Generation of personalized microvasculature from interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for generating personalized microvasculature ex vivo. A block may be configured to, under dynamic conditions, cultivate microvascular formation when seeded with at least one line of endothelial cells isolated from at least one first specimen and at least one line of mesenchymal stem cells isolated from at least one second specimen. A system may comprise at least one block so configured. A method may comprise seeding at least one block with at least one line of endothelial cells isolated from at least one first specimen and at least one line of mesenchymal stem cells isolated from at least one second specimen and exposing the cell types to dynamic culture conditions.

Description

[0001] GENERATION OF PERSONALIZED MICROVASCULATURE FROM 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 / 665,813, 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.

[0004] TECHNICAL FIELD

[0005] The subject matter disclosed herein is generally directed to the cultivation of mammalian endothelial cells (ECs) in combination with mammalian mesenchymal stem cells (MSCs) and additional cell lines, which may include pericytes (PCs), myocytes, fibroblasts, and / or other connective tissue cell types, within interconnecting porous hydrogel blocks (IPHBs). When a minimum ratio of mammalian ECs and MSCs are co-cultured in IPHBs, the system may facilitate the formation of mammalian vasculature, which may include capillaries and veins. The co-cultivation of additional mammalian cell types, such as PCs, myocytes, and / or fibroblasts, with ECs and / or MSCs, may further enhance the development of comprehensive vasculature, encompassing capillaries, venules, arterioles, veins, and / or arteries.

[0006] BACKGROUND

[0007] Current blood vessel tissue engineering techniques, such as scaffold-based approaches, cell-based techniques, bioprinting, and microfluidic systems, face several limitations, including difficulties in mimicking the physiological microenvironment and complexity in replicating the hierarchical structure of native blood vessels.

[0008] The scaffold-based approach can involve the use of biomaterial scaffolds that provide a structural framework for cell attachment and growth. Common materials may include natural polymers (e.g., collagen, fibrin, laminin) and synthetic polymers (e.g., polylactic acid, polycaprolactone). Advantages to this approach may include providing a three-dimensional (3D) structure that mimics the extracellular matrix, being tailored to have specific mechanical properties, and supporting cell adhesion, proliferation, and differentiation. However, the scaffold-based approach faces important limitations. This technique may have difficulty achieving uniform cell distribution. It also may have limited ability to mimic the dynamic environment of native tissues. Lastly, it faces issues with scaffold degradation and biocompatibility.

[0009] Cell-based techniques can involve the direct use of endothelial cells, mesenchymal stem cells, and other supportive cells to form vascular structures without additional scaffolding. These techniques may have potential for using autologous cells, reducing the risk of immune rejection. Cell-based techniques may also have the ability to form functional vascular networks through cell self-assembly. The disadvantages to these techniques may include challenges in sourcing sufficient quantities of cells, difficulties in maintaining cell viability and functionality over time, and complexity in replicating the hierarchical structure of native blood vessels.

[0010] 3D bioprinting can involve the layer-by-layer deposition of bioinks containing cells and biomaterials to create vascular structures. The advantages to this technique could include precision in creating complex geometries and hierarchical structures, ability to incorporate multiple cell types and gradients of biomaterials, and customization for patient-specific treatments. 3D bioprinting has several disadvantages. It experiences technical challenges in ensuring cell viability during and after printing. 3D bioprinting also experiences limitations in the resolution and scalability of printed constructs, as well as high costs and technical expertise required.

[0011] Microfluidic systems use channels and flow dynamics to mimic blood vessels on a small scale. Microfluidic systems may provide control over fluid flow and shear stress. However, microfluidic systems lack larger scale production and better tissue integration into clinical applications.

[0012] Despite advancements in technologies, several critical gaps and barriers need to be addressed to generate blood vessels, e.g. for tissue grafting and / or drug modeling. Achieving maturation of engineered blood vessels to resemble physiological and functional characteristics of native vessels, and promoting appropriate biochemical and mechanical cues to promote vessel maturation and function, are problems existing technologies face. The ability to integrate engineered vessels with host vasculature to promote proper blood flow and functionality, overcoming immune rejection, and promoting stable anastomosis with host vessels, are also limitations that needed to be addressed. Variability in cell sources, scaffold materials, and bioprinting techniques make it difficult to develop scalable and reproducible methods for generating blood vessels. Replicating the hierarchical complexity of native vascular networks, including capillaries, arterioles, and venules, while addressing the technical challenges in designing and fabricating such intricate structures remains a gap in the tissue engineering technologies available. Achieving long-term stability and functionality of engineered vessels in vivo and addressing issues related to vessel degradation, thrombosis, and mechanical failure over time are yet other limitations with the known techniques in tissue engineering. And the high costs of materials, equipment, and technical expertise make them less accessible for widespread clinical use.

[0013] In conclusion, while current tissue engineering technologies have demonstrated potential, overcoming existing gaps and barriers is still needed for the successful generation of mature, functional blood vessels. Advances in biomaterials, cell biology, and engineering techniques may help in addressing these challenges and realizing fuller potential of engineered vascular tissues in regenerative medicine and drug development.

