Cell-free substrate and method for producing same

Abstract

Provided are acellular substrates that provide for continuous 3D growth of a variety of cells and / or tissues, and methods of making the same. The acellular substrates have a three-dimensional (3D) macrostructure defined by a continuous matrix of extracellular matrix (ECM) material associated with a first cell type of interest and a network of microporous channels and / or chambers that extend throughout the continuous matrix of ECM material associated with the first cell type of interest. Methods of forming personalized grafts using a patient's own cells are also provided.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Patent Application No. 63 / 296,274, filed January 4, 2022, which is expressly incorporated by reference in its entirety.

[0002] Inventive embodiments of the present disclosure generally relate to an acellular substrate that provides for continuous 3D growth of a variety of cells and / or tissues, and methods of making the same, the acellular substrate having a three-dimensional (3D) macrostructure defined by a continuous matrix of extracellular matrix (ECM) material associated with a first target cell type, and a network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material associated with the first target cell type. Methods of forming personalized grafts using a patient's own cells are also provided. [Background technology]

[0003] Connective tissues such as bone, cartilage, skin, nerve, lung, muscle, and many others contain cells that secrete and organize extracellular matrix (ECM) proteins to create a scaffold that allows cells to migrate, grow, and organize to form tissues. The interplay between the organization and structure of the ECM's composition is well documented. For example, each tissue has different composition and organization structures that instruct cell types how to arrange, organize, and function. Summary of the Invention [Problem to be solved by the invention]

[0004] There remains a need in the art for the development of scaffolds that allow for the creation of biological scaffolds from any connective tissue of interest, such as ECM scaffolds (e.g., acellular substrates) that can receive autologous cells from a patient to construct an individualized graft. [Means for solving the problem]

[0005] One or more embodiments of the present invention may address one or more of the problems described above. Certain embodiments according to the present invention provide an acellular substrate (e.g., scaffold) that includes a three-dimensional (3D) macrostructure defined by a continuous matrix of extracellular matrix (ECM) material associated with a first target cell type and a network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material associated with the first target cell type. The 3D macrostructure may include a thickness defined by a top surface, a bottom surface, and at least one lateral edge extending from the top surface to the bottom surface.

[0006] In another aspect, certain embodiments according to the present invention provide a method of forming an acellular substrate comprising: (i) forming or disposing a network of microstrands and / or micropods comprising a degradable hydrogel material within a mold; (ii) seeding the network of microstrands and / or micropods by adding initial culture medium comprising cells of a target cell type within the mold containing the network of microstrands and / or micropods; (iii) supplying the cells by perfusing fresh culture medium through the mold to nourish the cells until the tissue grows and expands to fill the mold; (iv) performing a decellularization operation on the tissue located within the mold to form a continuous matrix of ECM material associated with the target cell type; and (v) degrading and removing the network of microstrands and / or micropods to form a network of microporous channels and / or chambers spanning the continuous matrix of ECM material associated with the target cell type to obtain an acellular substrate.

[0007] In another aspect, certain embodiments according to the present invention provide a method of forming an individualized graft comprising: (i) preparing or forming one or more acellular substrates as described and disclosed herein, wherein a first target cell type is associated with a patient's tissue having an abnormality, such as a particular organ tissue; (ii) seeding the one or more acellular substrates with healthy native cells associated with the patient's tissue having an abnormality; and (iii) providing a culture medium to the healthy native cells and allowing the healthy native cells to proliferate throughout the network of microporous channels and / or chambers of the acellular substrate to form the individualized graft.

[0008] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein but rather these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 illustrates a cell-free substrate according to certain embodiments of the present invention. [Diagram 2] FIG. 1 shows a cube-like mold with a network of microchannels and / or micropods 3D printed or placed within it. [Diagram 3] FIG. 1 shows how a cell type of interest is seeded onto the network of microchannels and / or micropods and an extracellular matrix of the cell type of interest is grown to fill the cube-like mold. [Figure 4] FIG. 1 shows the decellularized tissue and subsequent degradation or dissolution of the hydrogel material to form an intermediate acellular matrix. [Diagram 5] FIG. 13 shows how the intermediate acellular substrate is freeze-dried and subsequently removed from the cube-like mold and then sterilized. [Figure 6] ~ [Figure 9] FIG. 1 illustrates various configurations of bonded multiple acellular substrates according to certain embodiments of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present invention will now be described in more detail below with reference to the accompanying drawings, in which some, but not all, embodiments of the present invention are shown. Indeed, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0011] The disclosed invention generally relates to a three-dimensional (3D) acellular matrix that contains structural components and cues that direct cells how to behave to form tissues. In this regard, each tissue has a variety of configurations and organizational structures that direct cell types how to arrange, organize, and function. Accordingly, certain embodiments of the present invention include acellular matrices and methods of making same that allow for the creation of biological scaffolds from any connective tissue of interest. As described in more detail below, degradable inverted hydrogel molds can be sterilized and placed into, for example, cube-like molds that can range in volume from cubic centimeters to cubic decimeters. Cells from a particular tissue are added to the mold and cultured by perfusing the mold with culture medium to nourish the cells until a complete tissue is formed. Once a complete tissue is formed, the cells are removed through decellularization by applying a mild detergent and dialyzing the ECM. The ECM can then be subjected to multiple freeze-thaw cycles to remove any remaining cellular debris. The ECM can then be washed with a mild detergent and dialyzed again. The hydrogel inversion mold can then be dissolved, for example, via photolysis. The ECM can then be lyophilized and stored until use. The ECM scaffold (e.g., acellular substrate) can be used, for example, as a building block to create specific tissues. For example, if an ECM scaffold (e.g., acellular substrate) is first created from keratinocytes, and induced pluripotent stem cells from a patient are added to the ECM scaffold (e.g., acellular substrate), the ECM scaffold (e.g., acellular substrate) can be used to form a new autologous epidermal graft. According to certain embodiments, for example, the present invention provides for the use of ECM scaffolds (e.g., acellular substrates) to produce an ECM scaffold (e.g., acellular substrate) that can receive autologous cells from a patient to construct an individualized graft.

