Parallel co-culture of mammalian cells in interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for co-cultivation of a plurality of cell types. 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 co- cultivate the cell types when seeded with at least one cell of each type. A method may comprise seeding at least one block with at least one cell of each type.

Description

[0001] PARALLEL CO-CULTURE OF MAMMALIAN CELLS IN INTERCONNECTING POROUS HYDROGEL BLOCKS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 63 / 666,294, 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 co-cultivation of multiple cell types, e.g. including primary, immortalized, stem cells, and / or progenitor cells, and using interconnecting porous hydrogel blocks (IPHBs). IPHBs and embodiments herein may facilitate a versatile and dynamic platform for a variety of response assays, enhancing research and development in the biological and / or related fields.

[0006] BACKGROUND

[0007] Tissue engineering and organ engineering are interdisciplinary fields that combine principles from biology, engineering, and / or material science to develop biological substitutes that restore, maintain, and / or improve tissue and organ function. These fields may have the potential to revolutionize modem medicine by providing solutions for organ failure, tissue damage, and / or various chronic diseases. Current technologies and strategies in tissue and organ engineering include two-dimensional (2D) cell cultures, three-dimensional (3D) cell cultures, scaffold-based approaches, decellularized organ scaffolds, hydrogel-based systems, 3D bioprinting, and microfluidic organ-on-a-chip models. Traditional cell culture techniques may involve growing cells in a 2D monolayer on flat surfaces, such as petri dishes or multiwell plates. This technique may be simple, cost-effective, and used for basic research and high- throughput screening. A major drawback to 2D cell cultures is being limited in mimicking the complex 3D architecture of tissues and organs, which may result in less physiologically relevant data. Cells in 2D cultures often exhibit altered morphology, polarity, and / or function compared to those in vivo. 3D cell cultures are more advanced than 2D cultures and may use scaffolds, spheroids, or organoids to provide a more realistic tissue environment. This technique may better mimic the in vivo environment, providing more relevant data for drug testing and disease modeling. However, it often suffers from limitations in nutrient and oxygen diffusion, which may lead to necrotic cores. Standardization and reproducibility may also be challenging. Traditional scaffolds and 3D culture systems often focus on either vertical or horizontal nutrient and signal diffusion but rarely if ever integrate both in a cohesive manner.

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

[0009] Decellularized organ scaffolds may involve removing cellular components from donor organs, leaving behind the extracellular matrix (ECM), which may then repopulate with patient-derived cells. This approach can maintain natural architecture and / or mechanical properties and may reduce immune rejection when using patient cells. Drawbacks to this method include limited availability of suitable donor organs, challenges in achieving uniform cell repopulation, and potential for residual immunogenicity. Hydrogels are water-swollen, crosslinked polymeric networks that may mimic the ECM and may provide a hydrated environment for cell growth. Advantages to hydrogel-based systems may include high biocompatibility, tunable properties, and / or the ability to be injected or molded into complex shapes. Mechanical weakness, potential for rapid degradation, and / or challenges in integrating with native tissues may be setbacks.

[0010] 3D bioprinting may use layer-by-layer deposition of bioinks containing cells and / or biomaterials to create 3D tissue constructs. Advantages to 3D bioprinting may include high precision, customization potential, and / or ability to create complex architectures. Technical complexity, challenges in ensuring cell viability and function, and / or high cost are challenges to 3D bioprinting.

[0011] Microfluidic organ-on-a-chip models may use microfluidic technology to create miniature, physiologically relevant models of human organs. Relative control of the microenvironment, ability to mimic dynamic biological processes, and / or usefulness for drug testing and / or disease modeling may be some of the benefits of this approach to tissue and organ engineering. However, limited scalability, challenges in integrating multiple organ systems, and high technical expertise required are drawbacks. There are many gaps and / or barriers in the field of tissue and organ engineering. One of the major challenges in tissue engineering is the creation of a functional vascular network that may supply nutrients and / or remove waste. Difficulty in replicating the complex architecture and function of native blood vessels and / or limited success in integrating engineered vessels with host vasculature make the creation of a functional vascular network difficult. Native tissues and organs have highly complex structures and functions that are difficult to replicate in vitro and simplified models do not capture the multi-scale organization and interactions present in vivo, leading to less predictive data. Immune rejection remains a significant hurdle. The lack of effective strategies to prevent immune responses and / or the potential for chronic inflammation and graft rejection remain a barrier to preventing immune rejection. Successful integration of engineered tissues and organs with the host's native tissues is crucial for functionality. Differences in mechanical properties, lack of vascularization, and / or immune responses may impede integration and functionality. Variability in materials, cells, and methods used remains a gap in developing a standardized and scalable approach. Lack of standardized protocols and scalable manufacturing processes limits the widespread application of tissue engineering solutions. There is difficulty translating preclinical research into effective clinical therapies in current tissue engineering technologies. Current models do not accurately predict human responses, leading to high failure rates in clinical trials. Additionally, ethical and / or regulatory challenges may complicate the development and approval of new therapies. High costs associated with advanced tissue engineering technologies and materials may limit accessibility. There is a need for cost-effective and scalable solutions that may be widely adopted in clinical practice.

