Apparatus for culturing cells or tissues, or for testing in in vitro muscle tissue.

Abstract

An apparatus for testing in vitro muscle tissue in a microwell plate includes a base and two posts each protruding from the base. The apparatus also includes a wall having inner and outer wall surfaces surrounding the posts, the wall including at least two rib elements extending outwardly from the outer wall surface of the apparatus.

Description

Technical Field

[0001] The present invention relates to an apparatus for cell culture, tissue culture, or for testing in vitro cells and tissues. In particular, the present invention relates to an apparatus for testing the response of in vitro muscle tissue to external electrical stimulation.

Background Art

[0002] In the field of drug research, many drugs are being tested for cardiotoxicity. Many (new) drugs affect myocardial contraction and profile, and thus show toxic effects. Drug-induced cardiotoxicity is a major adverse effect detected for several clinically important drugs. This toxicity has hitherto led to the post-marketing withdrawal of a number of pharmacologically effective drugs and limited the effectiveness of other clinically useful drugs. Almost 10% of drugs in the past 40 years have been recalled from the clinical market worldwide due to concerns about cardiovascular safety.

[0003] Drug-induced cardiotoxicity is an important cause for rejecting compounds in preclinical and clinical development. This is one of the most serious side effects associated with new drug development and one of the major toxic effects induced by some types of drugs. The assessment of the risk of drug-induced cardiotoxicity, including QT interval prolongation, is considered today an essential element of the standard preclinical evaluation of new chemical entities.

[0004] Cardiotoxicity can be tested in animal models, for example in rats. However, these models have drawbacks and do not always accurately predict the effects in humans. This may potentially discard beneficial agents and tolerate potentially toxic agents in field trials in humans.

[0005] Another way to evaluate cardiotoxicity is to test the effect of drugs in vitro. The drug is added to an aggregate of cardiomyocytes and grown in vitro. Thereafter, the effect on electrical stimulation is studied.

[0006] An example of such state-of-the-art equipment is the HeartDyno® apparatus. This apparatus consists of an elliptical well with two small pillars at the bottom. These apparatuses are manufactured using thin-film technology. The molds are fabricated on a wafer by SU-8 photolithography, resulting in a shape with a depth of 700 μm. PDMS is cast onto these shapes and cured. After removing the PDMS from the wafer, a 6 mm diameter sample is punched out.

[0007] These samples are placed, for example, in the wells of a 96-well plate and attached to the bottom using silicone adhesive. A mixture of cardiomyocytes, cardiac fibroblasts, collagen, DMEM, NaOH, and Matrigel is added to the wells. Cardiomyocyte tissue is formed over several days. The tissue exhibits spontaneous contraction, but also contracts during electrical stimulation. During contraction, the two columns flex, and this flexure is analyzed using a video analysis algorithm.

[0008] US2019 / 316068 discloses a device for generating 3D cardiac tissue. [Overview of the project] [Problems that the invention aims to solve]

[0009] Adhering the device to the wells of a well plate is a cumbersome process. Performing this aseptically is difficult and requires extra manufacturing steps. Furthermore, the device must be aligned so that the columns are oriented similarly in each well, which requires special attention.

[0010] The proposed solution is to "clamp" the device within the wells of the well cell plate. This can be done by manufacturing the device from an elastomer material with an outer diameter slightly larger than the wells, and thus held in place by shear stress. However, doing so would deform the negative space of the device, which could render the device unusable. [Means for solving the problem]

[0011] The present invention is defined by the claims. According to an example of one aspect of the present invention, an apparatus for testing in vitro muscle tissue is provided, the apparatus having a base, two columns projecting from the base, and a wall having an inner wall surface and an outer wall surface surrounding the columns, the wall having at least two rib elements extending outward from the outer wall surface to the outer edge of the apparatus.

[0012] In vitro cardiomyocyte tissue can be electrically stimulated to contract in order to simulate the behavior of in vivo cardiac tissue. The tissue can be grown between and around two columns so that the two columns flex when the tissue contracts. The displacement of the columns caused by the flexing can be measured to determine the force from the tissue's contraction.

[0013] Typically, the apparatus for testing in vitro muscle tissue is placed in the wells of a microwell plate. To hold the apparatus within the well, the outer edge of the test unit can be made slightly larger than the well diameter so that shear stress holds the apparatus in place. However, this can deform the inner diameter of the wall.

[0014] To avoid or reduce deformation of the inner wall surface, rib elements are added to the wall to prevent or reduce deformation of the inner wall surface while maintaining sufficient shear stress height to hold the device in place during use without the use of adhesive. The outer diameter of the rib elements (at the outer edge of the device) may be larger than the outer diameter of the base to further reduce deformation of the device.

