Apparatus and method for generating and evaluating an engineering organization

Abstract

The present invention relates to methods and systems for generating three-dimensional (3D) engineered tissue (ET), and methods and systems for electrically stimulating ET. The present invention provides an ET assembly including an ET lid, wherein a rigid post assembly and a flexible post assembly are coupled to the ET lid. The present invention also provides a casting assembly including an ET assembly and a casting plate. The present invention also provides a stimulation method and system for stimulating tissue constructs.

Description

Technical Field

[0001] The present application relates to methods and systems for generating three-dimensional (3D) engineered tissue (ET), and methods and systems for electrically stimulating and measuring the contractility and various physiological properties of ET.

Background Art

[0002] Tissues thicker than single cell layers produced or enhanced by three-dimensional engineered tissue (referred to herein as ET) or artificial processes are of great interest in biomedical research due to their cost, availability, and remarkable ability to simulate human physiological tissue, compared to competing physiological models. Means for manufacturing, maintaining, and characterizing these ETs have accumulated much academic interest. A particularly popular ET system is called a two-post system, where the tissue is cast between two posts, at least one of which has sufficient flexibility such that the attached tissue curves when it contracts. The curvature of the post can be related to the force generated by the tissue, which can provide meaningful information regarding the health and function of the tissue. Although there have been attempts to commercialize such devices, these methods are prototypical in academic laboratories and are not efficient, industry-centered tools.

[0003] U.S. Publication No. 2019 / 0029549 by Sniadecki et al. disclosed such a two-post ET system. The magnet embedded in one post of Sniadecki's two-post ET system provides detection based on a magnetic sensor of the contractile force of the ET built into the multi-well plate. In this non-optical contraction measurement system, it is possible to measure the force applied by a 3DET suspended between a flexible post and a rigid post in which a magnet is embedded, and to detect a change in the magnetic field due to the deflection of the flexible post when the ET performs a contraction operation using a magnetometer. Other commercially available two-post systems provide, for example, an optical reading of post curvature under ET loading, as described by Hensen et al. Development of a Drug Screening Platform Based on Engineered Heart Tissue, Circulation Research, July 9, 2010, is disclosed and can be downloaded from http: / / circres.ahajournals.org.

[0004] However, the previously disclosed ET systems have limitations in terms of manufacturing throughput, ease of use, and ergonomics. The manufacture of the non-biological components of the system requires complex molding and demolding processes over several days. Such manufacturing processes are not always compatible with industrial standard manufacturing techniques such as compression or injection molding, or non-standard modifications to said techniques are required to achieve expandability.

[0005] The process for generating 3D tissue itself is a significant limiting factor. In many two - post systems, the ET is cast from a suspension of cells and extracellular matrix, and the extracellular matrix gels around the post after deposition in the mold. The operator has to cast the mold of the tissue itself in the wells of a tissue culture plate from a temperature - sensitive gel material such as gelatin or agarose, which has an adverse effect on the reproducibility and throughput of the 3D tissue construct. Other materials such as silicone resin can be used, but require time - consuming surface treatment so that the mold does not adhere to the tissue. The handle to which these posts for holding the tissue are attached does not have alignment features to ensure consistent placement within the groove, resulting in a high variability in the morphology of the cast tissue. Also, when cast, these gel molds have grooves with vertical walls, and the grooves make it difficult to remove the tissue construct due to friction between the tissue construct and the mold side.

[0006] The newly formed tissue is fragile and any minor adhesion to the casting substrate can cause a fatal impairment of the cast tissue. The member to which two posts are attached on top does not have features for efficient and reproducible interface connection with industrial standard culture devices and, due to its heavy shape, has limited ability to be integrated into an automated processing system. These members can easily and detrimentally misalign with any sensor for evaluating the tissue. A slight misalignment of the device on the microplate can crush the tissue between the top surface of the microplate and the substrate to which the upper post is attached. The parts cannot be easily handled in groups of more than six at a time and lack features that can be easily grasped by a human or robotic operator.

[0007] ET performance can be improved in many ways by electrical stimulation, which is very interesting but also presents challenges. Conventional electrical stimulation devices for cell culture generally relate to graphite rods or platinum wires permanently fixed to an electrical stimulation module, which is attached to the top of a microplate, where the electrodes protrude into the substrate bath of each well in the microplate. Since small molecules in the substrate bath can easily penetrate and adhere to the graphite electrodes, it is necessary to replace the graphite electrode assembly after only one drug screening experiment to avoid cross-contamination. However, such available graphite stimulation devices are very expensive as disposable consumables or are very difficult to assemble and integrate into industrial standard tissue culture incubators, thus limiting their commercial availability in high-throughput stimulation research. Platinum electrodes have advantages in terms of reusability because they do not have the porosity of graphite electrodes, but they have a higher tendency to produce cytotoxic electrolytic by-products and require extensive cleaning after use, which adversely affects their utility in high-throughput biological research. Platinum wires are also very expensive, thus limiting the commercial expandability of devices employing platinum wires. Also, there is no two-post system that combines means for stimulating and recording from tissue using the post itself as an electrode. Electrophysiology is another important metric of tissue function and can provide a highly informative view of the state of a given ET in combination with the contractile behavior, so the combination of recording functions is very beneficial. Since the tissue is in the vicinity of the electrode surface, using the post to stimulate the tissue can further reduce the voltage required to stimulate the tissue. This can reduce the possibility of generating toxic by-products resulting from other more remote stimulation methods. Summary of the Invention

[0008] The present invention relates to a method and system for generating three-dimensional (3D) engineered tissues (such as engineered heart tissue (EHT), engineered skeletal muscle tissue (EMT), and other types of engineered tissues such as excitable or contractile cells), and a method and system for electrically stimulating and measuring the contractility and various physiological properties of ET. An ET assembly is provided that includes an ET lid to which a plurality of pairs of post assemblies are coupled. A casting assembly is provided that includes the ET assembly and a casting plate. A stimulation lid and plate are provided.

