Organ generation from guided instructions in interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for ex vivo tissue generation. A block may comprise a 3D continuous polymeric matrix with a network of microporous cavities, and may be configured to interconnect with at least one other block. A system may comprise at least one block and be configured to cultivate one or more tissue components when seeded with at least one cell. A method may comprise seeding at least one block with at least one cell type.

Description

[0001] ORGAN GENERATION FROM GUIDED INSTRUCTIONS IN INTERCONNECTING POROUS HYDROGEL BLOCKS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 63 / 666,291, filed July 1, 2024, which is hereby incorporated by reference in its entirety for all that it contains (including all references therein) for all purposes as if restated and set forth fully herein to the maximum extent allowable by law.

[0004] TECHNICAL FIELD

[0005] The subject matter disclosed herein is generally directed to the cultivation of cells within interconnecting porous hydrogel blocks (IPHBs). These IPHBs may enable cells to communicate, coordinate, organize, secrete extracellular matrix, and / or develop organized tissue layers that may mature into functional organ components with vascularization when the appropriate mammalian cells are co-cultured. IPHBs may facilitate the co-cultivation of multiple cell types that self-regulate through various signaling mechanisms, including autocrine, paracrine, endocrine signaling, and / or direct communication via gap junctions.

[0006] BACKGROUND

[0007] Tissue engineering and organ engineering are interdisciplinary fields that combine principles from biology, engineering, and material science to develop biological substitutes that restore, maintain, or improve tissue and organ function. These fields may have the potential to revolutionize modem medicine by providing solutions for organ failure, tissue damage, and / or various chronic diseases. Current technologies and strategies in tissue and organ engineering may include scaffold-based approaches, decellularized organ scaffolds, hydrogelbased systems, three-dimensional (3D) bioprinting, and microfluidic organ-on-a-chip models.

[0008] The scaffold-based approach may use biomaterials to create scaffolds that support cell attachment, growth, and / or differentiation. Scaffolds may be made from natural materials such as collagen or gelatin and / or synthetic polymers such as poly(lactic-co-glycolic acid) (PLGA) or polycaprolactone (PCL). This approach may provide structural support, may be functionalized with bioactive molecules, and may be relatively easy to manufacture. However, it often lacks the complexity and / or dynamic nature of native tissues, may have limited vascularization, and may have the potential for immune rejection.

[0009] Decellularized organ scaffolds may involve removing cellular components from donor organs, leaving behind the extracellular matrix (ECM), which may then repopulate with patient-derived cells. This approach can maintain natural architecture and / or mechanical properties and may reduce immune rejection when using patient cells. Drawbacks to this method include limited availability of suitable donor organs, challenges in achieving uniform cell repopulation, and potential for residual immunogenicity.

[0010] Hydrogels are water-swollen, crosslinked polymeric networks that may mimic the ECM and provide a hydrated environment for cell growth. Advantages to hydrogel-based systems include high biocompatibility, tunable properties, and / or the ability to be injected or molded into complex shapes. Mechanical weakness, potential for rapid degradation, and / or challenges in integrating with native tissues may be setbacks to certain such systems.

[0011] 3D bioprinting may use layer-by-layer deposition of bioinks containing cells and biomaterials to create 3D tissue constructs. Advantages to 3D bioprinting may include higher precision, customization potential, and / or ability to create complex architectures. Technical complexity, challenges in ensuring cell viability and function, and high cost are among the challenges of 3D bioprinting.

[0012] Microfluidic organ-on-a-chip models may use microfluidic technology to create miniature, physiologically relevant models of human organs. More control of the microenvironment, ability to mimic dynamic biological processes, and / or usefulness for drug testing and / or disease modeling may be some of the benefits of this approach to tissue and organ engineering. However, limited scalability, challenges in integrating multiple organ systems, and high technical expertise required are among the drawbacks.

[0013] There are many gaps and / or barriers in the field of tissue and organ engineering. One of the major challenges in tissue engineering is the creation of a functional vascular network that may supply nutrients and / or remove waste. Difficulty in replicating the complex architecture and function of native blood vessels and / or limited success in integrating engineered vessels with host vasculature make the creation of a functional vascular network difficult. Native tissues and organs have highly complex structures and functions that are difficult to replicate in vitro and current technologies often fail to capture the multi-scale organization and / or dynamic nature of tissues, which may lead to limited functionality and integration. Immune rejection remains a significant hurdle, especially when using allogenic or xenogenic materials. The lack of effective strategies to prevent immune responses and / or the potential for chronic inflammation and graft rejection remain a barrier to preventing immune rejection. Successful integration of engineered tissues and organs with the host's native tissues is important for functionality. Differences in mechanical properties, lack of vascularization, and / or immune responses may impede integration. Lack of standardized protocols and / or scalable manufacturing processes may limit the widespread application of tissue engineering solutions. Variability in materials, cells, and methods used, as well as challenges in scaling up from lab to clinical settings may cause a barrier in developing a standardized and scalable approach. High costs associated with advanced tissue engineering technologies and materials may limit accessibility. There remains a need for cost-effective and scalable solutions that may be widely adopted in clinical practice.

[0014] Organ shortages, chronic diseases, personalized medicine, drug testing, and / or disease modeling may create the need for tissue and organ engineering. There remains a critical shortage of donor organs for transplantation, which may lead to long wait times and high mortality rates for patients with organ failure. Tissue engineering may provide solutions for chronic diseases that involve tissue degeneration and / or damage, such as heart disease, diabetes, and / or osteoarthritis. Engineered tissues may be tailored to individual patients, allowing for personalized treatment strategies and improved outcomes. Tissue and organ models may provide more accurate platforms for drug testing and / or disease modeling compared to traditional cell cultures and / or animal models, which may lead to better prediction of human responses.

[0015] While significant progress has been made in tissue engineering and organ engineering, several gaps and barriers may still hinder the successful engineering and integration of tissues and / or organs into modem medicine.

[0016] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present disclosure.

[0017] SUMMARY

[0018] One or more embodiments of the disclosure may address one or more of the aforementioned problems. Certain embodiments according to the disclosure provide a system for generating tissue and organ components ex vivo, including at least one interconnecting porous hydrogel block (IPHB) designed to provide a three-dimensional (3D) environment. A network of microchannels within the at least one IPHB may facilitate vertical and / or horizontal transfer of nutrients, gases, and signals, and adjustable physical properties of the at least one IPHB, including stiffness and / or microchannel diameter, may mimic the mechanical properties of native tissues. Further, the at least one IPHB may be configured to support the co-cultivation of multiple cell types, including endothelial cells, mesenchymal stem cells (MSCs), fibroblasts, epithelial cells, podocytes, and / or other connective tissue cells. Further still, the at least one IPHB may facilitate cell communication through autocrine, paracrine, endocrine signaling, and / or direct cell-to-cell contact via gap junctions. Yet again, the porous network within the at least one IPHB may allow for the transmission of extracellular vesicles and / or other biological signals. Still yet further, the hydrogel matrix may serve as a transport network for chemical, mechanical, physical, electrical, and / or biological signals between cells in different channels within at least one IPHB or across multiple interconnected IPHBs. Again, cells within the at least one IPHB may secrete extracellular matrix (ECM) components to alter the micromechanics of their environment and / or promote tissue organization. Still further, the microchannel and / or pore diameters of the at least one IPHB may be controlled by changing osmotic gradients. Still again, media perfusion may be applied to the at least one IPHB to simulate blood flow, enhance nutrient delivery, waste removal, and / or modulate other parameters of cell behavior.