[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 provide a system for generating personalized microvasculature ex vivo, including at least one interconnecting porous hydrogel block (IPHB) designed to provide a three-dimensional (3D) environment, wherein the at least one IPHB may be configured to provide a highly biomimetic environment that closely resembles the natural extracellular matrix (ECM). Endothelial cells (ECs) and mesenchymal stem cells (MSCs) may be co-cultivated within the at least one IPHB, and cultivation of additional cell types including pericytes (PCs), myocytes, and / or fibroblasts may enhance microvascular formation. Further, the at least one IPHB may be configured to form micro-vasculature, such as capillaries, venules, arterioles, veins, and / or arteries. Further still, the at least one IPHB may be configured to be modular and interconnected to another IPHB. Still yet, the at least one IPHB interconnected to another IPHB may utilize perfusion systems to simulate full organ systems. Yet again, the at least one IPHB interconnected to another at IPHB may be configured to be loaded as multiple blocks into standard well-plates. Still yet further, the at least one IPHB may be configured to enable the distribution of growth factors, nutrients, and other bioactive molecules in liquid format. Again, the at least one IPHB may be configured to support various diagnostic procedures including histological analysis, molecular profiling, and the collection of secreted byproducts for further analysis. Still further, the at least one IPHB may be cost-effective and comprehensible. Yet again further, the at least one IPHB may be configured to reduce human labor, media consumption by up to 90%, and / or plastic consumption by up to 85%. Yet even further, the at least one IPHB may be configured to closely simulate in vivo conditions by facilitating the formation of realistic microvascular structures and invasive behaviors by ECs and / or supportive cells. Still again, the at least one IPHB may be configured to use primary cells from patient biopsies to develop personalized microvascular models.

[0017] In another aspect, the present disclosure provides a method for generating personalized microvasculature ex vivo, including isolating primary ECs and MSCs from a patient biopsy, cultivating the ECs and MSCs within at least one IPHB that provides a 3D environment and provides a highly biomimetic environment that closely resembles the natural ECM, cocultivating the primary cells with additional cell types within the at least one IPHB to enhance microvascular formation, and utilizing dynamic culturing conditions, such as static and perfusion, to enhance the physiological relevance of the microvascular model. Further, the distribution of growth factors, nutrients, and other bioactive molecules in liquid format across the at least one IPHB may create gradients for comprehensive evaluation. Even further, the connection of at least one IPHB to another IPHB may utilize perfusion systems to simulate full organ systems. Again, the generated microvasculature within the at least one IPHB may allow for diagnostic evaluations, including histological analysis and molecular profiling. Still yet, the generated microvasculature in the at least one IPHB may be configured for high-throughput screening of therapeutic compounds. Again still, the secreted byproducts from the media or perfusate surrounding the at least one IPHB may be collected and analyzed. Yet further, the at least one IPHB may be configured to develop personalized microvascular models that reflect the unique characteristics of each patient’s tissue. Still, the expansion of the microvascular network by interconnecting at least one IPHB to additional IPHBs may accommodate growth and study progression. Even still, media and plastic consumption may be reduced by up to about 85-90%.

[0018] In another aspect, certain embodiments herein provide a method for generating personalized microvasculature ex vivo, including: isolating primary ECs and MSCs from a patient biopsy; cultivating the ECs and MSCs within at least one IPHB; wherein the at least one IPHB comprises a 3D macrostructure and the at least one IPHB comprises a highly biomimetic environment that closely resembles the natural ECM; co-cultivating the primary cells with additional cell types within the at least one IPHB to enhance microvascular formation; and utilizing dynamic culturing conditions including static and perfusion, to enhance the physiological relevance of the microvascular model.

[0019] In certain embodiments, a system for generating personalized microvasculature ex vivo may comprise at least one IPHB under dynamic culture conditions configured to cultivate microvascular formation when seeded with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen.

[0020] In some embodiments, the dynamic culture conditions may be configured to achieve nutrient and / or waste exchange for at least one line of cells.

[0021] In some embodiments, the dynamic culture conditions may comprise exposing at least one cell type to a culture media via perfusion and / or static solution.

[0022] In some embodiments, the culture media may comprise one or more nutrients for the cells. The dynamic culture conditions may further comprise varying the culture media’s nutrient type and / or concentration.

[0023] In some embodiments, at least one first specimen, at least one second specimen, or both, may derived from a human, a subject, and / or a biopsy. 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 source; examples include multiple first specimens from the same source, multiple second specimens from the same source, a first specimen from the same source as a second specimen, and / or multiple first specimens from the same source as one or more second specimens. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0024] In some embodiments, at least one first specimen and at least one second specimen may be derived from the same human, subject, and / or biopsy.

[0025] In some embodiments, at least one second specimen may consist of at least one first specimen.

[0026] In some embodiments, microvascular formation may comprise forming microvasculature, veins, venules, capillaries, arterioles, and / or arteries. Some embodiments may comprise fistulae and / or anastomoses.

