3D bioprinting of tumor tissue for mechanism research and drug screening

Abstract

Three-dimensional (3D) bioprinted cell culture systems of tumor tissue, methods of making and methods of using the same are described. The 3D cell culture system provides a plurality of compartments and vasculature as well as cell interfaces, including a tumor / non-tumor interface, a tumor-vasculature interface, to reconstruct a tumor immune microenvironment. The hardness of the 3D bio-printed stent can be adjusted to reflect different stages of canceration, including fibrosis, cirrhosis and fatty degeneration; and establishing chemical gradients, such as oxygen levels, to simulate oxidation pressure and hypoxic conditions. The manufacture of 3D cell cultures is achieved by bioprinting, such as micro-forming, injection exclusion, and suspension bioprinting techniques.

Description

[0001] This international patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 490,077, filed March 14, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0002] This invention generally relates to a three-dimensional cell culture system for the tumor microenvironment. Background Technology

[0003] Liver cancer (HCC) was the sixth leading cause of cancer diagnosis and the third leading cause of cancer death globally in 2020, with 905,677 new cases and 830,180 deaths (Sung H, et al., CA Cancer J Clin 71:209-49 (2021)). It remains the second leading cause of cancer death in China (Jiang D, et al., Cancer Communications 41:1024-36 (2021)). The main histological subtypes of HCC include hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (ICC), and complex hepatocellular cholangiocarcinoma (CHC). HCC has a very high metastasis rate and mortality rate (overall mortality to morbidity ratio >90%). Traditional risk factors include viral infection, alcohol consumption, high-fat diets, cirrhosis, and toxin exposure (Mittal S, El-Serag HB. Journal of clinical gastroenterology 47 Suppl:S2-S6 (2013)). Liver stiffness, measured by elastography based on non-invasive ultrasound or magnetic resonance imaging (MRI), directly reflects the fibrosis and inflammatory state of an organ and has been shown to be associated with an increased risk of HCC (Mueller S, Sandrin L. Hepatic medicine: evidence and research 2:49-67 (2010)). It represents a major indication for screening and monitoring disease progression. Curative treatment options, including surgery and radiofrequency ablation, can only be used in patients with a limited tumor burden (Hartke J, Johnson M, Ghabril M. Seminars in Diagnostic Pathology 34:153-9 (2017)). Systemic and immunotherapies have shown promising results in some patients; however, low response rates and limited survival benefits have led to their being considered ineffective, and further experimental evidence and clinical trials are needed (Zhong C, et al., Frontiers in Oncology 11 (2021)).

[0004] Currently available platforms for cancer mechanism research and drug screening are summarized in Figure 1 Traditional two-dimensional (2D) and 3D co-culture methods have proven practical, easier to manage, and capable of high-throughput replication. However, applying these methods to translational medicine presents many challenges and uncertainties (Law AMK, et al., Frontiers in Oncology 11(2021)):

[0005] 1. Due to static conditional dependence on unrealistic supplementation and nutrient waste, there is a lack of physical and biological relevance;

[0006] 2. Underestimating the potential value of the spatial architecture of the tumor immune microenvironment (TIME) in influencing disease progression and treatment response;

[0007] 3. Using a single type of cancer and immune cell failed to adequately elucidate the cellular complexity of TIME;

[0008] 4. It is impossible to accurately replicate the mechanisms and immunological characteristics specific to patients with different liver pathologies, especially regarding stiffness; and

[0009] 5. There is insufficient use of patient-derived materials, including cells and extracellular matrix, which are crucial for the development of personalized medicine.

[0010] Some of these drawbacks can be mitigated through in vivo models and clinical studies, which capture the complexity and pathophysiological relevance of diseases. However, significant limitations of human and animal experimental models remain, posing major obstacles to successful translational research. These include, but are not limited to:

[0011] 1. Inconsistency and tumor heterogeneity complicate inferences across different models and patient populations; and

[0012] 2. The large amount of resources required and the extended experimental period may limit the feasibility and scalability of these methods.

[0013] In recent years, 3D in vitro models have attracted attention for their ability to replicate tissue-like structures and cell interactions observed in the tumor immune microenvironment. Despite this progress, current 3D experimental models still fall short in capturing the full complexity and clinical relevance required for effective personalized drug screening and mechanistic studies in cancer research.

[0014] Therefore, the purpose of this invention is to provide three-dimensional, engineered, biological, highly clinically relevant, culturable and perfusionable bioprinted liver tumor tissue constructs and methods for their preparation, in order to reconstruct the tumor architecture and the liver tumor immune microenvironment.

[0015] Another objective is to provide clinically relevant 3D experimental models applicable to mechanism studies, drug screening, immunotherapy testing, personalized medicine, and / or biomarker discovery in liver cancer. Invention Overview

[0017] A three-dimensional (3D) cell culture system for engineered tumor tissue constructs, along with methods for its manufacture and use, is described. A preferred “miniature liver tumor” three-dimensional (3D) cell culture system for engineered liver tumor tissue constructs, along with methods for its manufacture and use, is provided. The 3D cell culture system is prepared by incorporating cells of interest obtained from patients or cell lines into natural (alginate) or synthetic (PEGDA, GelMA, F127, polyacrylamide) hydrogels. The 3D cell scaffold can be heterogeneous or homogeneous, and the cultured cells can include, but are not limited to, tumor cells, immune cells, or non-pathological liver parenchymal cells and non-parenchymal cells. When used to culture two or more types of heterogeneous cells, the 3D cell scaffold can be used, for example, to study cell-cell interactions and cell-ECM interactions between different cell types, for mechanistic studies in heterogeneous cellular environments, and for drug screening.

[0018] The disclosed 3D liver tumor scaffold uses 3D bioprinting technology to recreate “miniature liver tumors.” By precisely reconstructing each distinct tumor compartment containing relevant immune cells, the highly clinically simulated model (including the interaction between innate and adaptive tumor immune components and tumor architecture) allows for the identification of novel HCC-related tumor-promoting mechanisms and personalized drug screening.

[0019] Typically, a 3D cell culture system includes a first compartment containing tumor cells, a second compartment containing non-tumor cells, and a third compartment containing the vascular system. In some forms, the tumor cells are liver cancer cells, and / or the non-tumor cells are hepatocytes and / or endothelial cells. In a preferred form, the first and second compartments further include one or more types of immune cells, such as innate and adaptive immune cells. Exemplary innate and adaptive immune cells include macrophages, neutrophils, natural killer cells, and T lymphocytes. In some forms, the macrophages are tumor-associated macrophages (TAMs) and are CD68+CD163+. In other forms, the first and second compartments include macrophages that are CD68+CD206+. In some forms, the T lymphocytes are CD3+CD4+ T cells and / or CD3+CD8+ T cells.

[0020] Preferably, one or more of the first, second, and third compartments further comprise an extracellular matrix and / or hydrogel. The 3D cell culture system contains a vascular system that allows for chemical gradients. Thus, in some forms, the 3D bioprinted scaffold incorporates normoxic (with a vascular system) and hypoxic (without a vascular system) compartments to reconstruct the tumor architecture and complexity observed in patient tumors. Typically, tumor cells, non-tumor cells, and / or extracellular matrix are derived from liver tumor tissue of the same subject, e.g., a subject with hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma. In some forms, the hydrogel is a natural or synthetic hydrogel, such as alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, or combinations thereof. To better mimic patient-specific liver conditions, cirrhosis can be simulated in the 3D cell culture system, for example, by adjusting the stiffness of the hydrogel. Thus, in a preferred form, the hydrogel has a stiffness similar to that of liver tissue from a subject with hepatocellular carcinoma. Typically, a 3D cell culture system includes at least one inlet and at least one outlet for microfluidic connection, as well as at least one microfluidic channel. In some forms, the 3D cell culture system includes 2 to 20, preferably 3 to 5, microfluidic channels. The inlet and outlet are fluidly connected to one or more microfluidic channels to mimic the vascular system in tumor tissue. The thickness of the 3D cell culture system is typically at least about 10 mm, for example, at least about 10 mm x 10 mm x 0.5 mm in size.

[0021] Methods for fabricating three-dimensional (3D) cell culture systems are also provided. Typically, the method includes steps a) isolating tumor cells, non-tumor cells, and extracellular matrix (ECM) from a tumor sample; b) mixing one or more of the tumor cells, non-tumor cells, and ECM with a hydrogel to provide one or more mixtures; and c) 3D bioprinting the one or more mixtures from step b) to assemble a 3D cell culture system. Tumor samples are typically collected from a subject prior to step a). In some forms, the tumor sample is a liver biopsy from a subject with liver cancer such as HCC. The method typically prints five or more layers to form the 3D cell culture system. In some forms, the method uses a hydrogel formed by photochemical crosslinking of a photosensitive polymer and one or more photoinitiators. In some forms, the photosensitive polymer includes alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, methacrylamide polycaprolactone triol (PCLMA), methacrylamide hyaluronic acid (HAMA), and combinations thereof. In some forms, the photoinitiator includes lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, lithium acylphosphinic acid, Irgacure 2959, and camphorquinone. Photochemical crosslinking allows control over the stiffness of the hydrogel, and in a preferred form, the hydrogel has a stiffness similar to that of liver tissue from a subject with hepatocellular carcinoma. Typically, methods for manufacturing 3D cell culture systems introduce at least one inlet and at least one outlet for microfluidic connections, which are fluidly connected to microfluidic channels. In a preferred form, better mimicking the vascular system of liver tumor tissue, the 3D cell culture system includes 2 to 20, preferably 3 to 5, microfluidic channels. The manufactured 3D cell culture system has a thickness of at least about 10 mm, for example, at least about 10 mm x 10 mm x 0.5 mm in size. In some forms, bioprinting uses suspension, extrusion, microforming, digital light processing, stereolithography, and combinations thereof. The manufactured 3D cell culture system includes at least one compartment containing tumor cells, at least one compartment containing non-tumor cells, and at least one compartment containing a vascular system.

[0022] Methods using 3D cell culture systems are also described. Applications include mechanistic studies, drug screening, immunotherapy testing, personalized medicine, and / or biomarker discovery. In preferred forms, cell culture systems are well-suited for screening the efficacy of cancer therapies. Cancer therapies include conventional chemotherapeutic agents such as alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents; and / or immunotherapeutic agents, such as one or more immune checkpoint modulators, including PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists. In some forms, the methods include identifying cancer therapies with optimal efficacy in reducing or inhibiting the proliferation, migration, invasion, motility, and / or metastasis of tumor cells in the 3D cell culture system, optionally further including the step of administering the optimally effective cancer therapy to a subject in an amount that effectively reduces or inhibits tumor growth, tumor burden, and / or increases subject survival. In other forms, 3D cell culture systems are suitable for mechanistic studies of tumor biology, for example, providing mechanistic information on liver cancer metastasis and novel therapeutic targets. Attached Figure Description

[0023] Figure 1 It is a diagram showing the platforms currently available for mechanism research and drug screening, including 2D single culture, 3D co-culture on inserts, organ / tumor on chips, organoid / tumor-like organisms, animal models, and human patients.

[0024] Figure 2 It is a diagram that outlines the different components and characteristics of a three-dimensional scaffold.

[0025] Figure 3 This is a table comparing the features of different platforms, including 2D single culture, 3D co-culture on inserts, organ / tumor on a chip, organoid / tumoroid, animal models, human patients, and tumors in cubes (exemplary three-dimensional scaffolds described herein).

[0026] Figures 4A-4C It is a diagram that outlines and compares the characteristics of liver tumors and published clinical simulation models.

[0027] Figure 5A It is a diagram showing vascular systems and multicellular interfaces (e.g., tumor and non-tumor interfaces, and tumor and vascular system interfaces) manufactured through 3D microforming / bioprinting. Figure 5B Exemplary 3D scaffolds (top figure) printed using suspension bath and sacrificial extrusion are shown, as well as exemplary 4D scaffolds as described herein, such as a 4D stimulus-activated programmable platform (bottom figure).

[0028] Figure 6A and 6BThe diagram shows four exemplary constructs, including construct 1, bioprinted via microforming; construct 2, bioprinted via suspension, sacrificial extrusion technology; construct 3, bioprinted via stereolithography; and construct 4, bioprinted via 4D assembly of a multifunctional module.

[0029] Figures 7A-7B It is the preparation of the "tumor in a cube" of construct 1. Figure 7A ) and the "tumor in the cube" of Construct 2 ( Figure 7B An example program.

[0030] Figures 8A-8F This is a diagram outlining the characteristics of a 3D scaffold "tumor in a cube," including the operation of the "tumor in a cube" ( Figure 8A ); Hypoxia zone generated by microfluidics ( Figure 8B Long-term cell viability Figure 8C Patient-derived ECM promotes cell proliferation. Figure 8D Cell migration ability () Figure 8E ) and intercellular interactions ( Figure 8F A schematic diagram of ).

[0031] Figure 9A and 9B This demonstrates an exemplary method for simultaneously extracting and maintaining patient-derived ECM and cells. Figure 9A This is a diagram outlining the steps of an exemplary method for simultaneously extracting and maintaining patient-derived ECM and cells. Figure 9B These are exemplary photographs and micrographs of different cell populations from non-tumor (top) and tumor (bottom) samples.

[0032] Figures 10A-10C This demonstrates 2D constructs and exemplary 3D scaffolds (as described herein) for use with two exemplary drugs commonly used to treat hepatocellular carcinoma (HCC): sorafenib (…). Figure 10A and 10B ) and cisplatin Figure 10C Comparison between the reactions.

[0033] Figure 11 This is a diagram showing an exemplary 3D scaffold (as described herein) as an ex vivo model for screening CAR-T cell in situ programming. As a preclinical model for testing the efficacy and toxicity of CAR-T cell in situ programming, the exemplary 3D scaffold (as described herein) was combined with patient-derived tumor and immune cells for reprogramming as a viral platform (pLV-scFV-GPC3-CD28-41BB-CD3ζ-GFP). Invention Details

[0035] I. Definition

[0036] The term "bioprinting" refers to layer-by-layer bioprinting technology. Various biomaterials and cell types can be simultaneously printed onto a cell-compatible biomaterial substrate to construct 3D composite structures with good spatial resolution and reproducibility. Bioprinting has different modes, such as fused deposition modeling (FDM), stereolithography (SLA) bioprinting, inkjet bioprinting, and laser-assisted bioprinting.

