Tissue generation for drug discovery and BIO-therapeutic modeling in human models

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for cell culture. 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures. A method may comprise exposing at least one block to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

Description

[0001] TISSUE GENERATION FOR DRUG DISCOVERY AND BIO-THERAPEUTIC MODELING IN HUMAN MODELS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 63 / 666,296, 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 field of tissue engineering and drug discovery, and to the development and utilization of embodiments relating to interconnecting porous hydrogel blocks. These blocks may enable the co-culture of multiple cell types and the formation of complex tissue structures and organ components in vitro. Embodiments address the limitations of current in vitro technologies, including by providing scalable, modular, and physiologically relevant platforms for high-throughput drug screening, biotherapeutic development, and / r personalized medicine applications. They may facilitate the study of healthy and / or diseased tissues, tumor formation, and / or metastasis, providing versatile tools for advancing research and / or improving the translational accuracy of preclinical models.

[0006] BACKGROUND

[0007] One of the major challenges in drug discovery and biotherapeutic development lies in the ability to utilize and test representative models that accurately mimic human physiological systems. Current in vitro technologies for cultivating cells lack the complexity to replicate the cell behavior, tissue organization, and functional attributes of human organs. Cells do not behave in a cell culture plastic dish, plate, or flask as they would in a native tissue environment, leading to inaccurate or false results. Furthermore, in vitro technologies often lack the adaptability, flexibility, and versatility required to mimic human tissues effectively. Therefore, there is a need for advanced technologies that enable cells to organize into tissue structures and form components of organs, thereby increasing the confidence of results for benchtop testing in drug discovery and biotherapeutic development. Traditional in vitro cell culture techniques primarily involve growing cells on flat, rigid surfaces such as plastic dishes, plates, or flasks. Where these methods may be relatively cost- effective and accessible to use, they fail to replicate the complex three-dimensional (3D) architecture and microenvironment of human tissues. Cells grown in two-dimensional (2D) environments often exhibit altered morphology, reduced functionality, and atypical gene expression profiles, leading to inaccurate or misleading results in drug screening and disease modeling.

[0008] Addressing limitations of 2D cultures, development of 3D cell culture systems led to spheroids and organoids. Spheroids are aggregates of cells that form spontaneously in suspension or low-adhesion conditions, mimicking the 3D structure of tissues more closely than 2D cultures. 3D cell culture systems may offer enhanced prediction of drug efficacy and toxicity. However, spheroids lack the structural organization and functional complexity of native tissues, and their growth is often limited by nutrient and oxygen diffusion constraints.

[0009] Organoids are more advanced 3D cell culture models derived from stem cells or primary tissues that self-organize into miniature, organ-like structures. Organoids may be useful for studying diseases and testing drug responses. Some models have shown promise in recapitulating the cellular diversity and architecture of their in vivo counterparts. Despite their potential, organoids still face several challenges, including variability in formation, limited scalability, high costs, and difficulty in integrating multiple tissue types or vasculature.

[0010] Microfluidic systems and organ-on-a-chip technologies emerged as alternatives for creating more physiologically relevant in vitro models. These devices use microfabrication techniques to create channels and chambers that mimic the microenvironment and fluid flow conditions of tissues and organs. Organ-on-a-chip platforms may incorporate multiple cell types and simulate dynamic processes such as blood flow and mechanical forces. However, they are often complex to design and operate, require specialized equipment, and may not be easily scalable for high-throughput applications.

[0011] 3D bioprinting involves using a printer to create complex tissue structures by depositing layers of bioink containing cells and biomaterials. This approach may allow for higher precision in creating complex tissue structures and may be used to create a wider range of tissues and organ components. However, the disadvantages to 3D bioprinting include expensive equipment and materials and requiring specialized training and expertise.

[0012] Organotypic cultures involve culturing tissue slices or explants that retain the architecture and cellular composition of the original tissue. This method may maintain the native tissue structure and cell-cell interactions and may be useful for studying tissue-specific functions and responses. Disadvantages include limited longevity and scalability issues.

[0013] Despite several advancements, current in vitro models still struggle to replicate the complexity and functionality of human tissues and organs. Key limitations include lack of vascularization, inability to mimic tissue microenvironments, scalability issues, and cost and complexity. Most 3D culture systems and organoids lack functional vasculature, leading to inadequate nutrient and oxygen supply in larger constructs. Existing models often fail to accurately reproduce the extracellular matrix composition, mechanical properties, and cellular interactions found in native tissues. Many advanced in vitro models are challenging to scale up for high-throughput screening or industrial applications. High costs and technical complexity can limit the accessibility and widespread adoption of technologies like organ-on-a-chip devices.

[0014] The field of tissue engineering and drug discovery has seen a number of advancements over the past few decades. However, several challenges persist in developing reliable and physiologically relevant in vitro models for studying human diseases and screening potential therapeutic agents.

[0015] 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.

