Mammalian tissue generation using interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for generating mammalian tissue. A block may comprise a three-dimensional 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 a plurality of blocks configured to interconnect to form a scaffold configured to cultivate one or more cell types organized into stratified layers. A method may comprise acquiring a plurality of blocks, interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers, and seeding at least one block with at least one cell type adherent to the block. Embodiments may comprise perfusing at least one block with culture media and / or harvesting mammalian tissue generated.

Description

[0001] MAMMALIAN TISSUE GENERATION USING INTERCONNECTING POROUS

[0002] HYDROGEL BLOCKS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 63 / 665,798, filed June 28, 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.

[0005] TECHNICAL FIELD

[0006] The subject matter disclosed herein is generally directed to the cultivation of various cell types, individually or in combination, using interconnecting porous hydrogel blocks (IPHBs). IPHBs may provide comprehensive support to cells, which may include structural, mechanical, chemical, electrical, osmotic, nutrient, gas, and / or genetic support, and may include facilitating the organization of cells into stratified layers that resemble mature tissue. These tissue-like layers may comprise cells and extracellular matrix, and may create a positive feedback mechanism for generating additional tissue-like components. Such components may be used for constructing tissue grafts, including for humans, animals, and / or plants.

[0007] BACKGROUND

[0008] Current tissue engineering techniques, such as two-dimensional (2D) cell cultures, three-dimensional (3D) cultures, scafibld-based systems, bioprinting technologies, and organ- on-a-chip devices, face several limitations, including the inability to consistently generate mature tissues, challenges in scaling up tissue production, and difficulties in mimicking the physiological microenvironment.

[0009] Traditional 2D cell cultures do not adequately replicate the complex interactions found in vivo. 2D cell culture involves growing cells on flat surfaces such as petri dishes, flasks, or well plates. This method has been widely used for decades due to its simplicity and ease of use. However, cells in 2D cultures often exhibit different morphology and behavior compared to their in vivo counterparts. They lack the complex interactions with other cell types and the extracellular matrix, which are important for developing mature tissue structures.

[0010] 3D cultures, scaffold-based systems, and bioprinting technologies may face issues related to nutrient diffusion, waste removal, and maintaining cell viability over extended periods. 3D cell culture systems aim to replicate the in vivo environment more closely by allowing cells to grow in three dimensions. This could be via scaffolds, hydrogels, or spheroid cultures. These systems may provide better cell-cell and cell-matrix interactions, and may lead to more physiologically relevant tissues. However, challenges such as nutrient diffusion, waste removal, and uniform cell distribution still need to be addressed.

[0011] Scaffold-based approaches may use natural or synthetic materials to provide a structural framework for cell attachment and growth. These scaffolds may be engineered to have specific properties such as porosity, stiffness, and biodegradability. While scaffolds may support complex tissue architectures, issues like scaffold biocompatibility, degradation rates, and mechanical properties matching the target tissue remain significant challenges.

[0012] 3D bioprinting is a technique that uses computer-aided design to print cells, biomaterials, and growth factors layer-by-layer to create complex tissue structures. This technology may allow control over cell placement and creation of intricate tissue architectures. Despite its potential, bioprinting faces obstacles like ensuring cell viability during the printing process, achieving vascularization in printed tissues, and scaling up for clinical applications.

[0013] Organ-on-a-chip devices are microfluidic cell culture platforms that may mimic the physiological functions of human organs. These devices may use microchannels to simulate blood flow and may provide a controlled environment for studying cell behavior, drug responses, and disease mechanisms. Organ-on-a-chip devices may provide control over the microenvironment and mimicking dynamic physiological conditions on a small scale. They may be valuable for high-throughput screening and real-time monitoring. However, they are limited by their complexity, cost, and the small volume of tissue they can culture.

[0014] There remains a need in the art for simplified and efficient platforms that enable 3D continuous growth of a variety of cells and / or tissues, particularly platforms that more closely mimic or resemble in vivo conditions associated with a natural cell and / or tissue of interest. By addressing these critical challenges and offering significant advantages, interconnecting porous hydrogel blocks and particularly the embodiments herein represent a major advancement in tissue engineering, paving the way for the reliable production of mature tissues for various biomedical applications.