[0019] In another aspect, the present disclosure may provide a method for generating functional organ components ex vivo. Certain embodiments may comprise isolating primary cells from a mammalian tissue biopsy, cultivating the primary cells within the at least one IPHB that provide a 3D environment, co-cultivating the primary cells with additional cell types within the at least one IPHB to enhance tissue formation, and utilizing dynamic culturing conditions, including static and / or perfusion, to enhance the physiological relevance of the tissue model. Further, comprising the co-cultivation of endothelial cells and MSCs within at least one IPHB may support the formation of vascular networks, including capillaries, venules, arterioles, veins, and / or arteries. Some embodiments comprise the distribution of growth factors, nutrients, and / or other bioactive molecules, e.g. in liquid format, which could be across the at least one IPHB. Some embodiments comprise connecting multiple IPHBs using perfusion systems to simulate full organ systems, e.g. for more comprehensive studies. Some embodiments comprise diagnostic evaluations, such as histological analysis and / or molecular profiling, which could be performed on the generated tissues, e.g. within the at least one IPHB. Some embodiments comprise utilizing generated tissues, e.g. within the at least one IPHB, for high-throughput screening of therapeutic compounds. Some embodiments comprise collecting and / or analyzing secreted byproducts, e.g. from media and / or perfusate, which could be surrounding the at least one IPHB. Some embodiments comprise using at least one IPHB to develop one or more personalized tissue models that may reflect the unique characteristics of each patient’s tissue. Some embodiments comprise expanding the tissue model by interconnecting at least one IPHB to one or more IPHB(s). Some embodiments comprise reducing media consumption by up to 90% and plastic consumption by up to 85% compared to other cultivation formats. Some embodiments comprise forming the at least one IPHB to enable the study and modeling of disease progression, cancer, and / or tissue injuries within the 3D environment provided by the at least one IPHB. In some embodiments, therapeutic and / or cytotoxic agents may be (e.g., directly and / or indirectly) administered and / or incorporated into the hydrogel matrix, e.g. to interact with cells and / or tissues, including those cultivated within the IPHB(s).

[0020] In certain embodiments, a system for ex vivo tissue generation may comprise at least one IPHB. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block. The system may be configured to cultivate one or more tissue components when seeded with at least one cell type.

[0021] In some embodiments, at least one cell type may comprise endothelial cell, MSC, fibroblast, epithelial cell, podocyte, and / or connective tissue cell.

[0022] In some embodiments, at least one component may comprise a component of an organ, organ-like tissue, or a combination thereof.

[0023] In some embodiments, one or more cell types may be derived from a specimen, human, subject, tissue, organ, and / or biopsy. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. The subject could be a patient. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. Additional permutations will be readily apparent to those of ordinary skill in the art. Some embodiments may be configured to mimic one or more ECMs. At least one mimicked ECM may be that of a specimen, and / or an ECM from the derivative source (e.g. human, subject, biopsy).

[0024] Some embodiments may be configured to match at least one nutrient and / or physical property of the ECM.

[0025] In some embodiments, one or more ECM physical properties to be mimicked may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity. A physical property may be quantified, such as with a measured average and / or range. The physical property may be emulated and / or targeted within a goal threshold, including without limitation, only by way of example / for purposes of illustration, within 20% more or less than the measured average and / or range.

[0026] Some embodiments blocks may be configured to facilitate cell communication.

[0027] In some embodiments, cell communication may comprise autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, and / or gap junctions.

[0028] Some embodiments may be configured to transmit one or more extracellular vesicles and / or biological, electrical, physical, mechanical, and / or chemical signals.

[0029] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0030] In some embodiments, at least one cavity size, dimension, and / or diameter may be alterable by at least one electrochemical and / or osmotic gradient and / or differential.

[0031] Some embodiments may comprise dynamic culture conditions.

[0032] Some embodiments may comprise at least one compound. A compound may comprise one or more drugs, nutrients, growth factors, bioactive molecules, cytotoxic agents, and / or therapeutic agents.

[0033] Some embodiments may comprise one or more compounds distributed with geographic and / or temporal variation in concentration via at least one differential and / or gradient.

[0034] Some embodiments may be configured to cultivate microvascular formation.

[0035] In some embodiments, microvascular formation may comprise forming microvasculature and / or one or more vessels, veins, venules, capillaries, arterioles, and / or arteries. Some embodiments may comprise fistulae and / or anastomoses. In some embodiments, at least one block may be configured for diagnostic evaluation, histological analysis, molecular profiling, and / or therapeutic compound screening. Some examples may comprise immunohistochemical staining and / or testing.

[0036] In some embodiments, at least one block may be configured to collect and / or analyze, in any order, one or more secreted byproducts. For any secreted byproduct, collection may precede analysis, and vice versa. A collected secreted byproduct need not necessarily be analyzed, and vice versa.

[0037] In some embodiments, at least one secreted byproduct may be collected from a culture media and / or perfusate.

[0038] In certain embodiments, a method for ex vivo tissue generation may comprise seeding at least one IPHB with at least one cell type. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block.

[0039] In some embodiments, at least one cell may be isolated from at least one subject. One or more subjects may comprise a human, mammal, animal, bird, reptile, amphibian, and / or fish. At least one cell may be isolated from one or more organs and / or tissues.

[0040] Some embodiments may further comprise cultivating the cells into a personalized model and subjecting the model to analysis to guide diagnosis and / or therapy tailored for the subject.

[0041] In certain embodiments, an IPHB for ex vivo tissue generation may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The block may be configured to interconnect with at least one other block. The block may be further configured to cultivate one or more tissue components when seeded with at least one cell. At least one component may comprise an organ or organ-like component. At least one cell may be derived from a specimen, subject, human, tissue, organ, and / or biopsy.

[0042] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.

[0043] BRIEF DESCRIPTION OF THE DRAWINGS

[0044] An understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure may be utilized, and the accompanying drawings of which:

[0045] FIG. 1 illustrates two separate interconnecting porous hydrogel blocks IPHB(s) in accordance with certain embodiments;

[0046] FIG. 2 illustrates three interconnected IPHBs in accordance with certain embodiments; and

[0047] FIG. 3 illustrates a method for ex vivo tissue generation in accordance with certain embodiments.

[0048] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0049] DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0050] The disclosure now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments are shown. Indeed, what is claimed 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 the specification, and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise.

[0051] Certain presently disclosed embodiments relate to the development and application of interconnecting porous hydrogel blocks (IPHBs), for tissue and organ engineering. IPHBs may provide a novel and versatile platform that may overcome many of the limitations associated with current tissue engineering technologies. They enable the cultivation of complex, functional tissues and organ components by providing a dynamic and customizable three- dimensional (3D) microenvironment.