[0015] The columns may be taller than the walls, or the walls may be taller than the columns. Alternatively, the columns and walls may be of the same height. Similarly, the walls may be of the same height as the rib elements, or the rib elements may be of a different height (e.g., taller or shorter) than the walls. Each rib element may be of the same height or a different height. Within the virtual plane containing the base, there may be at least two non-parallel rib elements, two, three, four or more. However, in one preferred example, there are four rib elements. A symmetrical design has rib elements that extend radially, spaced 90 degrees apart from each other.

[0016] However, an asymmetric design may be used in which there are two pairs of rib elements facing each other in the diametrical direction, and the two pairs are not orthogonal to each other (and thus form a non-orthogonal cross).

[0017] This forms a cross shape of rib elements to provide a structure that does not deform under shear stress from any direction.

[0018] The inner wall surface may be elliptical, rectangular, and therefore may have an aspect ratio other than 1.

[0019] This non-1 aspect ratio promotes tissue growth in a contraction direction along the separation gap between columns.

[0020] The direction of contraction is the direction in which the tissue contracts. Therefore, by having a direction of contraction along the spacing between the columns, the tissue causes the columns to bend toward the center point between the two columns.

[0021] The height of the rib element may be up to the same as the height of the wall. The height of the wall can be up to 2000 μm.

[0022] The height of the column may be at least 500 μm and up to 2000 μm.

[0023] The device can be manufactured based on injection molding, injection compression, transfer molding, or embossing.

[0024] By bulk manufacturing the pillars using these methods, it is possible to reduce the variation in pillar rigidity caused by the manufacturing method and improve the manufacturing speed compared to hand casting.

[0025] The device (or only the pillar, base part, and / or rib element) can be manufactured from silicone rubber. The silicone rubber can include Elastosil 3040 / 30.

[0026] The pillar may be tapered towards the distal end, and the distal end of the pillar may be rounded or partially rounded.

[0027] By tapering the pillar, the mold release process (of the manufacturing process) can be made more reliable. Similarly, the rounded distal end of the pillar further enhances the reliability in the mold release process.

[0028] The Young's modulus of the material used to manufacture the pillar can be at least 0.5 MPa and at most 5 MPa. For example, the Young's modulus is 1 MPa - 2 MPa. The rigidity of the pillar depends on the Young's modulus and geometric shape of the pillar.

[0029] However, there are other material properties related to the function of the pillar. Tensile strength (e.g., 9.0 N / mm 2 ) and maximum elongation (e.g., 600% at the breaking point) are two of such related properties. The combination of these properties enables the release of pillar structures with an aspect ratio close to 5 (or more), and also enables the release of a mushroom shape.

[0030] Therefore, using silicone injection molding grades provides reliable final properties after manufacturing, facilitates scaling up, and is also suitable for shapes with aspect ratios of 5 or more for release from industrial molds, which is not possible with other casting solutions.

[0031] The present invention also provides a set of apparatus for testing in vitro muscle tissue in microwell plates, the set of apparatus being manufactured using multi-cavity injection molding, where each cavity corresponds to one apparatus.

[0032] Multi-injection molding not only increases the production speed of the equipment but also ensures a sufficient ratio of mold flow to wall thickness.

[0033] The present invention also provides a system for testing in vitro muscle tissue, the system comprising an array base and an array of devices for testing in vitro muscle tissue in microwell plates on the array base, wherein the array base and the array of devices are formed as a single unit.

[0034] For example, the devices can be arranged at equal intervals on an array base in a rectangular array. This allows for the manufacture of multiple devices without requiring a multi-cavity injection molding machine.

[0035] The system may further include a microwell plate having multiple wells, and multiple devices on the array base are arranged such that each device fits into one of the wells.

[0036] For example, by using a microwell plate with an open bottom, a system with an array of devices can be positioned beneath the microwell plate so that all the devices fit within the wells of the microwell plate. The system is manufactured to fit the microwell plate (i.e., the outer diameter of the devices fits within the wells, and the spacing between the devices is the same as the spacing between the wells).

[0037] Muscle tissue can then be grown within the wells and walls of the microwell plate around the column of the apparatus.

[0038] The apparatus may be manufactured from an elastic material, and the outer edge of each apparatus may be up to 5% larger than the inner circumference of the wells of the microwell plate.

[0039] If the device is slightly larger than the well and manufactured from a flexible material, the device can be fitted into the well, and the difference in diameter provides shear stress to the device. The difference in diameter depends on the material used to manufacture the device, the shear stress required to hold the device in the well during muscle contraction, and so on.

[0040] The system may further comprise a pair of electrodes for stimulating muscle tissue. The present invention also provides a method for manufacturing an apparatus for testing muscle tissue in vitro, the method comprising injection molding the apparatus using a mold, the mold defining a base portion, two columns projecting from the base portion, and a wall portion having an inner wall surface and an outer wall surface surrounding the columns, the wall portion including at least two rib elements extending outward from the outer surface to the outer edge of the apparatus.