[0009] One embodiment relates to an engineered tissue (ET) assembly, the engineered tissue assembly including an ET mounting lid, at least one flexible post assembly, and a selectable rigid post assembly. The ET mounting lid includes a plate, the plate including a first side, a second side, and a plurality of through holes. At least one rigid post assembly includes a first body, the first body including a plurality of rigid posts. At least one flexible post assembly includes a second body, the second body including a plurality of flexible posts. The plurality of rigid posts and flexible posts are arranged to include post pairs of rigid posts and flexible posts. The post pairs are received within corresponding through holes of the ET mounting lid or are located on the sides of the through holes.

[0010] In another embodiment, the ET assembly may be coupled to a casting plate that includes a plurality of casting holes. The post pairs are received within corresponding through holes of the plurality of through holes of the ET mounting lid and corresponding casting holes of the plurality of casting holes of the casting plate.

[0011] In another embodiment, a stimulation plate for stimulating a tissue construct is provided. The stimulation plate includes a body that includes a plurality of bottomless holes having an upper surface and a lower surface. The bottomless holes of the body are configured to accommodate the tissue construct on the upper surface. The stimulation plate further includes a back plate connected to the lower surface of the body. The back plate includes electrode pairs, each electrode pair corresponding to one of the plurality of bottomless holes and configured to be in electrical communication with the tissue construct within the corresponding bottomless hole.

[0012] Another embodiment relates to a stimulation lid that includes a first substrate and a second substrate. The first substrate includes a plurality of holes arranged in pairs. The second substrate is connected to the first substrate and includes a plurality of sockets, each socket including two pogo pins. The stimulation lid further includes a plurality of electrode rods arranged in pairs. Each of the plurality of rods extends through a corresponding one of the plurality of holes in the first substrate. The rod pairs are removably connected to the two pogo pins of each socket. The first substrate is configured to be connected to the surface of a casting plate, the casting plate including a plurality of holes having tissue constructs such that the distal ends of the electrode rod pairs are configured to extend into corresponding casting holes of a tissue culture microplate.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0014] FIG. 1A is a top isometric view of an ET mounting lid according to an embodiment. FIG. 1B is a bottom isometric view of the ET mounting lid of FIG. 1A. FIG. 1C is a top view of the ET mounting lid of FIG. 1A. FIG. 1D is an exploded top view of a part of the ET mounting lid shown in FIG. 1C. The ET mounting lid is configured to be used in combination with a casting plate (or microplate) for growing engineering tissue.

[0015] The ET mounting cover 100 includes a lattice structure formed of one or more rigid plates 102. A skirt portion 103 extends from the second side 102b so as to surround the rigid plate 102, which provides an area for a human or a robot operator to grip the EMT mounting cover 100, and further provides an area for placing barcodes or other important information for identifying a specific device. The ET mounting cover 100 has an array of through-holes 104 extending from the first side 102a of the plate 102 to the second side 102b of the plate 102. In the array of through-holes 104 in FIGS. 1A to 1C, an array of 4 columns and 6 rows is shown, however, any array size can be used, and an arrangement other than the array can be used.

[0016] The size and shape of each through-hole 104 may be different between the first side 102a and the second side 102b of the ET mounting cover 100. In one embodiment, the walls at the through-hole 104 are inclined at a certain angle between the first side 102a and the second side 102b. In another embodiment, the walls at the through-hole 104 extend perpendicularly between the first side 102a and the second side 102b.

[0017] The through-hole 104 is configured to overlap with the holes of the microplate such as the casting plate in FIG. 4A, as described below. Thereby, the arrangement of the through-holes 104 of the ET mounting cover 100 corresponds to the arrangement of the casting holes of the casting plate, so that at least a part of the through-holes 104 overlaps with a part of the casting holes of the casting plate. The size and shape of the through-holes 104 of the ET mounting cover may be any shape or size that enables the post to extend into the casting holes of the casting plate, as described below.

[0018] The ET mounting cover 100 includes connecting members 112 on the opposing sides of each row. The connecting members 112 of the ET mounting cover 100 are located outside the through-opening 104. The connecting members 112 are configured to join with the connecting elements of the post assembly of FIGS. 2A and / or 2B, as described below. In one embodiment, referring to FIGS. 1C and 1D, the connecting member 112a can have a different size and / or shape than the connecting member 112b. In the illustrated embodiment, the connecting members 112 of multiple rows are receiving members such as slots or openings, which are configured to receive the connecting elements of the post assembly such as pins. In another embodiment, the row 112 is a pin and the post assembly has a receiving member.

[0019] The ET mounting cover 100 has a plurality of rigid strips 110 that extend between the through-openings 104 of each row and are located at the opposing ends of the ET mounting cover 100. The flat rigid strips 110 provide rigidity to the ET mounting cover 100 and contribute to aligning and fixing various components fixed to the ET mounting cover 100 and preventing disturbance by processing. In the illustrated embodiment, each row includes four through-openings 104 separated by corresponding rigid strips 110. The rigid strips 110 are located on the first side 102a of the ET mounting cover 100, but the rigid strips 110 may be located on the second side 102b. The rigid strips 110 are integrally formed with a single rigid plate 102, but in other embodiments, the rigid strips 110 may be separable components connected to the surface of the plate 102.

[0020] Referring to FIG. 1B, the through-opening 104 located on the second side 102b of the ET mounting cover 100 includes a protruding ring 113 that extends from the second side of the ET mounting cover 100. The protruding ring 113 provides an alignment feature when connecting the ET mounting cover 100 to a multi-well tissue culture plate (including a casting plate), and will be described in more detail with reference to FIGS. 5A - 5C.

[0021] The second side 102b of the ET mounting cover 100 includes a plurality of pins 114 that extend orthogonally from the second side 102b. The pins 114 are rigid and are configured to removably secure the ET mounting cover to the casting plate by fitting with a receiving portion of the ET mounting cover 100 having a casting plate, which will be described in more detail with reference to FIGS. 5A-5C below. Accordingly, the size and shape of the pins 114 correspond to the receiving portion and the gaps of the casting plate. Although four pins 114 are shown, the second side of the ET mounting cover 100 can have any number of pins, including more or fewer pins.