[0052] In certain embodiments, the porous network within the IPHBs may run perpendicular to the microchannels, which allows cells to transmit signals such as extracellular vesicles. In some embodiments, the hydrogel matrix may serve as a transport network for chemical, mechanical, physical, electrical, and / or biological signals, enabling communication between cells in different channels within a single IPHB or across multiple joined IPHBs. Cells may modify their environment by secreting extracellular matrix (ECM) components, altering micromechanics, and / or controlling the constriction and relaxation of microchannel and pore diameters through osmotic gradients. These gradients, along with signal transmission, may be further modulated by applying media perfusion to simulate blood flow or influence other cellular behaviors.

[0053] In certain embodiments, IPHBs may support the creation of complex microenvironments that lead to the formation of sophisticated tissue structures and functional organ components in mammalian organisms. Cells within the IPHB(s) may adjust the system to form natural structures and / or components. The formation of healthy tissues may be achieved when mesenchymal stem cells (MSCs) and endothelial cells are paired with at least one other connective tissue cell type. As more cell types are introduced, they work together to organize, self-regulate, and maintain high levels of metabolism, viability, activity, migration, and / or proliferation. Waste products may be secreted into the luminal space of the microchannels and are removed by gravity, promoting a clean environment.

[0054] The ability of certain IPHBs and embodiments herein to support vascular system development may facilitate the maturation of tissue and organ components that grow outward from the IPHB(s) over time. The versatility of IPHBs and embodiments herein may allow users to manipulate various parameters to guide the formation of mammalian tissues and organ components. IPHBs and embodiments herein may be used to model healthy tissue formation, disease progression, cancer, and / or tissue injuries. Additionally, cytotoxic and / or therapeutic agents may be (e.g., directly and / or indirectly) administered and / or incorporated, such as into the hydrogel matrix, including to interact with cells and / or tissues, such as those cultivated within the IPHB(s).

[0055] Figure 1, for instance, illustrates two (2) separate IPHBs 1 in accordance with certain embodiments. Each of these IPHBs include a top surface 12, a bottom surface 14, and at least one side edge 16. The particular IPHBs 1 shown in Figure 1 include at least one interlockingmale component 50 and at least one interlocking-female component 60. Figure 2 illustrates three (3) interconnected IPHBs 1 in accordance with certain embodiments. The IPHBs 1 shown in Figure 2 each include a first interlocking-male component 51, a second interlocking-male component 52, a first interlocking-female component 61, and a second interlocking-female component 62. IPHBs 1 may interconnect via first interlocking-male component 51 or a second interlocking-male component 52 interfacing with a first interlocking-female component 61 or a second interlocking-female component 62, or vice versa. In certain embodiments, one or more interlocking-male components (50, 51, 52) comprise a protrusion. In certain embodiments, one or more interlocking-female components (60, 61, 62) comprise a recess. In certain embodiments, one or more interlocking-female components (60, 61, 62) is a recess configured complimentary to a protrusion of one or more interlocking-male components (50, 51, 52). Persons of ordinary skill will readily appreciate additional variations. For example, one or more interlocking-male components (50, 51, 52) may protrude from an IPHB l’s top surface 12, bottom surface 14, one or more side edges 16, or any combination thereof. One or more interlocking-female components (60, 61, 62) may recess into an IPHB 1 ’s top surface 12, bottom surface 14, one or more side edges 16, or any combination thereof. One or more interlocking-male components (50, 51, 52) may comprise a quadrangular protrusion, or any other angled or rounded shape, or any combination thereof. One or more interlocking-female components (60, 61, 62) may comprise a quadrangular recess, or any other angled or rounded shape, or any combination thereof.

[0056] In certain embodiments, IPHBs may feature a network of adjustable cavities such as microchannels and / or pores, e.g. perpendicularly to one another, and may facilitate vertical and / or horizontal transfer of nutrients, gases, and / or cellular signals. In certain embodiments, this dynamic microenvironment may closely mimic in vivo conditions, promoting cell viability, differentiation, and / or function by helping cells within the IPHBs to receive adequate nutrition and may effectively remove waste products. Traditional scaffold designs typically focus on either vertical or horizontal transport but rarely integrate both in a cohesive manner. The realization that combining these features could enhance cellular communication and nutrient delivery in 3D together with other features herein are not an obvious extension of existing designs.

[0057] In certain embodiments, IPHB stiffness and / or microchannel diameter may be modulated to mimic the mechanical properties of various tissues. This customization may allow researchers to create optimal environments for different cell types, enhancing cell growth, differentiation, and / or function. Traditional scaffolds and / or hydrogels often lack the flexibility to adjust their mechanical properties after fabrication. The concept of creating a dynamic and tunable hydrogel system that may be tailored to specific tissue requirements represents a significant departure from static scaffold designs, offering enhanced customization that is not typically considered in conventional approaches.

[0058] In certain embodiments, IPHBs may be designed to be modular, allowing them to be interconnected horizontally and / or vertically. This modularity may support the creation of larger and more complex tissue constructs, which may enable customization of the microenvironment for specific research needs and / or patient-specific conditions. In some embodiments, the scalability may support the generation of large tissue constructs and the creation of integrated organ systems, which may be important for advanced tissue engineering applications. Traditional tissue engineering methods, such as microfluidic models and / or traditional scaffolds, often struggle with scalability and integration. The modular design of IPHBs and embodiments herein may support seamless expansion and / or complex assembly, with features herein representing a novel approach to scaling tissue constructs that would not be readily apparent to someone skilled in the art.

[0059] In some embodiments, IPHBs may facilitate communication between cells through autocrine, paracrine, endocrine signaling, and / or direct cell-to-cell contact via gap junctions, in addition to the use of the hydrogel matrix and / or microchannels for signal transmission. The porous network and hydrogel matrix may enable the transmission of chemical, mechanical, physical, electrical, and / or biological signals which may promote coordinated cellular behavior. In certain embodiments, this comprehensive communication network may support the coordination and organization of cells into functional tissues and organ components, a feature not fully realized in conventional scaffolds. Where individual signaling mechanisms may be understood, integrating multiple signaling pathways within a single scaffold system to promote coordinated cellular behavior and other features herein represent sophisticated and non-obvious improvement over existing technologies. In some embodiments, the use of the hydrogel matrix as a medium for signal transmission may further enhance this communication network in ways not typically considered in traditional scaffolds.

[0060] In certain embodiments, cells within IPHBs may secrete their own ECM components, which may alter the micromechanics of their environment and promote tissue organization. In some embodiments, cells may control the constriction and relaxation of microchannel and / or pore diameters through osmotic gradients, further modulated by media perfusion. This selfregulation may allow cells to construct tissue structures that closely resemble their natural counterparts, enhancing the functionality of the engineered tissues.

