Ex VIVO tumor genesis of patient cancers using patient tissues cultivated in interconnecting porous hydrogel blocks

Abstract

Described herein are systems, methods, and interconnecting porous hydrogel blocks for cultivating tumor cells ex vivo. A block may comprise a three-dimensional continuous polymeric matrix with a network of microporous cavities, and may be configured to interconnect with at least one other block. A system may be configured to cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. A method may include seeding at least one block with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types and exposing the cells to dynamic culture conditions.

Description

EX VIVO TUMOR GENESIS OF PATIENT CANCERS USING PATIENT TISSUES CULTIVATED IN INTERCONNECTING POROUS HYDROGEL BLOCKSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of and priority to U.S. Provisional Application Serial No. 63 / 665,826, 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.TECHNICAL FIELD

[0002] The subject matter disclosed herein is generally directed to the cultivation of cancer cells, which could be isolated from patients suffering from connective tissue cancers, such as adenocarcinomas, bladder, bone, breast, brain, colon and / or rectal, endometrial, intestinal, kidney, liver, lung, melanoma, neuroblastomas, pancreatic, prostate, testicular, throat, thyroid, and / or other cancers, including with interconnecting porous hydrogel blocks (IPHBs). When patient cancer cells are cultivated in one or more IPHBs, the cells may exhibit distinct morphological and physiological behaviors compared to traditional models. Primary or other cancer cells in IPHB related embodiments may form aggregates, nodules, and / or complex structures instead of flat, spread-out phenotypes, and may demonstrate invasive behaviors by migrating between microchannels within the IPHB(s) as well as extending outside of IPHB(s). Introducing additional cell types, such as endothelial cells or other connective tissue cells, may result in cancer cells commandeering the behavior of these non-cancer cells, which may reflect a more realistic tumor microenvironment.BACKGROUND

[0003] Cancer research encompasses a wide range of methodologies, which may include modeling, tumor generation, diagnostic evaluation, drug discovery, radiation therapy, immunotherapy, and tissue engineering strategies. Each approach has its own set of advantages and disadvantages.

[0004] In vivo animal models may provide a comprehensive understanding of cancer biology within the context of a living organism. Animal models may provide a whole-organism context for studying cancer progression and treatment. These models may allow for the study of tumorhost interactions, metastasis, and systemic responses to therapies and may enable long-termstudies of cancer progression and treatment efficacy. However, in vivo animals create ethical concerns and regulatory constraints regarding animal use, incur high costs due to the timeconsuming nature of animal studies, and may have limited relevance to human cancer due to species-specific differences.

[0005] Traditional in vitro strategies involve culturing cancer cells in two-dimensional (2D) or three-dimensional (3D) systems. In vitro culture models may be more cost-effective and relatively simple to set up. They also may allow for controlled studies of cellular and molecular mechanisms or facilitate high-throughput screening of potential therapeutic compounds. However, in vitro cell culture models lack the complex tumor microenvironment present in vivo and have limited ability to model tumor heterogeneity and metastatic behavior. 2D cultures also often fail to mimic the 3D architecture of tumors.

[0006] Ex vivo tumor models are another approach to cancer research. This type of model may use patient-derived cells to provide a more personalized approach to cancer research. Ex vivo tumor models may better mimic the tumor microenvironment compared to 2D cultures and enable the study of specific cancer types and patient-specific responses to therapies. However, the drawbacks to these studies include limited scalability and variability in obtaining patient samples and complexity in maintaining the viability and functionality of primary cells.

[0007] Organ-on-a-chip models are microfluidic cell culture platforms that may mimic the physiological functions of human organs. These models may provide a more physiologically relevant environment by mimicking the architecture and functions of human organs. They also may allow for the study of cell-cell and cell-matrix interactions in a controlled setting and enable the integration of multiple organ systems to study systemic effects. The disadvantages of this approach include high cost, technical complexity, and limited scalability for high- throughput screening. Organ-on-a-chip models are also still in developmental stages, with variability in standardization and reproducibility.

[0008] Drug discovery and screening advantages include high-throughput screening that may allow for the rapid identification of potential therapeutic compounds, use of diverse libraries of small molecules, biologies, and natural products, and integration of computational methods (e.g., virtual screening) to enhance efficiency. However, high attrition rate of compounds from initial screening to clinical use and limited predictive power of in vitro assays for in vivo efficacy and toxicity are some of the setbacks to this approach. Drug discovery and screening is also expensive and a time-consuming process to bring a drug to market.

[0009] Radiation therapy is a cancer treatment that is currently used. The advantages to this treatment may include targeting localized tumors effectively, combining this treatment with other therapies (e.g., chemotherapy, immunotherapy) for enhanced efficacy, and improving targeting and minimizing damage to healthy tissues with advances in technology (e.g., precision radiation). There are drawbacks to radiation, including potential for severe side effects and damage to surrounding healthy tissues, limited efficacy against metastatic disease, and variability in patient responses and resistance development as main disadvantages to radiation therapy.

[0010] Immunotherapy is another cancer treatment that is currently utilized. Immunotherapy may harness the body’s immune system to target and eliminate cancer cells and has potential for long-lasting responses and remission. Advances in personalized immunotherapies (e.g., CAR-T cells) show promise for treating specific cancers. However, there is a risk of significant immune-related side effects (e.g., cytokine release syndrome). It is very costly and complex to develop and administer immunotherapies. There is also a variability in patient responses and potential for resistance.

[0011] Tissue engineering strategies may involve creating 3D tissue constructs using scaffolds and bioprinting techniques. This strategy may enable the creation of 3D models that better mimic the tumor microenvironment. They may also facilitate studies of tumor-stroma interactions and the impact of the extracellular matrix (ECM) on cancer progression. And they may provide platforms for personalized medicine and testing of patient-specific therapies. The disadvantages to tissue engineering strategies include technical complexity and high cost of creating tissue-engineered models, limited scalability and reproducibility, and challenges in maintaining long-term viability and functionality of engineered tissues.