[0027] Some embodiments may be configured to fluidically distribute nutrients, growth factors, and / or bioactive molecules.

[0028] In some embodiments, at least one block may be configured for compatibility with histological analysis and / or molecular profiling. This may comprise one or more forms of histological analysis, one or more forms of molecular profiling, or both. In some embodiments, at least one block may be configured to collect secreted byproducts from the microvascular formation, at least one line of ECs, at least one line of MSCs, or a combination thereof.

[0029] In some embodiments, at least one block may comprise a 3D macrostructure.

[0030] In some embodiments, one or more blocks may be configured to mimic an ECM. At least one mimicked ECM may be that of a first and / or second specimen, and / or an ECM from the derivative source (e.g. human, subject, biopsy).

[0031] In some embodiments, one or more blocks may be configured to mimic the ECM of at least one first specimen, at least one second specimen, or a combination thereof.

[0032] In some embodiments, one or more blocks may be configured to match at least one physical property of the ECM.

[0033] In some embodiments, one or more ECM physical properties to be mimicked may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity. A physical property may be quantified, such as with a measured average and / or range. The physical property may be emulated and / or targeted within a goal threshold, including without limitation, only by way of example / for purposes of illustration, within 20% more or less than the measured average and / or range.

[0034] In some embodiments, the culture media may be configured to mimic an ECM in at least one nutrient and / or physical property.

[0035] In some embodiments, one or more blocks may be configured for loading into one or more standard well-plates.

[0036] In certain embodiments, a method for generating personalized microvasculature ex vivo may comprise seeding at least one IPHB with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen and exposing the cell types to dynamic culture conditions.

[0037] Some embodiments may further comprise seeding at least one block with one or more additional cell types.

[0038] In some embodiments, one or more additional cell types may comprise PCs, myocytes, and / or fibroblasts.

[0039] In certain embodiments, an IPHB may be configured to under dynamic culture conditions cultivate microvascular formation ex vivo when seeded with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen.

[0040] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.

[0041] BRIEF DESCRIPTION OF THE DRAWINGS

[0042] An understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure may be utilized, and the accompanying drawings of which:

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

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

[0045] FIG. 3 illustrates a method for generating personalized microvasculature ex vivo in accordance with certain embodiments.

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

[0047] DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0048] The disclosure now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments are shown. Indeed, what is claimed may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. As used in the specification, and in the appended claims, the singular forms “a”, “an”, and “the”, include plural referents unless the context clearly dictates otherwise.

[0049] The presently disclosed embodiments relate to interconnecting porous hydrogel blocks (IPHBs), and designs for the ex vivo generation of personalized microvasculature. IPHBs may provide a three-dimensional (3D), biomimetic environment, and / or could support the formation of complex vascular structures. Embodiments herein may facilitate advanced research in tissue engineering, drug discovery, and / or personalized medicine. Certain embodiments herein may support the co-culturing of multiple adherent cell types to form mature tissues and organ components, including extracellular matrix (ECM) and vascular systems, which may be used to perfuse tissue grafts with nutrients. Certain embodiments comprise dynamic culture conditions. Cells may be cultured individually or in combination within IPHBs under static or perfusion conditions, where the speed, pressure, and volume of perfusate may be modulated to stimulate vascular formation and enhance the physiological strength and integrity of the vasculature. The physiological complexity of the blood vessel may be modulated by modifying the bulk stiffness of one or more IPHBs and by changing the size or diameter of the cavities (e.g. microchannels) in the IPHB(s).

[0050] When endothelial cells (ECs) and mesenchymal stem cells (MSCs) are co-cultivated within IPHBs, they may align along the IPHB microchannels and collaborate to form blood vessels, e.g. at least in part through paracrine signaling and cellular interactions. These blood vessels may extend outward from the IPHB(s) once established. As more IPHBs are interconnected and populated with at least ECs and MSCs, the density and complexity of the blood vessels may increase. The addition of further or additional cell types may contribute to greater physiological complexity and functionality of the blood vessels.

[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 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.

[0052] Embodiments herein may provide a controlled 3D microenvironment that mimics an ECM. Embodiments may promote the natural maturation of blood vessels through better cell attachment, proliferation, and / or differentiation. The porous IPHB structure may facilitate cell alignment and / or interaction, e.g. for vessel maturation. In some embodiments IPHBs are designed to be modular, allowing them to be interconnected horizontally and / or vertically to form larger and more complex tissue constructs. The interconnecting design of IPHBs can allow for the creation of extensive and complex vascular networks that may be scaled, e.g. by adding more blocks. This modularity enables the customization of the microenvironment and supports scalability for various research and / or therapeutic applications. Current technologies either focus on small-scale models or face significant challenges in scaling up. The innovative features of the embodiments herein and the modularity of IPHBs, which allow for expansion and increased complexity of a vascular network, address scalability issues in a manner not previously considered in traditional scaffold or bioprinting techniques. The ability to create physiologically relevant microvascular structures allows for the modeling of various vascular diseases, including cancer, diabetes, and cardiovascular conditions. Embodiments herein provide valuable tools for studying disease mechanisms and developing new therapies.