[0012] Better research systems are needed to develop models that more accurately predict human responses, reducing the high failure rates of clinical trials. Enhanced models may lead to more reliable data, improving the efficiency of drug discovery and development processes. Models that closely mimic the complexity and dynamics of native tissues may provide more relevant insights into disease mechanisms and / or therapeutic responses. This may facilitate the development of more effective and targeted therapies, which may advance precision medicine. Standardized and scalable systems are important for reproducibility and widespread adoption. This may streamline research processes, reduce variability, and / or enhance collaboration across different laboratories and institutions. Systems that support the seamless integration and / or functionalization of engineered tissues with host tissues may improve clinical outcomes. Better integration may lead to more successful tissue grafts and organ transplants, which may reduce the risk of complications and may improve patient outcomes. Cost-effective and accessible research systems may democratize tissue engineering and / or regenerative medicine. This may expand the reach of advanced therapies, which may make them available to a broader range of patients and healthcare providers. Systems that support the development of personalized tissue and organ models may enhance the precision and / or effectiveness of treatments. Personalized models may provide tailored therapeutic strategies, improving patient-specific outcomes and reducing adverse effects.

[0013] Advances in the field and current technologies and strategies for tissue and organ engineering continue to have significant limitations that hinder their effectiveness in research and / or clinical applications.

[0014] 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.

[0015] SUMMARY

[0016] One or more embodiments of the disclosure may address one or more of the aforementioned problems. Certain embodiments according to the disclosure may provide a system for the parallel co-cultivation of multiple cell types, which may comprise at least one interconnecting porous hydrogel block (IPHB) designed to provide a three-dimensional (3D) environment. A network of microchannels within the IPHB(s) may facilitate multidirectional migration and interaction of cells. Modular design may allow the IPHB(s) to be joined vertically, horizontally, or both for expansion. Further, the IPHB(s) may co-cultivate multiple cell types, e.g. including primary cells, immortalized cells, stem cells, and / or progenitor cells. Further still, the physical properties of the IPHB(s), e.g. including stiffness and microchannel diameter, may be modulated to match the mechanical properties of various tissues. Yet again, the microchannel architecture of the IPHB(s) may create a “highway tunnel” system, e.g. for efficient nutrient and / or signal transfer. Still yet further, the IPHB(s) may support static and / or perfusion-based culturing conditions, e.g. with perfusion applicable vertically and / or horizontally. In some implementations, at least one empty IPHB may serve as a spacer between experiments to observe the migration of cells, agents, and / or secretory factors. Still further, dynamic culturing conditions may allow for modulation of rate, pressure, volume, viscosity, and / or osmotic pressure, e.g. of perfusate. Still again, the IPHB(s) may be configured with semipermeable barriers, e.g. to facilitate paracrine signaling experiments such as between different cell populations. Even further, gradients of cytotoxic and / or therapeutic agents may be established within the IPHB(s), e.g. to test multiple targets simultaneously. Even further still, cells within the IPHB(s) may secrete their own extracellular matrix (ECM) components), e.g. to alter the micromechanics of their environment and / or facilitate tissue organization. Yet again, the IPHB(s) may be configured to be loaded into standard well-plates, e.g. to support high-throughput testing, such as of therapeutic compounds. Further still, the IPHB(s) may enable the study of disease progression, cancer, and / or tissue injuries, e.g. within its 3D environment. Yet further still, the IPHB(s) may be used to observe the effects of mechanical, electrical, and / or biological stimuli, e.g. on cell behavior and / or tissue organization.

[0017] In another aspect, the present disclosure may provide a method for the parallel cocultivation of multiple cell types, which may comprise isolating primary cells from a mammalian tissue biopsy, cultivating the primary cells within at least one IPHB e.g. to provide a 3D environment, co-cultivating the primary cells with additional cell types within the IPHB(s), and utilizing static and / or perfusion-based culturing conditions e.g. to enhance the physiological relevance of the tissue model(s). Further, gradients of cytotoxic and / or therapeutic agents within the IPHB(s) may be established, e.g. to test multiple targets, which could be simultaneously. Again, the IPHB(s) with semipermeable barriers may be configured to facilitate paracrine signaling experiments. Again still, the secretory output of cells within the IPHB(s) may be analyzed, including growth factors, cytokines, chemokines, and / or extracellular vesicles. Yet still, individual and / or multiple cells from the IPHB(s) may be extracted, e.g. for downstream characterization and / or analysis. Still yet, multiple cell types within the IPHB(s) may be co-cultivated, e.g. to generate complex tissue structure(s) and / or organ components). Still yet again, the IPHB(s) may be perfused, e.g. to enhance nutrient delivery, waste removal, and / or cellular organization.