[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. SUMMARY

[0016] One or more embodiments of the disclosure may address one or more of the aforementioned problems. Certain embodiments according to the disclosure provide a modular system for generating mammalian tissue, comprising at least one interconnecting porous hydrogel block (IPHB), with the at least one IPHB comprising a three-dimensional (3D) macrostructure defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers, wherein the at least one IPHB is configured to interconnect with another IPHB, forming a scaffold, and wherein the at least one IPHB is configured to cultivate one or more cell types, individually or in combination, to facilitate the organization of cells into stratified layers that mimic mature mammalian tissue. Further, the microporous channels and / or chambers of the at least one IPHB may be configured to facilitate vertical and horizontal cell communication to form organized tissue layers and the extracellular matrix. Further still, the microporous channels and / or chambers of the at least one IPHB may be preformed and optionally standardized across batches. Still yet, the at least one IPHB may comprise a biocompatible polymeric material. Yet again, the biocompatible polymeric material may comprise one or more natural polymers, such as plant-derived polymers and / or animal- derived polymers. Still yet further, the biocompatible polymeric material may comprise poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), or combinations thereof. Again, the at least one IPHB may be configured to support the cultivation of various cell types, including primary mammalian cell lines, mesenchymal stem cells (MSCs), induced pluripotent stem cells (IPSCs), immortalized cell lines, or any combination thereof. Even further, the at least one IPHB may be configured to co-culture multiple cell types to allow cells to interact and regulate into mature mammalian tissue. Still, the at least one IPHB may be configured to maintain the stem-like properties of MSCs over extended culture periods. Still further, the at least one IPHB may be configured to permeate the culture media. Yet again, the at least one IPHB may be configured to enable perfusion horizontally through the at least one IPHB when the at least one IPHB is interconnected with another IPHB. Yet again further, the at least one IPHB may be configured to enable perfusion vertically through the at least one IPHB when the at least one IPHB is stacked on top of another IPHB. Even further still, the at least one IPHB may be configured be enzymatically dissolved to leave behind intact mature mammalian tissue; wherein the continuous polymeric matrix material may be enzymatically dissolvable. Again, the at least one IPHB may be configured to reduce media consumption by up to 90%, plasticware usage by up to 85%, and human labor. In another aspect, the present disclosure provides a method of generating mature mammalian tissue, comprising providing an initial scaffolding comprising at least one IPHB comprising a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers, seeding the at least one IPHB with one or more types of adherent cells, and allowing the cells to propagate and form mature mammalian tissue within the at least one IPHB. Further, the at least one IPHB may include a first IPHB and a second IPHB, the method further comprising 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. Again, the adherent cells may comprise primary mammalian cell lines, MSCs, IPSCs, immortalized cell lines, or combinations thereof. Again still, the at least one IPHB may be seeded with at least two different cell types, and co-cultured to allow cells to interact and regulate into mature tissue. Yet still, the at least one IPHB may be perfused with the culture media to provide consistent nutrient delivery and waste removal throughout the generation process. Still yet, the at least one IPHB may be configured to be enzymatically dissolved to retrieve mature mammalian tissue. Even further, a microenvironment within the at least on IPHB may be configured to mimic the native physiological conditions from a biological, chemical, electrical, genetic, physical, structural, and mechanical perspective, facilitating the generation of physiologically relevant tissues. Again further, the method may comprise harvesting the mammalian tissue generated from the at least one IPHB, and the mammalian tissue generated from the at least one IPHB may be suitable for applications in research modeling, drug testing, and the development of tissue grafts for treating patients.

[0017] In certain embodiments, a modular system for generating mammalian tissue may comprise a plurality of IPHBs. 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 wherein the blocks interconnect to form a scaffold configured to cultivate one or more cell types organized into stratified layers.

[0018] In some embodiments, the cavities may comprise one or more channels and / or chambers. A cavity may comprise a well, recess, pore, pocket, and / or enclosure.

[0019] In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in a vertical axis relative to gravity. In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in an axis orthogonal to gravity.

[0020] In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in a horizontal axis relative to gravity.

[0021] In some embodiments, the network of microporous cavities may be predetermined before blocks are interconnected.

[0022] In some embodiments, the network of microporous cavities may be standardized across blocks.

[0023] In some embodiments, at least one block may comprise a biocompatible polymeric material.

[0024] In some embodiments, the biocompatible polymeric material may comprise at least one natural polymer, plant derived polymer, animal derived polymer, PLGA, and / or PEG.

[0025] In some embodiments, at least one block comprises an enzymatically dissolvable material.

[0026] In some embodiments, the 3D continuous polymeric matrix consists of enzymatically dissolvable material.

[0027] Some embodiments may be configured to cultivate at least one primary mammalian cell line, MSC, IPSC, and / or immortalized cell line.

[0028] Some embodiments may be configured to cultivate at least two different cell types contemporaneously.

