Cellular scaffold comprising electrical circuit

Abstract

The present invention relates generally to nano-scale lines and nano-electronic devices, which in some aspects can be embedded in biological structures, such as tissues, organoids, organs, organisms, and the like. For example, one aspect generally relates to a stretchable cytoskeleton that can be connected to an electrical circuit. In some cases, a biological structure may be formed around the scaffold and deformed when formed, thereby causing the cell scaffold to be embedded in the biological structure. In certain embodiments, the scaffold may be connected to an external device, for example, to determine a property (e.g., an electrical property) of the cellular scaffold, and / or to apply a stimulus (e.g., an electrical stimulus) to the biological structure. Other aspects of the invention generally relate to methods of making or using such cell scaffolds, kits comprising such cell scaffolds, biological structures comprising such cell scaffolds, and the like.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202080046017.4, with the application date of June 23, 2020 and the invention name of “Organoids, Embryos and Other Tissues Containing Electronic Devices and Methods Thereof”.

[0002] Related Applications

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 865,648, filed by Liu et al. on June 24, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” and U.S. Provisional Patent Application Serial No. 62 / 872,031, filed by Liu et al. on July 9, 2019, entitled “Organoids Containing Electronics, and Methods Thereof,” each of which is incorporated herein by reference in its entirety. Technical Field

[0004] The present invention generally relates to nanoscale wires and nanoelectronic devices, which in some aspects can be embedded in biological structures such as tissues, organoids, organs, organisms, etc. Technical Background

[0005] Complex organs (e.g., heart and brain) rely on spatiotemporally coordinated communication of heterogeneous cells to generate whole-organ function. Therefore, understanding the development, mechanisms, and diseases of these organs requires systems-level mapping of cellular activity with high spatiotemporal resolution over their entire 3D volume over a considerable time window. Recently, tremendous progress has miniaturized state-of-the-art electrophysiological tools to the micro / nanoscale with flexible and multiplexed electronic units, which significantly reduces tissue disruption while maintaining unparalleled spatiotemporal resolution for recording and manipulation. However, most of these devices either contact the organ superficially or penetrate locally and invasively via microneedle injection. Uniform implantation and distribution of large numbers of interconnected and individually addressable sensors / stimulators throughout 3D organs while minimizing damage for long-term recordings remains a key challenge.

[0006] Notably, 3D organs are derived from 2D embryonic germ layers during organogenesis in vivo. For example, both the neural and cardiac tubes originate from 2D cell layers in the embryo and eventually develop into the brain, spinal cord, and heart through a series of cell proliferation and folding processes. In addition, this 2D to 3D transition in organogenesis in vivo has recently been reproduced in vitro for human organoid culture, where cell contraction from human mesenchymal stem cells (hMSCs) drives human induced pluripotent stem cells (hiPSCs) to fold into 3D organoids for transplantation and drug screening. SUMMARY OF THE INVENTION

[0008] The present invention generally relates to nanoscale wires and nanoelectronic devices, which in some aspects can be embedded in biological structures, such as tissues, organoids, organs, organisms, etc. In some cases, the subject matter of the present disclosure relates to interrelated products, alternative technical solutions to a particular problem, and / or multiple different uses of one or more systems and / or articles.

[0009] In one aspect, the present invention generally relates to an article comprising a flexible cell scaffold comprising a grid of one or more nanoelectronic pathways. In another aspect, the present invention generally relates to an article comprising a stretchable cell scaffold forming a circuit that can extend outside the cell scaffold. In yet another aspect, the present invention generally relates to an article comprising a stretchable cell scaffold defining at least a portion of a circuit.

[0010] In one aspect, the present invention generally relates to methods comprising determining the electrical properties of an organoid using a stretchable cell scaffold disposed inside the organoid at a resolution of at least 1 mm. In some embodiments, the present invention generally relates to methods comprising determining the properties of a biological structure using a stretchable cell scaffold disposed inside the biological structure at a resolution of at least 1 mm. The property can be, for example, electrical, chemical, mechanical, or any other type of property, including those discussed herein. The biological structure can be, for example, a tissue, an organoid, an organ, an organism, etc., including those discussed herein.

[0011] On the other hand, the present invention generally relates to methods, the methods comprising determining the electrical properties of an organism with a resolution of at least 1 mm using a stretchable cell scaffold disposed inside the organism. In some embodiments, the present invention generally relates to methods, the methods comprising determining the properties of a biological structure with a resolution of at least 1 mm using a stretchable cell scaffold disposed inside the biological structure. The property can be, for example, electrical, chemical, mechanical, or any other type of property, including those discussed herein. The biological structure can be, for example, a tissue, an organoid, an organ, an organism, etc., including those discussed herein.

[0012] In one aspect, the present invention generally relates to articles. In one set of embodiments, the article comprises a biostructure at least partially embedding a stretchable cell scaffold described herein. In some cases, the cell scaffold defines at least a portion of an electrical circuit.

[0013] In another set of embodiments, the article comprises a cell scaffold defining at least a portion of an electrical circuit. In some cases, the cell scaffold is stretchable by at least 20% in a linear direction at a force of no more than 1 micronewton.

[0014] In another aspect, the invention generally relates to methods. In one set of embodiments, the method includes exposing cells to a stretchable cell scaffold defining at least a portion of a circuit, and growing the cells to form a biostructure at least partially encapsulating the cell scaffold therein.

[0015] In another set of embodiments, the method comprises exposing an embryo to a stretchable cell scaffold defining at least a portion of a circuit, and growing the embryo to form an organism embedding the cell scaffold.

[0016] In yet another set of embodiments, the method comprises determining electrical properties of a biological structure at a resolution of at least 1 mm using a stretchable cell scaffold embedded in the biological structure.

[0017] According to yet another set of embodiments, the method includes applying an electrical signal to at least a portion of the biological structure using a stretchable cell scaffold embedded in the biological structure.

[0018] In another set of embodiments, the method includes patterning a first layer comprising a first photoresist onto a sacrificial layer, patterning a metal path onto the first layer, depositing a second layer comprising a second photoresist onto the first layer, and removing at least a portion of the sacrificial layer to produce a cell scaffold comprising the first layer, the metal path, and the second layer, wherein the cell scaffold is stretchable.

[0019] In another aspect, the present invention encompasses methods of making one or more embodiments described herein, e.g., biostructures that embed flexible cell scaffolds containing electronic devices (such as nanoelectronic meshes). In yet another aspect, the present invention encompasses methods of using one or more embodiments described herein, e.g., biostructures that embed flexible cell scaffolds containing electronic devices (such as nanoelectronic meshes).

[0020] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identical component shown is generally represented by a single numeral. For clarity, not every component is labeled in every drawing, nor is every component of every embodiment of the invention shown, where illustration is not necessary to allow one of ordinary skill in the art to understand the invention. In the drawings:

[0022] Figures 1A to 1H A cell scaffold embedded in an organoid (e.g., a "cyborg organoid") according to certain embodiments of the present invention is shown;

[0023] FIG. 2A to FIG. 2H shows the implantation and distribution of cell scaffolds within organoids during organogenesis in certain embodiments of the invention;

[0024] FIG. 3A to FIG. 3N shows a human cardiac organoid comprising a cell scaffold in other embodiments of the present invention;

[0025] FIG. 4A to FIG. 4I shows electrophysiological mapping of an organoid in another embodiment of the present invention;

[0026] Figure 5 A method for producing a cell scaffold according to one embodiment of the present invention is shown;

[0027] FIG. 6A to FIG. 6B Various buckling modes of interconnectors in cell scaffolds in certain embodiments of the present invention are shown;

[0028] 7A to 7C Various strain distributions of interconnectors in a cell scaffold in certain embodiments of the present invention are shown;

[0029] Figure 8 shows the reaction force on the interconnector in another embodiment of the present invention;

[0030] FIG. 9A to FIG. 9B shows electrophysiological data of a cardiac organoid comprising a cell scaffold in yet another embodiment of the present invention;

[0031] FIG. 10A to FIG. 10C It is shown that according to another embodiment of the present invention, the cell scaffold is embedded in a living body; and

[0032] FIG. 11A to FIG. 11D It is shown that in yet another embodiment of the present invention, a cell scaffold is embedded in a brain organoid. DETAILED DESCRIPTION

[0033] The present invention generally relates to nanoscale wires and nanoelectronic devices, which in some aspects can be embedded in biological structures, such as tissues, organoids, organs, organisms, etc. For example, one aspect generally relates to a stretchable cell scaffold that can be connected to a circuit. In some cases, the biological structure can be formed around the support, and the cell scaffold is deformed when it is formed, thereby causing the cell scaffold to be embedded in the biological structure. In certain embodiments, the support can be connected to an external device, for example, to determine the properties of the cell scaffold (for example, electrical properties, chemical properties, mechanical properties, etc.), and / or to apply stimulation (for example, electrical stimulation) to the biological structure. Other aspects of the present invention generally relate to methods for making or using such cell scaffolds, test kits including such cell scaffolds, biological structures including such cell scaffolds, etc.

[0034] Reference now Figure 1A One aspect of the invention is described. In this figure, a cell scaffold is shown, some or all of which may be stretchable. For example, the cell scaffold may include multiple nodes connected by various interconnects (e.g., forming a grid structure), wherein the interconnects have a certain shape and / or are formed of materials that allow the interconnects to be manipulated or twisted, such as during stretching, compression, folding, etc., without destroying their connection. For example, the interconnects may have an "S" shape or a serpentine shape. In some cases, the interconnects may be formed of materials that can be manipulated by cells within the scaffold. For example, when organized into a larger biological structure, the cells may cause the cell scaffold to form a bowl-shaped or spherical structure, such as Figure 1A shown.

[0035] In some cases, cells can form a biostructure comprising a cell scaffold, which can be partially or completely embedded in the structure. For example, in some cases, the biostructure can be a tissue, an organoid, an organ, or a whole organism. Thus, in contrast to techniques in which a cell scaffold is implanted into a biostructure (i.e., a pre-existing biostructure), the biostructure can be formed around the cell scaffold. In some cases, as part of the growth process, the biostructure is able to manipulate or distort the cell scaffold, for example, to form a more suitable embedding shape of the cell scaffold within the biostructure. For example, Figure 1A As shown, the cell scaffold can initially be substantially planar and seeded with cells that form a substantially spherical organoid; as the organoid forms, the cells cause the cell scaffold to adopt a more spherical configuration, thereby producing an organoid embedded with the cell scaffold. As another non-limiting example, the cell scaffold can be introduced into an embryo, which can develop into an organism embedded with the cell scaffold. The biological structure can also be a tissue, such as a 3D tissue, or other structures such as those discussed herein.

[0036] In certain embodiments, the cell scaffold may also include components that can form an electrical circuit or portion thereof, including nanoelectronic components. For example, some or all of the interconnects may include metal or other conductive paths, and / or there may be sensors, stimulators, nanoscale wires, etc. within the cell scaffold. In some cases, the cell scaffold may define a self-contained electrical circuit, and / or a portion of the cell scaffold may be interfaced or connected to an external electrical device (such as a computer) using a suitable connector (such as a cable).

[0037] In various embodiments, the cell scaffold in the biostructure can be connected to an electronic circuit extending outside the cell scaffold. Nanoscale wires can form an integral part of the biostructure, and can be measured or controlled, for example, using an external electrical device. Due to high degree of electronic control, this allows the creation of a completely new type of functionalized biostructure. Therefore, such cell scaffolds can be measured and / or controlled with high resolution. In some cases, such cell scaffolds can be used for a variety of new applications, including applications in tissue engineering, prostheses, pacemakers, implants, etc.

[0038] The above discussion is a non-limiting example of one embodiment of the present invention, which relates to a cell scaffold, including a cell scaffold (partially or completely) embedded in a biological structure such as a tissue, an organoid, an organ, an organism, etc. However, other embodiments are also possible. Therefore, more generally, various aspects of the present invention relate to various cell scaffolds and other devices.

[0039] Therefore, some aspects of the present invention generally relate to flexible or stretchable grids or networks that can be used as cell scaffolds to grow structures such as organoids, tissues or even organisms. In general, a cell scaffold is a structure to which cells can attach and grow thereon, for example to form biological tissues and other biological structures. The cell scaffold may comprise biocompatible and / or biodegradable materials, and in some embodiments may also comprise growth factors, such as growth hormone, extracellular matrix proteins, specific metabolites or nutrients, etc. The cell scaffold is typically porous, for example, so that cells are inoculated therein, and / or for example nutrients, waste, etc. diffuse into and diffuse out of the cell scaffold.

[0040] In one set of embodiments, the scaffold may be shaped and / or may be formed of one or more materials that allow the scaffold to be flexible or stretchable. For example, the scaffold may be formed of an extendable shape such as a serpentine. In some cases, the cell scaffold may be formed of components that are not straight and may extend, for example, when pulled. For example, the cell scaffold may include one or more nodes connected by various interconnects, for example, forming a grid or network. The nodes may be evenly or unevenly distributed within the cell scaffold, and the interconnects may connect them in a regular pattern (e.g., in a rectangular or triangular array of nodes) or an irregular pattern. As a non-limiting example, Figure 1A Panel 1 in FIG. 1 shows a grid of nodes (points) in a square array connected by multiple interconnects between pairs of nodes (shown as wiggly lines). These nodes can represent points of connectivity, or there can be one or more electronic components, such as conductive paths, nanoscale wires, sensors, etc., at some or all of the nodes. The same or different electronic components can exist independently at different nodes within the grid or network.

[0041] The interconnector connecting two (or more) nodes together can have the same or different shapes or structures within a grid or network, and different interconnectors within a grid can independently have the same or different shapes. In some cases, the interconnector can have an extendable shape. For example, the interconnector can have a straight or linear shape, or a nonlinear shape, such as an S-shape, a serpentine (e.g., with two, three, four or more bends or inflection points), a zigzag (e.g., with two, three, four or more vertex angles), a spiral, etc. Such interconnector shapes can allow, for example, various operations to be performed without interrupting the connection of the interconnector to the node during stretching, compression, folding, etc.

[0042] In one set of embodiments, the interconnect may include one or more metal leads and one or more polymers, such as those discussed below. The polymer may include a photoresist polymer (such as SU-8) and / or a biocompatible polymer (such as Matrigel TM ). Other examples of photoresist polymers include, but are not limited to, those described below.

[0043] In some cases, the cell scaffold may have a total filling rate or area of ​​less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. The filling rate or area is the area of ​​the physical components of the cell scaffold compared to the total area of ​​the cell scaffold (including void space). Therefore, this is a measure of the "porosity" ("porosity") on the two-dimensional scale of the cell scaffold. For example, in some cases, the cell scaffold may have a grid structure or layout as described above, wherein the grid is relatively open. Therefore, a cell scaffold with a smaller filling rate will have a larger "open space", for example, to allow cell infiltration.

[0044] In some cases, the cell scaffold can be limited by one or more holes. Too small holes can hinder or limit cell entry. Therefore, in some embodiments, the cell scaffold can have at least about 100 microns, at least about 200 microns, at least about 300 microns, at least about 400 microns, at least about 500 microns, at least about 600 microns, at least about 700 microns, at least about 800 microns, at least about 900 microns or at least about 1mm average pore size. However, in other embodiments, too large holes can prevent cells from being able to use or even enter the pore volume satisfactorily. Therefore, in some cases, the cell scaffold can have no more than about 1.5mm, no more than about 1.4mm, no more than about 1.3mm, no more than about 1.2mm, no more than about 1.1mm, no more than about 1mm, no more than about 900 microns, no more than about 800 microns, no more than about 700 microns, no more than about 600 microns or no more than about 500 microns average pore size. The combination of these holes is also possible, for example, in one embodiment, the average pore size is at least about 100 microns and no more than about 1.5mm. Additionally, in some cases, pores larger or smaller than these may also be used in the cell scaffold. The pore size may be determined using any suitable technique, such as by visual inspection, BET measurement, and the like.