[0018] In certain embodiments, a system for co-cultivation of a plurality of cell types 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 co-cultivate the cell types when seeded with at least one cell of each type.

[0019] In some embodiments, at least one cell type may comprise a primary cell, immortalized cell, stem cell, and / or progenitor cell.

[0020] Some embodiments may be configured to match at least one physical property of at least one tissue of interest. A tissue of interest may comprise one or more ECMs, vessels, and / or organs.

[0021] In some embodiments, one or more physical properties may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity.

[0022] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0023] In some embodiments, at least one cavity size, dimension, and / or diameter may be modulated to match at least one physical property.

[0024] In some embodiments, at least one cavity may be configured for communication between two or more interconnected blocks in one or more vertical, horizontal, and / or orthogonal axes relative to gravity.

[0025] Some embodiments may comprise dynamic culture conditions.

[0026] In some embodiments, dynamic culture conditions may comprise exposing at least one cell type to a culture media via perfusion and / or static solution.

[0027] Some embodiments may comprise one or more perfusates. Some embodiments may be configured to allow modulation of at least one perfusate’s flow rate, pressure, volume, viscosity, and / or osmotic pressure.

[0028] In some embodiments, at least one block may be unseeded with cells, located between other interconnected blocks, and configured as spacing between at least two different environments.

[0029] Some embodiments may be configured to enable direct and / or indirect observation of migration of one or more cells, agents, and / or secretory factors.

[0030] Some embodiments may comprise at least one semipermeable barrier to facilitate paracrine signaling. Some embodiments may be configured to permit cell communication, autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, and / or gap junctions.

[0031] Some embodiments may be configured to permit transmission of one or more extracellular vesicles and / or biological, electrical, physical, mechanical, and / or chemical signals.

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

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

[0034] Some embodiments may be configured to permit observing at least one effect on tissue organization by one or more mechanical, electrical, and / or biological stimuli.

[0035] In certain embodiments, a method for co-cultivation of a plurality of cell types may comprise seeding at least one IPHB with at least one cell of each type. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block.

[0036] In some embodiments, at least one cell may be derived from at least one specimen, subject, human, tissue, organ, and / or biopsy. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. The subject could be a patient. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0037] In certain embodiments, an IPHB for co-cultivation of a plurality of cell types 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 co-cultivate the cell types when seeded with at least one cell of each type.

[0038] 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. 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 IPHB(s) in accordance with certain embodiments;

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

[0042] FIG. 3 illustrates a method for co-cultivation of a plurality cell types in accordance with certain embodiments.

[0043] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

[0044] DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0045] 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.

[0046] Certain presently disclosed embodiments may relate to the parallel co-cultivation of multiple cell types, e.g. including primary, immortalized, stem cells, and / or progenitor cells, which may be using Bio-Blocks, also known as interconnecting porous hydrogel blocks (IPHBs). IPHBs and embodiments herein may facilitate a versatile and dynamic environment for a variety of response assays, significantly enhancing research and development in the biological and related fields.

[0047] In certain embodiments, the IPHB(s) may feature a microchannel architecture that creates a “highway tunnel” system, which may enable cells to migrate and / or interact efficiently. As more IPHBs are joined together vertically and / or horizontally, such a system expands, which may allow for greater cell expansion and / or sophisticated co-culture of greater cell types. In some embodiments, IPHBs may facilitate the generation of tissues, organ components, and / or potentially entire organs. Certain embodiments and IPHBs may enable the monitoring of healthy tissue development, disease progression, cancer, and / or injury; and / or the effects of treatments on repairing, healing, and / or regenerating tissues ex vivo.

[0048] One of the important innovations disclosed is the ability to combine different cell types and expose them to cytotoxic and / or therapeutic agents in accordance with embodiments herein. Gradients may be established to test multiple targets in parallel, which may allow for complex analyses of cellular stimuli and responses over time. Furthermore, “empty” IPHBs can serve as spacers between experiments, enabling researchers to monitor and observe the migration of cells, agents, and / or secretory factors across different cell types and conditions.

[0049] In certain embodiments, the IPHBs can be joined together or separated by semipermeable barriers to facilitate paracrine signaling experiments. This might help ensure that only secreted signals interact between different IPHBs. Certain embodiments support both static media conditions and / or perfusion conditions, with perfusion applicable vertically and / or horizontally. This may allow for the modulation of liquid flow, gas exchange, mechanical stimuli, and / or electrical stimuli (e.g. through a hydrogel medium), which may give end users control over parameters such as rate, pressure, volume, viscosity, and / or osmotic pressure (e.g. of the perfusate). This flexibility may allow for more precise study of biological phenomena, including the formation of tissues and organ components, signaling gradients, and / or various other gradients.

[0050] In some embodiments, the “plug-and-play” nature of IPHBs and embodiments herein may provide exceptional versatility for studying physiological processes. It may enable the design and execution of experiments to develop and / or probe the formation of healthy, diseased, and / or custom mammalian and / or plant tissues. The IPHB system and embodiments herein may support a range of analytical methods, including immunolabeling, histological staining, supernatant analysis, and / or extraction of bulk and / or individual cells, e.g. for downstream characterization and / or analysis.