[0012] Despite significant advancements in cancer research, several gaps and barriers remain. Existing models often fail to capture the genetic, phenotypic, and microenvironmental heterogeneity of tumors, but developing models that accurately represent the complexity of human tumors remains challenging, hindering the prediction of therapeutic responses. Many models lack the inclusion of a realistic tumor microenvironment and vascularization, which are important for studying tumor progression and drug responses, but creating models that incorporate dynamic interactions between cancer cells, stromal cells, and the ECM is technically challenging. Current approaches do not adequately allow for personalized treatment strategies based on individual patient tumors, however variability in patient-derived samples and the complexity of developing personalized models limit the ability to tailor therapieseffectively. Many advanced models may not be amenable to high-throughput screening due to technical and cost constraints, but balancing the complexity of models with the need for scalability and reproducibility is difficult. In vitro and ex vivo models may often fail to predict in vivo efficacy and toxicity accurately, however, bridging the gap between model systems and clinical outcomes requires better integration of physiological relevance and predictive biomarkers. Further, high costs and technical complexity may limit the accessibility of advanced models and therapies, but costs and simplifying technologies are necessary to make advanced cancer research tools widely available.

[0013] In summary, while current approaches in cancer research offer insights into therapeutic opportunities, significant gaps and barriers remain. Advancements in modeling, particularly in creating more realistic and personalized tumor models, are important for improving the prediction of therapeutic responses and the development of effective treatments. Addressing these challenges require innovation and collaboration across multiple disciplines in cancer research.

[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.SUMMARY

[0015] 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 cultivating primary tumor cells ex vivo, comprising at least one interconnecting porous hydrogel block (IPHB) with the at least one IPHB including a three-dimensional (3D) macrostructure defined by a continuous polymeric matrix material with a network of microporous cavities (e.g., channels and / or chambers); cancer cells (e.g., primary cancer cells) derived from a specimen (e.g., patient biopsies) cultured within the at least one IPHB; cocultivation of cancer cells with supportive cell types (e.g., endothelial cells) within the at least one IPHB; and dynamic culturing capabilities configured for static and perfusion conditions to simulate physiological environments. Further, the at least one IPHB may be configured to support the formation of micro-vasculature within the tumor model. Further still, the at least one IPHB may be modular and designed to be interconnected to one or more other IPHB(s). Still yet, the at least one IPHB may allow for the loading of multiple IPHBs into standard wellplates. Yet again, the at least one IPHB may enable the distribution of drugs in liquid format. Again, the at least one IPHB may be connected using perfusion systems to simulate full organsystems. Still further, the at least one IPHB may support various diagnostic procedures, including histological analysis, molecular profiling, and the collection of secreted byproducts for further analysis. Yet further, the at least one IPHB may be cost-effective and easy to use. Even further, the at least one IPHB may be designed to reduce human labor, media consumption by up to 90%, and / or plastic consumption by up to 85%. Further still, the at least one IPHB may facilitate the formation of realistic tumor structures and invasive behaviors by primary cancer cells. Even further still, the at least one IPHB may enable the creation of personalized cancer models by using primary cells from patient biopsies.

[0016] In another aspect, the present disclosure provides a method for cultivating primary tumor cells ex vivo, which may comprise isolating cancer cells (e.g., primary cancer cells) from a specimen (e.g., patient biopsy); cultivating the cancer cells within the at least one IPHB that includes a 3D environment defined by a continuous polymeric matrix material with a network of microporous cavities (e.g., channels and / or chambers); co-cultivating the cancer cells with supportive cell types within the at least one IPHB; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the tumor model. Further, therapeutic compounds in liquid format may be distributed across the at least one IPHB. Again, the at least one IPHB may be connected to another IPHB using perfusion systems to simulate full organ systems. Again still, diagnostic evaluations, including histological analysis and molecular profiling, may be performed on the cultivated tumor cells within the at least one IPHB. Yet still, cultivated tumor cells and / or the at least one IPHB may be configured for high-throughput screening of therapeutic compounds. Still yet, secreted byproducts from the media and / or perfusate surrounding the at least one IPHB may be collected and / or analyzed. Still yet again, the at least one IPHB may be configured to create personalized cancer models that reflect the unique characteristics of each patient’s cancer for tailored treatment strategies. Even further, the tumor model may be expanded by interconnecting the at least one IPHB to one or more additional IPHB(s), e.g. to accommodate tumor growth and study progression. Even further still, media and plastic consumption may be reduced by up to 90% and 85% compared to other cultivation formats.

[0017] In another aspect, the present disclosure provides methods, including of treating a patient having a cancer. Some embodiments may comprise the following: (i) isolating cancer cells (e.g., primary cancer cells) from a specimen (e.g., patient biopsy); (ii) cultivating the cancer cells within at least one IPHB, the at least one IPHB having a 3D macrostructure defined by a continuous polymeric matrix material and a network of microporous cavities (e.g.,channels and / or chambers) extending throughout the continuous polymeric matrix material configured to provide a 3D environment; and (iii) co-cultivating the cancer cells with supportive cell types within the at least one IPHB, wherein co-cultivating may include feeding the cancer cells with supportive cell types with one or more culture media, and allowing the cancer cells with supportive cell types to propagate through the network of microporous channels and / or chambers; and wherein the co-cultivating comprises utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of a resulting tumor model. Some embodiments may comprise (iv) harvesting the tumor model from the at least one IPHB. Some embodiments may comprise (v) subjecting one or more portions of the tumor model to one or more respective therapeutic compound(s) or therapeutic treatments, such as in a parallel timeline. Some embodiments may comprise (vi) evaluating the effectiveness of each therapeutic compound and / or therapeutic treatment, and / or selecting one or more viable therapeutic compound and / or therapeutic treatment based on mitigation of growth of the tumor model and / or extent of cancer cell destruction. Some embodiments may comprise (vii) subjecting the patient to a treatment with one or more of the selected viable therapeutic compound(s) and / or therapeutic treatments). In some embodiments, the step of subjecting the patient to a treatment may start with about 10 days of the step of isolating cancer cells from the specimen. In some embodiments, the minimum such time interval may be about 10, 12, 14, 16, 18, 20, 22, 25, 30, 35, 40, 45, and / or 50 days. In some embodiments, the maximum such time interval may be about 180, 150, 120, 100, 90, 80, 70, 60, and / or 50 days.

[0018] In certain embodiments, a system for cultivating tumor cells ex vivo may comprise at least one IPHB configured to under dynamic culture conditions cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. 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.

[0019] In some embodiments, the cavities may include one or more channels and / or chambers. A cavity may comprise a well, recess, pore, pocket, and / or enclosure.

