Kidney-like formation from immortalized and patient cell cultivation in interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen. A method may comprise seeding at least one block with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen

Description

[0001] KIDNEY-LIKE FORMATION FROM IMMORTALIZED AND PATIENT CELL CULTIVATION 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 / 666,287, 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 HEK293 cells and / or patient-derived kidney cells within Bio-Blocks, also known as interconnecting porous hydrogel blocks (IPHBs). These modular hydrogels may be pre-formed with microchannels running perpendicularly, while the hydrogel formulations may create pores that facilitate vertical and / or horizontal transfer of nutrients, gases, and / or signals between cells. The stiffness of the IPHBs may be modulated to mimic the native tissue of the cell of interest, and the diameter of the microchannels may be adjusted to influence cell behavior.

[0006] BACKGROUND

[0007] Tissue engineering has made advances in developing models to study kidney function, disease progression, and drug discovery. Various strategies have been employed to create kidney-like structures and mimic kidney functions ex vivo.

[0008] Traditional two-dimensional (2D) cell cultures may be used for their relative simplicity and / or cost-effectiveness. These culture systems fail to replicate the complex three- dimensional (3D) architecture and microenvironment of the kidney, but developing systems that can accurately mimic the in vivo conditions of the kidney, including cell-cell and / or cellmatrix interactions, may be difficult and complex.

[0009] Scaffold-based approaches may use biomaterials such as collagen, gelatin, and / or synthetic polymers to create scaffolds that support the growth and organization of kidney cells. They provide structural support and can be functionalized with bioactive molecules to promote cell differentiation and maturation. However, they often lack the complexity and dynamic nature of the native kidney microenvironment, limiting their physiological relevance. Decellularized organ scaffolds can involve removing cellular components from donor organs, leaving behind the extracellular matrix, which is then repopulated with patient-derived cells. This approach may maintain the natural architecture and mechanical properties of the kidney, but it may have limited availability of suitable donor organs and / or challenges in achieving uniform cell repopulation and function.

[0010] Microfluidic organ-on-a-chip models may use microfluidic technology to create miniature, physiologically relevant models of kidney function. These models may allow for more control of the microenvironment, including fluid flow, shear stress, and / or nutrient delivery. However, downfalls to this approach include technical complexity and scalability issues, as well as the limited ability to replicate the full complexity of kidney tissue.

[0011] 3D bioprinting may use layer-by-layer deposition of bioinks containing kidney cells and biomaterials to create 3D tissue constructs. 3D bioprinting may have higher precision and customization potential, enabling the creation of complex tissue architectures. This technique, however, may face technical challenges in ensuring cell viability and function during and / or after the printing process.

[0012] HEK293 cells, derived from human embryonic kidney cells, may be used in biomedical research due to their relative ease of culture and transfection efficiency. Patient-derived kidney cells, on the other hand, may offer a more physiologically relevant model for studying kidney function, disease progression, and / or drug responses. HEK293 cells are typically grown in 2D monolayer cultures using standard tissue culture techniques. They are used in high-throughput screening assays, gene expression studies, and / or protein production. HEK293 cells may be relatively easy to culture, robust, and may be highly transfectable. However, they lack the complexity and specific characteristics of primary kidney cells, therefore limiting their relevance for certain types of research.

[0013] Patient derived kidney cells may be grown from biopsy samples and / or induced pluripotent stem cells differentiated into kidney cell types. These cells are typically cultivated in 2D cultures, organoids, or scaffold-based systems. Patient-derived kidney cells may provide a more accurate representation of patient-specific kidney function and disease states. Their disadvantages include limited availability, variability between samples, and challenges in maintaining cell viability and function over extended periods. Despite advancements in tissue engineering and cell culture techniques, several gaps and barriers exist that hinder the efficient generation and use of HEK293 cells and / or patient- derived kidney cells for drug discovery and disease progression research.