[0029] In some embodiments, at least one block may be configured to permit permeation of culture media.

[0030] Some embodiments may be configured to permit permeation of culture media vertically, horizontally, and / or orthogonally.

[0031] In certain embodiments, a method for generating mammalian tissue may comprise acquiring a plurality of IPHBs, interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers, and seeding at least one block with at least one cell type adherent to the block. 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.

[0032] In some embodiments, the blocks may be interconnected vertically, horizontally, and / or orthogonally. Some embodiments may further comprise seeding at least one block with at least one second cell type.

[0033] Some embodiments may further comprise perfusing at least one block with culture media.

[0034] Some embodiments may further comprise harvesting mammalian tissue generated after the seeding.

[0035] In some embodiments, at least one block may comprise an enzymatically dissolvable material. Some embodiments may further comprise enzymatically dissolving the enzymatically dissolvable material.

[0036] In certain embodiments, an IPHB for generating mammalian tissue 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 to form a scaffold configured to cultivate one or more cell types organized into stratified layers.

[0037] 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.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] 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:

[0040] FIG. 1 illustrates two separate interconnecting porous hydrogel blocks (IPHBs) in accordance with certain embodiments;

[0041] FIG. 2 illustrates three interconnected IPHBs in accordance with certain embodiments; and

[0042] FIG. 3 illustrates a method of generating mammalian tissue in accordance with certain embodiments.

[0043] The figures herein are for illustrative purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0044] 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.

[0045] The presently disclosed embodiments relate to interconnecting porous hydrogel blocks (IPHBs), and provide a novel approach to generating mature mammalian tissues. IPHBs may comprise modular, interconnecting units made of a continuous polymeric matrix material with a network of microporous cavities such as channels and / or chambers. These features may enable IPHBs to support the consistent and repeated generation of mature tissues using a wide range of adherent cell types, either individually or in combination as a co-culture.

[0046] The disclosure is cell agnostic and serves as an enabling technology in that it may allow for the generation of many types of soft connective tissue found in mammalian organisms. Additionally, the disclosure supports the synergistic cultivation of cells from a single cell type or multiple cell types together. IPHBs may enable different cell types to communicate and regulate each other, forming organized layers of tissue and extracellular matrix (ECM) based on the specific cells introduced into the IPHB. The formulation of tissue can be modified by altering the cell inputs seeded into the IPHB, allowing for customized tissue engineering solutions.

[0047] IPHBs and the embodiments herein represent a significant advancement in tissue engineering, providing a more scalable and versatile approach. They support the formation of mature tissues by allowing cells to create their own microenvironment within a three- dimensional (3D) structure. The modularity, reduced resource usage, and potentially the ability to dissolve the hydrogel to retrieve intact tissues could be significant advantages. The perfusion capabilities of IPHBs, potentially allowing for both horizontal and vertical nutrient delivery, may further enhance their functionality compared to traditional bioreactor systems. Additionally, the ability to scale embodiments herein from bench top to industrial applications, which could be combined with easy use and pre-optimized conditions for various cell types, can make them uniquely positioned to meet the demands of both research and commercial production. In summary, embodiments herein including IPHBs, as volumetric microfluidic devices, may offer a comprehensive solution for mammalian tissue generation, overcoming many limitations of traditional organ-on-a-chip or other devices and techniques by providing a scalable, versatile, and / or physiologically relevant environment for tissue development.

[0048] A major challenge in tissue engineering is creating vascular networks within engineered tissues to ensure adequate nutrient supply, gas perfusion, and waste removal. Without proper vascularization, tissues larger than a few hundred micrometers may struggle to maintain viability. In certain embodiments herein, the network of microporous channels within each IPHB may promote better nutrient diffusion and waste removal, addressing the issue of vascularization. This design may support the survival and growth of larger, more complex tissue constructs.

[0049] Finding reliable sources of cells and directing their differentiation into desired cell types remains challenging. Stem cells offer potential but controlling their differentiation and ensuring the formation of mature, functional tissues is complex. In certain embodiments herein, IPHBs may be configured to maintain the stem-like properties of mesenchymal stem cells (MSCs) over extended culture periods. This may be achieved by mimicking the mechanical properties of native tissues, which may help in retaining non-senescent cell populations and enhancing their secretory activity.