[0053] In certain embodiments, IPHBs may be pre-formed with a network of adjustable cavities such as pores and microchannels running throughout the hydrogel matrix, e.g. perpendicularly to one another. These microchannels may facilitate the vertical and / or horizontal transfer of nutrients, gases, and cellular signals, creating a dynamic and interactive microenvironment that closely mimics in vivo conditions, promoting cell viability and function. Embodiments herein may provide a robust platform for generating functional microvasculature, contributing to advancements in tissue engineering and regenerative medicine. The ability to create complex vascular networks supports the development of engineered tissues and organs.

[0054] In certain embodiments, IPHB stiffness may be modulated to mimic one or more mechanical properties of one or more tissues, which may promote an environment for cellular growth and differentiation. In certain embodiments, cavity size and / or microchannel diameter may be adjusted, which could influence cell behavior, and / or fluid dynamics (e.g. of perfusate). Modulating the physical conditions may closely replicate those found in vivo.

[0055] When seeded into IPHB(s) in accordance with embodiments herein, ECs and / or MSCs may aggregate and organize into sophisticated vascular structures, including capillaries, venules, arterioles, veins, and / or arteries. In some embodiments, the co-cultivation of these cell types may lead to the formation of capillaries, venules, arterioles, veins, and / or arteries, which may replicate the natural hierarchy of the vascular system. This may result in more physiologically relevant blood vessels that extend beyond the initial structure (e.g. scaffold, hydrogel matrix, or any confines). Vascular structures herein may facilitate the delivery of nutrients and / or removal of waste products, which may promote the health and / or functionality of cultured tissues. Certain embodiments may leverage paracrine signaling and / or natural cellular interactions in a controlled environment. In certain embodiments, the synergistic effects of co-cultivating diverse cell types within a modular hydrogel structure better replicates the natural cellular environment and enhances vascular formation through complex cell-cell interactions and signaling mechanisms.

[0056] Certain embodiments support both static and perfusion-based culturing conditions. In certain embodiments, modulation of perfusion speed, pressure, and / or volume may help optimize vascular formation and / or physiological integrity. Perfusion may enhance nutrient delivery and / or waste removal, accelerate cellular organization, and / or increase secretory output. Cells cultured under perfusion conditions may exhibit more rapid and / or coordinated formation of vascular structures. When perfusion is used instead of static culture, secretory output may be enhanced. Perfusion may support better nutrient exchange, waste exchange, organization of cells, and / or functional maturation of cells. The ability to cultivate cells under static or perfusion conditions within IPHBs may allow for fine-tuning of mechanical and / or biochemical stimuli, and / or may lead to more physiologically relevant vascular structures. Where perfusion bioreactors and dynamic culturing may be known concepts, the capabilities herein, especially integrated into a modular hydrogel system designed specifically for vascular tissue engineering, is innovative. The ability to precisely control the dynamic environment within interconnected hydrogel blocks to enhance vascular maturation and stability is not an obvious extension of existing technologies, especially as perfusion is typically separate from scaffold design. Embodiments and dynamic culture conditions discussed herein help vessels remain functional over extended periods, addressing issues related to degradation and mechanical failure.

[0057] Cultivation within IPHBs can result in increased secretory output, including growth factors, cytokines, chemokines, extracellular vesicles, and / or other bioactive molecules. This enhanced secretory activity can be valuable for studying vascular biology and / or evaluating therapeutic effects.

[0058] In some embodiments, the modular design of IPHBs may allow them to be loaded into standard well-plates, supporting high-throughput testing of therapeutic compounds. For example, a standard 6-well plate may accommodate up to about 24, 42, and / or 96 of certain IPHBs. Embodiments herein can align with high-throughput workflows and / or allow for the testing of multiple drugs and their effects on vascular tissues. The enhanced secretory output and physiological relevance of models in accordance with embodiments herein may significantly improve the accuracy and / or predictive power of drug testing.

[0059] Certain embodiments may be loaded with drugs and / or growth factors, which may interact with the cells seeded. In certain embodiments, compounds may be distributed in at least one differential and / or gradient, which may promote evaluation of therapeutic effects) and optimal dosing strategies.

[0060] Certain embodiments use primary cells from patient biopsies and may enable the creation of personalized vascular models that reflect the unique characteristics of a patient’s tissue. This personalized approach supports precision medicine and may allow for tailored treatment strategies and / or more accurate predictions of therapeutic efficacy.

[0061] Vascular structures formed within IPHBs may be joined horizontally and / or vertically or otherwise, by interconnecting blocks, e.g. within a biological cartridge. The versatility of embodiments herein may allow for the creation of integrated tissue systems and / or simulating full organ functions such as in a "System-on-a-Chip" format. Perfusion may be applied both horizontally and vertically (and / or otherwise), which may enhance the functionality and / or realism of tissue constructs.