[0051] 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 l’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.

[0052] The unique design of IPHBs and embodiments herein may feature a network of adjustable cavities (such as microchannels, chambers, and / or pores), e.g. perpendicularly to one another, and may facilitate multidirectional cell migration, interaction, and / or communication. This architecture may create a “highway tunnel” system (e.g. within the hydrogel matrix), which may promote efficient nutrient and / or signal transfer. In some embodiments, microchannels may be designed to mimic the natural extracellular matrix (ECM) environment, e.g. promoting natural cell behavior and / or interactions. The stiffness and / or microchannel diameter of the IPHB(s) in may be modulated to better match the mechanical properties of various tissues, such as ECM(s). This customization may allow researchers to create optimal environments for different cell types, enhancing their growth, differentiation, and / or function. The physiological relevance of the cell culture environment may promote more natural cell behavior and / or interactions compared to traditional two-dimensional cultures and / or even many existing three-dimensional (3D) cultures, and may result in more accurate and / or predictive data for biological research and / or drug discovery. The modular nature of IPHBs and embodiments herein may allow them to be joined vertically, horizontally, or both, supporting cell expansion and the creation of complex, large-scale tissue constructs. This scalability addresses current limitations, e.g. of scaffold-based and organ-on-a-chip models, and may enable researchers to build extensive and / or sophisticated tissue and / or organ systems, e.g. that better mimic in vivo conditions. The ability to integrate multiple blocks to create extensive tissue networks and organ systems and other features herein represent significant advancement and departure from conventional methods, which are typically limited in scale and complexity.

[0053] In certain embodiments, IPHBs may support both static and / or perfusion-based culturing conditions, with perfusion applicable vertically and / or horizontally. This may allow better control over the rate, pressure, volume, viscosity, and / or osmotic pressure of the perfusate. This dynamic environment may closely mimic physiological conditions, promoting the development of functional tissues and organ components. The ability to create dynamic and customizable environments may enhance the physiological relevance of the models and allow for the study of various biological phenomena, such as signaling gradients, mechanical stimuli, and / or electrical gradients. Where perfusion systems may be known, the integration of such dynamic control within a modular hydrogel system and the features of embodiments herein are novel. The capacity to modulate conditions to simulate physiological environments adds a level of customization not typically available in traditional culture systems.

[0054] IPHBs and embodiments herein may enable the co-cultivation of multiple cell types, including primary, immortalized, stem cells, and / or progenitor cells, within the same system. This versatility may allow for sophisticated co-culture experiments, where different cell types may be combined within a single system and exposed to cytotoxic and / or therapeutic agents, e.g. in parallel. The ability to perform these complex response assays may facilitate the study of cellular interactions and responses in a more realistic and integrated manner, improving the predictive power of preclinical research.

[0055] Certain embodiments support cultivation of healthy tissues, monitoring disease progression, studying cancer, and / or evaluating injury and / or repair mechanisms. Existing coculture systems often face limitations in maintaining diverse cell populations and controlling interactions within a shared environment. Embodiments herein may support versatile and / or dynamic co-culture, e.g. to promote natural cell interactions and / or complex response assays, and their features represent significant innovation that is not evident from prior art. In certain embodiments, IPHBs may be configured with semipermeable barrier(s), e.g. to study paracrine signaling between different cell populations. Gradients) and / or differential(s) of cytotoxic and / or therapeutic agent(s) may be established, e.g. to test multiple targets simultaneously, enabling complex analysis of cellular stimuli and / or responses, e.g. over time. Empty IPHB(s) may serve as spacer(s) between experiments, which may allow researchers to observe the migration of cells, agents, and / or secretory factors, e.g. across different conditions. These features may enable more detailed investigation(s) into cell signaling mechanisms and / or the effects of various stimuli, which may advance the understanding of complex biological processes. Features herein, such as semipermeable barriers to isolate and study paracrine signaling and gradient formation, represent sophisticated advancement. Embodiments herein may enable detailed investigations into cell signaling mechanisms that are not possible with conventional models.

[0056] Cells within certain embodiments and IPHBs may secrete their own ECM components, which may alter the micromechanics of their environment and facilitate tissue organization. This self-regulation may allow cells to construct tissue structures that closely resemble their natural counterparts, enhancing the functionality of the engineered tissues.

[0057] Certain embodiments and IPHBs may be designed for ease of use, allowing researchers to quickly set up and / or modify experiments, e.g. as needed. This may include the ability to introduce one or more “empty” blocks as spacers and / or customize the setup for specific research goals. The plug-and-play nature of IPHBs and embodiments herein offer a level of flexibility and user-friendliness that is not common in existing tissue engineering platforms. Features herein, including ability to adapt for a wide range of applications (e.g. from basic research to high-throughput screening), represent novel advancements to tissue and organ engineering. This plug-and-play versatility and other features herein may make IPHBs and embodiments herein accessible for a wide range of research applications, from basic biological research to high-throughput screening.