[0037] The term "effective dose" or "therapeutic effective dose" means a dose sufficient to treat, suppress, or alleviate one or more symptoms of a treated disease state, or otherwise provide the desired pharmacological and / or physiological effect. In some forms, an effective dose refers to an amount that, for example, compared to a matched subject not receiving the compound, is capable of treating one or more symptoms of hepatocellular carcinoma (HCC), reversing the progression of one or more symptoms of HCC, stopping the progression of one or more symptoms of HCC, or preventing the occurrence of one or more symptoms of HCC in a subject administering the formulation. The precise dose will vary depending on a variety of factors, such as subject dependent variables (e.g., lesion size / type, age, joint health, immune system health, etc.), disease or condition, and the treatment administered. An effective dose can be relative to a control. Such a control is known in the art and discussed herein, and can be, for example, the condition of the subject before or without administration of the drug.

[0038] The terms “treatment” or “prevention” mean improving, reducing, or otherwise preventing the occurrence or progression of a disease, symptom, or condition in animals that may be susceptible to the disease, symptom, and / or condition but have not yet been diagnosed with it; inhibiting the disease, symptom, or condition, for example, preventing its progression; and alleviating the disease, symptom, or condition, for example, causing its remission. Treating a disease or condition includes improving at least one symptom of the disease or condition, even if the underlying pathophysiology is not affected, such as treating a subject’s pain by administering an analgesic, even if the agent does not treat the cause of the pain. The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and alleviating or improving prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with HCC are reduced or eliminated, including but not limited to, reducing and / or inhibiting the rate of cancer cell proliferation / growth, improving the quality of life of individuals with the disease, reducing the dosage of other medications required to treat the disease, delaying disease progression, and / or prolonging the individual’s survival.

[0039] The terms "pharmaceutically acceptable" or "biocompatible" refer to compositions, polymers, and other materials and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" refers to pharmaceutically acceptable materials, compositions, or media, such as liquid or solid fillers, diluents, solvents, or encapsulating materials involved in carrying or transporting any subject composition from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable" in the sense of compatibility with the other components of the subject composition and harmlessness to the patient.

[0040] The terms “inhibition” or “reduction” in the context of inhibition mean a reduction or decrease in activity and amount. This can be a complete inhibition or reduction of activity or amount, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or standard level. Inhibition can be measured as a percentage value, for example, from 1% up to 100%, such as 5%, 10%, 25%, 50%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%. For example, a composition containing a neutralizing antibody against S100A10 can inhibit or reduce the activity and / or amount of the same S100A10 in subjects who have never received or been treated with the composition by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99%. In some forms, inhibition and reduction are compared based on the levels of mRNA, protein, cells, tissues, and organs.

[0041] The terms “contact” or “culture with” are intended to include incubating components and cells / tissues together in vitro (e.g., adding a compound to cells in a culture), and the “contact” or “culture with” step can be performed in any suitable manner. For example, cells can be processed in adherent, suspension, or 3D cultures; components can be added substantially simultaneously or sequentially in time (e.g., one hour, one day, or longer after the addition of the first component) (e.g., added together to a mixture). Cells may also be contacted with another agent, such as a growth factor or other differentiation agent, or with the environment to stabilize or further differentiate the cells, and include culturing cells under conditions known in the art.

[0042] II. 3D Bioprinted Scaffold

[0043] This paper describes the fabrication of a three-dimensional (3D) bioprinted scaffold to mimic the tumor microenvironment, which includes various immune cells, stromal cells, blood vessels, and the extracellular matrix. The described 3D bioprinted scaffold can replicate the tumor immune microenvironment and architecture, providing a model highly similar to clinical conditions for drug discovery and liver pathology research.

[0044] While not intended to be restrictive, exemplary features of the disclosed 3D bioprinting simulation models are shown in Figures 4A-4C The 3D bioprinted scaffolds are designed with clinical relevance and are well-suited for tumor biomimicry. In one exemplary form, this is achieved by incorporating patient-specific biomaterials (such as patient-derived extracellular matrix (ECM)) and patient-specific cells (such as tumor and immune cells from the patient). Furthermore, the disclosed 3D bioprinted scaffolds can mimic patient-specific liver conditions, such as cirrhosis, by adjusting the stiffness of the hydrogel and by simulating inflammation through the deposition of immune cells. Moreover, the disclosed 3D bioprinted scaffolds are capable of mimicking tumor architecture by creating multiple compartments and interfaces (including vascular systems, tumor and non-tumor compartments), thereby closely replicating the complexity of the human body.

[0045] In an exemplary form, the disclosed 3D bioprinted scaffold is a 3D liver tumor scaffold (also known as a "tumor in a cube" or "miniature liver tumor"). The 3D liver tumor scaffold utilizes 3D bioprinting technology to create "miniature liver tumors" that precisely reconstruct different tumor compartments, each embedded with associated immune cells. This 3D liver tumor scaffold closely mimics clinical scenarios, including the interaction of innate and adaptive tumor immune components and tumor architecture, and is suitable for identifying novel hepatocellular carcinoma (HCC)-related tumor-promoting mechanisms and personalized drug screening. For details on the benefits of the 3D bioprinted scaffold relative to current platforms, see [link to relevant documentation]. Figure 2 A direct comparison between the disclosed 3D bioprinted scaffolds and other experimental tools is summarized in [the following text is missing from the original] Figure 3 Unlike traditional 2D and 3D co-culture methods, the disclosed 3D bioprinted scaffold provides a matrix-rich 3D environment that supports multi-cell interactions across diverse interfaces, such as tumor / non-tumor interfaces and tumor-vascular system interfaces. Furthermore, the nutrient gradients generated within the system to support cell growth are highly biomimetic. Compared to lengthy, expensive, and often inefficient in vivo models, the disclosed 3D bioprinted scaffold offers a faster, more direct, consistent, and efficient platform for studying cell-cell interactions, dissecting disease mechanisms, and identifying novel biomarkers, and provides a reliable platform for drug development and personalized medicine. Figure 3 ).

[0046] In some forms, the disclosed 3D bioprinted scaffold is capable of regenerating specific cell differentiation and reorganization of multiple cell clusters, as observed in tumor tissue collected from patients. In one exemplary form, the disclosed 3D bioprinted scaffold regenerates tumor heterogeneity, a characteristic of tumor tissue obtained from patients.

[0047] In some forms, the disclosed 3D bioprinted scaffolds regenerate the complexity of tumors with different functional units, such as different cell types, extracellular matrix (ECM), vascular system, and various chemical factors. In exemplary forms, the disclosed 3D bioprinted scaffolds mimic physiologically relevant tumor-promoting mechanical forces, such as shear stress from dynamic flow in the vascular system, tension from solid tumors, and changes in ECM stiffness.

[0048] 3D bioprinted scaffolds typically contain multiple compartments. In one form, a 3D bioprinted scaffold contains three compartments: a first compartment containing tumor cells, a second compartment containing non-tumor cells, and a third compartment containing the vascular system. In some forms, the first compartment contains tumor cells. In some forms, the second compartment contains tumor cells. In some forms, the second compartment contains non-tumor cells. Typically, the compartment containing tumor cells does not contain non-tumor cells. An exemplary 3D bioprinted scaffold is shown in... Figure 2 (See “Product Design”). Figure 2 The illustrated embodiment has a first compartment containing non-tumor cells (e.g., hepatocytes); a second compartment containing tumor cells; and a third compartment containing the vascular system (e.g., endothelial cells). In some forms, the 3D bioprinted scaffold includes additional compartments, such as a fourth compartment, containing tumor cells from the same patient but at different stages of disease progression or from different time points during treatment.

[0049] In some forms, both tumor cells and non-tumor cells are derived from the same patient's liver tumor tissue. In other forms, tumor cells, non-tumor cells, and extracellular matrix are derived from the same subject's liver tumor tissue.

[0050] In some forms, the tumor cells are liver cancer cells. In some forms, the non-tumor cells are hepatocytes and / or endothelial cells. In some forms, the first and second compartments further contain one or more types of immune cells. In some forms, the immune cells are innate and adaptive immune cells selected from the group consisting of macrophages, neutrophils, natural killer cells, and T lymphocytes.

[0051] A. Extracellular matrix and biomaterials

[0052] ECM (Extracellular cellular matrix) is considered a key regulator of tissue homeostasis and organ function. It constitutes a complex network of proteins that collectively build the overall architecture of an organ. ECM involves the dynamic assembly of various ECM molecules, activating numerous intracellular signaling pathways crucial for regulating cellular behavior. ECM provides structural support and imparts mechanoelastic properties to tissues, thereby influencing their physical behavior under various physiological conditions and delivering environmental cues that affect cellular activity, such as the proliferation, survival, shape, migration, and differentiation of cells encapsulated in ECM. Furthermore, ECM maintains cellular homeostasis, thereby influencing cell adhesion, cell proliferation, cell differentiation, cell migration, and the establishment of cell polarity. ECM is important for preserving tissue mechanical integrity, promoting cell signaling pathways, guiding morphogenesis, enhancing intercellular communication, and regulating environmental interactions. ECM also participates in developmental processes and regeneration, where its components and interactions with cells guide tissue formation and healing. ECM also acts as a critical barrier and filter, regulating the passage of substances between different tissue compartments.

[0053] Alterations in the composition of the ECM and disruption of its regulatory functions are associated with a wide range of diseases. Dysregulation of ECM dynamics can lead to pathological conditions characterized by excessive ECM deposition (e.g., fibrosis) or insufficient ECM (which can impair tissue integrity). These changes contribute to the development and progression of various diseases, including cardiovascular diseases, which may be caused by the destruction of ECM in blood vessels; dermatological diseases, where ECM alterations affect skin elasticity and repair mechanisms; fibrosis, characterized by excessive ECM accumulation that affects organ function; and cancer, where ECM remodeling promotes tumor progression and metastasis. The cellular and molecular architecture of the ECM and its role in liver pathology are further described in the following review articles: Wells, Clin Liver Dis., 12(4):759-68 (2008); Arriazu et al., Antioxid Redox Signal., 21(7):1078-1097 (2014); Ortizet et al., Chronic Liver Disease, 2:41-52 (2021), all of which are incorporated herein by reference in their entirety.

[0054] Currently, existing ex vivo cell scaffolds do not include patient-derived ECM proteins, which are essential for simulating and studying different liver pathologies. The lack of these customized ECM proteins in the scaffolds means that current models may not be able to fully replicate the unique microenvironmental conditions of liver disease, as these occur in individual patients.

[0055] Therefore, the first and second compartments of the disclosed 3D bioprinted scaffold contain extracellular matrix (ECM). Typically, the ECM incorporated into the 3D bioprinted scaffold is derived from the patient. In some forms, the compartment containing tumor cells also contains ECM derived from tumor tissue (also referred to as "tumor ECM"). In some forms, the first compartment contains both tumor cells and tumor ECM. In some forms, the second compartment contains both tumor cells and tumor ECM. In some forms, the compartment containing non-tumor cells also contains ECM derived from non-tumor tissue (also referred to as "non-tumor ECM"). In some forms, the first compartment contains both non-tumor cells and non-tumor ECM. In some forms, the second compartment contains both non-tumor cells and non-tumor ECM. Typically, the compartment containing tumor cells and tumor-ECM does not contain non-tumor cells and non-tumor ECM. An exemplary 3D bioprinted scaffold is shown in... Figure 2 (See “Product Design”). Figure 2 The embodiment shown has a first compartment containing non-tumor cells (e.g., hepatocytes) and non-tumor ECM; a second compartment containing tumor cells, tumor-associated immune cells and tumor ECM; and a third compartment containing the vascular system (e.g., endothelial cells).

[0056] Although the hepatic extracellular matrix (ECM) comprises a relatively small portion of the total liver area, it provides crucial support and structure for the organ, facilitates the physical scaffold of hepatocytes, and, as mentioned above, aids in cell signaling and liver homeostasis. The concept of the “hepatic matrix” encompasses all complement-related ECM proteins and factors present in the liver; advances in omics technologies have propelled the development of this concept, allowing for a more comprehensive understanding of the composition of the ECM and its changes during disease (see Arteel GE, Naba A. JHEP Rep 2:100115 (2020)). Major components of the hepatic ECM include, but are not limited to, fibronectin, type IV collagen, and various molecules such as type I and type III collagen, laminin, and proteoglycans. Type I and type III collagen become particularly abundant during fibrosis. Most resident liver cells, including hepatocytes, bile duct cells, sinusoidal endothelial cells (non-epithelial cells), and Kupffer cells, contribute to the ECM. They are involved in both the secretion and remodeling processes of ECM components. In particular, hepatic stellate cells play a crucial role in ECM deposition during fibrosis. Liver fibrosis and cirrhosis are classic examples of ECM dysregulation, characterized by the excessive accumulation of ECM components, particularly collagen. This process is a response to chronic liver injury and inflammation. Both qualitative (changes in the types of ECM components) and quantitative (increased ECM production) alterations in the liver ECM contribute to the pathogenesis of liver fibrosis and cirrhosis.

[0057] Therefore, the disclosed 3D bioprinted scaffolds incorporate patient-derived ECM proteins to accurately replicate and study various liver pathologies. In some forms, the 3D bioprinted scaffolds contain collagen, non-collagen, or both.

[0058] In some forms, the disclosed 3D bioprinted scaffolds contain collagen. Collagen is the main fibrillary protein present in the ECM, accounting for about 30% of the total protein content in the body; therefore, it constitutes the main structural protein in mammalian tissues (reviewed in Frantz C, et al., J Cell Sci 123:4195–4200 (2010)).