[0041] These and other aspects of the present invention will become apparent from and be described with reference to the embodiments described below. [Brief explanation of the drawing]

[0042] To better understand the present invention and to more clearly illustrate how it can be put into practice, refer to the accompanying drawings as merely examples. [Figure 1] A diagram of a device having two tapered columns. [Figure 2] A diagram showing a model column for calculating the displacement of a tapered column caused by applied forces. [Figure 3] A diagram showing a simulated column with a tapered, rounded top. [Figure 4] A 3D representation of a device with columns. [Figure 5] Top view of a device manufactured by injection molding. [Figure 6] A diagram showing an injection-molded device containing myocardial tissue. [Figure 7] Results of the first experiment. [Figure 8] Results from the second experiment. [Figure 9] Top view of the device. [Figure 10] A diagram showing a device having rib elements around its outer edge. [Figure 11] A diagram showing a device having rib elements manufactured by injection molding. [Figure 12] A diagram showing an array of devices to be incorporated into a Wellcell plate. [Figure 13] A diagram showing a 96-well cell plate with an open bottom. [Figure 14] A diagram showing multiple devices manufactured using multi-cavity injection molding. [Figure 15] A diagram showing a device with inverted pillars. [Figure 16] A diagram showing a device having a stepped structure for determining the height position of muscle tissue. [Figure 17] This figure shows a plan view of the apparatus in Figure 16. [Figure 18] A diagram showing a device with a conical structure for determining height. [Modes for carrying out the invention]

[0043] The present invention will be described with reference to the drawings. Detailed descriptions and specific examples illustrate exemplary embodiments of the apparatus, systems, and methods, but should be understood to be for illustrative purposes only and not intended to limit the scope of the invention. These and other features, aspects, and advantages of the apparatus, systems, and methods of the present invention will be better understood from the following description, the appended claims, and the appended drawings. It should be understood that the drawings are purely schematic and not drawn to a specific scale. Also, it should be understood that the same reference numerals are used throughout the drawings to indicate the same or similar parts.

[0044] The present invention provides, for example, a device for testing in vitro muscle tissue in a microwell plate, the device comprising a base and two columns, each protruding from the base. The device also comprises a wall portion having an inner wall surface and an outer wall surface surrounding the columns, the wall portion comprising at least two rib elements extending outward from the outer wall surface to the outer edge of the device.

[0045] Figure 1 shows a diagram of a apparatus 100 having two tapered columns 102. The columns 102 are made from a flexible material, and the shape of the columns 102 is designed for mass production. The shape and height of the columns 102 are adjusted to have the material properties necessary for testing tissue samples 108. The design of the columns 102 is adapted to ensure demolding. The columns 102 are tapered, with a wider base and a smaller apex.

[0046] The column 102 extends from the base portion 104 and is enclosed within the inner surface of the wall portion 106. The column 102 is designed so that cardiac tissue 108 can grow between and around the column 102. The cardiac tissue 108 can be contracted with electrodes, and the contraction of the cardiac tissue 108 from electrical signals can be measured by the displacement of the column 102.

[0047] Figure 1(a) shows a cross-section of the device 100 where the cardiac tissue 108 between the columns 102 is not contracted. Figure 1(b) shows the same device 100, but the cardiac tissue 108 is contracted. The columns 102 are displaced due to the contraction of the cardiac tissue 108.

[0048] Figure 1C) shows the same apparatus 100 viewed from above when the cardiac tissue 108 is not contracting. In this example, the inner wall surface is elliptical. By growing cardiac cells within the elliptical cavity, the tissue 108 forms a direction of contraction along the spacing between the columns 102.

[0049] The apparatus 100 can be manufactured by injection molding, 3D printing, urethane casting, or press casting. The tapered column 102 allows for more robust demolding methods for injection molding, urethane casting, and press casting, and also allows for the printing of more stable structures by 3D printing methods. These methods enable mass production of the apparatus 100. The following embodiments are described in relation to injection molding. However, any features or advantages disclosed may also apply to other manufacturing methods. In this example, the base is shown at the bottom. However, this is not necessarily the case, and the base may have any orientation.

[0050] The column 102 can have a rectangular parallelepiped shape, and one, two, three, or four of its faces can be tapered. For example, two sides of the column 102 (e.g., facing inward toward the tissue and facing outward toward the tissue) may be tapered, while the other two faces are perpendicular to the base portion 104. The exact taper of the column 102 depends on the dimensions of the column 102 itself, but can range from a 1-degree taper to a 20-degree taper relative to a perpendicular line from the base portion 104.

[0051] Column 102 may instead be cylindrical, and therefore one side may be tapered. Similarly, the exact taper of column 102 depends on the dimensions of column 102 itself, but can range from a 1-degree taper to a 20-degree taper relative to the vertical line from the base portion 104. Exemplary tapers are 0.5° to 2°.