[0014] Difficulty in scaling up kidney tissue models for high-throughput drug screening and / or reproducibility across different labs is another gap in the known approaches to tissue engineering. There is a barrier in creating standardized and scalable tissue engineering platforms that can be widely adopted. It is difficult to maintain the viability and function of primary kidney cells over long-term cultures. Designing culture systems that may provide adequate nutrient delivery, waste removal, and mechanical cues to support cell health and function also remains difficult. Another gap in the approaches to tissue engineering is the limited ability to co-culture multiple cell types in a controlled manner to replicate the diverse cell populations found in the kidney. However, developing systems that allow for the cocultivation of endothelial cells, fibroblasts, epithelial cells, and / or other relevant cell types in a physiologically relevant manner is also difficult. Current kidney models are often not compatible with high-throughput screening technologies, and designing kidney models that can be easily integrated into high-throughput screening platforms for drug discovery is challenging. There is limited ability to create personalized kidney models that reflect the genetic and phenotypic diversity of patients. Developing personalized tissue engineering approaches that can be tailored to individual patients for more accurate disease modeling and drug testing is important.

[0015] In summary, while progress has been made in tissue engineering and cell culture techniques for kidney research, there remains substantial gaps and barriers that need to be addressed.

[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 kidney-like organ components ex vivo, including at least one interconnecting porous hydrogel block (IPHB) designed to provide a three-dimensional (3D) environment; HEK293 cells and patient-derived kidney cells cultured within the at least one IPHB; the ability to co-cultivate additional cell types within the at least one IPHB; and wherein the additional cell types may include endothelial cells, fibroblasts, epithelial cells, podocytes, mesangial cells (MSGs), and / or mesenchymal stem cells (MSCs). Further, the at least one IPHB may be configured to support the formation of complex kidney structures. Further still, the kidney structures may include the renal cortex, glomerulus, nephrons, Bowman’s capsule, renal medulla, proximal tubule, and / or the loop of Henle. Yet again, the at least one IPHB may be configured to facilitate the formation of realistic kidney structures and functional components by HEK293 cells and / or patient-derived kidney cells. Still yet further, the at least one IPHB may be configured to enable the creation of personalized kidney models by using primary cells from patient biopsies. Again, the at least one IPHB may be configured to be modular and / or interconnected to one or more IPHBs. Still further, the at least one IPHB may be configured to connect to another IPHB using perfusion systems to simulate full organ systems. Still again, the at least one IPHB may be configured to allow for the loading of a plurality of the at least one IPHB into standard well-plates. Yet further, the at least one IPHB may be configured to enable the distribution of growth factors, nutrients, and / or other bioactive molecules in liquid format. Even further, the at least one IPHB may be configured to support various diagnostic procedures for further analysis. Even further still, the diagnostic procedures may include histological analysis, molecular profiling, and / or the collection of secreted byproducts. Still, the at least one IPHB may be cost-effective and easy to use. Yet still, the at least one IPHB may be configured to reduce media consumption by up to 90% and / or plastic consumption by up to 85% compared to other formats.

[0019] In another aspect, the present disclosure provides a method for generating kidney-like organ components ex vivo, including isolating primary kidney cells from a patient biopsy and / or HEK293 cells; cultivating the primary kidney cells within the at least one IPHB that may provide a 3D environment; co-cultivating the primary cells with additional cell types within the at least one IPHB to enhance kidney tissue formation; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the kidney model. Further, growth factors, nutrients, and / or other bioactive molecules in liquid format may be distributed across the at least one IPHB. Again, the at least one IPHB may be configured to be connected to another IPHB using perfusion systems to simulate full organ systems. Again still, the diagnostic evaluations may include histological analysis and / or molecular profiling. Yet still, the generated kidney tissue components in the at least one IPHB may be configured to be used for high-throughput screening of therapeutic compounds. Still yet, the secreted byproducts from the media or perfusate surrounding the at least one IPHB may be collected and / or analyzed. Still yet again, the at least one IPHB may be configured to develop personalized kidney tissue models that reflect the unique characteristics of each patient’s kidney. Even further, the kidney tissue model may be expanded by interconnecting the at least one IPHB to one or more other IPHBs to accommodate tissue growth and study progression. Even further still, media consumption may be reduced by up to 90% and plastic consumption may be reduced by up to 85% compared to other cultivation formats.