[0016] SUMMARY

[0017] 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 three-dimensional (3D) cell culture and tissue engineering, which may comprise at least one interconnecting porous hydrogel block (IPHB); wherein the at least one IPHB may have a hydrogel matrix embedded with interconnected microchannels that form a pseudo- vasculature network; wherein the at least one IPHB may be configured to interconnect with another IPHB horizontally and / or vertically to expand the available volume and / or surface area for cell growth; and a mechanism for perfusing liquids, nutrients, and / or gases through the microchannels under controllable pressures and flow rates. In some embodiments, the hydrogel matrix may comprise biocompatible polymeric material.

[0018] In some embodiments, the biocompatible polymeric material may comprise one or more natural polymers, such as plant-derived polymers and / or animal derived polymers. In some embodiments, the biocompatible polymeric material may comprise polyethylene glycol (PEG), alginate, collagen, and / or hyaluronic acid. In some embodiments, the hydrogel matrix may be oxygen-permeable and / or swellable, which could be based on salinity (e.g. of a perfused liquid). In some embodiments, an otherwise closed vessel with two or more openings for perfusion may be configured to apply a uniform, bifurcating, and / or other flow of liquids, nutrients, and / or gases, e.g. through the IPHB(s). In some embodiments, the IPHB(s) may comprise a plurality of different cell types, which could include primary cells, immortalized cell lines, stem cells, and / or progenitor cells. In some embodiments, cells may be co-cultured, e.g. within the microchannels of the IPHB(s).

[0019] In another aspect, the present disclosure provides a method for generating 3D tissue structures, which may comprise providing a plurality of IPHBs; wherein at least one IPHB has a hydrogel matrix embedded with interconnected microchannels that form a pseudovasculature network; seeding the microchannels with one or more types of cells; joining a plurality of the IPHBs to expand the available volume and / or surface area, e.g. as needed; perfusing the IPHB(s) with at least one medium to support cell growth and / or tissue formation; and wherein the medium / media contain(s) nutrients, gases, and / or other factors, which could be under controllable conditions.

[0020] In some embodiments, cells may include endothelial cells and / or mesenchymal stem cells (MSCs), e.g. to form vascularized tissue structures. Some embodiments may comprise dissolving the hydrogel matrix enzymatically to release cells for analysis. In some embodiments, the IPHB(s) may be configured to be fixed, embedded, and / or sectioned for histological and / or immunohistochemical studies. In some embodiments, the medium / media may be perfused through the IPHB(s) and may be configured to be oxygen-permeable and / or adjusts its properties based on salinity (e.g., of the liquid). In some embodiments, metabolites and / or signaling molecules may be collected, e.g. for biochemical analysis, possibly from the medium / media (which may have been perfused, e.g. through the IPHB(s)). In some embodiments, one or more electrophysiological properties of cells, such as those forming tissue constructs, e.g. within the IPHB(s), may be assessed. Some embodiments may be configured to allow for gravity-assisted waste removal, e.g. to maintain a healthier environment for cell growth. Some embodiments may be configured to be utilized for personalized medicine applications, which could be by creating patient-specific tissue models, e.g. to optimize treatment protocols. Some embodiments may be configured to be utilized for cancer research, such as by using cancer cell lines to facilitate tumor formation, growth, and / or metastasis studies. In another aspect, the present disclosure provides a 3D cell culture system, which may comprise providing a plurality of IPHBs; wherein at least one IPHB has a hydrogel matrix embedded with interconnected microchannels that form a pseudo-vasculature network; wherein at least one IPHB may be configured to interconnect with another IPHB horizontally and / or vertically; and a dynamic culturing environment configured to support both static and perfusion-based culture conditions. In some embodiments, the pseudo-vasculature network, e.g. within the IPHB(s), may be configured to facilitate efficient transport of nutrients, gases, and / or signaling molecules. Some embodiments may comprise a mechanism for controlling the rate, pressure, volume, viscosity, and / or osmotic pressure of the perfusion medium. Some embodiments may be configured to be fixed within a closed vessel with controllable perfusion parameters to simulate dynamic in vivo conditions. In some embodiments, the mechanical stiffness and / or material composition of the IPHB(s) may be configured to be tunable to mimic one or more parameters of one or more tissues and / or structures, e.g. in a body (such as a human). In some embodiments, IPHB form factor may be consistent across research and / or industrial scales, which could enable high-throughput screening and / or large-scale tissue production. Some embodiments may support co-culture of multiple cell types. In some embodiments, cell types may include primary cells, immortalized cell lines, stem cells, and / or progenitor cells.

[0021] In certain embodiments, a system for cell culture 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

[0022] In some embodiments, polymeric matrix may comprise one or more biocompatible polymeric materials.

[0023] In some embodiments, biocompatible polymeric material may comprise at least one natural polymer, plant derived polymer, animal derived polymer, polyethylene glycol, alginate, collagen, laminin, and / or hyaluronic acid. A natural polymer may comprise laminin. A gelatin may comprise porcine, bovine (cow), ovine (sheep), shellfish, and / or fish gelatin.

[0024] In some embodiments, at least one block may be oxygen permeable.