[0022] The ET mounting cover 100 may be manufactured from one or more molded, milled, or 3D printed parts, where injection molded parts are preferred process parts. The molding material may be a rigid thermoplastic, and in some embodiments, one or more parts may be manufactured from an elastomeric thermosetting plastic or a thermoplastic.

[0023] The through-opening 104 at the first side 102a of the ET mounting cover 100 is configured to receive a plurality of post assemblies, such as the post assemblies of FIGS. 2A, 2B, and 2C.

[0024] FIG. 2A shows a rigid post assembly 200, and FIGS. 2B and 2C show alternative flexible post assemblies 202, 204. Both the rigid post assembly 200 and the flexible post assemblies 202, 204 include a base 208, a plurality of posts 210a, 210b, 210c extending from the base 208, and a plurality of connecting elements 212a, 212b, which are pins in the illustrated embodiment. Each row of the ET mounting cover 100 is configured to receive the rigid post assembly 200 of FIG. 2A on the first side proximate the first rigid strip 110 in that row and to receive the flexible post assembly of FIG. 2B (or FIG. 2C) on the second side proximate the other rigid strip 110 in that row.

[0025] The proximal ends of posts 210a, 210b, 210c are connected to the base 208, and the distal ends of posts 210a, 210b, 210c extend away from the base 208. The corresponding plurality of posts 210a, 210b, 210c and the base 208 form a single integral part, which may be formed of an elastomeric material. The base 208 has a size that provides adequate rigidity to support the posts 210a, 210b, 210c, but the base 208 may be flexible. The distal ends 211 of the posts can incorporate additional anchor features to firmly connect any overmolded material (such as tissue) to the posts.

[0026] Generally, the posts 210a, 210b, 210c are typical posts made of a polymeric material and are configured to allow tissue to grow therebetween. The number and position of the posts 210a, 210b, 210c extending from each base 208 correspond to the number and position of the through openings 104 in the ET attachment lid 100. More specifically, the plurality of posts 210a, 210b, 210c are spaced apart from each other to correspond to the through openings 104 of the ET attachment lid 100, such that when attaching the post assemblies 200, 202, 204 to the ET attachment lid 100, the posts 210a, 210b, 210c extend into the through openings 104 of the ET attachment lid 100 as optimally shown in FIG. 3E.

[0027] Between adjacent posts 210a, 210b, 210c of post assemblies 200, 202, 204 are connecting elements, and these connecting elements are pins 212a, 212b extending from base 208. Pins 212a, 212b are configured to mechanically connect post members 200, 202, 204 to ET mounting cover 100. In particular, pins 212a, 212b are arranged and sized to be inserted into corresponding slots in each row located on the first side of ET mounting cover 100, thereby fixing the post assembly to ET mounting cover 100. Pin 212a of rigid post assembly 200 may be different from pin 212b of flexible post assemblies 202, 204 to assist the assembler. Thus, as shown in FIG. 1D, connecting member 112a of ET mounting cover 100 can have a first dimension and is used to accommodate rigid post assembly 200, and connecting member 112b of ET mounting cover 100 has a different second dimension.

[0028] Pins 212a, 212b can include locking features such as lips, for example, which assist in fixing post assemblies 200, 202, 204 to ET mounting cover 100 by being pressed against the second side 102b of ET mounting cover 100. The above-described connection mechanism between ET mounting cover 100 and post assemblies 200, 202, 204 includes a male member and a female member. Although ET mounting cover 100 is shown as having the female member and post assemblies 200, 202, 204 are shown as having the male member, it is understood that ET mounting cover 100 may have the male member and post assemblies 200, 202, 204 may have the female member. Also, any other mechanical connection between the ET mounting cover and the post assembly can be used.

[0029] The differences between the flexible post assemblies 202, 204 and the rigid post assembly 200 are as follows. That is, the flexible post assemblies 202, 204 include a plurality of flexible posts 210b, 210c that are flexible with respect to the tissue construct and the forces associated with its electrical stimulation, and the rigid post assembly 200 includes a plurality of rigid posts 210a that are rigid with respect to the tissue construct and the forces associated with its electrical stimulation.

[0030] The flexible post 210b in FIG. 2B has a large diameter (or size) at its proximal end but a small diameter along most of its length. This allows adjustment of the post bending stiffness for a given application without changing the tip shape. The flexible post 210b may be configured to bend in all planes (including vertical planes). The flexible post 210c in FIG. 2C has a flat rectangular shape along most of its length from the proximal end and gradually becomes smaller in a narrow region towards its distal end. The flexible post 210c in FIG. 2C is configured to maintain rigidity in the lateral plane while bending in a single plane in response to one or more forces associated with the grown tissue construct and its electrical stimulation.

[0031] The rigid posts 210a are several orders of magnitude stiffer than the flexible posts 210b, 210c, as is well known in the art. The rigid posts 210a are manufactured with an appropriate shape and material such that they retain their rigidity when exposed to tissue forces (e.g., tissue growth and forces during the testing period), and the flexible posts 210b, 210c are manufactured with an appropriate shape and material such that they will bend in at least one or more directions when exposed to tissue forces. For example, the rigid posts 210a may be manufactured from a rigid thermoplastic, such as, but not limited to, polystyrene or polycarbonate.

[0032] The flexible posts 210b, 210c can include magnets at their distal ends 211. The magnets may be embedded within the elastomeric material or coupled to the outer surface. The flexible posts 210b, 210c may be manufactured from an elastomeric material. Both the rigid post and the flexible post can include caps at their distal ends to securely connect any overmolded biological tissue to the post. The flexible post is used as a transducer of the forces generated by the tissue. By knowing the mechanical properties of the post and the curvature of the post loaded by the tissue, the forces generated by the tissue at any given time can be estimated, which can generate significant information regarding the health and function of the tissue.