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

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

[0022] In some embodiments, the culture media may include 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, one or more specimens 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 patient (e.g. including differing in the location and / or time biopsied), and / or different patients. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0024] In some embodiments, one or more specimens may be derived from a site of primary tumor, metastasis, metastatic disease, lymphangitic disease, lymphoid tissue, lymph node, lymphadenopathy, adenopathy, or a combination thereof.

[0025] In some embodiments, at least one additional cell type may include endothelial cells.

[0026] Some embodiments may be configured to fluidically distribute nutrients, growth factors, and / or bioactive molecules.

[0027] 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.

[0028] In some embodiments, at least one block may be configured to collect secreted byproducts, e.g. from the tumor cells.

[0029] Some embodiments may be configured to cultivate microvascular formation.

[0030] Some embodiments may be configured to fluidically distribute at least one drug across the cultivated tumor cells, the seeded tumor cells, or a combination thereof.

[0031] Some embodiments may include one or more drugs, which could be distributed across the system, e.g. with geographic, temporal, and / or other variation in concentration via one or more differentials) and / or gradients).

[0032] In some embodiments, the one or more drugs may be selected from the group consisting of: medicine, medication, pharmaceutical, biologic, immunologic, immunotherapy, chemotherapy, radiotherapy, radiotracer, tracer, stain, diagnostic agent, theranostic agent, therapeutic agent, treatment agent, or any combination thereof.

[0033] In some embodiments, the one or more blocks may be configured for loading into one or more standard well-plates.

[0034] In certain embodiments, a method for cultivating tumor cells ex vivo may comprise seeding at least one IPHB with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types and exposing the cells to dynamic culture conditions. 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.

[0035] Some embodiments may comprise exposing 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).

[0036] Some embodiments 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, and / or tumor cells).

[0037] In some embodiments, the 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.

[0038] In certain embodiments, an IPHB for cultivating tumor cells ex vivo may be configured to under dynamic culture conditions cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. 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.

[0039] 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

[0040] 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:

[0041] FIG. 1 illustrates two separate interconnecting porous hydrogel blocks (IPHBs) in accordance with certain embodiments;

[0042] FIG. 2 illustrates three interconnected IPHBs in accordance with certain embodiments; and

[0043] FIG. 3 illustrates a method for cultivating tumor cells ex vivo in accordance with certain embodiments.

[0044] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.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 relate to the development and application of interconnecting porous hydrogel blocks (IPHBs), for the ex vivo cultivation of primary cancer cells isolated from patients. IPHBs may provide a three-dimensional (3D), biomimetic environment that could support the formation of tumor-like structures and facilitate advanced research in cancer modeling, drug discovery, and / or disease progression.

[0047] As noted above, tissue engineering strategies involve creating complex 3D tissue constructs using scaffolds and bioprinting techniques. This strategy enables the creation of 3D models that better mimic the tumor microenvironment. It also facilitates studies of tumorstroma interactions and the impact of the extracellular matrix (ECM) on cancer progression. Lastly, it provides platforms for personalized medicine and testing of patient-specific therapies. The disadvantages to tissue engineering strategies may include technical complexity and high cost of creating tissue-engineered models, limited scalability and reproducibility, and challenges in maintaining long-term viability and functionality of engineered tissues. In accordance with certain embodiments. However, IPHBs and embodiments herein may be simpler to set up and use compared to bioprinting techniques. The dynamic culturing capabilities of embodiments herein may allow for better simulation of physiological conditions, enhancing the relevance of the models, including with the advent of modular design to make them more scalable and reproducible compared to custom-engineered tissue constructs. IPHBsand embodiments herein may be relatively accessible to use for tissue formation and new assay development.

[0048] One of the important advantages of certain embodiments herein is the ability to support the formation of micro-vasculature, which can facilitate the development of a more realistic tumorigenic model. This vascularization can enable the cancer cells to receive adequate nutrient supply and waste removal, mimicking in vivo conditions more closely than traditional models. Additionally, IPHBs may allow for perfusion, which can accelerate the growth and progression of seeded and / or cultivated cancer cells. Embodiment herein may enhance the physiological relevance of a tumor model and / or provide a platform to study the dynamics of cancer progression and metastasis under more natural conditions.

[0049] Embodiments herein may offer a versatile platform for developing and / or testing new therapies. By cultivating cancer cells from patients, researchers may model the progression of cancers on a personalized level and test various therapies in parallel at different doses within a standardized format. Initial therapeutic treatments, including radiation exposure, may be applied to patient-specific cancer cells in embodiments herein to establish an effective baseline. Once a promising therapeutic candidate and dose are identified, the cancer may be expanded using additional IPHBs in certain embodiments for larger-scale evaluation, e.g. to evaluate that the therapeutic protocol is effective and / or safe for the patient.

[0050] Moreover, embodiments herein may enable cross-comparisons between cancers originating from the same tissue but with different genotypes. This capability may help in understanding the genetic and / or molecular basis of cancer heterogeneity and / or for developing targeted therapies that may address specific genetic profiles. The tumors grown in IPHBs and / or embodiments herein may be preserved for future comparison, e.g. against other cancers, which could include from the same patient, the patient’s family members, and / or defined populations.

[0051] The unique design of IPHBs and embodiments herein may allow for the collection of secreted byproducts, such as extracellular vesicles, growth factors, cytokines, chemokines, and / or other oncogenic factors, including from the media in which the IPHB(s) could be submerged. In certain embodiments, the block(s) may be fixed, flash-frozen, cryo-sectioned, and / or labeled with antibodies and / or histological stains, which could include pursuant to current or other clinical protocols, e.g. to monitor cancer progression. In certain embodiments, the media and / or perfusate may be analyzed for secreted factors. In certain embodiments,enzymatic dissolution of the IPHB(s) may release cells, e.g. for further probing, such as via flow cytometry, RT-qPCR, RNA sequencing, bioassay(s), and / or otherwise.

[0052] In some embodiments, mature tumors may form within seven days post-seeding, with complex cancer environments emerging rapidly once other cell types are introduced. Tumors may be maintained for extended periods or indefinitely in certain embodiments, e.g. including by adding IPHBs to accommodate microenvironment expansion and / or supplying media, which could be in a static and / or perfusion format. Certain embodiments reduce media consumption by 90% and / or plastic consumption by 85% compared to other formats, highlighting efficiency and sustainability e.g. in cancer research and / or therapy development.