[0020] In certain embodiments, a system for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

[0021] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0022] In some embodiments, one or more specimens may be derived from a 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.

[0023] In some embodiments, the specimen may comprise a living human’s kidney.

[0024] In some embodiments, the specimen may be derived from the kidney in vivo.

[0025] In some embodiments, at least one component may comprise renal cortex, glomerulus, nephron, Bowman’s capsule, renal medulla, proximal tubule, loop of Henle, distal convoluted tubule, and / or collecting duct.

[0026] In some embodiments, one or more blocks may be configured for loading into one or more standard well-plates.

[0027] Some embodiments may further comprise dynamic culture conditions. In some embodiments, dynamic culture conditions may be configured to achieve nutrient and / or waste exchange for at least one cell.

[0028] In some embodiments, dynamic culture conditions may comprise exposing at least one cell to a culture media via perfusion and / or static solution.

[0029] In some embodiments, culture media may comprise one or more nutrients for at least one cell. In some embodiments, dynamic culture conditions may comprise varying the culture media’s nutrient type and / or concentration.

[0030] Some embodiments may be further configured to fluidically distribute one or more nutrients, growth factors, and / or bioactive molecules.

[0031] In some embodiments, the system may be configured to be seeded with at least one additional cell type.

[0032] In some embodiments, an additional cell type may comprise endothelial cell, epithelial cell, podocyte, MSG, and / or MSC.

[0033] In some embodiments, at least one block may be configured for compatibility with histological analysis and / or molecular profiling. Some examples may comprise immunohistochemical staining and / or testing.

[0034] In some embodiments, at least one block may be configured to collect secreted byproducts.

[0035] In certain embodiments, a method for ex vivo kidney tissue generation may comprise seeding at least one IPHB with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen. 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.

[0036] Some embodiments may comprise cultivating cells under dynamic culture conditions.

[0037] In some embodiments, at least one kidney cell may be isolated from at least one vertebrate subject. One or more vertebrate subjects may comprise a human, mammal, animal, bird, reptile, amphibian, and / or fish.

[0038] 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.

[0039] In certain embodiments, an IPHB for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

[0040] 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.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] 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:

[0043] FIG. 1 illustrates two separate interconnecting porous hydrogel blocks (IPHB) in accordance with certain embodiments;

[0044] FIG. 2 illustrates three interconnected IPHBs in accordance with certain embodiments; and

[0045] FIG. 3 illustrates a method for ex vivo kidney tissue generation in accordance with certain embodiments.

[0046] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0047] DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0048] 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.

[0049] Certain presently disclosed embodiments relate to the development and application of interconnecting porous hydrogel blocks (IPHBs) for the ex vivo cultivation of kidney-like organ components using HEK293 cells and patient-derived kidney cells. Certain embodiments and IPHBs may be designed to provide a three-dimensional (3D), biomimetic environment that could support the formation of complex kidney structures and facilitate advanced research in drug discovery, drug testing, and / or disease progression.

[0050] 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.

[0051] 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 run throughout the hydrogel matrix and facilitate the vertical and / or horizontal transfer of nutrients, gases, and / or cellular signals. In certain embodiments, IPHBs may have adjustable stiffness and microchannel diameter, e.g. to support the formation of complex kidney structures and / or promote physiological relevance. This dynamic 3D microenvironment may closely mimic in vivo conditions, promoting cell viability and function. In some embodiments, the stiffness of the IPHBs can be modulated to mimic the mechanical properties of native kidney tissue, e.g. to provide an optimal environment for cellular growth and / or differentiation. In certain embodiments, the diameter of the microchannels can be adjusted to influence cell behavior, e.g. to ensure that the physical conditions closely replicate those found in vivo.