[0033] Sniadecki's U.S. Publication No. 2019 / 0029549 describes another rigid post, flexible post, and tissue growth details, the full text of which is incorporated herein for all purposes. The application further disclosed an apparatus using a rigid / flexible post pair, but the rigid posts described herein are made of a high-rigidity thermoplastic and attached to a high-rigidity thermoplastic, so higher rigidity than the present disclosure can be achieved. The above disclosure uses a rigid insert embedded in another flexible post, but the rigid insert projects from the base of the flexible material, so that the post can still be displaced by the load generated by the ET due to the curvature of the unsupported elastomeric base protruding therefrom. The rigid post assembly 200 in our apparatus is firmly fixed to the rigid ET mounting lid 100, whereby they do not displace due to ET contraction.

[0034] In one embodiment, using various conventional cell culture and tissue generation techniques known in the art, a post pair including one rigid post and one flexible post is configured to hold an ET construct therebetween. In other embodiments, the post pair includes two flexible posts, which are configured to hold an ET construct therebetween.

[0035] FIG. 3A is a top isometric view of a partially assembled ET assembly 300 according to an embodiment, the assembly including the ET mounting lid 100 of FIG. 1A and post assemblies 200 and 202. For simplicity and ease of explanation, only a pair of rigid post assemblies 200 and a flexible post assembly 202 are connected in a single row on the first side 102a of the ET mounting lid 100.

[0036] FIG. 3B shows an exploded view of the partially assembled ET assembly 300 of FIG. 3A with the pair of rigid post assemblies 200 and the flexible post assembly 202 not connected to the ET mounting lid 100. FIG. 3C is a plan view of the partially assembled ET assembly 300 of FIG. 3A. FIG. 3C shows the cross-sectional line positions of the cross-sections shown in FIGS. 3D and 3E. Only a single rigid post assembly and a single flexible post assembly are attached to a single row of the ET mounting lid, but corresponding rigid and flexible post assemblies are attached to all rows to form a fully assembled ET assembly as shown in FIG. 3F.

[0037] As best shown in FIG. 3D, the connecting member 112a of the ET mounting lid 100 houses the pins 212a, 212b of the post assemblies 200, 202 and fixes the post assemblies to the ET mounting lid by a press-fit structure. As best shown in FIG. 3E, each pair of posts including one rigid post 210a and one flexible post 210b extends into corresponding through openings on opposite sides of the through openings. The rigid post assembly 200 and the flexible post assembly 202 are spaced apart from each other when attached to the ET mounting lid 100, as best shown in FIG. 3A. The pair of posts 210a, 210b are spaced apart from each other by a specific distance with respect to their central axes when attached to the ET mounting lid 100 to form the ET assembly, and in some embodiments, the specific distance may be between 4 mm and 20 mm so as to allow a tissue construct to grow therebetween, as is commonly used in the art.

[0038] As shown in FIGS. 3D and 3E, the pins 114 of the ET mounting lid 100 are longer than the rigid and flexible posts 210a, 210b. That is, the distal ends of the pins 114 are farther from the second side 102b of the ET mounting lid 100 than the distal ends of the flexible and rigid posts 210a, 210b. Each pin 114 is used as an alignment guide for connecting the ET assembly 300 to the casting plate, as will be described with reference to FIGS. 5A - 5C below. In particular, the pins 114 are received in the receiving members of the casting plate.

[0039] FIG. 3F shows the assembled ET assembly 300, which, when fully assembled, each row includes the rigid post assembly 200 and the flexible post assembly 202 as described above, and it is prepared for connection to the casting plate 400.

[0040] In another embodiment, the rigid post assembly 200 in each post pair can be exchanged with the flexible post assembly 202 (or 204), in which case the flexible post pairs are positioned relative to each other and used to grow tissue constructs. This may be preferable in some scenarios and can improve the signal strength using rigid and flexible pairs. However, one flexible post deflects by the full length of tissue contraction, which is the opposite of two flexible posts where each flexible post deflects by half the length. In particular, using a rigid - flexible pair is advantageous (such a design can improve the signal strength of ET contractility measured when combined with a magnetometer - based contractility measurement system (e.g., disclosed by Sniakdecki et al.), which is the opposite of two flexible posts (where each post deflects by half the length) and a single flexible post deflects by the full length of tissue contraction). The two - flexible - post structure is useful when it can be suitably performed in a more flexible in - vivo environment, such as in optical - imaging - based measurement means or other types of measurements.

[0041] In another embodiment, the ET mounting lid 100 and the rigid post assembly 200 are manufactured from the same material that forms an integral part (or body), thereby improving manufacturing efficiency. And while suitable means for attaching the rigid post assembly 200 and the flexible post assemblies 202, 204 is by inserting pins 212a or 212b, the rigid post assembly 200 and the flexible post assemblies 202, 204 may be fixed to the ET mounting lid 100 using any means including adhesives, sealants, fixtures, mechanical interlocks of overmolding, etc. or any combination thereof.

[0042] FIG. 4A is a top isometric view of a casting plate 400 (or microplate) according to one embodiment. FIG. 4B is a plan view of the casting plate 400 of FIG. 4A, which shows the cross-sectional line used in the cross-sectional view of FIG. 4C. The casting plate 400 includes an array of casting holes 402, which are used to cast tissue to produce an ET adhered between a pair of rigid and flexible posts of the ET assembly 300 of FIG. 3F. The array of casting holes 402 corresponds to an array of through openings in the ET mounting lid of the ET assembly.

[0043] The casting plate 400 includes casting holes formed by a first portion 404 having an array of bottomless holes 406 and a second portion 408 having an array of recessed holes 410. The bottomless holes 406 are circular in plan view, and the recessed holes 410 are rectangular in plan view. In the illustrated embodiment, the first portion 404 and the second portion 408 are separate members. However, in another embodiment, the bottomless holes 406 and the recessed holes 410 may be formed in a single member, as shown in FIG. 4D.