[0045] In some cases, the interconnect can have a minimum dimension or a maximum cross-sectional dimension of less than about 100 microns, less than about 50 microns, less than about 40 microns, less than about 30 microns, less than about 20 microns, less than about 10 microns, less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 700 nanometers, less than about 600 nanometers, less than about 500 nanometers, less than about 300 nanometers, less than about 200 nanometers, less than about 100 nanometers, less than about 80 nanometers, less than about 50 nanometers, less than about 30 nanometers, less than about 10 nanometers, less than about 5 nanometers, less than about 2 nanometers, etc. The interconnect can also have any suitable cross-sectional shape, such as circular, square, rectangular, polygonal, elliptical, regular, irregular, etc.

[0046] In addition, in one set of embodiments, one or more materials within the cell scaffold are stretchable or flexible. For example, in some cases, the cell scaffold may include a mesh or portion thereof (e.g., an interconnector) that may be stretchable or flexible, or may be manipulated or twisted in some manner. It should be understood that the flexibility of a material is not purely an intrinsic material property; a thinner piece of material may provide greater flexibility than a relatively thicker piece of the same material. In addition, in some cases, the flexibility of a material may also be a function of its shape, for example, as discussed above.

[0047] In some cases, the cell scaffold may have components (such as interconnects) that are sufficiently flexible or stretchable such that, for example, the cell scaffold (or components thereof, such as interconnects) can be stretched in a linear direction by at least 10%, at least 20%, at least 30%, at least 50%, at least 75%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, etc., before catastrophic failure, breakage, interruption of connection of the interconnect to a node, loss of electrical connection, etc. of the cell scaffold.

[0048] In addition, in some cases, the cell scaffold can also show a certain degree of elasticity, for example, so that the cell scaffold can be (at least partially) restored to its original structure before stretching. For example, starting from the time when the stretching of the material stops, the cell scaffold (or its components, such as interconnectors) can be restored to at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% (completely elastic) of its original structure. Therefore, for example, a 1cm material stretched to 2cm experiences 100% stretching in a linear direction, and if it is subsequently contracted to 1.5cm, it shows 50% recovery to its original structure (0.5cm from the stretching of its 1cm). However, it should be understood that in some embodiments, the cell scaffold is not elastic.

[0049] In some embodiments, the scaffold can have components (such as interconnects) that are sufficiently flexible or stretchable so that the cell scaffold (or its components, such as interconnects) can have at least 0.01 n·Nm, at least 0.02 n·Nm, at least 0.03 n·Nm, at least 0.04 n·Nm, at least 0.05 n·Nm, at least 0.06 n·Nm, at least 0.07 n·Nm, at least 0.08 n·Nm, at least 0.09 n·Nm, at least 0.10 n·Nm, at least 0.11 n·Nm, at least 0.12 n·Nm, at least 0.13 n·Nm, at least 0.14 n·Nm, at least 0.15 n·Nm, at least 0.16 n·Nm, at least 0.17 n·Nm, at least 0.18 n·Nm, at least 0.19 n·Nm, at least 0.20 n·Nm, at least 0.21 n·Nm, at least 0.22 n·Nm, at least 0.23 n·Nm, at least 0.24 n·Nm, at least 0.25 n·Nm, at least 0.26 n·Nm, at least 0.27 n·Nm, at least 0.28 n·Nm, at least 0.29 n·Nm, at least 0.30 n·Nm, at least 0.31 n·Nm, at least 0.32 n·Nm, at least 0.33 n·Nm, at least 0.34 n·Nm ·Nm, at least 0.1n·Nm, at least 0.2n·Nm, at least 0.3n·Nm, at least 0.4n·Nm, at least 0.5n·Nm, at least 0.7n·Nm, at least 1n·Nm, at least 1.5n·Nm, at least 2n·Nm, at least 2.5n·Nm, at least 3n·Nm, at least 3.5n·Nm, at least 4n·Nm, at least 4.5n·Nm, at least 5n·Nm, etc. have an effective bending stiffness. In some embodiments, the interconnector can have an effective bending stiffness of less than 5n·Nm, less than 4.5n·Nm, less than 4n·Nm, less than 3.5n·Nm, less than 3n·Nm, less than 2.5n·Nm, less than 2n·Nm, less than 1.9n·Nm, less than 1.8n·Nm, less than 1.5n·Nm, less than 1.3n·Nm, less than 1n·Nm, less than 0.9n·Nm, less than 0.8n·Nm, less than 0.5n·Nm, less than 0.3n·Nm, etc. Any combination of these is also possible; for example, the cell scaffold or interconnector can exhibit an effective bending stiffness between 0.090n·Nm and 1.9n·Nm. For examples of determining the effective bending stiffness of a material, see the following examples.

[0050] In certain embodiments, the cell scaffold may have components (such as interconnects) that are sufficiently flexible or stretchable so that the cell scaffold (or its components, such as interconnects) can be compressed in a linear direction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, etc., without catastrophic failure of the cell scaffold, fracture, interruption of the connection of the interconnect to the node, loss of electrical connection, etc.

[0051] Additionally, in certain embodiments, the cell scaffold may have components (such as interconnects) that are sufficiently flexible or stretchable such that the cell scaffold (or its components, such as interconnects) can be folded at least 30°, at least 45°, at least 90°, at least 135°, at least 150°, at least 180°, etc. from an initial planar structure.

[0052] In various embodiments, the cell scaffold may include a variety of materials. For example, the cell scaffold may include one or more polymers, such as a photoresist, which defines an interconnector or other component in the cell scaffold. In some cases, one or more portions of the cell scaffold may include components that can form a circuit in the cell scaffold, such as a nanoelectronic component. For example, the cell scaffold may include a metal or other conductive path, for example, which defines a circuit, and / or may be connected to an external electrical device.

[0053] Therefore, in certain embodiments, the cell scaffold comprises one or more polymers, such as photoresists, biocompatible polymers, biodegradable polymers, etc., as discussed herein. For example, in various embodiments, one or more of the polymers can be photoresists. Although not commonly used in cell scaffolds, photoresists are commonly used in lithographic techniques, which can be used as discussed herein. For example, a photoresist can be selected based on the ability of the photoresist to react with light to become substantially insoluble in (or substantially soluble in, in some cases) a photoresist developer.

[0054] Photoresists that can be used include, but are not limited to, SU-8, S1805, LOR 3A, poly (methyl methacrylate), poly (methyl glutarimide), phenol formaldehyde resin (naphthoquinone diazide / phenolic varnish), naphthoquinone diazide (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400-37, etc. These and many other photoresists are commercially available. Other examples of photoresist polymers include, but are not limited to, those described below, and those described in International Patent Application Publication No. WO 2019 / 084498 (which is incorporated herein by reference). In some cases, the photoresist can be a soft material, such as a hydrogel. In some embodiments, the photoresist includes a polymer formed by photocuring a fluorinated monomer containing a crosslinkable functional group using a photosensitive initiator (photoinitiator). One example of such a polymer is perfluoropolyether dimethacrylate (PFPE-DMA). Other examples are discussed in more detail below.

[0055] In some cases, one or more polymers can be biocompatible and / or biodegradable. Examples of such biocompatible and / or biodegradable polymers include, but are not limited to, poly(lactic-glycolic acid) copolymers, polylactic acid, polyglycolic acid, poly(methyl methacrylate), poly(trimethylene carbonate), collagen, fibrin, polysaccharide materials such as chitosan or glycosaminoglycans, hyaluronic acid, polycaprolactone, etc. Some photoresists are also biocompatible and / or biodegradable in some cases.

[0056] Generally, biocompatible materials are materials that will not induce an immune response or induce a relatively low immune response, such as will not damage the cell scaffold or the cells therein to continue to play the material of its intended use. In some embodiments, biocompatible materials can perform its desired function, and will not, for example, when present in a subject, cause any undesirable local or systemic effect in the subject. In some cases, the presence of the material will not cause any undesirable local or systemic effect, or make any biological response of the subject substantially not affect the ability of the material to continue to play its intended use. For example, in a cell scaffold, when implanted in a subject, the cell scaffold may be able to support appropriate cell or tissue activity, such as, including promoting molecules and / or mechanical signaling systems, and will not cause undesirable effects in those cells, or undesirable local or systemic reactions, or will not cause the reaction that causes the cell scaffold to stop playing its intended use.

[0057] When a biodegradable material is exposed to a biological system, it will usually degrade over time, such as by oxidation, hydrolysis, enzyme attack, phagocytosis, etc. For example, when a biodegradable material is exposed to water (e.g., hydrolysis) or an enzyme, it will degrade over time. In some cases, a biodegradable material is a material that exhibits degradation (e.g., loss of quality and / or structure) when exposed to physiological conditions for at least about one month, at least about 6 months, or at least about one year. For example, a biodegradable material can exhibit at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% mass loss. In some cases, some or all of the degradation products can be reabsorbed or metabolized, such as, entering cells or tissues. For example, some biodegradable materials can release substances that can be metabolized by cells or tissues during degradation.

[0058] In some embodiments, in addition to the polymers described herein, the cell scaffold may also include other materials. Non-limiting examples include other polymers, growth hormones, extracellular matrix proteins, specific metabolites or nutrients, etc. For example, in one embodiment, one or more reagents that can promote cell growth can be added to the cell scaffold, for example, hormones such as growth hormones, extracellular matrix proteins, pharmaceutical agents, vitamins, etc. Many such growth hormones are commercially available and can be easily selected by those of ordinary skill in the art based on the specific type of cells or tissues used or desired. Similarly, non-limiting examples of extracellular matrix proteins include gelatin, laminin, fibronectin, heparan sulfate, proteoglycans, entactin, hyaluronic acid, collagen, elastin, chondroitin sulfate, keratan sulfate, Matrigel TM Many such extracellular matrix proteins are commercially available and can also be readily identified by one of ordinary skill in the art based on the particular type of cells or tissue used or desired.

[0059] As another example, in some embodiments, additional scaffold materials can be added to the cell scaffold, for example, to control the size of the pores in the cell scaffold, promote cell adhesion or growth in the cell scaffold, enhance the structural stability of the cell scaffold, control the flexibility of the cell scaffold, etc. For example, additional fibers or other suitable polymers can be added to the cell scaffold, for example, electrospun fibers can be used as the second scaffold. Additional scaffold materials can be formed by any material (e.g., photoresist or biocompatible and / or biodegradable polymers, or other polymers described herein) described herein with reference to the cell scaffold. As another non-limiting example, a glue such as silicone elastomer glue can be used to control the shape of the cell scaffold.

[0060] In one set of embodiments, the cell scaffold may, for example, contain metal or other conductive paths within the interconnector or node within the cell scaffold. Examples of metals for metal leads or paths that can be used include, but are not limited to, platinum, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, etc., and any combination of these and / or other metals. Other examples include conductive polymers such as poly (3,4-ethylenedioxythiophene) (PEDOT), polyacetylene, polyphenylene vinylene, polypyrrole, polythiophene (e.g., poly (3,4-ethylenedioxythiophene)), polyphenylene sulfide, etc.

[0061] In some cases, materials can be selected that are easily introduced into the cell scaffold, for example, using techniques compatible with photolithography. For example, in one set of embodiments, photolithography techniques such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography can be used to stack or deposit one or more metals on a substrate. In some cases, additional processing steps can also be used to define or register a path.

[0062] In some embodiments, more than one metal can be used in the path. For example, two, three or more metals can be used in the path. The metals can be deposited in different regions or alloyed together, or in some cases, the metals can be stacked on top of each other, for example, using various photolithography techniques. If different types of metals are stacked on top of each other, in some embodiments, they can be stacked in a "stressed" configuration (although in other embodiments, they are not necessarily stressed). For example, in some embodiments, a chromium / palladium / chromium deposition process can form a prestressed arrangement that can spontaneously form a three-dimensional structure after being released from the substrate. See, for example, U.S. Patent Nos. 9,457,128 or 9,786,850, each of which is incorporated herein by reference in its entirety.

[0063] In some embodiments, the conductive path can be relatively narrow. For example, the conductive path can have a minimum dimension or maximum cross-sectional dimension of less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 700nm, less than about 600nm, less than about 500nm, less than about 300nm, less than about 200nm, less than about 100nm, less than about 80nm, less than about 50nm, less than about 30nm, less than about 10nm, less than about 5nm, less than about 2nm, etc. The conductive path can have any suitable cross-sectional shape, such as annular, square, rectangular, polygonal, elliptical, regular, irregular, etc. As discussed in detail below, such conductive paths can be realized using photolithography or other techniques.

[0064] In some cases, the conductive path can define a circuit that is internally contained within the cell support and / or extends outside the cell support, for example, so that the circuit is in electrical communication with an external electrical system, such as a computer or a transmitter (e.g., a radio transmitter, a wireless transmitter, an Internet connection, etc.). In some embodiments, the cell support can include components such as nanoelectronic components. Non-limiting examples of such components include nanoscale wires, sensors such as nanosensors, transistors such as field effect transistors, resistors, capacitors, inductors, diodes, integrated circuits, batteries, power supplies, RFID tags, antennas, transmitters, etc., which can be present in one or more circuits within the cell support.

[0065] In addition, in some cases, the components in the cell scaffold can include electrodes. The electrode can include any suitable material, such as carbon, or metal, such as gold, platinum, silver, etc. In some cases, the electrode can be used to determine the properties (e.g., electrical properties, chemical properties, mechanical properties, etc.) of the cell scaffold, and / or apply stimulation (e.g., electrical stimulation) to the biostructure. In some cases, conductive polymers can also be used together with electrodes. The non-limiting examples of conductive polymers include poly (3,4-ethylenedioxythiophene) (PEDOT), polyacetylene, polyphenylene vinylene, polypyrrole, polythiophene (e.g., poly (3,4-ethylenedioxythiophene)), polyphenylene sulfide or other conductive polymers such as those described herein.

[0066] In some cases, the assembly can be used to determine properties of a cell scaffold, such as when it is embedded in a biological structure, such as described herein. For example, one or more locations within a cell scaffold (e.g., electrodes or nanoscale wires, etc.) can be located to determine properties, such as chemical properties, electrical properties, mechanical properties, etc. Other examples include sensing Ca 2+Spikes, voltage changes, cell signaling pathways, ion concentrations, pH changes, sensing biomolecules or reactive entities, etc. In some cases, a location is defined as one or more nodes within a cell scaffold, some or all of which can be individually addressable. For example, a node within a cell scaffold can include nanoscale wires, such as those discussed in more detail below.

[0067] In certain embodiments, the conductive path can define a circuit (using a suitable connector) that can be interfaced or connected to an external electrical device such as a computer. For example, the cell scaffold can be directly connected to an external device (for example, using an interface such as described in U.S. Patent Application Publication No. 2018 / 0328884 (which is incorporated by reference in its entirety)). In some cases, a suitable connector, such as a cable, can be used to establish this connection between the circuit in the cell scaffold and an external device. The non-limiting examples of a cable include commercially available cables, such as ribbon cables, flexible flat cables, 8-pin cables, 16-pin cables, etc. or other cables. However, external connection is not always required, and in some cases, the scaffold can be an independent circuit. For example, the circuit can be capable of wirelessly transmitting information to an external device, storing information for later access (for example, after sacrificing an organoid, an organ, or an organism), etc.

[0068] Additionally, in certain embodiments, in addition to and / or in lieu of electrical connection, the cell support may be capable of communicating with an external device using wireless communication. For example, the cell support may include a transmitter (e.g., a radio transmitter, a wireless transmitter, an Internet connection, etc.) and / or a receiver, for example, which may communicate with a transmitter and / or a receiver on an external device.

[0069] In some embodiments, more than one circuit and / or more than one conductive path can be used within a cell support. For example, multiple conductive paths or circuits can be used so that some or all nodes can be individually electronically addressed within a cell support. However, in other embodiments, more than one node can be addressed via a specific conductive path.

[0070] In various aspects, cells can be cultured on a cell scaffold and allowed to grow into a biological structure, such as an organoid or tissue, or the cell scaffold can be exposed to a pre-existing biological structure, such as an embryo. The cell scaffold can be partially or completely embedded in the structure, for example, during the growth of the structure. The biological structure can be, for example, a tissue, an organoid, an organ, an organism, etc.