[0053] Figure 1, for instance, illustrates two (2) separate IPHBs 1 in accordance with certain embodiments. Each of these IPHBs include a top surface 12, a bottom surface 14, and at least one side edge 16. The particular IPHBs 1 shown in Figure 1 include at least one interlockingmale component 50 and at least one interlocking-female component 60. Figure 2 illustrates three (3) interconnected IPHBs 1 in accordance with certain embodiments. The IPHBs 1 shown in Figure 2 each include a first interlocking-male component 51, a second interlocking-male component 52, a first interlocking-female component 61, and a second interlocking-female component 62. IPHBs 1 may interconnect via first interlocking-male component 51 or a second interlocking-male component 52 interfacing with a first interlocking-female component 61 or a second interlocking-female component 62, or vice versa. In certain embodiments, one or more interlocking-male components (50, 51, 52) comprise a protrusion. In certain embodiments, one or more interlocking-female components (60, 61, 62) comprise a recess. In certain embodiments, one or more interlocking-female components (60, 61, 62) is a recess configured complimentary to a protrusion of one or more interlocking-male components (50, 51, 52). Persons of ordinary skill will readily appreciate additional variations. For example, one or more interlocking-male components (50, 51, 52) may protrude from an IPHB l’s top surface 12, bottom surface 14, one or more side edges 16, or any combination thereof. One or more interlocking female components (60, 61, 62) may recess into an IPHB 1 ’s top surface 12, bottom surface 14, one or more side edges 16, or any combination thereof. One or more interlocking-male components (50, 51, 52) may comprise a quadrangular protrusion, or any other angled or rounded shape, or any combination thereof. One or more interlocking-female components (60, 61, 62) may comprise a quadrangular recess, or any other angled or rounded shape, or any combination thereof.

[0054] In certain embodiments, IPHBs may feature a network of adjustable cavities such has microchannels and / or pores, e.g. perpendicularly to one another, and may facilitate vertical and / or horizontal transfer of nutrients, gases, and / or cellular signals. In certain embodiments, IPHB stiffness may be modulated to mimic the mechanical properties of various tissues, e.g. to provide an optimal environment for cellular growth and / or 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 physical conditions may closely replicate those found in vivo. The 3D porous structure of IPHBs may support the formation of complex tumor architectures, including nodules and bulbous structures, which may not be achievable in traditional two dimensional (2D) cultures. In some embodiments, the dynamic and interactive environment within IPHBs may allow cancer cells to exhibit behaviors similar to those observed in vivo, including better cell-cell and cell-matrix interactions, and may create a more realistic tumor microenvironment. When additional cell types such as endothelial cells (ECs) and / or other connective tissue cells are introduced, cancer cells may influence the behavior of these non-cancer cells, which may further enhance the complexity of the tumor microenvironment in certain embodiments. Traditional models often fail to capture the genetic, phenotypic, and microenvironmental heterogeneity of tumors. IPHBs and embodiments herein may facilitate the growth of primary tumor cells in a 3D environment, allowing for the preservation of tumor heterogeneity. This may lead to more accurate representations of tumor complexity and better predictive models for therapeutic responses.

[0055] In certain embodiments, IPHBs may be designed to be modular, allowing them to be interconnected horizontally and / or vertically. This modularity may support the creation of larger and more complex tissue constructs, which may enable customization of the tumor microenvironment for specific research needs or patient-specific conditions. The interconnecting design of certain embodiments and IPHBs may allow for expansion and / or customization, and / or may make them suitable for a wide range of research and / or clinical applications.

[0056] When seeded into IPHB(s) in accordance with certain embodiments herein, primary cancer cells from patient biopsies may aggregate and / or organize into sophisticated tumor-like structures. In some embodiments, these structures may exhibit distinct morphological and / or physiological behaviors compared to traditional 2D cultures, including the formation of nodules and / or invasive growth patterns.

[0057] Certain embodiments and / or IPHBs may be configured to support the co-cultivation of various cell types, which may include ECs, fibroblasts, epithelial cells, and / or mesenchymal stem cells (MSCs). This co-cultivation may lead to the formation of complex, multi-cellular tumor microenvironments that closely mimic the natural tumor environment. The specific integration of primary cancer cells with other supportive cell types, such as ECs, within a modular hydrogel system and the features of embodiments herein are novel in concept. While the individual use of these cell types in cancer research is known, the idea of co-cultivating them in accordance with embodiments herein to mimic the natural tumor microenvironment and promote realistic tumor formation is unique. Traditional approaches often use single cell types or simple combinations in 2D cultures or fixed scaffolds. In certain embodiments, the synergistic effects of co-cultivating multiple cell types within a dynamic, modular hydrogel structure better replicates the complex interactions and environment of in vivo tumors.

[0058] Certain embodiments support both static and perfusion-based culturing conditions. In certain embodiments, perfusion enhances nutrient delivery, waste removal, and cellular organization, resulting in more rapid and coordinated formation of tumor structures. The ability to cultivate cells under both static and perfusion conditions may allow for more accurate simulation of physiological conditions, enhancing the relevance of the tumor model for studying cancer progression and therapeutic responses. When perfusion is used instead of static culture, the secretory output may be further enhanced. Cells cultured under perfusion conditions may also exhibit more rapid and coordinated formation across interconnected IPHBs. Perfusion may support better nutrient and / or waste exchange, accelerate cellular organization, and / or increase secretory output, including within and / or from the IPHB(s). Cells cultured under perfusion conditions may exhibit more rapid and coordinated formation across interconnected IPHBs. The capability to cultivate cells under both static and perfusion conditions within the IPHBs and other features of embodiments herein represent significant advancement. In certain embodiments, modulation of perfusion speed, pressure, and / or volume may help optimize tumor growth and / or physiological integrity. Where perfusion bioreactors and dynamic culturing may be known concepts, the capabilities herein, especially integrated into a modular hydrogel system designed specifically for ex vivo tumor generation, is innovative. The ability to precisely control the dynamic environment within interconnected hydrogel blocks to enhance tumor formation and behavior is not an obvious extension of existing technologies, especially as perfusion is typically separate from scaffold design.

[0059] Cultivation of cancer cells within IPHBs can result in a sustained increase in the secretory output. This may include one or more proteins, extracellular vesicles, growth factors, cytokines, chemokines, antibodies, oncogenic factors, and / or others. These secreted factors may be important for studying tumor biology and / or evaluating the effects of various treatments.

