The present application is directed to methods and systems for generating three-dimensional (3D) engineered tissues (ETs), and methods and systems for electrical stimulation and measurement of contractility and various physiological characteristics of same.
3-Dimensional engineered tissues (referred here as ETs), or tissues thicker than one-cell layer that are manufactured or augmented by an artificial process, are of considerable interest in biomedical research for their cost, availability, and remarkable ability to mimic human physiological tissues, when compared with competing physiological models. Means fabricating, maintaining, and characterizing these ETs have accumulated much academic interest. A particularly sought-after ET system is referred to as a 2 post system, where a tissue is cast between two posts, at least one of which is flexible enough to bend under the contractions of the tissue to which it is adhered. The bending of the post can be related to the force the tissue generates, which can provide meaningful information regarding the health and function of the tissue. Attempts to commercialize this sort of device exist, but the methods resemble more something native to an academic lab than an efficient, industry-focused tool.
U.S. Publication No. 2019/0029549 by Sniadecki et al. discloses such a two-post ET system. A magnet embedded into one post of the two-post ET system of Sniadecki provides magnetic sensor-based detection of contractile forces of ETs residing in a multiwell plate. This non-optical contractile assay system enables the measurement of a force exerted by a 3D ET suspended between a magnet-embedded flexible post and a rigid post, and uses a magnetometer to detect a change in the magnetic field resulting from a deflection of the flexible post as the ET undergoes contractile actions. Other commercially available 2 post systems provide optical readouts of post bending under ET load, as described in Hensen et al. The publication Development of a Drug Screening Platform Based on Engineered Heart Tissue, Circulation Research; Jul. 9, 2010 downloadable from http://circres.ahajournals.org.
Previously disclosed ET systems, however, have limitations terms of manufacturing throughput, ease of use, and ergonomics. Fabrication of non-biological components of the system require complicated molding and demolding processes that could take several days. Such fabrication processes are often times non compatible with industry standard fabrication techniques such as compression or injection molding, or require unconventional modifications to said techniques to achieve scalability. The process for generating the 3D tissues themselves is a significantly limiting factor. In many 2 post systems, ETs are cast from a suspension of cells and extracellular matrix that gels around the posts after being deposited in a mold. The operator is required to cast the mold for the tissues themselves within the wells of a tissue culture plate out of a highly temperature-sensitive gel material, such as gelatin or agarose, which detrimentally affects reproducibility and throughput of the 3D tissue constructs. Other materials such as silicone can be used, but require laborious surface treatments to render the molds nonadherent to tissues. The handles on which theses posts that hold the tissues are mounted have no alignment features that ensure their consistent placement within the trenches, leading to highly variability in the morphologies of cast tissues. Additionally, once cast, these gel molds have trenches with vertical walls that make removal of the tissue constructs difficult due to friction between the tissue constructs and the sides of the mold.
Newly-formed tissues are delicate and any slight adherence to the casting substrate can cause catastrophic failure of the tissue cast. The components on which the two posts are mounted lack features for efficiently and reproducibly interfacing with industry standard culture devices and have limited ability to integrate into automated handling systems due to their unwieldly shapes. They may be easily and deleteriously misaligned with any sensors intended to evaluate the tissue. Slight misplacement of the device on a microplate may squish the tissue between the top surface of the microplate and the substrate on which the posts are mounted. They cannot be readily handled in groups of more than 6 at a time, and lack features by which a human or robotic operator can grip easily.