[0052] In addition, the distal end of the tapered column 102 from the base (i.e., the tip of the tapered column) may have a head portion. The head portion of the column has a larger diameter (one-dimensional or two-dimensional) than the rest of the column 102 and prevents tissue from sliding toward the distal end of the column and detaching from it.

[0053] In the case of the apparatus 100 having a head portion that is injection molded, it is preferable that the column 102 (below the head) is also tapered. The tapering of the column reduces friction between the column 102 and the mold when demolding the apparatus 100. The tensile strength (e.g., 7 N / mm²) of the material used to manufacture the column (i.e., silicone rubber) is 2 ~11N / mm 2 Preferably 9.0 N / mm 2 The column structure having a head portion can be released from the mold by the force and the maximum elongation (for example, 400% to 800%, preferably 600%).

[0054] By reducing friction between the tapered column 102 and the mold, the only major force required to remove the tapered column 102 with its head is the force required to elastically deform the head of the column so that it passes through the smallest portion of the mold. The properties of silicone rubber (e.g., tensile strength and maximum elongation) allow the silicone rubber to deform elastically in such a manner and to retain its original shape once removed from the mold. Other materials with similar properties could also be used.

[0055] The shape of the head can also be designed to reduce the force required to pull it out of the mold. The shape of the head also depends on the material properties, the shape of the column 102, and the taper used on the column. For example, the head can be rounded, or it may be spherical with a diameter greater than (or equal to) the width of the widest side of the column 102. A column with a head can also have a mushroom-shaped geometric form.

[0056] Therefore, using silicone injection molding grades provides reliable final characteristics with the potential for easy scale-up after manufacturing, and is also suitable for tapered columns 102 with head sections, which is not possible with other casting solutions.

[0057] Figure 2 shows a model column 102 for calculating the displacement of a tapered column 102 due to applied forces. The taper of column 102 affects its stiffness and elasticity, which should be compensated for. When converting the design to an injection molding design, changes in material and geometric shape should be compensated for to maintain the correct stiffness and elasticity of column 102. The effects of design changes to column 102 can be calculated using simulation techniques.

[0058] A common soft material for injection molding is Elastosil 3040 / 30, which has a Shore hardness of 40A. Using this material to manufacture the apparatus 100 affects the elastic properties of the column 102. Simulations were performed to estimate these effects, and an optimized design was proposed.

[0059] Using injection molding improves the reproducibility of rigidity and results in much better hardness. Sylgard184 silicone is less reliable due to temperature variations during curing and insufficient precision (and therefore weight difference) in mixing the two components, silicone A and crosslinking agent B, in the desired ratio, e.g., 9:1. In injection molding, components A and B are mixed in much larger quantities in a 1:1 ratio, minimizing chemical metric discrepancies. Furthermore, it can be easily scaled up.

[0060] After injection molding, the reaction of Elastosil 3040 / 30 begins after heating to over 150°C in the mold. The problem with Sylgard 184 is that it begins to harden at room temperature. Also, with Sylgard 184, the idle time is short so it hardens quickly, making it impossible to pour into small cavities. In injection molding, the mixture is heated in a heated mold, and the viscosity is low at high pressure (i.e., 30 MPa to 60 MPa), resulting in all cavities for column 102 being filled.

[0061] Figure 2(a) shows a simulation of a tapered column 102 to measure the strain of the column 102 when a force is applied. The typical modulus of elasticity for Sylgard 184 is 1.45 MPa, and for Elastosil it is 1.4 MPa. A bulk modulus of 2.2 GPa is used for both materials. The Neo-Hookian approximation was used in the calculations.

[0062] Figure 2(b) shows a simplified representation of displacement measurement 204. Measurement point 202 on column 102 is at a height of 3 / 4. The apparatus using Slygard 184 has a stiffness of 500 μm / N at a displacement of 100 μm, and therefore the stiffness of column 102 of Elastosil relative to column of Slygard 184 is estimated at a displacement of 100 μm.

[0063] In the simulation shown in Figure 2(a), a distributed force is applied to a defined area of ​​column 102, affecting a region of 1 / 2 to 3 / 4 of the column's height. This force is increased until the horizontal displacement at the monitoring point (at 3 / 4 of the column's height) reaches 100 μm. The height of column 102 is 800 μm.

[0064] The stiffness of the Sylgard 184 column is derived at a displacement of 100 μm and compared to the stiffness of the Elastosil 3040 / 30 column 102. The stiffness of the tapered column design is approximately twice as high as that of the Sylgard 184 column. This can be addressed with new column shapes (i.e., taller column 102, different taper angles, etc.). The choice of stiffness depends on the needs of any particular user. For example, a taller column 102 can be used to compensate for the modified stiffness, and as a result, column 102 can be manufactured using injection molding technology, making its design suitable for mass production.

[0065] Figure 3 shows a simulated column 102 having a tapered, rounded top. Figure 3(a) shows a pair of columns 102 with a height of 800 μm. Figure 3(b) shows a column 102 with a height of 900 μm.