[0071] A variety of cells can be used together with the scaffold. The cell can be a separated cell, a cell aggregate, a tissue construct comprising the cell, etc. The example of the cell includes but is not limited to bacteria or other unicellular organisms, or eukaryotic cells, such as plant cells or animal cells. If the cell is from a multicellular organism, the cell can be from any part of the organism. For example, if the cell is from an animal, the cell can be a cardiomyocyte, a fibroblast, a keratinocyte, a hepatocyte, a chondrocyte, a nerve cell, an osteocyte, an osteoblast, a muscle cell, a blood cell, an endothelial cell, an immune cell (e.g., a T cell, a B cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), etc. In some cases, the cell can be a cancer cell.

[0072] Examples of cells that can form suitable organoids or organs include, but are not limited to, brain cells, cardiac (heart) cells, nephron (kidney) cells, etc. In some cases, the cells are, for example, cancer cells that can grow to form tumors. In certain embodiments, the cells are stem cells, such as pluripotent stem cells. In some cases, the cells can also be exposed to other compounds, such as drugs, to determine their effects on the growth of cells into organoids, organs, or organisms. This can be useful, for example, for drug testing.

[0073] For example, in one group of embodiments, cells such as stem cells can be seeded on a cell scaffold and allowed to grow or self-assemble into a biostructure, such as an organoid, an organ or even an organism. In some cases, the cell can form a "ball" shape when it forms a biostructure, and the flexibility of the scaffold can enable the cell to keep the cell scaffold embedded in the biostructure, thereby causing the cell scaffold to be embedded in the biostructure. For example, the cell scaffold can be flexible enough so that the cells in the biostructure can manipulate or distort the cell scaffold when it forms a biostructure. For example, the cell can be able to distort the cell scaffold at least 20% in a linear direction, or other distortions as described herein. In contrast, many other scaffolds lack this flexibility and are therefore unable to be embedded therein when the biostructure is formed. Therefore, in certain embodiments, the cell scaffold can be embedded in the biostructure without being inserted or injected into the biostructure, such as after its formation.

[0074] In some cases, the cell scaffold can comprise at least a portion of a biostructure. For example, the cell scaffold can be manipulated or distorted, for example, by a cell, to form a three-dimensional structure (e.g., such that the cell scaffold is not a two-dimensional planar structure) that defines an internal volume, wherein the cell scaffold is at least partially embedded in the biostructure.

[0075] The cell support can be partially or completely embedded in the biostructure. For example, the cell support can be completely embedded in the inside of the biostructure so that any part of the cell support is not exposed to the outside of the biostructure. In another group of embodiments, the cell support is only partially embedded in the biostructure, and at least a portion of the cell support is exposed to the outside of the biostructure. For example, the outside of the cell support can be used to electrically connect the cell support to an external electrical device, such as a computer. For example, a suitable connector can be connected to the exposed portion of the cell support, for example, to form a connection between the circuit in the cell support and an external device.

[0076] In some cases, the cell scaffold comprises a relatively large part of the biostructure. For example, the cell scaffold may comprise at least 30% by volume, at least 40% by volume, at least 50% by volume, at least 60% by volume, at least 70% by volume, at least 80% by volume, at least 90% by volume or at least 95% by volume of the biostructure. In addition, in some cases, the cell scaffold may be distributed in a relatively large part of the biostructure. For example, the cell scaffold may be manipulated or distorted so that at least 10% by volume of the biostructure is no more than 5 microns, 10 microns or 30 microns from the cell scaffold embedded therein, and in some cases, at least 20% by volume, at least 30% by volume, at least 40% by volume, at least 50% by volume, at least 60% by volume, at least 70% by volume, at least 80% by volume, at least 90% by volume or at least 95% by volume of the biostructure is no more than 5 microns, 10 microns or 30 microns from the cell scaffold.

[0077] In some cases, the cell scaffold can be manipulated or twisted by, for example, cells to stretch the cell scaffold. For example, the biostructure can grow and / or expand, and when it expands, the embedded cell scaffold can be stretched along with the expansion of the biostructure. In certain embodiments, when the cell scaffold is expanded by the biostructure, it can show a lower filling rate. For example, the cell scaffold can have a first initial filling rate (for example, before adding cells) of less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc., and a second filling rate less than the initial filling rate after expansion. For example, the second filling rate can be less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of the initial filling rate. In some cases, the second filling ratio can be less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, etc. In addition, in some cases, the cells can generate strain on the cell scaffold during expansion. For example, at least a portion of the cell scaffold can exhibit a tensile strain of at least 10% or at least 20%.

[0078] A variety of biological structures are contemplated, such as tissues, organoids, organs, whole organisms, etc. Non-limiting examples of tissues or organs include brain, heart, kidney, etc. In some cases, tissues or organs may also arise from cancer cells or tumor cells.

[0079] In some embodiments, the biostructure can be an organoid. In some cases, an organoid is a miniaturized and simplified form of an organ produced in vitro. They can be derived from various sources, such as one or more cells from a tissue, embryonic stem cells, induced pluripotent stem cells, etc. In some cases, such cells can self-organize in three-dimensional culture due to their self-renewal and differentiation abilities. Therefore, in certain embodiments, such cells can be added to a cell scaffold, and the cells can form an organoid that embeds the cell scaffold.

[0080] In addition, in some cases, the biological structure can be an organism, i.e., a whole organism. The organism can be any suitable organism, including plants or animals, such as invertebrates or vertebrate organisms. The organism can be, for example, an invertebrate (e.g., fruit fly), a fish (e.g., zebrafish), an amphibian (e.g., a frog), a reptile, a bird, or a human or non-human mammal, such as a monkey, a cow, a sheep, a goat, a horse, a rabbit, a pig, a rodent such as a mouse or a rat, a dog or a cat. In some cases, a cell scaffold can be added to a partially formed biological structure such as an embryo, and the partially formed biological structure can grow into an organism. Non-limiting examples include plant cells, animal cells, etc. For example, as the embryo develops into an organism, the growing cells can manipulate and embed the cell scaffold so that the cell scaffold is partially or completely embedded in the organism. See, for example, FIG. 10A to FIG. 10C Shown is the development and embedding of a cell scaffold within an embryo according to certain embodiments.

[0081] In addition, in one set of embodiments, a cell scaffold (with or without a biostructure) can be implanted into an organism. For example, an organoid or organ containing a cell scaffold can be implanted into an organism. The organism can be a human or non-human mammal, such as a monkey, a cow, a sheep, a goat, a horse, a rabbit, a pig, a mouse, a rat, a dog, or a cat. The organoid or organ can be from the same or different species as the organism, and can be from the same individual or a different individual.

[0082] Certain aspects of the invention generally relate to systems and methods for preparing cell scaffolds, such as those described herein. Cell scaffolds can be fabricated, for example, using well-known photolithographic techniques, such as those discussed below.

[0083] In various embodiments, cell scaffold is constructed by assembling various polymers, metals and other components (for example, nanoscale wires) together on substrate. For example, lithographic techniques such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. can be used for patterning polymers, metals, etc. on substrate. After assembly, at least a portion of substrate (for example, sacrificial material) can be removed to allow the scaffold to be partially or completely removed from the substrate. Other materials can also be added to the scaffold, for example, to help stabilize the structure, add additional reagents to enhance its biocompatibility, etc. The scaffold can be used in vivo, for example, by implanting it in a subject, and and / or using it in vitro, for example, by seeding cells, etc. on the scaffold. In addition, in some cases, cells can be initially grown or cultured on the scaffold, for example, to form a biological structure, such as tissue, organoid, organ, organism, etc. In some cases, as discussed, the cell scaffold can have enough flexibility so that the cell scaffold is embedded therein when the biological structure is formed.

[0084] For example, in one set of embodiments, a cell scaffold can be constructed by providing a substrate, depositing a sacrificial layer on the substrate, then patterning a first photoresist on the sacrificial layer, patterning a conductive path on the first photoresist and a second photoresist on the conductive path, and removing the sacrificial layer to produce a cell scaffold. See, e.g. Figure 5 The first and second photoresists may comprise the same or different materials. Optionally, other components such as electrode assemblies, nanoscale wires, connectors such as cables, etc. may also be added to the cell scaffold prior to or during formation.

[0085] The substrate may be selected to be a substrate that can be used for lithography techniques such as electron beam lithography or photolithography, or other lithography techniques including those discussed herein. For example, the substrate may include or consist essentially of a semiconductor material such as silicon, although other substrate materials (e.g., metals) may also be used. Typically, the substrate is a substantially planar substrate, for example, so that polymers, metals, etc. can be patterned on the substrate. In some cases, a portion of the substrate may be oxidized, for example, to form SiO2 and / or Si3N4 on a portion of the substrate, which can facilitate subsequent addition of materials (metals, polymers, etc.) to the substrate.

[0086] In certain embodiments, one or more polymers may also be deposited before depositing sacrificial material or otherwise formed into one or more polymers. In some cases, polymers may be deposited as material layers or otherwise formed into material layers on substrates. Any suitable technology may be used, for example, using lithography techniques such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, etc. to deposit. In some cases, some or all of polymers may be biocompatible and / or biodegradable. In some embodiments, the deposited polymer may also include methyl methacrylate and / or poly (methyl methacrylate).

[0087] Next, a sacrificial material can be deposited. The sacrificial material can be selected to be a material that can be removed without substantially changing other materials (e.g., polymers, other metals, nano-sized wires, etc.) deposited thereon. For example, in one embodiment, the sacrificial material can be a metal, such as an easily etched metal. For example, the sacrificial material can include germanium or nickel, which can be etched or otherwise removed, for example, using a peroxide (e.g., H2O2) or a nickel etchant (many of which can be easily commercially available) to remove. In some cases, the sacrificial material can be deposited on an oxidized portion or polymer previously deposited on a substrate. In some cases, the sacrificial material is deposited as a layer. The thickness of the layer can be less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 900nm, less than about 800nm, less than about 700nm, less than about 600nm, less than about 500nm, less than about 400nm, less than about 300nm, less than about 200nm, less than about 100nm, etc.

[0088] In some embodiments, the first photoresist can be deposited on, for example, a sacrificial material. The photoresist can include one or more polymers that can be deposited as one or more layers. Examples of photoresists include, but are not limited to, SU-8, S1805, LOR 3A, poly(methyl methacrylate), poly(methyl glutarimide), phenol formaldehyde resin (naphthoquinone diazide / phenolic varnish), naphthoquinone diazide (DNQ), Hoechst AZ 4620, Hoechst AZ 4562, Shipley 1400-17, Shipley 1400-27, Shipley 1400-37, etc., and any other photoresist discussed herein.

[0089] Photoresist can be used to at least partially define cell scaffold.In one group of embodiments, photoresist can be deposited as material layer so that the part of photoresist can be removed subsequently.For example, photoresist can use photolithography such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography or other techniques known to those of ordinary skill in the art for removing polymer deposition.In some cases, use more than one photoresist, for example, be deposited as more than one layer (for example, sequentially), and each layer can independently have less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 900nm, less than about 800nm, less than about 700nm, less than about 600nm, less than about 500nm, less than about 400nm, less than about 300nm, less than about 200nm, less than about 100nm etc. thickness. For example, in some embodiments, portions of the photoresist can be exposed to light (visible light, UV, etc.), electrons, ions, X-rays, etc. (e.g., projected onto the photoresist), and the exposed portions can be etched away (e.g., using a suitable etchant, plasma, etc.) to produce a pattern.

[0090] Thus, the photoresist can be formed into a specific pattern, such as in a grid or mesh, for example, as discussed herein. For example, the pattern can include a grid and interconnects having a shape that allows the interconnects to be manipulated or twisted without destroying their connection, such as during stretching, compression, folding, etc. The pattern can be regular or irregular.

[0091] Next, a metal or other conductive material can be deposited, for example, on one of the aforementioned materials to form a conductive path within the cell scaffold. More than one metal can be used, which can be deposited as one or more layers. For example, a first metal can be deposited, and a second metal can be deposited on at least a portion of the first metal. Optionally, more metals can be used, for example, a third metal can be deposited on at least a portion of the second metal, and the third metal can be the same as or different from the first metal. In some cases, each metal can independently have a thickness of less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, less than about 80 nm, less than about 60 nm, less than about 40 nm, less than about 30 nm, less than about 20 nm, less than about 10 nm, less than about 8 nm, less than about 6 nm, less than about 4 nm, or less than about 2 nm, etc., and the layers can have the same or different thicknesses.

[0092] Any suitable technique can be used to deposit the metal, and if more than one metal is used, the technique used to deposit each metal can be independently the same or different. For example, in one set of embodiments, a deposition technique such as sputtering can be used. Other examples include, but are not limited to, physical vapor deposition, vacuum deposition, chemical vapor deposition, cathode arc deposition, evaporative deposition, electron beam PVD, pulsed laser deposition, ion beam sputtering, reactive sputtering, ion-assisted deposition, high-target-utilization sputtering, high-power impulse magnetron sputtering, gas flow sputtering, etc.

[0093] In some cases, metal can be selected so that the deposition process, for example, produces a prestressed arrangement due to atomic lattice mismatch, which causes subsequent metal leads (lead) to be warped or bent once released from substrate. Although such processes are generally undesirable in the prior art, in certain embodiments of the present invention, such prestressed arrangements can be used to form a three-dimensional structure (spontaneous in some cases) when the resulting cell scaffold is released from substrate. See, for example, U.S. Patent Application Publication Nos. 2014 / 0073063, 2014 / 0074253, 2017 / 0069858, 2017 / 0072109, each of which is incorporated herein by reference in its entirety. However, it should be understood that in other embodiments, it is not necessary to deposit metal with a prestressed arrangement.

[0094] Examples of metals that may be deposited (stressed or unstressed) include, but are not limited to, aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, palladium, and any combination of these and / or other metals. For example, a chromium / gold / chromium deposition process may be used, such as Figure 5 shown.

[0095] In certain embodiments, the second photoresist may be deposited on the previous material. The second photoresist may be the same or different from the first photoresist, and may include any photoresist material discussed herein, including any material described with reference to the first photoresist. The second photoresist may include one or more polymers, which may be deposited as one or more layers. In some embodiments, the second photoresist may be deposited, for example, as a material layer on one or more portions of the substrate, so that portions of the second photoresist may be subsequently removed, for example, using lithographic techniques such as electron beam lithography, photolithography, X-ray lithography, extreme ultraviolet lithography, ion projection lithography, or other techniques known to those of ordinary skill in the art for removing photoresists. In some cases, more than one photoresist may be used, for example, deposited as more than one layer (e.g., sequentially), and each layer may independently have a thickness of less than about 5 microns, less than about 4 microns, less than about 3 microns, less than about 2 microns, less than about 1 micron, less than about 900 nm, less than about 800 nm, less than about 700 nm, less than about 600 nm, less than about 500 nm, less than about 400 nm, less than about 300 nm, less than about 200 nm, less than about 100 nm, etc.

[0096] After the cell scaffold is formed, in some cases, some or all of the sacrificial materials can be removed. For example, in one set of embodiments, at least a portion of the sacrificial material is exposed to an etchant capable of removing the sacrificial material. For example, if the sacrificial material is a metal such as nickel, a suitable etchant (e.g., a metal etchant such as a nickel etchant, acetone, etc.) can be used to remove the sacrificial metal. Many such etchants can be easily commercially available. In addition, in some embodiments, the cell scaffold can also be dried, for example, in air (e.g., passively) by using a heat source, by using a critical point dryer, etc.

[0097] Other materials may also be added to the cell scaffold, for example before or after it forms a three-dimensional structure, for example to help stabilize the structure, add additional agents to enhance its biocompatibility (e.g., growth hormone, extracellular matrix protein, Matrigel TM etc.), so that it forms a suitable three-dimensional structure, controls the pore size, etc. Non-limiting examples of such materials have been previously discussed above, and include other polymers, growth hormones, extracellular matrix proteins, specific metabolites or nutrients, additional scaffold materials, etc.