The performance of ETs can be enhanced in many ways by electrical stimulation, while of great interest, this also presents challenges. Conventional electrical stimulation devices used in cell culture typically involve graphite rods or platinum wires permanently affixed to an electrical stimulation module that fits over the top of a microplate with the electrodes protruding into the media bath of each well in the microplate. As small molecules in the media bath permeate and attach to graphite electrodes easily, the graphite electrode assembly needs to be replaced after only a single drug-screening experiment to avoid cross-contamination. However, available such graphite stimulation devices are either too costly as a single-use consumable, or quite challenging to assemble and integrate into industry standard tissue cultureware, limiting the commercial utility of such devices in high-throughput stimulation studies. While platinum electrodes lack the porosity of graphite electrodes, giving them an edge in terms of reusability, they have a higher propensity to generate cytotoxic electrolytic bi-products and require extensive cleaning after use, which negatively impacts their utility in high-throughput biological studies. Platinum wire is also quite expensive, limiting the commercial scalability of devices that employ it. Additionally, no two post system incorporates means of stimulating and recording from tissues using the posts themselves as electrodes. Since electrophysiology is another important metric of tissue function, and can be combined with contractile behavior to give a highly informative view the state of a given ET, incorporation of recording functionality would be of great benefit. Stimulation of the tissue using the posts can also reduce the voltage required to stimulate the tissue due to the tissue's proximity to the electrode surface. This reduces the potential for toxic byproduct formation that would be incurred by other more remote stimulation delivery methods.
The present application is directed to methods and systems for generating three-dimensional (3D) engineered tissues (ETs), such as engineered heart tissues (EHT), engineered skeletal muscle tissues (EMT) and other types of engineered tissues of excitable or contractile cells, and methods and systems for electrical stimulation and measurement of contractility and various physiological characteristics of same. Provided is an ET assembly comprising an ET lid with pairs of post assemblies coupled thereto. Provided is a casting assembly comprising the ET assembly and a casting plate. Provided are stimulation lids and plates.
One embodiment is directed to an engineered tissue (ET) assembly comprising an ET mounting lid, at least one flexible post assembly and an optional rigid post assembly. The ET mounting lid comprises a plate comprising a first side, a second side, a plurality of through holes. The at least one rigid post assembly comprises a first body comprising a plurality of rigid posts. The at least one flexible post assembly comprising a second body comprising a plurality of flexible posts. The plurality of rigid posts and flexible posts are arranged in pairs of posts comprising a rigid post and a flexible post. The pairs of posts are received in respective through holes of the ET mounting lid or are at sides of the through holes.
In another embodiment, the ET assembly may be coupled to a casting plate comprising a plurality of casting wells. The pairs of posts are received in respective through holes of the plurality of through holes of the ET mounting lid and respective casting wells of the plurality of casting wells of the casting plate.
In another embodiment a stimulation plate for stimulating tissue constructs is provided. The stimulation plate comprises a body comprising a plurality of bottomless wells having an upper surface and a lower surface. The bottomless wells of the body are configured to receive the tissue constructs at the upper surface. The stimulation plate further includes a backing plate coupled to the lower surface of the body. The backing plate comprising pairs of electrodes, each pair of electrodes corresponding with a well of the plurality of bottomless wells and configured to electrically communicate with the tissue constructs in the respective bottomless wells.
Another embodiment is directed to a stimulation lid comprising first and second substrates. The first substrate comprising a plurality of holes arranged in pairs. The second substrate is coupled to the first substrate and comprises a plurality of sockets, each socket including two pogo pins. The stimulation lid further includes a plurality of electrode rods arranged in pairs. Each rod of the plurality of rods extending through respective holes of the plurality of holes of the first substrate. The pairs of rods are removably coupled to the two pogo pins of each socket. The first substrate is configured to couple to a surface of a casting plate comprising a plurality of wells having tissue constructs such that distal ends of pairs of the electrode rods are configured to extend into respective casting wells of a tissue culture microplate.