[0059] In some forms, 3D bioprinted scaffolds contain one or more fibrillation-forming or fibrillary collagens, fibrillary-associated collagens with interrupted triple helices (FACIT), network-forming collagens, transmembrane collagens, and / or multiplexins. Facillation-forming or fibrillary collagens include type I, II, III, XI collagens, and the recently discovered types XXIV and XXVII. They are the most abundant and widely distributed collagens in the body. Their function is primarily mechanical, as they provide tensile strength to both tissues and organs (reviewed in Ricard-Blum S. and Ruggiero, Pathol Biol (Paris) 53:430–442, (2005)). Facillary-associated collagens with interrupted triple helices (FACIT) constitute the largest collagen subclass, including types IX, XII, XIV, XVI, XIX, XX, XXI, and XXII. FACITs themselves do not form fibrils, but they bind to the surface of pre-existing collagen fibrils, facilitating fibrillary expansion. Anchoring fibrils are primarily composed of type VII collagen, extending from the basement membrane of epithelial cells. They attach to the reticular lamina by wrapping around bundles of type III collagen in the reticular fibrous network and forming the basement membrane (reviewed in Ricard-Blum S. Cold Spring Harb Perspect Biol 3:a004978, 2011). Network-forming collagen contains multiple breaks in its triple helix, providing flexibility and enabling it to form linear, axial, and transverse associations within the protein network. Type IV collagen is the most important structural component of the basement membrane (Gelse, et al., Adv Drug Deliv Rev 55:1531–1546, 2003; Knupp and Squire, Adv Protein Chem, 70:375–403, (2005)). Type VI collagen is a heterotrimeric molecule that aggregates into a filamentous network and binds to various stromal cell proteins. Type VIII and X collagens are highly homologous heterotrimeric short-chain molecules that form a hexagonal network; however, they exhibit distinct localizations (Gelse, et al., Adv Drug Deliv Rev 55:1531–1546, 2003). Multiplexins include type XV and XVIII collagens. They are basement membrane collagens, endostatin precursors, endostatin XVIII, and endostatin XV, and are secreted via proteolytic processes (reviewed in Ricard-Blum S. and Ruggiero, Pathol Biol (Paris) 53:430–442, (2005)).

[0060] In some forms, 3D bioprinted scaffolds contain proteins that make up the basement membrane (referred to as "basement membrane proteins"). The basement membrane is a highly specialized type of ECM that acts as a reservoir of growth factors that guide cell function, provide cell adhesion, and control cell organization and differentiation (Karsdal MA, et al. Assay Drug Dev Technol. 2013 Mar;11(2):70–92). In some forms, 3D bioprinted scaffolds contain laminin, nidogen / entactin, heparan sulfate proteoglycans, and nonfibrillary collagens such as type IV collagen.

[0061] In some forms, 3D bioprinted scaffolds contain proteins that make up the interstitial matrix (IM) (called "IM proteins"). The IM is produced by fibroblasts and surrounds cells, constituting the majority of the extracellular matrix (ECM) in the body. In some forms, 3D bioprinted scaffolds contain one or more types I, III, and / or V collagens. In some forms, 3D bioprinted scaffolds contain one or more of elastin, fibronectin, and tendinin (Karsdal MA, et al. Adv. DrugDeliv Rev. 2017;121:43–56).

[0062] In some forms, the disclosed 3D bioprinted scaffolds incorporate a combination of extracellular matrix (ECM) and bioactive hydrogels. The integration of ECM with certain forms of bioactive hydrogels allows for more precise replication of the liver tumor microenvironment, thereby facilitating new discoveries about mechanisms of liver cancer progression and regression. In some forms, ECM is encapsulated within a hydrogel as a tissue engineering scaffold for 3D cell culture. The hydrogel can be polymerized using light, UV radiation, redox agents (e.g., a combination of sodium thiosulfate and sodium persulfate), pH variations, or by using other suitable polymerization initiators (such as divalent cations like calcium).

[0063] Polymerizable agents may comprise monomers, macromonomers, oligomers, polymers, or mixtures thereof. Polymer compositions may consist solely of covalently cross-linked polymers, or mixtures of covalently and ionicly cross-linked or hydrophilic polymers.

[0064] Suitable hydrophilic polymers include synthetic polymers such as poly(ethylene glycol), poly(ethylene oxide), partially or fully hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyl oxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamer and meroxaprol), poloxamine, carboxymethyl cellulose and hydroxyalkylated cellulose such as hydroxyethyl cellulose and methyl hydroxypropyl cellulose, and natural polymers such as polypeptides, polysaccharides or sugars such as FICOLL™, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin or copolymers thereof. "Cellulose" includes cellulose and its derivatives of the types described above; "dextran" includes dextran and its similar derivatives.

[0065] Examples of materials that can be used to form hydrogels include modified alginates. Alginates are carbohydrate polymers isolated from seaweed that can be crosslinked to form hydrogels upon exposure to divalent cations such as calcium. Alginates undergo ionic crosslinking in water at room temperature in the presence of divalent cations to form a hydrogel matrix. Modified alginate derivatives with improved hydrogel-forming abilities can be synthesized. Using alginates as starting materials is advantageous because they are available from more than one source and can be obtained with good purity and characterization. The term "modified alginate" refers to chemically modified alginates that possess modified hydrogel properties. Naturally occurring alginates can be chemically modified to produce alginate polymer derivatives that degrade more rapidly.

[0066] Polysaccharides are also useful, being very viscous liquids or thixotropic, and forming gels over time through slow structural evolution. For example, hyaluronic acid can be used to form injectable gels with a hairspray-like consistency. Modified hyaluronic acid derivatives are particularly useful. The term "hyaluronic acid" refers to both natural and chemically modified hyaluronic acid. Modified hyaluronic acid can be designed and synthesized using pre-selected chemical modifications to modulate the rate and extent of cross-linking and biodegradation. For example, modified hyaluronic acid can be designed and synthesized by esterifying it with relatively hydrophobic groups such as propionic acid or benzyl esters to make the polymer more hydrophobic and form a gel, or by grafting it with amines to promote electrostatic self-assembly. Therefore, injectable modified hyaluronic acid can be synthesized because it flows under pressure but retains a gel-like structure when not under pressure.

[0067] Other available materials include proteins such as fibrin, collagen, and gelatin. Other polymeric hydrogel precursors include polyethylene oxide-polypropylene glycol block copolymers, such as PLURONICS™ or TETRONICS™, which are crosslinked by hydrogen bonding and / or by temperature changes, as described in Steinleitner et al., Obstetrics & Gynecology, 77:48-52 (1991); and Steinleitner et al., Fertility and Sterility, 57:305-308 (1992). Polymer mixtures can also be used. For example, a mixture of polyethylene oxide and polyacrylic acid can be used, which forms a gel by hydrogen bonding upon mixing. In one embodiment, a mixture of 5% w / w polyacrylic acid solution and 5% w / w polyethylene oxide (polyethylene glycol, polyoxyethylene) 100,000 can be combined to form a gel over time (e.g., as quickly as within a few seconds).

[0068] Water-soluble polymers with charged side groups can be crosslinked by reacting the polymer with an aqueous solution containing ions of opposite charge. If the polymer has acidic side groups, it is a cation; if it has basic side groups, it is an anion. Examples of cations used to crosslink polymers with acidic side groups to form hydrogels are monovalent cations such as sodium, divalent cations such as calcium, and polyvalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, as well as di-, tri-, or tetrafunctional organic cations such as alkylammonium salts. Aqueous solutions of these cation salts are added to the polymer to form soft, highly swollen hydrogels and films. The higher the cation concentration, or the higher the valence, the greater the degree of crosslinking of the polymer. Additionally, polymers can be enzymatically crosslinked, for example, fibrin with thrombin.

[0069] Suitable ionic crosslinking groups include phenolic, amine, imine, amide, carboxylic acid, sulfonic acid, and phosphate groups. Aliphatic hydroxyl groups are not considered reactive groups in the chemistry disclosed herein. Negatively charged groups, such as carboxylates, sulfonates, and phosphate ions, can be crosslinked with cations such as calcium ions. The crosslinking of alginate with calcium ions is an example of this type of ionic crosslinking. Positively charged groups, such as ammonium ions, can be crosslinked with negatively charged ions (such as carboxylates, sulfonates, and phosphate ions). Preferably, the negatively charged ion contains more than one carboxylate, sulfonate, or phosphate group.

[0070] Preferred anions for crosslinking polymers to form hydrogels are monovalent, divalent, or trivalent anions, such as low molecular weight dicarboxylic acids, for example, terephthalic acid, sulfate ions, and carbonate ions. Aqueous solutions of salts of these anions are added to the polymer to form soft, highly swollen hydrogels and films, as described with respect to cations.

[0071] A variety of polymeric cationic compounds can be used to stabilize composite polymer hydrogels as semi-permeable surface films. Examples of materials that can be used include polymers with basic reactive groups such as amine or imine groups, having a preferred molecular weight of 3,000 to 100,000, such as polyethyleneimine and polylysine. These are commercially available. One polymeric cationic compound is poly(L-lysine); examples of synthetic polyamines are polyethyleneimine, poly(ethyleneamine), and poly(allylamine). There are also natural polymeric cationic compounds such as polysaccharides and chitosan.

[0072] Polymer anionic polymers and copolymers containing acrylic acid, methacrylic acid, and other derivatives of acrylic acid, which can react with basic surface groups on polymer hydrogels to form semipermeable membranes, and those with side chains SO 3 Polymers with H groups (such as sulfonated polystyrene) and polystyrene having carboxylic acid groups. These polymers can be modified to contain polymerizable groups and / or ionic crosslinking groups. Methods for modifying hydrophilic polymers to include these groups are well known to those skilled in the art.

[0073] Polymers can be inherently biodegradable but preferably have low biodegradability (for predictability of dissolution) but a sufficiently low molecular weight to allow excretion. The maximum molecular weight permissible for excretion in humans (or other species intended for use therein) will vary depending on the polymer type but is typically around 20,000 Daltons or lower. Available, but less suitable for general use due to their inherent biodegradability, are water-soluble natural polymers and synthetic equivalents or derivatives, including peptides, polynucleotides, and degradable polysaccharides.

[0074] The polymer can be a monoblock having a molecular weight of at least 600, preferably 2000 or greater, preferably at least 3000. Alternatively, the polymer can comprise two or more water-soluble blocks linked by other groups. Such linking groups can comprise biodegradable bonds, polymerizable bonds, or both. For example, unsaturated dicarboxylic acids (such as maleic acid, fumaric acid, or aconitic acid) can be esterified with a hydrophilic polymer containing hydroxyl groups (such as polyethylene glycol) or amidated with a hydrophilic polymer containing amine groups (such as poloxamine).

[0075] Covalently crosslinkable hydrogel precursors are also useful. For example, water-soluble polyamines (such as chitosan) can be crosslinked with water-soluble diisothiocyanates (such as polyethylene glycol diisothiocyanate). The isothiocyanate will react with the amine to form a chemically crosslinked gel. The reaction of aldehydes with amines, such as with polyethylene glycol dialdehyde, can also be utilized. Hydroxylated water-soluble polymers can also be used.

[0076] Alternatively, polymers containing substituents can be used, which crosslink via a free radical reaction upon contact with a free radical initiator. For example, polymers containing photochemically crosslinkable olefinically unsaturated groups can be utilized. In this embodiment, a water-soluble macromonomer comprising at least one water-soluble region, a biodegradable region, and at least two free radical polymerizable regions is provided. The macromonomer is polymerized by exposing the polymerizable regions to free radicals generated, for example, by photosensitive chemicals and / or light. Examples of such macromonomers are PEG-oligolactic acid-acrylates, wherein the acrylate groups are polymerized using a free radical initiation system (such as eosin dye) or by brief exposure to ultraviolet or visible light. Additionally, water-soluble polymers comprising photochemically crosslinkable cinnamoyl groups, as disclosed in Matsuda et al., ASAIDTrans., 38:154-157 (1992), can be used.

[0077] Typically, the polymer is at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions. Methods for synthesizing the other polymers described above are known to those skilled in the art. See, for example, *Concise Encyclopedia of Polymer Science and Polymeric Amines and Ammonium Salts*, E. Goethals, editor (Pergamen Press, Elmsford, NY 1980). Many polymers, such as poly(acrylic acid), are commercially available. Naturally occurring and synthetic polymers can be modified using chemical reactions available in the art and described, for example, in *March, “Advanced Organic Chemistry,” 4*. th Edition, 1992, Wiley-Interscience Publication, New York.

[0078] For example, a hydrogel solution is prepared by mixing 10% by weight / volume (w / v) of a polymerizable polymer into sterile phosphate-buffered saline (PBS), where sterile PBS is a suitable solvent, and adjusting the pH to approximately 7.4. In some embodiments, the polymer is photopolymerizable poly(ethylene glycol) diacrylate (PEGDA) or photopolymerizable poly(ethylene oxide) diacrylate (PEODA).

[0079] Optionally, the hydrogel solution may include various additives, such as 100 U / ml penicillin and 100 μg / ml streptomycin, to inhibit microbial contamination. However, these are not the only bioactive additives that can be included in the hydrogel solution. For example, bioactive additives may include, alone or in combination, growth factors, cell differentiation factors, other cell mediators, nutrients, antibiotics, anti-inflammatory agents, and other drugs. While not limiting, depending on the cell type to be encapsulated in the same or adjacent hydrogel layers, some suitable cell growth factors include heparin-binding growth factor (HBGF), transforming growth factor (TGFα or TGFβ), α-fibroblast growth factor (FGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), various angiogenesis factors, nerve growth factor (NGF), and muscle morphology growth factor.

[0080] Additionally, the hydrogel solution optionally includes a suitable non-toxic polymerization initiator, thoroughly mixed to achieve a final concentration of 0.05% w / v. When PEGDA or PEODA is selected as the polymer, a polymerization initiator is preferably added, and the photoinitiator Igracure 2959 (available from Ciba Specialty Chemicals Corp., Tarrytown, NY) is preferred, although other suitable photoinitiators may also be used.

[0081] Exemplary photopolymerizable polymers are PEGDA and PEODA. Suitable hydrophilic polymers include synthetic polymers such as partially or fully hydrolyzed poly(vinyl alcohol), poly(vinylpyrrolidone), poly(ethyl oxazoline), poly(ethylene oxide)-co-poly(propylene oxide) block copolymers (poloxamer and meroxaprol), poloxamine, carboxymethyl cellulose and hydroxyalkylated cellulose such as hydroxyethyl cellulose and methyl hydroxypropyl cellulose, and natural polymers such as peptides, polysaccharides or sugars such as Ficoll®, sucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin or copolymers thereof. The term "cellulose" includes cellulose and derivatives of the types described above; "dextran" includes dextran and similar derivatives.

[0082] An exemplary photoinitiator is Igracure 2959. Other photoinitiators include HPK, which is commercially available from Polysciences. Additionally, various dyes and amine catalysts are known to form active substances upon exposure to external radiation. Specifically, the absorption of light by the dye causes it to present a triplet state, which subsequently reacts with an amine to form the active substance that initiates polymerization. Typically, polymerization can be initiated by irradiation with light of wavelengths between about 200-700 nm, most preferably in the long-wavelength ultraviolet or visible light range, 320 nm or higher, and most preferably between about 365 nm and 514 nm.