[0062] Embodiments herein consume significantly less media and plasticware compared to traditional cultivation methods. Certain embodiments may require only 10% of the media needed for 2D cultivation of ECs, and may reduce plastic consumption by 85%. Efficiency reduces costs and improves the sustainability of cell culture processes. Notwithstanding their advanced capabilities, embodiments herein can be accessible to use and fabricate using available materials such as hydrogel, which may be employed by researchers with varying levels of expertise. This accessibility makes them suitable for a wide range of research institutions, clinical applications, and / or educational settings. Existing high-end bioprinting and scaffold fabrication techniques are often cost-prohibitive and require specialized equipment. The ability for a modular hydrogel system and / or other embodiments herein to achieve similar or superior outcomes in vascular engineering at reduced cost is a non-obvious and innovative solution that addresses economic and practical challenges in the field.

[0063] This disclosure is particularly suited for applications in tissue engineering and regenerative medicine, drug discovery and testing, personalized medicine, and disease modeling. Embodiments herein provide a robust platform for generating functional micro vasculature, contributing to advancements in engineered tissues and organs. They may also enable a high-throughput screening of therapeutic compounds with enhanced predictive power. They may support the development of tailored treatments based on patient-specific vascular models and facilitate the study of various vascular diseases, including cancer, diabetes, and cardiovascular conditions. The consistent and reproducible nature of IPHB fabrication supports vascular networks to be reliably produced at various scales, including from small laboratory settings to large-scale clinical applications.

[0064] Embodiments herein introduce several novel and non-obvious advancements in the field of blood vessel tissue engineering. For example, integrating multiple cell types within a modular and dynamically controlled hydrogel system may address key limitations of existing technologies. These innovations may enable the formation of complex, scalable, and / or functional vascular networks. They may provide a cost-effective and / or accessible solution for tissue grafting and / or regenerative medicine. While there may be trade-offs in initial setup complexity and / or precision compared to other technologies like bioprinting and microfluidic systems, the overall benefits of embodiments herein make them highly promising technology for advancing tissue engineering, patient grafts, and / or drug discovery.

[0065] Certain embodiments may be 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 measured goal or threshold. Examples may include deviation by about 15%, 10%, 8%, 5%, 3%, or 1%, from the natural tissue of interest. The one or more physical properties may include, for example, softness and / or tension strength, and / or elasticity. One or more physical properties may comprise porosity, stiffness, and / or elastic modulus. Additionally or alternatively, the network of microporous cavities (e.g. channels and / or chambers) may be structured to mimic the morphology of a natural tissue of interest, such as by varying the geometry and / or dimensions of a network of microporous channels and / or chambers to mirror the morphology of the natural tissue of interest. In certain examples, a 3D network of microporous channels and / or chambers defines a 3D scaffolding for propagation of one or more cell types of interest. In accordance with certain embodiments, an average diameter of one or more microporous cavities may comprise from about 100 to about 800 microns. The minimum diameter of one or more microporous cavities may comprise 100, 120, 150, 180, 200, 220, and / or 250 microns, in certain embodiments. The maximum diameter of one or more microporous cavities may comprise 800, 780, 750, 720, 700, 680, 650, 620, 600, 580, 550, 520, 500, 480, 450, 420, 400, 380, 350, 320, 300, 280, and / or 250 microns, in certain embodiments. Additionally or alternatively, microporous channels and / or chambers may comprise at least about 40% by volume of the 3D macrostructure of an IPHB. In certain embodiments, microporous cavities may comprise at least about 40, 50, 60, and / or 70% by volume of the 3D macrostructure. In certain embodiments, microporous cavities may comprise at most about 90, 85, 80, 75, and / or 70% by volume of the 3D macrostructure.

[0066] 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.

[0067] 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 further comprise a synthetic polymer, such as a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or any combination(s) thereof. The continuous polymeric matrix material, in accordance with certain embodiments, may comprise a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

[0068] 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%.

[0069] In certain embodiments, a continuous polymeric matrix material, as noted above, 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.

[0070] 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 50%.

[0071] In accordance with certain embodiments, an interface between the network of microporous cavities (e.g., channels and / or chambers) and the continuous polymeric matrix material may comprise a coating of at least one compatibilizer selected to promote adhesion of at least one cell of interest. This coating may be applied subsequent to IPHB formation. By way of example, the coating comprising the compatibilizer(s) may comprise a biological coating, including, for example, collagen I (e.g., from or for human MSCs [e.g., from or for adipose, bone barrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); laminin I (e.g., from or for human induced pluripotent stem cells [IPSCs] and / or mouse dorsal root ganglia); hyaluronan (e.g., from porcine hepatocytes and / or human dermal adult fibroblasts); gelatin (e.g., from human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human myocytes, human osteoblasts, human osteocytes, human chondrocytes, human CD8+ T cells, Human CD4+ T cells, human macrophages, bovine myocytes, porcine hepatocytes, porcine chondrocytes, porcine osteocytes, and / or equine muscle derived stem cells); fibrin (e.g., from or for human keratinocytes); fibronectin (e.g., from or for human MSCs [e.g., from or for adipose, bone marrow, and / or umbilical cord tissues], human neonatal dermal fibroblasts, human adult dermal fibroblasts, human keratinocytes, human osteoblasts, human osteocytes, and / or human chondrocytes); or any combination(s) thereof.

