Contractile tissue-based analysis device

Abstract

To provide a multi-well plate being a contractile tissue-based analysis device.SOLUTION: There is provided a multi-well plate comprising a substantially planar top surface and bottom surface and a plurality of wells opening in the top surface. Therein all wells of the multi-well plate each comprise a support structure 100. The support structure comprises: a substantially planar base element 10; and a first support pillar 20 and a second support pillar 30. Each of the first support pillar and the second support pillar extends from the base element in a direction substantially perpendicular to a plane of the base element. Each support pillar comprises a stem portion 21, 31 and a head portion 22, 32. Each stem portion extends between the base element and each of the head portions. At least one of the support pillars is capable of flexing along an axis Y-Y extending between the head portions of the support pillars. Each support structure further comprises a receptacle.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present technology relates to a contractile tissue-based analytical device in which a strip of natural or engineered contractile tissue is supported by a support structure comprising a substantially planar base element and first and second support pillars, and movement of one or both support pillars caused by the strip of contractile tissue can be captured from below, i.e., through the planar base element. [Background technology]

[0002] Contractile tissues, such as artificial muscle tissue strips (MTS), are of widespread interest in applications such as drug screening, personalized medicine, disease modeling, and tissue transplantation. In particular, drug candidates in the drug development pipeline need to be screened for adverse cardiac effects, which is most conveniently performed in vitro in artificial myocardial tissue.

[0003] One of the most important and best-understood features of myocardial tissue is its contractile properties. Many studies have been performed on the in vitro growth of 3D, strip-format, force-generating engineered cardiac tissue (EHT). Analysis of the contractile properties of in vitro-grown myocardial tissue allows for testing changes in human cardiac function without the need for human trials. Among other things, such testing can provide a rapid and early indication of the potential for drugs to cause cardiac-related side effects, such as drug-induced atrial fibrillation.

[0004] Compliant materials made from biological or synthetic polymers have widespread applications in life sciences, including analytics and advanced cell culture. For example, hydrogels offer tunable protein and cell adhesion properties, widely varying mechanical properties, and controllable diffusivity of dissolved compounds. Compliant materials such as hydrogels have traditionally been cast into their desired final 3D shape, limiting the design freedom that can be achieved. The recent emergence of 3D printing methods has enabled the direct and rapid fabrication of highly complex 3D shapes. The primary 3D printing methods for compliant materials include mechanical extrusion of polymer solutions (bioprinting) and spatially selective photochemical crosslinking of polymers (stereolithography). Summary of the Invention [Problem to be solved by the invention]

[0005] A major challenge in contractile microtissue engineering is the robustness of the engineered tissue against "necking," which can lead to subsequent failure. Previous studies have demonstrated the importance of pillar stiffness and matrix composition in the robustness of engineered tissues. However, the influence of the geometric features of the pillars themselves has not been widely studied, likely due to the limited 3D design freedom afforded by traditional molding approaches (e.g., Hansen et al., Circulation Research, July 9, 2010, pp. 35-44).

[0006] Other publications on similar techniques include US2015 / 0313704 and Lari et al., Experimental Eye Research, 94 (2012) 128-135.

[0007] At least some of the above problems are addressed by the techniques of this disclosure. [Means for solving the problem]

[0008] The present invention provides a contractile tissue-based assay device, said assay device comprising at least one support structure: The support structure includes: a substantially planar base element; a first support pillar and a second support pillar, each support pillar extending from the base element in a direction substantially perpendicular to a plane of the base element; Equipped with each support pillar includes a stem portion and a head portion, each stem portion extending between the base element and each head portion; at least one of the support pillars, preferably both of the support pillars, is bendable along an axis YY extending between head portions of the support pillars; a strip of contractile tissue extending between the head portions of the first support pillar and the second support pillar; the analytical apparatus further comprising an optical detection device disposed on a side of the base element opposite the support pillar; a head portion of at least one support pillar, preferably both support pillars, comprising at least one fiducial marker detectable by said optical detection device; At least one of the fiducial markers has an extension from a head portion in at least a direction substantially parallel to the plane of the base element; at least one of the fiducial markers is optically identifiable by the optical detection device from a side of the base element opposite the support pillar; The optical detection device is configured to acquire image data from at least one of the at least one fiducial markers on the head portion.

[0009] Also provided is a method for analyzing the response of a strip of contractile tissue to a drug, said method comprising: providing an analytical device according to the present invention; acquiring, by the optical detection device, a first set of image data from at least one fiducial marker on at least one of a head portion of the first support pillar and a head portion of the second support pillar; introducing a drug into the assay device so that the drug contacts the strip of contractile tissue; acquiring a second set of image data from at least one of the first support pillar and the head portion of the second support pillar by the optical detection device; Includes.