[0061] The modular nature of IPHBs and embodiments herein may allow for the integration of different tissue types to simulate full organ systems. Perfusion may be applied horizontally and / or vertically, enhancing the functionality and realism of the tissue constructs. In some embodiments, IPHBs may support both static and perfusion-based culturing conditions, allowing for dynamic modulation of the cellular microenvironment. Perfusion may enhance nutrient delivery, waste removal, and / or cellular organization, which may result in more rapid and coordinated formation of functional vascular networks and / or tissue structures. Perfusion may mimic physiological conditions better than static cultures, which may promote the development of functional tissues and vascular networks. Conventional scaffolds often rely on static culturing, which limits nutrient delivery and waste removal. Features herein, including the dual capability of certain IPHBs and embodiments to operate under both static and perfusion conditions, thus enhancing cell viability and function, represent novel feature that significantly advance the field.

[0062] Cultivation within IPHBs and embodiments herein may result in increased secretory output of important biomolecules, which may include growth factors, cytokines, chemokines, and / or extracellular vesicles. This enhanced secretory activity may be valuable for studying cellular interactions and / or evaluating therapeutic effects, which can offer a more dynamic environment than traditional static cultures and may provide deeper insights into tissue and / or organ function. The enhanced secretory activity achieved within IPHBs and embodiments herein may indicate a highly supportive and / or interactive environment for cells, which is not easily achievable with traditional scaffolds. The ability to create such an environment through the specific design and material properties of IPHBs and other features of embodiments herein again represent non-obvious advancement.

[0063] IPHBs and embodiments herein may support the generation of vascular systems, e.g. when endothelial cells and MSCs are co-cultured. The formation of capillaries, venules, arterioles, veins, and / or arteries may be facilitated, which can promote nutrient delivery and / or waste removal. There is a current gap in creating functional vascular networks in engineered tissues. The dynamic microchannel network in IPHBs may support the co-cultivation of endothelial cells and MSCs, which may promote the formation of capillaries, venules, arterioles, veins, and / or arteries. Perfusion may further enhance the development of these vascular structures, which may ensure adequate nutrient delivery and / or waste removal.

[0064] In some embodiments, the modular design of IPHBs may allow them to be loaded into standard well-plates, supporting high-throughput testing of therapeutic compounds and facilitating large-scale experimentation. For example, a standard 6-well plate may accommodate up to about 24, 42, and / or 96 of certain IPHBs. Embodiments herein can align with high-throughput workflows. Integrating high-throughput compatibility into a hydrogel- based scaffold system may require careful consideration of size, scalability, and / or ease of use. This feature may allow IPHBs and / or embodiments herein to be seamlessly integrated into existing laboratory workflows, among other features herein representing novel approaches that address key limitations in current tissue engineering methods.

[0065] Certain embodiments may be loaded with drugs and / or growth factors, which may interact with the cells seeded. In certain embodiments, compounds may be distributed in at least one differential and / or gradient, which may promote evaluation of therapeutic effect(s) and / or optimal dosing strategies.

[0066] By using primary cells from patient biopsies, certain embodiments may enable the creation of personalized organ models that reflect the unique characteristics of each patient’s tissue. This approach may support precision medicine, allowing for tailored treatment strategies and / or more accurate predictions of therapeutic efficacy, which is a significant improvement over generic models. The concept of creating highly personalized and functional tissue models within a modular hydrogel system that may support complex cell interactions and / or dynamic conditions is a sophisticated innovation. This personalized approach to tissue engineering, tailored to individual patient needs, represents significant improvement beyond conventional methods.

[0067] High costs may be associated with advanced tissue engineering technologies and / or materials may limit accessibility. However, IPHBs may be designed to be cost-effective and easy to use, which may reduce the need for specialized equipment and expertise. Their efficient use of media and plasticware further lowers costs, making advanced tissue engineering more accessible.

[0068] Despite their advanced capabilities, IPHBs and embodiments herein may be relatively simple to use and accessible to researchers with varying levels of expertise. This accessibility makes them suitable for a wide range of research institutions, clinical applications, and / or educational settings.

[0069] In some embodiments, this disclosure may be particularly suited for applications in organ generation and / or tissue engineering, drug discovery and / or testing, personalized medicine, and / or disease modeling. IPHBs and embodiments herein may provide a robust platform for generating functional organ components, which may contribute to advancements in tissue engineering and / or regenerative medicine. Some embodiments may enable high- throughput screening of therapeutic compounds with enhanced predictive power. IPHBs and embodiments herein may support the development of tailored treatments based on patientspecific organ models and / or facilitate the study of various diseases and conditions, e.g. cancer, diabetes, and / or cardiovascular diseases, such as by creating physiologically relevant organ models.

[0070] Current tissue and organ engineering approaches experience challenges in integrating engineered tissues with host tissues. The modular design of IPHBs and embodiments herein may allow for the creation of tissue constructs that may be seamlessly integrated with host tissues. The dynamic culturing conditions and / or enhanced vascularization of certain embodiments may support the functional integration of engineered tissues. Immune rejection of engineered tissues and organs remains another challenge. By using patient-derived cells, IPHBs and embodiments herein may minimize the risk of immune rejection. The customizable environment within IPHBs may support the cultivation of patient-specific tissues that are more likely to be accepted by the host's immune system.

[0071] In some embodiments, IPHBs may introduce a versatile and scalable platform that integrates advanced features for tissue and organ engineering. Their ability to support complex cell communication, dynamic culturing conditions, and / or high-throughput testing are among features herein setting them apart from existing technologies. By overcoming key limitations such as vascularization, tissue complexity, and / or immune rejection, certain IPHBs and embodiments herein enable more accurate and functional tissue models. This advancement may enhance the capabilities of researchers in studying disease mechanisms, evaluating drug efficacy, and / or developing new therapies. The ability to create personalized tissue and organ models using patient-derived cells may support the development of tailored treatments and / or precision medicine. Some embodiments may provide a platform for testing patient-specific responses to therapies, improving treatment outcomes and / or reducing adverse effects. They may be designed to be efficient in terms of media and plasticware consumption, reducing costs and environmental impact. Their accessibility and ease of use may make advanced tissue engineering technologies more widely available to researchers and clinicians. Their modular and customizable nature may position them for further advancements in tissue and organ engineering, such as the integration of additional cell types, the creation of more complex organ systems, and / or the exploration of new applications in regenerative medicine and tissue repair.

[0072] Certain embodiments may be configured to mimic and / or resemble in vivo conditions associated with a natural cell and / or tissue of interest. For example, a continuous polymeric matrix material may mimic a natural tissue of interest by matching one or more physical properties of said tissue within about 20% of a measurable goal or threshold. Examples may include deviation by about 15%, 10%, 8%, 5%, 3%, or 1%, from the natural tissue of interest. The one or more physical properties may include, for example, softness and / or tension strength, and / or elasticity. One or more physical properties may comprise porosity, stiffness, and / or elastic modulus. Additionally or alternatively, the network of microporous cavities (e.g. channels and / or chambers) may be structured to mimic the morphology of a natural tissue of interest, such as by varying the geometry and / or dimensions of a network of microporous channels and / or chambers to mirror the morphology of the natural tissue of interest. In certain examples, a 3D network of microporous channels and / or chambers defines a 3D scaffolding for propagation of one or more cell types of interest. In accordance with certain embodiments, an average diameter of one or more microporous cavities may comprise from about 100 to about 800 microns. The minimum diameter of one or more microporous cavities may comprise 100, 120, 150, 180, 200, 220, and / or 250 microns, in certain embodiments. The maximum diameter of one or more microporous cavities may comprise 800, 780, 750, 720, 700, 680, 650, 620, 600, 580, 550, 520, 500, 480, 450, 420, 400, 380, 350, 320, 300, 280, and / or 250 microns, in certain embodiments. Additionally or alternatively, microporous channels and / or chambers may comprise at least about 40% by volume of the 3D macrostructure of an IPHB. In certain embodiments, microporous cavities may comprise at least about 40, 50, 60, and / or 70% by volume of the 3D macrostructure. In certain embodiments, microporous cavities may comprise at most about 90, 85, 80, 75, and / or 70% by volume of the 3D macrostructure.