[0050] It can be advantageous for engineered tissues to match the mechanical properties of the target tissues to function correctly in vivo. Achieving this balance is difficult, especially for load-bearing tissues like bone and cartilage. In certain embodiments herein, the hydrogel matrix of each IPHB may mimic the natural ECM, providing a supportive environment for cells to attach, grow, and differentiate. This may enhance the physiological relevance of the cultured tissues compared to traditional 2D cultures. In certain embodiments herein, IPHBs may be perfused with media, to help efficient nutrient delivery and waste removal. The unique design of certain embodiments herein may allow for perfusion to be applied horizontally through horizontally interconnected IPHBs and / or vertically through vertically interconnected (e.g., vertically stacked) IPHBs. This is a significant improvement over systems that rely on stir-tank bioreactors, wave bioreactors, roller bottles, or hollow-fiber bioreactors, which often struggle with uniform nutrient distribution and waste removal. The horizontal and / or vertical perfusion capabilities of certain embodiments herein may provide more consistent and controlled culture conditions, enhancing the quality and reliability of the engineered tissues.

[0051] Scaling up tissue engineering processes for commercial production while maintaining consistency and quality is a significant hurdle. This includes standardizing cell culture conditions, materials, and manufacturing processes. In certain embodiments herein, IPHBs feature a modular design that may allow for easy expansion and customization of the cell culture environment. This modularity may enable the creation of larger tissue constructs by simply adding more blocks, facilitating scale-up without compromising cell viability. This scalability and other features herein set these embodiments apart from other technologies on the market.

[0052] Helping engineered tissues integrate seamlessly with a host tissue when implanted is advantageous. This may involve matching the biological, mechanical, and / or immunological properties to help avoid rejection and promote functionality. In certain embodiments herein, the interconnecting feature of IPHBs may allow for the creation of customized tissue architectures, and / or may enable the co-culture of different cell types to form complex organoids or micro-tissues. This flexibility may support various tissue engineering applications, and may include research modeling, drug testing, and / or the development of grafts for treating patients.

[0053] 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.

[0054] Embodiments herein are designed to overcome the limitations of current tissue engineering technologies, including by providing a scalable and customizable 3D culture environment. IPHBs may be designed as modular units that can interconnect with one another, allowing for easy expansion and customization of the cell culture environment. The modular design may allow for easy expansion and assembly of larger tissue constructs by simply adding additional blocks, while preformed and standardized microchannels may ensure consistent and reliable cell culture conditions. Certain embodiments herein may facilitate physiological-like signaling and communication between cells through ECM and hydrogel material, enabling the formation of organized, stratified tissue layers. Unlike other tissue culture systems, IPHBs and embodiments herein may provide a scalable platform that can grow with the needs of the experiment or application without compromising consistency and maturity, making them suitable for both small-scale laboratory research and large-scale industrial production.

[0055] Embodiments herein can be highly versatile and can support the cultivation of a wide range of cell types, including primary mammalian cell lines, MSCs, induced pluripotent stem cells (IPSCs), and immortalized cell lines from various connective tissues. This versatility can be suitable for a variety of tissue engineering applications, including research modeling, drug testing, and the development of grafts for treating patients. The ability to customize the tissue formulation by altering the cell inputs may further enhance their utility. In certain embodiments herein, IPHBs may facilitate the generation of mature tissues in manners unlike techniques in the prior art. The ability of embodiments herein to support a wide range of cell types in a standardized manner and other novel features address important gaps in the field.

[0056] In certain embodiments herein, IPHBs may be designed as modular units that can interconnect to form larger and more complex tissue constructs. Each block may comprise a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous cavities, which may include channels and / or chambers. This design may support the generation of large and complex tissue constructs by interconnecting additional blocks. The ability to scale up tissue generation without compromising consistency and maturity and other unique features set embodiments herein apart from traditional methods. The modularity may allow for easier expansion and customization of the cell culture environment, may help support both small-scale laboratory research and large-scale industrial production, and may make embodiments versatile for various research and commercial needs. The format of IPHBs in certain embodiments herein may allow for the generation of mature tissues efficiently, and / or may support the transition from laboratory research to industrial-scale production.