[0052] 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 tumor microenvironment for specific research needs and / or patient-specific conditions. In some embodiments, the modular design of IPHBs may allow them to be loaded into standard wellplates, supporting high-throughput testing of therapeutic compounds. For example, a standard 6-well plate may accommodate up to about 24, 42, and / or 96 of certain IPHBs. The interconnecting design of certain embodiments and IPHBs may allow for expansion and / or customization, and / or may make them suitable for a wide range of research and / or clinical applications.

[0053] When seeded into IPHB(s) in accordance with certain embodiments herein, HEK293 cells and / or patient-derived kidney cells may aggregate and / or organize into sophisticated kidney-like structures. In some embodiments, identifiable components such as the renal cortex, glomerulus, nephrons, Bowman’s capsule, renal medulla, proximal tubule, and / or the loop of Henle may be observed within 14 days of cultivation.

[0054] Certain embodiments and / or IPHB(s) may support the co-cultivation of various cell types, which may include endothelial cells, fibroblasts, epithelial cells, podocytes, mesangial cells (MSGs), and / or mesenchymal stem cells (MSCs). This co-cultivation may lead to the formation of complex, multi-cellular tissue structures that closely mimic the natural kidney microenvironment.

[0055] Certain embodiments may be capable of supporting both static and perfusion-based culturing conditions. In certain embodiments, perfusion may enhance nutrient delivery and waste removal, accelerate cellular organization, and increase secretory output. Cells cultured under perfusion conditions may also exhibit more rapid and coordinated formation across interconnected IPHBs. When perfusion is used instead of static culture, the secretory output is further enhanced. Perfusion may support better nutrient and / or waste exchange and promote quicker organization and / or functional maturation of the cells within the IPHB(s). Cultivation of HEK293 cells and / or patient-derived kidney cells within IPHBs can result in a sustained increase in the secretory output. This may include proteins, extracellular vesicles, growth factors, cytokines, chemokines, antibodies, and / or other secretory components. These secreted factors may be important for drug discovery and testing, e.g. to provide valuable insights into cellular responses to various treatments.

[0056] 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 optimal dosing strategies.

[0057] By using primary cells from patient biopsies, certain embodiments may enable the creation of personalized kidney models that reflect the unique characteristics of each patient’s tissue. This personalized approach may support precision medicine, allowing for tailored treatment strategies and more accurate predictions of therapeutic efficacy.

[0058] Kidney-like components formed in certain embodiments, including within IPHBs can grow outward and be joined horizontally by interconnecting blocks and / or vertically or otherwise, e.g. within a biological cartridge. This versatility of embodiments herein may allow for the creation of integrated organ systems and / or simulating full organ functions such as in a “System-on-a-Chip” format. Perfusion can be applied both horizontally and vertically(and / or otherwise), which may enhance the functionality and realism of the tissue constructs.

[0059] Embodiments herein consume significantly less media and plasticware compared to traditional cultivation methods. Certain embodiments may require only 10% of the media needed for two-dimensional (2D) cultivation of HEK293 cells, and may reduce plastic consumption by 85%. Efficiency reduces costs and improves the sustainability of cell culture processes. IPHBs and embodiments herein are also simple to use and accessible to researchers with varying levels of expertise, making them suitable for a wide range of research institutions, clinical applications, and / or educational settings.

[0060] This disclosure may be particularly suited for applications in personalized medicine, drug discovery and testing, disease modeling, and / or regenerative medicine. Generating kidney components in embodiments herein and IPHBs may support the development of tailored treatments based on patient-specific kidney models. Embodiments herein may provide a robust platform for high-throughput drug screening, which may allow for the testing of multiple compounds and their effects on kidney tissue. The enhanced secretory output and physiological relevance of the tissue models provided herein may improve the accuracy and / or predictive power of drug testing. Embodiments herein may create personalized kidney models which enable researchers to study kidney biology, disease progression and / or the effects of various treatments on specific patient tissues. This approach may facilitate the development of targeted therapies and improve our understanding of kidney-related diseases. Embodiments herein may support the generation of functional kidney tissue components, which may contribute to advancements in regenerative medicine. They hold potential for developing new treatments and therapies for kidney disease and injury.