[0060] 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. The modular and scalable nature of IPHBs and embodiments herein makes them suitable for high-throughput applications. For example, researchers may expand and / or customize tumor models to suit various experimental needs, which may enable efficient screening of multiple therapies and / or dosages.

[0061] Certain embodiments may be loaded with drugs and / or growth factors, which may interact with the cells seeded. In certain embodiments, compounds may be distributed in at least one differential and / or gradient, which may promote evaluation of therapeutic effect(s) and optimal dosing strategies.

[0062] Embodiments herein may provide a versatile platform for diagnostic and / or therapeutic evaluation. They may be used for a wide array of diagnostic procedures, which may include histological analysis and / or molecular profiling, as well as for testing various therapeutic interventions, including radiation, immunotherapy, and / or drug discovery. Traditional models often lack the versatility and adaptability required for comprehensive diagnostic and therapeutic evaluations. The integrations of diagnostic and / or therapeutic capabilities in embodiments herein, including within a single, modular hydrogel system, represent novel and non-obvious advancements over existing methods.

[0063] By using primary cancer cells from patient biopsies, certain embodiments may enable the creation of personalized tumor models that reflect the unique characteristics of each patient’s cancer, supporting precision medicine. Using patient-derived cells may enable personalized cancer models, which may facilitate tailored treatment strategies and / or more accurate predictions of therapeutic responses. The modular design can allow for scaling and / or customization, which may be important for studying patient-specific tumor behaviors and / or testing personalized therapies. Personalized medicine approaches typically rely on simpler models that do not fully replicate the tumor microenvironment. Embodiments herein, includinga modular hydrogel system, which may be used to create scalable and personalized tumor models, and / or provide a more accurate representation of individual cancers, represent non- obvious and innovative solutions. In vitro and ex vivo models often fail to predict in vivo efficacy and toxicity accurately. By providing a more physiologically relevant environment, IPHBs and embodiments herein may improve the predictive power of ex vivo tumor models. The inclusion of dynamic culturing conditions and / or micro-vasculature may enhance the relevance of models, e.g. for studying therapeutic efficacy and / or safety, and may help bridge the gap between preclinical studies and clinical outcomes.

[0064] Tumor-like structures formed in certain embodiments, including within IPHBs, may grow outward and / or be joined horizontally and / or vertically or otherwise, e.g. within a biological cartridge. The versatility of embodiments herein may allow for the creation of integrated tumor systems and / or simulating full tumor progression and metastasis 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 realism of the tumor constructs.

[0065] 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 cancer cells, and may reduce plastic consumption by 85%. Efficiency reduces costs and improves the sustainability of cell culture processes. IPHBs and embodiments herein are designed to be cost-effective and relatively easy to produce, reducing barriers to entry for research institutions and / or clinical applications.

[0066] IPHBs and embodiments herein may be simple to use and accessible to researchers with varying levels of expertise, making them suitable for a wide range of research institutions, clinical applications, and / or educational settings. The simplicity and ease of use of certain IPHBs and embodiments herein may make them accessible to a wide range of users, which may include high school or other students, highlighting their educational potential.

[0067] The ability of certain IPHBs and embodiments herein to support micro-vasculature formation is an important advancement. Vascularization may support nutrient supply and waste removal, promoting the growth and maintenance of viable, functional tumor tissues that more closely resemble in vivo tumors. Current models often lack realistic vascularization, which limits their physiological relevance. The integration of micro-vasculature formation within a modular hydrogel system may allow for better nutrient supply and waste removal, representing a novel advancement that is not readily apparent in existing technologies. The porous structure of IPHBs may support the formation of micro-vasculature and complex tumor architectures,providing a more accurate mimic of the in vivo microenvironment. This may enable the study of tumor-stroma interactions and / or the impact of the ECM on cancer progression.

[0068] There are many benefits to embodiments herein in comparison with current tissue engineering techniques readily available. IPHBs and embodiments herein may avoid, reduce, and / or eliminate the ethical concerns associated with animal testing, may use human cells to avoid the interspecies differences that may limit the relevance of animal models, and / or may offer a controlled environment to study specific variables and / or interactions without the complexity of whole-organism systems. IPHBs and embodiments herein may be connected using perfusion systems like Organ-on-a-Chip to simulate full organ systems, and may create and / or contribute to a "System-on-a-Chip" for comprehensive studies.

[0069] Certain embodiments may provide a 3D environment that supports the formation of complex tumor structures and micro-vasculature, closely mimicking in vivo conditions. They may also have the ability to use both static and perfusion conditions that allow for better simulation of physiological conditions. They may be used to cultivate patient-derived cells, enabling personalized cancer modeling and treatment testing. They may be loaded with different drugs formulated in a liquid format, including distributed as a gradient, and / or may be tested in high-throughput formats. For example, a standard 6-well plate may accommodate up to about 24, 42, and / or 96 of certain IPHBs.

[0070] IPHBs and embodiments herein may offer greater modularity and / or customization, allowing for scalable tumor models. They may support a wider range of diagnostic and / or therapeutic applications, including histological analysis and / or molecular profiling. The ability of certain IPHBs and embodiments herein to support micro- vasculature formation adds a level of physiological relevance relative to traditional organ-on-a-chip devices. IPHBs may be connected to form integrated systems, enhancing their functionality in simulating whole organ systems.

[0071] This disclosure may be particularly suited for applications in cancer modeling and disease progression, drug discovery and testing, and regenerative and personalized medicine. Embodiments herein may provide a robust platform for studying tumor biology, cancer progression, and metastasis. Their ability to create complex and physiologically relevant tumor microenvironments may enhance the understanding of cancer behavior and the development of new therapeutic strategies. They may enable high-throughput screening of therapeutic compounds, which may allow for the testing of multiple drugs and their effects on patientspecific tumor models. Their enhanced secretory output and physiological relevance mayimprove the accuracy and predictive power of drug testing. They may support the generation of personalized tumor models, contributing to advancements in precision medicine and development of new cancer therapies. They hold potential for developing new treatments and therapies tailored to individual patients’ cancers, improving treatment outcomes and reducing side effects.