[0010] Further aspects of the invention are provided by the dependent claims, the drawings and the following description. [Brief explanation of the drawings]

[0011] [Figure 1A] 1A-1C illustrate an embodiment of a support structure without contractile tissue. [Figure 1B] FIG. 10 shows another embodiment of a support structure without contractile tissue. [Figure 1C] FIG. 10 shows another embodiment of a support structure without contractile tissue. [Figure 1D] FIG. 10 shows an embodiment of a support structure without contractile tissue, viewed along the YY axis extending between the head portions of the pillars. [Figure 2] FIG. 1B shows an analytical device based on the support structure of FIG. 1A. [Figure 3]Figures 1A and 1B show the design of support structures with wells with integrated pillars for tissue formation: (a) a microwell support structure with 170 microwells using rectangular pillars; (b) a miniwell support structure with 10 miniwells using rectangular pillars; and (c) a support structure with 10 miniwells with teardrop-shaped pillar heads designed to minimize local stress during muscle microtissue formation. This design includes the addition of 3D triangular fiducial markers on the heads, which facilitate automated optical tracking of pillar end displacement. Scale bars are 2 mm (top) and 500 μm (bottom). [Figure 4] This figure shows the results of observing tissue formation over a period of 4 days after cells were loaded onto the support structure designs shown in Figures 3(a), (b), and (c), until compacted tissue was observed on day 3. The white scale bar is 250 μm, and the black scale bar is 500 μm. [Figure 5a] 5a , which shows a top view of an alternative design of the analytical device of FIG. 3c , in which three identical support structures are arranged in a “three-leaf clover” configuration (only one of which is fully shown). Each support structure includes a pair (first and second) of support pillars, the centrally located one of which can bend along an axis YY extending between the head portions 22, 32 of the support pillar. Each head portion 22, 32 of FIG. 5a includes two fiducial markers 26, 36 that protrude at an angle of approximately 45 degrees from the vertical. The fixed fiducial marker is defined as the reference point (the central cross) where the three support structures join. FIG. 5 also shows the planar base element 10 and the receptacle 40. [Figure 5b] Figure 5a shows a bottom view (inverted microscope configuration) of the 3D-printed muscle tissue analysis device, in which muscle tissue consisting of C2C12 mouse myoblasts is formed between a pair of pillars. Both the tilted moving fiducial marker and the horizontal fixed fiducial marker exhibit optically distinct characteristics in the inverted microscope image, even after the introduction of muscle tissue. [Figure 6]Figure 1 shows fiducial markers on the head automatically identified by the tracking software and markers at three time points (a, b, c) during muscle contraction. Scale bar = 500 μm. [Figure 7a] 7a is a top view of an alternative design of the analytical device of Figures 3c and 5a, showing a support structure comprising a pair of (first and second) support pillars, each capable of bending along an axis YY extending between the head portions of the support pillars. Each head portion of Figure 7a includes two fiducial markers projecting at an angle of approximately 45 degrees from vertical and an embossed fiducial marker on the head. [Figure 7b] Figure 7a shows a bottom view of the 3D-printed analytical device. Muscle tissue consisting of C2C12 mouse myoblasts has formed between the support pillars after 3 days of culture. The focal plane of the microscope objective has been adjusted to coincide with the end point of the protruding fiducial marker, clearly showing the outline of the protruding fiducial marker. [Figure 7c] Shown is the same device at the same time point as in Figure 7b, with the focal plane adjusted to coincide with the plane of the embossed marker on the support pillar, and the embossed marker is barely detectable. [Figure 7d] This image shows the same device after 4 days of culture with further compression of the muscle tissue and resulting stronger deflection of the support pillars. The focal plane was adjusted to coincide with the end of the clearly outlined protruding fiducial marker. At this level of tissue compression and deflection of the support pillars, the embossed fiducial markers on the support pillars are not discernible. The scale bar is 500 μm. DETAILED DESCRIPTION OF THE INVENTION

[0012] Thus, a contractile tissue-based assay device is provided that can be used to rapidly assay the contractile properties of contractile tissue, particularly its response to drugs.

[0013] The present invention enables automated analysis of the contractile behavior of contractile tissue, such as muscle tissue, leading to the performance of in vitro screening of drug candidate toxicity and efficacy in industrial and clinical settings. The present invention includes a design and fabrication method that allows for optical tracking of contractile behavior in an inverted microscope configuration. This is an industrially significant improvement over prior art methods that employ tracking contractions in an upright microscope configuration, as it allows for monitoring of contractions without compromising tissue sterility and does not impede access to the tissue under study.

[0014] The focus of this technique is on "inverse geometry," in which the deflection of the support pillars is observed from the side (i.e., underneath) of the support structure to which they are fixed. The results of Hansen et al. (Circulation Research, July 9, 2010, pp. 35-44) clearly demonstrate the challenges of tracking the position of the posts in that configuration. Therefore, according to Hansen et al., it is necessary to track the visible portion of the muscle strip itself, which is clearly not optimal in an automated industrial environment.

[0015] (Support structure) The analysis device 200 comprises at least one support structure 100. A support structure is a component of the device that supports contractile tissue, the deflection of which can be detected by optical means. The support structure allows for the in-situ growth and support of contractile tissue, such as myocardial tissue, while allowing the contractile tissue to contract autonomously or in response to a stimulus.