[0025] In some embodiments, at least one block may be oxygen permeable based on the salinity of at least one perfusate. In some embodiments, at least one block may be swellable.

[0026] In some embodiments, at least one block may be swellable based on the salinity of at least one perfusate.

[0027] In some embodiments, at least one block may be configured to be enzymatically dissolvable.

[0028] In some embodiments, at least one block may be configured to be fixed, embedded, and / or sectioned for histological and / or immunohistochemical study.

[0029] Some embodiments may comprise a vessel with two or more openings configured to apply uniform, bifurcating, and / or other flow of perfusate.

[0030] In some embodiments, at least one perfusate may comprise one or more liquids, nutrients, and / or gases.

[0031] Some embodiments may be configured to cultivate one or more cell types when seeded with at least one cell of each type.

[0032] In some embodiments, at least one cell type may comprise primary cells, immortalized cell lines, stem cells, progenitor cells, endothelial cells, and / or MSCs.

[0033] Some embodiments may be configured to permit co-cultivation of a plurality of cell types within at least one block.

[0034] Some embodiments may be configured to permit collection and / or biochemical analysis of one or more metabolites and / or signaling molecules.

[0035] Some embodiments may be configured to permit assessment of one or more electrophysiological properties of cultivated cells.

[0036] Some embodiments may be configured to permit gravity assisted removal of waste.

[0037] In certain embodiments, a method for cell culture may comprise exposing at least one IPHB to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures. 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. One or more cavities may be configured to allow perfusion.

[0038] Some embodiments may comprise seeding at least one block with at least one cell type.

[0039] Some embodiments may comprise co-cultivation of a plurality of cell types.

[0040] Some embodiments may comprise assessment of one or more electrophysiological properties of cultivated cells. Some embodiments may comprise collection and / or biochemical analysis of one or more metabolites and / or signaling molecules.

[0041] In certain embodiments, an IPHB for cell culture 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

[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 IPHBs 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 cell culture 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 embodiments relate to interconnecting porous hydrogel blocks (IPHBs). They may offer a novel and advanced platform for 3D cell culture, tissue engineering, drug discovery, and / or personalized medicine. They may be designed to replicate the complexity and / or functionality of native tissues and / or organs, overcoming the limitations of traditional in vitro models. Certain of their key features and / or functionalities are detailed below, illustrating how they provide a scalable, modular, and / or physiologically relevant environment for various biological applications.

[0052] Certain embodiments and IPHBs may comprise a pseudo- vasculature network of microchannels within a hydrogel matrix. One or multiple cell types may be deposited into these microchannels. As cells require more space to proliferate, migrate, and / or organize into tissue layers, additional IPHBs may be joined to expand the available volume and / or surface area. IPHBs may be connected horizontally and / or vertically, e.g. to align microchannels in different configurations, which may direct how cells expand and / or build tissues.

[0053] In certain embodiments, IPHBs may be fixed within a closed vessel with two or more openings to allow perfusion of defined liquids, nutrients, and / or gases, e.g. in a uniform and / or bifurcating direction(s), which could be under controllable pressure(s) and / or flow rate(s). In some embodiments, hydrogel matrix may be oxygen-permeable and / or swellable, which could be based on salinity (e.g., of perfused liquid(s)). Under such conditions, cells may organize into distinct tissue layer(s) and / or form well-defined tissue structure(s). Introducing additional cell type(s), such as those derived from epithelial, mesothelial, and / or endothelial germ layers, may lead to formation of more complex tissue(s) and / or specific organ components). Certain embodiments may, such as in cancer research, facilitate tumor formation, growth, and / or metastasis studies, e.g. using cancer cell lines.

[0054] Alongside and / or in addition to formation of human tissues and / or organ components, certain embodiments may allow researchers to enzymatically dissolve hydrogel, e.g. to study and / or profile bulk and / or individual cells. In some embodiments, the IPHB(s) may be fixed, embedded (e.g. in paraffin), cryopreserved, sectioned, and / or labeled (such as with one or more histological stains and / or antibodies), e.g. for tissue and / or organ characterization. Cells forming tissue constructs within IPHBs can also be assessed for electrophysiological properties. Features herein make certain embodiments suitable for high-throughput drug screening and / or biotherapeutic development, e.g. tailored such as to specific human populations and / or individual patients. In certain embodiments, the IPHB(s) maintain the same or similar form factor at both research and industrial scales, essentially differing only in the number of blocks connected to generate the required tissue volumes. Certain embodiments allow waste secreted by cells and / or tissues to fall away, e.g. by gravity, which may promote healthier growth. Metabolites and / or biological signals may be collected, such as from perfused media, e.g. for analysis to track disease, cancer, injury, and / or recovery progression. Some embodiments can be easily scaled to produce large quantities of tissues for drug discovery, biotherapeutic development, and / or tissue graft engineering.