[0083] Many dyes can be used for photopolymerization, including erythrosine, phloxime, rosemary, thionine, camphorquinone, ethyl eosin, eosin, methylene blue, riboflavin, 2,2-dimethyl-2-phenylacetylphenyl, 2-methoxy-2-phenylacetylphenyl, 2,2-dimethoxy-2-phenylacetylphenyl, other acetylphenyl derivatives, and camphorquinone. Suitable cocatalysts include amines such as N-methyldiethanolamine, N,N-dimethylbenzylamine, triethanolamine, triethylamine, dibenzylamine, N-benzylethanolamine, and N-isopropylbenzylamine. Triethanolamine is a preferred cocatalyst for one of these dyes. The photopolymerization of these polymer solutions is based on the finding that a combination of polymer and photoinitiator (at a cell-nontoxic concentration, less than 0.1% by weight, more preferably 0.05% to 0.01% by weight of initiator) crosslinks upon exposure to light equivalent to 1 to 3 mWatt / cm².

[0084] While photopolymers are preferred for preparing hydrogels because it is convenient to control polymerization using external radiation provided by a surgical endoscope, the present invention can be implemented using other polymeric materials and polymerization initiators. Examples of other materials that can be used to form hydrogels include (a) modified alginates, (b) polysaccharides (e.g., gellan gum and carrageenan) that gellenate by exposure to monovalent cations, (c) polysaccharides (e.g., hyaluronic acid) that are very viscous liquids or thixotropic and can form gels over time through slow structural evolution, and (d) polymeric hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins).

[0085] B. Patient-specific tissue stiffness

[0086] Besides the composition of the extracellular matrix (ECM), tissue stiffness is partly determined by the stiffness of the underlying ECM. Studies have shown that changes in tissue stiffness can significantly affect cellular behavior within tissues, particularly in the context of liver disease (Wong GL, et al., J Hepatol 60: 339-45, 2014; Wells RG, Hepatology 47: 1394-400, 2008). For example, studies on Kupffer cells, which constitute the second largest cell population in the liver, have highlighted the crucial role of basal stiffness in guiding macrophage activity. This finding is key to the development of immunologically based biomaterials that can modulate macrophage responses after implantation (Sridharan R, et al., Materials Today 18:313-25, 2015).

[0087] Furthermore, given that cirrhosis is a known independent risk factor for hepatocellular carcinoma (HCC), studies on liver stiffness have explored its potential to predict HCC risk. Foucher et al. identified a threshold of 53.7 kPa for the presence of HCC, which is significantly higher than the stiffness of healthy liver tissue (2–7 kPa) (Foucher J, et al., Gut 55:403–408, 2006). Other studies have confirmed that liver stiffness exceeding 8 kPa significantly increases the risk of HCC (Jung KS, et al., Hepatology 53(3): 885-94, 2010). Increased liver stiffness is also observed in patients with other liver diseases such as hepatitis B and C, as well as non-alcoholic steatohepatitis (NASH) (Mueller S, Sandrin L, Hepat Med 2:49-67, 2010).

[0088] In a preferred form, the 3D bioprinted scaffold is engineered to precisely mimic the stiffness characteristics of a patient's liver, thereby facilitating the replication of biophysical parameters found in clinical liver samples for advanced studies of cell behavior. This customization allows for the exploration of cellular responses under conditions that closely reflect the physiological environment, providing valuable insights into the mechanisms controlling cell interactions within liver tissue.

[0089] Furthermore, the 3D bioprinted scaffolds are designed to incorporate stiffness profiles representing various stages of cancer development, including fibrosis, cirrhosis, and steatosis. This approach allows for a detailed examination of liver disease progression, contributing to a deeper understanding of the transitions between these stages and their impact on cell dynamics and pathology.

[0090] In some forms, the hydrogel used in the disclosed 3D bioprinted scaffolds has a hardness ranging from about 2 kPa to about 75 kPa. This range is intentionally chosen to cover a wide range of liver pathologies, ensuring that the model can be customized to faithfully simulate specific conditions of interest. In some forms, the hydrogel used in the disclosed 3D bioprinted models has a hardness of about 5 kPa to about 75 kPa, about 20 kPa to about 60 kPa, or about 30 kPa to about 50 kPa. In some forms, the hydrogel used in the disclosed 3D bioprinted models has a hardness of about 2 kPa, about 5 kPa, about 10 kPa, about 15 kPa, about 20 kPa, about 25 kPa, about 30 kPa, about 35 kPa, about 40 kPa, about 45 kPa, about 50 kPa, about 55 kPa, about 60 kPa, about 65 kPa, about 70 kPa, or about 75 kPa.

[0091] In some forms, the hydrogel used in the disclosed 3D bioprinted scaffold has different hardness depending on the compartments of the hydrogel. For example, in some forms, the compartments containing tumor cells have a hardness of about 20 kPa to about 75 kPa to reproduce the hardness of diseased liver tissue extracted from a patient. In some forms, the compartments containing non-tumor cells have a hardness of about 2 kPa to about 19 kPa to reproduce the hardness of healthy tissue extracted from a patient.

[0092] C. Cellular compositions for the immune microenvironment of tumor cells

[0093] Tumor cells stimulate significant molecular, cellular, and physical changes within their host tissues to support tumor growth and progression. Many risk factors for liver cancer have an immunological basis, such as infection with hepatitis viruses and conditions like non-alcoholic steatohepatitis (NASH), which lead to persistent and imbalanced immunological changes as the tumor develops and progresses. The tumor immune microenvironment (TIME) plays a role in tumor immune surveillance and immune evasion mechanisms; therefore, the development of new insights into checkpoint inhibitor therapy is of great significance (Tang T, et al., Signal Transduction and Targeted Therapy 6: 72 (2021)). A single-cell sequencing study involving more than 100 patients with hepatocellular carcinoma (HCC) identified five distinct immune subtypes, each characterized by a unique composition of immune cells and extracellular matrix (Xue, R., Zhang, Q., Cao, Q. et al., Nature 612, 141–147 (2022)). This heterogeneity in the immune spectrum leads to diverse somatic mutations and transcriptomic patterns in tumor cells, significantly impacting the effectiveness of immunotherapy. Although immune cell diversity and TIME play a crucial role in understanding liver tumors, current models have not yet integrated or demonstrated this immune variant spectrum in liver cancer research.

[0094] The disclosed 3D bioprinted scaffold is designed to preserve the unique immunological profile of each individual patient. This approach enables a more accurate representation of the intrastent hepatocellular carcinoma tumor immune microenvironment (TIME), thereby enhancing personalized drug screening. Such precise mimicking of patient-specific immunological characteristics within the scaffold allows for customized assessment of treatment response. Figure 4A and 4BAs shown, different immune cell types, including macrophages, dendritic cells, B regulatory cells, T regulatory cells, and epithelial progenitor cells, contribute to tumor development, progression, and recurrence before and after treatment (Yeung OWH, Lo CM, Ling CC, et al., Journal of Hepatology 62: 607-16 (2015); Pang L, Ng KT-P, Liu J, et al., Cancer Letters 522: 80-92 (2021); Li CX, Ling CC, Shao Y, et al., JHepatol 65: 944-52 (2016); Shao Y, Lo CM, Ling CC, et al., Cancer Letters 355:264-72 (2014); and Ling CC, Ng KTP, Shao Y, et al., Journal of Hepatology 60:103-9 (2014)).

[0095] In some forms, the disclosed 3D bioprinted scaffolds include tumor cells, cancer-associated fibroblasts, endothelial cells, immune cells, etc. In some forms, the immune cells include innate immune cells and adaptive immune cells. Exemplary innate and adaptive immune cells include macrophages, neutrophils, natural killer cells, and T lymphocytes. In some forms, the disclosed 3D bioprinted scaffolds include malignant hepatocytes, healthy hepatocytes, and non-parenchymal cells, including macrophages, lymphocytes, and endothelial cells. In some exemplary forms, the disclosed 3D bioprinted scaffolds include macrophages and T lymphocytes abundant in hepatocellular carcinoma. In further forms, the disclosed 3D bioprinted scaffolds are suitable for studying the interaction between adaptive and innate immunity.

[0096] In one exemplary embodiment, the disclosed 3D bioprinted scaffold preserves the unique immunological characteristics of an individual patient, enabling accurate replication of the liver cancer-specific tumor microenvironment. In another embodiment, the 3D bioprinted scaffold is customized for enhanced personalized drug screening, providing improved compatibility and efficacy in therapeutic testing.

[0097] D. Vascular system in tumor compartments

[0098] Tumors are not only aggregates of malignant cells but also complex, well-organized vascular systems. Primary tumors are divided into a tumor core, tumor stroma, and invasive periphery based on tumor compartments. The vascular system within a tumor is abnormal, which impairs blood flow and restricts the delivery of oxygen, nutrients, and therapeutic agents (including antibodies and immune cells). In addition to nutrient supply, the vascular system also allows for molecular gradients of waste metabolites.

[0099] The vascular system (also known as the blood vessel system) comprises structures composed of vascular endothelial cells and their supporting cells. The vascular system not only maintains tissues but also plays a crucial role in tissue maturation. In some forms, vascular structures provide tissues with the oxygen and nutrients necessary for their survival. Even before blood flows into tissues, reconstructing the three-dimensional tissue structure with vascular and cellular polarity is important for cell differentiation, proliferation, and maintenance.

[0100] Therefore, 3D and 4D bioprinted scaffolds contain vascular systems or blood vessels. Vascular systems or blood vessels include populations of vascular cells. In a preferred form, the vascular cells of the 3D and 4D bioprinted scaffolds are derived from the patient's own vascular tissue. In other forms, the vascular cells of the 3D and 4D bioprinted scaffolds can be derived from pluripotent or totipotent cells (e.g., induced pluripotent stem cells and embryonic stem cells) through induced differentiation. The vascular cells contained in bioprinted scaffolds are typically vascular endothelial cells. Vascular endothelial cells include cells that constitute the vascular endothelium or cells capable of differentiating into such cells, such as vascular endothelial progenitor cells and vascular endothelial stem cells. Whether a cell is a vascular endothelial cell can be determined by examining whether the cell expresses marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3, and CD31. If any or more of the aforementioned marker proteins are expressed, the cell can be safely considered a vascular endothelial cell. Furthermore, markers for vascular endothelial progenitor cells include, but are not limited to, c-kit and Sca-1. If the cells of interest express these markers, then the cells can be identified as vascular endothelial progenitor cells (Fang, et al., PLOS Biology, 2012; 10(10): e1001407).

[0101] Because chemical forces influence the tumor microenvironment (TME) and directly affect cancer growth, oxygen is a key factor in this situation. Oxygen deficiency primarily occurs when the tumor's oxygen demand exceeds the supply provided by the adjacent vascular system, a condition known as hypoxia. Most healthy organs exist in an oxygen content of 3-6%, while a level below 3% is described as hypoxia. Hypoxia occurs extensively in malignant tumors and is known to contribute to tumor progression through a variety of mechanisms. Responses typically include abnormal growth of the vascular system during angiogenesis and EMT in cancer cells, which ultimately leads to metastasis.

[0102] Using the vascular system, oxygen gradients can be generated in 3D bioscaffolds with hypoxic regions. The resulting hypoxia (low oxygen concentration) and low pH, in turn, can induce the production of immunosuppressive molecules such as transforming growth factor-β (TGFβ), vascular endothelial growth factor (VEGF), and adenosine in the tumor microenvironment (TME). Hypoxia is a common phenomenon in intratumoral regions of HCC patients. Abnormal microvascular systems and uncontrolled proliferation of HCC cells lead to oxygen deficiency. Hypoxia is involved in multiple biological processes in HCC and promotes tumor invasiveness, chemotherapy resistance, and immunotherapy resistance (Cramer T, Vaupel P. Journal of Hepatology 76: 975-80 (2022)).

[0103] Therefore, in some forms, 3D bioprinted scaffolds incorporate one or more normoxic (with vascular systems) compartments to reconstruct the tumor architecture and complexity observed in patient tumors. In other forms, 3D bioprinted scaffolds incorporate one or more hypoxic (without vascular systems) compartments to reconstruct the tumor architecture and complexity observed in patient tumors.

[0104] In tumor tissue, chronic hypoxia occurs when the tumor expands beyond 70 μm from pre-existing nutrient vessels, preventing adequate oxygen delivery (Emami Nejad, et al., Cancer Cell Int 21, 62 (2021)). In some forms, regions with different oxygen and nutrient gradients are generated based on their distance from the vascular system within the 3D bioprinted scaffold. In some forms, the distance between tumor cells and the vascular system with oxygen and nutrient supply ranges from approximately 0.5 μm to approximately 400 μm, simultaneously generating normoxic and hypoxic cells, such as... Figure 8BAs shown in the diagram. In some forms, the distance between tumor cells and the vascular system is approximately 0.5 µm, approximately 50 µm, approximately 100 µm, approximately 150 µm, approximately 200 µm, approximately 250 µm, approximately 300 µm, approximately 350 µm, or approximately 400 µm. In some forms, the distance between non-tumor cells and the vascular system is approximately 0.5 µm to approximately 1400 µm. In some forms, the distance between non-tumor cells and the vascular system is approximately 0.5 µm, approximately 50 µm, approximately 100 µm, approximately 200 µm, approximately 300 µm, approximately 500 µm, approximately 700 µm, approximately 900 µm, approximately 1000 µm, approximately 1100 µm, approximately 1200 µm, approximately 1300 µm, or approximately 1400 µm. The average size of a single tumor or non-tumor cell is approximately 5 µm to approximately 40 µm. In some forms, the average size of a single tumor or non-tumor cell is about 5 µm, about 10 µm, about 15 µm, about 20 µm, about 25 µm, about 30 µm, about 35 µm, or about 40 µm.

[0105] E. Geometry of bioprinted scaffolds

[0106] Bioprinted scaffolds can have a 3D configuration, i.e., a 3D bioprinted scaffold, or a 4D configuration, i.e., a 4D bioprinted scaffold.

[0107] 1. 3D bioprinted scaffold

[0108] The disclosed 3D scaffold is composed of a bioprinted living three-dimensional construct simulating liver tumor tissue. The 3D scaffold contains one or more bioprinted compartments, including at least one compartment containing tumor cells and macrophages, a second compartment containing non-tumor cells such as hepatocytes and T lymphocytes, and a third compartment containing the vascular system.