The ET mounting lid 100 comprises a lattice structure formed from one or more rigid plates 102. Around rigid plates 102 extends a skirt 103 from second side 102b that provides a region for a human or robotic operator to grasp EMT mounting lid 100, as well as providing a region for placement of a barcode or other information important for identification of a specific device. The ET mounting lid 100 includes an array of through openings 104 extending from a first side 102a of the plate 102 to a second side 102b of the plate 102. The array of the through openings 104 in
The size and shape of each through opening 104 may be different at the first and second sides 102a, 102b of the ET mounting lid 100. In one embodiment, the walls at the through openings 104 slope at an angle between the first and second sides 102a, 102b. In another embodiment, the walls at the through openings 104 extend perpendicularly between the first and second sides 102a, 102b.
The through openings 104 are configured to overlap with wells of a microplate, such as the casting plate of
The ET mounting lid 100 includes coupling members 112 at opposing sides of each row. The coupling members 112 of the ET mounting 100 are located outwardly of the through openings 104. The coupling members 112 are configured to engage with coupling elements of post assemblies of
The ET mounting lid 100 includes a plurality of rigid strips 110 extending between each row of through openings 104 and at opposing ends of the ET mounting lid 100. The flat rigid strips 110 provide stiffness to the ET mounting lid 100 and help align and secure various components affixed to ET mounting lid 100 and protect against perturbations due to handling. In the illustrated embodiment, each row includes four through openings 104 separated by respective rigid strips 110. The rigid strips 110 are located at the first side 102a of the ET mounting lid 100, however, the rigid strips 110 may be located at a second side 102b. The rigid strips 110 are integrally formed in a single rigid plate 102; however, in other embodiments, the rigid strips 110 may be separable components coupled to a surface of the plate 102.
With reference to
The second side 102b of the ET mounting lid 100 includes a plurality of pegs 114 extending orthogonally from the second side 102b. The pegs 114 are rigid and configured to mate with receiving portions of the ET mounting lid 100 with a casting plate to removably secure the ET mounting lid to the casting plate as will be explained in more detail below with reference to
The ET mounting lid 100 may be made from one or more molded, milled, or 3D printed, with injection molding being the preferred process parts. The molding material may be rigid thermoplastics, and in some embodiments one or more parts may be made from elastomeric thermoset or thermoplastic.
The through openings 104 at the first side 102a of the ET mounting lid 100 are configured to receive a plurality of post assemblies, such as the post assemblies of
Proximal ends of the posts 210a, 210b, 210c are coupled to the base 208 and distal ends of the posts 210a, 210b, 210c extend away from the base 208. The respective plurality of posts 210a, 210b, 210c and base 208 form a single monolithic component, which may be formed from elastomeric material. The base 208 has dimensions to provide suitable rigidity for supporting the posts 210a, 210b, 210c; however, the base 208 may be flexible. The distal ends 211 of the posts may incorporate additional anchoring features to securely couple any over-molded material, such as tissue, to the posts.
Generally described, the posts 210a, 210b, 210c are conventional posts, made from a polymeric material, configured to allow tissue to grow therebetween. The number and positions of posts 210a, 210b, 210c extending from each base 208 correspond to the number and position of through openings 104 in the ET mounting lid 100. More particularly, the plurality of posts 210a, 210b, 210c are spaced apart from each other in a manner that corresponds to the through openings 104 of the ET mounting lid 100 such that when the post assemblies 200, 202, 204 are installed on the ET mounting lid 100, the posts 210a, 210b, 210c extend into the through openings 104 of the ET mounting lid 100 as best shown in
Between adjacent posts 210a, 210b, 210c of the post assemblies 200, 202, 204 are coupling elements, which are pins 212a, 212b extending from the base 208. The pins 212a, 212b, are configured to mechanically couple the post assemblies 200, 202, 204 to the ET mounting lid 100. In particular, the pins 212a, 212b are arranged and dimensioned to be inserted into respective slots of each row at the first side of the ET mounting lid 100 to thereby secure the post assembly to the ET mounting lid 100. The pins 212a of the rigid post assembly 200 may be different from the pins 212b of the flexible post assemblies 202, 204 to aid the assembler. Accordingly, the coupling members 112a of the ET mounting lid 100 may be of a first size, as shown in
The pins 212a, 212b may include a locking feature, such as a lip, that presses against the second side 102b of the ET mounting lid 100 to aid in securing post assemblies 200, 202, 204 to the ET mounting lid 100. The above described coupling mechanism between the ET mounting lid 100 and the post assemblies 200, 202, 204 are male and female components. Although the ET mounting lid 100 is shown as having the female component and the post assemblies 200, 202, 204 are shown as having the male component, it is to be appreciated that the ET mounting lid 100 can have the male component and the post assemblies 200, 202, 204 can have the female component. Furthermore, any other mechanical coupling between the ET mounting lid and the post assemblies may be used.