[0055] There is no similar technology on the market. Embodiments herein function differently from commercial technologies such as microfluidic devices and organoids. In certain embodiments herein, cells may form their own microenvironments using the pseudovasculature system. When appropriate endothelial cell types are applied, tissues with vascular systems may develop following the pseudo-vasculature microchannels, forming vascularized tissues. Unlike other technologies with predefined physiologies, IPHBs and embodiments herein may allow cells to replicate and build their own physiology based on cellular memory.

[0056] Additionally, IPHBs and embodiments herein can be modular. For certain embodiments and IPHBs, and their microstructure, macrostructure, mechanical stiffness, and / or material composition may be tuned and / or modified, such as to mimic specific parameters, e.g. of defined tissues and / or tissue structures, which could be from a human body. Among other features herein, adaptability and versatility make embodiments herein a promising tool for advancing drug discovery, tissue engineering, and personalized medicine.

[0057] The foregoing background highlights the innovative nature of IPHBs and embodiments herein and their potential to revolutionize the field of drug discovery and tissue engineering.

[0058] 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.

[0059] In certain embodiments, the IPHB(s) may comprise a hydrogel matrix embedded with interconnected microscopic cavities (e.g., microchannels, chambers, and / or pores). A hydrogel material may be selected based on biocompatibility, permeability, and / or one or more mechanical properties. Certain embodiments may comprise polyethylene glycol (PEG), alginate, collagen, and / or hyaluronic acid. Certain embodiments may be designed to mimic at least one extracellular matrix (ECM) found in one or more native tissues, e.g. providing a supportive environment for cell growth and / or organization.

[0060] IPHBs may be embedded with interconnected microchannels that simulate a pseudovasculature system, facilitating the efficient transport of nutrients, gases, and / or signaling molecules. This design may support the growth and / or maintenance of larger, more complex tissue constructs, e.g. by mimicking natural blood flow. Where microfluidic devices and organ- on-a-chip systems may incorporate microchannels to mimic blood flow, the pseudovasculature network within IPHBs and embodiments herein is distinct. The interconnected microchannels of IPHBs and embodiments herein not only facilitate nutrient and / or signal exchange, but also allow for scalable, modular expansion. This design may support larger tissue constructs and more complex tissue architectures, which are not typically achievable with existing technologies.

[0061] The modular nature of IPHBs and embodiments herein may allow for easy expansion and / or customization. Multiple blocks may be joined horizontally and / or vertically, creating varied configurations and / or expanding the available volume and / or surface area. This scalability may support the construction of sophisticated tissue architectures and / or organ components, which is not feasible with traditional 2D or static 3D culture systems. IPHBs and embodiments herein may be customized to suit specific experimental needs, enabling researchers to create tailored tissue models for various applications. This and other features herein are non-obvious because, among other things, traditional 3D culture systems and organoid models lack such flexibility and scalability. The integration of additional blocks to increase surface area and / or volume for cell growth is among novel features herein addressing limitations of fixed-size culture systems.

[0062] Certain embodiments provide a supportive extracellular environment that closely mimics the properties of native tissues. This may promote natural cell behavior, organization, and / or function, e.g. leading to more accurate and / or reliable experimental results. IPHBs and embodiments herein may support the co-culture of various cell types, e.g. including primary cells, immortalized cell lines, stem cells, and / or progenitor cells. They may enable formation of complex, multi-tissue structures that better replicate in vivo conditions. For example, endothelial cells may be co-cultured with mesenchymal stem cells (MSCs) and / or other connective tissue cell types to form vascularized tissues. Certain embodiments and IPHBs may be used in both static and perfusion-based cultures. Perfusion systems may allow for the controlled delivery of liquids, nutrients, and / or gases, e.g. simulating in vivo conditions and / or enhancing tissue organization and / or functionality. Certain embodiments may be oxygen- permeable and / or swellable, e.g. based on salinity (such as of a perfused liquid), which could further support cell viability and / or function. Certain embodiments and IPHBs may enable the formation of well-defined tissue structures and organ components. Cells may organize into distinct layer(s) of tissue(s), and with the addition of specific cell types, more complex structure(s), such as nephron(s), glomeruli, and / or other kidney components), may be generated. In cancer research, certain implementations may facilitate tumor formation, growth, and / or metastasis studies. Traditional 2D cultures and static 3D cultures lack the complexity and physiological relevance of in vivo environments. Organoids and organ-on-a-chip systems offer some physiological relevance but often face limitations in scalability and integration of multiple tissue types. Microfluidic systems and organ-on-a-chip devices may mimic dynamic conditions but often require specialized equipment and technical expertise.

[0063] IPHBs and embodiments herein may be suitable for diverse applications, e.g. including drug discovery, biotherapeutic development, cancer research, regenerative medicine, and / or personalized medicine. This versatility and the features herein make them an invaluable tool for advancing research across multiple fields. In some embodiments, consistent form factor of IPHBs across research and industrial scales may allow for high-throughput drug screening and / or large-scale tissue production, and may make them a cost-effective solution for both laboratory studies and industrial applications. Scalability is important for identifying potential therapeutics efficiently and effectively. Many advanced in vitro models are challenging to scale up for high-throughput screening or industrial applications.