[0044] As optimally shown in FIG. 4C, the side walls in the recessed holes 410 are inclined at a certain angle, so that the area of the opening in the recessed holes 410 close to the bottomless holes 406 is larger than the area at the base of the recessed holes 410. Also, the corners at the bottom of the recessed holes 410 are curved or chamfered. The inclined side walls and / or the curved or chamfered corners of the recessed holes 410 can contribute to allowing the removal of the as-cast structure and / or suppressing the formation of air bubbles during the structure casting process. In some embodiments, the casting plate 400 is an injection-molded part made of polypropylene, thereby further suppressing the adhesion of the structure to the hole walls. The recessed holes are rectangular in this embodiment, but they can be of other shapes, such as oval, dog-bone shape, or any other suitable shape, depending on the situation.

[0045] FIG. 4C is a cross-sectional view showing an alternative embodiment of the casting plate 400a. The casting plate 400a is the same as the casting plate 400 of FIGS. 4A - 4C, except that the bottomless holes 406 and the recessed holes 410 of the casting holes are formed throughout the body 407.

[0046] The upper surface of the casting plate 400 (or 400a) is configured to abut against or be removably connected to the ET mounting assembly 300 to form a casting assembly 500 as shown in FIGS. 5A - 5C. FIGS. 3A - 3E show a partially formed ET mounting assembly to avoid confusion with the details of the present invention. However, it should be understood that the fully assembled mounting assembly of FIG. 3F is generally connected to the casting plate 400 to form the casting assembly 500.

[0047] FIG. 5A is a plan view of the casting assembly 500 including the ET assembly 300 of FIG. 3A, and the ET assembly is removably connected to the casting plate 400 of FIG. 4A. FIG. 5B is a cross-sectional view of the casting assembly 500 of FIG. 5A along the cross-section line shown in FIG. 5A. FIG. 5C is an enlarged view of a part of the casting assembly 500 of FIG. 5B.

[0048] As optimally shown in FIG. 5B, the second side 102b of the ET assembly 300 is removably coupled to the upper surface of the casting plate 400, whereby the outer surface portion of the ET assembly 300 is disposed within the recess of the outer surface portion of the casting plate 400.

[0049] The casting holes (i.e., the bottomless hole 406 and the recessed hole 410) of the casting plate 400 are aligned with the through openings of the ET assembly 300, whereby the pair of rigid posts 210a and the flexible posts 210b are received within the casting holes and extend through the bottomless hole 406 and into the recessed hole 410. Only a pair of rigid and flexible posts are shown, but it will be understood that each casting hole includes a corresponding pair of rigid and flexible posts.

[0050] As described above, the protruding ring 113 of the ET mounting lid 100 provides an alignment feature for connecting the ET mounting lid 100 to the casting plate 400. In particular, the protruding ring 113 can achieve more consistent alignment by aligning the ET mounting lid with the casting holes of the casting plate such that the posts of the ET assembly are aligned with the central regions of the casting holes, enabling automatic placement and resulting in a more consistent form from tissue to tissue. By improving tissue alignment in this way, more reproducible magnetic sensing, imaging, and any other processes requiring consistent placement can be provided. These protruding rings 113 enable the ET assembly 300 to be placed more accurately and consistently with the appropriate shape factor not only on the casting plate 400 but also on other tissue culture plates, so that the tissues fixed to the ET assembly 300 can be aligned more consistently with any sensor for evaluating these tissues, and more consistent and reproducible data can be obtained.

[0051] Although not shown in the cross-sectional views of FIGS. 5B and 5C, pin 114 provides alignment features that assist in aligning ET assembly 300 when it is placed on casting plate 400. Since pin 114 is longer than rigid posts 210a and flexible posts 210b, it can protect the ends of rigid posts 210a and flexible posts 210b or any tissue formed therebetween from striking casting plate 400, which is because the ends of pin 114 are received in corresponding openings in casting plate 400 before the ends of rigid posts 210a and flexible posts 210b are received in blind holes 406 of casting plate 400.

[0052] In operation, tissue solution is provided through through-opening 104 of ET assembly 300, which is received within recess 410 where the tissue is cast between post pairs within each casting. After the tissue solution has sufficiently gelled, ET assembly 300 can be removed from the casting plate by removing it from the casting plate. ET assembly 300 can then be placed on any other suitable tissue culture microplate for long-term culturing of the tissue fixed thereon. There may be some situations where the tissue must gel within recess 410 for an extended period of time, and in such situations, blind holes 406 can provide a reservoir for additional nutrient medium to maintain the tissue during this extended period.

[0053] Each member of casting assembly 500 allows for improving manufacturability, including automated processing of each member that improves the manufacturability of manufacturing tissue casts that include muscle tissue, nerve tissue, or combinations thereof.

[0054] Electrical stimulation can be used to induce more maturation or physiological development of ET. After growing in the corresponding recess 410 (or bottomless hole 406 in some cases) during the casting process and being removed from the casting plate 400, the cells and tissues attached to the ET assembly 300 can be exposed to electrical stimulation. FIGS. 6A and 6B show the opposite side of a stimulation plate 600 (or electrical plate assembly) for applying electrical stimulation to the cells and tissues of a 3D ET (e.g., engineered heart tissue (EHT)). FIG. 6A shows a top isometric view of the stimulation plate 600, FIG. 6B shows a bottom isometric view, and FIG. 6C shows a cross-section of the stimulation plate 600.

[0055] The stimulation plate 600 includes a backplate 604 and a body 605, the body including a plurality of bottomless holes 606 that correspond to the through openings of the ET assembly 300. The backplate 604 is a rigid backplate that is connected to the body 605 by, for example, any suitable adhesive or bonding material. In the illustrated embodiment, the backplate 604 includes a printed circuit board (PCB) having conductive electrodes 610a, 610b (FIG. 7B), such as gold, electrically connected to connectors on a first side. The conductive electrodes 610a, 610b on the first side of the body 605 are aligned with the bottomless holes 606 of the body 605. The PCB can include viewing windows 612a and 612b, thereby enabling observation of the contents of the bottomless holes 606 from the lower surface of the stimulation plate 600. The viewing windows 612a, 612b and the conductive electrodes 610a, 610b are shown with different sizes and shapes, but they may all be of the same size and shape, or of other sizes and shapes different from those shown.