[0058] The enhanced physiological relevance and / or dynamic culturing conditions of IPHBs and embodiments herein may improve the predictive power of preclinical research. This may lead to more reliable data, reducing failure rates in clinical trials and / or supporting the development of more effective and / or targeted therapies.

[0059] Some embodiments may support a range of analytical methods, e.g. including immunolabeling, histological staining, supernatant analysis, and / or the extraction of bulk and / or individual cells, such as for downstream characterization. Researchers may study the secretory output of cells, such as including growth factors, cytokines, chemokines, and / or extracellular vesicles, e.g. providing valuable insights into cellular interactions and / or therapeutic effects.

[0060] In certain embodiments, IPHBs may be loaded into standard well-plates, e.g. supporting high-throughput testing of therapeutic compounds. This compatibility with existing lab infrastructure may allow for efficient and / or scalable testing, e.g. facilitating large-scale experimentation and / or accelerating the drug discovery process.

[0061] Current tissue and organ engineering approaches experience challenges in integrating engineered tissues with host tissues. Vascularization, physiological relevance, standardization and reproducibility, translation to therapies, integration with host tissues, immune rejection, and cost and accessibility are some of the limitations of current technologies that embodiments herein can help overcome.

[0062] Creating functional vascular networks in engineered tissues is challenging. The dynamic microchannel network in certain embodiments and IPHBs may support the formation of capillaries and / or other vascular structures, which may enhance nutrient delivery and / or waste removal. Perfusion may further promote vascularization, e.g. addressing a major limitation of existing models.

[0063] Existing models often fail to fully replicate the complexity and dynamic nature of native tissues. In some embodiments, IPHBs may provide a more physiologically relevant environment by facilitating natural cell behavior, interactions, and / or signaling within a 3D structure. This may lead to more accurate and predictive data for biological research and drug discovery.

[0064] Variability in materials, cells, and methods used across different laboratories makes it difficult to standardize and reproduce tissue engineering techniques. In certain embodiments, IPHBs may offer a standardized platform that may be easily customized and / or scaled. This consistency may improve reproducibility and reliability, which may facilitate more robust and comparable research outcomes.

[0065] Difficulty translating preclinical research into effective clinical therapies is another gap in current tissue engineering approaches. The enhanced physiological relevance and / or predictive power of IPHBs and embodiments herein may improve the reliability of preclinical data, supporting the development of more effective and / or targeted therapies. This may facilitate the translation of research findings into clinical applications.

[0066] Challenges in integrating engineered tissues with host native tissues is yet another problem current tissue engineering technologies face. Features of IPHBs and embodiments herein, including modular design and / or dynamic culturing conditions, may support the creation of tissue constructs that may integrate more seamlessly with host tissues, which may improve clinical outcomes, e.g. for tissue graft(s) and / or organ transplants).

[0067] Immune rejection of engineered tissues and organs remains a significant hurdle. By using patient-derived cells and customizable environments, certain IPHBs and embodiments herein may reduce the risk of immune rejection. This approach may support the development of one or more personalized tissue models and / or therapies, which may be more likely to be accepted by a host, such as by the immune system.

[0068] Advanced tissue engineering technologies and materials are typically associated with high costs. IPHBs and embodiments herein may be designed to be cost-effective and easy to use, which may reduce the need for specialized equipment and / or expertise. Their efficient use of media and plasticware further lowers costs, which may make advanced tissue engineering more accessible.

[0069] IPHBs and embodiments herein may enable the creation of personalized tissue and / or organ models using patient-derived cells. This may support more tailored treatment strategies and / or more accurate predictions of therapeutic efficacy. In some embodiments, the compatibility of IPHBs and embodiments herein with high-throughput testing platforms may facilitate large-scale drug screening and / or accelerate the drug discovery process. IPHBs and embodiments herein may support detailed studies of disease progression, cancer, and / or tissue injuries, e.g. in a more physiologically relevant environment, and may enhance understanding of complex biological process(es). The ability to create scalable and / or integrated tissue constructs) and / or organ system(s) may pave the way for advancements) in regenerative medicine and / or tissue engineering. The cost-effective nature of IPHBs and embodiments herein, combined with features herein such as versatility and ease of use, may make advanced tissue engineering technologies accessible to a broader range of researchers and / or clinicians.

[0070] IPHBs and embodiments herein represent significant advancement in tissue and organ engineering. By addressing limitations of current technologies, and e.g. providing a versatile, scalable, and / or physiologically relevant platform, they may enhance the predictive power of preclinical research, support the development of personalized therapies, and / or facilitate the translation of research findings into clinical applications. This innovation may advance the field of tissue engineering, which may create new opportunities and pathways for research and development.