[0098] In addition, in some cases, the cell scaffold is exposed to cells, and the cells can be cultured or allowed to grow, for example, to form a biostructure. In some cases, the cells are plated or inoculated as single cells, although in some cases, larger cell collections (cell assembly) (tissues, embryos, etc.) can be used. In one group of embodiments, the cell scaffold can be exposed to cells in vitro, and and / or the cell scaffold can be exposed or even immersed in a suitable cell growth medium. Such culture medium is widely available commercially. In some embodiments, the cell scaffold can be implanted in the subject in vivo, for example, when tissues, organs, organoids, etc. grow. However, it should be understood that in all embodiments, for example, in the case where the whole organism develops from cells, it is not necessary to implant.

[0099] In addition, it should be understood that in all embodiments, exposure to cells is not necessary. For example, in one set of embodiments, the cell scaffold can be prepared in the absence of cells. For example, the cell scaffold can be sold as part of a kit, and the user can expose the cell scaffold to cells (or use it for other purposes).

[0100] Additionally, in some embodiments, the cell scaffold can be interfaced with one or more electronic devices, e.g., an external electrical system, such as a computer or a transmitter (e.g., a radio transmitter, a wireless transmitter, etc.), e.g., as discussed herein. The interface can occur at any suitable time, e.g., before or after exposure to cells, before or after formation of a biological structure (e.g., an organoid or an organism), before or after sale to a user, etc.

[0101] For example, in some cases, electronic testing of cytoscaffolds can be performed.Cytoscaffolds or a portion thereof can be connected to an external circuit, for example, electronically interrogating or otherwise determining the electronic state of cytoscaffolds. For example, cytoscaffolds can include one or more nanometer-scale wires or other nanometer electronic components, which can be used as sensors. Depending on the application, such determinations can be performed quantitatively and / or qualitatively, and such determinations can involve all or only a portion of cytoscaffolds, for example, as discussed herein.

[0102] Therefore, as mentioned above, in some aspects, the cell scaffold can include one or more nanoscale wires.For example, one or more nodes can include nanoscale wires, and / or nanoscale wires can be included in interconnectors, etc. In some cases, the cell scaffold in an organoid, an organ, or an organism can include one or more sensors or stimulators, which are interconnected with a stretchable mesh interconnector to form a network, for example, as shown in Figure 1. In some embodiments, a sensor or stimulator can include nanoscale wires, such as those described herein. Such sensors can be monitored, for example, individually or collectively.

[0103] Non-limiting examples of suitable nanoscale wires include carbon nanotubes, nanorods, nanowires, organic and inorganic conductive and semiconducting polymers, metal nanoscale wires, semiconductor nanoscale wires (e.g., formed from silicon), etc. If carbon nanotubes are used, they may be single-walled and / or multi-walled, and may be metallic and / or semiconducting in nature. Other conductive or semiconducting elements may also be used within the cell scaffold, which elements may not be nanoscale wires, but have various small nanoscale dimensions.

[0104] In general, a "nanoscale wire" (also referred to herein as a "nanoscopic-scale wire" or "nanoscopic wire") is typically a wire or other nanoscale object having at least one cross-sectional dimension at any point along its length, and in some embodiments, having two orthogonal cross-sectional dimensions (e.g., diameters) of less than 1 micron, less than about 500 nm, less than about 200 nm, less than about 150 nm, less than about 100 nm, less than about 70 nm, less than about 50 nm, less than about 20 nm, less than about 10 nm, less than about 5 nm, less than about 2 nm, or less than about 1 nm. In some embodiments, the nanoscale wire is typically cylindrical. However, in other embodiments, other shapes are also possible; for example, the nanoscale wire can be faceted, i.e., the nanoscale wire can have a polygonal cross-section. The cross-section of the nanoscale wire can be any arbitrary shape, including but not limited to circular, square, rectangular, annular, polygonal, or elliptical, and can be regular or irregular in shape. The nanoscale wire can also be solid or hollow.

[0105] In some cases, one dimension of the nanoscale wire is substantially longer than other dimensions of the nanoscale wire. For example, the nanoscale wire can have a longest dimension that is at least about 1 micron, at least about 3 microns, at least about 5 microns, or at least about 10 microns, or about 20 microns in length, and / or in some cases the nanoscale wire can have an aspect ratio (longest dimension to shortest orthogonal dimension) of greater than about 2:1, greater than about 3:1, greater than about 4:1, greater than about 5:1, greater than about 10:1, greater than about 25:1, greater than about 50:1, greater than about 75:1, greater than about 100:1, greater than about 150:1, greater than about 250:1, greater than about 500:1, greater than about 750:1, or greater than about 1000:1 or more.

[0106] In some embodiments, the nanoscale wire is substantially uniform, or the average diameter of the nanoscale wire varies by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%. For example, the nanoscale wire can be grown from substantially uniform nanoclusters or particles (e.g., colloidal particles). See, for example, U.S. Patent 7,301,199, entitled "Nanoscale Wires and Related Devices" issued by Lieber et al. on November 27, 2007, which is incorporated herein by reference in its entirety. In some cases, the nanoscale wire can be one of a population of nanoscale wires in which the average diameter of the nanoscale wire population varies by less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%.

[0107] In some embodiments, the nanoscale wire has the conductivity of any semiconductor or any metal or the same or similar order of conductivity as any semiconductor or any metal. The nanoscale wire can be formed of suitable materials such as semiconductors, metals, etc., and any suitable combination thereof. In some cases, the nanoscale wire will have the ability to transfer charge, such as being conductive. For example, the nanoscale wire can have a relatively low resistivity, such as less than about 10 -3 Ohm m, less than about 10 -4 Ohmm, less than about 10 -6 Ohm m or less than about 10 -7 Ohm m. In some embodiments, the nanoscale wires can have a conductance of at least about 1 microSiemens, at least about 3 microSiemens, at least about 10 microSiemens, at least about 30 microSiemens, or at least about 100 microSiemens.

[0108] In various embodiments, nanoscale wires can be solid or hollow. As used herein, "nanotubes" are hollow or hollow-core nanoscale wires, including those nanotubes known to those of ordinary skill in the art. As another example, nanotubes can be produced by creating core / shell nanowires and then etching away at least a portion of the core to leave a hollow shell. Therefore, in one set of embodiments, nanoscale wires are non-carbon nanotubes. In contrast, "nanowires" are nanoscale wires that are generally solid (i.e., not hollow). Therefore, in one set of embodiments, nanoscale wires can be semiconductor nanowires, such as silicon nanowires.

[0109] For example, in one embodiment, the nanoscale wire may comprise or consist essentially of a metal. Non-limiting examples of potentially suitable metals include aluminum, gold, silver, copper, molybdenum, tantalum, titanium, nickel, tungsten, chromium, or palladium. In another set of embodiments, the nanoscale wire comprises or consists essentially of a semiconductor. Typically, a semiconductor is an element having semiconducting or semimetallic properties (i.e., between the properties of a metal and a non-metal). An example of a semiconductor is silicon. Other non-limiting examples include elemental semiconductors such as gallium, germanium, diamond (carbon), tin, selenium, tellurium, boron, or phosphorus. In other embodiments, more than one element may be present in the nanoscale wire as a semiconductor, such as gallium arsenide, gallium nitride, indium phosphide, cadmium selenide, etc. Other examples include Group II-VI materials (which include at least one member of Group II and at least one member of Group VI of the periodic table, such as ZnS, ZnSe, ZnSSe, ZnCdS, CdS or CdSe), or Group III-V materials (which include at least one member of Group III and at least one member of Group V, such as GaAs, GaP, GaAsP, InAs, InP, AlGaAs or InAsP).

[0110] In certain embodiments, the semiconductor can be undoped or doped (e.g., p-type or n-type). For example, in one set of embodiments, the nanoscale wire can be a p-type semiconductor nanoscale wire or an n-type semiconductor nanoscale wire, and can be used as a component of a transistor such as a field effect transistor ("FET"). For example, the nanoscale wire can serve as the "gate" of a source-gate-drain arrangement of a FET, while metal leads or other conductive paths (as discussed herein) are used as source and drain electrodes.

[0111] In some embodiments, the dopant or semiconductor may include a mixture of Group IV elements, such as a mixture of silicon and carbon, or a mixture of silicon and germanium. In other embodiments, the dopant or semiconductor may include a mixture of Group III and Group IV elements, such as BN, BP, BAs, AlN, AlP, AlAs, AlSb, GaN, GaP, GaAs, GaSb, InN, InP, InAs, or InSb. Mixtures of these may also be used, such as a mixture of BN / BP / BAs or BN / AlP. In other embodiments, the dopant may include an alloy of Group III and Group V elements. For example, the alloy may include a mixture of AlGaN, GaPAs, InPAs, GaInN, AlGaInN, GaInAsP, etc. In other embodiments, the dopant may also include a mixture of Group II and Group VI semiconductors. For example, the semiconductor may include ZnO, ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, BeS, BeSe, BeTe, MgS, MgSe, etc. Alloys or mixtures of these dopants are also possible, for example, (ZnCd)Se or Zn(SSe), etc. In addition, alloys of semiconductors of different groups are also possible, for example, a combination of Group II-VI and Group III-V semiconductors, for example, (GaAs) x (ZnS) 1-x . Other examples of dopants may include combinations of Group IV and Group VI elements, such as GeS, GeSe, GeTe, SnS, SnSe, SnTe, PbO, PbS, PbSe, or PbTe. Other semiconductor mixtures may include combinations of Group I and Group VII, for example, CuF, CuCl, CuBr, CuI, AgF, AgCl, AgBr, AgI, etc. Other dopant compounds may include different mixtures of these elements, such as BeSiN2, CaCN2, ZnGeP2, CdSnAs2, ZnSnSb2, CuGeP3, CuSi2P3, Si3N4, Ge3N4, Al2O3, (Al, Ga, In)2(S, Se, Te)3, Al2CO, (Cu, Ag)(Al, Ga, In, Tl, Fe)(S, Se, Te)2, etc.

[0112] In certain embodiments, semiconductor doping can be achieved by bulk-doping to produce p-type or n-type semiconductors, although in other embodiments, other doping techniques (such as ion implantation) can be used. Many such doping techniques that can be used are familiar to those of ordinary skill in the art, including bulk doping and surface doping techniques. A bulk-doped article (e.g., an article, or a portion or region of an article) is an article in which a dopant is substantially incorporated into the entire lattice of the article, which is different from an article in which a dopant is only incorporated into a specific region of the lattice at the atomic scale, such as only on the surface or outside. For example, some articles are typically doped after substrate growth, so that the dopant extends only from the surface or outside to a limited distance inside the lattice. It should be understood that "bulk doping" does not define or reflect the concentration or amount of doping in a semiconductor, nor does it necessarily mean that doping is uniform. "Heavy doping" and "light doping" are terms whose meanings are clearly understood by those of ordinary skill in the art. In some embodiments, one or more regions contain a single monolayer of atoms ("delta doping"). In some cases, the region may be less than the thickness of a single monolayer (e.g., if some atoms in the monolayer do not exist). As a specific example, the regions may be arranged in a layered structure within the nanoscale wire, and one or more regions may be delta-doped or partially delta-doped.

[0113] Thus, in one set of embodiments, the nanoscale wire may include a heterojunction, for example, a heterojunction of two regions with dissimilar materials or elements and / or the same material or element but with different proportions or concentrations. The regions of the nanoscale wire may be different from each other, with minimal cross-contamination, or the composition of the nanoscale wire may gradually change from one region to the next. The regions may be arranged longitudinally relative to each other, or radially (e.g., as in a core / shell arrangement) on the nanoscale wire. Each region may have any size or shape within the wire. The junction may be, for example, a p / n junction, a p / p junction, an n / n junction, a p / i junction (where i refers to an intrinsic semiconductor), an n / i junction, an i / i junction, etc. In some embodiments, the junction may also be a Schottky junction. The junction may also be, for example, a semiconductor / semiconductor junction, a semiconductor / metal junction, a semiconductor / insulator junction, a metal / metal junction, a metal / insulator junction, an insulator / insulator junction, etc. The junction may also be a junction of two materials (a doped semiconductor and a doped or non-doped semiconductor) or a junction between regions with different doping concentrations. The junction may also be a defect region to a perfect single crystal, an amorphous region to a crystal, a crystal to another crystal, an amorphous region to another amorphous region, a defect region to another defect region, an amorphous region to a defect region, and the like. There may be more than two regions, and these regions may have unique compositions or may contain the same composition. As an example, a wire may have a first region containing a first composition, a second region containing a second composition, and a third region containing a third composition or a composition identical to the first composition. Non-limiting examples of nanoscale wires containing heterojunctions (including core / shell heterojunctions, longitudinal heterojunctions, and the like, and combinations thereof) are described in U.S. Patent No. 7,301,199, entitled "Nanoscale Wires and Related Devices," issued by Lieber et al. on November 27, 2007 (which is incorporated herein by reference in its entirety).

[0114] In some embodiments, the nanoscale wire is a bent or kinked nanoscale wire. A kink is typically a relatively sharp transition or turn between a first substantially straight portion of the wire and a second substantially straight portion of the wire. For example, a nanoscale wire may have 1, 2, 3, 4, or 5 or more kinks. In some cases, the nanoscale wire is formed of a single crystal and / or includes or consists essentially of a single crystal orientation, such as <110> Crystal orientation, <112> Crystal orientation or Crystallographic orientation. It should be noted that the kink region does not need to have the same crystallographic orientation as the rest of the semiconductor nanoscale wire. In some embodiments, the kink in the semiconductor nanoscale wire can be at an angle of about 120 ° or its multiple. In some cases, the kink can be intentionally positioned along the nanoscale wire. For example, the catalyst particles can be exposed to various gaseous reactants to form one or more kinks in the nanoscale wire to grow nanoscale wires from catalyst particles. Non-limiting examples of kinked nanoscale wires and suitable techniques for making such wires are disclosed in international patent application number PCT / US2010 / 050199 entitled "Bent Nanowires and Related Probing of Species" filed by Tian et al. on September 24, 2010, which was disclosed in WO 2011 / 038228 on March 31, 2011, which is incorporated herein by reference in its entirety.

[0115] In one set of embodiments, the nanoscale wire is formed of a single crystal, for example, a single crystal nanoscale wire comprising a semiconductor. A single crystal item can be formed by covalent bonding, ionic bonding, etc. and / or a combination thereof. Although such a single crystal item may contain defects in the crystal in some cases, a single crystal item is different from an item that includes one or more crystals that are not ionically bonded or covalently bonded, but are only in close proximity to each other.

[0116] In some embodiments, the nanoscale wires used herein are separate or free-standing nanoscale wires. For example, a "separate" or "free-standing" nanoscale wire may not be attached to another item at some point in its lifetime, such as to another nanoscale wire, or the free-standing nanoscale wire may be in solution. This is in contrast to nanoscale features etched onto the surface of a substrate (e.g., a silicon wafer) (wherein the nanoscale features have never been removed from the substrate surface as separate items). This is also in contrast to the conductive portion of an article, which is different from the surrounding material only by in-situ chemical or physical changes, that is, a portion of a uniform item is different from its surrounding portion by selective doping, etching, etc. A "separate" or "free-standing" nanoscale wire is a functional device that can (but is not required to) be removed from the location where it is manufactured as a separate item and transported to a different location and combined with different components to manufacture such as those described herein and those that can be envisioned by a person of ordinary skill in the art when reading this disclosure.

[0117] In various embodiments, more than one nanoscale wire may be present in a cell scaffold. The nanoscale wires may be independently the same or different. For example, a cell scaffold may contain at least 5 nanoscale wires, at least about 10 nanoscale wires, at least about 30 nanoscale wires, at least about 50 nanoscale wires, at least about 100 nanoscale wires, at least about 300 nanoscale wires, at least about 1000 nanoscale wires, etc. The nanoscale wires may be uniformly or non-uniformly distributed throughout the cell scaffold. In some cases, the nanoscale wires may be at least about 10 nanoscale wires / mm. 3 , at least about 30 nanometer scale lines / mm 3 , at least about 50 nanometer scale lines / mm 3 , at least about 75 nanometers / mm 3 or at least about 100 nanometers / mm 3 In some embodiments, the nanoscale wires are distributed within the cell scaffold such that the average spacing between a nanoscale wire and its nearest neighboring nanoscale wire is less than about 2 mm, less than about 1 mm, less than about 500 microns, less than about 300 microns, less than about 100 microns, less than about 50 microns, less than about 30 microns, or less than about 10 microns.