[0056] Figure 6C shows a cross-section of the stimulation plate 600. The plurality of bottomless holes 606 in the main body 605 are configured to accommodate tissue constructs. The cells or tissue constructs are electrically communicated with the conductive electrodes 610a, 610b and are arranged to be capacitively coupled, for example. Electrical stimulation is used to induce an electrophysiological response in the cell culture or to characterize the response to its drug compounds.

[0057] The conductive electrodes 610a and 610b are configured to be interfaced with a stimulator by a general method in this field, such as contact pads and connectors in a PCB, various traces and via holes, and the provision of electrical signals by an external power source. The back plate 604 can further include a transparent film, which is connected to the first side 602a of the PCB by, for example, any suitable adhesive or bonding material to seal the bottomless holes of the stimulation plate.

[0058] Figures 7A and 7B show the PCB of Figures 6A and 6B in more detail. As shown in Figure 7B, the bottom side of the PCB has a ground plane and a connector, but in some embodiments, the connector may be located on the upper side. The connector may be a flexible connector for connecting with the connector of the stimulator. In other embodiments, instead of the connector, the exposed contact pads on the second side 6B are used to connect the conductive electrodes 610a and 610b to the stimulator to provide sufficient current to penetrate each of the corresponding rows of holes in the stimulation plate.

[0059] FIG. 8A shows an alternative backplate for the stimulation plate 600. FIG. 8A is a plan view of the first surface 802a of the backplate 800 of the stimulation plate, and FIG. 8B is a plan view of the second surface 802b of the backplate 800. In this embodiment, the stimulation plate 800 includes a transparent plastic film 802, but in other embodiments, the film may be made of a translucent or opaque film such as polyimide. The first surface 802a is composed of a transparent plastic film 802 and a conductive electrode 803 arranged in pairs. The first surface 802a is configured to be directly adhered to the main body 605 by any suitable adhesive or bonding material such that the conductive electrode 803 is aligned with the bottomless hole 606 of the main body 605.

[0060] The second surface 802b of the backplate 800 includes electrical contacts 805, which are connected to the conductive electrodes 803 of the first surface 802a via vias and traces known in the art. The transparent film is used for sealing the tissue construct, and the tissue construct may be provided in the bottomless hole 606 of the main body 605. Each bottomless hole 606 of the main body 605 may be electrically insulated, or a plurality of holes (e.g., a row of holes) may be electrically connected via traces. The conductive electrode 803 may have a geometric shape optimized for a given application, such as the shown semi-circular pattern, and serpentine and fractal geometric shapes may also be used. The conductive material may be a metal, ceramics, polymer material, conductive paste, or a mixture or layer thereof. Some examples of these materials include gold, platinum, graphite, carbon nanotubes, PEDOT, PEDOT PSS, indium tin oxide, and sputtered iridium oxide, which can improve the charge injection ability of the electrode.

[0061] To perform the test, the 3D tissue constructs formed on the paired rigid and flexible posts in the ET assembly 300 are inserted into each bottomless hole 606 of the main body 605 of the stimulation plate 600. Stimulating the tissue culture from the bottom of the tissue culture plate provides an improved test method that is superior to the prior art, which can be efficiently manufactured using industrial standard processes and is easy to replace.

[0062] Another embodiment provides a direct substrate-based stimulation plate that can be used in place of the backplate. The size and / or layout of the stimulation electrodes in the backplate may be configured in any form, which is designed to optimize the electrical stimulation of various types, sizes, shapes, or tissue components. In yet another embodiment, the electrode pattern on the backplate can have a direct substrate-based electrical stimulation design that covers a large area of the substrate with a conductive material having a very low electrical impedance, such as PEDOT:PSS.

[0063] Conventional electrical ET stimulation devices are stimulation caps that use rod electrodes that project downward from the top of the tissue culture microplate into the substrate bath of each well to stimulate the tissue culture therein. FIGS. 9A-9C illustrate an improvement to the electrical stimulation cap 900, which uses a rod electrode 904 that projects downward into the microplate. The stimulation cap 900 applies electrical stimulation to the cells and tissue constructs fixed to the ET element 300. FIG. 9A shows an isometric exploded view of the various components of the stimulation cap 900, and FIG. 9C shows a bottom view of the fully assembled stimulation cap 900.

[0064] Referring to FIG. 9A, the stimulation cap 900 includes a printed circuit board (PCB) 902 configured to be coupled to a connector of an external electrical stimulator or to incorporate an on-board electrical stimulation module.

[0065] The stimulation lid 900 further includes a plurality of removable rod electrodes 904, such as graphite rod electrodes or any other suitable material, which are arranged in pairs and electrically connected to the PCB 902. In one embodiment, the diameter of the rod electrode 904 is 3 / 16 inch. However, in other embodiments, electrodes of smaller or larger diameter or non-rod-shaped electrodes may be used.

[0066] The first end of the electrode 904 is connected to the PCB 902 by contacting and pressing against the pogo pin 912 on the PCB 902. FIG. 9B shows a partial enlarged view of a pair of pogo pins 912 located within the corresponding socket 914 of the PCB 902 for accommodating the electrode 904. In particular, the first end of the electrode 904 is pressed against the pogo pin 912 housed within the socket 914 and biased by a spring to provide an electrical connection to the electrical components, such as, for example, the contacts, pads, vias, and traces of the PCB 902 commonly used in this field. The pogo pin 912 attached to the PCB 902 is frictionally fitted into the socket 914 to ensure electrical connection with the rod electrode 904. The rod electrode 904 is removable within the socket 914 of the PCB 902. Thus, the rod electrode 904 can be replaced individually as needed, thereby improving the commercial usability of the stimulation lid 900 in high-throughput electrical stimulation research.

[0067] The second ends of the pair of rod electrodes are arranged and configured to be inserted into the holes of the microplate to stimulate the tissue construct. The stimulation lid 900 further includes an aligner 906 that is mechanically connected to the PCB 902. The aligner 906 includes a through-opening that is arranged to correspond to or overlap with the holes of the microplate, such that the second ends of each pair of rod electrodes extend into the corresponding microplate holes. In operation, the stimulator provides a signal to charge the rods, and the signal stimulates the cells and tissue cultures within the holes of the microplate.