[0016] 1A-1C , an embodiment of the support structure is shown. Generally, the support structure comprises a substantially planar base element 10, a first support pillar 20, and a second support pillar 30. Additional support pillars may be provided as needed, and in one embodiment the support structure may further comprise one or more, for example, two, three, or four, additional support pillars.

[0017] The substantially planar base element of the support structure provides a base on which the support pillars are disposed. By "substantially planar," the base element extends between substantially parallel upper and lower surfaces, the extent between which is much less than the extent of these surfaces. The upper and lower surfaces of the base element may be, for example, rectangular, square, circular, oval, or any other suitable shape. Typically, the thickness of the base element is 0.1-1.0 mm. Typically, the base element has a maximum extension in its plane of 2-40 mm. The base element is suitably formed from a polymer matrix, preferably the same polymer matrix as the support pillars.

[0018] The support pillars 20, 30 of the support structure extend from the base element 10 in a direction substantially perpendicular to the plane of the base element. This direction is generally defined as "above" the plane of the base element. Each support pillar includes a stem portion 21, 31 and a head portion 22, 32, with each stem portion 21, 31 extending between the base element 10 and the respective head portion 22, 32. Preferably, the head portion of each support pillar has a different shape that distinguishes it from the stem portion, although the head and stem portions may have the same shape. Importantly, at least one of the first and second support pillars, and preferably both the first and second support pillars, can bend along an axis YY extending between the head portions of the support pillars. Due to the flexibility of the support pillars, contractions of the contractile tissue, either autonomously or in response to a stimulus, are transmitted to the head portions of the support pillars and detected by an analysis device. Suitably, the axis YY is disposed substantially parallel to the plane of the base element. This allows for easy optical tracking of the head portion and subsequent analysis.

[0019] The stiffness of the support pillars is preferably in the range of 0.01-10 N / m, for example 0.1-1 N / m, of the flexibility (compliance) of the support pillars. Suitably, the head portions of the first and second support pillars are separated from each other along axis YY by a distance of 3 mm or less, for example less than 2 mm, or less than 1 mm. This configuration improves the viability of contractile tissue cultured between the head portions, as the path length for essentially continuous diffusion of oxygen to all parts of the contractile tissue is shorter in tissues of smaller dimensions.

[0020] The first support pillar and the second support pillar typically have the same three-dimensional shape. In the embodiment shown in Figure 1A, the head portion is substantially spherical and the stem portion is substantially cylindrical. Suitably, in this embodiment, the axis of each cylindrical stem portion extends through the geometric center of each spherical portion of the head portion.

[0021] In the preferred embodiment shown in Figure 1C, each head portion has a three-dimensional teardrop shape with a substantially spherical body extending to an apex 25, 35. The head portion of each support pillar is arranged such that the apex of each head portion and the geometric center of each spherical body lie along the same axis YY that extends between the head portions. The apex of each head portion lies inside the geometric center of each spherical body along the axis YY.

[0022] The teardrop-shaped pillar head according to Figure 1C has the advantage over rectangular pillars in that it does not introduce the same degree of stress concentration around sharp corners. This reduces the risk of tissue thinning and eventual fracture seen in the tissues of Figures 4a and 4b. Muscle tissue strips (MTS) created with support structures according to the present invention using teardrop-shaped heads with soft-edged biomechanical cues show no tissue damage at 3 days of culture. The MTS also show more pronounced tissue formation, as seen in Figure 4C.

[0023] The support pillars are suitably formed from a polymer matrix, preferably the same polymer matrix as the base element.

[0024] The analytical device of the present invention includes fixed or moving optical markers (fiducial markers) that are introduced during the fabrication of the two force-responsive pillars, and muscle tissue forms and holds between the two force-responsive pillars. Fiducial markers can be three-dimensional objects defined either by selectively removing material in 3D (embossed structures) or by selectively adding material (protruding structures). Fiducial markers can be defined as having a long axis perpendicular to the plane of the base element, or their long axis or axes can be defined such that they are neither parallel nor perpendicular to the plane of the base element. For example, they can be two rectangular prisms protruding at opposite angles from a support pillar.

[0025] Thus, the support structure can be equipped with fiducial markers 26, 36 for optical tracking. The incorporation of fiducial markers allows for accurate contraction analysis of muscle tissue. When tissue forms around the pillars at a defined position relative to the head, the height of tissue attachment is also clearly defined. This allows for reliable calculation of contractile force based on pillar stiffness and deflection. The length of deflection can be determined by optical tracking of the incorporated fiducial markers, allowing for calculation of the force exerted by cells within the strip of artificial muscle tissue.

[0026] High-resolution 3D printing allows for the introduction of such non-vertically protruding fiducial markers of sub-millimeter dimensions during device fabrication. The ability to fabricate such fiducial markers by 3D printing on relevant length scales and in sufficiently compliant materials forms part of the present invention. This is a further advantage over the prior art of Hansen et al., where pillars are molded.