[0012] According to certain embodiments of the invention, the acellular substrate may include a custom scaffold designed to culture cells that are used to form a custom ECM scaffold for a particular patient. For example, cells are removed from the ECM scaffold and the patient's own cells are added to the ECM scaffold designed for that cell. That is, if a patient has a tissue defect, a custom mold (e.g., a 3D network of microstrands and / or micropods formed from a hydrogel material based on the tissue morphology) can be created to repair the defect and a custom scaffold (e.g., dissolution or degradation of the hydrogel material to form a 3D network of microstrands and / or micropods) personalized graft can be created for that patient. In this regard, the tissue microstructure can be used to differentiate stem cells into the required cells without the need for growth factor cocktails. That is, according to certain embodiments of the invention, custom scaffold designs can be provided to direct the differentiation fate of stem cells to create a personalized tissue graft for a patient to correct the tissue defect. For example, by using magnetic resonance imaging and 3D printing, certain embodiments of the present invention may provide for preparing a custom hydrogel mold (a 3D network of microstrands and / or micropods formed from hydrogel material based on the tissue morphology of the tissue) to create a custom ECM scaffold (e.g., an acellular substrate). Until now, structure alone has not been considered sufficient to differentiate and control cell fate to form tissue. The main mechanism behind cell fate control has always been thought to be based on growth factors and signals. However, certain embodiments of the present invention have revealed that substrates and structures control cell fate, which can be used to create novel scaffolds to build custom implants to treat tissue abnormalities in patients.

[0013] According to certain embodiments of the present invention, the method of forming an acellular substrate allows for custom scaffolds to be created from a patient's own anatomical structures to form implants from the application of the patient's own cells. In this regard, for example, magnetic resonance imaging and stereolithography (3D printing) can be used to print an inverted hydrogel scaffold mold into which cells can be seeded to form a custom extracellular matrix scaffold based on the patient's own tissue. For example, the inverted hydrogel scaffold mold is a network of microstrands and / or micropods structured based on the tissue morphology of the target cell lineage. Furthermore, the method according to certain embodiments of the present invention allows for tailor-made scaffolds to be created from various compositions of extracellular matrix based on the cell volume and ratio seeded on the hydrogel scaffold mold. In this regard, a single cell-free substrate may be seeded with multiple cell lines of interest, or one cell-free substrate may be seeded with a first cell line of interest while an adjacent cell-free substrate is seeded with a second cell line of interest and allowed to grow together, forming an interface between the two cell lines of interest.One or more subject cells may be used to detect and characterize glycosaminoglycans (GAGs), proteoglycans, hyaluronic acid (HA), heparin sulfate (HS), chondroitin sulfate (CS), keratan sulfate (KS), collagen types (e.g., fibrillar (I, II, III, V, XI), facilit (IX, XII, XIV), short chain (VIII, X), basement membrane (IV), and others (VI, VII, XIII), elastin, DNA, RNA, fibronectin, and glycoproteins, laminin-111 (laminin-1), laminin-211 (laminin-2), laminin-121 (laminin-3), laminin-221 (laminin-4), laminin-332 / laminin-3 The cells can form a continuous extracellular matrix comprising individual extracellular matrix components such as A32 (laminin-5 / laminin-5A), laminin-3B32 (laminin-5B), laminin-311 / laminin-3A11 (laminin-6 / laminin-6A), laminin-321 / laminin-3A21 (laminin-7 / laminin-7A), laminin-411 (laminin-8), laminin-421 (laminin-9), laminin-511 (laminin-10), laminin-521 (laminin-11), laminin-213 (laminin-12), laminin-423 (laminin-14), laminin-522, laminin-523 (laminin-15), or combinations thereof.