The difference between the flexible post assemblies 202, 204 and the rigid post assembly 200 is that the flexible post assemblies 202, 204 include a plurality of flexible posts 210b, 210c that are flexible to forces associated with tissue constructs and electrical stimulation of same, while the rigid post assembly 200 includes a plurality of rigid posts 210a, which are rigid to the forces associated with tissue constructs and the electrical stimulation of same.
The flexible posts 210b of
The rigid posts 210a are orders of magnitude stiffer than the flexible posts 210b, 210c as is well known in the art. The rigid posts 210a are made of suitable shapes and material such that they remain rigid when exposed to tissue forces, such as forces during tissue growth and testing, while the flexible posts 210b, 210c are made of suitable shapes and materials such that they flex in at least one or more directions when exposed to the tissue forces. For example, the rigid posts 210a may be made of rigid thermoplastics such as, but not limited to polystyrene or polycarbonate.
The flexible posts 210b, 210c may include a magnet at the distal end 211. The magnet may be embedded inside the elastomeric material or coupled to the outer surface. The flexible posts 210b, 210c may be made of an elastomeric material. Both the rigid posts and the flexible posts may include a cap at the distal end to securely couple any over-molded biological tissue to the post. The flexible posts act as a transducer for the force generated by this tissue; by knowing the mechanical properties of the post, as well as the post bends loaded by the tissue, one can estimate the force the tissue is generating at any given time, which can yield meaningful information about the health and function of the tissue.
U.S. Publication No. 2019/0029549 by Sniadecki describes further details of rigid and flexible posts and tissue growth and is incorporated herein in its entirety for all purposes. While this patent also discloses device using a rigid/flexible post pair, the rigid post described herein can achieve higher rigidity than in that disclosure due to being made of high rigidity thermoplastic and mounted to a high rigidity thermoplastic. While the aforementioned disclosure utilizes a rigid insert embedded into an otherwise flexible post, protruding from a base of flexible material, such that the post can still displace under loads sometimes generated by ETs due to flexure of the unsupported elastomeric base from which it protrudes. The rigid post assemblies 200 in our device are securely fixtured to the rigid ET mounting lid 100 such that they will not displace due to ET contraction.
In one embodiment, pairs of posts comprising one rigid post and one flexible post are configured to hold an ET construct therebetween, using various cell culture and tissue generation techniques that are generally known in the art. In other embodiments, the pairs of posts comprise two flexible posts configured to hold an ET construct therebetween.
As best shown in
As shown in
It should be noted that in another embodiment, the rigid post assembly 200 in each pair of posts may be exchanged for a flexible post assembly 202 (or 204) such that pairs of flexible posts are positioned opposite each other for growing tissue constructs. This may be preferable in some scenarios, although use of a rigid and flexible pair may improve signal strength since the one flexible post deflects the entire length of the tissue contraction, as opposed to with two flexible posts, where each flexible post deflects only half the length. In particular, although the use of a rigid—flexible pair can be advantageous as such a design improves the signal strength of measured contractility of ETs when used in conjunction with a magnetometer-based contractility measurement system (as disclosed by Sniakdecki et all) as the single flexible post deflects the entire length of the tissue contraction, as opposed to with two flexible posts where each post deflects only half the length, two flexible post configuration can be useful in optical imaging-based assay schemes or when other types of assays could be preferably carried out in a more pliable in-vivo like environment.