[0061] Embodiments herein may generate kidney components and cultivate HEK293 cells and / or patient-derived kidney cells. This presents several non-obvious aspects that may distinguish it from existing technologies and would not be readily apparent to someone skilled in the art of tissue engineering and cell culture.

[0062] Certain embodiments may combine several functional features within a single modular system, including 3D structure, adjustable stiffness, microchannel design, and / or dynamic nutrient and signal exchange. While individual features like 3D scaffolds or perfusion systems are known, integrating these features into a cohesive, scalable, and / or modular system specifically designed for kidney tissue engineering may be unique. Traditional approaches often focus on single aspects of tissue engineering, such as scaffold design or bioreactors, without integrating multiple functionalities in a modular format. The realization that combining these features can create a more physiologically relevant and versatile platform for kidney tissue generation represents a novel advancement.

[0063] The ability of certain IPHBs and embodiments herein to modulate the stiffness of the hydrogel and / or the diameter of the microchannels to mimic the native tissue environment and influence cell behavior is an important innovation. This level of customization may allow more precise control over the cellular microenvironment, which is not a common feature in existing tissue engineering systems. Current systems often lack the capability to fine-tune physical properties to this degree. The fact that adjusting these parameters can significantly enhance cell viability, organization, and / or function within a 3D hydrogel matrix is a non-obvious improvement over standard methods.

[0064] Certain embodiments may support both static and perfusion-based culturing conditions, which may allow for a dynamic environment that may promote rapid and / or physiologically relevant tissue formation. This dual capability may enhance the versatility and applicability of the system for different research and / or therapeutic purposes. While perfusion systems are known, their integration into a modular hydrogel system that also may support static culture and / or facilitate the vertical and / or horizontal exchange of nutrients and signals and other features herein are innovative. Embodiments herein may leverage the benefits of both static and dynamic cultures in a single platform and are not an obvious extension of existing technologies.

[0065] The ability of IPHBs and embodiments herein to support the aggregation and / or organization of HEK293 cells and / or patient-derived kidney cells into sophisticated kidney components such as the renal cortex, glomerulus, nephrons, Bowman’s capsule, renal medulla, proximal tubule, and / or the loop of Henle is a novel achievement. Achieving such complex tissue structures typically requires advanced and specialized techniques. The fact that IPHBs and embodiments herein can facilitate this level of tissue organization through their unique design and features highlights an important and non-obvious advancement in tissue engineering.

[0066] IPHBs and embodiments herein may significantly increase the secretory output of cultured cells, including proteins, extracellular vesicles, growth factors, cytokines, chemokines, and / or antibodies. This enhanced functionality may be particularly valuable for drug discovery and / or testing. Traditional 2D cultures and even some 3D cultures may not achieve the same level of secretory activity. The design and functionality of IPHBs and embodiments herein may promote such a high level of secretory output.

[0067] The modular design of IPHBs and embodiments herein may allow for easy integration into standard well-plates and / or support high-throughput testing. The potential to load multiple IPHBs into a standard well-plate format for parallel testing of therapeutic compounds and other features herein are novel. Existing kidney models often face challenges in scalability and / or high-throughput compatibility. The realization that a modular, hydrogel-based system can overcome these challenges and provide a scalable solution for high-throughput applications and other features herein are not obvious extensions of current technologies.

[0068] Embodiments herein may offer novel and superior platforms for generating kidney-like organ components and cultivating HEK293 cells and / or patient-derived kidney cells. By addressing key limitations of existing models and providing a more realistic and personalized approach to kidney research, they may advance the fields of tissue engineering, drug discovery, and / or disease progression research. Their modular, scalable, and / or dynamic nature, and / or their cost-effectiveness and / or ease of use, make them a transformative tool for researchers and / or clinicians.