[0014] According to certain embodiments of the present invention, the custom-formed extracellular matrix facilitates the organization and alignment of the patient's stem cells on the extracellular matrix scaffold. Furthermore, the extracellular matrix scaffold allows the patient's stem cells to differentiate into cells necessary for tissue formation. As described above, the method according to certain embodiments of the present invention allows for the custom formation of tissue grafts using the patient's own cells that are biocompatible with a particular patient, rather than relying on synthetically produced tissue or donor tissue.

[0015] According to certain embodiments of the invention, the methods disclosed herein allow for the creation of bioengineered patient tissues or delivery of therapeutic cells for regenerative applications without relying on synthetic tissue, donor tissue, or bioengineered tissue created from donor tissue. For example, certain embodiments of the invention provide methods for creating custom autologous grafts using a patient's own stem cells and / or somatic cells. According to certain embodiments of the invention, the invention advantageously eliminates the need for patients to take immunosuppressant medications because the tissue is generated from the patient rather than from a donor or synthetic material.

[0016] Certain embodiments according to the invention provide an acellular substrate (e.g., scaffold) comprising a three-dimensional (3D) macrostructure defined by a continuous matrix of extracellular matrix (ECM) material associated with a first cell type of interest and a network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material associated with the first cell type of interest. The 3D macrostructure may include a top surface, a bottom surface, and a thickness defined by at least one lateral edge extending from the top surface to the bottom surface. For example, FIG. 1 shows an acellular substrate 1 having a 3D macrostructure defined by a continuous matrix of ECM material 10 and a network of microporous channels and / or chambers 30 extending throughout the continuous matrix of ECM material. The continuous matrix of ECM material 10 in FIG. 1 is rendered transparent to show the network of microporous channels and / or chambers 30 extending throughout it.

[0017] According to certain embodiments of the invention, the acellular substrate may further comprise at least one male connecting component and / or at least one female connecting component, each of which may be used to connect or join two or more individual acellular substrates together. As an example, the at least one male connecting component (if present) may comprise a first male connecting component extending outwardly from at least one side edge. For example, the at least one side edge may comprise a first side edge and a second side edge, wherein the at least one male connecting component comprises a first male connecting component extending outwardly from the first side edge and a second male connecting component extending outwardly from the second side edge. Additionally or alternatively, the at least one male connecting component comprises a third male connecting component extending outwardly from the top surface. According to certain embodiments of the invention, the acellular substrate may comprise a first connecting female component extending inwardly from at least one side edge towards the interior of the 3D macrostructure. For example, the at least one side edge comprises a third side edge and a fourth side edge, wherein the at least one connecting female component comprises a first connecting female component extending inwardly from the third side edge towards the interior of the 3D macrostructure and a second connecting female component extending inwardly from the fourth side edge towards the interior of the 3D macrostructure. According to certain embodiments of the invention, the first side edge and the third side edge define a first pair of opposing side edges, while the second side edge and the fourth side edge define a second pair of opposing side edges. Additionally or alternatively, the at least one connecting female component comprises a third connecting female component extending inwardly from the lower surface towards the interior of the 3D macrostructure.

[0018] According to certain embodiments of the invention, the 3D macrostructure, excluding the at least one interlocking male component and the at least one interlocking female component (if present), defines a cube, a rectangular prism, or a triangular prism. According to certain embodiments of the invention, the 3D macrostructure, excluding the at least one interlocking male component and the at least one interlocking female component (if present), defines a polygonal prism having 3-12 side edges, such as at least about 3 sides, at least about 4 sides, at least about 5 sides, at least about 6 sides, at least about 7 sides, and at least about 8 sides, and / or any of the following: up to about 12 sides, up to about 11 sides, up to about 10 sides, up to about 9 sides, and up to about 8 sides.

[0019] According to certain embodiments of the present invention, the at least one side edge includes a first side edge, a second side edge, and an arched side edge adjacent to and located between the first side edge and the second side edge. For example, the first side edge may include at least one interlocking male component extending outward from the first side edge, and the second side edge may include at least one interlocking female component extending inward from the second side edge toward the interior of the 3D macrostructure. Additionally or alternatively, the at least one interlocking male component includes a second interlocking male component extending outward from the top surface. Additionally or alternatively, the at least one interlocking female component includes a second interlocking female component extending inward from the bottom surface toward the interior of the 3D macrostructure. By way of example only, the 3D macrostructure, excluding the at least one interlocking male component and the at least one interlocking female component, defines a half cylinder, such as ⅛ cylinder to ½ cylinder, e.g., ⅛ cylinder, ¼ cylinder, ⅓ cylinder, or ½ cylinder.