[0109] In a preferred embodiment, the 3D scaffold is fabricated using microfluidic channels and a dynamic flow system that mimics physiological systems such as blood flow. In some embodiments, the disclosed 3D bioprinted scaffold includes at least one inlet and one outlet for microfluidic connections. The construct is combined with multi-branched microfluidic channels to increase their contact area with tumor and non-tumor regions for efficient material exchange. To mimic the different diameters of blood vessels in a vascular system, the width of the microfluidic channels in the construct ranges from about 0.5 mm to about 1.0 mm. In some embodiments, the width of the microfluidic channels is about 0.1 mm, about 0.2 mm, about 0.3 mm, about 0.4 mm, about 0.5 mm, about 0.6 mm, about 0.7 mm, about 0.8 mm, about 0.9 mm, or about 1.0 mm. In some embodiments, to mimic blood vessels, all microfluidic channels are encapsulated with endothelial cells (see Figure 5).

[0110] In other forms, 3D scaffolds can control the chemical microenvironment to provide physiologically relevant methods, such as generating well-controlled chemical gradients across multicellular systems in 3D ECM and ECM, whose spatiotemporal distribution successfully mimics in vivo conditions.

[0111] In some forms, the disclosed 3D bioprinted scaffold can be constructed using any of three different designs, as shown in Figure 6, each containing inlets and outlets for microfluidic connections. Each layer of engineered liver tissue comprises multiple cells along the X, Y, and Z axes and has a thickness of at least about 10 mm. The hydrogel matrix can be generated in one or more layers. In some forms, the disclosed 3D bioprinted scaffold comprises five or more layers. In a preferred form, the dimensions of the construct are at least 10 mm x 10 mm x 0.5 mm. Suitable ranges for the dimensions of the construct are length (about 20 to about 40 mm), width (about 20 to about 40 mm), and height (about 10 to about 30 mm).

[0112] 2. 4D bioprinted scaffold

[0113] In general, the physiological systems within the human body exhibit complex and dynamic environments, with microenvironmental conditions (such as temperature, pH, enzymes, and potential) fluctuating in real time across different tissues (see Kavand, et al., Adv. Mater. 2022, 34, 2107876). To meet the real-time demands of this dynamic microenvironment in tissues and organs, innovative paradigms of 4D bioprinting (3D bioprinting + time) have emerged, aiming to generate intelligent scaffolds for tissue engineering (S. Miao, et al., Sci. Rep. 2016, 6, 27226). In recent years, the concept of 4D printing has evolved to allow the shape, properties, or function of 3D-printed objects to dynamically change over time in response to external stimuli (X. Kuang, et al., Adv. Funct. Mater. 2019, 29, 1805290). For example, 4D bioprinting has been applied to create smart tissue and organ scaffolds (Wang, et al., Adv. Mater. 2022,34, 2109198; Miao, et al., Mater.Today 2017, 20, 577; and Chen, et al., Int. J.Extreme Manuf.2023, 5, 032007).

[0114] Therefore, bioprinted scaffolds with a four-dimensional (4D) configuration have also been disclosed. In some forms, the 4D bioprinted scaffolds incorporate various types of stimuli, such as humidity, temperature, pH, light, and magnetic fields.

[0115] 4D printing technology has been used to create 4D programmable modules that respond to stimuli, providing an innovative approach to constructing architectural structures with both superior structural and mechanical properties. The disclosed 4D bioprinted scaffold enables precise control of environmental conditions within a culture system, allowing for accurate observation of interactions between various cell types in vitro. The system comprises a porous scaffold combined with a stimulus-responsive platform, designed to mimic the architecture of a liver plate.

[0116] Leveraging advancements in 3D printing and smart biomaterials, 4D bioprinted scaffolds more accurately target tumor cells, immune cells, stromal cells, and vascular cells, aligning them with their natural physiological functions. These 4D bioprinted scaffolds include microchannels serving as potential endothelialized vascular systems and fluid conduits, as well as 4D-printed modules made from stimulus-responsive hydrogels that act as sources of biosignal release. These modules are designed to be stackable and interlocking, similar to traditional building blocks, offering customizable and flexible options for creating a variety of functional geometries. This feature is particularly beneficial for reconstructing patient-specific tumor structures, providing a new level of controllability and adaptability in medical modeling and therapeutic applications.

[0117] Biomaterials that can be incorporated into 4D bioprinted scaffolds include, but are not limited to, shape memory polymers (SMPs) and liquid crystal elastomers. In some forms, the biomaterials incorporated into 4D bioprinted scaffolds are alloys, such as shape memory alloys (SMAs). When subjected to mechanical stress or temperature changes, shape memory alloys (SMAs) undergo a phase transformation (between austenite and martensite). In particular, SMAs “remember” their initial form and revert to their initial form after conditions normalize. Exemplary SMAs that can be used include copper-aluminum-nickel alloys and nickel-titanium (NiTi) alloys.

[0118] In some forms, the biomaterial incorporated into the 4D bioprinting scaffold is ceramic. An exemplary ceramic that can be used is a “ceramic ink” made of an elastomeric poly(dimethylsiloxane) matrix nanocomposite material, which can be printed, deformed, and then transformed into a silicon carbide (SiOC)-based ceramic nanomaterial (G. Liu, Y. Zhao, G. Wu, J. Lu, Sci. Adv. 2018, 4: eaat0641).

[0119] In some forms, the biomaterials incorporated into 4D bioprinted scaffolds are polymers, such as shape memory polymers (SMPs). SMPs represent the most widely used smart materials in 4D printing (Zhou, et al., Macromol. RapidCommun., 2021, 42, 2100176). SMPs can retain a temporary shape and recover their initial shape upon exposure to external stimuli (Kuang, et al., Adv.Funct.Mater.2019, 29, 1805290). When the temperature exceeds the transition temperature, SMPs can temporarily deform. Upon cooling below this threshold and unloading, the SMP retains its deformed shape. This process can be repeated to achieve reversible deformation and has been applied in many TE (Technical Engineering) instances.

[0120] In some forms, SMPs are hydrogels used for 3D printing as described above. Hydrogels are 3D polymer networks permeated with water. Their unique properties, such as wettability, responsiveness, softness, biocompatibility, and bioactivity, make them promising materials for tissue engineering. Furthermore, hydrogels exhibit stimulus-responsive behavior, making them promising materials for 4D bioprinting. The ability of hydrogel structures to dynamically change and adapt is crucial for tissue engineering (TE) applications. Hydrogel structures can exhibit deformability, respond to specific physiological cues, and even undergo controlled degradation as they integrate with their surrounding biological environment. For example, Gladman et al. proposed biomimetic 4D printing using hydrogels (Gladman, et al. Nat. Mater. 2016, 15, 413). During the bioprinting process, cellulose fibrils are oriented along the printing direction by shear forces. When the hydrogel is immersed in water, anisotropic swelling behavior is observed, resulting in programmable shape changes and complex 3D morphologies. In another example, Ding et al. developed a cell-loaded bio-ink for 4D bioprinting (Ding, et al., Adv. Mater. 2022, 34, 2109394). The printed cell-loaded constructs exhibited high cell viability and programmable deformation under external stimuli. These exemplary 4D live-cell bioprinting techniques can be used to fabricate 4D bioprinting scaffolds as disclosed herein. 4D bioprinting materials and fabrication methods are further described in Chen et al., Advanced Materials, 2307686 (2023).

[0121] III. Preparation Method

[0122] The composition of the tumor microenvironment varies depending on the tumor type, but key features include immune cells, stromal cells, blood vessels, and the extracellular matrix. To create highly clinically relevant models, one or more methods are employed to incorporate clinical components into the bioprinting process.

[0123] A method for preparing 3D in vitro cell cultures simulating liver cancer and related tumor microenvironments (TMEs) in simulated subjects is provided. In some embodiments, one or more of the following steps are used: biomaterial preparation and 3D bioprinting technology.

[0124] (a) Simultaneously isolate and preserve patient-derived cells and ECM;

[0125] (b) Patient-derived cell maintenance for bioprinting;

[0126] (c) Combining micro-molding with 3D multi-material injection exclusion and suspension bioprinting technologies to prepare “tumors in cubes” or “miniature liver tumors”.

[0127] A. Simultaneously isolate and preserve patient-derived cells and ECs

[0128] Due to its clinical nature, ECM is a tissue-derived biomaterial that can be used as a bioactive component for tissue engineering applications (Kim YS, Majid M, Melchiorri AJ, Mikos AG. Bioengineering & translationalmedicine 4: 83-95 (2018)). The versatility of ECM makes it an excellent candidate for a variety of applications, from whole tissue scaffolds to digestive fluids that can be used as bioinks for 3D printing.

[0129] Current methods for isolating tissue ECM result in low reproducibility and high variability. The utilization of ECM in tissue engineering applications is still under development, and a large part of current work remains focused on exploring the effects of different decellularization methods. More importantly, all current protocols are designed to extract ECM or cells from clinical samples. Therefore, in a preferred form, the disclosed method simultaneously preserves both of these key components for in vitro regeneration and bioprinting of TIME.

[0130] In the exemplary extraction method, fresh patient liver tissue, including both tumor and non-tumor cells, measuring approximately 1-3 cm in diameter and removed during surgery, is first washed with 1xHBSS containing 1x penicillin-streptomycin to remove blood and potential contaminants. It is then cut into small pieces with a diameter of 1-2 mm using sterile surgical scissors. To simultaneously preserve the extracellular matrix compartments of HCC tissue, primary cells are first isolated with a combination of 1 mM EDTA and other components and mechanically disrupted at 150 rpm for 30 minutes in an orbital shaker at 37°C. The supernatant containing various cell types is then harvested and separated using a newly developed protocol. For enrichment of primary hepatocytes and HCC cells, the supernatant is centrifuged at 50 g for 5 minutes, and the cell pellet is then resuspended. For enrichment of smaller hepatic nonparenchymal cell (NPC) fractions, a) hepatic endothelial cells were centrifuged at 300 g for 5 min, then covered with a double layer (25% / 50%) density gradient to remove dead cells, and then purified by FACS sorting for CD31+; (b) macrophages and Kupffer cells were first centrifuged at 300 g for 5 min, then centrifuged at 650 g for 7 min, and then enriched by FACS; c) immune cells were enriched by Ficoll in patient-derived blood PBMCs and then FACS sorted for the desired immune population.

[0131] To extract extracellular matrix from HCC tissue, 1% Triton X-100 and other components were added to cell-deficient cubes to deplete any remaining cells. The cubes were then incubated overnight at 300 rpm in an orbital shaker at 37°C. The supernatant was discarded, and the remaining cubes were then freeze-dried for 24 hours using a Thermo ModulyoD freeze dryer to obtain a lyophilized powder of tissue-derived ECM.

[0132] 1. Origin of liver tissue

[0133] Early-stage liver cancer can be cured through local ablation, surgical resection, or liver transplantation. Treatment options depend on tumor characteristics, the severity of underlying liver dysfunction, age, other medical comorbidities, and available medical resources and local expertise. Catheter-based local therapies are used for patients with intermediate-stage cancer. Kinases and immune checkpoint inhibitors have proven to be effective treatment options for patients with advanced HCC.

[0134] In some formats, the subjects are undergoing standard treatments for liver cancer. For example, in some formats, additional therapies or procedures include surgery, transplantation, radiation therapy, or chemotherapy.

[0135] In some forms, subjects are undergoing immunotherapy, such as the use of one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists) to inhibit checkpoint proteins (e.g., components of the PD-1 / PD-L1 axis or CD28-CTLA-4 axis), adoptive T-cell therapy, and / or cancer vaccines.

[0136] In some cases, samples are collected during surgical removal of the tumor. In other cases, the sample is from metastatic liver cancer.

[0137] In some forms, the cancer may have developed resistance to previously administered chemotherapy agents. Therefore, in some forms, the subjects from whom the biomaterials used for 3D bioprinted scaffolds are derived are subjects whose cancers were resistant to or insensitive to one or more conventional chemotherapy agents prior to sample collection.

[0138] i. Hepatocellular carcinoma

[0139] In some forms, liver tissue is surgically removed from patients with hepatocellular carcinoma (HCC). HCC is the most common primary malignant tumor of the liver and a leading cause of cancer-related deaths worldwide.

[0140] Chronic liver disease and cirrhosis remain the most important risk factors for the development of HCC, with viral hepatitis and excessive alcohol intake being the major risk factors worldwide.

[0141] Chronic viral hepatitis can lead to cirrhosis and / or hepatocellular carcinoma (HCC). Hepatitis B and C are the most common causes of chronic hepatitis worldwide. Hepatitis B virus (HBV) is a double-stranded circular DNA molecule with eight genotypes (A through H). Genotypes A and D are more common in Europe and the Middle East, while genotypes B and C are more common in Asia. Hepatitis B is transmitted through contaminated blood transfusions, intravenous injections, and sexual contact. Mother-to-child transmission is the leading cause of HBV infection globally. Five percent of the world's population is infected with hepatitis B.

[0142] Several epidemiological studies have demonstrated that chronic HBV infection has a significant hepatocarcinogenicity. Hepatitis B carriers have a 10%–25% lifetime risk of developing HCC. Unlike other causes of chronic hepatitis, HBV is unique in that HCC can develop without evidence of cirrhosis. Genotype C is associated with a higher risk of HCC compared to genotypes A, B, and D. Active HBV infection carries an independent risk of HCC, with HBV DNA levels >10⁵ / mL viral copies associated with a 2.5–3-fold increased risk of developing HCC during 8–10 years of follow-up. Hepatitis B surface antigen (HBsAg) is not the only hematological marker of significant risk of developing HCC. Patients with positive hepatitis B core antibodies (who are HBsAg negative) still have a risk of developing HCC. Antiviral therapy for hepatitis B can significantly reduce the hepatocarcinogenicity of HBV. Viral suppression can lead to a significant 5-year reduction in HCC incidence from 13.7% (control) to 3.7%, with the largest reduction observed in patients with cirrhosis. 10. HBV vaccination has led to a significant decrease in the incidence of HBV-related HCC. The East Asian Newborn Vaccination Program is estimated to reduce the incidence of hepatitis B-related HCC by 70%-85%. Despite perinatal immunization, 5%-10% of infants remain at risk of hepatitis B infection. Treatment of mothers with chronic hepatitis B in the third trimester of pregnancy with nucleoside analogues has proven superior to vaccination alone in preventing neonatal transmission.