[0068] A gasket 908 made of an elastomeric material is provided between the PCB 902 and the aligner 906. The gasket provides a seal to ensure the sterility of the tissue culture within the microplate and below the stimulation lid 900 when connecting the stimulation lid to the microplate. The gap between the PCB and the aligner can be further sealed with a curable polymer material.

[0069] FIG. 10A shows the stimulation lid 900 of FIG. 9C attached to the ET assembly 300 with the lid partially assembled and the assembly positioned at the top of a microplate device suitable for tissue culture. The aligner 906 includes a peripheral lip that mates with a recess surrounding the perimeter of the microplate device. FIG. 10B shows the stimulation lid 900 of FIG. 9C fully attached to the ET assembly 300, where the lip of the aligner 906 is attached to the recess of the ET assembly 300. As shown in one microplate well, the second end of the rod electrode extends through the microplate well toward the tissue construct. The cells or tissue construct are in electrical communication with the rod electrode and are capacitively coupled, for example. Electrical stimulation is used to induce an electrophysiological response in the cell culture or to characterize its response to a drug compound.

[0070] FIG. 11 shows a post 210d according to another embodiment. The post is the same as the post 210a described above, however, one or more layers of conductive material 230 are applied to the post 210d, for example, a first layer of gold and a second layer of conductive polymer. The conductive layer can extend to the base 208 to form an electrical contact pad for interfacing with external equipment. Only the rigid post is shown, but the flexible post may be coated with a layer of conductive material.

[0071] The post 210a coated with 230 may be used to transmit an electrical signal to the tissue or record an electrical signal from the tissue in order to manipulate and evaluate the behavior of the tissue. The portion of the conductive coating layer 230 can be further coated with an insulating layer 232, thereby restricting the conductive regions exposed to the tissue construct. By performing additional patterning of the conductive layer 230, multiple traces can be formed that can record or stimulate electrical activity ET independently from multiple dots.

[0072] One or more conductive layers of the post (more specifically, a flexible post) or a trace of such a conductive layer can be formed of a flexible electrode material. Examples of such flexible electrode arrays that can be formed on the post are disclosed on the website of the commercial company BMSEED (www.bmseed.com), which website is hereby incorporated by reference. Also, such a conductive layer or a trace thereof may operate with an external system for measuring the electrophysiology of ET (with or without electrical stimulation) and may be configured to operate alone or in conjunction with the measurement of the contractility of ET.

[0073] The consumable device of the present invention is uniquely designed to reduce variability in the ET casting process. Current tissue casting means are essentially academic. These means are technically strong and have extensive expertise, but it is difficult to transfer this technology to other users in a reproducible manner. The ET assembly and casting assembly of the present invention enable the production of tissues with well-matching shapes, and these tissues are aligned with the center of each casting hole. This enables the robust formation of 3D ETs, and these 3D ETs completely wrap themselves around each post and reduce the variability associated with misalignment during the tissue casting process. The unique design of the grooves in the casting holes enables successful transfer from the casting holes that enable their gelation to tissue culture plates containing their respective growth substrates. This is of absolute importance because the newly formed tissue is fragile and any minor adhesion to the casting substrate can cause a fatal obstacle to the cast ET. Also, the design of the pins extending from the grid (or ET plate) contributes to handling the tissue when the grid is transferred to a new culture plate for substrate exchange, drug testing, imaging purposes, etc. If these pins are not in the appropriate position, it is difficult to transfer the grid without accidentally colliding with the fragile tissue and potentially destroying the cultured cells. All of these advantages are apparent upon viewing the disclosure and drawings provided herein.

[0074] Various embodiments of the present invention provide methods and systems for realizing high-throughput tissue growth in both contractility measurements by optical imaging and contractility measurements by magnetic sensors. In particular, when used with the compatible casting plates described herein, the ET assembly described herein has significant advantages in cardiac and other ET contractility measurements. Also, a two-post multi-well ET system assembly having flexible magnetic posts can be made compatible with or made part of a contractile force measurement system using the magnetic sensors disclosed in Sniadecki's US Publication No. 2019 / 0029549.

[0075] In addition, various features of the present invention improve the manufacturability, reproducibility, and utility of consumable devices for casting and generating ET constructs, and achieve high-throughput measurements that are cost-effective for the contraction forces of various ET constructs. Similarly, various features of the stimulation plates and stimulation caps of the present invention have advantages in electrically stimulating various cell or tissue constructs in a high-throughput manner, while simultaneously improving the manufacturability, reproducibility, and utility of such consumable stimulation devices.

[0076] Also, the combination of the stimulation cap and the compatible ET assembly provides an advantageous means for monitoring or measuring the contraction force of tissue constructs under simultaneous or non-simultaneous electrical stimulation.

[0077] As described above, an exemplary tissue construct growing within the well may be engineered heart tissue (EHT), which is formed and suspended between two posts. These 3D heart tissues can be generated by cardiomyocytes cast with the casting plates of the present disclosure and can be constructed with the two-post ET assemblies of the present disclosure.

[0078] Exemplary 3D heart tissues were made from the WTC11 cell line of cardiomyocytes using the devices of the present disclosure. The following images are diagrams showing ET assemblies with fixed tissues.

[0079] [Photo 1] TIFF0007702420000001.tif126120

[0080] The following photographic images show enlarged images of tissues fixed to two posts of the flexible and rigid posts 200, 202, or 204.

[0081] [Photo 2] TIFF0007702420000002.tif130141

[0082] The various embodiments described above can be combined to provide other embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification and / or listed in the application data table are hereby incorporated by reference in their entirety. If necessary, each aspect of the embodiments can be modified to adopt concepts from various patents, applications, and disclosures to provide other embodiments.

[0083] Based on the above detailed description, these and other changes can be made to the embodiments. In general, in the appended claims, the terms used should not be construed as limiting the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to cover all possible embodiments and the full scope of all equivalents permitted by these claims. Accordingly, the claims are not to be limited to the present disclosure.