[0073] Embodiments herein may be suitable for a variety of applications, such as producing and / or growing cell cultures, bacteria cultures, yeast cultures, biologies, exosomes, extracellular vesicles, growth factors, monoclonal antibodies, peptides, proteins, viral particles, oligonucleotides, and / or organelles; organoid formation, plant growth, drug delivery, tissue formation, ex vivo modeling, electrical conduction, wound healing, cellular reprogramming, filtration, optics, and / or microfluidics; helping construct and / or form a custom network of microchannels, custom scaffold architecture, custom ECM derived scaffold, dissolvable hydrogel, custom tissue, custom tissue formation, and / or custom configuration; accepting patient cells; and / or microenvironment manipulation.

[0074] In accordance with certain embodiments of the disclosure, the continuous polymeric matrix material may be generally non-degradable. In certain examples, the cells and / or tissue produced in the IPHB may need to be flushed out of the interior network of the network of microporous channels and / or chambers for further analysis, purification, and / or development. Additionally or alternatively, the continuous polymeric matrix material may be selectably degradable (e.g., enzymatically dissolved). For example, hydrogel formulations may be rendered biodegradable, such as by insertion of enzyme-sensitive sequences and / or utilization of native matrix-derived compounds. For example, the continuous polymeric matrix material may comprise a selectably degradable hydrogel material comprising one or more degradable polymers, such as one or more biopolymers derived from a living organism. The one or more biopolymers derived from a living organism, for example, may comprise a polynucleotide, polysaccharide, polypeptide, or any combination thereof. In accordance with certain embodiments, the one or more biopolymers may comprise collagen, gelatin (e.g., porcine, bovine [cow], ovine [sheep], shellfish, fish, etc.), laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and / or proteins that can be gently degraded, such as with the use of specific enzymes, ionic solvents, neutral detergents, weak acids, and / or peroxides to disrupt the biopolymer chains. In accordance with certain embodiments, the one or more biopolymers may comprise degradable monomers comprising esters, such as hydroxybutyrate, lactic acid, glycolic acid, and / or caprolactone; anhydrides, such as adipic acid and / or sebacic acid; saccharides, such as cellulose, alginate, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, and / or heparin; proteins; nucleotides, such as DNA and / or RNA; peptides, such as collagen, gelatin, silk, and / or fibrin; urethanes; phosphates; carbonates; and / or vinyl chlorides. Additionally or alternatively, the biopolymer may comprise poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and / or other suitable hydrogels. In accordance with certain embodiments, the selectably degradable hydrogel material may comprise a synthetic polymer, such as a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or any combinations) thereof. The continuous polymeric matrix material, in accordance with certain embodiments, may comprise a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

[0075] In certain embodiments, one or more biopolymers may comprise anywhere between approximately 10 and 100% of the dry weight of an IPHB and / or its continuous polymeric matrix material. In certain embodiments, the minimum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more biopolymers may be about 10, 15, 20, 25, 30, 35, 40, 45, and / or 50%. In certain embodiments, the maximum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more biopolymers may be about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, and / or 50%.

[0076] In certain embodiments, a continuous polymeric matrix material may comprise a swellable hydrogel material. The swellable hydrogel material may comprise a radically mediated reaction product of at least a first monomer including acrylate or methacrylate functional group(s) and a second monomer or oligomer including at least two (2) free-radically polymerizable functional groups. For example, the at least two (2) free-radically polymerizable functional groups may independently from each other comprise an acrylate or methacrylate group, an allylic group, an alkynyl, a vinyl nitrile, a vinyl ether, a vinyl ester, a vinyl amide, a styrenic group, a maleate group, a fumarate group, and / or a norbomene group. In accordance with certain embodiments, at least one of the first monomer and / or the second monomer may comprise polyethylene glycol functionality (e.g., — O(C2H4O)nH; where n has a value from 1 to 100), polypropylene glycol functionality (e.g., — O(C3HeO)nH; where n has a value from 1 to 100), and / or glycerol functionality incorporated into a backbone of the monomer and / or grafted onto the monomer as a side-chain or a component of a side chain. By way of example, at least one of the first monomer and / or second monomer may comprise 2-Hydroxyethyl acrylate (HEA), Poly(ethylene glycol) methyl ether acrylate (MPEGA), N-Methyl acetamide (NMA), or Poly(ethylene glycol) diacrylate (PEGDA). In accordance with certain embodiments, non-limiting examples of non-degradable monomers that may be utilized in hydrogel materials may include polyolefins (e.g., ethylene, propylene), styrene, nylon (e.g., amides), and / or acrylics. In accordance with certain embodiments, non-limiting examples of degradable monomers that may be utilized in hydrogel materials may include esters (e.g., hydroxybutyrate, lactic acid, glycolic acid, caprolactone), anhydrides ( e.g., adipic acid, sebacic acid), saccharides (e.g., cellulose, alginate, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, heparin), proteins, nucleotides (e.g., DNA, RNA), peptides (e.g., collagen, gelatin, silk, fibrin), urethanes, phosphates, carbonates, and / or vinyl chlorides. Additionally or alternatively, a third monomer comprising a cross-linking agent may be incorporated in continuous polymeric matrix material. Additionally or alternatively, the swellable hydrogel material may comprise one or more natural polymers, such as plant-derived polymers (e.g., cellulosic- polymers) and / or animal-derived polymers. A natural polymer herein may comprise laminin. Further, a gelatin herein may comprise porcine, bovine (cow), ovine (sheep), shellfish, and / or fish gelatin. In certain embodiments, one or more thermoplastic polymers may comprise between about 10 to 100% of the dry weight of an IPHB and / or its continuous polymeric matrix material. In certain embodiments, the minimum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more thermoplastic polymers may be about 10, 15, 20, 25, 30, 35, 40, 45, and / or 50%. In certain embodiments, the maximum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more thermoplastic polymers maybe about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, and / or 50%.