[0143] Hepatitis C virus (HCV) is a small, single-stranded RNA virus that exhibits high genetic variability. Six different genotypes of HCV have been isolated. Genotypes I, II, and III are predominant in Western countries and the Far East, while genotype IV is predominant in the Middle East. The highest rate of chronic hepatitis C infection is found in Egypt (18%), with lower rates in Europe (0.5%–2.5%), the United States (1.8%), and Canada (0.8%).16 Once infected with HCV, 80% of patients will progress to chronic hepatitis, and approximately 20% will develop cirrhosis. In hepatitis C, the development of hepatocellular carcinoma (HCC) almost exclusively occurs in livers with established cirrhosis; however, in the HALT-C trial, 8% of HCC occurred in patients with only advanced fibrosis. Co-infection with HBV and HCV in patients with cirrhosis increases the risk of HCC, with an odds ratio (OR) of 165, compared to 17 for hepatitis C alone and 23 for hepatitis B alone. Compared to HCV-HCC alone, the synergistic effect of alcohol increased the incidence of HCC by 1.7 to 2.9 times. In patients who achieved a sustained virological response after HCV treatment, the risk of HCC was significantly reduced, with a 54% reduction in all-cause mortality. Although recent advances in medication have made HCV treatment easier, a vaccine against the virus remains a challenge.

[0144] Alcohol consumption remains a significant risk factor for the development of hepatocellular carcinoma (HCC). The relationship between alcohol and liver disease is related to lifetime alcohol consumption, with heavy drinking rather than social drinking being the primary risk factor for HCC. The prevalence of heavy drinking in the United States is five times higher than that of hepatitis C. Heavy drinking accounts for 40-50% of all HCC cases in Europe. European studies report an increased relative risk of developing liver disease for women who drink more than 7-13 times per week and for men who drink more than 14-27 times per week. In the United States, studies have shown that people who consume more than 60 g / day of ethanol daily have a two- to four-fold increased risk of liver cancer. A meta-analysis of 19 prospective studies showed that drinking alcohol three or more times a day increases the risk of liver cancer by 16%, and drinking six or more times a day increases the risk by 22%.

[0145] 60% of patients over 50 years of age with diabetes or obesity are considered to have NASH with advanced fibrosis. Chronic diseases such as diabetes and obesity increase the risk of HCC. Diabetes directly affects the liver because of its crucial role in glucose metabolism. It can lead to chronic hepatitis, fatty liver, liver failure, and cirrhosis. Diabetes is an independent risk factor for HCC. Patients with diabetes have a 1.8 to 4 times increased risk of developing HCC. Obesity is well-known to be associated with many hepatobiliary diseases, including non-alcoholic fatty liver disease (NAFLD), steatosis, and cryptogenic cirrhosis, all of which can contribute to the development of HCC.

[0146] In some forms, cells and ECM are extracted from clinical samples, including those from patients with HCC and intrahepatic cholangiocarcinoma.

[0147] B. Patient-derived cell maintenance for bioprinting

[0148] Compared to cell lines or stem cell-derived cells, patient-derived cells (PDCs), including tumor and immune cells generated from patient samples, directly reflect the patient's tumor characteristics and clinical response (Kim SY, Lee JY, Kim DH, et al., Scientific Reports 9: 19909 (2019)). They are also considered a key target for successful personalized medicine development. Practical challenges in primary cell culture involve the limited availability of tumor samples, the restoration of the tumor microenvironment for signaling stimulation, stromal cell growth, and tumor cell senescence.

[0149] Both innate and adaptive immune components are typically present in the tumor microenvironment of a patient's tumor tissue, including macrophages, lymphocytes, and tumor cells. All cells are derived from one or more of the following sources: adult liver tissue; established liver-derived and mononuclear cell lines. Macrophages can be characterized based on staining for CD68, CD163, or CD206. Lymphocytes can be characterized based on staining for CD3, CD4, or CD8. To maintain consistency, all isolated primary cells were genetically immortalized.

[0150] In a preferred form, the disclosed method involves extracting different cell populations from tumor samples and maintaining their viability in a bioprinted scaffold for further mechanistic and drug screening studies.

[0151] C. 3D bioprinting of patient-derived ECM and cells

[0152] Currently, bioprinting of specific tissues and organs with high cellular heterogeneity and complex structures remains a major challenge, primarily due to the need for vascularization for adequate tissue oxygenation, nutrient delivery, and waste removal (Bertassoni LE. Advanced Materials 34: 2101321 (2022)).

[0153] The disclosed methods involve combining bioprinting technologies (including extrusion, microforming, and digital light processing (DLP)) with hydrogels containing sacrificed, cell-loaded materials to construct and assemble different tumor compartments with multiple cell-material interaction interfaces (tumor / non-tumor, tumor / vascular system, and non-tumor / vascular system) for reconstructing the complexity of tumor architecture in HCC. Exemplary 3D bioprinted scaffolds include vascular systems and multiple cellular interfaces (e.g., tumor-non-tumor interfaces and tumor-vascular system interfaces) (Figure 5).

[0154] Based on bioprinting technology, bio-inks can create cell aggregates comprising different cell types, including cancer-associated fibroblasts, immune cells, and endothelial cells, to form vascular networks. Furthermore, bioprinting can create biomimetic microenvironments for the heterogeneous distribution of bio-associated proteins and growth factors, which is crucial for controlling tumor cell signaling, proliferation, and migration. Bioprinting technology enables the direct printing / patterning of cells in microfluidic devices, mimicking vascular systems and biological barriers. Vascularization is essential for maintaining tissue viability and can be used to separate different tissue compartments. The vascular system of tumors differs significantly from the blood vessels supplying healthy tissue, particularly in terms of heterogeneity, permeability, multidirectional blood flow, and disordered distribution throughout the tumor.

[0155] In a preferred form, bioprinting technology is used to manufacture miniature liver 3D cell culture systems to mimic the heterogeneous microenvironment and complex 3D microstructures of tumors.

[0156] 1. Manufacturing of patient-derived ECM hydrogels

[0157] Freeze-dried patient-derived ECM can be dissolved in a hydrogel. In one form, freeze-dried patient-derived ECM is dissolved in a PEGDA, alginate-gelatin, and methacrylamide gelatin (GelMA) hydrogel to generate a 0.1–0.5% solution, and then the patient-derived cells are loaded in hydrogen gas prior to bioprinting.

[0158] 2. 3D bioprinting of hydrogel compartments

[0159] All designs comprise three compartments: the tumor, the tumor matrix, and the vascular system. The first two compartments are 3D bioprinted using bio-ink 1 and bio-ink 2 via designed pathways. The hydrogel matrix may also include a first cell type and a second cell type embedded therein. In some respects, the hydrogel compartments may include the first cell type embedded therein.

[0160] Photosensitive polymers that can be used include (e.g., but not limited to) poly(ethylene glycol) diacrylate (PEDGA), gelatin methacrylate (GelMA), polyacrylamide (PMA), and alginate gelatin. Photosensitive polymers can also be modified by conjugation with peptides such as collagen and fibronectin. Biomaterials that highly mimic the liver tumor microenvironment and vascular-specific ECM, such as alginate, gelatin, type IV collagen, and fibronectin, will be used as biomaterials to mimic HCC tissue. These natural extra-ECM proteins have been reported to be highly present in both healthy and tumor liver tissues, in addition to the patient's ECM. GelMA and PMA can be used as biomaterials to mimic blood vessels because they have recently been used to assemble many properties of blood and lymphatic vessels. To mimic the tumor microenvironment, different cell types are loaded with ECM-mixed hydrogels, hepatocytes in non-tumor compartments, HCC, immune cells in tumor compartments, and endothelial cells in vascular system compartments.

[0161] The 3D construct includes inlets and outlets for connecting microfluidic devices to supply cells, nutrients, and remove waste. In areas lacking a vascular system, hypoxia can be induced in compartments 1 and 2.

[0162] 3. Hydrogel matrix

[0163] Following bioprinting, a photosensitive hydrogel solution is cast onto the printed hydrogel matrix. The photosensitive hydrogel solution consists of a photosensitive polymer, a photoinitiator, and various cells. This photosensitive polymer can include, for example, but not limited to, Gelma, PEGDA, polycaprolactone trimethylol methacrylate (PCLMA), and hyaluronic acid methacrylate (HAMA). This photoinitiator can include, for example, but not limited to, lithium phenyl-2,4,6-trimethylbenzoylphosphinic acid, lithium acylphosphinic acid, Irgacure 2959, and camphorquinone, which are typically used with ethyl 4-N,N-dimethylaminobenzoate or TEA and the photosensitizer isopropylthioxanthone. High concentrations of photoinitiators can be used to achieve increased z-resolution by limiting the penetration depth of incident light; however, these high concentrations can disrupt the photopolymerization reaction and are cytotoxic. The system is then formed by curing the hydrogel matrix under blue light. In some forms, the hydrogel solution is 0.5%–10% hydrogel. In some formulations, the concentration of the photoinitiator is 0.05%–0.5%. In another embodiment, the cell density is 0–5.0 x 10⁻⁶. 6 Cells / mL hydrogel solution. In some forms, the curing time is 10-100 s.

[0164] 4. 3D Bioprinting Methods for Hydrogels

[0165] Two schemes for designing and manufacturing 3D bioprinted scaffolds are summarized in ( Figures 7A-7B In one embodiment, the bio-inks were printed using a Bioscaffold BS5.3 3D bioprinter from GeSim, Germany. A 410 µm diameter nozzle was selected for smooth printing. The printing speed was optimized to 8 mm·s⁻¹ to obtain perfectly aligned structures. The printing pressures for the PEGDA and PAM bio-inks were optimized to 0.08–0.095 MPa and 0.09–0.1 MPa, respectively. The spacing was optimized to 800 µm. After printing, the samples were irradiated with a 365 nm UV lamp (350 mW·cm⁻² / 2 min) to polymerize and crosslink the hydrogel. The printed structures were then placed in water overnight to completely remove uncrosslinked monomers and achieve equilibrium shapes in a swollen state.

[0166] In some forms, the bioprinting techniques used include extrusion, microforming, digital light processing (DLP) using hydrogels constructed with sacrificial load cells, bioprinting in suspension, and any combination thereof. Bioprinting incorporates different tumor compartments to reconstruct the integrity and complexity of tumor architecture in liver tumors.

[0167] In some forms, 3D bioprinting scaffolds are bioprinted using stereolithography.

[0168] 5. Stiffness determination of clinical samples and 3D bioprinted scaffolds

[0169] In a preferred embodiment, a 3D bioprinted scaffold is fabricated to mimic the stiffness of a patient's liver, replicating the biophysical parameters of a clinical sample used to study cell behavior. In other embodiments, an ECM stiffness gradient is generated, for example, by controlling the degree of photopolymerization.

[0170] Measurements of fresh liver tissue and bioprinted scaffolds were performed on samples embedded in 4% low-melting-point agarose and cut into 50 μm sections using a Leica VT1000S vibratory microtome (Buffalo Grove, IL). Measurements of frozen liver tissue embedded in OCT compounds (Sakura, Torrance, CA) were performed by rapid freezing via direct immersion in liquid nitrogen and cut into 50 μm sections using a Leica CM1900-13 cryostat. Measurements were performed using an MFP3D-BIO inverted optical atomic force microscope (AFM) (Asylum Research, Santa Barbara, CA) mounted on a Nikon TE200-U inverted fluorescence microscope (Melville, NY). Indentation was performed using a silicon nitride cantilever (k=0.06 N / m) modified with a 5 μm diameter borosilicate glass spherical tip (Novascan Tech, Ames, IA). The cantilever was calibrated using a thermal oscillation method for each experiment. AFM force mapping was performed on a 90 μm × 90 μm field. Gradients were generated after acquisition. Each experimental group included at least 3 different mice, with 3 slices per mouse, and 2 gradients generated for each slice (one orthogonal to the fibrils and one parallel to the fibrils). Data analysis was performed using the Hertz model in Igor Pro v. 6.22A (WaveMetrics, Lake Oswego, OR) with a Poisson's ratio of 0.5.

[0171] To replicate the biophysical characteristics of clinical samples onto a 3D bioprinted scaffold, the stiffness values ​​of both tumor and non-tumor tissues from the patient were first measured using AFM. Based on the results, the 3D scaffold was then constructed by varying the percentage of the hydrogel (Figure 4a).

[0172] 6. Microfluidic perfusion

[0173] In some implementations, after biomanufacturing, the hydrogel is connected to a microfluidic system for the delivery of nutrients, chemicals, proteins, and cells in culture models accompanied by time-dependent diffusion. Figure 8AThe use of a microfluidic continuous perfusion model extended cell viability and culture duration. Due to the diffusive properties of the hydrogel used in the model, a nutrient gradient could be generated, thus creating a hypoxic region similar to that observed in the core of a patient's tumor. Figure 8B Importantly, screening of novel immunotherapies, such as pembrolizumab (anti-PD1), monalizumab (anti-NKG2a), and ipilimumab (anti-CTLA-4), can also be performed in the system. Microfluidics with flow rates ranging from 0 to 50 μL / min can be used in constructs for the delivery of nutrients, chemicals, proteins, and cells.

[0174] 7. Cell characteristics and viability

[0175] In the preferred form, high cell viability is achieved in the hydrogel.

[0176] In some forms, after biomanufacturing, all cell-loaded hydrogels are cultured for 14 days under standard conditions, with nutrients replenished via microfluidics. Confocal microscopy confirms the invention's support as a long-term 3D tumor culture system. Figure 8C Furthermore, the presence of patient-derived ECM is essential for stimulating tumor cell proliferation. Figure 8D ).

[0177] 8. Microstructure analysis of 3D bioprinted scaffolds using scanning electron microscopy.

[0178] Standard SEM microscopy can be applied to the lyophilized hydrogels. Hydrogel samples were dried by lyophilization (samples of 2 mm × 2 mm × 2 mm were first frozen at -30°C for 3 hours, then lyophilized at -90°C for 12 hours at 0.1 mbar using a Gregor Instrument lyophilizer). The samples were then attached to an aluminum sample holder using conductive tape and covered with a thin layer of platinum (vacuum sputter coater SCD 050; Leica, Austria). The samples were then inserted into an SEM microscope and observed in high vacuum using a secondary electron detector with an accelerating voltage of 10 kV. Figure 8E The presence of pores allows for cell interactions and biological functions, such as cell migration and phagocytosis. Figure 8F ).

[0179] D. Stimulus-activated programmable building module manufacturing in 4D printing

[0180] The fabrication of 4D bioprinted scaffolds involves creating 3D structures with integrated vascular networks. Exemplary techniques for incorporating these networks involve printing vascular systems using an aqueous volatile ink made from Pluronic F127, which can then be easily removed under mild conditions. This triblock copolymer was chosen because of its dual advantages: its printability facilitates the printing of synthetic microvascular networks, and, importantly, it exhibits bioinertness to a variety of cell types during the short period required for scaffold fabrication.