[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 mesenchymal stem cells {MSCs} [e.g., from or for adipose, bone barrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); laminin I (e.g., from or for human induced pluripotent stem cells {IPSCs} and / or mouse dorsal root ganglia); hyaluronan (e.g., from porcine hepatocytes and / or human dermal adult fibroblasts); gelatin (e.g., from human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, human CD8+ T cells, Human CD4+ T cells, human macrophages, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); fibrin (e.g., from or for human keratinocytes); fibronectin (e.g., from or for human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human osteoblasts, human osteocytes, and / or human chondrocytes); or any combination(s) thereof.

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

[0079] In another aspect, the present disclosure provides methods for the parallel co-cultivation of multiple cell types. Some embodiments may comprise isolating primary cells from a mammalian tissue biopsy; cultivating the primary cells within IPHB(s) e.g. to provide a 3D environment; co-cultivating the primary cells with additional cell types within the IPHB(s); and utilizing static and / or perfusion-based culturing conditions to enhance the physiological relevance of the tissue model(s). Gradients) and / or differential(s) of cytotoxic and / or therapeutic agent(s) within the IPHB(s) may be established, e.g. to test multiple targets simultaneously. The IPHB(s) may be configured with semipermeable barrier(s), e.g. to facilitate paracrine signaling experiments. The secretory output of cells within the IPHB(s), e.g. including growth factors, cytokines, chemokines, and / or extracellular vesicles, may be analyzed. Individual and / or multiple cells may be extracted from the IPHB(s), e.g. for downstream characterization and / or analysis. Multiple cell types within the IPHB(s) may be co-cultivated, e.g. to generate complex tissue structures and / or organ components). The IPHB(s) may be perfused, e.g. to enhance nutrient delivery, waste removal, and / or cellular organization.

[0080] In certain embodiments, a system for co-cultivation of a plurality of cell types 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 co-cultivate the cell types when seeded with at least one cell of each type.

[0081] In some embodiments, at least one cell type may comprise a primary cell, immortalized cell, stem cell, and / or progenitor cell.

[0082] Some embodiments may be configured to match at least one physical property of at least one tissue of interest. A tissue of interest may comprise one or more ECMs, vessels, and / or organs.

[0083] In some embodiments, one or more physical properties may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity.

[0084] In some embodiments, at least one cavity may comprise one or more channels, chambers, and / or pores.

[0085] In some embodiments, at least one cavity size, dimension, and / or diameter may be modulated to match at least one physical property.

[0086] In some embodiments, at least one cavity may be configured for communication between two or more interconnected blocks in one or more vertical, horizontal, and / or orthogonal axes relative to gravity.

[0087] Some embodiments may comprise dynamic culture conditions.

[0088] In some embodiments, dynamic culture conditions may comprise exposing at least one cell type to a culture media via perfusion and / or static solution.

[0089] Some embodiments may comprise one or more perfusates. Some embodiments may be configured to allow modulation of at least one perfusate’s flow rate, pressure, volume, viscosity, and / or osmotic pressure.

[0090] In some embodiments, at least one block may be unseeded with cells, located between other interconnected blocks, and configured as spacing between at least two different environments. Some embodiments may be configured to enable direct and / or indirect observation of migration of one or more cells, agents, and / or secretory factors.

[0091] Some embodiments may comprise at least one semipermeable barrier to facilitate paracrine signaling.

[0092] Some embodiments may be configured to permit cell communication, autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, and / or gap junctions.

[0093] Some embodiments may be configured to permit transmission of one or more extracellular vesicles and / or biological, electrical, physical, mechanical, and / or chemical signals.

[0094] Some embodiments may comprise at least one compound. A compound may comprise one or more drugs, nutrients, growth factors, bioactive molecules, cytotoxic agents, and / or therapeutic agents.

[0095] Some embodiments may comprise one or more compounds distributed with geographic and / or temporal variation in concentration via at least one differential and / or gradient.

[0096] Some embodiments may be configured to permit observing at least one effect on tissue organization by one or more mechanical, electrical, and / or biological stimuli.

[0097] In certain embodiments, a method for co-cultivation of a plurality of cell types may comprise seeding at least one IPHB with at least one cell of each type. One or more blocks may comprise a 3D continuous polymeric matrix with a network of microporous cavities. One or more blocks may be configured to interconnect with at least one other block.

[0098] In some embodiments, at least one cell may be derived from at least one specimen, subject, human, tissue, organ, and / or biopsy. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. The subject could be a patient. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0099] In certain embodiments, an IPHB for co-cultivation of a plurality of cell types 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 co-cultivate the cell types when seeded with at least one cell of each type. Described herein are systems, methods, and IPHBs for co-cultivation of a plurality of cell types. 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 co-cultivate the cell types when seeded with at least one cell of each type. A method may comprise seeding at least one IPHB with at least one cell of each type.