[0077] In accordance with certain embodiments, an interface between the network of microporous cavities (e.g., channels and / or chambers) and the continuous polymeric matrix material may comprise a coating of at least one compatibilizer selected to promote adhesion of at least one cell of interest. This coating may be applied subsequent to IPHB formation. By way of example, the coating comprising the compatibilizer(s) may comprise a biological coating, including, for example, collagen I (e.g., from or for human MSCs [e.g., from or for adipose, bone barrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); laminin I (e.g., from or for human induced pluripotent stem cells [IPSCs] and / or mouse dorsal root ganglia); hyaluronan (e.g., from porcine hepatocytes and / or human dermal adult fibroblasts); gelatin (e.g., from human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, human CD8+ T cells, Human CD4+ T cells, human macrophages, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); fibrin (e.g., from or for human keratinocytes); fibronectin (e.g., from or for human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human osteoblasts, human osteocytes, and / or human chondrocytes); or any combination(s) thereof.

[0078] By way of example, in certain embodiments one or more IPHBs may be seeded and / or configured to enable cell growth of one or more of the following: human stem cells, such as human Wharton’s Jelly cells, human umbilical cord derived MSCs, human bone marrow derived MSCs, human adipose derived MSCs, human skin derived IPSCs, human blood cell derived IPSCs, human CD4+ T cells, and human CD8+ T cells; primary mammalian cells, such as HepG2 cells or other liver carcinoma cells, human adult dermal fibroblasts, human neonatal dermal fibroblasts, human adult keratinocytes, mouse dorsal root ganglia or other primary neural cells, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, equine muscle derived MSCs, snail cells, and human macrophages; and immortalized mammalian cell lines, such as UB-OC2 or mouse cochlear epithelium cells, human myoblastoma or other muscle tumor cells, PC3 or other prostate cancer cells, CHO or Chinese hamster ovary cells, HEK293 or other human embryonic kidney cells, SHSY5Y or other neuronal tumor cells, PANC-1 or other human pancreatic cancer cells, HeLa or other cervical cancer cells, A549 or other lung cancer cells, and A673 or other muscle cancer cells; and primary plant cells, such as rosemary, tobacco, and tomato.

[0079] In another aspect, the present disclosure provides methods for generating functional organ components ex vivo. Some embodiments may comprise isolating primary cells from a mammalian tissue biopsy; cultivating the primary cells within the IPHB(s), which may provide a 3D environment; co-cultivating the primary cells with additional cell types within the IPHB to enhance tissue formation; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the tissue model. The co-cultivation of endothelial cells and MSCs within the IPHB(s) may support the formation of vascular networks including capillaries, venules, arterioles, veins, and / or arteries. Growth factors, nutrients, and / or other bioactive molecules, e.g. in liquid format, may be distributed, such as across the IPHB(s). One IPHB may be connected to one or more IPHB(s) using perfusion systems, e.g. to simulate full organ systems, such as for more comprehensive studies. Diagnostic evaluations, including histological analysis and / or molecular profiling, may be performed, such as on the generated tissues, e.g. within the IPHB(s). Generated tissues, including within the IPHB(s), may be utilized for high-throughput screening of therapeutic compounds. Secreted byproducts, such as from the media and / or perfusate, e.g. surrounding the IPHB(s), may be collected and / or analyzed. The utilization of the IPHB(s) may develop one or more personalized tissue models that reflect the unique characteristics of each patient’s tissue. The tissue model may be expanded by interconnecting the IPHB to one or more IPHB(s). Media consumption may be reduced by up to 90% and plastic consumption may be reduced by up to 85% compared to other cultivation formats. In certain embodiments, a system for ex vivo tissue generation may comprise at least one IPHB. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block. The system may be configured to cultivate one or more tissue components when seeded with at least one cell type.

[0080] In some embodiments, at least one cell type may comprise endothelial cell, MSC, fibroblast, epithelial cell, podocyte, and / or connective tissue cell.

[0081] In some embodiments, at least one component may comprise a component of an organ, organ-like tissue, or a combination thereof.

[0082] In some embodiments, one or more cell types may be derived from a specimen, human, subject, tissue, organ, and / or biopsy. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. The subject could be a patient. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0083] Some embodiments may be configured to mimic one or more ECMs. At least one mimicked ECM may be that of a specimen, and / or an ECM from the derivative source (e.g. human, subject, biopsy).

[0084] Some embodiments may be configured to match at least one nutrient and / or physical property of the ECM.

[0085] In some embodiments, one or more ECM physical properties to be mimicked may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity. A physical property may be quantified, such as with a measured average and / or range. The physical property may be emulated and / or targeted within a goal threshold, including without limitation, only by way of example / for purposes of illustration, within 20% more or less than the measured average and / or range.

[0086] Some embodiments blocks may be configured to facilitate cell communication.

[0087] In some embodiments, cell communication may comprise autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, and / or gap junctions. Some embodiments may be configured to transmit one or more extracellular vesicles and / or biological, electrical, physical, mechanical, and / or chemical signals.

[0088] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0089] In some embodiments, at least one cavity size, dimension, and / or diameter may be alterable by at least one electrochemical and / or osmotic gradient and / or differential.

[0090] Some embodiments may comprise dynamic culture conditions.

[0091] Some embodiments may comprise at least one compound. A compound may comprise one or more drugs, nutrients, growth factors, bioactive molecules, cytotoxic agents, and / or therapeutic agents.

[0092] Some embodiments may comprise one or more compounds distributed with geographic and / or temporal variation in concentration via at least one differential and / or gradient.

[0093] Some embodiments may be configured to cultivate microvascular formation.

[0094] In some embodiments, microvascular formation may comprise forming microvasculature and / or one or more vessels, veins, venules, capillaries, arterioles, and / or arteries. Some embodiments may comprise fistulae and / or anastomoses.

[0095] In some embodiments, at least one block may be configured for diagnostic evaluation, histological analysis, molecular profiling, and / or therapeutic compound screening. Some examples may comprise immunohistochemical staining and / or testing.

[0096] In some embodiments, at least one block may be configured to collect and / or analyze, in any order, one or more secreted byproducts. For any secreted byproduct, collection may precede analysis, and vice versa. A collected secreted byproduct need not necessarily be analyzed, and vice versa.

[0097] In some embodiments, at least one secreted byproduct may be collected from a culture media and / or perfusate.

[0098] In certain embodiments, a method for ex vivo tissue generation may comprise seeding at least one IPHB with at least one cell type. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block.

[0099] In some embodiments, at least one cell may be isolated from at least one subject. One or more subjects may comprise a human, mammal, animal, bird, reptile, amphibian, and / or fish. At least one cell may be isolated from one or more organs and / or tissues. Some embodiments may further comprise cultivating the cells into a personalized model and subjecting the model to analysis to guide diagnosis and / or therapy tailored for the subject.

[0100] In certain embodiments, an IPHB for ex vivo tissue generation may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The block may be configured to interconnect with at least one other block. The block may be further configured to cultivate one or more tissue components when seeded with at least one cell. At least one component may comprise an organ or organ-like component. At least one cell may be derived from a specimen, subject, human, tissue, organ, and / or biopsy.

[0101] Described herein are systems, methods, and IPHBs for ex vivo tissue generation. A block may comprise a 3D continuous polymeric matrix with a network of microporous cavities, and may be configured to interconnect with at least one other block. A system may comprise at least one IPHB and be configured to cultivate one or more tissue components when seeded with at least one cell. A method may comprise seeding at least one IPHB with at least one cell type.