[0020] According to one particular embodiment of the present invention, the upper surface is approximately 0.25 cm 2 ~about 25cm 2 For example, any of the following: at least about 0.25 cm 2 , at least about 0.5 cm 2 , at least about 0.75 cm 2 , at least about 1 cm 2, at least about 1.5 cm 2 , at least about 2 cm 2 , at least about 5 cm 2 , at least about 8 cm 2 , at least about 10 cm 2 , and at least about 12 cm 2 , and / or any of the following: approx. 25 cm 2 , about 22cm 2 , about 20cm 2 , about 18cm 2 , about 15cm 2 , and about 12 cm 2 Additionally or alternatively, the lower surface comprises a macroscopic surface area of ​​about 0.25 cm 2 ~about 25cm 2 For example, any of the following: at least about 0.25 cm 2 , at least about 0.5 cm 2 , at least about 0.75 cm 2 , at least about 1 cm 2 , at least about 1.5 cm 2 , at least about 2 cm 2 , at least about 5 cm 2 , at least about 8 cm 2 , at least about 10 cm 2 , and at least about 12 cm 2 , and / or any of the following: approx. 25 cm 2 , about 22cm 2 , about 20cm 2 , about 18cm 2 , about 15cm 2 , and about 12 cm 2 Additionally or alternatively, the thickness of the 3D macrostructure is from about 0.5 cm to about 3 cm, e.g., any of the following: at least about 0.5 cm, at least about 0.75 cm, at least about 1 cm, at least about 1.25 cm, and at least about 1.5 cm, and / or any of the following: up to about 3 cm, up to about 2.5 cm, up to about 2 cm, and up to about 1.5 cm. Additionally or alternatively, each of the at least one female connecting component is configured to receive a corresponding at least one male connecting component of the second acellular substrate.

[0021] According to certain embodiments of the invention, the network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material is between about 100 microns and about 800 microns, e.g., any of the following: at least about 100 microns, at least about 120 microns, at least about 150 microns, at least about 180 microns, at least about 200 microns, at least about 220 microns, and at least about 250 microns, and / or any of the following: up to about 800 microns, up to about 780 microns, up to about 750 microns. In some embodiments, the average diameter may be up to about 720 microns, up to about 700 microns, up to about 680 microns, up to about 650 microns, up to about 620 microns, up to about 600 microns, up to about 580 microns, up to about 550 microns, up to about 520 microns, up to about 500 microns, up to about 480 microns, up to about 450 microns, up to about 420 microns, up to about 400 microns, up to about 380 microns, up to about 350 microns, up to about 320 microns, up to about 300 microns, up to about 280 microns, and up to about 250 microns.

[0022] In another aspect, certain embodiments according to the present invention provide a method of forming an acellular substrate comprising: (i) forming or disposing a network of microstrands and / or micropods comprising a degradable hydrogel material within a mold; (ii) seeding the network of microstrands and / or micropods by adding initial culture medium comprising cells of a target cell type within the mold containing the network of microstrands and / or micropods; (iii) supplying the cells by perfusing fresh culture medium through the mold to nourish the cells until the tissue grows and expands to fill the mold; (iv) performing a decellularization operation on the tissue located within the mold to form a continuous matrix of ECM material associated with the target cell type; and (v) degrading and removing the network of microstrands and / or micropods to form a network of microporous channels and / or chambers spanning the continuous matrix of ECM material associated with the target cell type to obtain an acellular substrate.

[0023] According to certain embodiments of the present invention, the formation or arrangement of the network of microstrands and / or micropods includes the implementation of additive manufacturing techniques such as 3D printing of digital photosynthetic printing. The structure of the microstrand and / or micropod network is selected based on the cell morphology of the target cell type. For example, the cell morphology of the target cell type has a target matrix structure and a target microporous network of channels and / or chambers, where the structure of the microstrand and / or micropod network is similar or identical to the target microporous network of channels and / or chambers. Additionally or alternatively, the microstrand and / or micropod network can be between about 100 microns and about 800 microns, such as any of the following: at least about 100 microns, at least about 120 microns, at least about 150 microns, at least about 180 microns, at least about 200 microns, at least about 220 microns, and at least about 250 microns, and / or any of the following: up to about 800 microns, up to about 780 microns, up to about 750 microns, up to about 720 microns. up to about 700 microns, up to about 680 microns, up to about 650 microns, up to about 620 microns, up to about 600 microns, up to about 580 microns, up to about 550 microns, up to about 520 microns, up to about 500 microns, up to about 480 microns, up to about 450 microns, up to about 420 microns, up to about 400 microns, up to about 380 microns, up to about 350 microns, up to about 320 microns, up to about 300 microns, up to about 280 microns, and up to about 250 microns.