[0118] In the cell scaffold, some or all of the nanoscale wires may be individually electronically addressable. For example, in some cases, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or substantially all of the nanoscale wires in the cell scaffold may be individually electronically addressable. In some embodiments, the electrical properties of the nanoscale wires may be individually determined (e.g., partially or completely distinguishable without including the electrical properties of other nanoscale wires), and / or such that the electrical properties of the nanoscale wires may be individually controlled (e.g., by applying a desired voltage or current to the nanoscale wires without simultaneously applying a voltage or current to other nanoscale wires). However, in other embodiments, at least some of the nanoscale wires may be controlled within the same electronic circuit (e.g., by incorporating nanoscale wires in series and / or parallel), such that the nanoscale wires may still be electronically controlled and / or determined.

[0119] In some embodiments, the nanoscale wire can respond to external properties of the nanoscale wire, such as chemical properties, electrical properties, physical properties, etc. This determination can be qualitative and / or quantitative. For example, in one set of embodiments, the nanoscale wire can respond to voltage. For example, the nanoscale wire can exhibit a voltage sensitivity of at least about 5 micro-Siemens / volt; by measuring the conductivity of the nanoscale wire, the voltage around the nanoscale wire can be determined. In other embodiments, the voltage sensitivity can be at least about 10 micro-Siemens / volt, at least about 30 micro-Siemens / volt, at least about 50 micro-Siemens / volt, or at least about 100 micro-Siemens / volt. Other examples of electrical properties that can be measured include resistance, resistivity, conductance, conductivity, impedance, etc.

[0120] As another example, the nanoscale wire can respond to the chemical properties of the environment surrounding the nanoscale wire. For example, the electrical properties of the nanoscale wire can be affected by the chemical environment surrounding the nanoscale wire, and the electrical properties can be determined to determine the chemical environment surrounding the nanoscale wire. As specific non-limiting examples, the nanoscale wire can be sensitive to pH or hydrogen ions. Further non-limiting examples of such nanoscale wires are discussed in U.S. Patent No. 7,129,554, entitled "Nanosensors," filed on October 31, 2006, which is incorporated herein by reference in its entirety.

[0121] For example, the nanoscale wire may have the ability to bind to an analyte that is indicative of the chemical properties of the environment surrounding the nanoscale wire (e.g., hydrogen ions for pH, or the concentration of a target analyte), and / or the nanoscale wire may be partially or fully functionalized, i.e., contain surface functionalized moieties to which the analyte can bind, thereby resulting in a determinable change in the property of the nanoscale wire, such as a change in the resistivity or impedance of the nanoscale wire. The binding of the analyte may be specific or non-specific. The functional moiety may include simple groups selected from, but not limited to, -OH, -CHO, -COOH, -SOH, -CN, -NH, -SH, -COSH, -COOR, halides; biomolecular entities including, but not limited to, amino acids, proteins, sugars, DNA, antibodies, antigens, and enzymes; grafted polymer chains having a chain length less than the diameter of the nanowire core, selected from a group of polymers including, but not limited to, polyamides, polyesters, polyimides, polyacrylic acids; shells of materials including, for example, metals, semiconductors, and insulators, which may be metallic elements, oxides, sulfides, nitrides, selenides, polymers, and polymer gels.

[0122] In some embodiments, the reaction entity can be bound to the surface of the nanoscale wire, and / or positioned relative to the nanoscale wire so that the analyte can be determined by determining the change in the properties of the nanoscale wire. Depending on the application, "determination" can be quantitative and / or qualitative. The term "reaction entity" refers to any entity that can interact with the analyte in such a way that the properties of the nanoscale wire (such as electrical properties) can be detected. The reaction entity can enhance the interaction between the nanowire and the analyte, or produce a new chemical species that has a higher affinity for the nanowire or to enrich the analyte around the nanowire. The reaction entity can include a binding partner to which the analyte binds. When the reaction entity is a binding partner, a specific binding partner of the analyte can be included. For example, the reaction entity can be a nucleic acid, an antibody, a sugar, a carbohydrate or a protein. Alternatively, the reaction entity can be a polymer, a catalyst or a quantum dot. As a catalyst, the reaction entity can catalyze a reaction involving the analyte, producing a product that causes a detectable change in the nanoscale wire, for example, by binding to an auxiliary binding partner of the product electrically coupled to the nanowire. Another exemplary reaction entity is a reactant that reacts with the analyte to produce a product that can cause a detectable change in the nanowire. The reactive entity may include a shell on the nanowire, such as a polymer shell, which recognizes molecules in, for example, a gas sample, causing a change in the conductivity of the polymer, which in turn causes a detectable change in the nanowire.

[0123] The term "binding partner" refers to a molecule that can bind to a specific analyte or its "binding partner", including specific, semi-specific and non-specific binding partners known to those of ordinary skill in the art. The term "specific binding", when referring to a binding partner (e.g., protein, nucleic acid, antibody, etc.), refers to a reaction that determines the presence and / or identity of one or other member of a binding pair in a mixture of heterogeneous molecules (e.g., proteins and other biological products). Thus, for example, in the case of a receptor / ligand binding pair, the ligand will specifically and / or preferentially select its receptor from a complex mixture of molecules, and vice versa. An enzyme will specifically bind to its substrate, a nucleic acid will specifically bind to its complementary sequence, and an antibody will specifically bind to its antigen. Other examples include nucleic acids that specifically bind (hybridize) to its complementary sequence, antibodies that specifically bind to its antigen, etc. Binding can be through one or more of a variety of mechanisms, including but not limited to ionic interactions, and / or covalent interactions, and / or hydrophobic interactions, and / or van der Waals interactions, etc.

[0124] In addition, as discussed, the cell scaffold may include a photoresist in some aspects, such as a soft photoresist. For example, in some embodiments, the photoresist may include a polymer formed by photocuring a fluorinated monomer containing a crosslinkable functional group using a photoinitiator. For example, this can promote the stretchability of the cell scaffold. An example of such a polymer is perfluoropolyether dimethacrylate (PFPE-DMA). In addition, in some cases, the photoresist may be a photocurable composition. In some embodiments, the photocurable composition includes: a fluorinated monomer containing a crosslinkable functional group; and a photoinitiator. Other non-limiting examples of photoresists can be found in International Patent Application Publication No. WO 2019 / 084498 (which is incorporated herein by reference in its entirety).

[0125] Some embodiments of the present disclosure relate to photocurable compositions that can be cured to form elastomers that exhibit high stretchability and are chemically orthogonal to various developer solvents used in lithography and are therefore compatible with lithography. In addition, the elastomer can be patterned (with fine feature resolution) and the elastomer can be used as a photoresist for patterning various materials, including electrically (or electronically) active materials. Examples of applications of such photopatternable compositions include forming stretchable and transparent substrates, stretchable and transparent dielectric / passivation / encapsulation films or layers for elastic or stretchable microelectronics, and photoresists for material patterning, such as in the case of implantable medical devices, wearable electronic devices, and soft electronic devices; other biomedical devices; cosmetics; prosthetics; and other applications involving interfaces with the human body, animal body, or other biological tissues, where it is desired that mechanical properties match those of the biological tissue.

[0126] In some embodiments, a test kit for example comprising a cell scaffold as described herein may be provided. The cell may be provided together with the test kit or may not be provided together with the test kit. The test kit may include packaging or components, including the cell scaffold, and other components optionally associated with the cell scaffold, such as cells. Examples of other components include, but are not limited to, solvents, surfactants, diluents, salts, buffers, emulsifiers, chelating agents, fillers, antioxidants, adhesives, fillers, preservatives, desiccants, antimicrobial agents, needles, syringes, packaging materials, pipes, bottles, flasks, beakers, culture dishes, glass frits, filters, rings, clips, wrappers, patches, containers, etc., for example, for use, application, modification, assembly, storage, packaging, preparation, mixing, dilution and / or preservation of the cell scaffold.

[0127] The kit may include instructions of any form, which are provided in conjunction with the components of the kit (in a manner that one of ordinary skill in the art will recognize that the instructions are associated with those components). For example, the instructions may include instructions for the use, modification, mixing, dilution, preservation, administration, assembly, storage, packaging and / or preparation of the cytoscaffold. The instructions may be provided in any form that one of ordinary skill in the art would recognize as a suitable medium containing such instructions, such as written or published, oral, audible (e.g., telephone), digital, optical, visual (e.g., videotape, DVD, etc.) or electronic communication (including Internet or network-based communication), provided in any manner.

[0128] The following documents are incorporated herein by reference in their entirety: U.S. Provisional Patent Application Serial No. 62 / 865,648, filed on June 24, 2019, by Liu et al., entitled “Organoids Containing Electronics, and Methods Thereof”; U.S. Provisional Patent Application Serial No. 62 / 872,031, filed on July 9, 2019, by Liu et al., entitled “Organoids Containing Electronics, and Methods Thereof”; Lieber et al., filed on May 1, 2007 release No. 7,211,464, entitled “Doped Elongated Semiconductors, Growing Such Semiconductors, Devices Including Such Semiconductors, and Fabricating Such Devices,” to Lieber et al.; No. 7,301,199, entitled “Nanoscale Wires and Related Devices,” issued on November 27, 2007 to Lieber et al.; and International Patent Application No. PCT / US2010 / 050199, entitled “Bent Nanowires and Related Probing of Species,” filed on September 24, 2010 by Tian et al., which was published on March 31, 2011 as WO 2011 / 038228. In addition, each of the following is incorporated herein by reference in its entirety: U.S. Patent Nos. 9,786,850 and 9,457,128; U.S. Patent Application Publication Nos. 2017 / 0069858, 2014 / 0073063, 2017 / 0072109, and 2014 / 0074253; and International Patent Application Publication No. WO2019 / 084498.

[0129] The following examples are intended to illustrate certain embodiments of the invention, but are not intended to exemplify the full scope of the invention.

[0130] Example 1

[0131] Inspired by natural organogenesis, this example illustrates a new approach to achieve 3D implantation and distribution of nanoelectronic devices within organoids ( Figure 1A ). The first stage involves transferring and laminating the grid-like planar nanoelectronics along with their input / output (I / O) interconnectors onto a 2D sheet of stem cells (Stage I). During cell aggregation, proliferation, and migration, cellular attractions between stem cells gradually shrink the cell sheet into a cell-dense sheet, which simultaneously compresses the nanoelectronics into a tightly packed structure and covers the nanoelectronics with stem cells (Stage II). This interwoven cell / nanoelectronic structure then shrinks and curls up due to organogenesis-induced self-folding, first into a bowl-like geometry (Stage III) and then into a 3D spherical morphology (Stage IV). In this process, the grid-like device seamlessly reconfigures with the cell sheet due to its soft mechanical structure while maintaining a uniform spatial distribution throughout the tissue, thereby generating fully grown 3D organoids with embedded sensor / stimulator arrays in a minimally invasive and globally distributed manner (Stage IV) - hence the name "cyborg organoid". Finally, stem cells in the formed cyborg organoids can be further differentiated into targeted types of functional cells (such as cardiomyocytes), while their electrophysiological activities can be monitored long-term using embedded nanoelectronic devices (Stage V).

[0132] Several important design features of nanoelectronics allow for seamless, tissue-wide integration ( Figure 1B ). First, the device in this embodiment uses a serpentine grid layout with a total filling rate of less than 11%, which leads to significantly improved in-plane stretchability of up to 30% and compressibility of up to several times its initial volume due to the out-of-plane buckling of the grid network, thus being able to accommodate dramatic volume changes (mainly compressive) during organogenesis. Second, two sets of grid dimensions are implemented (ribbon width / thickness = 20 microns / 0.8 microns or 10 microns / 2.8 microns), which results in a tiny device mass of less than 15 micrograms and an effective bending stiffness of 0.090n·Nm and 1.9n·Nm, respectively, which is essentially imperceptible to the surrounding tissue. Third, the sensor with low impedance and an average diameter of 20 microns, which is close to the typical size of a single cell, allows non-invasive, localized single-cell electrophysiological recordings. The detailed fabrication procedure can be found below and Figure 5 middle.

[0133] Figure 1CThe mesh nanoelectronic device is shown about to be released from the fabrication substrate, while the inset shows a single platinum electrode electrochemically deposited with poly(3,4-ethylenedioxythiophene) (PEDOT) to further reduce the interfacial impedance. The released device was folded (up to 180°) and stretched (biaxially, up to 30%) in a water-filled chamber for 100 cycles ( Figures 1D to 1F ) showed no visible damage ( Figure 1D to Figure 1E ) nor did it show significant changes in electrode impedance ( Figure 1F Impedance measurements also showed a >10-fold decrease in impedance at 1 kHz and a phase shift toward resistance-dominated behavior after coating the electrodes with PEDOT. In addition, the longevity of the device was tested using an immersion test in 1x phosphate-buffered saline (PBS) at 37°C, where the impedance values ​​of three random channels at 1 kHz remained stable for 30 days ( Figure 1H ), showing the potential of this type of sensing platform for long-term studies in physiological environments.

[0134] FIG1 illustrates the concept and design of the cyborg organoid of this particular embodiment. Figure 1A is a schematic diagram illustrating the step-by-step assembly of stretchable mesh electronics into organoids through organogenesis. Part (I) shows the lamination of stretchable mesh electronics onto on a continuous sheet seeded with human induced pluripotent stem cells (hiPSC) or hiPSC-derived progenitor cells. Part (II) shows that cell proliferation and migration induced by cell-cell attraction, the cell sheet aggregates into a cell-dense plate, which embeds the stretchable mesh electronics into the cell sheet and folds it into a tightly packed structure. Part (III) shows the organogenesis of 2D-to-3D self-organization to fold the 2D cell sheet / nanoelectronic device hybrid into a 3D structure. Part (IV) shows that the organogenesis unfolds the tightly packed nanoelectronic devices and distributes their structures throughout the 3D organoid. Part (V) shows that the further development and differentiation of stem / progenitor cells into different cell types with electrophysiological behaviors can be continuously monitored by 3D embedded nanoelectronic devices.

[0135] Figure 1B An exploded view of a stretchable mesh nanoelectronic device design is shown, with (from top to bottom) a 400 nm thick top SU-8 encapsulation layer, a 50 nm thick platinum (Pt) electrode layer coated with poly(3,4-ethylenedioxythiophene) (PEDOT), a 40 nm thick gold (Au) interconnector layer, and a 400 nm thick bottom SU-8 encapsulation layer. Note that in addition to SU-8, other materials such as those described herein and those described in International Patent Application Publication No. WO 2019 / 084498 (which is incorporated herein by reference) may be used. The inset shows a serpentine layout of the mesh. Figure 1C An optical image of a mesh electronics device prior to release from the fabrication substrate is shown. The inset shows a magnified view of a single Pt electrode coated with PEDOT. Figure 1D and Figure 1E is bent by optical tweezers ( Figure 1D ) and stretching ( Figure 1E ) Optical image of a stretchable mesh electronic device released from water. Figure 1F Impedance and phase from 0.1 to 10 kHz are shown for a representative channel with and without PEDOT. Figure 1G shows that after 100 cycles of folding (reaching Figure 1D shown) and stretched for 100 cycles (to Figure 1E The average impedance (n=5) of each channel at 1 kHz in its original state after biaxial expansion (approximately 30%) is shown. Figure 1H Impedance at 1 kHz is shown for three representative channels as a function of incubation time in PBS at 37°C. Figure 5 Schematic diagram showing the overall fabrication process of stretchable mesh nanoelectronic devices.