[0064] Certain embodiments and IPHBs herein may be used to create patient-specific tissue model(s), e.g. enabling personalized drug testing and / or therapy development. By comparing healthy and diseased tissue(s), they may help identify specific drug target(s) and / or optimize treatment protocol(s), e.g. for individual patient(s).

[0065] Certain embodiments and IPHBs herein may enable more comprehensive analysis of cultured tissue(s). In some examples, hydrogel may be enzymatically dissolved to release cells for profiling, and / or the block(s) may be fixed, embedded, and / or sectioned for histological and / or immunohistochemical studies. In some examples, the IPHB(s) may be non-degradable. In some embodiments, one or more metabolites and / or signaling molecules may be collected, such as from the perfusion media, e.g. for biochemical analysis. In some embodiments, cells forming tissue constructs (e.g. within the IPHB(s)) may be assessed, such as for electrophysiological properties, which may provide insight(s) into tissue functionality and / or potential therapeutic target(s). The features herein and high level of detailed analysis is non- obvious compared to existing technologies, which often have limitations in accessing the cultured cells for downstream applications. Even the advanced models of other existing options still may require complex and costly analytical techniques.

[0066] Some embodiments and IPHBs allow waste products secreted by cells to fall away by gravity, maintaining a healthier environment for cell growth and reducing the risk of contamination. The ability to remove waste efficiently and maintain a supportive extracellular environment enhances cell viability and function, leading to more reliable experimental outcomes. This gravity-assisted waste removal might not be a feature found in traditional cell culture methods or even in advanced 3D culture systems.

[0067] The microstructure, macrostructure, mechanical stiffness, and / or material composition of IPHBs and embodiments herein may be tuned and / or modified, e.g. to mimic specific parameters, such as of defined tissues and / or tissue structures, which could be in or from a human body. This level of customization and adaptability is not available in other in vitro models, among the features making IPHBs and embodiments herein a unique and powerful tool for researchers.

[0068] Unlike microfluidic devices, organ-on-a-chip platforms, or traditional organoids, IPHBs and embodiments herein offer a unique combination of modularity, scalability, physiological relevance, and versatility. High costs and technical complexity limit the accessibility and widespread adoption of other technologies like organ-on-a-chip devices. Variability in the formation and behavior of 3D cultures and organoids may lead to inconsistent experimental results. Among other features, the ability of cells to form their own microenvironments and / or build their own physiology based on cellular memory sets IPHBs and embodiments herein apart from other technologies.

[0069] By addressing key limitations, IPHBs and embodiments herein have the potential to revolutionize tissue engineering, drug discovery, and / or personalized medicine, enabling more accurate and / or reliable preclinical models and / or accelerating the development of new therapeutics. IPHBs and embodiments herein address important limitations of existing in vitro models, providing a scalable, cost-effective, and / or physiologically relevant platforms that enhance the accuracy and / or reliability of preclinical studies. By mimicking the complexity and / or functionality of native tissues, certain embodiments and IPHBs enable more accurate prediction of drug efficacy and / or toxicity, accelerating the development of new therapeutics. Certain embodiments and IPHBs may support the creation of patient-specific tissue model(s), allowing for personalized drug testing and / or therapy development. By comparing healthy and diseased tissue(s), certain embodiments and IPHBs may help identify specific drug target(s) and / or optimize treatment protocol(s), e.g. for individual patient(s), which may advance the field of personalized medicine. The ability of certain IPHBs and embodiments herein to provide reliable and / or physiologically relevant in vitro models may reduce the need for animal testing, which may help address ethical concerns and / or potentially lower research costs. By increasing confidence in benchtop testing, certain IPHBs and embodiments herein may enable researchers to design more intentional and / or specific animal experiments, and may help minimize the number of animals required. IPHBs and embodiments herein offer versatile platforms that may be used across various fields, including drug discovery, biotherapeutic development, cancer research, regenerative medicine, and / or toxicology. The modularity and / or adaptability of IPHBs and embodiments herein make them an invaluable tool for advancing research and improving the translational accuracy of preclinical models.

[0070] Embodiments herein represent significant advancements, including in the fields of tissue engineering and drug discovery, by addressing key limitations of existing technologies. They provide versatile, scalable, and / or physiologically relevant platforms, e.g. that enhance the accuracy and / or reliability of preclinical models, accelerate the development of new therapeutics, and / or reduce the need for animal testing. Their modularity and / or adaptability contribute to them being invaluable tools, e.g. for researchers and / or developers, such as in the life sciences. Embodiments herein may enable the translation of discoveries into effective and personalized medical therapies.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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%.

[0075] 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.