[0057] Certain embodiments herein may help create a microenvironment that closely mimics the native physiological conditions from a biological, chemical, electrical, genetic, physical, structural, and / or mechanical perspective. This may include the formation of stratified tissue layers and / or the facilitation of physiological-like signaling and / or communication between cells through ECM and hydrogel material. An IPHB may be composed of a continuous polymeric matrix material with a network of microporous cavities, such as channels and / or chambers. The interconnected microporous channels may enable vertical and / or horizontal communication among cells, and may allow for the formation of organized layers of tissue and / or ECM. Cells may communicate with one another within the same IPHB and / or across different IPHBs, which could be directly or indirectly interconnected. Embodiments herein and such features may support the development of mature tissues with a higher degree of physiological relevance compared to conventional methods. The design of certain embodiments herein may mimic the natural ECM, and / or may provide a supportive environment for cells to attach, grow, and / or differentiate. In certain embodiments, the 3D structure of IPHBs may allow cells to interact more naturally with their surroundings, and / or may promote the formation of tissue-like structures. Features of embodiments herein present significant advancement over 2D culture systems, which do not adequately replicate the complex interactions found in vivo. Certain embodiments may allow for the co-culture of multiple cell types, and may enable them to interact and regulate each other to form organized layers of tissue and ECM. This co-culture capability may help create more complex and / or physiologically relevant tissue models. The ability to use IPHBs for co-culturing different cell types to generate mature tissues successfully and other features of embodiments herein are not evident in existing technologies.

[0058] In certain embodiments, IPHBs may be perfused with media to promote efficient nutrient delivery and waste removal. Perfusion may be applied horizontally and / or vertically through interconnected IPHBs, which may be stacked vertically and / or horizontally. Such perfusion may provide more consistent and controlled culture conditions than traditional bioreactor systems, such as stir-tank bioreactors, wave bioreactors, roller bottles, and hollowfiber bioreactors, which often struggle with uniform nutrient distribution. This and other features herein may enhance the functionality and reliability of the engineered tissues. Additionally, IPHBs may be designed to maintain the stem-like properties of MSCs and / or other cell types over extended culture periods, which may avoid, decrease, and / or eliminate the need for frequent subculturing. This could not only reduce labor and the risk of cell senescence, but also could support continuous and long-term studies, which could make a system ideal for generating mature tissues in accordance with embodiments herein.

[0059] As noted herein, certain embodiments may comprise IPHBs configured to mimic 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.

[0060] In certain embodiments, one or more IPHBs may be enzymatically dissolved, which may leave a formed tissue intact. This feature may be advantageous for downstream applications, such as tissue transplantation and / or analysis; and / or it may allow for easier retrieval of the engineered tissues without compromising their integrity. Alternatively or additionally, one or more IPHBs may be non-degradable.

[0061] In certain embodiments, the microchannels in one or more IPHBs may be preformed and / or standardized across batches, which may promote consistent and / or reliable cultivation of cells and tissues. An IPHB may be optimized for different cell types, which could make for easier use by one or more end-users. Standardization may simplify the setup process and / or promote reproducibility, which can be important for research and / or clinical applications. The ability to produce standardized mature tissues repeatedly and consistently poses a significant advancement that is not obvious from existing technologies.

[0062] The innovative design of embodiments herein may allow for a dramatic reduction in media consumption (e.g. up to 90%), plasticware usage (e.g. up to 85%), and / or human labor. Embodiments herein may lower operational costs. Embodiments herein may support sustainable laboratory practices, including by minimizing waste and / or resource usage. This efficiency may address practical barriers in tissue engineering and / or may support widespread adoption for research institutions and / or industrial applications.

[0063] Embodiments herein may be suited for applications in research modeling, drug testing, and / or the development of tissue grafts such as for treating patients. They may be helpful in creating physiologically relevant tissue models, such as for studying cell behavior, disease mechanisms, and / or drug responses. They may be used to develop more accurate and / or reliable models, such as for high-throughput screening and / or toxicity testing. They may be utilized to generate tissue grafts, e.g. for transplantation, which could be to treat injuries, defects, and / or diseases, including in humans and / or animals. They may be utilized to tailor tissue constructs, e.g. to specific research and / or clinical needs, such as by adjusting cell types and / or culture conditions. They may provide a versatile, scalable, and / or physiologically relevant platform. They may enable reliable production, e.g. of mature tissues, which could be for various biomedical applications. Embodiments herein represent significant advancement in tissue engineering.

[0064] The non-obviousness of embodiments herein is evident, including, for example, through the unprecedented outcomes they achieve. IPHBs can enable the repeated and consistent generation of mature tissues using many an adherent cell type, including separately or in combination with multiple cell types (co-culture). The potential to produce standardized, mature tissues reliably and consistently using a versatile, scalable, and physiologically relevant platform represents a significant and non-obvious advancement in the field of tissue engineering. Such outcomes would not be readily apparent to someone skilled in the art based on existing knowledge and technologies, further underscoring the non-obvious nature of embodiments herein. Embodiments herein overcome limitations of existing technologies and pave the way for reliable production of mature tissues for various biomedical applications, from research and drug testing to clinical therapies.