In another embodiment, ET mounting lid 100 and rigid post assembly 200 are made from the same material formed from a monolithic part (or body), thereby improving manufacturing efficiency. Also, while the preferred means of mounting the rigid and flexible post assemblies 200, 202, 204, is by insertion of the pins 212a or 212b, rigid and flexible post assemblies 200, 202, 204 may be affixed to the ET mounting lid 100 using any means, including adhesives, sealants, clamps, overmolded mechanical interlocks, among others, or any combination thereof.
The casting plate 400 comprises casting wells formed by a first portion 404 having an array of bottomless wells 406 and a second portion 408 having an array of recessed wells 410. The bottomless wells 406 are circular shaped in plan view, and the recessed wells 410 are rectangular shaped in plan view. In the illustrated embodiment, the first portion 404 and the second portion 408 are separated components; however, in another embodiment, the bottomless wells 406 and the recessed wells 410 may be formed in a single component, as shown in
As best shown in
The upper surface of the casting plate 400 (or 400a) is configured to abut with or removably coupled to the ET mounting assembly 300 to form a casting assembly 500 as shown in
As best shown in
The casting wells, that is, the bottomless and recessed wells 406, 410, of the casting plate 400 are aligned with the through openings of the ET assembly 300 such that the pairs of rigid and flexible posts 210a, 210b are received in the casting wells, extending through the bottomless wells 406 and into the recessed wells 410. Although only a single pair of rigid and flexible posts are shown, it is to be understood that each casting well would include a respective pair of rigid and flexible posts.
As previously mentioned, the protruding rings 113 of the ET mounting lid 100 provide an alignment feature for coupling the ET mounting lid 100 to the casting plate 400. In particular, the protruding rings 113 align the ET mounting lid to the casting wells of the casting plate such that posts of the ET assembly are aligned in a center region of the casting wells, thereby providing a more consistent alignment and allowing for automated placement, as well as more consistent morphology from tissue to tissue. This improved tissue consistency provides for more reproducible magnetic sensing, imaging, and anything else that requires consistent placement. Since these protruding rings 113 allow more accurate and consistent placement of ET assembly 300, not only on the casting plate 400, but also on any other tissue culture plate, with the proper form factor, they more consistently align tissues affixed to ET assembly 300 with any sensor used to evaluate these tissues, giving more consistent and reproducible data.
Although not shown in cross sectional view of
In operation, tissue solution is provided at the through opening 104 of the ET assembly 300, which is received in recessed wells 410, wherein tissue casts between the pairs of the posts in each casting. After the tissue solution has gelled sufficiently, it can be removed from the casting plate by removing ET assembly 300 from the casting plate. ET assembly 300 may then be placed upon any other appropriate tissue culture microplate for long-term culture of the tissues affixed thereupon. There may be some situations in which the tissue must gel for long periods of time within a recessed well 410, in which case bottomless wells 406 may provide a reservoir for additional nutrient medium to sustain the tissue over this duration.
The various components of the casting assembly 500 allow for improved manufacturability, including automated handling, of the various components improving the manufacturability of tissue casting, including muscle tissue, nervous tissue, or combinations thereof.
Electrical stimulation can be used to induce more maturation or physiological development of the ETs. Cells and tissue that have grown in the casting process in each respective recessed well 410 (and in some cases in the bottomless well 406) and remain adhered to ET assembly 300 after removal from the casting plate 400, may be exposed to electrical stimulation.