[0136] Example 2

[0137] In this example, the stretchable mesh nanoelectronic device described in Example 1 was integrated with hiPSCs or hiPSC-derived cardiac progenitor cells (hiPSC-CPCs) co-cultured with human mesenchymal stem cells (hMSCs) ( FIG. 2 ). The original flat sheet of cells sandwiching nanoelectronics ( Figure 2A , I) spontaneous packaging of nanoelectronic devices into highly compact structures through hMSC-driven condensation into cell sheets ( Figure 2A , II). Further 3D reorganization converts the cell sheets into 3D organoids in approximately 72 hours ( Figure 2A , III). Phase images illustrate the initial compression of the nanoelectronic device together with the cell sheet / plate at each step ( Figure 2C , I to II) and subsequent folding ( Figure 2C , III). With further growth and expansion, the cyborg organoids fully unfolded and the nanoelectronic devices were distributed throughout the organoid. Phase and false color images after approximately 480 hours of organogenesis showed that the devices were evenly distributed on and within the organoid as it grew and expanded ( Figure 2D Importantly, the identical morphology of nanoelectronic devices from hMSC / hiPSC and hMSC / hiPSC-CPC assemblies further demonstrates the versatility of this approach for different types of cyborg organoids ( FIG. 2A to FIG. 2C ).

[0138] Imaging results showed that effective strains (defined as the relative change in the distance between adjacent nodes of the mesh) of up to about 50% compression and about 20% tension were applied to the nanoelectronic devices during organogenesis. Simulations performed by finite element analysis (FEA, see below for more details) showed that large deformations were accommodated by out-of-plane buckling of the mesh ribbon, resulting in a maximum nominal strain of less than 1.2%, which is below the fracture limit of SU-8 and gold ( Figure 2F to Figure 2G , FIG. 6A to FIG. 6B and 7A to 7C ), indicating the robustness of this nanoelectronic structure design. Remarkably, the buckling modes in compression and tension observed in the experiments can be qualitatively reproduced by simulation. Additionally, simulations show that the end-to-end forces required to deform the nanoelectronic device are comparable to the forces required to deform cells at the same scale, which indeed suggests a minimally invasive implantation procedure ( Figure 2H and Figure 8 ).

[0139] Figure 2 shows the 3D implantation and distribution of stretchable mesh-like nanoelectronics via organogenesis. Figure 2A In the optical image, the Figure 1A Representative steps for assembling a stretchable mesh nanoelectronic device based on organoid development corresponding to the schematic diagram in FIG. 1 , which includes laminating the stretchable mesh nanoelectronic device on hiPSC-CPCs and hMSCs were co-cultured. The dotted circles highlight the boundaries of the cell plates at different stages. Figure 2B In this magnified phase image, stretchable mesh-like nanoelectronics are shown in Figure 2. Figure 2A The region highlighted by the dashed box deforms at different steps under the action of mechanical forces from organogenesis. Figure 2C In the further magnified phase image, the Figure 2B Figure 3. Cell-nanoelectronic device interaction at different steps with the region highlighted by the dashed box. SU-8 ribbons from the nanoelectronic device and stem cells are shown. White arrows highlight the region where the ribbon is embedded in the cell. Figure 2D Bright-field phase image of a representative fully assembled cyborg organoid, with stretchable mesh-like nanoelectronics deployed and integrated throughout the organoid. The inset shows the Figure 2C Magnified phase image of the area highlighted by the dashed box. Figure 2Eis the representative effective strain of the device, calculated from phase images as a function of culture time. Grey points and lines show individual data points, while black points and lines show average results. (Values ​​= mean + / - sem, n = 10). Figure 2F Two compression modes of a repeating unit cell in a mesh electronics device obtained from finite element analysis (FEA) are shown. Merged simulation and optical images show representative modes in real samples. The scale represents the nominal strain in the cell. Figure 2G Shown is the stretching pattern of a repeating unit cell in a mesh electronics device obtained from FEA. Merged simulation and optical images show representative patterns in real samples. The grayscale represents the nominal strain in the cell. Figure 2H The 2F and Figure 2G Force magnitude as a function of strain for the three deformation modes.

[0140] Fig. 6A and Figure 6B A linear perturbation analysis is shown showing the buckling mode of a unit strip in compression. The deformation in the Z direction is magnified by a factor of 50 for convenience. Fig. 6A In the case of W / H=10 microns / 2.8 microns, Figure 6B In the example, W / H=20 μm / 0.8 μm.

[0141] exist 7A to 7C In Figure 2, FEA shows the maximum principal nominal strain distribution during the belt buckling process. Fig. 7A Compression (mode I) is shown with an effective compressive strain of 0 to 50%, W / H = 10 microns / 2.8 microns. Figure 7B Compression (mode II) is shown with an effective compressive strain of 0 to 50%, W / H = 10 microns / 2.8 microns. Figure 7C The stretching (mode I) is shown with an effective tensile strain of 0 to 20%, W / H = 10 microns / 2.8 microns.

[0142] Figure 8 are simulation results showing the magnitude of the end-to-end reaction force on a unit strip as a function of the applied effective in-plane strain. As expected, the magnitude of the force is larger for thicker devices. The bend in each curve corresponds to the onset of buckling in the simulation.

[0143] On day 40 of tissue development and differentiation, optical images showed that the nanoelectronics had been fully embedded in the organoids, with the expanded mesh strips covering the entire tissue area ( Figure 3A). The flexibility and stretchability of the fully embedded nanoelectronics enabled them to adapt to the contraction of the cardiac organoids. To further reveal the encapsulation of nanoelectronics within the 3D organoids, the cyborg cardiac organoids were cleared at day 40 of differentiation. 3D reconstructed fluorescence images revealed 3D interwoven nanoelectronic / cellular structures across the entire organoid ( Figure 3B ). A zoomed-in view further shows that the nanoelectronics are sandwiched between the outer layers of the organoid with migrating hMSCs ( Figure 3C ,I) and the core of the organoid with mature cardiomyocytes. Importantly, as the planar nanoelectronics developed alongside the 2D cell sheet, the images showed tight coupling between differentiated cardiomyocytes and the nanoelectronics throughout the organoid ( Figure 3C ), which is rarely observed in other methods of implantation of mature living organoids and organs.

[0144] To investigate the effects of integrated nanoelectronics on organoid differentiation, hiPSC-CPC / hMSC cardiac semi-mechanical organoids at different stages (days 7, 20, and 40 of differentiation) were fixed, sectioned, and immunostained for stage-specific marker expression ( Figures 3D to 3F ) and compared them with those from control cardiac organoids. Protein markers including TNT, α-actinin, and actin were imaged to quantify cardiomyocyte maturation, while NKX2.5 was imaged in hiPSC-CPCs ( Figure 3G to Figure 3J The average fluorescence intensity during the culture period showed that the fluorescence intensity of TNT and α-actinin increased significantly ( Figure 3K and Figure 3M ), while no statistically significant differences were found between semi-mechanical and control organoids at all stages. In addition, fluorescence intensity plots along the axis of individual cells within organoids showed the same level of increased sarcomere alignment in semi-mechanical and control organoids from day 7 to day 40 of differentiation ( Figure 3L and Figure 3N ). These results essentially demonstrated that the integrated nanoelectronics did not disrupt the 3D differentiation of iPSC-derived cardiac organoids.

[0145] FIG3 shows the development of the human heart cyborg organoid in this example. Figure 3A Top view (I) and bottom view (II) of cardiac cyborg organoids at differentiation day 40. Arrows highlight the folding and stretching of the stretchable mesh device through 3D reorganization of the cell sheet. Figure 3BTop (I) and bottom (II) views of 3D reconstructed fluorescent imaging of cleared, immunostained cardiac cyborgs at day 40 of differentiation. The different shades correspond to TNT, CD44, and DAPI. SU-8 in the device was labeled with R6G for imaging in this and the following panels. Figure 3C In, from Figure 3B Magnified views of the area highlighted by the dark dashed box show that migrating hMSCs were observed to form the outer layer of the organoid (I) and tight coupling between iPSC-derived cardiomyocytes and the nanoelectronic devices (white arrows) (I-IV). Figure 3D Schematic diagram showing the slicing of cardiac cyborg organoids for characterization at different stages of organogenesis. Figure 3E and Figure 3F shows a 30 μm thick cyborg organoid ( Figure 3E ) and control organoids ( Figure 3F ). The shadows correspond to TNT, device, and DAPI, respectively, and are shown at the top of the image panels for this and subsequent images. Figure 3G to Figure 3N Immunostaining and fluorescence imaging characterized the maturation of cardiomyocytes in organoids at day 7 (I), day 20 (II), and day 40 (III-IV) of differentiation. Figure 3G to Figure 3H ), and α-actinin and actin ( FIG. 3I to FIG. 3J ) was used to characterize the maturation level of organoids with and without embedded nanoelectronic devices. The enlarged view (IV) further shows the subcellular localization of TNTs and α-actinin at day 40 of differentiation. Figure 3K ) and α-actinin ( Figure 3M ) (Value = mean + / - sem, n = 6. **, P < 0.01; ***, P < 0.001; ***, P < 0.0001, two-tailed unpaired t-test.) Fluorescence intensity graphs showing the arrangement of myomes in control and cyborg organoids at days 7, 20, and 40 of differentiation ( Figure 3L ) and α-actinin arrangement ( Figure 3N ).

[0146] Example 3

[0147] The strong coupling between stem / progenitor cells and nanoelectronics provides a unique opportunity to map the evolution of cellular electrophysiology during organogenesis. In this example, electrophysiological recordings were performed on a cardiac cyborg that typically forms in about 48 hours. FIG. 4A to FIG. 4B ). Figure 4CVoltage traces from 14-channel (out of 16 channels) electrophysiological recordings on cardiac cyborgs at day 35 of differentiation are shown. Magnified view of a single spike ( Figure 4D ) show the inhomogeneous electrophysiological behavior of cells distributed across organoids and reveal the temporal latency of tissue-wide propagation of local field potentials (LFPs). Long-term tracking of LFPs with millisecond temporal resolution during organogenesis ( Figure 4E ) revealed a dynamic change from an initial slow waveform to the emergence of repolarization and then to a spike of rapid depolarization. The mean amplitude of the fast component associated with depolarization remained undetectable until day 31 of differentiation and then increased monotonically ( Figure 4F ). It was found that the field potential duration (FPD) remained relatively stable, between 0.7 and 0.8 s ( Figure 4G ) and slightly increased. Activation mapping confirmed that LFP propagation across cardiac organoids gradually became synchronized from day 26 to day 35 of differentiation ( Figure 4H , Fig. 4I These recordings suggest that functional maturation of cardiac organoids is not marked by bursting frequency or FPD, but rather by the synchronization of the bursting phase.

[0148] In summary, these embodiments show the generation of human cardiac semi-mechanical organoids through 2D to 3D tissue reconstruction of organogenesis, and the evolution of electrophysiological patterns during organogenesis is studied. These can be scaled to integrate a large number of sensors made by light or electron beam lithography. Cardiac semi-mechanical organoids can be used to study cardiac development, disease and treatment. Further development and promotion of semi-mechanical organoids can serve as a paradigm shifting platform for spatially resolved high-fidelity and long-term electrophysiological recordings of many other types of organoids and animal embryos, as well as for monitoring and controlling cell therapies implemented by organoids.

[0149] Figure 4 shows long-term, multiplexed, tissue-wide electrophysiological mapping of cardiac organoids during organogenesis. Figure 4A is a schematic diagram showing the setup for connecting a cyborg cardiac organoid to external recording equipment for multiplexing electrophysiology during organoid development. Figure 4B In Figure 2, optical images show representative processes of ligating 3D tissues of cardiac cyborg organoids in a culture chamber for electrophysiological recordings (0, 24, and 48 hours). Figure 4C In Figure 1, a 14-channel voltage trace recorded from a cardiac cyborg organoid at day 35 of differentiation. Figure 4D middle, Figure 4C The enlarged view of the highlighted box in shows the single-spike field potential recording. Figure 4E Shows Figure 4C The dashed box in the middle shows the magnified images of the channel at three different culture days (day 26, day 31, and day 35 of differentiation). FIG. 4F to FIG. 4I Shows Figure 4E The amplitude of the fast peak as a function of differentiation time ( Figure 4F ) and field potential duration ( Figure 4G ). The gray line shows a single channel. The black line shows the average result of 14 channels. (Value = mean + / - sem, n = 14.) Figure 4H and Fig. 4I In the 26th day of differentiation ( Figure 4H ) and day 35 ( Fig. 4I ) isochronous mapping shows FIG. 9A to FIG. 9B Delay in activation time (maximum -dV / dt) of three consecutive peak events marked in .

[0150] Figure 9 shows that on the 26th day of culture ( Fig. 9A ) and day 35 ( Fig. 9B ) showing the transition from irregular activity patterns to synchronized beating. This dataset was used to construct the three consecutive spike events within the dashed red rectangle. Figure 4H The dashed line segment on day 26 represents the activation time of peak #1 for each channel.

[0151] Example 4

[0152] The following are various materials and methods used in the above examples.

[0153] Fabrication of soft and stretchable mesh nanoelectronic devices. Figure 5 As shown, the important steps to fabricate ultrathin, stretchable mesh nanoelectronics include:

[0154] (1) A silicon wafer on which thermal oxide (500-nm thickness) was grown was cleaned with acetone, isopropyl alcohol, and water.

[0155] (2) A 100-nm-thick nickel (Ni) was deposited as a sacrificial layer using an electron beam evaporator.

[0156] (3) SU-8 precursor (SU-8 2000.5, MicroChem) was spin-coated at 4000 rpm and pre-baked at (65°C, 95°C) for 2 min each. 2Exposure to 365nm ultraviolet (UV), post-baking at (65°C, 95°C) for 2 minutes each, developing with SU-8 developer (MicroChem) for 60 seconds, and hard baking at 180°C for 40 minutes to define a grid SU-8 pattern (400-nm thick) for bottom encapsulation. For fluorescent imaging, 0.004wt‰ of Rhodamine 6G powder (Sigma-Aldrich) was added to the SU-8 precursor. Please note that in addition to SU-8, other materials may be used, including those described herein and those in International Patent Application Publication No. WO 2019 / 084498 (which is incorporated herein by reference in its entirety).

[0157] (4) LOR3A photoresist (MicroChem) was spin-coated at 4000 rpm, followed by pre-baking at 180°C for 5 min; S1805 photoresist (MicroChem) was spin-coated at 4000 rpm, followed by pre-backing at 115°C for 1 min; the samples were then heated at 40 mJ / cm 2 Expose to 405 nm UV and develop using CD-26 developer (Micropost) for 70 seconds to define the interconnector pattern.

[0158] (5) 5 / 40 / 5-nm-thick chromium / gold / chromium (Cr / Au / Cr) was deposited by electron beam evaporator, followed by a standard lift-off procedure in a Remover PG (MicroChem) overnight to define gold interconnects.

[0159] (6) repeating step (4) to define an electrode array pattern in the LOR3A / S1805 double-layer photoresist;

[0160] (7) 5 / 50-nm thick chromium / platinum (Cr / Pt) was deposited by electron beam evaporator, followed by a standard lift-off procedure in a remover PG (MicroChem) for 10 min to define the electrode array;

[0161] (8) Repeat step (3) for the top SU-8 package (400-nm thickness).

[0162] (9) Poly(3,4-ethylenedioxythiophene) (PEDOT) was electrochemically polymerized on a Pt electrode array using a precursor consisting of 14 mM 3,4-ethylenedioxythiophene (EDOT, Sigma-Aldrich) in a 1x phosphate buffered saline (PBS) solution. The precursor was drop-cast onto the device, and a 1 V DC voltage (Dr.Meter DC power supply PS-305DM) was then passed for 200 seconds using the device electrode as the anode and an external Pt wire as the cathode. The device was then rinsed with DI water for 30 seconds and dried.

[0163] (10) A 16-channel flexible flat cable (Molex) was soldered to the input / output pads using a flip-chip bonder (Finetech Fineplacer).

[0164] (11) The chamber was bonded to the substrate wafer using a biocompatible adhesive (Kwik-Sil, WPI) to completely enclose the grid portion of the device.