[0065] 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.

[0066] 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.

[0067] 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%.

[0068] 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, or a norbomene group. In accordance with certain embodiments, at least one of the first monomer 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 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 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.

[0069] In certain embodiments, one or more thermoplastic polymers 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%.

[0070] 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 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.

[0071] 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.

[0072] In certain embodiments, a method of generating 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., 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. Certain embodiments 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 may comprise primary mammalian cell lines, MSCs, IPSCs, immortalized cell lines, or any combinations) thereof. One or more IPHBs may be seeded with at least two different cell types, and co-cultured to allow cells to interact and regulate into mature tissue. One or more IPHBs may be perfused with culture media to promote consistent nutrient delivery and waste removal throughout the generation process. One or more IPHBs may be configured to be enzymatically dissolved to retrieve mature mammalian tissue. A microenvironment within an IPHB may be configured to mimic native physiological conditions from a biological, chemical, electrical, genetic, physical, structural, and / or mechanical perspective, facilitating the generation of physiologically relevant tissues. Mammalian tissue generated may be harvested, and the mammalian tissue generated may be suitable for applications in research modeling, drug testing, and / or the development of tissue grafts, e.g. for treating patients.

[0073] Throughout embodiments herein, the number of interconnected IPHBs may vary. For example, certain embodiments may comprise between about 2 to 200 interconnected IPHBs. In certain embodiments, the minimum number of interconnected IPHBs may be about 2, 3, 5, 10, 20, 40, 50, 60, 80, and / or 100. In certain embodiments, the maximum number of interconnected IPHBs may be about 200, 180, 150, 120, and / or 100. Additional variations will be readily apparent to those of skill in the art, including more or fewer IPHBs.

[0074] In certain embodiments, a modular system for generating mammalian tissue may comprise a plurality of IPHBs. 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 wherein the blocks interconnect to form a scaffold configured to cultivate one or more cell types organized into stratified layers.

[0075] In some embodiments, the cavities may comprise one or more channels and / or chambers. A cavity may comprise a well, recess, pore, pocket, and / or enclosure.

[0076] In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in a vertical axis relative to gravity.

[0077] In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in an axis orthogonal to gravity.

[0078] In some embodiments, at least one channel may be configured for communication between at least two interconnected blocks in a horizontal axis relative to gravity.

[0079] In some embodiments, the network of microporous cavities may be predetermined before blocks are interconnected.

[0080] In some embodiments, the network of microporous cavities may be standardized across blocks. In some embodiments, at least one block may comprise a biocompatible polymeric material.

[0081] In some embodiments, the biocompatible polymeric material may comprise at least one natural polymer, plant derived polymer, animal derived polymer, PLGA, and / or PEG.

[0082] In some embodiments, at least one block comprises an enzymatically dissolvable material.

[0083] In some embodiments, the 3D continuous polymeric matrix consists of enzymatically dissolvable material.

[0084] Some embodiments may be configured to cultivate at least one primary mammalian cell line, MSC, IPSC, and / or immortalized cell line.

[0085] Some embodiments may be configured to cultivate at least two different cell types contemporaneously.

[0086] In some embodiments, at least one block may be configured to permit permeation of culture media.

[0087] Some embodiments may be configured to permit permeation of culture media vertically, horizontally, and / or orthogonally.

[0088] In certain embodiments, a method for generating mammalian tissue may comprise acquiring a plurality of IPHBs, interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers, and seeding at least one block with at least one cell type adherent to the block. 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.

[0089] In some embodiments, the blocks may be interconnected vertically, horizontally, and / or orthogonally.

[0090] Some embodiments may further comprise seeding at least one block with at least one second cell type.

[0091] Some embodiments may further comprise perfusing at least one block with culture media.

[0092] Some embodiments may further comprise harvesting mammalian tissue generated after the seeding.

[0093] In some embodiments, at least one block may comprise an enzymatically dissolvable material. Some embodiments may further comprise enzymatically dissolving the enzymatically dissolvable material. In certain embodiments, an IPHB for generating mammalian tissue 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 to form a scaffold configured to cultivate one or more cell types organized into stratified layers

[0094] Described herein are systems, methods, and IPHBs for generating mammalian tissue. A 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 a plurality of blocks configured to interconnect to form a scaffold configured to cultivate one or more cell types organized into stratified layers. A method may comprise acquiring a plurality of blocks, interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers, and seeding at least one block with at least one cell type adherent to the block. Embodiments may comprise perfusing at least one block with culture media and / or harvesting mammalian tissue generated.