[0024] According to certain embodiments of the invention, the network of microstrands and / or micropods comprises a selectively degradable hydrogel material comprising one or more degradable polymers, such as one or more biopolymers of biological origin. For example, the one or more biopolymers of biological origin may comprise polynucleotides, polysaccharides, polypeptides, or any combination thereof. According to certain embodiments of the invention, the one or more biopolymers include collagen, gelatin, laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and proteins that can be gently degraded, such as by using protein-specific enzymes, ionic solvents, neutral detergents, weak acids, and peroxides to break the biopolymer chains. According to certain embodiments of the invention, the one or more biopolymers include degradable monomers including esters such as hydroxybutyrate, lactic acid, glycolic acid, and caprolactone, anhydrides such as adipic acid and sebacic acid, cellulose, alginate, pectin, dextrin, sugars such as chitosan, hyaluronan, chondroitin sulfate, and heparin, proteins, nucleotides (DNA, RNA), peptides such as collagen, gelatin, silk, and fibrin, urethanes, phosphates, carbonates, and vinyl chlorides. Additionally or alternatively, the selectively degradable hydrogel material further includes synthetic polymers such as polyesters, polyanhydrides, polycarbonates, polyurethanes, polyphosphates, or combinations thereof. According to certain embodiments of the invention, the selectively degradable hydrogel material comprises 0% to 25% by weight of synthetic polymer, for example, any of the following: at least about 0% by weight, at least about 1% by weight, at least about 3% by weight, at least about 5% by weight, at least about 8% by weight, and at least about 10% by weight of synthetic polymer, and / or any of the following: up to about 25% by weight, up to about 20% by weight, up to about 15% by weight, and up to about 12% by weight of synthetic polymer.

[0025] According to certain embodiments of the invention, performing a decellularization procedure on the tissue disposed within the mold includes treating the tissue with a detergent, followed by dialysis of the continuous matrix of ECM material associated with the cell type of interest. In this regard, primary cells can be selectively removed while maintaining the structure of the ECM material. After decellularization, the method can include lyophilizing the continuous matrix of ECM material associated with the cell type of interest. The method can further include removing the acellular substrate from the mold and / or sterilizing the acellular substrate, such as by an electron beam or gamma irradiation procedure.

[0026] 2-5 show a general method of making an acellular substrate. FIG. 2 shows a cube-like mold 100 with a network of microstrands and / or micropods 130 3D printed or placed therein. The network of microstrands and / or micropods 130 may be formed from a degradable or dissolvable hydrogel material such as those described and disclosed herein. FIG. 3 shows seeding a cell type of interest 33 onto the network of microchannels and / or micropods 130 and growing an extracellular matrix of the cell type of interest to fill a cube-like mold that may include at least one male connecting component and / or at least one female connecting component (not shown). After the cube-like mold 100 is filled with the extracellular matrix material, not only is a decellularized tissue 10 produced as shown in FIG. 4, but the hydrogel material (e.g., the network of microstrands and / or micropods) 30 is subsequently degraded or dissolved to form an intermediate acellular substrate. Figure 5 shows the intermediate acellular substrate being freeze-dried and subsequently removed from the cube-like mold and then sterilized. Figure 1 shows the resulting acellular matrix formed according to certain embodiments of the present invention.

[0027] In another aspect, certain embodiments according to the present invention provide a method of forming a personalized graft comprising: (i) preparing or forming one or more acellular substrates as described and disclosed herein, where a first target cell type is associated with a patient's tissue having an abnormality, such as a particular organ tissue; (ii) seeding the one or more acellular substrates with healthy native cells associated with the patient's tissue having an abnormality; and (iii) providing a culture medium to the healthy native cells, allowing the healthy native cells to proliferate throughout the network of microporous channels and / or chambers of the acellular substrate to form a personalized graft. For example, cells of a first cell type can be removed from the ECM material and the patient's own cells can be added to the acellular substrate designed for that cell. That is, if a patient has a tissue defect, one can create a custom mold (e.g., a 3D network of microstrands and / or micropods formed from a hydrogel material based on the histomorphology of the tissue) to repair the defect and create a custom scaffold (e.g., dissolution or degradation of the hydrogel material to form the 3D network of microstrands and / or micropods) personalized implant for that patient. Thus, methods according to certain embodiments of the present invention provide for creating a custom scaffold from a patient's own anatomy and forming an implant from the application of the patient's own cells.

[0028] According to certain embodiments of the invention, the one or more acellular substrates include a first acellular substrate and a second acellular substrate, where the first acellular substrate and the second acellular substrate are the same. For example, the first acellular substrate may be bonded to the second acellular substrate, thus resulting in a multi-layer acellular scaffold. The method may include growing healthy native cells throughout the aggregate network of microporous channels and / or chambers of the multi-layer acellular scaffold to form an individualized graft. According to certain embodiments of the invention, the one or more acellular substrates include at least two acellular substrates bonded together to define the multi-layer acellular scaffold, or any of the following: at least about 2, at least about 3, at least about 5, 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, and at least about 100 acellular substrates. Figures 6-9 show non-limiting examples of various configurations 200 of joined acellular substrates according to certain embodiments of the present invention. As shown in Figures 6-9, the acellular substrates may be linked or otherwise joined together in a number of different 3D geometric shapes. EXAMPLES

[0029] The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way, i.e., the specific features described in the following examples are merely illustrative and not limiting.