The stimulation plate 600 includes a backing plate 604 and a body 605 comprising a plurality of bottomless wells 606 that correspond to the through openings of the ET assembly 300. The backing plate 604 is a rigid backing plate that is coupled to the body 605, such as by any suitable bonding or adhesive material. In the illustrated embodiment, the backing plate 604 comprises a printed circuit board (PCB) with conductive electrodes 610a, 610b (
The conductive electrodes 610a and 610b are configured to interface with a stimulator, by conventional ways in the art, such as by contact pads or a connector and various traces and vias on the PCB, and an external power source to provide electrical signals thereto. The backing plate 604 may further include a transparent film coupled to the first side 602a of the PCB, such as by any suitable bonding or adhesive material, to thereby seal the bottomless wells of the stimulation plate.
The second surface 802b of the backing plate 800 includes electrical contacts 805 that are coupled to the conductive electrodes 803 of the first surface 802a by vias and traces as is well known in the art. The transparent film acts as a seal for the tissue constructs which may be provided within the bottomless well 606 of the body 605. Each bottomless well 606 of the body 605 may be electrically isolated or many wells, such a row of wells, may be electrically coupled together by traces. The conducting electrodes 803 may have geometries best suited for the given application, such as the shown semicircular pattern, but also serpentine and fractal geometries may be used. The conductive material may be metal, ceramic, polymeric material, conductive paste, or a mixture or layering thereof. Some examples of these materials include Gold, Platinum, Graphite, Carbon Nanotubes, PEDOT, PEDOT PSS, Indium Tin Oxide, and Sputtered Iridium Oxide, which can enhance charge injection capabilities of the electrodes.
For testing, the 3D tissue constructs formed on the pairs of the rigid and flexible posts on the ET assembly 300 are inserted into each bottomless wells 606 of the body 605 of the stimulation plate 600. Stimulating the tissue cultures from the bottom of a tissue culture plate provides an improved testing method over the prior art, in that they can be efficiently manufactured using industry standard processes and are easily replaceable.
Another embodiment provides a direct substrate-based stimulation plate that can be used to replace the backing plates. The sizes and/or layout of stimulation electrodes on the backing plate can be configured in any format that are designed to optimize electrical stimulation of various types, sizes, shapes or assemblies of tissues. In one further embodiment, the electrode pattern on the backing plate could have a direct substrate-based electrical stimulation design with electrically conductive material of very low electrical impedance, such as PEDOT:PSS, that covers a large surface area of the substrate.
Conventional electrical ET stimulation devices are stimulation lids that utilize rod electrodes protruding downward from the top of a tissue culture microplate into the media bath of each well to stimulate tissue cultures therein.
With reference to
The stimulation lid 900 further comprises a plurality of removable rod electrodes 904, such as graphite rod electrodes or any other suitable material, arranged in pairs and in electrical connection with the PCB 902. In one embodiment, the rod electrodes 904 are 3/16 inch in diameter; however, smaller or larger diameter electrodes, or non-rod-shaped electrodes may be used in other embodiments.
First ends of the electrodes 904 are coupled to the PCB 902 by contacting and pressing against pogo pins 912 on the PCB 902.
Second ends of the pair rod electrodes are arranged and configured to be inserted into wells of a microplate for stimulating the tissue constructs. The stimulation lid 900 further includes an aligner 906 mechanically coupled to the PCB 902. The aligner 906 includes through openings that are arranged to correspond with or overlap the wells of a microplate so that second ends of each pair of rod electrodes extend into a respective microplate well. In operation, the stimulator provides signals to charge the rods, the signals stimulate the cells and tissue cultures in the wells of the microplate.
Between the PCB 902 and aligner 906 is a gasket 908 made of elastomeric material. The gasket provides a seal to ensure sterility of the tissue cultures within the microplate and below the stimulation lid 900 when the stimulation lid is coupled to the microplate. Gaps between the PCB and aligner may be additionally sealed with curable polymeric material.
The post 210a coated in 230 may be used to send or record electrical signals to or from the tissue in order to manipulate and evaluate its behavior. Portions of conductive coating 230 may be further covered in an insulating layer 232, thereby limiting the conductive area exposed to the tissue construct. Additional patterning of the conductive layer 230 can be performed to form multiple tracks that can record or stimulate electrical activity ETs from multiple points independently.