[0095] Turning to Figure 3, illustrated is an example embodiment of a method 300 for generating mammalian tissue. In certain embodiments, method 300 may comprise step 310 of acquiring blocks. Step 310 may comprise acquiring a plurality of IPHBs. At least one block may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The cavities may comprise one or more channels and / or chambers. At least one block may be configured to interconnect with at least one other block. In certain examples, a block may comprise IPHB 1 of FIGS. 1 and / or 2. Method 300 may comprise step 320 of interconnecting blocks. Step 320 may comprise interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers. Method 300 may comprise step 330 of seeding. Step 330 may comprise seeding at least one block with at least one cell type adherent to the block. Certain embodiments may comprise additional steps. For example, method 300 may comprise step 340 of additional seeding, step 350 of perfusing, and / or step 360 of harvesting. Step 340 may comprise seeding at least one block with at least one second cell type. Step 350 may comprise perfusing at least one block with culture media. Step 360 may comprise harvesting mammalian tissue, which could have been generated, and which could have been generated as a result of or subsequent to seeding or earlier process (e.g. steps 310, 320, and / or 330). 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 acquiring blocks 310, interconnecting blocks 320, seeding 330, and harvesting 360 (not necessarily including additional seeding 340 or perfusing 350). Some embodiments may comprise acquiring blocks 310, interconnecting blocks 320, seeding 330, perfusing 350, and harvesting 360. Some embodiments may comprise additional seeding 340, which could be after or subsequent to harvesting 360 (and / or perfusing 350). Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of seeding 330 (with or without additional seeding 340), perfusing 350, and / or harvesting 360. Further, other steps, like acquiring blocks 310 and / or interconnecting blocks 320, could be executed (and / or re- executed) before, during, and / or after other steps (i.e. seeding 330, and optionally additional seeding 340, perfusing 350, and / or harvesting 360). Such embodiments may include changing the configuration of existing blocks, removing blocks, and / or adding blocks.

[0096] 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.

[0097] 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.

[0098] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0099] Clause 1. A modular system for generating mammalian tissue, comprising: at least one IPHB, with the at least one IPHB comprising a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers; wherein the at least one IPHB is configured to interlock with another IPHB, forming a scaffold; and wherein the at least one IPHB is configured to cultivate one or more cell types, individually or in combination, to facilitate the organization of cells into stratified layers that mimic mature mammalian tissue.

[0100] Clause 2. The system of clause 1, wherein the microporous channels and / or chambers of the at least one IPHB are configured to facilitate vertical and horizontal cell communication to form organized tissue layers and the extracellular matrix.

[0101] Clause 3. The system of clause 2, wherein the microporous channels and / or chambers of the at least one IPHB are preformed and optionally standardized across batches.

[0102] Clause 4. The system of clause 1, wherein the at least one IPHB comprises a biocompatible polymeric material.

[0103] Clause 5. The system of clause 4, wherein the biocompatible polymeric material comprises one or more natural polymers, such as plant-derived polymers and / or animal-derived polymers.

[0104] Clause 6. The system of clause 4-5, wherein the biocompatible polymeric material comprises PLGA, PEG, or combinations thereof.

[0105] Clause 7. The system of clause 4-6, wherein the at least one IPHB is configured to support the cultivation of various cell types, including primary mammalian cell lines, MSCs, IPSCs, immortalized cell lines, or any combination thereof. Clause 8. The system of clause 7, wherein the at least one IPHB is configured to coculture multiple cell types to allow cells to interact and regulate into mature mammalian tissue.

[0106] Clause 9. The system of clause 1, wherein the at least one IPHB is configured to maintain the stem-like properties of MSCs over extended culture periods.

[0107] Clause 10. The system of clause 1, wherein the at least one IPHB is configured to permeate the culture media.

[0108] Clause 11. The system of clause 10, wherein the at least one IPHB is configured to enable perfusion horizontally through the at least one IPHB when the at least one IPHB is interconnected with another IPHB.

[0109] Clause 12. The system of clauses 10-11, wherein the at least one IPHB is configured to enable perfusion vertically through the at least one IPHB when the at least one IPHB is stacked on top of another IPHB.

[0110] Clause 13. The system of clause 1, wherein the at least one IPHB is configured be enzymatically dissolved to leave behind intact mature mammalian tissue; wherein the continuous polymeric matrix material is enzymatically dissolvable.

[0111] Clause 14. The system of clause 1 , wherein the at least one IPHB is configured to reduce media consumption by up to 90%, plasticware usage by up to 85%, and human labor.