[0027] Thus, at least one support pillar, preferably the head portion 22, 32 of each support pillar 20, 30, suitably comprises at least one fiducial marker 26, 36 that can be detected by an optical detection device. To improve optical tracking of the head portions, each head portion may include two or more fiducial markers. When two or more fiducial markers are present on a head portion, they preferably extend in different directions from the head portion. In one embodiment, the head portion of each support pillar suitably comprises at least one fiducial marker. In another embodiment, the head portion of a support pillar includes at least one fiducial marker whose movement is tracked relative to another fixed fiducial marker present elsewhere on the support structure or analytical apparatus.

[0028] Optical tracking of fiducial markers from "below" the plane of the base element is possible if the fiducial markers extend from the head portion and are visible from below the plane of the base element. Visibility of the fiducial markers and head portion from below the plane may be further hindered by the presence of contractile tissue surrounding a portion of the head portion. Thus, at least one fiducial marker 26, 36 has an extension from (the respective) head portion 22, 32 (located thereon) at least in a direction substantially parallel to the plane of the base element 10, and preferably in a direction substantially perpendicular to the axis YY.

[0029] In the specific embodiment shown in FIG. 1D , the fiducial marker and the head portion of each support pillar form a “Y” shape when viewed along axis YY extending between the two head portions. Preferably, the fiducial marker, head portion, and stem portion of each support pillar are essentially coplanar in a plane extending perpendicular to the plane of the base element. This design allows for good visibility of the fiducial marker from “below” the plane of the base element. At the same time, if the marker and pillar are “Y” shaped and initial focus is at the bottom of the marker, bending of the pillar brings the higher portion of the marker into sharp focus. In this way, the “Y” shaped configuration allows for sharp focus to be maintained over a wide range of constrictions.

[0030] In the analytical device according to this embodiment, the at least one fiducial marker may have a main extension from the head portion that is aligned at an angle of 30-60 degrees, preferably 40-50 degrees, more preferably 45 degrees, relative to an axis extending along each support pillar substantially perpendicular to the plane of the base element. In particular, the at least one fiducial marker suitably extends from the head portion in a direction away from the base element.

[0031] The support structure of the analytical device may include a translucent slide 110 disposed on the side of the base element opposite the support pillars (i.e., "below" the support structure). An optical detection device 220 is disposed on the side of the translucent slide opposite the substantially planar base element (i.e., below the translucent slide). The translucent slide serves as additional support for the support structure. The support structure may be formed on the translucent slide, and the support structure including the translucent slide may then be incorporated into the analytical device. Suitably, at least the planar base element and the translucent slide (if present) are at least partially transparent to visible light. Preferably, the support pillars and fiducial markers are also at least partially transparent to visible light. In the context of this specification, "visible light" means electromagnetic radiation having a wavelength between 400 nm and 700 nm.

[0032] As shown in Figures 1B and 1C, the support structure can further include a receptacle 40. The receptacle includes a bottom wall 41 and at least one side wall 42. The bottom wall has at least two openings, with the stem portion of each support pillar extending through one of the openings. The at least one side wall extends from the bottom wall away from the base element such that the receptacle defines an interior volume, with the head portion disposed entirely within the interior volume. The receptacle supports stem cells, contractile progenitor cells, or contractile tissue cells, as well as associated stromal cells and pre-tissue gel-forming components in a growth medium.

[0033] The support structure can include microstructures that promote tissue formation. In one aspect, the microstructures can induce directionality in the tissue forming along the axis between the pillars. In a second aspect, the microstructures can induce tissue formation at predetermined locations on the support pillars by the presence of localized reductions or expansions in the cross-section of the pillars to limit tissue sliding. In a third aspect, the microstructures can reduce excessive tensile stress in the forming tissue by providing dimensionally gradual support along the axis between the pillars, for example, in the form of teardrop-shaped heads with their long axes parallel to the axis between the pillars.

[0034] The support structure can be fabricated using 3D printing, which allows for a broader design spectrum and further exploits the possibilities of mechanically guided tissue differentiation. Thus, in one embodiment, the base element, support pillars, fiducial markers, and receptacles are preferably formed by 3D printing from the same polymer matrix. The polymer matrix is preferably a compliant polymer matrix, more preferably a hydrogel polymer matrix, e.g., a poly(ethylene glycol)-based polymer matrix.

[0035] (contractile tissue) A strip of contractile tissue 210 extends between the head portions 22, 32 of the first and second struts 20, 30. The contractile tissue is muscle tissue, such as cardiac muscle tissue, skeletal muscle tissue, or smooth muscle tissue, of which cardiac muscle tissue is preferred.