[0165] (12) The device surface was treated with a light oxygen plasma (Anatech 106 oxygen plasma bucket asher), followed by the addition of 3 mL of Ni etchant (TFB type, Transene) into the chamber for 2 to 4 hours to completely release the mesh electronics from the substrate wafer. The device was then prepared for a subsequent sterilization step prior to cell culture.

[0166] Organoid Culture. Cell Culture Materials. Human induced pluripotent stem cells (hiPSC-(IMR90)-1) were purchased from WiCell Research Institute (Madison, WI, USA). Mycoplasma-free identification and testing were performed by WiCell Research Institute. Upon receipt of the cells, a cell bank was established. For the experiments, each aliquot from the cell bank was used for less than 5 additional passages. Human mesenchymal stem cells (hMSC; Pt-2501) were obtained from Lonza (Walkersville, MD, USA) and used for less than 5 additional passages. Reagents and their suppliers: Essential 8 TM (E8) culture medium (Catalog No. A1517001, Life Technologies); MSCGM Bullet Kit (Catalog No. PT-3238 & PT-4105, Lonza); RPMI1640 Medium (Catalog No. 11875093, life technologies); B-27 Supplement (Catalog No. 17504044, life technologies); B-27 TM Supplement, minus insulin (Catalog No. A1895601, Life Technologies); CHIR 99021, (Catalog No. 44-231-0, Tocris Bioscience TM); IWR-1 (Catalog No. CAYM-13659-5, Cayman Chemical); Normal donkey serum (Catalog No. 017-000-121, Jackson ImmunoResearch); Accutase (Catalog No. A1110501, Life Technologies); Trypsin-EDTA 0.05% (Catalog No. 25-300-054, Invitrogen); EDTA 0.5M pH 8.0 (Catalog No. AM9260G, Invitrogen); Y-27632 (Catalog No. 129830-38-2, Sigma); Matrigel (Catalog No. 08-774-552, Corning); DAPI (Catalog No. D9542, Sigma). Phalloidin-iFluor 647 (Catalog No. ab176759, Abcam); Poly-D-lysine hydrobromide (Catalog No. P7280-5MG, Sigma); Photoinitiator 2,20-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (Catalog No. VA-044, Wako Chemicals). Antibodies used in these examples: Nanog (Santa Cruz, 1:100); CD44 (Abcam, 1:100); Cardiac troponin T (TNT) (Invitrogen, 1:100); α-actinin (Sigma, 1:100); NKX2.5 (Abcam, 1:100); Alexa 594 donkey anti-mouse (Jackson ImmunoResearch, 1:500); Alexa 647 donkey anti-rabbit (Invitrogen, 1:500).

[0167] Two-dimensional (2D) cell culture. hiPSCs-(IMR90)-1 were maintained in 6-well plates coated with Matrigel in Essential 8 TM The steps are as follows:

[0168] (1) Coat a 6-well plate with Matrigel at 37°C for at least 1 hour.

[0169] (2) Aspirate the Matrigel from the wells and replace with 2 mL of E8 medium.

[0170] (3) Incubate hiPSCs with 0.5 mM EDTA at room temperature for 5 minutes.

[0171] (4) Aspirate the EDTA solution and replace it with 1 mL of E8 medium. The cells are pipetted into clusters.

[0172] (5) 100-200 μl of cells were seeded in a 6-well plate coated with matrix gel.

[0173] (6) The culture medium was changed every day. Cells were passaged every 4 days.

[0174] hMSCs were cultured in MSCGM medium in T-75 flasks. The steps are as follows:

[0175] (1) 5000-6000 cells / cm 2 Inoculate in a T-75 flask containing approximately 30 ml of MSCGM medium.

[0176] (2) The culture medium was changed every 3-4 days.

[0177] (3) When hMSCs reached about 90% confluence, the cells were dissociated using trypsin-EDTA 0.05%. The cells were used at the fifth passage.

[0178] 2D hiPSC-derived cardiomyocyte differentiation. The cardiomyocyte differentiation steps are as follows:

[0179] (1) Culture hiPSCs in a 6-well plate containing E8 medium for 3-4 days.

[0180] (2) Remove the E8 medium. Add RPMI 1640 medium + 1% B27-insulin and 12 micromolar CHIR99021 for differentiation. This day is defined as day 0.

[0181] (3) On day 1, the culture medium was changed to RPMI 1640 medium + 1% B27-insulin.

[0182] (4) On day 3, the culture medium was changed to RPMI 1640 medium + 1% B27-insulin and 5 micromolar IWR1.

[0183] (5) On day 5, the culture medium was changed to RPMI 1640 medium + 1% B27-insulin.

[0184] (6) On day 7, the culture medium was changed to RPMI 1640 medium + 1% B27.

[0185] (7) The medium was then replaced with fresh RPMI 1640 medium + 1% B27 every other day. Cells began to beat from day 8 or day 10.

[0186] 3D culture of cyborg organoids. Integration of stretchable mesh nanoelectronics with cells to generate cyborg organoids followed by immediate fabrication and release of the device in the chamber, which includes the following steps:

[0187] (1) Released devices were sterilized by 1) DI water rinses three times, and 2) 70% ethanol for at least 15 minutes.

[0188] (2) The device was washed three times with PBS and then incubated with poly-D-lysine hydrobromide (0.01% w / v) overnight.

[0189] (3) Remove the poly-D-lysine hydrobromide solution and add 300 μL of liquid Matrigel (10 mg / mL) to the chamber from the side without the device on ice. The device is incubated at 37°C for at least 30 minutes to solidify the Matrigel layer.

[0190] (4) hiPSC or hiPSC-derived cardiac progenitor cells (hiPSC-CPC) (2-3x10 6 cells) and hMSCs (2-4x10 5 Cells) were suspended in a mixture of E8 or RPMI 1640 (supplemented with 1% B27-insulin or 1% B27) and MSCGM medium, then transferred onto solidified Matrigel in a chamber and cultured at 37°C, 5% CO2.

[0191] (5) For electrophysiological measurements, the mixture of hiPSC-CPC and hMSC on day 24 was transferred to Matrigel and cultured at 37°C and 5% CO2. hiPSC-derived semimechanical cardiac organoids formed within 48-72 hours. The electrical activity of the organoids was recorded from 48 hours after their formation, corresponding to day 26 of cardiac differentiation in Figure 4.

[0192] Characterization was performed as follows.

[0193] Organoid clearing, sectioning, immunostaining and imaging. For whole organoid imaging, the program was adapted from the tissue clearing technology CLARITY and the passive clearing technology (PACT). The organoids were fixed with 4% PFA at 4°C overnight and incubated with a hydrogel solution (0.25% (w / v) VA-044 and 4% (w / v) acrylamide in PBS) at 4°C for 24 hours. The samples were placed in an X-CLARITY hydrogel polymerization device at 37°C and -90kPa vacuum for 3-4 hours, then washed overnight in PBS, and then electrophoretic lipid extraction was performed in the X-CLARITY electrophoretic tissue clearing (ETC) chamber for 24 hours. Immunostaining was then performed by staining the primary antibody for 3-5 days and the secondary antibody for 2-4 days. The samples were immersed in an optically transparent solution overnight and embedded in a 2% agarose gel, then imaged using a Zeiss two-photon time-lapse confocal microscope at the Harvard Center for Biological Imaging (HCBI). To characterize the semimechanical cardiac organoids, organoids at different stages were fixed with 4% PFA at 4°C overnight and immersed in 30% sucrose for at least 12 h. The samples were then embedded in optimal cutting temperature (OCT) compound and cryosectioned at 30 μm thickness. Cardiac organoids without device integration were used as controls. For staining, primary antibodies were incubated overnight at 4°C and secondary antibodies were stained for 3–4 h at room temperature. For 2D culture, cells were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT). Cells were incubated with primary antibodies at 4°C overnight and secondary antibodies were stained for 1–2 h at room temperature. Finally, 4′,6-diamidino-2-phenylindole (DAPI) was stained for 10 min. Cells were washed three times with PBS before imaging. All samples were imaged at HCBI using a Zeiss 880 confocal microscope. Images were analyzed by Zen (Blue version) and Fiji. Fluorescence intensity was calculated by Fiji. Data analysis and statistical tests were performed by Graphpad Prism.

[0194] Device impedance characterization. The electrochemical impedance spectra of the electrodes from each device were measured using a three-electrode apparatus. Platinum wire (300 microns in diameter, 1.5 cm in immersion length) and a standard silver / silver chloride electrode were used as counter and reference electrodes, respectively. During the measurement, the device was immersed in a 1xPBS solution. The measurements were performed using an SP-150 potentiostat (Bio-logic) and its commercial software EC-lab. For each measurement, at least three frequency sweeps were measured from 1 MHz to 1 Hz to obtain statistical results. A 100 mV peak-to-peak sinusoidal voltage was applied. For each data point, the responses of 10 consecutive sinusoids (10% of the interval period duration) were accumulated and averaged.

[0195] Electrophysiological measurements and data analysis. A Blackrock CerePlex DC voltage amplifier and a 32-channel Blackrock μ(mu) digital headstage connected to the device were used to record the electrical activity of the organoids. The connector from the headamplifier to the device (16 channels only) was homemade. The organoid culture medium was grounded, and the reference electrode was also inserted into the culture medium, away from the device (more than 1 cm away). Platinum wire was used as ground and reference electrode. A sampling rate of 2000 samples per second was used. A bandpass filter (Butterworth, 4th order) from 0.3 to 1000 Hz was applied to reduce high-frequency noise. Matlab code provided by Blackrock was used to convert the raw data files into an accessible format. The data were then transferred to GraphpadPrism for post-processing.

[0196] Numerical mechanical simulations Abaqus 6.12 was used to analyze the structure. The purpose of the simulation was to evaluate the strain and stress concentration, as well as the effective stiffness of the unit strip during buckling. Figure 2E showed that during organoid development, the mesh undergoes a maximum compression of about 50% of its initial dimensions, and a maximum stretching of up to 20%. Buckling of the ribbon structures has also been observed experimentally.

[0197] The buckling modes of the unit strip in compression were analyzed using the “Linear Perturbation, Buckling” module of Abaqus for two device geometries (width / thickness = 10 μm / 2.8 μm or 20 μm / 0.8 μm). The first five buckling modes are as follows Fig. 6A and Figure 6B One end of the ribbon was subjected to an end-fixed boundary condition, while the second end was subjected to an in-plane displacement (rotation was constrained). Since modes I and II in compression were most extensively observed in the recorded optical images, these modes were simulated only in large deformations. Similarly, mode I was simulated during device stretching. For this purpose, a “Static, General” step was run to apply either 50% end-to-end compression or 20% stretching. A small concentrated force (magnitude of about 10 -6 -10 -7 To check convergence, the seeding size of the mesh (hexagonal elements) was varied from 0.5 micron to 1 micron.

[0198] As expected, the strain is higher in the thicker device (10 μm wide and 2.8 μm thick). Nevertheless, for compressive mode I ( Figure 2F and Fig. 7A ) and Mode II ( Figure 2F and Figure 7B ) and stretch mode I ( Figure 2G and Figure 7C ), the maximum principal nominal strains of the SU-8 layer and the gold layer were both below 1.2%, which is well below the fracture limit of the two materials. This result confirms that the device in this embodiment can resist compression and tension during organogenesis.

[0199] FIG. 6A to FIG. 6B A linear perturbation analysis is shown showing the buckling mode of a unit strip in compression. The deformation in the Z direction is magnified by a factor of 50 for convenience. Fig. 6A In the case of W / H=10 microns / 2.8 microns, Figure 6B In the example, W / H=20 μm / 0.8 μm.

[0200] 7A to 7C An FEA is shown showing the maximum principal nominal strain distribution during buckling of a ribbon (W / H = 10 microns / 2.8 microns). Fig. 7A Compression (mode I) from 0 to 50% effective compressive strain is shown. Figure 7B Compression (mode II) from 0 to 50% effective compressive strain is shown. Figure 7C Stretching with an effective tensile strain of 0 to 20% (mode I) is shown.

[0201] Figure 8 Simulation results showing the magnitude of the end-to-end reaction force on a unit strip as a function of the applied effective in-plane strain are shown. As expected, the magnitude of the force is higher for thicker devices. The bend in each curve corresponds to the onset of buckling in the simulation.

[0202] FIG. 9A to FIG. 9B The results show that on the 26th day of culture ( Fig. 9A ) and day 35 ( Fig. 9B ) showing the transition from irregular activity patterns to synchronized beating. This dataset was used to construct the three consecutive spike events within the dashed rectangle. Figure 4H The dashed line segment on day 26 represents the activation time of peak 1 of each channel.

[0203] Example 5

[0204] This example illustrates the implantation and distribution of flexible nanoelectronics through embryogenesis during animal development for brain-wide electrophysiological mapping.

[0205] Real-time recording of brain-wide cellular activity with single-cell-single-spike spatiotemporal resolution in behaving animals is important for understanding brain function, where local cellular activity should be studied in conjunction with behavior occurring throughout the brain and the environment as a whole. So far, several techniques have been developed to achieve in vivo brain-wide imaging. For example, functional magnetic resonance imaging (fMRI) can image whole-brain activity, but it is limited by its low spatiotemporal resolution. Combining microscopic imaging with genetically encoded reporters allows tissue-wide, cell-specific imaging, but tissue scattering and slow three-dimensional (3D) volume scanning limit fast-optical access to cells from multiple and deep brain regions in non-transparent living species. Electrophysiological tools, especially implanted microelectrodes, can allow rapid interrogation of single-cell activity within 3D tissues. State-of-the-art multi-electrode arrays driven by transistor-driven multiplexing circuits can probe 100-1000 cells in the brain and are expected to increase significantly in the near future through the integration of advanced circuits. However, current technologies cannot distribute and integrate these individually addressable microelectrodes in a minimally invasive manner to simultaneously address neurons throughout the 3D living brain of a behaving animal. Importantly, high-resolution imaging results show that cells in the mature brain are innervated at the nanoscale. Increasing the number of electrodes implanted into the brain of an adult animal will greatly increase tissue damage. Therefore, it is important to develop new biointegration methods that enable the implantation and distribution of large numbers of microelectrodes throughout the brain for brain-wide electrophysiological mapping with high spatiotemporal resolution.

[0206] In this embodiment, micron-thin, tissue-level flexible mesh electronics (called "tissue-like electronics") that fully mimic tissue scaffold structures can form long-term stable and seamless integration with neural networks for single-cell, 3D electrophysiological mapping in vitro and in vivo, which opens up opportunities for the following biointegration methods.

[0207] From the perspective of developmental biology ( Fig. 10A ), the 3D structure of the brain is differentiated from a single cell layer on the ectoderm (called the neural plate) in the embryo through cell division, proliferation and neurulation (a folding process). By integrating a layer of fully stretchable mesh electronics ( Fig. 10B ), exploiting this unique 2D to 3D cytoarchitecture transition during brain development, allowing the expansion and folding process of organogenesis to fully distribute and seamlessly integrate interconnected electrode arrays throughout the mature brain of adult animals.

[0208] In this example, electrode arrays containing 100 microelectrodes are densely packed and individually addressed into a grid-like network via microfabricated interconnectors. These interconnectors contain hierarchical and highly stretchable (>1000% stretchability) microstructures with feature sizes comparable to those of individual cells. These tissue-like electronics can be 3D reorganized by developing brain tissue to allow noninvasive implantation of electrodes throughout the 3D structure of the brain of the final adult animal ( Fig. 10C ), thereby constructing a "cyborg" for multiplexed electrophysiological mapping. Initial tests of this technology have been successfully demonstrated in brain organoids cultured in vitro.

[0209] In one example, frog (Xenopus laevis) embryos were used. They were exposed to a solution during development. Before the neural plate began to fold, the input / output (I / O) area of ​​the electronics was first fixed to a substrate to ensure that it would not fold into the embryo, and then removed from the substrate and mounted on the surface of the embryo. After the electronics grew into the embryo, a post-binding method was used to connect the I / O of the electronics to an external measurement device for recording neuronal activity.