[0076] 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] A method of generating mature mammalian tissue may comprise providing an initial scaffolding comprising an IPHB that comprises a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous cavities (e.g., pores, channels, and / or chambers), seeding the IPHB with one or more types of adherent cells, and allowing the cells to propagate and form mature mammalian tissue within the IPHB. Some embodiments may include a first IPHB and a second IPHB, interconnecting at least the first IPHB and the second IPHB to expand an initial scaffolding to expand the culture environment and generate larger mature mammalian tissue. Adherent cells comprise primary mammalian cell lines, MSCs, IPSCs, immortalized cell lines, or combinations thereof. The IPHB(s) may be seeded with at least two different cell types, and co-cultured to allow cells to interact and regulate into mature tissue. The IPHB(s) may be perfused with the culture media, e.g. to provide consistent nutrient delivery, and / or waste removal throughout the generation process. The IPHB(s) may be configured to be enzymatically dissolved to retrieve mature mammalian tissue. A microenvironment within the IPHB(s) may be configured to mimic the native physiological conditions from a biological, chemical, electrical, genetic, physical, structural, and / or mechanical perspective, and / or may facilitate the generation of physiologically relevant tissue(s).

[0080] Tissue generated from any embodiment herein may be harvested, and for example may be suitable for application(s) in research modeling, drug testing, and / or the development of tissue graft(s), such as for treating patient(s). A method for generating 3D tissue structure(s) may comprise providing a plurality of IPHBs. At least one IPHB may have a hydrogel matrix embedded with interconnected microchannels that form a pseudo-vasculature network. Some embodiments comprise seeding the microchannels with one or more types of cells, joining a plurality of IPHBs to expand the available volume and / or surface area (e.g., as needed), and / or perfusing at least one IPHB (such as with a medium, e.g. to support cell growth and / or tissue formations)). The medium in some embodiments may contain one or more nutrients, gases, and / or other factors, which could be under controllable condition(s). The cells in some embodiments may include endothelial cells and / or MSCs, e.g. to form vascularized tissue structure(s). The hydrogel matrix in some embodiments may be enzymatically dissolved, e.g. to release cells, such as for analysis. The IPHB in some embodiments may be configured to be fixed, embedded, and / or sectioned, e.g. for histological and / or immunohistochemical studies. The medium, which may be perfused through the IPHB(s), in some embodiments, may be configured to be oxygen-permeable and / or adjust its properties based on salinity (e.g. of the liquid). Metabolites an / or signaling molecules in some embodiments may be collected, such as for biochemical analysis, e.g. from the medium (which may have perfused, such as through the IPHB(s)). In some embodiments, electrophysiological properties of cells forming tissue constructs may be assessed. Some embodiments may be configured to allow for gravity-assisted waste removal, e.g. maintaining a healthier environment for cell growth. Some embodiments may be configured to be utilized for personalized medicine applications, such as by creating patient-specific tissue models, e.g. to optimize treatment protocols. Some embodiments may be configured to be utilized for cancer research, such as by using cancer cell lines, e.g. to facilitate tumor formation, growth, and / or metastasis studies.

[0081] In certain embodiments, a system for cell culture 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

[0082] In some embodiments, polymeric matrix may comprise one or more biocompatible polymeric materials.

[0083] In some embodiments, biocompatible polymeric material may comprise at least one natural polymer, plant derived polymer, animal derived polymer, polyethylene glycol, alginate, collagen, laminin, and / or hyaluronic acid. A natural polymer may comprise laminin. A gelatin may comprise porcine, bovine (cow), ovine (sheep), shellfish, and / or fish gelatin.

[0084] In some embodiments, at least one block may be oxygen permeable.

[0085] In some embodiments, at least one block may be oxygen permeable based on the salinity of at least one perfusate.

[0086] In some embodiments, at least one block may be swellable.

[0087] In some embodiments, at least one block may be swellable based on the salinity of at least one perfusate.

[0088] In some embodiments, at least one block may be configured to be enzymatically dissolvable.

[0089] In some embodiments, at least one block may be configured to be fixed, embedded, and / or sectioned for histological and / or immunohistochemical study.

[0090] Some embodiments may comprise a vessel with two or more openings configured to apply uniform, bifurcating, and / or other flow of perfusate.

[0091] In some embodiments, at least one perfusate may comprise one or more liquids, nutrients, and / or gases.

[0092] Some embodiments may be configured to cultivate one or more cell types when seeded with at least one cell of each type.

[0093] In some embodiments, at least one cell type may comprise primary cells, immortalized cell lines, stem cells, progenitor cells, endothelial cells, and / or MSCs.

[0094] Some embodiments may be configured to permit co-cultivation of a plurality of cell types within at least one block.

[0095] Some embodiments may be configured to permit collection and / or biochemical analysis of one or more metabolites and / or signaling molecules.

[0096] Some embodiments may be configured to permit assessment of one or more electrophysiological properties of cultivated cells.

[0097] Some embodiments may be configured to permit gravity assisted removal of waste.

[0098] In certain embodiments, a method for cell culture may comprise exposing at least one IPHB to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures. 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. One or more cavities may be configured to allow perfusion. Some embodiments may comprise seeding at least one block with at least one cell type. Some embodiments may comprise co-cultivation of a plurality of cell types.

[0099] Some embodiments may comprise assessment of one or more electrophysiological properties of cultivated cells.