[0036] The muscle tissue is seeding muscle tissue progenitor cells or muscle tissue cells onto the support structure described herein in a culture medium containing components that support cell survival and components that induce gelation of the medium, the culture medium being either a mixture of fibrinogen, aprotinin, and thrombin, or a mixture of gelatin methacryloyl and a free radical photoinitiator, or a mixture of type I collagen and bicarbonate; incubating or illuminating the support structure and muscle tissue cells until tissue is formed; Culturing the muscle tissue cells, suitably for 2-40 days, and stimulating the formed tissue via one or more combinations of electrical, metabolic, and mechanical stimulation; and providing a support structure as described herein having a strip of muscle tissue extending between head portions of the first and second support pillars; Including, The method can be formed on the support structure described above.

[0037] The cells used in this technology are preferably mammalian stem cells, progenitor cells, or muscle tissue cells, and preferably human stem cells, progenitor cells, or muscle tissue cells. When the cells are derived from stem cells, the stem cells may be of fetal or non-fetal origin, preferably non-fetal origin, such as dermal skin cells (fibroblasts). The cells used in the following examples are either human induced pluripotent stem cell-derived cardiac progenitor cells (hiPSC-cardiomyocytes) or mouse myoblasts (C2C12).

[0038] (Optical detection device) The analysis apparatus 200 further comprises an optical detection device 220 arranged on the side of the base element opposite the support pillar (i.e., below the support structure). The analysis apparatus is configured to acquire image data from at least one of the head portions of the first support pillar and the second support pillar. Suitably, the optical detection device is a camera, such as a video camera, preferably a CMOS or CCD-based image sensor with a 1x objective lens. The image data is preferably video image data. The image data can be processed using computer software to analyze muscle tissue contractions.

[0039] Locating the optical detection device below the support structure offers the advantage that muscle tissue can be grown in situ within the device and analyzed directly without disturbing the tissue or moving the support structure. Furthermore, the optical detection and associated electronics can be located away from the "wet" topside of the support structure (where cell growth media and drugs are introduced to the support structure).

[0040] (Analyzer) A schematic diagram of an analytical device based on the support device of FIG. 1A is shown in FIG.

[0041] The analyzer may further comprise a pair of electrodes connected to a power source and configured to apply electrical stimulation to the strip of myocardial tissue, which may be used to induce contraction of the muscle tissue when measuring response to drugs. Additionally, electrical stimulation may be used when maturing muscle tissue from stem cells.

[0042] The analytical device may further comprise at least one administration means for providing at least one drug to said strip of muscle tissue.

[0043] (Array) An array, for example a 96-well platform, is provided for the preparation, culture, and analysis of muscle tissue.

[0044] Thus, the analytical devices described herein can include a plurality of support structures arranged in a planar array, with the planar base elements of all support structures in the array being substantially coplanar within the plane of the array, and the support pillars of all support structures in the array being located on the same side of the planar array. Such an array provides for rapid screening of muscle tissue response to drugs.

[0045] In one aspect, the analytical device is a multiwell plate having substantially planar upper and lower surfaces and comprising a plurality of wells open at the upper surface, at least one well, preferably all wells, of the multiwell plate comprising a support structure as defined herein.

[0046] The array can have one optical detection device positioned on the side of the planar array opposite the support pillar, and the optical detection device can be moved within the plane of the array. In this way, one optical detection device can be used to analyze multiple strips of muscle tissue.

[0047] Alternatively, the array may have a plurality of optical detection devices arranged on the side of the planar array opposite the support pillars, each optical detection device arranged to acquire image data from at least one of the head portions of the first and second support pillars of each support structure.

[0048] Also provided is a method for analyzing the response of a strip of contractile tissue to a drug, said method comprising: Providing an analytical device as described herein; acquiring, by the optical detection device, a first set of image data from at least one fiducial marker on at least one of a head portion of a first support pillar and a second support pillar; introducing a drug into the assay device so that the drug contacts the strip of myocardial tissue; acquiring a second set of image data from at least one of the head portions of the first support pillar and the second support pillar by the optical detection device; Includes.

[0049] Advantageously, the strip of contractile tissue is paced by application of a pulsed electrical signal during acquisition of at least the first and second sets of image data.

[0050] The "response" analyzed above may be the contractile response of a strip of contractile tissue and the second image data set may be characteristic of the contractile response of the strip of contractile tissue to the drug. The first image data set and / or the second image data set may be video image data.

[0051] (Example) Materials and Methods (Stereolithography 3D printing and printing solution configuration) Poly(ethylene glycol) diacrylate Mn 700g mol -1 3D support structures made from (PEGDA, 455008, Sigma-Aldrich) hydrogel were obtained by projection stereolithography using a high-resolution 3D printer. The printer used a one-to-one projection of a dynamic image displayed on a digital mirror structure (DMD) with a pixel pitch of 10.8 μm in the lateral dimension. The aqueous printing solution contained 200 mg / mL PEGDA (20% PEGDA) or 500 mg / mL PEGDA (50% PEGDA) in ultrapure MilliQ water (MQ, Merck-Millipore), 5 mg / mL of photoinitiator (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, LAP, Allevi or 900889, Sigma-Aldrich), and 9 mg / mL of photoabsorber (quinoline yellow, QY, 309052, Sigma-Aldrich). The solution components were mixed at room temperature and degassed for 30 min to avoid air bubbles that could interfere with the initiator light and cause deformation of the 3D printed object.