[0072] 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.

[0073] A method for generating personalized microvasculature ex vivo may comprise isolating primary ECs and MSCs from a patient biopsy, cultivating the ECs and MSCs within an IPHB which comprises a 3D macrostructure and a highly biomimetic environment that closely resembles the natural ECM, co-cultivating the primary cells with additional cell types within the at least one IPHB to enhance microvascular formation, and utilizing dynamic culturing conditions including static and perfusion, to enhance the physiological relevance of the microvascular model. In some embodiments, distribution of growth factors, nutrients, and / or other bioactive molecules in liquid format across the IPHB may create gradient(s) for comprehensive evaluation. In some embodiments, connection of the IPHB to another IPHB may use perfusion systems to simulate full organ systems. In some embodiments, interconnecting at least the first IPHB and the second IPHB may expand the microvascular network to accommodate growth and study progression. In some embodiments, diagnostic evaluations may be performed on the generated micro vasculature within the IPHB. These may comprise histological analysis and / or molecular profifing. In some embodiments, IPHB(s) and / or generated microvasculature may be configured for high-throughput screening of therapeutic compounds. In some embodiments, secreted byproducts, such as from culture media and / or perfusate, which may be surrounding the IPHB(s), may be collected and / or analyzed. Some embodiments may develop one or more personalized microvascular models, which may reflect one or more unique characteristics of a patient’s tissue. Some embodiments may be configured to reduce human labor, media consumption by up to about 90%, and / or plasticware usage by up to about 85%.

[0074] In certain embodiments, a system for generating personalized microvasculature ex vivo may comprise at least one IPHB under dynamic culture conditions configured to cultivate microvascular formation when seeded with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen.

[0075] In some embodiments, the dynamic culture conditions may be configured to achieve nutrient and / or waste exchange for at least one line of cells.

[0076] In some embodiments, the dynamic culture conditions may comprise exposing at least one cell type to a culture media via perfusion and / or static solution. In some embodiments, the culture media may comprise one or more nutrients for the cells. The dynamic culture conditions may further comprise varying the culture media’s nutrient type and / or concentration.

[0077] In some embodiments, at least one first specimen, at least one second specimen, or both, may derived from a human, a subject, and / or a biopsy. 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 source; examples include multiple first specimens from the same source, multiple second specimens from the same source, a first specimen from the same source as a second specimen, and / or multiple first specimens from the same source as one or more second specimens. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0078] In some embodiments, at least one first specimen and at least one second specimen may be derived from the same human, subject, and / or biopsy.

[0079] In some embodiments, at least one second specimen may consist of at least one first specimen.

[0080] In some embodiments, microvascular formation may comprise forming microvasculature, veins, venules, capillaries, arterioles, and / or arteries. Some embodiments may comprise fistulae and / or anastomoses.

[0081] Some embodiments may be configured to fluidically distribute nutrients, growth factors, and / or bioactive molecules.

[0082] In some embodiments, at least one block may be configured for compatibility with histological analysis and / or molecular profiling. This may comprise one or more forms of histological analysis, one or more forms of molecular profiling, or both.

[0083] In some embodiments, at least one block may be configured to collect secreted byproducts from the microvascular formation, at least one line of ECs, at least one line of MSCs, or a combination thereof.

[0084] In some embodiments, at least one block may comprise a 3D macrostructure.

[0085] In some embodiments, one or more blocks may be configured to mimic an ECM. At least one mimicked ECM may be that of a first and / or second specimen, and / or an ECM from the derivative source (e.g. human, subject, biopsy).

[0086] In some embodiments, one or more blocks may be configured to mimic the ECM of at least one first specimen, at least one second specimen, or a combination thereof. In some embodiments, one or more blocks may be configured to match at least one physical property of the ECM.

[0087] In some embodiments, one or more ECM physical properties to be mimicked may comprise porosity, stiffness, hardness, capacitance, elasticity, elastic modulus, density, conductivity, thermal conductivity, electric conductivity, electric charge, solubility, hydrophilicity, and / or lipophilicity. A physical property may be quantified, such as with a measured average and / or range. The physical property may be emulated and / or targeted within a goal threshold, including without limitation, only by way of example / for purposes of illustration, within 20% more or less than the measured average and / or range.

[0088] In some embodiments, the culture media may be configured to mimic an ECM in at least one nutrient and / or physical property.

[0089] In some embodiments, one or more blocks may be configured for loading into one or more standard well-plates.

[0090] In certain embodiments, a method for generating personalized microvasculature ex vivo may comprise seeding at least one IPHB with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen and exposing the cell types to dynamic culture conditions.

[0091] Some embodiments may further comprise seeding at least one block with one or more additional cell types.

[0092] In some embodiments, one or more additional cell types may comprise pericytes (PCs), myocytes, and / or fibroblasts.