[0030] A unique closed system is designed in which liquids are applied and aspirated as needed. An inverted hydrogel mold (e.g., a network of microstrands and micropods) is first deposited in a cube-like mold. Sterile saline is then applied twice for 5 minutes each. Then, cell culture medium containing 1 million cells per mL is applied to each mold. The medium is completely replaced by perfusion every 24 hours. After a minimum of 7 days, the cell culture medium is removed and the mold is washed with sterile saline. Sodium dodecyl sulfate (SDS) and sodium palmitate (SP) are applied to each mold for up to 72 hours with gentle agitation. The SDS and SP are aspirated and the ECM is dialyzed against sterile deionized water (DI) for up to 72 hours. The ECM is then cycled from -170°C to 37°C for 6 days. SDS and SP are applied to each mold for up to 72 hours with gentle agitation. SDS and SP were aspirated and the ECM was dialyzed against sterile DI water for up to 72 hours. The inverted mold (e.g., microstrand and micropod network) was then dissolved by photolysis. The ECM was washed three times with sterile saline and 10% antibiotics for 60 minutes under UV light. The ECM was washed with sterile saline. The ECM was then packaged, lyophilized and stored.

[0031] Those skilled in the art will appreciate that these and other modifications and variations may be made to the present invention without departing from the spirit and scope of the present invention, which is more particularly set forth in the appended claims. It should further be understood that aspects of the various embodiments may be interchanged in whole or in part. Moreover, those skilled in the art will appreciate that the foregoing description is for illustrative purposes only and is not intended to limit the invention as further described in such appended claims. Accordingly, the spirit and scope of the appended claims should not be limited to the exemplary descriptions of the interpretations contained herein. [Explanation of symbols]

[0032] 1 Cell-free substrate 10. Continuous Matrix of ECM Materials 30 Network of microporous channels and / or chambers 100 Cube-like mold 130 Network of microstrands and / or micropods 133 Target Cell Types 200 Various Configurations

Claims

1. 1. A cell-free substrate comprising: a three-dimensional (3D) macrostructure defined by a continuous matrix of extracellular matrix (ECM) material associated with a first cell type of interest and a network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material associated with said first cell type of interest. Including, wherein the 3D macrostructure comprises a thickness defined by an upper surface, a lower surface, and at least one side edge extending from the upper surface to the lower surface. Cell-free substrate.

2. 10. The cell-free substrate of claim 1, further comprising at least one male connecting component and at least one female connecting component.

3. 3. The cell-free substrate of claim 2, wherein the at least one male connecting element comprises a first male connecting element extending outwardly from the at least one side edge.

4. 3. The cell-free substrate of claim 2, wherein the at least one side edge comprises a first side edge and a second side edge, and wherein the at least one connecting male component comprises a first connecting male component extending outwardly from the first side edge and a second connecting male component extending outwardly from the second side edge.

5. 5. The cell-free substrate of claim 2, wherein the at least one male connecting element comprises a third male connecting element extending outwardly from the top surface.

6. 5. The cell-free substrate of claim 2, wherein the at least one female connecting element comprises a first female connecting element extending inwardly from the at least one side edge toward an interior of the 3D macrostructure.

7. 7. The cell-free substrate of any one of claims 4 to 6, wherein the at least one lateral edge comprises a third lateral edge and a fourth lateral edge, and wherein the at least one connecting female component comprises a first connecting female component extending inwardly from the third lateral edge toward an interior of the 3D macrostructure and a second connecting female component extending inwardly from the fourth lateral edge toward an interior of the 3D macrostructure.

8. 8. The cell-free substrate of claim 7, wherein the first side edge and the third side edge define a first pair of opposing side edges.

9. 9. The cell-free substrate of claim 7 or 8, wherein the second side edge and the fourth side edge define a second pair of opposing side edges.

10. 10. The cell-free substrate of claim 7, wherein the at least one female connecting component comprises a third female connecting component extending inwardly from the lower surface toward an interior of the 3D macrostructure.

11. 11. The cell-free substrate of claim 1, wherein the 3D macrostructure, excluding the at least one male interlocking component and the at least one female interlocking component, defines a cube, a rectangular prism, or a triangular prism.

12. 11. The cell-free substrate of any one of claims 1 to 10, wherein the 3D macrostructure, excluding the at least one male connecting component and the at least one female connecting component, defines a polygonal prism having 3 to 12 lateral edges, e.g., at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, and at least about 8 lateral edges, and / or any of the following: up to about 12, up to about 11, up to about 10, up to about 9, and up to about 8 lateral edges.

13. 2. The cell-free substrate of claim 1, wherein the at least one side edge comprises a first side edge, a second side edge, and an arched side edge located adjacent and between the first side edge and the second side edge.

14. 14. The cell-free substrate of claim 13, wherein the first side edge comprises the at least one male connecting element extending outwardly from the first side edge and the second side edge comprises the at least one female connecting element extending inwardly from the second side edge toward an interior of the 3D macrostructure.