[0072] Embodiments herein may offer novel and superior platforms for generating ex vivo tumor models from patient-derived cancer cells. By addressing key limitations of existing models and providing a more realistic and personalized approach to cancer research, they may advance the fields of cancer modeling, drug discovery, and disease progression research. Their modular, scalable, and dynamic nature, combined with cost-effectiveness and ease of use, make them a transformative tool for researchers and clinicians alike.

[0073] 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% byvolume of the 3D macro structure. In certain embodiments, microporous cavities may comprise at most about 90, 85, 80, 75, and / or 70% by volume of the 3D macrostructure.

[0074] 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.

[0075] In accordance with certain embodiments of the disclosure, the continuous polymeric matrix material may be generally non-degradable. In certain examples, the cells and / or tissue produced in the IPHB may need to be flushed out of the interior network of the network of microporous channels and / or chambers for further analysis, purification, and / or development. Additionally or alternatively, the continuous polymeric matrix material may be selectably degradable (e.g., enzymatically dissolved). For example, hydrogel formulations may be rendered biodegradable, such as by insertion of enzyme-sensitive sequences and / or utilization of native matrix-derived compounds. For example, the continuous polymeric matrix material may comprise a selectably degradable hydrogel material comprising one or more degradable polymers, such as one or more biopolymers derived from a living organism. The one or more biopolymers derived from a living organism, for example, may comprise a polynucleotide, polysaccharide, polypeptide, or any combination thereof. In accordance with certain embodiments, the one or more biopolymers may comprise collagen, gelatin (e.g., porcine, bovine (cow), ovine (sheep), shellfish, fish, etc.), laminin, alginate, glycosaminoglycans, oligonucleotides (e.g., DNA, RNA), carbohydrates, lipids, cellulose, alginate, and / or proteins that can be gently degraded, such as with the use of specific enzymes, ionic solvents, neutral detergents, weak acids, and / or peroxides to disrupt the biopolymer chains. In accordance with certain embodiments, the one or more biopolymers may comprise degradable monomers comprising esters, such as hydroxybutyrate, lactic acid, glycolic acid, and caprolactone; anhydrides, such as adipic acid and / or sebacic acid; saccharides, such as cellulose, alginate, pectin, dextrin, chitosan, hyaluronan, chondroitin sulfate, and / or heparin; proteins; nucleotides, such as DNA and / or RNA; peptides, such as collagen, gelatin, silk, and / or fibrin; urethanes;phosphates; carbonates; and / or vinyl chlorides. Additionally or alternatively, the biopolymer may comprise poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), and / or other suitable hydrogels. In accordance with certain embodiments, the selectably degradable hydrogel material may further comprise a synthetic polymer, such as a polyester, a polyanhydride, a polycarbonate, a polyurethane, a polyphosphate, or any combination(s) thereof. The continuous polymeric matrix material, in accordance with certain embodiments, may comprise a 3D crosslinked polymer network, a non-crosslinked polymer network, or a combination thereof.

[0076] In certain embodiments, one or more biopolymers may comprise anywhere between approximately 10 and 100% of the dry weight of an IPHB and / or its continuous polymeric matrix material. In certain embodiments, the minimum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more biopolymers may be about 10, 15, 20, 25, 30, 35, 40, 45, and / or 50%. In certain embodiments, the maximum dry weight of an IPHB and / or its continuous polymeric matrix material to consist of one or more biopolymers may be aboutlOO, 95, 90, 85, 80, 75, 70, 65, 60, 55, and / or 50%.

[0077] 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 groups 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.

[0078] 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%.

[0079] 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.

[0080] 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.

[0081] A method for cultivating primary tumor cells ex vivo may comprise isolating primary cancer cells from a patient biopsy; cultivating the primary cancer cells within the IPHB that comprises a 3D environment defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers; co-cultivating the primary cancer cells with supportive cell types within the IPHB; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the tumor model. In some embodiments, therapeutic compounds in liquid format may be distributed across the IPHB. In some embodiments, the IPHB may be connected to another IPHB using perfusion systems to simulate full organ systems. In some embodiments, diagnostic evaluations, including histological analysis and molecular profiling, may be performed on the cultivated tumor cells within the IPHB. In some embodiments, cultivated tumor cells in the IPHB may be configured for high-throughput screening of therapeutic compounds. In some embodiments, secretedbyproducts from the media or perfusate surrounding the at least one IPHB may be collected and / or analyzed. In some embodiments, an IPHB may be configured to create personalized cancer models that reflect the unique characteristics of each patient’s cancer for tailored treatment strategies. In some embodiments, the tumor model may be expanded by interconnecting the at least one IPHB to additional IPHBs to accommodate tumor growth and study progression. In some embodiments, media and plastic consumption may be reduced by up to 90% and 85% compared to other cultivation formats.

[0082] In another aspect, the present disclosure provides methods, including of treating a patient having a cancer. Some embodiments may comprise the following: (i) isolating cancer cells (e.g., primary cancer cells) from a specimen (e.g., patient biopsy); (ii) cultivating the cancer cells within at least one IPHB, the at least one IPHB having a 3D macrostructure defined by a continuous polymeric matrix material and a network of microporous cavities (e.g., channels and / or chambers) extending throughout the continuous polymeric matrix material configured to provide a 3D environment; and (iii) co-cultivating the cancer cells with supportive cell types within the at least one IPHB, wherein co-cultivating may include feeding the cancer cells with supportive cell types with one or more culture media, and allowing the cancer cells with supportive cell types to propagate through the network of microporous channels and / or chambers; and wherein the co-cultivating comprises utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of a resulting tumor model. Some embodiments may comprise (iv) harvesting the tumor model from the at least one IPHB. Some embodiments may comprise (v) subjecting one or more portions of the tumor model to one or more respective therapeutic compound(s) or therapeutic treatments, such as in a parallel timeline. Some embodiments may comprise (vi) evaluating the effectiveness of each therapeutic compound and / or therapeutic treatment, and / or selecting one or more viable therapeutic compound and / or therapeutic treatment based on mitigation of growth of the tumor model and / or extent of cancer cell destruction. Some embodiments may comprise (vii) subjecting the patient to a treatment with one or more of the selected viable therapeutic compound(s) and / or therapeutic treatments). In some embodiments, the step of subjecting the patient to a treatment may start with about 10 days of the step of isolating cancer cells from the specimen. In some embodiments, the minimum such time interval may be about 10, 12, 14, 16, 18, 20, 22, 25, 30, 35, 40, 45, and / or 50 days. In some embodiments, the maximum such time interval may be about 180, 150, 120, 100, 90, 80, 70, 60, and / or 50 days.