[0093] In certain embodiments, an IPHB may be configured to under dynamic culture conditions cultivate microvascular formation ex vivo when seeded with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen.

[0094] Described herein are systems, methods, and IPHBs for generating personalized microvasculature ex vivo. A block may be configured to, under dynamic conditions, cultivate microvascular formation when seeded with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen. A system may comprise at least one block so configured. A method may comprise seeding at least one block with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen and exposing the cell types to dynamic culture conditions. Turning to Figure 3, illustrated is an example embodiment of a method 300 for generating personalized microvasculature ex vivo. In certain embodiments, method 300 may comprise step 310 of seeding and step 320 of dynamic culture conditions. Step 310 may comprise seeding at least one IPHB with at least one line of ECs isolated from at least one first specimen and at least one line of MSCs isolated from at least one second specimen. The IPHB may comprise a 3D continuous polymeric matrix with a network of microporous cavities. The cavities may comprise one or more channels and / or chambers. Step 320 may comprise exposing the cells to dynamic culture conditions. Dynamic culture conditions 320 may be configured to achieve nutrient and / or waste exchange for at least one line of cells. Dynamic culture conditions 320 may comprise exposing at least one cell type to a culture media via perfusion and / or static solution. The culture media may comprise one or more nutrients for the cells. 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.

[0095] In some embodiments of step 310, at least one first specimen, at least one second specimen, or both, may derived from a human, a subject, and / or a biopsy. 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 source; examples include multiple first specimens from the same source, multiple second specimens from the same source, a first specimen from the same source as a second specimen, and / or multiple first specimens from the same source as one or more second specimens. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0096] Certain embodiments may comprise additional steps. For example, method 300 may comprise step 330 of additional seeding and / or step 340 of harvesting. Step 330 may comprise seeding at least one block with at least one additional cell type. Examples may include, without limitation, PCs, myocytes, and / or fibroblasts. Step 340 may comprise harvesting microvasculature, which could have been generated, and which could have been generated as a result of or subsequent to seeding or other process (e.g. steps 310 and / or 320).

[0097] 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 harvesting 340 (not necessarily including additional seeding 330). Some embodiments may comprise seeding 310, dynamic culture conditions 320, and additional seeding 330. In some embodiments, additional seeding 330 could be after or subsequent to harvesting 340. Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of seeding 310 (with or without additional seeding 330), dynamic culture conditions 320, and / or harvesting 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, additional seeding 330, and / or harvesting 340). Such embodiments may include changing the configuration of existing blocks, removing blocks, and / or adding blocks.

[0098] 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, ECs, 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.

[0099] 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.

[0100] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0101] Clause 1. A system for generating personalized micro vasculature ex vivo, comprising: at least one IPHB configured to provide a 3D environment and to provide a highly biomimetic environment that closely resembles the natural ECM; ECs and MSCs seeded within the at least one IPHB and co-cultivated within the at least one IPHB; and optionally additional cell types seeded within the at least one IPHB and cultivated therein, wherein the cultivation of additional cell types including PCs, myocytes, and fibroblasts to enhance microvascular formation.

[0102] Clause 2. The system of clause 1, wherein the at least one IPHB is configured to form micro-vasculature, such as capillaries, venules, arterioles, veins, and arteries.

[0103] Clause 3. The system of clause 1, wherein the at least one IPHB is configured to be modular and interconnected or interlocked to one or more additional IPHB(s) to expand the microvascular network size from an initial microvascular network size and / or customize the microenvironment for specific research needs or patient-specific conditions.

[0104] Clause 4. The system of clause 3, wherein the at least one IPHB interconnected to another IPHB utilizes perfusion systems to simulate full organ systems, whereby creating a "system-on-a-chip" for comprehensive studies.

[0105] Clause 5. The system of clause 3, wherein the at least one IPHB comprises a plurality of individual IPHBs interlocked together and configured to be loaded into standard well-plates, and enabling high -throughput testing of one or more therapeutic compounds.

[0106] Clause 6. The system of clause 1, wherein the at least one IPHB is configured to enable the distribution of growth factors, nutrients, and other bioactive molecules in liquid format, and optionally creating gradients across the IPHB(s) for comprehensive evaluation.

[0107] Clause 7. The system of clause 1, wherein the at least one IPHB is configured to support various diagnostic procedures including histological analysis, molecular profiling, and the collection of secreted byproducts for further analysis. Clause 8. The system of clause 1, wherein the at least one IPHB is cost-effective and comprehensible.

[0108] Clause 9. The system of clause 1, wherein the at least one IPHB is configured to reduce media consumption by up to 90% and plastic consumption by up to 85% and human labor.

[0109] Clause 10. The system of clause 1, wherein the at least one IPHB is configured to closely simulate in vivo conditions by facilitating the formation of realistic microvascular structures and invasive behaviors by ECs and supportive cells.

[0110] Clause 11. The system of clause 1, wherein the at least one IPHB is configured to use primary cells from patient biopsies to develop personalized microvascular models; wherein the IPHB(s) enable the creation of personalized microvascular models by using primary cells from patient biopsies, reflecting the unique characteristics of each patient’s tissue.