[0112] Clause 15. A method of generating mature mammalian tissue, comprising: providing an initial scaffolding comprising at least one IPHB; wherein the at least one IPHB comprises a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers; seeding the at least one IPHB with one or more types of adherent cells; and allowing the cells to propagate and form mature mammalian tissue within the at least one IPHB.

[0113] Clause 16. The method of clause 15, wherein the at least one IPHB includes a first IPHB and a second IPHB, the method further comprising interlocking 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.

[0114] Clause 17. The method of clause 15, wherein the adherent cells comprise primary mammalian cell lines, MSCs, IPSCs, immortalized cell lines, or combinations thereof.

[0115] Clause 18. The method of clause 17, wherein the at least one IPHB is seeded with at least two different cell types, and co-cultured to allow cells to interact and regulate into mature tissue. Clause 19. The method of clause 15, wherein the at least one IPHB is perfused with the culture media to provide consistent nutrient delivery and waste removal throughout the generation process.

[0116] Clause 20. The method of clause 15, wherein the at least one IPHB is configured to be enzymatically dissolved to retrieve mature mammalian tissue.

[0117] Clause 21. The method of clause 15, wherein a microenvironment within the at least one IPHB is configured to mimic the native physiological conditions from a biological, chemical, electrical, genetic, physical, structural, and mechanical perspective, facilitating the generation of physiologically relevant tissues.

[0118] Clause 22. The method of clause 15, further comprising harvesting the mammalian tissue generated from the at least one IPHB, and the mammalian tissue generated from the at least one IPHB is suitable for applications in research modeling, drug testing, and the development of tissue grafts for treating patients.

[0119] 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 modular system for generating mammalian tissue, comprising: a plurality of interconnecting porous hydrogel blocks, wherein each 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, wherein the blocks interconnect to form a scaffold configured to cultivate one or more cell types organized into stratified layers.

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

3. The system of claim 2, wherein at least one channel is configured for communication between at least two interconnected blocks in a vertical axis relative to gravity.

4. The system of claim 2, wherein at least one channel is configured for communication between at least two interconnected blocks in an axis orthogonal to gravity.

5. The system of claim 4, wherein at least one channel is configured for communication between at least two interconnected blocks in a horizontal axis relative to gravity.

6. The system of claim 1, wherein the network of microporous cavities is predetermined before blocks are interconnected.

7. The system of claim 1, wherein the network of microporous cavities is standardized across blocks.

8. The system of claim 1, wherein at least one block comprises a biocompatible polymeric material.

9. The system of claim 8, wherein the biocompatible polymeric material is selected from the group consisting of: natural polymer, plant derived polymer, animal derived polymer, poly(lactic-co-glycolic acid), polyethylene glycol, or any combination thereof.

10. The system of claim 1, wherein at least one block comprises an enzymatically dissolvable material.

11. The system of claim 10, wherein the three-dimensional continuous polymeric matrix consists of enzymatically dissolvable material.

12. The system of claim 1, further configured to cultivate at least one cell type selected from the group consisting of: primary mammalian cell line, mesenchymal stem cell, induced pluripotent stem cell, immortalized cell line, or any combination thereof.

13. The system of claim 1, further configured to cultivate at least two different cell types contemporaneously.

14. The system of claim 1, wherein at least one block is configured to permit permeation of culture media.

15. The system of claim 14, further configured to permit permeation of culture media vertically, horizontally, orthogonally, or any combination thereof.

16. A method for generating mammalian tissue, comprising: acquiring a plurality of interconnecting porous hydrogel blocks, wherein each 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, interconnecting the blocks to form a scaffold configured to cultivate one or more cell types organized into stratified layers, and seeding at least one block with at least one cell type adherent to the block.

17. The method of claim 16, wherein the blocks are interconnected vertically, horizontally, orthogonally, or any combination thereof.

18. The method of claim 16, further comprising seeding at least one block with at least one second cell type.

19. The method of claim 16, further comprising perfusing at least one block with culture media.

20. The method of claim 16, further comprising harvesting mammalian tissue generated after the seeding.

21. The method of claim 20, at least one block comprising an enzymatically dissolvable material, the method further comprising enzymatically dissolving the enzymatically dissolvable material.

22. An interconnecting porous hydrogel block for generating mammalian tissue, comprising a three-dimensional continuous polymeric matrix with a network of microporous cavities, and configured to interconnect with at least one other block to form a scaffold configured to cultivate one or more cell types organized into stratified layers.

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