[0066] The rounded top design is 15% stiffer than the standard configuration shown in Figure 2. However, since this can be mass-produced, the variation in stiffness will be much smaller. It has been found that columns 102 with a height of 950-970 μm are suitable to fully compensate for the variation in stiffness. Columns 102 with rounded tops can distribute local stresses more effectively. Rectangular columns 102 have higher stress concentrations and cause larger variations.

[0067] A mold for apparatus 100 having a tapered, rounded top was designed and fabricated, and apparatus 100 was fabricated from this mold. It was found that demolding of a 900 μm column 102 made of Elastosil 3040 / 30 material was successful.

[0068] The mold has a base portion that defines the outside (bottom) of the base portion 104 and the outside (radially outward-facing wall) of the wall portion 106. The top portion defines the inside (top) of the base portion 104, the inside (radially inward-facing wall) of the wall portion 106 and the column 102, and has two cavities for forming the two column 102.

[0069] Inserts for defining the column are fitted into the cavity, and therefore the shape can be easily modified to meet various product requirements. Furthermore, this manufacturing method ensures a clearly defined top shape of the column 102. Different inserts can be used within the cavity to produce different column shapes (and therefore different stiffness values).

[0070] By growing cells within the elliptical cavity, the muscle tissue 108 forms a contraction direction along the spacing between the columns 102 (i.e., parallel to the line connecting the centers of the two columns), although initially the cells may not adhere to the columns 102. A crucial step is to promote uniform cell proliferation into a fusion layer of cells on the extracellular matrix within the apparatus 100 to form the muscle tissue 108.

[0071] To address this, the silicone column 102 can be modified with carboxyl groups to enable direct cell adhesion to the column 102. By bulk modifying the silicone with linoleic acid, carboxyl groups are introduced and activated on the surface, allowing cells to adhere to the column 102. In this way, cardiomyocytes can grow more effectively into muscle tissue 108 between the two columns 102.

[0072] Figure 4 shows a 3D representation of the apparatus 100 having columns 102. In this example, the inner surface of the wall 106 has an elliptical shape. The apparatus 100 is described as having two columns 102 within a cavity (i.e., the cavity is defined by the wall 106).

[0073] Furthermore, by adding a column head to the distal (upper) end of the column 102, it is possible to prevent the muscle tissue 108 from being separated from the column 102 by the movement of the muscle tissue 108 caused by contraction. If appropriate dimensions are selected, the elasticity of the material allows the column 102 to be demolded despite the column head.

[0074] Figure 5 shows a top view of the apparatus 100 manufactured by injection molding. Markings from the injection molding process can be seen near and around the column 102. Similarly, in this example, the inner surface of the wall 106 is elliptical.

[0075] Figure 6 shows a device 100 having myocardial tissue 108. The cavity is filled with gel (extracellular matrix), and myocardial cells are grown on the gel to form muscle tissue 108. When the tissue 108 contracts, the column 102 moves, and muscle function can be monitored when an appropriate indicator (e.g., a color applied to the top of the column to allow for more precise focusing of a microscope) is provided at the top of the column 102.

[0076] The results of the first experiment are shown in Figure 7. The graph shows a plot of pulse frequency (y-axis) versus pacing (stimulation) frequency. Pacing frequency is the frequency of the electrical signals used to stimulate muscle tissue 108, and pulse frequency is the frequency at which muscle tissue 108 is observed to contract.

[0077] The experiment involved stimulating myocardial tissue 108 within apparatus 100 and comparing the results with those of other apparatuses. The formed tissue 108 was electrically stimulated at a range of pacing frequencies with a pulse duration of 10 ms and an increment of 0.2 Hz at 35 V. Muscle contraction force was calculated using the following formula:

number

[0078] The results obtained are shown in Figures 7a) and 7b). Figure 7a) shows the pulsation frequency (y axis) as a function of pacing frequency (x axis) for five tapered columnar devices 100 manufactured by injection molding. Figure 7b) shows the pulsation frequency (y axis) as a function of pacing frequency (x axis) on five HeartDyno devices.

[0079] The tapered device was observed to perform comparably to the HeartDyno device, or to function better, allowing the cardiomyocyte tissue 108 to respond more consistently to higher-frequency pacing. Furthermore, the tapered device used in the results of Figure 7a) was even more rounded.

[0080] Figure 8 shows the results of the second experiment. This experiment also involves stimulating myocardial tissue 108 within the device 100. Figure 8 a) shows the beating frequency (y axis) as a function of the pacing frequency (x axis) of the seven tapered column device 100. Figure 8 b) shows the normalized beating frequency d (y axis) as a function of the pacing frequency (x axis) of the seven tapered column device 100. It can be seen that the contraction amplitude increases, which indicates a positive force-frequency relationship. This is characteristic of mature cardiomyocytes. These results indicate that the tapered column device 100 performs comparably to or slightly better than the HeartDyno device. Furthermore, they have the advantage of less variability in stiffness due to the use of mass production technology. Also, the use of mass production technology can reduce costs.