[0102] Turning to Figure 3, illustrated is an example embodiment of a method 300 for ex vivo tissue generation. In certain embodiments, method 300 may comprise step 310 of seeding and optionally step 320 of dynamic culture conditions. Step 310 may comprise seeding at least one IPHB with at least one cell type. The IPHB may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The cavities may comprise one or more channels, pores, and / or chambers. Step 320 may comprise exposing cells to dynamic culture conditions. Dynamic culture conditions 320 may be configured to achieve nutrient and / or waste exchange. Dynamic culture conditions 320 may comprise exposing at least one cell to a culture media via perfusion and / or static solution. The culture media may comprise one or more nutrients. Dynamic culture conditions 320 may comprise varying a culture media’s nutrient type and / or concentration. Dynamic culture conditions 320 may comprise perfusing at least one block with culture media. In certain embodiments, one or more cavities may be configured as pathway(s) for nutrient delivery, gas exchange, and / or waste removal.

[0103] In some embodiments of step 310, at least one cell may be derived from a human, a subject, a biopsy, or a combination thereof. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. One or more subjects may be mammals, animals, birds, reptiles, amphibians, and / or fish. At least one cell could be derived from one or more tissues and / or organs. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0104] Certain embodiments may comprise additional or different steps. For example, step 320 is optional. By way of further example, method 300 may comprise step 330 of at least one personalized model and / or step 340 of at least one analysis. Step 330 may comprise cultivating cells derived from one or more subjects into one or more personalized models. Step 340 may comprise subjecting cells (e.g., the personalized model(s)) to one or more analyses to guide diagnosis and / or therapy, which could be tailored for the subjects). Step 340 may comprise exposing one or more of the cells to at least one experimental variable, which could be across one or more geographic, temporal, and / or other differential(s) and / or gradients). An experimental variable may comprise at least one drug, thermal energy, radiation, or any combination thereof. A drug may comprise at least one medicine, medication, pharmaceutical, biologic, immunologic, immunotherapy, chemotherapy, radiotherapy, radiotracer, tracer, stain, diagnostic agent, theranostic agent, therapeutic agent, and / or treatment agent. Step 340 may comprise evaluating, measuring, and / or observing the effect of at least one experimental variable on the senescence, growth, and / or characteristic(s) of the cells (e.g., seeded, cultivated, primary, tumor, metastatic, tissue, and / or organ cells).

[0105] All steps may be incorporated in any combination and in any order, including repeating step(s). By way of example, certain method embodiments may comprise seeding 310, dynamic culture conditions 320, and analysis 340 (not necessarily including personalized model 330). In some embodiments, personalized model 330 could be after or subsequent to analysis 340. Some embodiments might not comprise dynamic culture conditions 320. Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of seeding 310, dynamic culture conditions 320, personalized model 330, and / or analysis 340. Further, the one or more blocks underlying step 310 (and / or being seeded) could be altered before, during, and / or after other steps (e.g. seeding 310, dynamic culture conditions 320, personalized model 330, and / or analysis 340). Such embodiments may include changing the configuration of existing blocks, removing blocks, and / or adding blocks.

[0106] Throughout embodiments of systems, methods, and blocks herein, cells may comprise one or more adipose derived MSCs, HEK 293 cells, HeLa cells, Henrietta Lacks cells, CHO cells, Chinese hamster ovary cells, Wharton jelly cells, bone marrow derived MSCs, Panc-1 cells, ASPC-1 cells, BcPc-3 cells, pancreatic cancer cells, human pancreatic cancer cells, endothelial cells, keratinocytes, keratocytes, CD4+ T cells, CD8+ T cells, IPSCs, PBMC cells, peripheral blood mononuclear cells, hepatocytes, porcine hepatocytes, muscle derived MSCs, equine muscle derived MSCs, MDA-MCB-231 cells, breast cancer cells, human breast cancer cells, triple negative breast cancer cells, bone marrow aspirate, human bone marrow aspirate, SNU-398 cells, HepG2 cells, liver cancer cells, human liver cancer cells, SHSY5Y cells, neuroblastoma cells, human neuroblastoma cells, murine neuroblastoma cells, N2a cells, mouse neuroblastoma cells, neuroprogenitor cells, Schwann cells, macrophages, dorsal root ganglia, murine dorsal root ganglia, myocytes, bovine myocytes, chondrocytes, porcine chondrocytes, osteocytes, porcine osteocytes, UB-OC2 cells, organ of Corti cells, murine organ of Corti cells, cochlear cells, prostate PDX cells, PC3 cells, prostate cancer cells, human prostate cancer cells, A549 cells, lung carcinoma cells, human lung carcinoma cells, DMS53 cells, H82 cells, epithelial like lung carcinoma cells, NIH-H1703 cells, squamous lung cancer cells, A673 cells, muscle cancer cells, soft tissue cancer cells, bone cancer cells, sarcoma cells, Ewing sarcoma cells, human Ewing sarcoma cells, MDCK cells, Madin-Darby canine kidney cells, planaria slurry, snail slurry, kidney slurry, murine kidney slurry, kidney explant, murine kidney explant, Vero cells, monkey Vero cells, epithelial cells, E. coli, S. aureus, trabecular meshwork cells, renal cells, human renal cells, C2C12 cells, myoblasts, myoblast cells, murine myoblast cells, MLO-A5 cells, IDG-SW3 cells, osteoblast cells, osteocyte-like cells, murine osteoblast cells, murine osteocyte-like cells, fibroblast cells, fibroblasts, human fibroblasts, porcine fibroblasts, avian fibroblasts, fish fibroblasts, murine fibroblasts, American Type Culture Collection (ATCC) fibroblasts, CRL fibroblasts, CRL-4061 fibroblasts, hTERT gingival fibroblasts, CRL-4058 fibroblasts, hTERT lung fibroblasts, CRL-2522 fibroblasts, BJ fibroblast cells, NIH / 3T3 cells, embryonic fibroblast cells, murine embryonic fibroblast cells, or any combination thereof.

[0107] Persons having ordinary skill in the art will readily appreciate that the various features of embodiments herein, whether discussed or mentioned or disclosed in a context of a system, method, or block, may be practiced in embodiments of other / different blocks, methods, and / or systems, and vice versa, all within the present scope.

[0108] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations. Clause 1. A system for generating tissue and organ components ex vivo, comprising: at least one IPHB designed to provide a 3D environment, wherein the at least one IPHB has a network of microchannels within the at least one IPHB to facilitate vertical and horizontal transfer of nutrients, gases, and signals; and adjustable physical properties of the at least one IPHB, including stiffness and / or microchannel diameter, to mimic the mechanical properties of native tissues of interest.

[0109] Clause 2. The system of clause 1, wherein the at least one IPHB is configured to support the co-cultivation of multiple cell types.

[0110] Clause 3. The system of clause 2, wherein the cell types include endothelial cells, MSCs, fibroblasts, epithelial cells, podocytes, and other connective tissue cells.