[0069] 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 measured 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.

[0070] 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 extracellular matrix derived scaffold, dissolvable hydrogel, custom tissue, custom tissue formation, and / or custom configuration; accepting patient cells; and / or microenvironment manipulation.

[0071] 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 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 further comprise a synthetic polymer, such as a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or any combination(s) 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. 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 aboutlOO, 95, 90, 85, 80, 75, 70, 65, 60, 55, and / or 50%.

[0072] 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.

[0073] 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 50%.

[0074] 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.

[0075] 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.

[0076] A method for generating kidney-like organ components ex vivo may comprise isolating primary kidney cells from a patient biopsy or HEK293 cells; cultivating the primary kidney cells within the IPHB(s) to provide a 3D environment; co-cultivating the primary cells with additional cell types within the IPHB(s) to enhance kidney tissue formation; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the kidney model. In some embodiments, growth factors, nutrients, and / or other bioactive molecules (e.g. in liquid format) may be distributed across at least one IPHB. In some embodiments, the IPHB may be configured to be connected to another IPHB using perfusion systems to simulate full organ systems. In some embodiments, diagnostic evaluations, including histological analysis and / or molecular profiling, may be performed on the generated kidney tissue components, e.g. within the IPHB(s). In some embodiments, generated kidney tissue components in the IPHB(s) may be configured to be used for high-throughput screening of therapeutic compounds. In some embodiments, the secreted byproducts from the media and / or perfusate, e.g. surrounding the IPHB(s), may be collected and / or analyzed. In some embodiments, the IPHB may be configured to develop personalized kidney tissue models that reflect the unique characteristics of each patient’s kidney. In some embodiments, the kidney tissue model may be expanded by interconnecting one IPHB to at least one or more IPHB(s) to accommodate tissue growth and study progression. In some embodiments, media and plastic consumption may be reduced by up to 90% and 85% compared to other cultivation formats.

[0077] In certain embodiments, a system for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

[0078] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0079] In some embodiments, one or more specimens may be derived from a 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.

[0080] In some embodiments, the specimen may comprise a living human’s kidney.

[0081] In some embodiments, the specimen may be derived from the kidney in vivo.

[0082] In some embodiments, at least one component may comprise renal cortex, glomerulus, nephron, Bowman’s capsule, renal medulla, proximal tubule, loop of Henle, distal convoluted tubule, and / or collecting duct.

[0083] In some embodiments, one or more blocks may be configured for loading into one or more standard well-plates.

[0084] Some embodiments may further comprise dynamic culture conditions.

[0085] In some embodiments, dynamic culture conditions may be configured to achieve nutrient and / or waste exchange for at least one cell.

[0086] In some embodiments, dynamic culture conditions may comprise exposing at least one cell to a culture media via perfusion and / or static solution. In some embodiments, culture media may comprise one or more nutrients for at least one cell. In some embodiments, dynamic culture conditions may comprise varying the culture media’s nutrient type and / or concentration.

[0087] Some embodiments may be further configured to fluidically distribute one or more nutrients, growth factors, and / or bioactive molecules.

[0088] In some embodiments, the system may be configured to be seeded with at least one additional cell type.

[0089] In some embodiments, an additional cell type may comprise endothelial cell, epithelial cell, podocyte, MSG, and / or MSC.

[0090] In some embodiments, at least one block may be configured for compatibility with histological analysis and / or molecular profiling. Some examples may comprise immunohistochemical staining and / or testing.

[0091] In some embodiments, at least one block may be configured to collect secreted byproducts.

[0092] In certain embodiments, a method for ex vivo kidney tissue generation may comprise seeding at least one IPHB with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen. 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.

[0093] Some embodiments may comprise cultivating cells under dynamic culture conditions.