[0081] Figure 9 shows a top view of the apparatus 100 (columns 102 are not shown). Figure 9a) shows the apparatus 100 properly inserted into the wells of a microwell plate (i.e., a well cell plate). Figure 9(b) shows the apparatus 100 when deformed due to improper insertion into the wells of a well cell plate. This deformation can occur if the apparatus is slightly larger than the wells in the well cell plate (to avoid the use of adhesive).

[0082] The deformation is exaggerated in Figure 9b) and occurs after the apparatus 100 is placed in a non-ideal well shape, causing unpredictable deformation of the inner surface, which can therefore affect partial differences in cell growth performance and reproducibility.

[0083] Figure 10 shows the apparatus 100 having a base portion 104, a wall portion 106, and rib elements 1002 around the outer circumference of the apparatus 100 (columns 102 are not shown). This allows for the use of a slightly larger outer diameter of the apparatus 100 compared to the inner surface of the wells of the well cell plate without deforming the inner surface of the apparatus surface 100. In this way, the shear stress of the apparatus 100 relative to the well cell plate can be increased to the point where adhesion of the apparatus 100 to the well cell plate is no longer required. Thus, the rib elements 1002 (in this case, cross ribs) can increase the shear stress and reduce the deformation of the apparatus 100, particularly the deformation of its inner surface.

[0084] The device 100 is made from an elastomer material that can deform when an external force is applied. By designing transverse ribs 1002 around this wall portion 106, deformation of the inner wall surface is prevented when inserted into the well, while maintaining a sufficiently high shear force to avoid the need for any form of adhesive between the device 100 and the well.

[0085] The flexible material and integrated cross ribs 1002 of the apparatus 100 offer further advantages, including overcoming tolerance issues in the shape of the apparatus 100 or array tolerances (overcoming variations between parts), in addition to eliminating the need to use adhesives. In some manufacturing methods, the demolding direction of either the apparatus 100 or the wellcell plate can help increase shear force or provide a complete mechanical lock.

[0086] In this example, a device 100 having four cross ribs 1002 is shown. However, the device 100 may also have two cross ribs 1002, three cross ribs 1002, or more than four cross ribs 1002.

[0087] Figure 11 shows apparatus 100 having rib elements 1002 manufactured by injection molding. Apparatus 100 is manufactured by injection molding of Wacker Elastosil LR 3040 / 40 (Shore hardness A 40).

[0088] The apparatus 100 can be punched out with a 6.4 mm punch and placed in the wells (e.g., 96 wells) of a well cell plate having an inner diameter of 6.4 mm.

[0089] The shear stress of the apparatus 100 within the wells of the well cell plate was found to be high enough to hold the apparatus 100 in place until the completion of a protocol to grow and test cardiac tissue for a period of up to 9 days. Figure 12 shows an array 1202 of the apparatus 100 for integration into a well cell plate. In addition to a single apparatus 100, the apparatus 100 can also be manufactured as an integrated array 1202 on a slab of material. For this purpose, a slab of material is manufactured having an array of apparatus 100 with cross ribs 1002. This slab can be placed beneath a well cell plate having an open bottom. In this way, the array 1202 closes the well cell plate and provides the apparatus 100 for cell culture. The array 1202 may be circular, rectangular, or any other ratio in between, depending on the requirements.

[0090] Figure 13 shows a 96-well cell plate with an open bottom. This well cell plate can be used for the array 1202 of the apparatus shown in Figure 12. Alternatively, the apparatus 100 of the array 1202, integrated onto a material slab, can be used directly without requiring a well cell plate. In this case, the outer wall defines the apparatus 100 of the array 1202 directly above the shared base, and this structure can be manufactured as a single unit. The outer wall acts as a substitute for the wells in the well cell plate.

[0091] Figure 14 shows multiple devices 100 manufactured by multi-cavity injection molding.

[0092] Manufacturing a single slab material (the apparatus for array 1202) requires a large injection molding machine to fill the large volume and the small details of the columns 102. Injection molding works best with uniform wall thickness (e.g., the thickness of the walls and columns 102). If they differ by a large ratio, the small details solidify more rapidly, even before the mold is completely filled, while the large volume solidifies most slowly by polymerization. An undesirable situation can arise if the smallest details have already solidified while a larger volume needs to be filled. This can lead to defects, and in this case, there is a risk that the mold portion for the columns 102 will not be completely filled.

[0093] This problem makes it difficult to manufacture small details (i.e., columns 102) within a large slab. This can be solved by balancing the amount of material with the wall thickness. Figure 14 shows several separate apparatuses 100, each with optimized wall thickness and flow path. As the volume to be solidified decreases, the production cycle time also improves.