[0083] In certain embodiments, a system for cultivating tumor cells ex vivo may comprise at least one IPHB configured to under dynamic culture conditions cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. 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.

[0084] In some embodiments, the cavities may include one or more channels and / or chambers. A cavity may comprise a well, recess, pore, pocket, and / or enclosure.

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

[0086] In some embodiments, the dynamic culture conditions may comprise exposing at least one cell type to a culture media, e.g. via perfusion and / or static solution.

[0087] In some embodiments, the culture media may include one or more nutrients for the cells. The dynamic culture conditions may further comprise varying the culture media’s nutrient type and / or concentration.

[0088] In some embodiments, one or more specimens 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 patient (e.g. including differing in the location and / or time biopsied), and / or different patients. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0089] In some embodiments, one or more specimens may be derived from a site of primary tumor, metastasis, metastatic disease, lymphangitic disease, lymphoid tissue, lymph node, lymphadenopathy, adenopathy, or a combination thereof.

[0090] In some embodiments, at least one additional cell type may include ECs.

[0091] Some embodiments may be configured to fluidically distribute nutrients, growth factors, and / or bioactive molecules.

[0092] 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.

[0093] In some embodiments, at least one block may be configured to collect secreted byproducts, e.g. from the tumor cells.

[0094] Some embodiments may be configured to cultivate microvascular formation.

[0095] Some embodiments may be configured to fluidically distribute at least one drug across the cultivated tumor cells, the seeded tumor cells, or a combination thereof.

[0096] Some embodiments may include one or more drugs, which could be distributed across the system, e.g. with geographic, temporal, and / or other variation in concentration via one or more differentials) and / or gradients).

[0097] In some embodiments, the one or more drugs may be selected from the group consisting of: medicine, medication, pharmaceutical, biologic, immunologic, immunotherapy, chemotherapy, radiotherapy, radiotracer, tracer, stain, diagnostic agent, theranostic agent, therapeutic agent, treatment agent, or any combination thereof.

[0098] In some embodiments, the one or more blocks may be configured for loading into one or more standard well-plates.

[0099] In certain embodiments, a method for cultivating tumor cells ex vivo may comprise seeding at least one IPHB with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types and exposing the cells to dynamic culture conditions. 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.

[0100] Some embodiments may comprise exposing 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).

[0101] Some embodiments 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, and / or tumor cells).

[0102] In some embodiments, the 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.

[0103] In certain embodiments, an IPHB for cultivating tumor cells ex vivo may be configured to under dynamic culture conditions cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. One ormore 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.

[0104] Described herein are systems, methods, and IPHBs for cultivating tumor cells ex vivo. 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 be configured to cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types. A method may include seeding at least one block with at least one line of tumor cells isolated from at least one specimen and one or more additional cell types and exposing the cells to dynamic culture conditions.

[0105] Turning to Figure 3, illustrated is an example embodiment of a method 300 for cultivating tumor cells 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 tumor cells isolated from at least one specimen and one or more additional cell types. 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.

[0106] In some embodiments of step 310, one or more specimens 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 patient (e.g. including differing in the location and / or time biopsied), and / or different patients. Additional permutations will be readily apparent to those of ordinary skill in the art.

[0107] Certain embodiments may comprise additional steps. For example, method 300 may comprise step 330 of at least one experimental variable and / or step 340 of evaluation. Step 330 may comprise exposing one or more of the cells to at least one experimental variable, whichcould 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, and / or tumor cells).

[0108] 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 evaluation 340 (not necessarily including experimental variable 330). In some embodiments, experimental variable 330 could be after or subsequent to evaluation 340. Some embodiments may comprise cyclical iterations, in any order, such as multiple instances of seeding 310, dynamic culture conditions 320, experimental variable 330, and / or evaluation 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, experimental variable 330, and / or evaluation 340). Such embodiments may include changing the configuration of existing blocks, removing blocks, and / or adding blocks.

[0109] 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.

[0110] 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.[OHl] As explained above and reiterated below, the present disclosure includes, without limitation, the following example implementations.

[0112] Clause 1. A system for cultivating primary tumor cells ex vivo, comprising: at least one IPHB with the at least one IPHB comprising a 3D macrostructure defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers configured to provide a 3D environment; and primary cancer cells derived from patient biopsies and supportive cell types, such as ECs, co-cultured within the at least one IPHB; wherein the at least on IPHB enables dynamic culturing capabilities configured for static and perfusion conditions to simulate physiological environments.

[0113] Clause 2. The system of clause 1, wherein the at least one IPHB is configured to support the formation of micro- vasculature within the tumor model.

[0114] Clause 3. The system of clause 1 , wherein the at least one IPHB is modular and designed to be interconnected to one or more additional IPHBs to expand the 3D environment, wherein the addition of one or more additional IPHBs increases the scale of the tumor model andprovides a customized microenvironment for specific research needs or patient-specific conditions.

[0115] Clause 4. The system of clause 3, wherein the at least one IPHB allows for the loading of multiple IPHBs into standard well-plates, which enables high-throughput testing of one or more therapeutic compounds and / or treatments.

[0116] Clause 5. The system of clause 1, wherein the at least one IPHB enables the distribution of drugs in liquid format, which may optionally create gradients across the blocks for comprehensive therapeutic evaluation.

[0117] Clause 6. The system of clause 1, wherein the at least one IPHB is connected using perfusion systems to simulate full organ systems, whereby creating a “system-on-a-chip” for comprehensive studies.

[0118] Clause 7. The system of clause 1, wherein the at least one IPHB supports various diagnostic procedures, including histological analysis, molecular profiling, and the collection of secreted byproducts for further analysis.

[0119] Clause 8. The system of clause 1, wherein the at least one IPHB is cost-effective and easy to use.

[0120] Clause 9. The system of clause 1, wherein the at least one IPHB is designed to reduce media consumption by up to 90%, plastic consumption by up to 85%, and human labor.

[0121] Clause 10. The system of clause 1, wherein the at least one IPHB facilitates the formation of realistic tumor structures and invasive behaviors by primary cancer cells.

[0122] Clause 11. The system of clause 1, wherein the at least one IPHB enables the creation of personalized cancer models by using primary cells from patient biopsies.