[0094] In some embodiments, at least one kidney cell may be isolated from at least one vertebrate subject. One or more vertebrate subjects may comprise a human, mammal, animal, bird, reptile, amphibian, and / or fish.

[0095] 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.

[0096] In certain embodiments, an IPHB for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen. Described herein are systems, methods, and IPHBs for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen. A method may comprise seeding at least one IPHB with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

[0097] Turning to Figure 3, illustrated is an example embodiment of a method 300 for ex vivo kidney 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 HEK293 cell and at least one kidney cell isolated from at least one specimen. 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.

[0098] In some embodiments of step 310, at least one kidney 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. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0099] 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).

[0100] 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.

[0101] 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.

[0102] 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.

[0103] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0104] Clause 1. A system for generating kidney-like organ components ex vivo, comprising: at least one IPHB designed to provide a 3D environment; HEK293 cells and patient-derived kidney cells cultured within the at least one IPHB; and optionally co-cultivation of one or more additional cell types within the at least one IPHB with the HEK293 cells and patient-derived kidney cells; wherein the one or more additional cell types comprise endothelial cells, fibroblasts, epithelial cells, podocytes, MSGs, and MSCs. Clause 2. The system of clause 1, wherein the at least one IPHB is configured to support the formation of complex kidney structures.

[0105] Clause 3. The system of clause 2, wherein the complex kidney structures comprise the renal cortex, glomerulus, nephrons, Bowman’s capsule, renal medulla, proximal tubule, and the loop of Henle.

[0106] Cause 4. The system of clauses 2-3, wherein the at least one IPHB is configured to facilitate the formation of realistic kidney structures and functional components by HEK293 cells and patient-derived kidney cells, optionally closely mimicking in vivo conditions.

[0107] Clause 5. The system of clauses 2-4, wherein the at least one IPHB is configured to enable the creation of personalized kidney models by using primary cells from patient biopsies, whereby reflecting the unique characteristics of each patient’s kidney issue.

[0108] Clause 6. The system of clause 1, wherein the at least one IPHB is configured to be modular and interconnected to one or more additional IPHBs, whereby a scale of the kidney model is enlarged and / or a microenvironment within the at least one IPHB is customized for a specific research need and / or patient-specific conditions.

[0109] Clause 7. The system of clause 6, wherein the at least one IPHB is configured to connect to the one or more additional IPHBs using perfusion systems to simulate full organ systems, whereby creating a “system-on-a-chip” for comprehensive studies.

[0110] Clause 8. The system of clause 6, wherein the at least one IPHB is configured to allow for the loading of a plurality of the at least one IPHB into standard well-plates, whereby supporting high-throughput testing of one or more therapeutic compounds.

[0111] Clause 9. The system of clause 1, wherein the at least one IPHB is configured to enable the distribution of growth factors, nutrients, and other bioactive molecules in liquid format, whereby optionally creating gradients across the at least one IPHB for comprehensive evaluation.

[0112] Clause 10. The system of clause 1, wherein the at least one IPHB is configured to support various diagnostic procedures for further analysis.

[0113] Clause 11. The system of clause 10, wherein the diagnostic procedures comprise histological analysis, molecular profiling, and the collection of secreted byproducts.

[0114] Clause 12. The system of clause 1, wherein the at least one IPHB is cost-effective and easy to use. Clause 13. The system of clause 1 , wherein the at least one IPHB is configured to reduce media consumption by up to 90% and plastic consumption by up to 85% compared to other formats.

[0115] Clause 14. A method for generating kidney-like organ components ex vivo, comprising: isolating primary kidney cells, wherein the primary kidney cells are from a patient biopsy or HEK293 cells; cultivating the primary kidney cells within at least one IPHB that provides a 3D environment; co-cultivating the primary cells with one or more additional cell types within the at least one IPHB to enhance kidney tissue formation; and utilizing dynamic culturing conditions, including static and / or perfusion, to enhance the physiological relevance of the kidney model.