The one or more electrically conductive layers, or the tracks of such conductive layer, of the posts (more particularly, flexible posts) may be formed with flexible electrode materials. One example of such flexible electrode arrays that can be formed on the posts is disclosed on the website of a commercial company, BMSEED (www.bmseed.com), which is incorporated herein by this reference. Additionally, such a conductive layer or the tracks thereof could be configured to work with an external system for measuring electrophysiology of the ETs (with electrical stimulation or without stimulation), separately or in conjunction with the measurement of contractility of the ETs.
The consumable device of the present invention is uniquely designed to reduce variability in the ET casting process. Current tissue-casting protocols are academic in nature. These protocols are highly technical and difficult to transfer the technology to other users in a way that is reproducible, even with extensive expertise. The ET assembly and the casting assembly of the present invention allows for the production of highly consistently-shaped tissues that are aligned in the center of each casting well. This allows for the robust formation of 3D ETs that completely wrap themselves around each post and reduces variability associated with misalignment during the tissue casting process. The unique design of the trenches in the casting wells also allows for the successful transfer from the casting wells, where they are allowed to gel, to their respective tissue culture plates containing medium for growth. This is absolutely critical, as newly-formed tissues are delicate and any slight adherence to the casting substrate can cause catastrophic failure of the ETs cast. Additionally, the design of the pegs extending from the lattice (or ET plate) aid in the handling of tissues when transferring the lattice to new culture plates for medium changes, drug testing, or imaging purposes, among others. Without these pegs in place, it would be difficult to transfer the lattice without accidentally bumping the fragile tissues and potentially ruining the cultured cells. All of these advantages are apparent when read and viewed with disclosures and figures provided herein.
The various embodiments described herein provide methods and systems for implementing tissue growth in a high-throughput manner for both optical imaging-based and magnetic sensor-based contractility assays. In particular, the ET assemblies described herein, when used with the compatible casting plate described herein, provide significant advantages in cardiac and other ET contractility assays. Furthermore, the two-post, multi-well ET system assembly with flexible magnetic posts can be made compatible or be a part of a magnetic sensor-based contractile force measurement system disclosed in the U.S. Publication No. 2019/0029549 by Sniadecki.
Additionally, the various features of the present invention improve the manufacturability, reproducibility and utility of the consumable devices used for casting and generating the ET constructs and enables cost-effective, high-throughput measurements of the contractile forces of various ET constructs. Likewise, the various features of the stimulation plate and stimulation lid of the present invention provide advantages in electrically stimulating in a high-throughput manner the various types of cells or tissue constructs while improving the manufacturability, reproducibility, and utility of such consumable stimulation devices.
Furthermore, the stimulation lid in combination with the compatible ET assembly offers an advantageous means for monitoring or measuring contractile forces of the tissue constructs under simultaneous or non-simultaneous electrical stimulation.
Exemplary tissue constructs grown in the wells as described above may be engineered heart tissues (EHTs), which would have formed and been suspended between the two posts. These 3D cardiac tissues can generate from cardiomyocytes cast with the casting plate of the present disclosure and may be constructed with the two-post ET assembly of the present disclosure.
Exemplary 3D cardiac tissues were generated using the devices of the present disclosure from a WTC11 cell line of cardiomyocytes. The images below show views of the ET assembly with affixed tissues.
The photo images below show magnified images of tissue affixed to two posts of flexible and rigid posts 200, 202, or 204.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
This application claims the benefit of priority to U.S. Provisional Application No. 62/981,446, filed Feb. 25, 2020, which application is hereby incorporated by reference in its entirety.
Filing Document | Filing Date | Country | Kind |
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PCT/US21/19748 | 2/25/2021 | WO |
Number | Date | Country | |
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62981446 | Feb 2020 | US |