[0181] A key feature of the Pluronic F127 is its ability to undergo thermally reversible gelation. Specifically, the volatile ink transforms into a liquid state with reduced elasticity at temperatures below approximately 4°C. This property significantly facilitates ink removal from the printed scaffold, allowing for the creation of precise internal voids or channels that mimic vascular structures. This approach enhances the fidelity and functionality of 3D bioprinted scaffolds, enabling the development of more complex and biologically relevant tissue models.

[0182] Engineered tissue modules can be fabricated using a layer-by-layer technique involving continuous printing with multiple bio-inks. In an exemplary method, the periphery of each module is outlined with PDMS (polydimethylsiloxane) ink, establishing defined boundaries for subsequent bio-ink deposition. After digesting tumor tissue, isolated hepatocellular carcinoma (HCC) cells are suspended and adjusted to a specific concentration. These cells are then combined with a bio-ink composed of methacrylamide gelatin (GelMA) and sodium alginate.

[0183] The HCC cell-loaded construct can then be stabilized by immersing it in a 100 mM calcium chloride (CaCl2) solution for one minute. This process enhances the structural integrity of the construct and utilizes calcium ions (CaCl2). 2+ The ability to promote crosslinking within the sodium alginate matrix was then enhanced. Subsequently, a volatile ink based on Pluronic F127 was patterned onto a glass substrate to define vascular system channels.

[0184] After the printing process is complete, the vascular system and cell-containing scaffold are encapsulated within a layer of pure GelMA bio-ink. The bio-ink is preheated to 37°C and then exposed to 405 nm blue light for at least 60 seconds to initiate cross-linking within the GelMA matrix to solidify the structure.

[0185] To complete the manufacturing process, the entire assembly is cooled to 4°C. This cooling step liquefies the Pluronic F127 ink, allowing it to be evacuated under a moderate vacuum. The removal of the volatile ink reveals carefully designed channels within the encapsulation structure, mimicking an interconnected network of blood vessels. This sophisticated procedure ensures the production of robust, biofunctional engineered tissue modules with potential applications in tissue engineering and regenerative medicine.

[0186] IV. Instructions for Use

[0187] A method for 3D in vitro cell culture using simulated hepatocellular carcinoma (HCC) and related tumor microenvironments (TME) from subjects is provided. The disclosed 3D bioprinted culture system simulates complex physiological architectures and provides integrated features including 3D scaffolds, multicellular compartments, and vascular systems to simulate dynamic in vivo flow. The disclosed 3D bioprinted culture platform also enables precise spatiotemporal control, which is well-suited for functional studies of metastasis mechanisms and drug responses.

[0188] 3D bioprinting culture systems are clinically relevant and biomimetic, making them suitable for in vitro assays such as personalized drug screening, drug discovery, drug testing, preclinical studies, three-dimensional biology research, and cell-based liver cancer screening.

[0189] Using a network of blood vessels and nutritional supplies, the system can last for at least two weeks, making it ideal for a variety of purposes, including screening for immunotherapeutic and chemopreventive drugs, testing of novel therapeutics, and research.

[0190] While not intended to be restrictive, 3D and 4D bioprinted culture systems can be used to (i) study the interaction between tumor and immune cells to discover new therapeutic targets; (ii) test the safety and efficacy of precision medicine drugs in patients with this need; and (iii) potentially be the first customized and patient-specific drug testing platform.

[0191] A. Mechanistic studies of tumor biology

[0192] A primary goal of cancer research is to understand tumor biology related to metastasis mechanisms and tumor microenvironment (TME), which can support the development of anticancer drugs and treatments. This is important because it is essential to understand how factors such as mechanical forces, shear stress, chemotaxis, and hypoxia influence tumor progression, invasion, metastasis, and ultimately, drug metabolism.

[0193] In microfluidic systems of 3D bioprinted scaffolds, the fabricated microchannels can be used to precisely control interstitial fluid flow or wall shear stress, pressure, etc. However, by integrating with various trace elements, the network can be made very similar to small capillaries in size and branching.

[0194] Metastasis remains the leading cause of death for most cancer patients and is increasingly becoming a major focus of cancer research (Hanahan D and Weinberg RA, Cell, 2011, 144, 646–674.). Cancer models, including in vitro cell cultures and animal models, have made significant contributions to the development of cancer diagnostics and treatments over the past few decades (Reddy BS, Lipids, 1992, 27, 807–813; Lee GY, Kenny PA, Lee EH and Bissell MJ, Nature Methods, 2007, 4, 359; Kashaninejad N, Nikmaneshi M, Moghadas H, Kiyoumarsi Oskouei A, Rismanian M, Barisam M, Saidi M and Firoozabadi B, Micromachines, 2016, 7, 130.). Most in vitro models utilize easy-to-use 2D platforms, but they are limited by their lack of ability to simulate the physiological in vivo environment (Tibbitt MW and Anseth KS, Biotechnology and Bioengineering, 2009, 103, 655–663). With scientists' continuous efforts to develop 3D systems, progress has been made in cancer research regarding 3D systems.

[0195] Compared to 2D platforms and other 3D platforms, the disclosed 3D bioprinted scaffold system is physiologically more relevant. It incorporates both normoxic (with a vascular system) and hypoxic (without a vascular system) compartments to reconstruct the tumor architecture and complexity observed in patient tumors. In a preferred embodiment, the method using a 3D bioprinted scaffold provides mechanistic information about liver cancer metastasis.

[0196] Solid TMEs generate numerous environmental stressors, such as acidic pH, nutrient depletion, hypoxia, or accumulated waste products, which weaken immune responses and inhibit the antitumor activity of NK and T cells. Microfluidic platforms for 3D bioprinted scaffolds facilitate the simulation and understanding of cancer metastasis processes and the interactions between tumor cells and immune cells. In a preferred form, the disclosed microfluidic-based 3D bioprinted scaffold systems can reveal cell-cell interactions, such as those between macrophages, tumor cells, and / or mesothelial cells. In some forms, tumor cell adhesion is studied under controlled static and shear conditions.

[0197] Cancer cells require extravasation, a process in which they migrate through blood vessels to secondary tissues or organs to form metastases. The endothelial barrier, which allows cancer cells to extravasate to reach metastatic sites, is a key regulator of metastasis. Cancer cells adhere to blood vessels, migrate through the endothelium, and eventually escape circulation to invade secondary sites. Therefore, in some forms, 3D bioprinted scaffolds aim to investigate the roles of surface adhesion molecules and endothelial permeability in cancer extravasation.

[0198] Studying angiogenesis, or blood vessel formation, remains a significant challenge. 3D bioprinted scaffolds comprise perfusion microvessels that can represent the inner lining of endothelial cells. Therefore, in some forms, 3D bioprinted scaffolds are used to study angiogenesis. Angiogenesis is the process by which new blood vessels grow from pre-existing vessels.

[0199] B. New therapeutic targets

[0200] There is a lack of effective treatments available for liver cancer. This system and method can be used for mechanistic studies of cancer biology in vitro under controlled conditions.

[0201] The disclosed 3D bioprinted scaffold system includes immune cells, thus enabling an understanding of the process of immunosuppression and, consequently, the development of novel immunotherapies by bridging the gap between in vitro and animal models.

[0202] In some forms, this method can identify novel therapeutic targets in liver cancer. In a preferred form, the method enables the validation of novel therapeutic targets on 3D bioprinted scaffolds. In a further form, two or more therapeutic targets are targeted simultaneously to enhance efficacy.

[0203] C. The efficacy of precision medicine in individual patients

[0204] This system and method can be used to investigate the efficacy of one or more existing therapies (including chemotherapeutic agents and immunotherapeutic agents) to identify the most effective treatments in reducing or inhibiting the proliferation and / or viability of cancer cells in patients from which the biomaterials of the 3D bioprinted scaffold are derived. For example, such as Figures 10A-10C As shown, the drug response of two exemplary drugs for HCC treatment, sorafenib and cisplatin, was verified using the disclosed 3D bioprinted scaffold. Figures 10A-10C Using a multi-compartment design, the cytotoxic effects of different drugs at different doses were simultaneously evaluated in regions with different cell populations (including healthy hepatocytes) using both live and dead cell assays. Figure 10A Compared to 2D culture, the response rates of both drug doses in the 3D construct showed similar clinical outcomes. Figure 10B and 10CIn another instance, the efficacy and toxicity of CAR-T cell in situ programming of a 3D bioprinted scaffold were tested. This invention, combined with patient-derived tumor and immune cells, was used for the reprogramming of a viral platform (pLV-scFV-GPC3-CD28-41BB-CD3ζ-GFP). Figure 11 A). The translational and antitumor efficacy of this treatment can be assessed using live-dead cell assays and used as a preclinical reference for further clinical applications. Therefore, the disclosed 3D bioprinted scaffold is suitable for in vitro assays, drug discovery, drug testing, preclinical studies, three-dimensional biology research, and cell-based screening for liver and cholangiocarcinoma.

[0205] In some forms, the disclosed methods are applicable to guiding treatment strategies for subjects identified as having HCC. These methods can aid in the risk stratification and effective management of patients with HCC.

[0206] In other forms, this method categorizes cancer patients as responders or non-responders to current therapies. In some forms, the patient is a non-responder to one or more existing therapies.

[0207] Screening the efficacy of exemplary conventional cancer therapeutics (including chemotherapeutic agents, cytokines, and chemokines) on 3D bioprinted scaffolds. Most chemotherapeutic agents can be categorized into alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents. These drugs affect cell division or DNA synthesis and function in some way. Other therapeutics include monoclonal antibodies and tyrosine kinase inhibitors, such as imatinib mesylate (GLEEVEC® or GLIVEC®), which directly targets molecular abnormalities in certain types of cancer (chronic myeloid leukemia, gastrointestinal stromal tumors).

[0208] In some forms, one or more therapies that will be screened for efficacy on 3D bioprinted scaffolds are immunotherapies, such as those using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists) to inhibit checkpoint proteins (e.g., components of the PD-1 / PD-L1 axis or CD28-CTLA-4 axis), adoptive T-cell therapy, and / or cancer vaccines. Exemplary immune checkpoint modulators for immunotherapy include pembrolizumab (anti-PD1 mAb), durvalumab (anti-PDL1 mAb), PDR001 (anti-PD1 mAb), atezolizumab (anti-PD1 mAb), nivolumab (anti-PD1 mAb), trimemumab (anti-CTLA4 mAb), avelumab (anti-PD1 mAb), and RG7876 (CD40 agonist mAb).

[0209] In a preferred embodiment, the method further includes the step of administering an effective amount of a therapeutic agent identified as effective for the patient using a 3D bioprinted scaffold to reduce or inhibit the proliferation, migration, invasion, motility, and / or metastasis of cancer cells in the patient. In a further preferred embodiment, the therapeutic agent identified as effective for the patient using a 3D bioprinted scaffold effectively reduces or inhibits tumor growth, tumor burden, and / or increases the survival rate of the subject.

[0210] D. Patient-specific drug screening and testing platform

[0211] This system and method can be used to investigate the activity or applicability of one or more test compounds in treating or alleviating or preventing one or more symptoms of liver cancer. This method can effectively screen for new therapeutic agents that reduce or inhibit the proliferation and / or viability of cancer cells within a 3D bioprinted scaffold, compared to untreated control cancer cells in a separate 3D bioprinted scaffold prepared in a similar manner using cells and biomaterials derived from the same patient.

[0212] In some forms, the method involves contacting cancer cells with a potential therapeutic agent, and selecting the therapeutic agent as a suitable new drug candidate if the therapeutic agent reduces or inhibits the proliferation and / or activity of cancer cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100% compared to untreated control cancer cells.

[0213] In a preferred embodiment, the method further includes administering a novel drug identified as effective for the 3D bioprinted scaffold to a patient in an amount that effectively reduces or inhibits the proliferation, migration, invasion, motility, and / or metastasis of cancer cells, wherein the cells and biomaterials of the 3D bioprinted scaffold are derived from the patient. In a further preferred embodiment, the novel drug identified as effective for the 3D bioprinted scaffold effectively reduces or inhibits tumor growth, tumor burden, and / or increases the survival rate of the subject.

[0214] E. Clinically relevant diagnostic platform

[0215] As mentioned above, liver cancer is characterized by cellular heterogeneity and a complex landscape of various tumor immune microenvironment (TIME) subtypes (Xue, et al., Nature 612:141-7 (2022)). This variability plays a crucial role in the heterogeneous responses observed in immunotherapy (Atkins et al., Br J Cancer 123:1496-501 (2020)). These findings highlight the importance of developing drug screening models that can accurately replicate patient-specific timeframes to effectively predict treatment outcomes.

[0216] To meet this need, the described 3D bioprinted scaffold emerges as a customized in vitro diagnostic tool, providing a high-fidelity platform for precision medicine in liver cancer. This scaffold is capable of classifying immune subtypes and mimicking the individual tumor immune environment, thereby facilitating precision medicine approaches. It is specifically designed to evaluate the effectiveness and safety of various treatment options, including conventional therapies, targeted molecular therapies, and immunotherapies.

[0217] The construction of the 3D bioprinted scaffold involves the use of bio-inks embedded with loaded cells and extracellular matrix (ECM) components. These bio-inks are fabricated using advanced bioprinting techniques based on detailed patient-specific TIME maps derived from proteomics, RNA sequencing, and spatial transcriptomics data. By integrating this comprehensive molecular and spatial information, the scaffold provides a precise, patient-specific platform for assessing treatment response, paving the way for more personalized and effective liver cancer treatment strategies.

[0218] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0219] 1. A three-dimensional (3D) cell culture system comprising a first compartment containing tumor cells, a second compartment containing non-tumor cells, and a third compartment containing a vascular system.

[0220] 2. The 3D cell culture system described in paragraph 1, wherein the tumor cells belong to liver cancer.

[0221] 3. The 3D cell culture system described in paragraph 1 or 2, wherein the non-tumor cells are hepatocytes and / or endothelial cells.

[0222] 4. The 3D cell culture system of any one of paragraphs 1-3, wherein the first compartment and the second compartment further comprise one or more types of immune cells.

[0223] 5. The 3D cell culture system of any one of paragraphs 1-4, wherein the immune cells are innate and adaptive immune cells selected from the group consisting of macrophages, neutrophils, natural killer cells and T lymphocytes.