[0052] Computer-aided design (CAD) structures were drawn using Autodesk Inventor Professional with dimensions matching multiples of the printer's DMD pixel pitch (10.8 μm). This ensured the greatest possible dimensional accuracy of the printed object compared to the CAD design dimensions. The CAD structures were sliced at a thickness of 20 μm using the open-source Slic3r software (www.slic3r.org). The sliced structures were then scanned at an intensity of 20 mW / cm for 50% PEGDA and 20% PEGDA, respectively. 2 The structures were 3D printed using 365 nm light at 1000 kJ / s for 3 or 5 seconds of exposure. The structures were 3D printed onto surface-treated glass coverslips (22 x 22 mm #4, Menzel-Glaser). The surface treatment provided a methacrylate layer on the coverslip, allowing chemical cross-linking between the printed object and the glass cover slide.

[0053] (Preparing the support structure) Five different support structures were fabricated to generate muscle tissue strips (MTS). Two designs were selected from the investigated cell-seeding platforms using support pillars (Figures 1a and 1b). The third design (Figure 1c, in accordance with the present invention) was developed to minimize stress concentrations around the vertical support pillars, promoting more robust MTS formation. The fourth design (Figure 5a, in accordance with the present invention) includes fiducial markers extending at opposing angles from the head portion of the pillar and a fixed fiducial marker. The fifth design (Figure 7a, in accordance with the present invention) has fiducial markers extending from the pillar heads, as in the fourth design, and also has embossed fiducial markers on the pillar heads.

[0054] The 3D-printed support structures were washed with phosphate-buffered saline (PBS) for at least 24 hours after printing. The liquid was changed twice to wash away residual printing solution from the crosslinked PEGDA network. Sterilization was performed by immersing the printed support structures in 70% v / v ethanol / water for 10 minutes, followed by exposure to UV-C (254 nm) for 15 minutes (Mini UV Sterilization Cabinet, Cleaver Scientific). The support structures were stored sterile in PBS until use, ensuring that the water was replaced with PBS before cell culture. Before seeding cells onto the support structures, the PBS was removed and the platform was blotted with sterile lint-free paper to ensure the wells were empty.

[0055] (Cell seeding and culture) C2C12 mouse myoblasts (C3H myoblasts, 91031101, Sigma-Aldrich) were used. Cells were maintained in culture using a growth medium consisting of DMEM high glucose (Sigma-Aldrich) containing 10% fetal bovine serum (FBS) (Sigma-Aldrich) and 1% penicillin / streptomyosin (P / S, Sigma-Aldrich). Tissue formation was performed by dissolving 10 × 10 cells in a solution of 10 mg / mL fibrinogen (F8630, Sigma-Aldrich), 0.5 μg / mL aprotinin (A1153, Sigma-Aldrich), 20% (v / v) Matrigel (354277, Corning), and 3 U / mL thrombin (T7513, Sigma-Aldrich). 6The process began with casting cells suspended at 100 cells / mL, placed in growth medium, and injected into the prepared support structure. The solution was kept on ice to prevent gelation until casting. The wells of the larger support structure (Figures 1b and 1c) were individually loaded with 3.5 μL of cell suspension. The wells of the smaller support structure (Figure 1a) were loaded in a two-step process. First, 200 μL of cell suspension was placed on top of all wells of the support structure. Next, the support structure was spun in a centrifuge at 200 g for 10 seconds to drive the suspension into the wells. The loaded support structure was incubated at 37°C for 30 minutes to allow a fibrin matrix to form before growth medium was added. After 2 days of culture, the medium was replaced with DMEM high glucose containing 2% FBS and 1% P / S to promote myoblast fusion into myotubes. Medium changes were performed every 2–3 days throughout the culture period.

[0056] (viability staining) Staining was performed with 2 μg / μL calcein AM (15560597, Fisher Scientific), 4 μg / mL propidium iodide (81845, Sigma-Aldrich), and 2 μg / mL Hoechst 34580 (H21486, Invitrogen) by incubation at 37°C for 1 hour. After washing the samples with medium, confocal imaging was performed on a Zeiss LSM700 using a Zeiss 10x / 0.3NA Epiplan Neofluar objective at excitation wavelengths of 405 nm, 488 nm, and 555 nm for Hoechst 34580, calcein AM, and propidium iodide, respectively. Recorded Z-stacks were merged into 2D images in FIJI / ImageJ by maximum intensity projection.

[0057] (Muscle Tissue Strip (MTS) Formation) Mouse myoblasts were cultured at 10 × 10 in a fibrin / Matrigel matrix in three different support structures. 6The cells were cast at 1000 cells / mL. Cells begin to differentiate when they are in close proximity to each other. Upon differentiation, myoblasts fuse to form myotubes. Cell elongation is a sign of differentiation and indicates the alignment of sarcomeres in response to cell contraction. Aligned sarcomeres allow cells to exert greater force in the direction of extension. Because the cell-laden fibrin / Matrigel matrix is embedded around pillars, the contraction of the cell matrix is restricted by the pillars, resulting in the formation of elongated structures and ultimately the formation of MTS.