[0094] By manufacturing a separate device 100, tolerance differences within the well cell plate can be overcome compared to an array 1202 of device 100 having column details. Manufacturing array 1202 may cause leakage within the well plate due to tolerance and shrinkage differences, but this can be overcome by manufacturing a separate device 100. The device can be positioned, for example, within a 96-well cell plate or any other target, manually or robotically.

[0095] One problem that may arise with these apparatuses 100 is knowing the exact height of the muscle tissue 108 on the column 102. As shown earlier (in Equation 1), the displacement of the column 102 depends on the height of the muscle tissue 108 on the column 102. Furthermore, this position may change during the experiment. It is known that the muscle tissue 108 can climb the column 102 by mechanical force. In addition, while the column is bending, the muscle tissue 102 may detach from the column 102, potentially causing the experiment to fail.

[0096] To avoid this problem, the apparatus 100 may be "inverted" so that the columns 102 protrude downward (relative to the well cell plate). Figure 15 shows the apparatus with inverted columns (inverted column apparatus). The inverted column apparatus has two columns 102 protruding from a base 104. The inverted column apparatus is mounted on a so-called ultra-low adhesion well in a well cell plate. The columns "hang" in the well, and muscle tissue 108 can grow around them. Motion analysis of the columns 102 can be performed from below the well (if the well cell plate is made of a transparent material).

[0097] The cell / collagen mixture is deposited at the bottom of an ultra-low adhesion well. The base portion 104 is positioned at the top of the well and acts as a lid for the well, while the column 102 protrudes into the cell / collagen mixture. When cardiomyocyte tissue 108 is formed, it grows around the column 102. It does not adhere to the well because an ultra-low adhesion well is used. As the cells begin to come into contact with each other, the column 102 begins to move. This movement is captured by a microscope placed below the well.

[0098] In the inverted column apparatus, the cell culture is always at the end of the column 102, and therefore the height position of the muscle tissue 108 is well determined. This increases the reproducibility of the force required to bend the column 102. Also, when the column 102 moves, the muscle tissue cannot detach from the column 102 due to gravity. Furthermore, the relative positions of the two columns 102 are determined and cannot spread apart.

[0099] A further advantage of this embodiment is that the inverted column device can be mass-produced by injection molding. Also, since the column 102 hangs down from the base 104, the range of possible material stiffness values ​​is larger. In the design of the upright device 100, the support column 102 requires a certain degree of stiffness to maintain an upright position.

[0100] This measurement system can be equipped with a microscope that looks up from below the well cell plate. This is comparable to current systems for measuring column displacement. In either system, placing anything at the bottom of the well will interfere with the measurement.

[0101] In this inverted design, the base can function as a plug for the well, preventing spills or sealing the well from contaminants (such as air).

[0102] Myocardial cells in muscle tissue 108 can be paced as described above. This is done using a voltage of, for example, 35 V at a specific frequency (to simulate a heartbeat). The myocardial cells contract in response to stimulation. This contraction can be monitored while adapting the pace frequency. Two electrodes 1502 are placed in the well to pace the cells.

[0103] These electrodes can be integrated into an inverted device, as shown in Figure 15. In this way, the placement of the electrodes does not obstruct the view for observing cells from the bottom of the well. Furthermore, since the electrode 1502 can be held by the base 104, it is always properly positioned and does not need to be opened to stimulate the muscle tissue 108 (i.e., the base 104 does not need to be removed). This prevents contamination of the cell culture.

[0104] In conventional upward-projecting columns, it is important that the height of the muscle tissue 108 can be determined above the column. Figure 16 shows a device 100 having a stepped structure 1602 positioned outside the cavity area. The stepped structure 1602 is used to determine the height position of the muscle tissue 1602 on the column 102. In one example, this staircase has steps of 50 μm each, but this dimension can of course be changed depending on the size of the device 100, column 102 and / or cellwell plate. For example, if the device 100 having the structure 1602 is manufactured using injection molding, the size of the steps can be changed by using different injection-molded inserts.

[0105] When using a microscope to observe muscle tissue 108, the microscope must be focused on the muscle tissue 108 itself. When focusing on muscle tissue 108 on column 102, the z position (i.e., height) can be determined by looking at the corresponding step of the staircase structure 1602, which is also in focus. Each step of the staircase structure 1602 can be marked (e.g., with numbers, letters, or other distinctive features) to facilitate reading the corresponding z position. The height of muscle tissue 108 can then be determined, and therefore the force on column 102 can be normalized by calculation (i.e., using Equation 1).

[0106] In this example, the wall portion 106 has a separate outer surface for the structure 1602 on the outside of the outer wall that defines the cavity. The structure 1602 can instead be located inside the outer wall 106, and the height-determining structure may be located inside the wall 106 near the column, in which case it is easily visible.

[0107] Figure 17 shows a plan view of the apparatus shown in Figure 16.