[0210] Brain tissue from different developmental stages was collected, sectioned, cleaned, and stained to test the effects of electronics on brain development. The location of sensors was imaged using micro-CT and photocurrent imaging techniques, showing their spatial association with cells. Single-cell RNA sequencing, immunostaining, pharmacology, and classical electrophysiology were used to assess cell differentiation, neuronal connectivity, and the vitality of neuronal signaling. The behavior of the "cyborg animals" was used to assess the effects of implanted electronics on central nervous system function after development.

[0211] Brain-wide single-cell electrophysiological mapping. Using multi-electrode arrays implanted throughout the brain of cyborg animals, multiplexed electrophysiological mapping across multiple brain regions with single-cell-single-spike spatiotemporal resolution is achieved. The evolution of action potentials of multiple neurons distributed across different brain regions can be tracked and mapped throughout brain development and aging in behaving animals.

[0212] These experiments provide new methods for implanting microelectrodes throughout the brain of a behaving animal to interrogate brain-wide neuronal activity on the basis of single cell single spikes. This approach using multiplexed circuits allows for scalable implantation of large numbers of microelectrodes that are statistically significant to the number of neurons in the animal's brain. Although Xenopus laevis is described here, this is also applicable to other aquatic animals and rodents. This approach is similar to that of transgenic animals, where tissue-wide gene expression of the entire organ is allowed, as opposed to methods such as viral injection techniques that express genes in limited areas of tissue. This will provide unprecedented opportunities for high-resolution access and control of brain-wide activity, which will have a huge impact not only on neuroscience, but also on expanded / enhanced perception and cognition.

[0213] FIG. 10 shows a schematic diagram of a "cyborg" for brain-wide electrophysiological mapping. Fig. 10A Shown that the vertebrate brain develops from the folding and expansion of a 2D cell layer called the neural plate. Fig. 10B It is shown that stretchable mesh electronics can be integrated with the neural plate in the embryo. Folding of the neural plate can deliver functional electrode arrays into the neural tube with interconnects, and the I / Os remain on the outer epidermal tissue layer. Fig. 10C The reorganization of the neural tube into the final 3D brain structure is shown, with microelectrodes fully distributed and seamlessly integrated throughout the brain of a behaving animal for electrophysiological interrogation.

[0214] FIG. 11 shows a demonstration of 3D folding of stretchable mesh electronics developed via in vitro brain organoids for tissue-wide bioelectric interfacing. Fig.11A Schematic diagrams showing the step-by-step merger of stretchable mesh electronics with sensor / stimulator arrays (integrated and distributed throughout 3D living tissue) during organogenesis are shown: I: 2D stretchable mesh electronics are laminated onto a 2D stem cell sheet. Proliferation of stem cells entraps the stretchable mesh electronics. II: The stem cell sheet folds the 2D stretchable mesh electronics into a 3D structure while its self-organized 2D to 3D transition during further organoid development; III: Final organoid with differentiated different types of cells. The stretchable mesh electronics have been fully folded and merged into the organoid, with sensors and stimulators fully distributed throughout the 3D organoid. Fig. 11B Shows the corresponding Fig.11A Schematic diagram of the optical photography images of the different steps. Fig. 11C yes Fig. 11B and Fig.11DBright-field microscopy images of the area highlighted by the dashed box show tightly packed stretchable mesh electronics (I) folding through the organoid (II) and fully expanded distributed sensors / stimulators throughout the 3D organoid (III). Fig.11D Bright-field microscopy images showing representative tissue-scale electronics innervating brain organoids are shown.

[0215] Although several embodiments of the present invention have been described and illustrated herein, a person of ordinary skill in the art will readily recognize various other means and / or structures for performing the functions described herein and / or obtaining the results and / or one or more advantageous aspects described herein, and each such variation and / or modification is considered to be within the scope of the present invention. More generally, it will be readily understood by those skilled in the art that all parameters, dimensions, materials, and configurations described herein are exemplary, and the actual parameters, dimensions, materials, and / or configurations will depend on one or more specific applications for which the teachings of the present invention are used. Those skilled in the art will recognize, or will be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experiments. Therefore, it should be understood that the foregoing embodiments are given only as examples, and within the scope of the appended claims and their equivalents, the present invention may be implemented in a manner different from that specifically described and required. The present invention relates to each individual feature, system, article, material, kit, and / or method described herein. In addition, if two or more such features, systems, articles, materials, kits, and / or methods do not contradict each other, any combination of two or more such features, systems, articles, materials, kits, and / or methods is included within the scope of the present invention.

[0216] In the event that this specification and the documents incorporated by reference include conflicting and / or inconsistent disclosures, this specification shall prevail. If two or more documents incorporated by reference contain conflicting and / or inconsistent disclosures, the document with the later effective date shall prevail.

[0217] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0218] As used in the specification and claims, the indefinite articles "a" and "an" should be understood to mean "at least one", unless explicitly indicated to the contrary.

[0219] As used in the specification and claims, the phrase "and / or" should be understood to mean "either or both" of the elements so connected, i.e., elements that are present in combination in some cases and separately in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the elements so connected. In addition to the elements specifically identified by the "and / or" clause, other elements may optionally be present, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, when used in conjunction with open language such as "comprising", in one embodiment, a reference to "A and / or B" may refer to only A (optionally including elements other than B); in another embodiment, to only B (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); and so on.

[0220] As used in this specification and in the claims, "or" should be understood to have the same meaning as "and / or" defined above. For example, when separating items in a list, "or" or "and / or" should be interpreted as inclusive, i.e., including at least one, but also more than one of a number or series of elements, and optionally, additional unlisted items. Only terms that explicitly indicate the contrary, such as "only one of" or "exactly one of", or when used in the claims, "consisting of..." will refer to including exactly one element of a plurality or series of elements. In general, as used herein, the term "or" should be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") only when preceded by an exclusive term, such as "either," "one of," "only one of," or "exactly one of."

[0221] As used in this specification and claims, when referring to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "at least one of A and / or B") may refer to at least one, optionally including more than one, A, without B (and optionally including elements other than B) in one embodiment; to at least one, optionally including more than one, B, without A (and optionally including elements other than A) in another embodiment; to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements) in yet another embodiment; and so on.

[0222] When the word "about" is used herein to refer to a number, it should be understood that another embodiment of the present invention includes the number not modified by the presence of the word "about".

[0223] It should also be understood that in any method claimed herein that includes more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described unless explicitly stated to the contrary.

[0224] In the claims, as well as in the above description, all transitional phrases, such as "comprising," "including," "carrying," "having," "containing," "involving," "maintaining," "consisting of," etc., are to be understood as open-ended, i.e., meaning including but not limited to. Only the transitional phrases "consisting of" and "consisting essentially of" are closed or semi-closed transitional phrases, respectively, as described in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. A product comprising: A biological structure at least partially embedding a stretchable cell scaffold therein, The cell scaffold defines at least a portion of an electrical circuit.

2. The article of claim 1, wherein the cell scaffold is stretchable by at least 10% in a linear direction.

3. The article of any one of claims 1 or 2, wherein the cell scaffold is stretchable by at least 20% in a linear direction.

4. The article of claim 3, wherein the cell scaffold is stretchable by at least 50% in a linear direction.

5. The article of claim 4, wherein the cell scaffold is stretchable by at least 100% in a linear direction.

6. The article of claim 5, wherein the cell scaffold is stretchable by at least 1000% in a linear direction.

7. The article of any one of claims 1-6, wherein the cell scaffold is foldable by at least 90°.

8. The article of claim 7, wherein the cell scaffold is foldable by at least 180°.

9. The article of any one of claims 1-8, wherein the stretchable cell scaffold has a first packing fraction of less than 40% in the absence of the biostructure.

10. The article of claim 9, wherein the first fill fraction is less than 20%.

11. The article of claim 10, wherein the first fill fraction is less than 10%.

12. The article of any one of claims 9-11, wherein the cell scaffold has a second filling rate of less than 10% when embedded in the biostructure.

13. The article of claim 12, wherein the second fill fraction is less than 5%.

14. The article of any one of claims 1-13, wherein the cell scaffold has a second filling rate when embedded in the biostructure that is less than 50% of the first filling rate.

15. The article of claim 14, wherein the cell scaffold has a second filling rate when embedded in the biostructure that is less than 30% of the first filling rate.

16. The article of any one of claims 1-15, wherein at least a portion of the cell scaffold exhibits a strain of at least 10%.

17. The article of claim 16, wherein at least a portion of the cell scaffold exhibits a strain of at least 20%.

18. The article of any one of claims 1-17, wherein the cell scaffold comprises at least 30% by volume of the biostructure.

19. The article of claim 18, wherein the cell scaffold comprises at least 50% by volume of the biostructure.

20. The article of claim 19, wherein the cell scaffold comprises at least 70% by volume of the biostructure.

21. The article of any one of claims 1-20, wherein at least 30% by volume of the biostructure is no more than 10 microns from the cell scaffold embedded therein.

22. The article of claim 21, wherein at least 50% by volume of the biostructure is no more than 10 microns from the cell scaffold embedded therein.

23. The article of claim 22, wherein at least 70% by volume of the biostructure is no more than 10 microns from the cell scaffold embedded therein.

24. The article of any one of claims 1-23, wherein the biostructure completely embeds the cell scaffold.

25. The article of any one of claims 1-24, wherein no portion of the cell scaffold is exposed to the exterior of the biological structure.

26. The article of any one of claims 1-25, wherein at least a portion of the circuitry of the cell scaffold is electrically connected to an electrical device external to the biological structure.

27. The article of claim 26, wherein the cell scaffold is electrically connected to the electrical device via a connector.

28. The article of claim 27, wherein the connector is a cable.

29. The article of claim 28, wherein the cable is a ribbon cable.

30. The article of claim 28, wherein the cable is a flexible flat cable.

31. The article of manufacture of claim 28, wherein the cable is an 8-pin cable.

32. The article of manufacture of claim 28, wherein the cable is a 16-pin cable.

33. The article of any one of claims 26-32, wherein the electrical device comprises a computer.

34. The article of any one of claims 26-33, wherein the electrical device comprises a transmitter.

35. The article of any one of claims 1-34, wherein the cell scaffold comprises a mesh comprising a plurality of nodes, at least some of the nodes being connected by interconnectors to form the mesh.

36. The article of claim 35, wherein the grid is a rectangular grid.

37. The article of claim 35, wherein the grid is an irregular grid.

38. The article of any one of claims 35-37, wherein at least some of the interconnects are serpentine.

39. The article of any one of claims 35-38, wherein at least some of the interconnects are stretchable.

40. The article of any one of claims 35-39, wherein at least some of the interconnects include at least one inflection point.

41. The article of any one of claims 35-40, wherein at least some of the interconnects have a maximum cross-sectional dimension of less than 50 microns.

42. The article of claim 41, wherein the largest cross-sectional dimension is less than 30 microns.

43. The article of any one of claims 1-42, wherein the cell scaffold comprises a photoresist.

44. The article of claim 43, wherein the photoresist comprises SU-8.

45. The article of any one of claims 43 or 44, wherein the photoresist comprises a polymer formed by photocuring a fluorinated monomer comprising a crosslinkable functional group using a photoinitiator.

46. ​​The article of any one of claims 43-45, wherein the photoresist comprises a hydrogel.

47. The article of any one of claims 43-46, wherein the photoresist comprises perfluoropolyether dimethacrylate (PFPE-DMA).

48. The article of any one of claims 1-47, wherein the cell scaffold comprises a metal pathway.

49. The article of claim 48, wherein at least some of the metal paths comprise gold.

50. The article of any one of claims 48 or 49, wherein at least some of the metal paths comprise chromium.

51. The article of any one of claims 48-50, wherein at least some of the metal paths are flexible.

52. The article of any one of claims 1-51, wherein the cell scaffold comprises an electrode.

53. The article of claim 52, wherein the electrode comprises a metal.

54. The article of claim 53, wherein the electrode comprises platinum.

55. The article of any one of claims 1-54, wherein the cell scaffold comprises a conductive polymer.

56. The article of claim 55, wherein the conductive polymer is in contact with the electrode.

57. The article of any one of claims 55 or 56, wherein the conductive polymer comprises poly(3,4-ethylenedioxythiophene).

58. The article of any one of claims 1-57, wherein the cell scaffold comprises an extracellular matrix protein.

59. The article of any one of claims 1-58, wherein the cell scaffold comprises nanoscale wires.

60. The article of claim 59, wherein the nanoscale wires are pH sensitive.

61. The article of any one of claims 59 or 60, wherein the nanoscale wires have a conductance of at least about 1 microSiemens.

62. The article of any one of claims 59-61, wherein the nanoscale wire is responsive to an electrical property external to the nanoscale wire.

63. The article of any one of claims 59-62, wherein the nanoscale wires exhibit a voltage sensitivity of at least about 5 microSiemens / Volt.

64. The article of any one of claims 1-63, wherein the biological structure is an organoid.

65. The article of any one of claims 1-63, wherein the biological structure is an organ.

66. The article of any one of claims 1-63, wherein the biological structure is a living organism.

67. The article of manufacture of claim 66, wherein the organism is a vertebrate.

68. The article of any one of claims 66 or 67, wherein the organism is a frog.

69. The article of any one of claims 66 or 67, wherein the organism is a rodent.

70. An article comprising: A cell scaffold defining at least a portion of an electrical circuit, wherein the cell scaffold is stretchable by at least 20% in a linear direction at a force not exceeding 1 micronewton.

71. A method comprising: exposing the cells to a stretchable cell scaffold defining at least a portion of the circuit; as well as The cells are grown to form a biological structure at least partially encapsulating the cell scaffold therein.

72. The method of claim 71, wherein the cells comprise stem cells.

73. The method of any one of claims 71 or 72, wherein the cell is part of an embryo.

74. The method of claim 73, wherein the embryo is a vertebrate embryo.

75. The method of any one of claims 71-74, wherein the cell comprises a cancer cell.

76. The method of any one of claims 71-75, wherein during the cell culturing, the cells distort the cell scaffold by at least 20% in a linear direction.

77. The method of any one of claims 71-76, further comprising electrically connecting the cell scaffold to an electrical device external to the biological structure.

78. A method comprising: exposing the embryo to a stretchable cell scaffold defining at least a portion of a circuit; as well as The embryo is grown to form an organism embedding the cell scaffold.

79. A method comprising: The electrical properties of the biostructures are determined with a resolution of at least 1 mm using a stretchable cell scaffold embedded within the biostructures.

80. A method comprising: An electrical signal is applied to at least a portion of the biological structure using a stretchable cell scaffold embedded within the biological structure.

81. A method comprising: patterning a first layer comprising a first photoresist onto the sacrificial layer; patterning metal paths into the first layer; depositing a second layer comprising a second photoresist onto the first layer; as well as At least a portion of the sacrificial layer is removed to produce a cell scaffold comprising the first layer, the metal path, and the second layer, wherein the cell scaffold is stretchable.

82. The method of claim 81 further comprising patterning electrodes on the metal paths.

83. The method of any one of claims 81 or 82, further comprising patterning a conductive polymer on the electrode.

84. The method of claim 83, wherein the conductive polymer comprises poly(3,4-ethylenedioxythiophene) (PEDOT).

85. The method of any one of claims 81-84, further comprising attaching a connector to the cell scaffold.

86. The method of claim 85, wherein the connector comprises a cable.

87. The method of any one of claims 81-86, further comprising exposing the cell scaffold to cells.

88. The method of any one of claims 81-87, further comprising exposing the cell scaffold to an embryo.

89. The method of any one of claims 81-88, wherein the sacrificial layer comprises nickel.

90. The method of any one of claims 81-89, comprising removing at least a portion of the sacrificial layer using an etchant.

91. The method of any one of claims 81-90, wherein the first photoresist and the second photoresist are substantially identical.

92. The method of any one of claims 81-91, wherein the first photoresist comprises SU-8.

93. The method of any one of claims 81-92, wherein the first photoresist comprises perfluoropolyether dimethacrylate (PFPE-DMA).

94. The method of any one of claims 81-93, wherein the metal path comprises gold.

95. The method of any one of claims 81-94, wherein the metal path comprises chromium.

Images