[0100] Some embodiments may comprise collection and / or biochemical analysis of one or more metabolites and / or signaling molecules.

[0101] In certain embodiments, an IPHB for cell culture 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

[0102] Described herein are systems, methods, and IPHBs for cell culture. 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures. A method may comprise exposing at least one IPHB to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures.

[0103] Turning to Figure 3, illustrated is an example embodiment of a method 300 for cell culture. In certain embodiments, method 300 may comprise step 310 of perfusate and optionally step 320 of seeding. Step 310 may comprise exposing at least one IPHB to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, and / or osmotic pressures. The IPHB may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more cavities may be configured to allow perfusion. One or more cavities may comprise one or more channels, pores, and / or chambers. Step 310 may comprise dynamic culture conditions. Dynamic culture conditions may be configured to achieve nutrient and / or waste exchange. Dynamic culture conditions may comprise exposing at least one IPHB to a culture media via perfusion and / or static solution. The culture media may comprise one or more nutrients. Dynamic culture conditions may comprise varying a culture media’s nutrient type and / or concentration. In certain embodiments, one or more cavities may be configured as pathway(s) for nutrient delivery, gas exchange, and / or waste removal. Some embodiments may further comprise a step 320 for seeding. Step 320 may comprise seeding at least one IPHB with at least one cell type. Some embodiments may comprise co-cultivation of a plurality of cell types. In some embodiments, 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 blocks and / or cells (e.g., the personalized model(s) or otherwise) to one or more analyses, which could guide diagnosis and / or therapy, and may be tailored (e.g., for the subjects) or otherwise). 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 senescence, growth, and / or characteristic(s) (e.g., of cells). Step 340 may comprise assessment of one or more electrophysiological properties of cultivated cells. Step 340 may comprise collection and / or biochemical analysis of one or more metabolites and / or signaling molecules.

[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 perfusate 310, seeding 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 seeding 320. Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of perfusate 310, seeding 320, personalized model 330, and / or analysis 340. Further, the one or more blocks underlying step 320 (and / or being seeded) could be altered before, during, and / or after other steps (e.g. perfusate 310, seeding 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. 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.

[0107] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0108] Clause 1. A system for 3D cell culture and tissue engineering, comprising: at least one IPHB, wherein each of the at least one IPHB has a hydrogel matrix embedded with interconnected microchannels that form a pseudo-vasculature network, and wherein the at least one IPHB is configured to interlock with one or more additional IPHBs horizontally and / or vertically to expand the available volume and surface area for cell growth; and a mechanism for perfusing liquids, nutrients, and gases through the microchannels under controllable pressures and / or flow rates.

[0109] Clause 2. The system of clause 1, wherein the hydrogel matrix comprises biocompatible polymeric material.

[0110] Clause 3. The system of clause 2, wherein the biocompatible polymeric material comprises one or more natural polymers, such as plant-derived polymers and / or animal derived polymers.

[0111] Clause 4. The system of clauses 2-3, wherein the biocompatible polymeric material comprises polyethylene glycol (PEG), alginate, collagen, and hyaluronic acid.

[0112] Clause 5. The system of clauses 2-4, wherein the hydrogel matrix is oxygen-permeable and swellable based on the salinity of the perfused liquid.

[0113] Clause 6. The system of clause 1, further comprising a closed vessel with two or more openings for the perfusion mechanism and being configured to apply a uniform or bifurcating flow of liquids, nutrients, and / or gases through the at least one IPHB.

[0114] Clause 7. The system of clause 1, wherein the at least one IPHB comprises a plurality of different cell types seeded therein, including primary cells, immortalized cell lines, stem cells, and progenitor cells.

[0115] Clause 8. The system of clause 6, wherein the plurality of different cell types are cocultured within the microchannels of the at least one IPHB.

[0116] Clause 9. A method for generating 3D tissue structures, comprising: providing a plurality of IPHBs, wherein each IPHB has a hydrogel matrix embedded with interconnected microchannels that form a pseudo- vasculature network; seeding the microchannels with one or more types of cells; joining at least a portion of the plurality of IPHBs to expand the available volume and surface area as needed; perfusing the portion of the plurality of IPHBs with a medium to support cell growth and tissue formation; and wherein the medium contains nutrients, gases, and other factors under controllable conditions to support cell growth and tissue formation.

[0117] Clause 10. The method of clause 9, wherein the cells include endothelial cells and MSCs to form vascularized tissue structures.

[0118] Clause 11. The method of clauses 9-10, further comprising dissolving the hydrogel matrix enzymatically to release cells for analysis.

[0119] Clause 12. The method of clauses 9-10, wherein the at least one IPHB is configured to be fixed, embedded, and sectioned for histological and immunohistochemical studies.

[0120] Clause 13. The method of clause 9, wherein the medium perfused through the at least one IPHB is configured to be oxygen-permeable and adjusts its properties based on the salinity of the liquid.

[0121] Clause 14. The method of clause 9, wherein metabolites and signaling molecules are collected for biochemical analysis from the medium perfused through the at least one IPHB.