[0100] Turning to Figure 3, illustrated is an example embodiment of a method 300 for cocultivation of a plurality of cell types. In certain embodiments, method 300 may comprise step 310 of seeding and optionally step 320 of dynamic culture conditions. Step 310 may comprise seeding at least one IPHB with at least one cell of each type. The IPHB may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The cavities may comprise one or more channels, pores, and / or chambers. Step 320 may comprise exposing cells to dynamic culture conditions. Dynamic culture conditions 320 may be configured to achieve nutrient and / or waste exchange. Dynamic culture conditions 320 may comprise exposing at least one cell to a culture media via perfusion and / or static solution. The culture media may comprise one or more nutrients. Dynamic culture conditions 320 may comprise varying a culture media’s nutrient type and / or concentration. Dynamic culture conditions 320 may comprise perfusing at least one block with culture media. In certain embodiments, one or more cavities may be configured as pathway(s) for nutrient delivery, gas exchange, and / or waste removal.

[0101] In some embodiments of step 310, at least one cell may be derived from a human, a subject, a biopsy, or a combination thereof. The biopsy could be from the subject or otherwise. The subject could be a human or a nonhuman. Multiple specimens could be from the same and / or different sources; examples include multiple specimens from the same biopsy, different biopsies from the same subject (e.g. including differing in the location and / or time biopsied), and / or different subjects. One or more subjects may be mammals, animals, birds, reptiles, amphibians, and / or fish. At least one cell could be derived from one or more tissues and / or organs. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0102] Certain embodiments may comprise additional or different steps. For example, step 320 is optional. By way of further example, method 300 may comprise step 330 of at least one personalized model and / or step 340 of at least one analysis. Step 330 may comprise cultivating cells derived from one or more subjects into one or more personalized models. Step 340 may comprise subjecting cells (e.g., the personalized model(s)) to one or more analyses to guide diagnosis and / or therapy, which could be tailored for the subjects). Step 340 may comprise exposing one or more of the cells to at least one experimental variable, which could be across one or more geographic, temporal, and / or other differential(s) and / or gradients). An experimental variable may comprise at least one drug, thermal energy, radiation, or any combination thereof. A drug may comprise at least one medicine, medication, pharmaceutical, biologic, immunologic, immunotherapy, chemotherapy, radiotherapy, radiotracer, tracer, stain, diagnostic agent, theranostic agent, therapeutic agent, and / or treatment agent. Step 340 may comprise evaluating, measuring, and / or observing the effect of at least one experimental variable on the senescence, growth, and / or characteristic(s) of the cells (e.g., seeded, cultivated, primary, tumor, metastatic, tissue, and / or organ cells).

[0103] All steps may be incorporated in any combination and in any order, including repeating step(s). By way of example, certain method embodiments may comprise seeding 310, dynamic culture conditions 320, and analysis 340 (not necessarily including personalized model 330). In some embodiments, personalized model 330 could be after or subsequent to analysis 340. Some embodiments might not comprise dynamic culture conditions 320. Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of seeding 310, dynamic culture conditions 320, personalized model 330, and / or analysis 340. Further, the one or more blocks underlying step 310 (and / or being seeded) could be altered before, during, and / or after other steps (e.g. seeding 310, dynamic culture conditions 320, personalized model 330, and / or analysis 340). Such embodiments may include changing the configuration of existing blocks, removing blocks, and / or adding blocks.

[0104] 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.

[0105] 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.

[0106] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0107] Clause 1. A system for the parallel co-cultivation of multiple cell types, comprising: at least one IPHB, wherein the at least one IPHB provides a 3D environment including a network of microchannels within the at least one IPHB to facilitate multidirectional migration and interaction of cells; wherein the at least one IPHB has a modular design allowing the at least one IPHB to be joined vertically, horizontally, or both for indefinite expansion.

[0108] Clause 2. The system of clause 1, wherein the at least one IPHB is configured to cocultivate multiple cell types. Clause 3. The system of clause 2, wherein the cell types include primary cells, immortalized cells, stem cells, progenitor cells, and any combination thereof.

[0109] Clause 4. The system of clause 1, wherein one or more physical properties of the at least one IPHB, including stiffness and / or microchannel diameter, are configured to be modulated to match the mechanical properties of various tissues of interest.

[0110] Clause 5. The system of clause 1, wherein the microchannel architecture of the at least one IPHB creates a “highway tunnel” system for efficient nutrient and signal transfer.

[0111] Clause 6. The system of clause 5, wherein the at least one IPHB support both static and perfusion-based culturing conditions, with perfusion applicable vertically or horizontally.

[0112] Clause 7. The system of clause 1, wherein at least one empty IPHB is configured and located within a group of interconnected IPHBs to serve as a spacer between experiments to observe the migration of cells, agents, and secretory factors.

[0113] Clause 8. The system of clause 1, wherein the dynamic culturing conditions are configured to allow for the modulation of rate, and / or pressure, and / or volume, and / or viscosity, and / or osmotic pressure of the perfusate.

[0114] Clause 9. The system of clause 1, wherein the at least one IPHB is configured with semipermeable barriers to facilitate paracrine signaling experiments between different cell populations.