[0108] Figure 18 shows an apparatus 100 having a conical structure 1602 for height determination. In this example, the diameter of the cone changes with the height of the structure 1602. Thus, the diameter of the cross-section of this cone, which is in focus when using a microscope, corresponds to the z-position of the muscle tissue 108. In this example, there are no separate steps in the measurement of height (as experienced in a staircase structure), and therefore the height can be measured at any position.

[0109] The structure 1602 can be any other shape (for example, a tapered 3D shape, a stepped shape, etc.) such that, when viewed from above, the cross-section of the structure 1602 changes with respect to the height of the structure 1602 from the base 104.

[0110] The structure 1602 may be taller than the column 102 and / or the wall 106, or may be the same height as either of them. If the structure 1602 is taller than the column 102, the structure 1602 may have a height mark above it indicating the height of the column 102 (or a height slightly lower than the column), which can then act as a warning mark for the microscope user to recognize that there is a risk of the muscle tissue 108 moving away from the column 102 when both the muscle tissue 108 and the height mark are in focus.

[0111] Alternatively, the structure 1602 may be lower than the column 102. In this case, if the user of the microscope can no longer focus on the structure 1602, this can act as a warning sign that there is a risk that the muscle tissue 108 may be detached from the column 102. Also, to facilitate height measurement, there may be multiple height marks at different height positions on the structure 1602. The above example is described using a microscope to image the column 102 and thus detect and measure its displacement. However, any other imaging system capable of measuring the column 102 (e.g., a camera) may also be used. Modifications of the disclosed embodiments can be understood and implemented by those skilled in the art in carrying out the claimed invention from the examination of the drawings, disclosures and appended claims. In the claims, the word “have” does not exclude other elements or steps, and the indefinite article “a” or “an” does not exclude plurality.

[0112] The mere fact that certain means are described in different dependent claims does not imply that combinations of these means cannot be used advantageously. Where the term “adapted to” is used in a claim or specification, it is intended to be equivalent to the term “configured to.” No reference numeral in a claim should be construed as limiting in scope.

Claims

1. An apparatus made of elastomer material for in vitro culturing of muscle tissue, which is used by being inserted into wells formed in a well cell plate, It has a base portion, two columns protruding from the base portion, and a wall portion having an inner wall surface and an outer wall surface surrounding the columns. The apparatus wherein the wall portion has at least two rib elements extending outward from the outer wall surface to the outer edge of the apparatus, and the outer diameter of the outer edge due to the rib elements is greater than the inner diameter of the well.

2. The apparatus according to claim 1, wherein the wall portion includes four rib elements extending radially at 90-degree angles from one another within a virtual plane including the base portion.

3. The apparatus according to claim 1 or 2, wherein the inner wall surface is elliptical or rectangular.

4. The apparatus according to any one of claims 1 to 3, wherein the height of the rib element is at most the same height as the wall portion, and the height of the wall portion is at most 2000 μm.

5. The apparatus according to any one of claims 1 to 4, wherein the height of the column is 500 μm or more and 2000 μm or less.

6. The apparatus according to any one of claims 1 to 5, wherein the Young's modulus of the material used to manufacture the column is at least 0.5 MPa and at most 5 MPa.

7. The apparatus according to any one of claims 1 to 6, obtained by injection molding, injection compression, transfer molding, or embossing.

8. The apparatus according to any one of claims 1 to 7, wherein the column, the base portion, the wall portion, and the rib element contain or are made of silicone rubber.

9. The apparatus according to any one of claims 1 to 8, wherein the column is tapered from the base portion toward the distal end, and the distal end of the column is rounded or partially rounded at the boundary between one or more side walls of the column and the distal end of the column.

10. A set of apparatus according to any one of claims 1 to 9, manufactured using multi-cavity injection molding, wherein each cavity corresponds to one apparatus.

11. A system for testing muscle tissue in vitro, comprising an array base and an array of devices according to any one of claims 1 to 9 on the array base, wherein the array base and the array of devices are formed as a single unit.

12. The system according to claim 11, further comprising a microwell cell plate having multiple wells, wherein the array of the devices on the array base is positioned such that each device fits into one of the wells.

13. The system according to claim 12, wherein the apparatus is manufactured from an elastic material, and the outer diameter of the outer edge of each apparatus is up to 5% larger than the inner diameter of the wells of the microwell cell plate.

14. The system according to any one of claims 11 to 13, further comprising a pair of electrodes for stimulating muscle tissue.

15. A method for manufacturing an apparatus for testing muscle tissue in vitro, the apparatus being used by being inserted into a well formed in a well cell plate, the method comprising the step of injection molding the apparatus using a mold defining a base portion, two columns projecting from the base portion, and a wall portion having an inner wall surface and an outer wall surface surrounding the columns, wherein the wall portion has at least two rib elements extending outward from the outer wall surface to the outer edge of the apparatus, the outer diameter of the outer edge due to the rib elements being greater than the inner diameter of the well, the method.

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