[0123] Clause 12. A method for cultivating primary tumor cells ex vivo, comprising: isolating primary cancer cells from a patient biopsy; cultivating the primary cancer cells within the at least one IPHB that comprises a 3D environment defined by a continuous polymeric matrix material with a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material configured to provide a 3D environment; co-cultivating the primary cancer cells with supportive cell types within the at least one IPHB; and utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of the tumor model.

[0124] Clause 13. The method of clause 12, wherein therapeutic compounds in liquid format are distributed across the at least one IPHB.

[0125] Clause 14. The method of clause 12, wherein the at least one IPHB is connected to another IPHB using perfusion systems to simulate full organ systems.

[0126] Clause 15. The method of clause 12, wherein diagnostic evaluations, including histological analysis and molecular profiling, are performed on the cultivated tumor cells within the at least one IPHB.

[0127] Clause 16. The method of clause 15, further comprising subjecting cultivated tumor cells from the at least one IPHB to a high-throughput screening of therapeutic compound(s).

[0128] Clause 17. The method of clause 12, wherein secreted byproducts from the media or perfusate surrounding the at least one IPHB is collected and analyzed.

[0129] Clause 18. The method of clause 12, wherein the at least one IPHB is configured to create personalized cancer models that reflect the unique characteristics of each patient’s cancer for tailored treatment strategies.

[0130] Clause 19. The method of clause 12, wherein the tumor model is expanded by interconnecting the at least one IPHB to additional IPHBs to accommodate tumor growth and study progression.

[0131] Clause 20. The method of clause 12, wherein media and plastic consumption are reduced by up to 90% and 85% compared to other cultivation formats.

[0132] Clause 21. A method of treating a patient having a cancer, comprising; isolating primary cancer cells from a patient biopsy; cultivating the primary cancer cells within the at least one IPHB, the at least one IPHB having a 3D macrostructure defined by a continuous polymeric matrix material and a network of microporous channels and / or chambers extending throughout the continuous polymeric matrix material configured to provide a 3D environment; cocultivating the primary cancer cells with supportive cell types within the at least one IPHB, wherein co-cultivating comprises feeding the primary cancer cells with supportive cell types with one or more culture media, and allowing the primary cancer cells with supportive cell types propagate through the network of microporous channels and / or chambers; and wherein the co-cultivating comprises utilizing dynamic culturing conditions, including static and perfusion, to enhance the physiological relevance of a resulting tumor model; harvesting the tumor model from the at least one IPHB; subjecting one or more portions of the tumor model to one or more respective therapeutic compound(s) or therapeutic treatments, such as in a parallel timeline; evaluating the effectiveness of each therapeutic compound and / or therapeutic treatment and selecting one or more viable therapeutic compound and / or therapeutic treatment based on mitigation of growth of the tumor model and / or extent of cancer cell destruction; andsubjecting the patient to a treatment with one or more of the selected viable therapeutic compound(s) and / or therapeutic treatments).

[0133] Clause 22. The method of clause 21, wherein the step of subjecting the patient to a treatment starts with about 10 days of the step of isolating primary cancer cells from the patient biopsy, such as at least about any of the following: 10, 12, 14, 16, 18, 20, 22, 25, 30, 35, 40, 45, and 50 days, and / or optionally at most about any of the following: 180, 150, 120, 100, 90, 80, 70, 60, and 50 days.

[0134] Clause 23. The method of clauses 21-22, wherein the harvested tumor model is physically divided into the one or more portions prior to subjecting them to one or more respective therapeutic compound(s) or therapeutic treatments, such as in a parallel timeline.

[0135] These and other modifications and variations to the embodiments may be practiced by those of ordinary skill in the art without departing from the spirit and scope of the embodiments, which is more particularly set forth in the appended 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 embodiments as further described in such appended claims. Therefore, the spirit and scope of the appended claims should not be limited to the exemplary description of the versions contained herein.

Claims

CLAIMSTHAT WHICH IS CLAIMED:

1. A system for cultivating tumor cells ex vivo, comprising: at least one interconnecting porous hydrogel block, wherein each block comprises a three-dimensional continuous polymeric matrix with a network of microporous cavities, and is configured to interconnect with at least one other block, wherein the system is configured to, under dynamic culture conditions, cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen, and one or more additional cell types.

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

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

4. 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.

5. The system of claim 4, 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.

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

7. The system of claim 6, one or more specimens are derived from a site of primary tumor, metastasis, metastatic disease, lymphangitic disease, lymphoid tissue, lymph node, lymphadenopathy, adenopathy, or a combination thereof.

8. The system of claim 1 , wherein at least one additional cell type comprises endothelial cells.

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 tumor cells.

12. The system of claim 1, further configured to cultivate microvascular formation.

13. The system of claim 1, further configured to fluidically distribute at least one drug across the cultivated tumor cells, the seeded tumor cells, or a combination thereof.

14. The system of claim 13, comprising one or more drugs 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.

15. The system of claim 13, the one or more drugs being selected from the group consisting of: medicine, medication, pharmaceutical, biologic, immunologic, immunotherapy, chemotherapy, radiotherapy, radiotracer, tracer, stain, diagnostic agent, theranostic agent, therapeutic agent, treatment agent, or any combination thereof.

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

17. A method for cultivating tumor cells ex vivo, comprising: seeding at least one interconnecting porous hydrogel block with at least one line of tumor cells isolated from at least one specimen, and one or more additional cell types, and exposing the cells to dynamic culture conditions, further wherein each blockcomprises a three-dimensional continuous polymeric matrix with a network of microporous cavities, and is configured to interconnect with at least one other block.

18. The method of claim 17, further comprising exposing the cells to at least one experimental variable across one or more geographic, temporal, or both geographic and temporal differentials, gradients, or any combination thereof.

19. The method of claim 18, further comprising evaluating, measuring, observing, or a combination thereof, the effect of at least one experimental variable on the senescence, growth, characteristics, or any combination thereof, of the seeded tumor cells, cultivated tumor cells, or both.

20. The method of claiml8, wherein the experimental variable comprises a drug, thermal energy, radiation, or any combination thereof.

21. An interconnecting porous hydrogel block for cultivating tumor cells ex vivo, 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, under dynamic culture conditions, cultivate tumor cells when seeded with at least one line of tumor cells isolated from at least one specimen, and one or more additional cell types.

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