[0122] Clause 15. The method of clauses 13-14, wherein the electrophysiological properties of the cells forming tissue constructs within the at least one IPHB are assessed.

[0123] Clause 16. The method of clause 9, wherein the at least one IPHB is configured to allow for gravity-assisted waste removal, maintaining a healthier environment for cell growth.

[0124] Clause 17. The method of clause 9, wherein the at least one IPHB is configured to be utilized for personalized medicine applications by creating patient-specific tissue models to optimize treatment protocols.

[0125] Clause 18. The method of clause 17, wherein the at least one IPHB is configured to be utilized for cancer research by using cancer cell lines to facilitate tumor formation, growth, and metastasis studies.

[0126] Clause 19. A 3D cell culture system, comprising: a plurality of IPHBs, wherein the plurality of IPHBs each has a hydrogel matrix embedded with interconnected microchannels that form a pseudo-vasculature network, and wherein each of the plurality of IPHBs is configured to interlock with one or more IPHBs horizontally and / or vertically; a dynamic culturing environment configured to support both static and perfusion-based culture conditions.

[0127] Clause 20. The system of clause 19, wherein the pseudo-vasculature network within the IPHBs is configured to facilitate efficient transport of nutrients, gases, and signaling molecules. Clause 21. The system of clause 19, further comprising a mechanism for controlling the rate, pressure, volume, viscosity, and / or osmotic pressure of the perfusion medium.

[0128] Clause 22. The system of clause 19, wherein the IPHBs are each configured to be fixed within a closed vessel with controllable perfusion parameters to simulate dynamic in vivo conditions.

[0129] Clause 23. The system of clause 22, wherein the mechanical stiffness and material composition of the IPHBs are each configured to be tunable to mimic specific parameters of defined tissues or tissue structures in the human body.

[0130] Clause 24. The system of clause 19, wherein the form factor of the IPHBs are consistent across research and industrial scales, enabling high-throughput screening and large-scale tissue production.

[0131] Clause 25. The system of clause 19, wherein the IPHBs each support the co-culture of multiple cell types.

[0132] Clause 26. The system of clause 25, wherein the cell types include primary cells, immortalized cell lines, stem cells, and progenitor cells.

[0133] 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

CLAIMSWHAT IS CLAIMED IS:

1. A system for cell culture, 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, osmotic pressures, or a combination thereof.

2. The system of claim 1 , wherein the polymeric matrix comprises one or more biocompatible polymeric materials.

3. The system of claim 2, wherein at least one biocompatible polymeric material comprises at least one natural polymer, plant derived polymer, animal derived polymer, polyethylene glycol, alginate, collagen, laminin, hyaluronic acid, or any combination thereof.

4. The system of claim 1 , wherein at least one block is oxygen permeable.

5. The system of claim 4, wherein at least one block is oxygen permeable based on the salinity of at least one perfusate.

6. The system of claim 1, wherein at least one block is swellable.

7. The system of claim 6, wherein at least one block is swellable based on the salinity of at least one perfusate.

8. The system of claim 1, wherein at least one block is configured to be enzymatically dissolvable.

9. The system of claim 1, wherein at least one block is configured to be fixed, embedded, or sectioned for histological or immunohistochemical study or any combination thereof.

10. The system of claim 1, further comprising a vessel with two or more openings configured to apply a uniform or bifurcating flow of perfusate or combination thereof.

11. The system of claim 1, wherein at least one perfusate comprises one or more liquids, nutrients, gases, or combination thereof.

12. The system of claim 1, further configured to cultivate one or more cell types when seeded with at least one cell of each type.

13. The system of claim 12, wherein at least one cell type comprises primary cells, immortalized cell lines, stem cells, progenitor cells, endothelial cells, mesenchymal stem cells, or a combination thereof.

14. The system of claim 12, configured to permit co-cultivation of a plurality of cell types within at least one block.

15. The system of claim 1, configured to permit collection, biochemical analysis, or a combination thereof, of one or more metabolites, signaling molecules, or a combination thereof.

16. The system of claim 12, configured to permit assessment of one or more electrophysiological properties of cultivated cells.

17. The system of claim 1, configured to permit gravity assisted removal of waste.

18. A method for cell culture, comprising exposing at least one interconnecting porous hydrogel block to at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, osmotic pressures, or a combination thereof,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, further wherein at least one cavity is configured to allow perfusion.

19. The method of claim 18, further comprising seeding at least one block with at least one cell type.

20. The method of claim 19, comprising co-cultivation of a plurality of cell types.

21. The system of claim 20, comprising assessment of one or more electrophysiological properties of cultivated cells.

22. The method of claim 18, further comprising collection, biochemical analysis, or a combination thereof, of one or more metabolites, signaling molecules, or a combination thereof.

23. An interconnecting porous hydrogel block for cell culture, 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 be perfused via at least one cavity by at least one perfusate under one or more controllable flow rates, pressures, volumes, viscosities, osmotic pressures, or a combination thereof.

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