[0111] Clause 4. The system of clause 1, wherein the at least one IPHB is configured to facilitate cell communication through autocrine, paracrine, endocrine signaling, and direct cell- to-cell contact via gap junctions.

[0112] Clause 5. The system of clause 1, wherein the porous network within the at least one IPHB is configured to allow for the transmission of extracellular vesicles and other biological signals.

[0113] Clause 6. The system of clause 1, wherein the hydrogel matrix serves as a transport network for chemical, mechanical, physical, electrical, and biological signals between cells in different channels within at least one IPHB or across multiple joined IPHBs.

[0114] Clause 7. The system of clause 1, wherein cells within the at least one IPHB secrete ECM components to alter the micromechanics of their environment and promote tissue organization.

[0115] Clause 8. The system of clause 1, wherein the microchannels and / or pore diameters of the at least one IPHB are controllable by changing osmotic gradients, whereby affecting the swelling of the hydrogel.

[0116] Clause 9. The system of clause 1, wherein media perfusion may be applied to the at least one IPHB to simulate blood flow, enhance nutrient delivery, waste removal, and modulate other parameters of cell behavior.

[0117] Clause 10. A method for generating functional organ components ex vivo, comprising: isolating primary cells from a mammalian tissue biopsy; cultivating the primary cells within the at least one IPHB that provide a 3D environment; co-cultivating the primary cells with one or more additional cell types within the at least one IPHB to enhance tissue formation; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the tissue model.

[0118] Clause 11. The method of clause 10, wherein the co-cultivation of endothelial cells and MSCs within the at least one IPHB supports the formation of vascular networks.

[0119] Clause 12. The method of clause 11, wherein the vascular networks include capillaries, venules, arterioles, veins, and arteries.

[0120] Clause 13. The method of clause 10, further comprising the distribution of growth factors, nutrients, and other bioactive molecules in liquid format across the at least one IPHB, whereby optionally creating gradients for comprehensive evaluation.

[0121] Clause 14. The method of clause 10, further comprising a step of connecting the at least one IPHB to one or more additional IPHBs using perfusion systems is configured to simulate full organ systems for comprehensive studies.

[0122] Clause 15. The method of clause 10, further comprising a step of performing one or more diagnostic evaluations, including histological analysis and molecular profiling, on the generated tissues within the at least one IPHB.

[0123] Clause 16. The method of clause 10, wherein the generated tissues within the at least one IPHB are further utilized for high-throughput screening of therapeutic compounds.

[0124] Clause 17. The method of clause 10, further comprising a step of collecting and / or analyzing secreted byproducts from the media or perfusate surrounding the at least one IPHB.

[0125] Clause 18. The method of clause 10, wherein the utilization of the at least one IPHB develops personalized tissue models that reflect the unique characteristics of each patient’s tissue, such as for a personalized or tailored treatment protocol or strategy.

[0126] Clause 19. The method of clause 10, wherein the tissue model is expanded by interconnecting the at least one IPHB to one or more additional IPHBs to accommodate tissue growth and / or study progression.

[0127] Clause 20. The method of clause 10, wherein media and plastic consumption is reduced by up to 90% and 85% compared to other cultivation formats.

[0128] Clause 21. The system of clause 10, wherein the at least one IPHB enables the study and modeling of disease progression, cancer, and tissue injuries within the 3D environment provided by the at least one IPHB. Clause 22. The system of clause 10, further comprising directly administering or incorporating one or more therapeutic and cytotoxic agents into the hydrogel matrix to interact with cells and tissues cultivated within the IPHBs.

[0129] These and other modifications and variations to the embodiments may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and it is not intended to limit the claims. Therefore, the spirit and scope of the appended claims should not be limited to the exemplary description of the versions contained herein.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A system for ex vivo tissue generation, comprising at least one interconnecting porous hydrogel block, which comprises a three-dimensional continuous polymeric matrix with a network of microporous cavities and is configured to interconnect with at least one other block, wherein the system is configured to cultivate one or more tissue components when seeded with at least one cell type.

2. The system of claim 1, wherein at least one cell type is selected from the group consisting of: endothelial cell, mesenchymal stem cell, fibroblast, epithelial cell, podocyte, and connective tissue cell.

3. The system of claim 1 , wherein at least one component is a component of an organ, organlike tissue, or a combination thereof.

4. The system of claim 1, wherein one or more cell types are derived from a specimen, a human, a subject, a biopsy, or a combination thereof.

5. The system of claim 1 , wherein one or more blocks are configured to mimic an extracellular matrix.

6. The system of claim 5, further configured to match at least one physical property of the extracellular matrix.

7. The system of claim 6, wherein one or more physical properties are selected from the group consisting of: porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, lipophilicity, or a combination thereof.

8. The system of claim 1, wherein one or more blocks are configured to facilitate cell communication.

9. The system of claim 8, wherein the cell communication comprises autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, gap junctions, or a combination thereof.

10. The system of claim 1, wherein one or more blocks are configured to transmit one or more extracellular vesicles, biological signals, electrical signals, physical signals, mechanical signals, chemical signals, or a combination thereof.

11. The system of claim 1, wherein the cavities comprise one or more channels, one or more chambers, one or more pores, or a combination thereof.

12. The system of claim 1, wherein at least one cavity size, dimension, diameter, or combination thereof is alterable by at least one electrochemical or osmotic gradient or differential or any combination thereof.

13. The system of claim 1, further comprising dynamic culture conditions.

14. The system of claim 1, further comprising at least one compound comprising one or more drugs, nutrients, growth factors, bioactive molecules, cytotoxic agents, therapeutic agents, or a combination thereof.

15. The system of claim 14, comprising one or more compounds distributed across the system with geographic, temporal, or both geographic and temporal variation in concentration via at least one differential, gradient, or a combination thereof.

16. The system of claim 1, further configured to cultivate microvascular formation.

17. The system of claim 16, wherein microvascular formation comprises forming microvasculature, veins, venules, capillaries, arterioles, arteries, or a combination thereof.

18. The system of claim 1, wherein at least one block is configured for diagnostic evaluation, histological analysis, molecular profiling, therapeutic compound screening, or a combination thereof.

19. The system of claim 1 , wherein at least one block is configured to collect, analyze, or collect and analyze one or more secreted byproducts.

20. The system of claim 19, wherein at least one secreted byproduct is collected from a culture media, perfusate, or combination thereof.

21. A method for ex vivo tissue generation, comprising seeding at least one interconnecting porous hydrogel block with at least one cell type, wherein at least one block comprises a three-dimensional continuous polymeric matrix with a network of microporous cavities and is configured to interconnect with at least one other block.

22. The method of claim 21, wherein at least one cell is isolated from a subject.

23. The method of claim 22, further comprising cultivating the cells into a personalized model and subjecting the model to analysis to guide diagnosis, therapy, or a combination thereof tailored for the subject.

24. An interconnecting porous hydrogel block for ex vivo tissue generation, comprising a three-dimensional continuous polymeric matrix with a network of microporous cavities, configured to interconnect with at least one other block, and further configured to cultivate one or more tissue components when seeded with at least one cell type.

Images