15. 15. The cell-free substrate of claim 13 or 14, wherein the at least one male connecting element includes a second male connecting element extending outwardly from the top surface.

16. 16. The cell-free substrate of any one of claims 13 to 15, wherein the at least one female connecting component comprises a second female connecting component extending inwardly from the lower surface toward an interior of the 3D macrostructure.

17. 17. The cell-free substrate of any one of claims 13 to 16, wherein the 3D macrostructure, excluding the at least one male connecting component and the at least one female connecting component, defines a half cylinder, such as ⅛ to ½ cylinder, e.g., ⅛ cylinder, ¼ cylinder, ⅓ cylinder, or ½ cylinder.

18. The upper surface is approximately 0.25 cm ２ ~Approx. 25cm ２ , e.g., any of the following: at least about 0.25 cm ２ , at least about 0.5 cm ２ , at least about 0.75 cm ２ , at least about 1 cm ２ , at least about 1.5 cm ２ , at least about 2 cm ２ , at least about 5 cm ２ , at least about 8 cm ２ , at least about 10 cm ２ , and at least about 12 cm ２ , and / or any of the following: about 25 cm ２ , about 22cm ２ , about 20cm ２ , about 18cm ２ , about 15cm ２ , and about 12 cm ２ The cell-free substrate according to any one of claims 1 to 17, comprising a macroscopic surface area of

19. The lower surface is approximately 0.25 cm ２ ~Approx. 25cm ２ , e.g., any of the following: at least about 0.25 cm ２ , at least about 0.5 cm ２ , at least about 0.75 cm ２ , at least about 1 cm ２ , at least about 1.5 cm ２ , at least about 2 cm ２ , at least about 5 cm ２ , at least about 8 cm ２ , at least about 10 cm ２ , and at least about 12 cm ２ , and / or any of the following: about 25 cm ２ , about 22cm ２ , about 20cm ２ , about 18cm ２ , about 15cm ２ , and about 12 cm ２ The cell-free substrate according to any one of claims 1 to 18, comprising a macroscopic surface area of

20. 20. The cell-free substrate of any one of claims 1 to 19, wherein the thickness of the 3D macrostructure is from about 0.5 cm to about 3 cm, such as any of the following: at least about 0.5 cm, at least about 0.75 cm, at least about 1 cm, at least about 1.25 cm, and at least about 1.5 cm, and / or any of the following: up to about 3 cm, up to about 2.5 cm, up to about 2 cm, and up to about 1.5 cm.

21. 23. The cell-free substrate of any one of claims 2 to 22, wherein each of the at least one female connecting component is configured to receive a corresponding at least one male connecting component of a second cell-free substrate.

22. The network of microporous channels and / or chambers extending throughout the continuous matrix of the ECM material may be between about 100 microns and about 800 microns, e.g., any of the following: at least about 100 microns, at least about 120 microns, at least about 150 microns, at least about 180 microns, at least about 200 microns, at least about 220 microns, and at least about 250 microns, and / or any of the following: up to about 800 microns, up to about 780 microns, up to about 750 microns, up to about 720 microns, up to about 70 ...50 microns, up to about 720 microns, up to about 700 microns, and / or any of the following: up to about 800 microns, up to about 750 microns, up to about 720 microns, up to about 700 microns, and / or any of the following: up to about 800 microns, up to about 7 22. The cell-free substrate of any one of claims 1-21, having an average diameter of up to about 680 microns, up to about 650 microns, up to about 620 microns, up to about 600 microns, up to about 580 microns, up to about 550 microns, up to about 520 microns, up to about 500 microns, up to about 480 microns, up to about 450 microns, up to about 420 microns, up to about 400 microns, up to about 380 microns, up to about 350 microns, up to about 320 microns, up to about 300 microns, up to about 280 microns, and up to about 250 microns.

23. 23. The cell-free substrate of any one of claims 1 to 22, wherein the network of microporous channels and / or chambers extending throughout the continuous matrix of ECM material comprises at least about 40% by volume of the 3D macrostructure, such as any of the following: at least about 40% by volume, at least about 50% by volume, at least about 60% by volume, and at least about 70% by volume of the 3D macrostructure, and / or any of the following: up to about 90% by volume, up to about 85% by volume, up to about 80% by volume, up to about 75% by volume, and up to about 70% by volume of the 3D macrostructure.

24. 1. A method of forming an acellular substrate, comprising: (i) forming or disposing in a mold a network of microstrands and / or micropods comprising a degradable hydrogel material; (ii) seeding the network of microstrands and / or micropods by adding an initial culture medium containing cells of a cell type of interest into the mold containing the network of microstrands and / or micropods; (iii) supplying the cells with fresh culture medium by perfusing the mold to nourish the cells until tissue grows and expands to fill the mold; (iv) performing a decellularization operation on the tissue located within the mold to form a continuous matrix of ECM material associated with the cell type of interest; (v) disintegrating and removing the network of microstrands and / or micropods to form a network of microporous channels and / or chambers extending throughout a continuous matrix of the ECM material associated with the cell type of interest, thereby obtaining the acellular substrate; A method comprising:

25. 25. The method of claim 24, wherein forming or disposing the network of microstrands and / or micropods comprises performing an additive manufacturing technique such as 3D printing of digital photosynthetic printing.