[0058] The rigidity of the pillars continued to provide an opposing force as the cells contracted. After 24 hours of culture, the cells began to contract the fibrin matrix, forming tissue surrounding the two pillars (Figure 4). Two days after seeding, clear tissue was visible in all designs.

[0059] In the support structure of Figure 3a, the formed tissue was surrounded by excess cells trapped in the matrix without contributing to tissue formation. The design of Figure 3a allowed for a low number of cells per well due to the small well size, but many cells were lost, necessitating a very inefficient cell seeding method. In both the support structures of Figures 3a and 3b, the tissue thinned due to necking, as seen in Figures 4a and 4b, and there was a risk of the tissue eventually breaking. Necking is known to destroy engineered tissues regardless of cell type, posing a serious challenge to tissue engineering. The teardrop-shaped pillar head of the present invention has an advantage over rectangular pillars by not introducing the same degree of stress concentration around the corners. This reduces the risk of tissue thinning and eventual breakage seen in the tissues of Figures 4a and 4b. Muscle tissue strips (MTS) created with the support structure of the present invention using teardrop-shaped heads with soft-edged biomechanical cues showed no tissue damage at day 3 of culture. This MTS also showed clearer tissue formation, as can be seen in Figure 4c. All engineered tissues showed high cell viability and a tight cellular structure with aligned cells.

[0060] As shown in Figures 5a and 7a, the support structure design with protruding fiducial markers on the pillar heads supports the formation of MTSs similarly to designs without protruding fiducial markers. Figure 5b shows a micrograph of the bottom view of an MTS formed with the design in Figure 5a, while Figures 7b–7d show bottom views of MTSs formed with the design in Figure 7a on days 3 (Figures 7b–7c) and 4 (Figure 7d), respectively. Figures 7b–7d show how the fiducial markers embossed on the pillar heads become less discernible over time due to optical distortion of the embossed profile caused by opaque tissue enveloping the pillar heads and increasing pillar head deflection. The profile and axial position of the protruding fiducial markers remained clearly visible at all time points. The selection of a protruding fiducial marker orientation that does not coincide with the base plane allows for high-quality imaging of at least a portion of the fiducial marker at a constant focal length for multiple pillar deflections.

[0061] (Optical analysis) Tissue contraction in the support structure design shown in Figure 3 was monitored using a custom-made stage incubator attached to a Motic stereomicroscope. Images were recorded every 5 minutes. Custom-made tracking software was used to track the optical markers on the pillar head in the acquired image sequences. Figure 6 shows fiducial markers on the head automatically identified by the tracking software and markers at three time points (a, b, c) during muscle tissue contraction. The scale bar is 500 micrometers.

Claims

1. A contractile tissue-based analytical device (200), said analytical device (200) comprising at least one support structure (100); The support structure (100) comprises: a substantially planar base element (10); a first support pillar (20) and a second support pillar (30), each of which extends from the base element (10) in a direction substantially perpendicular to the plane of the base element (10); Equipped with Each of the support pillars (20, 30) includes a stem portion (21, 31) and a head portion (22, 32), each stem portion (21, 31) extending between the base element (10) and each of the head portions (22, 32); at least one of the support pillars (20, 30), preferably both of the support pillars (20, 30), is bendable along an axis Y-Y extending between the head portions (22, 32) of the support pillars (20, 30); a strip of contractile tissue (210) extending between the head portions (22, 32) of the first and second support pillars (20, 30); The analytical apparatus (200) further comprises an optical detection device (220) arranged on a side of the base element (10) opposite the support pillars (20, 30), the head portion of at least one of the support pillars (20, 30), preferably the head portions (22, 32) of both of the support pillars (20, 30), is provided with at least one fiducial marker (26, 36) that can be detected by the optical detection device (220); At least one of the fiducial markers (26, 36) has an extension from the head portion (22, 32) at least in a direction substantially parallel to the plane of the base element (10); at least one of the fiducial markers (26, 36) is optically identifiable by the optical detection device (220) from a side of the base element (10) opposite the support pillars (20, 30); the optical detection device (220) is configured to acquire image data from at least one of the at least one fiducial markers (26, 36) of the head portion (22, 32); Analyzer (200).

2. At least one of the fiducial markers (26, 36) extends beyond the strip of contractile tissue (210) and the support pillars (20, 30) at least in a direction substantially parallel to the plane of the base element (10); The analytical device (200) of claim 1.

3. At least one of the fiducial markers (26, 36) further has an extension from the head portion (22, 32) at least in a direction substantially perpendicular to the axis YY.