[0084] This application claims the benefit of priority of U.S. Provisional Application No. 62 / 981,446, filed on February 25, 2020, which is hereby incorporated by reference in its entirety.

Claims

1. An engineering tissue (ET) assembly comprising: an ET mounting lid including a plate having a first side, a second side, and a plurality of through holes; a first post assembly including a first body including a plurality of rigid polymer first posts; a second post assembly including a second body including a plurality of flexible polymer second posts; wherein the plurality of rigid polymer first posts and the plurality of flexible polymer second posts are provided in post pairs, each post pair including one of the plurality of rigid polymer first posts and one of the plurality of flexible polymer second posts; the first post assembly and the second post assembly are aligned with the plurality of through holes by a plurality of connections provided in the ET mounting lid; the post pairs are received within corresponding through holes of the ET mounting lid, and for each through hole, the rigid polymer first post is disposed on a first side of the through hole and the flexible polymer second post is disposed on a second side of the through hole opposite the first side, and distal ends of the rigid polymer first post and the flexible polymer second post are spaced apart from each other by a distance such that a tissue construct can be accommodated, an engineering tissue (ET) assembly characterized by this.

2. The ET mounting lid includes a plurality of rigid strips extending between the plurality of through holes, and the first post assembly and the second post assembly are aligned with the plurality of rigid strips, the ET assembly according to claim 1, characterized by this.

3. Including a plurality of pins, the first post assembly and the second post assembly are connected to the first side of the plate, and distal ends of the plurality of pins extend further from the second side of the plate than distal ends of the plurality of rigid polymer first posts and the plurality of flexible polymer second posts, the ET assembly according to claim 1, characterized by this.

4. The plurality of connections include a first plurality of mechanical connections or adhesive connections and a second plurality of mechanical connections or adhesive connections, the first post assembly is connected to the ET mounting lid by the first plurality of mechanical connections or adhesive connections, and the second post assembly is connected to the ET mounting lid by the second plurality of mechanical connections or adhesive connections, the ET assembly according to claim 1, characterized by this.

5. The first post assembly is aligned with the second post assembly by the first plurality of mechanical or adhesive connections and the second plurality of mechanical or adhesive connections, the ET assembly according to claim 4, characterized in that.

6. The first post assembly and the ET mounting cover are manufactured as a single integral part, the ET assembly according to claim 1, characterized in that.

7. At least one of the plurality of rigid polymer first posts and the plurality of flexible polymer second posts is at least partially coated with at least one layer of conductive material, the ET assembly according to claim 1, characterized in that.

8. The at least one layer of the conductive material is partially coated with an electrical insulator, the ET assembly according to claim 7, characterized in that.

9. The periphery of the ET mounting cover includes a skirt portion extending beyond the second side of the plate, the ET assembly according to claim 1, characterized in that.

10. A casting assembly, comprising a casting plate including a plurality of casting holes and an engineering tissue (ET) assembly, The ET assembly is An ET mounting cover including a plate having a first side, a second side, and a plurality of through holes, A first post assembly including a first body including a plurality of rigid polymer first posts, A second post assembly including a second body including a plurality of flexible polymer second posts, The first post assembly and the second post assembly are aligned with the plurality of through holes by a plurality of connections provided in the ET mounting cover, The plurality of rigid polymer first posts and the plurality of flexible polymer second posts are provided in post pairs, each post pair including one of the plurality of rigid polymer first posts and one of the plurality of flexible polymer second posts, The post pair is received within a corresponding through-hole of the ET mounting cover, and for each through-hole, the rigid polymer first post is disposed on a first side of the through-hole and the flexible polymer second post is disposed on a second side of the through-hole opposite the first side, and distal ends of the rigid polymer first post and the flexible polymer second post are spaced apart from each other by a distance such as to accommodate a tissue construct, and each of the post pairs is received within a corresponding casting hole of the plurality of casting holes of the casting plate. A casting assembly characterized by this.

11. Each of the plurality of casting holes includes a bottomless hole disposed above the recessed hole, and ends of the post pair are received within a corresponding recessed hole. The casting assembly according to claim 10, characterized by this.

12. The size of each of the bottomless holes is larger than the size of each of the recessed holes. The casting assembly according to claim 11, characterized by this.

13. For each casting hole, the recessed hole is a container for a tissue solution. The casting assembly according to claim 11, characterized by this.

14. For each casting hole, the wall forming the recessed hole is inclined, and corners formed on the wall and bottom surface of the recessed hole are chamfered or curved. The casting assembly according to claim 11, characterized by this.

15. The ET mounting cover includes a plurality of rigid strips extending between the plurality of through-holes, and the first post assembly and the second post assembly are aligned with the plurality of rigid strips. The casting assembly according to claim 10, characterized by this.

16. The first post assembly is aligned with the second post assembly by a plurality of connections. The casting assembly according to claim 10, characterized by this.

17. An assembly including an engineering tissue (ET) assembly and a stimulation plate for stimulating a plurality of tissue constructs, The ET assembly is the ET assembly according to claim 1, The stimulation plate includes a body and a back plate, The body includes a plurality of bottomless holes having an upper surface and a lower surface, and each of the plurality of bottomless holes is configured to accommodate one of the plurality of tissue constructs on the upper surface. The backplate is connected to the lower surface of the body, the backplate includes electrode pairs, each electrode pair corresponds to one of the plurality of bottomless holes, and is configured to be in electrical communication with the tissue construct housed in the corresponding bottomless hole. An assembly characterized by this.

18. The assembly according to claim 17, wherein the backplate is a PCB, and the electrode pairs are conductive pad electrodes formed on the surface of the PCB.

19. The ET assembly is connected to the stimulation plate such that the post pairs extend into the corresponding bottomless holes, each post pair having one of the plurality of tissue constructs therebetween, and each of the electrode pairs is configured to be in electrical communication with the tissue construct housed in the corresponding bottomless hole. The assembly according to claim 17, characterized by this.

20. The assembly according to claim 17, wherein the electrode pairs are at least partially coated with one or more layers of conductive or insulating material.

Images