26. The method of claim 24 or 25, wherein the structure of the microstrand and / or micropod network is selected based on the cell morphology of the target cell type, the cell morphology having a target matrix structure and a target microporous network of channels and / or chambers, and wherein the structure of the microstrand and / or micropod network is similar to or identical to the target microporous network of the channels and / or chambers.

27. The network of microstrands and / or micropods may be from about 100 microns to about 800 microns, e.g., any of the following: at least about 100 microns, at least about 120 microns, at least about 150 microns, at least about 180 microns, at least about 200 microns, at least about 220 microns, and at least about 250 microns, and / or any of the following: up to about 800 microns, up to about 780 microns, up to about 750 microns, up to about 720 microns, up to about 700 microns, up to about 80 ...50 microns, up to about 720 microns, up to about 700 microns, up to about 800 microns, up to about 750 microns, up to about 750 microns, up to about 700 microns, up to about 800 microns, up to about 750 microns, up to about 750 microns, up to about 700 microns, up to about 800 microns, 27. The method of any one of claims 24 to 26, wherein the granular particles have an average diameter of about 680 microns, up to about 650 microns, up to about 620 microns, up to about 600 microns, up to about 580 microns, up to about 550 microns, up to about 520 microns, up to about 500 microns, up to about 480 microns, up to about 450 microns, up to about 420 microns, up to about 400 microns, up to about 380 microns, up to about 350 microns, up to about 320 microns, up to about 300 microns, up to about 280 microns, and up to about 250 microns.

28. 28. The method of any one of claims 24 to 27, wherein the network of microstrands and / or micropods comprises a selectively degradable hydrogel material comprising one or more degradable polymers, such as one or more biopolymers of biological origin.

29. 30. The method of claim 28, wherein the one or more biopolymers of biological origin comprise polynucleotides, polysaccharides, polypeptides, or any combination thereof.

30. 30. The method of claim 28 or 29, wherein the one or more biopolymers include collagen, gelatin, laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and proteins that can be gently degraded, such as by using protein-specific enzymes, ionic solvents, neutral detergents, weak acids, and peroxides to break the biopolymer chains.

31. 31. The method of any one of claims 28 to 30, wherein the one or more biopolymers include esters such as hydroxybutyrate, lactic acid, glycolic acid, and caprolactone, anhydrides such as adipic acid and sebacic acid, sugars such as cellulose, alginates, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, and heparin, proteins, nucleotides (DNA, RNA), peptides such as collagen, gelatin, silk, and fibrin, urethanes, phosphates, carbonates, and degradable monomers including vinyl chloride.

32. 32. The method of any one of claims 28 to 31, wherein the selectively degradable hydrogel material further comprises a synthetic polymer such as a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or a combination thereof.

33. 33. The method of any one of claims 24 to 32, wherein performing the decellularization operation on the tissue placed in the mold comprises treating the tissue with a detergent, followed by dialysis of the continuous matrix of ECM material associated with the target cell type.

34. 34. The method of any one of claims 24 to 33, further comprising the step of freeze-drying the continuous matrix of ECM material associated with the cell type of interest.

35. 35. The method of any one of claims 24 to 34, further comprising the step of sterilizing the cell-free substrate, such as by an electron beam or gamma irradiation procedure.

36. 36. The method of any one of claims 24 to 35, further comprising removing the acellular substrate from the mold.

37. 1. A method of forming an individualized implant, comprising: (i) providing or forming one or more cell-free substrates according to any one of claims 1 to 23, wherein the first cell type of interest is associated with a tissue of a patient having an abnormality, such as a specific organ tissue; (ii) seeding said one or more acellular substrates with healthy native cells associated with said patient's tissue having an abnormality; (iii) providing a culture medium to the healthy native cells and allowing the healthy native cells to proliferate throughout the network of microporous channels and / or chambers of the acellular substrate to form the individualized graft; A method comprising:

38. 38. The method of claim 37, wherein the one or more cell-free substrates comprise a first cell-free substrate and a second cell-free substrate, wherein the first cell-free substrate and the second cell-free substrate are the same.

39. 38. The method of claim 37, wherein the first acellular substrate is joined to the second acellular substrate to obtain a multi-layered acellular scaffold and the healthy native cells are allowed to proliferate throughout a collective network of microporous channels and / or chambers of the multi-layered acellular scaffold to form the individualized graft.

40. 40. The method of any one of claims 37-39, wherein the one or more acellular substrates comprise at least two acellular substrates joined together to define a multi-acellular scaffold, or any of the following: at least about 2, at least about 3, at least about 5, 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, and at least about 100 acellular substrates.

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