3. The analytical device (200) of claim 1 or 2.

4. At least one of the fiducial markers (26, 36) has a main extension from the head portion (22, 32); The main extensions are aligned along each support pillar (20, 30) at an angle of 30-60 degrees, preferably 40-50 degrees, more preferably 45 degrees, relative to an axis extending substantially perpendicular to the plane of the base element (10); The analytical device (200) of any one of claims 1 to 3.

5. At least one of the fiducial markers (26, 36) extends from the head portion (22, 32) in a direction away from the base element (10); The analytical device (200) of any one of claims 1 to 4.

6. a pair of electrodes (201, 202) connected to a power source and configured to apply electrical stimulation to the strip of myocardial tissue (210); The analytical device (200) of any one of claims 1 to 5.

7. the support structure (100) of the analytical device (200) comprises a translucent slide (110) arranged on a face of the substantially planar base element (10) opposite the support pillars (20, 30); the optical detection device (220) is disposed on a side of the translucent slide (110) opposite the substantially planar base element (10); The analytical device (200) of any one of claims 1 to 6.

8. said head portions (22, 32) being spaced apart from one another along said axis YY by a distance of no more than 2 mm; The analytical device (200) of any one of claims 1 to 7.

9. said axis Y-Y being arranged substantially parallel to the plane of said base element (10); The analytical device (200) of any one of claims 1 to 8.

10. at least the planar base element (10) and the translucent slide (110), preferably as well as the support pillars (20, 30) and the fiducial markers (26, 36), are at least partially transparent to visible light; The analytical device (200) of any one of claims 1 to 9.

11. the head portion (22, 32) has a three-dimensional teardrop shape with a substantially spherical body extending to an apex (25, 35); the head portion (22, 32) of each of the support pillars (20, 30) is arranged such that the apex (25, 35) of the head portion (22, 32) and the geometric center of each spherical body lie along the axis Y-Y; the vertex (25, 35) of each of the head portions (22, 32) is located inside the geometric center of each spherical body along the axis Y-Y; The analytical device (200) of any one of claims 1 to 10.

12. The receptacle (40) further comprises a bottom wall (41) and at least one side wall (42), the bottom wall (41) having at least two openings (41'); Each stem portion (21, 31) of the support pillars (20, 30) extends through one of the openings (41′); At least one of the side walls (42) extends from the bottom wall (41) in a direction away from the base element (10) such that the receptacle (40) defines an interior volume; The head portion (22, 32) is disposed entirely within the interior volume. The analytical device (200) of any one of claims 1 to 11.

13. and further comprising at least one administration means for providing at least one drug to said strip of muscle tissue. The analytical device (200) of any one of claims 1 to 12.

14. The base element (10), the support pillars (20, 30) and the receptacle (40) are formed from the same polymer matrix, preferably a compliant polymer matrix, more preferably a hydrogel polymer matrix, e.g., a poly(ethylene glycol)-based polymer matrix, and are preferably 3D printed. The analytical device (200) of any one of claims 1 to 13.

15. The analytical device (200) comprises a plurality of support structures (100) arranged in a planar array (250); the planar base elements (10) of all support structures (100) in the array (250) are substantially coplanar in the plane of the array (250); the support pillars (20, 30) of all support structures (100) in the array are arranged on the same plane of the planar array (250); The analytical device (200) of any one of claims 1 to 14.

16. 1. A multiwell plate having substantially planar upper and lower surfaces and including a plurality of wells open at said upper surface; At least one well, preferably all wells of the multi-well plate are provided with the support structure (100) according to any one of claims 1 to 13. The analytical device (200) of claim 15.

17. an optical detection device (220) disposed on the side of the planar array (250) opposite the support pillars (20, 30); The optical detection device (220) is movable in the plane of the array (250).

17. An analytical device (200) according to claim 15 or 16.

18. a plurality of optical detection devices (220) disposed on a side of the planar array (250) opposite the support pillars (20, 30); each said optical detection device (220) is configured to acquire image data from at least one of said head portions (22, 32) of said first support pillar (20) and said second support pillar (30) of each support structure (100); The analytical device (200) of any one of claims 15 to 17.

19. 1. A method for analyzing the response of a strip of myocardial tissue (210) to a drug, comprising: Providing the analytical device (200) of any one of claims 1 to 18; acquiring, by the optical detection device (220), a first set of image data from at least one fiducial marker (26, 36) of at least one of the head portions (22, 32) of the first support pillar (20) and the second support pillar (30); introducing the drug into the analytical device (200) so as to contact the strip of contractile tissue (210); acquiring a second set of image data from at least one of the head portions (22, 32) of the first support pillar (20) and the second support pillar (30) by the optical detection device (220); Including, method.

20. the strip of contractile tissue (210) is paced by application of a pulsed electrical signal during acquisition of at least the first set of image data and the second set of image data; 20. The method of claim 19.

21. the response being the contractile response of the strip of contractile tissue (210); the second set of image data being characteristic of the contractile response of the strip of contractile tissue to the drug; 21. The method of claim 19 or 20.

22. the first set of image data and / or the second set of image data is video image data; 22. The method of any one of claims 19 to 21.

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