[0111] Clause 12. A method for generating personalized microvasculature ex vivo, comprising: isolating primary ECs and MSCs from a patient biopsy; cultivating the ECs and MSCs within at least one IPHB; wherein the at least one IPHB comprises a 3D macrostructure; wherein the at least one IPHB comprises a highly biomimetic environment that closely resembles the natural ECM; co-cultivating the primary cells with additional cell types within the at least one IPHB to enhance microvascular formation; and utilizing dynamic culturing conditions including static and perfusion, to enhance the physiological relevance of the microvascular model.

[0112] Clause 13. The method of clause 12, further comprising a step of distributing growth factors, nutrients, and other bioactive molecules in liquid format across the at least one IPHB to create gradients for comprehensive evaluation.

[0113] Clause 14. The method of clause 12, further comprising a step of connecting a plurality of individual IPHBs together using perfusion systems to simulate full organ systems, whereby enabling comprehensive studies.

[0114] Clause 15. The method of clause 14, further comprising interconnecting a first IPHB and a second IPHB to expand the microvascular network to accommodate growth and study progression.

[0115] Clause 16. The method of clause 12, further comprising a step of performing diagnostic evaluations) on the generated micro vasculature within the at least one IPHB.

[0116] Clause 17. The method of clause 16, wherein the diagnostic evaluations comprise histological analysis and / or molecular profiling. Clause 18. The method of clause 12, wherein generated microvasculature in the at least one IPHB is configured for high-throughput screening of therapeutic compounds.

[0117] Clause 19. The method of clause 12, further comprising the collection and analyzation of secreted byproducts from the media or perfusate surrounding the at least one IPHB.

[0118] Clause 20. The method of clause 12, wherein the at least one IPHB develops personalized micro vascular models which reflect the unique characteristics of each patient’s tissue, whereby enabling patient specific tailored treatment protocols.

[0119] Clause 21. The method of clause 12, 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.

[0120] These and other modifications and variations may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the claims. In addition, it should be understood that aspects of the various embodiments may be interchanged in whole or in part. Furthermore, those of ordinary skill in the art will appreciate that the foregoing description is by way of example only, and it is not intended to limit the claims. Therefore, the spirit and scope of the appended claims should not be limited to the exemplary description of the versions contained herein.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A system for generating personalized micro vasculature ex vivo, comprising: at least one interconnecting porous hydrogel block, under dynamic culture conditions, configured to cultivate microvascular formation when seeded with at least one line of endothelial cells isolated from at least one first specimen, and at least one line of mesenchymal stem cells isolated from at least one second specimen.

2. The system of claim 1, wherein the dynamic culture conditions are configured to achieve nutrient exchange, waste exchange, or a combination thereof, for at least one line of cells.

3. The system of claim 1, wherein the dynamic culture conditions comprise exposing at least one cell type to a culture media via perfusion, static solution, or a combination thereof.

4. The system of claim 3, wherein the culture media comprises one or more nutrients for the cells, and the dynamic culture conditions further comprise varying the culture media’s nutrient type, nutrient concentration, or a combination thereof.

5. The system of claim 1, wherein at least one first specimen, at least one second specimen, or both, are derived from a human, a subject, a biopsy, or a combination thereof.

6. The system of claim 5, wherein at least one first specimen and at least one second specimen are derived from the same human, subject, or biopsy.

7. The system of claim 6, wherein at least one second specimen consists of at least one first specimen.

8. The system of claim 1, wherein microvascular formation comprises forming microvasculature, veins, venules, capillaries, arterioles, arteries, or a combination thereof.

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

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

11. The system of claim 1, wherein at least one block is configured to collect secreted byproducts from the microvascular formation, at least one line of endothelial cells, at least one line of mesenchymal stem cells, or a combination thereof.

12. The system of claim 1, with at least one block comprising a three-dimensional macrostructure.

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

14. The system of claim 13, further configured to mimic the extracellular matrix of at least one first specimen, at least one second specimen, or a combination thereof.

15. The system of claim 13, further configured to match at least one physical property of the extracellular matrix.

16. The system of claim 15, 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.

17. The system of claim 3, further wherein the culture media is configured to mimic an extracellular matrix in at least one nutrient, one or more physical properties, or a combination thereof.

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

19. A method for generating personalized microvasculature ex vivo, comprising: seeding at least one interconnecting porous hydrogel block with at least one line of endothelial cells isolated from at least one first specimen, and at least one line of mesenchymal stem cells isolated from at least one second specimen, and exposing the cell types to dynamic culture conditions.

20. The method of claim 19, further comprising seeding at least one block with one or more additional cell types.

21. The method of claim 20, wherein the one or more additional cell types comprise pericytes, myocytes, fibroblasts, or a combination thereof.

22. An interconnecting porous hydrogel block, configured to, under dynamic culture conditions, cultivate microvascular formation ex vivo when seeded with: at least one line of endothelial cells isolated from at least one first specimen, and at least one line of mesenchymal stem cells isolated from at least one second specimen.

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