structure and in which cardiomyocytes are oriented in the longitudinal direction, the method comprising the step of obtaining the artificial myocardial tissue by culturing cardiomyocytes on a surface having a contact angle with water of 95° or more.

6. The manufacturing method described in claim 5, wherein in the above process, an area (A) having a surface with a contact angle to water of 95° or more is bonded to two areas (B) having surfaces with a contact angle to water of 85° or less, and by culturing cardiomyocytes in the areas (A) and (B), the rod-shaped artificial myocardial tissue is formed on the line connecting the two areas (B).

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