[0116] Clause 15. The method of clause 14, further comprising a step of distributing growth factors, nutrients, and other bioactive molecules in liquid format across the at least one IPHB, such as to create gradients for comprehensive evaluation.

[0117] Clause 16. The method of clause 14, further comprising interconnecting the at least one IPHB to one or more additional IPHBs using perfusion systems to simulate full organ systems, such as for comprehensive studies.

[0118] Clause 17. The method of clause 14, wherein diagnostic evaluations are performed on the generated kidney tissue components within the at least one IPHB.

[0119] Clause 18. The method of clause 17, wherein the diagnostic evaluations comprise histological analysis and molecular profiling.

[0120] Clause 19. The method of clause 14, wherein the generated kidney tissue components in the at least one IPHB are configured to be used for high-throughput screening of therapeutic compounds.

[0121] Clause 20. The method of clause 14, further comprising a step of collecting and / or analyzing secreted byproducts from the media or perfusate surrounding the at least one IPHB.

[0122] Clause 21. The method of clause 14, wherein the at least one IPHB is configured to develop personalized kidney tissue models that reflect the unique characteristics of each patient’s kidney, whereby the personalized kidney tissue model may be subjected to further analysis for the development of personalized or tailored treatment protocols.

[0123] Clause 22. The method of clause 14, wherein the kidney tissue model is expanded by interconnecting the at least one IPHB to one or more additional IPHBs to accommodate tissue growth and study progression. Clause 23. The method of clause 14, wherein media and plastic consumption are reduced by up to 90% and 85% compared to other cultivation formats.

[0124] These and other modifications and variations 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

CLAIMSTHAT WHICH IS CLAIMED:

1. A system for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

2. 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.

3. The system of claim 1 , wherein one or more specimens are derived from a human, a subject, a biopsy, or a combination thereof.

4. The system of claim 3, wherein the specimen comprises a living human’s kidney.

5. The system of claim 4, wherein the specimen is derived from the kidney in vivo.

6. The system of claim 1 , wherein at least one component is selected from the group consisting of: renal cortex, glomerulus, nephron, Bowman’s capsule, renal medulla, proximal tubule, loop of Henle, distal convoluted tubule, and collecting duct.

7. The system of claim 1 , with the one or more blocks configured for loading into one or more standard well-plates.

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

9. The system of claim 8, wherein the dynamic culture conditions are configured to achieve nutrient exchange, waste exchange, or a combination thereof, for at least one cell.

10. The system of claim 8, wherein the dynamic culture conditions comprise exposing at least one cell to a culture media via perfusion, static solution, or a combination thereof.

11. The system of claim 10, wherein the culture media comprises one or more nutrients for at least one cell, and the dynamic culture conditions further comprise varying the culture media’s nutrient type, nutrient concentration, or a combination thereof.

12. The system of claim 1, further configured to fluidically distribute one or more nutrients, growth factors, bioactive molecules, or a combination thereof.

13. The system of claim 1, wherein the system is seeded with at least one additional cell type.

14. The system of claim 13, wherein at least one additional cell type is selected from the group consisting of: endothelial cell, epithelial cell, podocyte, mesangial cell, and mesenchymal stem cell.

15. The system of claim 1, wherein at least one block is further configured for compatibility with histological analysis, molecular profiling, or a combination thereof.

16. The system of claim 1, wherein at least one block is configured to collect secreted byproducts.

17. A method for ex vivo kidney tissue generation, comprising seeding at least one interconnecting porous hydrogel block with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen, 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.

18. The method of claim 17, further comprising cultivating the cells under dynamic culture conditions.

19. The method of claim 17, wherein at least one kidney cell is isolated from a vertebrate subject.

20. The method of claim 19, 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.

21. An interconnecting porous hydrogel block for ex vivo kidney 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 kidney-like organ components when seeded with at least one HEK293 cell and at least one kidney cell isolated from at least one specimen.

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