[0224] 6. The 3D cell culture system described in paragraph 5, wherein the macrophages are tumor-associated macrophages (TAMs), and wherein the macrophages are CD68+CD163+.

[0225] 7. The 3D cell culture system described in paragraph 5, wherein the macrophages are CD68+CD206+.

[0226] 8. The 3D cell culture system described in paragraph 5, wherein the T lymphocytes are CD3+CD4+ T cells and / or CD3+CD8+ T cells.

[0227] 9. The 3D cell culture system of any one of paragraphs 1-8, wherein one or more of the first, second and third compartments further comprises an extracellular matrix.

[0228] 10. The 3D cell culture system of any one of paragraphs 1-9, wherein one or more of the first, second and third compartments further comprises a hydrogel.

[0229] 11. The 3D cell culture system of any one of paragraphs 1-10, wherein the system comprises an oxygen gradient to form one or more normoxic zones and one or more hypoxic zones.

[0230] 12. The 3D cell culture system of any one of paragraphs 1-11, wherein the tumor cells and non-tumor cells are both derived from liver tumor tissue of the same subject.

[0231] 13. The 3D cell culture system of any one of paragraphs 9-12, wherein the tumor cells, non-tumor cells and extracellular matrix are derived from liver tumor tissue of the same subject.

[0232] 14. The 3D cell culture system described in paragraph 12 or 13, wherein the subject has hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma.

[0233] 15. The 3D cell culture system of any one of paragraphs 10-14, wherein the hydrogel is a natural or synthetic hydrogel selected from the group consisting of alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, and combinations thereof.

[0234] 16. The 3D cell culture system of any one of paragraphs 10-15, wherein the hydrogel has a stiffness similar to that of the liver tissue of a subject with hepatocellular carcinoma.

[0235] 17. The 3D cell culture system of any one of paragraphs 1-16, wherein the system comprises at least one inlet and at least one outlet for microfluidic connection.

[0236] 18. The 3D cell culture system of any one of paragraphs 1-17, wherein the vascular system of the 3D cell culture system comprises at least one microfluidic channel.

[0237] 19. The 3D cell culture system of any one of paragraphs 1-18, wherein the vascular system of the 3D cell culture system comprises 2 to 20, preferably 3 to 5, microfluidic channels.

[0238] 20. The 3D cell culture system of any one of paragraphs 17-19, wherein the at least one inlet and the at least one outlet are fluidly connected to a microfluidic channel.

[0239] 21. The 3D cell culture system of any one of paragraphs 1-20, wherein the 3D cell culture system has a thickness of at least about 10 mm.

[0240] 22. The 3D cell culture system of any one of paragraphs 1-21, wherein the 3D cell culture system has a size of at least about 10 mm x 10 mm x 0.5 mm.

[0241] 23. A method for preparing a three-dimensional (3D) cell culture system, the method comprising:

[0242] (a) Isolation of tumor cells, non-tumor cells and extracellular matrix (ECM) from tumor samples;

[0243] (b) Mixing one or more of tumor cells, non-tumor cells, and ECM with a hydrogel to provide one or more mixtures; and

[0244] (c) 3D bioprinting of one or more mixtures from step (b) to assemble a 3D cell culture system.

[0245] 24. The method described in paragraph 23, which further compares the step of collecting the tumor sample from the subject prior to step (a).

[0246] 25. The method described in paragraph 23 or 24, wherein the tumor sample is a liver biopsy of a subject with liver cancer.

[0247] 26. The method of any one of paragraphs 23-25, wherein the 3D bioprinting prints 5 or more layers.

[0248] 27. The method of any one of paragraphs 23-26, wherein the hydrogel is a natural or synthetic hydrogel.

[0249] 28. The method of any one of paragraphs 23-27, wherein the hydrogel is formed by photochemical crosslinking of a photosensitive polymer and one or more photoinitiators.

[0250] 29. The method of paragraph 28, wherein the photosensitive polymer is selected from the group consisting of: alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, methacrylamide polycaprolactone triol (PCLMA), methacrylamide hyaluronic acid (HAMA), and combinations thereof.

[0251] 30. The method of paragraph 28 or 29, wherein the photoinitiator is selected from the group consisting of: lithium phenyl-2,4,6-trimethylbenzoylphosphinate, lithium acylphosphinate, Irgacure 2959 and camphorquinone.

[0252] 31. The method of any one of paragraphs 28-30, wherein the hardness of the hydrogel is controlled by the degree of photochemical crosslinking.

[0253] 32. The method of any one of paragraphs 23-31, wherein the hydrogel has a stiffness similar to that of the liver tissue of a subject with liver cancer.

[0254] 33. The method of any one of paragraphs 23-32, wherein the 3D cell culture system comprises at least one inlet and at least one outlet for microfluidic connection.

[0255] 34. The method of any one of paragraphs 23-33, wherein the 3D cell culture system comprises at least one microfluidic channel.

[0256] 35. The method of any one of paragraphs 23-34, wherein the 3D cell culture system comprises 2 to 20, preferably 3 to 5, microfluidic channels.

[0257] 36. The method of any one of paragraphs 33-35, wherein the at least one inlet and the at least one outlet are fluidly connected to a microfluidic channel.

[0258] 37. The method of any one of paragraphs 33-36, wherein the 3D cell culture system has a thickness of at least about 10 mm.

[0259] 38. The method of any one of paragraphs 33-37, wherein the 3D cell culture system has a size of at least about 10 mm x 10 mm x 0.5 mm.

[0260] 39. The method of any one of paragraphs 33-38, wherein the bioprinting comprises suspension, extrusion, microforming, digital light processing, stereolithography, and combinations thereof.

[0261] 40. The method of any one of paragraphs 33-39, wherein the 3D cell culture system comprises at least one compartment containing tumor cells, at least one compartment containing non-tumor cells, and at least one compartment containing a vascular system.

[0262] 41. A method for using the 3D cell culture system described in any one of paragraphs 1-22 for mechanism studies, drug screening, immunotherapy testing, personalized medicine and / or biomarker discovery.

[0263] 42. The method described in paragraph 41, wherein the 3D cell culture system is used to screen the efficacy of cancer therapies.

[0264] 43. The method described in paragraph 41 or 42, wherein the cancer therapy includes conventional chemotherapy agents.

[0265] 44. The method described in paragraph 43, wherein the conventional chemotherapeutic agent is selected from one or more of the group consisting of: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents.

[0266] 45. The method described in paragraph 41 or 42, wherein the cancer therapy includes an immunotherapy agent.

[0267] 46. ​​The method described in paragraph 45, wherein the immunotherapeutic agent is one or more immune checkpoint modulators selected from the group consisting of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists.

[0268] 47. The method of any one of paragraphs 42-46, further comprising identifying a cancer therapy that is most effective in reducing or inhibiting the proliferation, migration, invasion, movement and / or metastasis of said tumor cells in said 3D cell culture system.

[0269] 48. The method of paragraph 47, further comprising administering to the subject a cancer therapy of optimal efficacy in an amount that effectively reduces or inhibits tumor growth, tumor burden and / or increases the subject's survival.

[0270] 49. The method described in paragraph 41, wherein the 3D cell culture system is used for mechanistic studies of tumor biology.

[0271] 50. The method described in paragraph 49, wherein the 3D cell culture system provides mechanistic information on liver cancer metastasis and novel therapeutic targets.

Claims

1. A three-dimensional (3D) cell culture system comprising a first compartment containing tumor cells, a second compartment containing non-tumor cells, and a third compartment containing a vascular system.

2. The 3D cell culture system of claim 1, wherein the tumor cells belong to liver cancer.

3. The 3D cell culture system of claim 1 or 2, wherein the non-tumor cells are hepatocytes and / or endothelial cells.

4. The 3D cell culture system of any one of claims 1-3, wherein the first compartment and the second compartment further comprise one or more types of immune cells.

5. The 3D cell culture system according to any one of claims 1-4, wherein the immune cells are innate and adaptive immune cells selected from the group consisting of macrophages, neutrophils, natural killer cells and T lymphocytes.

6. The 3D cell culture system of claim 5, wherein the macrophages are tumor-associated macrophages (TAMs), and wherein the macrophages are CD68+CD163+.

7. The 3D cell culture system of claim 5, wherein the macrophages are CD68+CD206+.

8. The 3D cell culture system of claim 5, wherein the T lymphocytes are CD3+CD4+ T cells and / or CD3+CD8+ T cells.

9. The 3D cell culture system of any one of claims 1-8, wherein one or more of the first, second, and third compartments further comprises an extracellular matrix.

10. The 3D cell culture system of any one of claims 1-9, wherein one or more of the first, second, and third compartments further comprises a hydrogel.

11. The 3D cell culture system of any one of claims 1-10, wherein the system comprises an oxygen gradient to form one or more normoxic zones and one or more hypoxic zones.

12. The 3D cell culture system of any one of claims 1-11, wherein the tumor cells and non-tumor cells are both derived from liver tumor tissue of the same subject.

13. The 3D cell culture system of any one of claims 9-12, wherein the tumor cells, non-tumor cells, and extracellular matrix are derived from liver tumor tissue of the same subject.

14. The 3D cell culture system of claim 12 or 13, wherein the subject has hepatocellular carcinoma and / or intrahepatic cholangiocarcinoma.

15. The 3D cell culture system of any one of claims 10-14, wherein the hydrogel is a natural or synthetic hydrogel selected from the group consisting of alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, and combinations thereof.

16. The 3D cell culture system of any one of claims 10-15, wherein the hydrogel has a stiffness similar to that of the liver tissue of a subject with liver cancer.

17. The 3D cell culture system of any one of claims 1-16, wherein the system comprises at least one inlet and at least one outlet for microfluidic connection.

18. The 3D cell culture system of any one of claims 1-17, wherein the vascular system of the 3D cell culture system comprises at least one microfluidic channel.

19. The 3D cell culture system according to any one of claims 1-18, wherein the vascular system of the 3D cell culture system comprises 2 to 20, preferably 3 to 5, microfluidic channels.

20. The 3D cell culture system of any one of claims 17-19, wherein the at least one inlet and the at least one outlet are fluidly connected to a microfluidic channel.

21. The 3D cell culture system according to any one of claims 1-20, wherein the 3D cell culture system has a thickness of at least about 10 mm.

22. The 3D cell culture system of any one of claims 1-21, wherein the 3D cell culture system has a size of at least about 10 mm x 10 mm x 0.5 mm.

23. A method for preparing a three-dimensional (3D) cell culture system, the method comprising: (a) Isolation of tumor cells, non-tumor cells and extracellular matrix (ECM) from tumor samples; (b) Mixing one or more of tumor cells, non-tumor cells, and ECM with a hydrogel to provide one or more mixtures; and (c) 3D bioprinting of one or more mixtures from step (b) to assemble a 3D cell culture system.

24. The method of claim 23, further comparing the step of collecting the tumor sample from the subject prior to step (a).

25. The method of claim 23 or 24, wherein the tumor sample is a liver biopsy of a subject suffering from liver cancer.

26. The method of any one of claims 23-25, wherein the 3D bioprinting prints 5 or more layers.

27. The method of any one of claims 23-26, wherein the hydrogel is a natural or synthetic hydrogel.

28. The method of any one of claims 23-27, wherein the hydrogel is formed by photochemical crosslinking of a photosensitive polymer and one or more photoinitiators.

29. The method of claim 28, wherein the photosensitive polymer is selected from the group consisting of: alginate, gelatin, polyethylene glycol diacrylate (PEGDA), methacrylamide gelatin (GelMA), F127, polyacrylamide, methacrylamide polycaprolactone triol (PCLMA), methacrylamide hyaluronic acid (HAMA), and combinations thereof.

30. The method of claim 28 or 29, wherein the photoinitiator is selected from the group consisting of: lithium phenyl-2,4,6-trimethylbenzoylphosphinate, lithium acylphosphinate, Irgacure 2959, and camphorquinone.

31. The method of any one of claims 28-30, wherein the hardness of the hydrogel is controlled by the degree of photochemical crosslinking.

32. The method of any one of claims 23-31, wherein the hydrogel has a stiffness similar to that of the liver tissue of a subject suffering from liver cancer.

33. The method of any one of claims 23-32, wherein the 3D cell culture system comprises at least one inlet and at least one outlet for microfluidic connection.

34. The method of any one of claims 23-33, wherein the 3D cell culture system comprises at least one microfluidic channel.

35. The method of any one of claims 23-34, wherein the 3D cell culture system comprises 2 to 20, preferably 3 to 5, microfluidic channels.

36. The method of any one of claims 33-35, wherein the at least one inlet and the at least one outlet are fluidly connected to a microfluidic channel.

37. The method of any one of claims 33-36, wherein the 3D cell culture system has a thickness of at least about 10 mm.

38. The method of any one of claims 33-37, wherein the 3D cell culture system has a size of at least about 10 mm x 10 mm x 0.5 mm.

39. The method of any one of claims 33-38, wherein the bioprinting comprises suspension, extrusion, microforming, digital light processing, stereolithography, and combinations thereof.

40. The method of any one of claims 33-39, wherein the 3D cell culture system comprises at least one compartment containing tumor cells, at least one compartment containing non-tumor cells, and at least one compartment containing a vascular system.

41. A method for using the 3D cell culture system of any one of claims 1-22 for mechanism studies, drug screening, immunotherapy testing, personalized medicine and / or biomarker discovery.

42. The method of claim 41, wherein the 3D cell culture system is used to screen the efficacy of cancer therapies.

43. The method of claim 41 or 42, wherein the cancer therapy comprises conventional chemotherapy agents.

44. The method of claim 43, wherein the conventional chemotherapeutic agent is selected from one or more of the group consisting of: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents.

45. The method of claim 41 or 42, wherein the cancer therapy comprises an immunotherapy agent.

46. ​​The method of claim 45, wherein the immunotherapeutic agent is one or more immune checkpoint modulators selected from the group consisting of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists.

47. The method of any one of claims 42-46, further comprising identifying a cancer therapy that is most effective in reducing or inhibiting the proliferation, migration, invasion, movement and / or metastasis of said tumor cells in the 3D cell culture system.

48. The method of claim 47, further comprising administering to the subject an amount of cancer therapy with optimal efficacy to effectively reduce or inhibit tumor growth, tumor burden, and / or increase the subject's survival.

49. The method of claim 41, wherein the 3D cell culture system is used for mechanistic studies of tumor biology.

50. The method of claim 49, wherein the 3D cell culture system provides mechanistic information on liver cancer metastasis and novel therapeutic targets.

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