[0115] Clause 10. The system of clause 1, wherein gradients of cytotoxic and therapeutic agents may be established within the at least one IPHB to test multiple targets simultaneously.

[0116] Clause 11. The system of clause 1, wherein cells within the at least one IPHB secrete their own ECM components to alter the micromechanics of their environment and facilitate tissue organization.

[0117] Clause 12. The system of clause 1, wherein the at least one IPHB is configured to be loaded into standard well-plates to support high-throughput testing of therapeutic compounds.

[0118] Clause 13. The system of clause 1 , wherein the at least one IPHB is configured to enable the study of disease progression, cancer, and tissue injuries within its 3D environment.

[0119] Clause 14. The system of clause 1, wherein the at least one IPHB is configured to observe the effects of mechanical, electrical, and biological stimuli on cell behavior and tissue organization.

[0120] Clause 15. A method for the parallel co-cultivation of multiple cell types, comprising: isolating primary cells from a mammalian tissue biopsy; cultivating the primary cells within the at least one IPHB, wherein the at least one IPHB provides a 3D environment; co-cultivating the primary cells with one or more additional cell types within the at least one IPHB; and utilizing static and perfusion-based culturing conditions to enhance the physiological relevance of the tissue model.

[0121] Clause 16. The method of clause 15, further comprising forming gradients of cytotoxic and therapeutic agents within the at least one IPHB to test multiple targets simultaneously.

[0122] Clause 17. The method of clause 15, wherein the at least one IPHB is configured with semipermeable barriers to facilitate paracrine signaling experiments.

[0123] Clause 18. The method of clause 15, further comprising a step of collecting and / or analyzing the secretory output of cells within the at least one IPHB, including growth factors, cytokines, chemokines, and extracellular vesicles.

[0124] Clause 19. The method of clause 15, further comprising a step of extracting bulk, such as multiple cells or individual cells from the at least one IPHB, optionally for downstream characterization and analysis.

[0125] Clause 20. The method of clause 15, wherein multiple cell types within the at least one IPHB are co-cultivated to generate complex tissue structures and / or organ components.

[0126] Clause 21. The method of clause 15, wherein the at least one IPHB is perfused to enhance nutrient delivery, waste removal, and cellular organization, thereby promoting the development of functional tissues and / or organ components.

[0127] These and other modifications and variations may be practiced by those of ordinary skill in the art without departing from 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 co-cultivation of a plurality of cell types, 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 co-cultivate the cell types when seeded with at least one cell of each type.

2. The system of claim 1, wherein at least one cell type is selected from the group consisting of: primary cell, immortalized cell, stem cell, progenitor cell, or a combination thereof.

3. The system of claim 1 , further configured to match at least one physical property of at least one tissue of interest.

4. The system of claim 3, wherein one or more physical properties are selected from the group consisting of: porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, lipophilicity, or a combination thereof.

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

6. The system of claim 4, wherein at least one cavity size, dimension, diameter, or combination thereof is modulated to match at least one physical property.

7. The system of claim 5, wherein at least one channel is configured for communication between two or more interconnected blocks in one or more vertical, horizontal, or orthogonal axes relative to gravity or any combination thereof.

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

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

10. The system of claim 8, comprising one or more perfusates, and configured to allow modulation of at least one perfusate’s flow rate, pressure, volume, viscosity, osmotic pressure, or any combination thereof.

11. The system of claim 1, wherein at least one block is not seeded with cells, located between other interconnected blocks, and configured as spacing between at least two different environments.

12. The system of claim 11, configured to enable direct or indirect observation of migration of one or more cells, agents, secretory factors, or a combination thereof.

13. The system of claim 1, comprising at least one semipermeable barrier to facilitate paracrine signaling.

14. The system of claim 1, configured to permit cell communication, autocrine signaling, paracrine signaling, endocrine signaling, direct cell-to-cell contact, gap junctions, or a combination thereof.

15. The system of claim 1, configured to permit transmission of one or more extracellular vesicles, biological signals, electrical signals, physical signals, mechanical signals, chemical signals, or a combination thereof.

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

17. The system of claim 16, comprising one or more compounds distributed across the system with geographic, temporal, or both geographic and temporal variation in concentration via at least one differential, gradient, or a combination thereof.

18. The system of claim 1, configured to permit observing at least one effect on tissue organization by one or more mechanical, electrical, or biological stimuli or any combination thereof.

19. A method for co-cultivation of a plurality of cell types, comprising seeding at least one interconnecting porous hydrogel block with at least one cell of each type, wherein at least one block comprises a three-dimensional continuous polymeric matrix with a network of microporous cavities and is configured to interconnect with at least one other block.

20. The method of claim 19, wherein at least one cell is derived from at least one specimen, subject, human, biopsy, or combination thereof.

21. An interconnecting porous hydrogel block for co-cultivation of a plurality of cell types, 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 co-cultivate the cell types when seeded with at least one cell of each type.

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