EP4680717A1

EP4680717A1 - 3d bioprinting of tumor tissue for mechanistic study and drug screening

Info

Publication number: EP4680717A1
Application number: EP24769980.4A
Authority: EP
Inventors: Kwan Man; Wai Ho Oscar YEUNG; Fredrick Yum Hung CHENG; Jiye ZHU; Jian Lu; Zhengyi Mao
Original assignee: University of Hong Kong HKU
Current assignee: University of Hong Kong HKU
Priority date: 2023-03-14
Filing date: 2024-03-14
Publication date: 2026-01-21
Also published as: WO2024188290A1; CN120936704A

Abstract

A 3-dimensional (3D) bioprinted cell culture system of tumor tissue, methods of making and methods of use thereof are described. The 3D cell culture system provides multiple compartments and vasculature as well as cellular interfaces including tumor/non-tumor interface, tumor-vasculature interface to recreate tumor immune microenvironment. 3D bioprinted scaffold enables adjustment of stiffness to reflect the different stages of carcinogenesis including fibrosis, cirrhosis, and steatosis; and establishing chemical gradients, for example, oxygen levels, to mimic oxidative stress and hypoxia condition. Fabrication of the 3D cell culture is achieved by bioprinting such as micro-molding, inject exclusion and suspension bioprinting techniques.

Description

3D BIOPRINTING OF TUMOR TISSUE FOR MECHANISTIC STUDY AND DRUG SCREENING

This international patent application claims the benefit of U.S. Provisional Patent Application No.: 63/490,077 filed on March 14, 2023, the entire content of which is incorporated by reference for all purpose.

FIELD OF THE INVENTION

The invention generally relates to a three-dimensional cell culture system of tumor microenvironment.

BACKGROUND OF THE INVENTION

Liver cancer is the sixth most diagnosed cancer and the third leading cause of cancer death worldwide 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 cause of cancer death in China (Jiang D, et al., Cancer Communications 41: 1024-36 (2021) ) . The major histo-types of liver cancer include hepatocellular carcinoma (HCC) , intrahepatic cholangiocarcinoma (ICC) , and combined hepatocellular cholangiocarcinoma (CHC) . HCC has a very high metastatic and fatality rate (overall mortality to incidence ratio >90%) . Traditional risk factors include viral infection, alcoholic consumption, fatty diet, cirrhosis. toxin exposure (Mittal S, El-Serag HB. Journal of clinical gastroenterology 47 Suppl: S2-S6 (2013) ) . Liver stiffness which directly reflect the fibrotic and inflammatory status of the organ, measured by non-invasive ultrasound-or magnetic resonance imaging (MRI) -based elastography, has also been shown to associate with increased risk of HCC (Mueller S, Sandrin L. Hepatic medicine: evidence and research 2: 49-67 (2010) ) . It represents the main indication for screening and surveillance of the disease development. Curative treatment options, including surgical and radiofrequency ablation, can only be applied to patients with limited tumor burden (Hartke J, Johnson M, Ghabril M. Seminars in Diagnostic Pathology 34: 153-9 (2017) ) . Systemic and immunotherapies show promising results in some patients, however, the low response rate and limited survival benefits are considered to be ineffective and require further experimental evidence and clinical trials (Zhong C, et al., Frontiers in Oncology 11 (2021) ) .
Current available platforms for mechanistic studies and drug screening in cancer are summarized in Figure 1. Traditional 2-dimensional (2D) and 3D co-culture methods have demonstrated practicality, being easier to manage and capable of replication with high throughput. However, applying these methods to translational medicine presents many challenges and uncertainties. (Law AMK, et al., Frontiers in Oncology 11 (2021) ) :
1. A lack of physical and biological relevance due to static conditions that rely on unrealistic supplementation and waste of nutrients;
2. An underestimation of the potential value of the spatial architecture of the tumor immune microenvironment (TIME) in influencing disease progression and treatment response;
3. The use of a single type of cancer and immune cells, which fails to fully elucidate the cellular complexity of the TIME;
4. An inability to precisely replicate the mechanical and immunological characteristics specific to patients with different liver pathologies, particularly in terms of stiffness; and
5. A deficiency in the use of patient-derived materials, including cells and the extracellular matrix, which are crucial for the development of personalized therapies.
Some of these disadvantages can be mitigated through in vivo models and clinical studies, which are capable of capturing the complexity and pathophysiological relevance of diseases. Nevertheless, significant drawbacks of both human and animal experimental models persist, presenting major obstacles to successful translational research. These include but are not limited to:
1. Inconsistency and tumor heterogeneity, which complicate the extrapolation of results across different models and patient populations; and
2. The extensive resources required and the prolonged duration of experimental periods, which can limit the feasibility and scalability of these approaches.
In recent years, 3D in vitro models have gained attention for their ability to replicate tissue-like structures and cellular 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.
Therefore, it is an object of the invention to provide three dimensional, engineered, biological, highly clinically relevant, culturable, and perfusable, bio-printed liver tumor tissue constructs and methods of making thereof to reconstitute tumor architecture and liver tumor immune microenvironment.
It is another object to provide clinically relevant 3D experimental models suitable for mechanistic studies, drug screening, immunotherapy testing, personalized medicine, and/or biomarker discovery in liver cancer.

SUMMARY OF THE INVENTION

3-dimensional (3D) cell culture systems of engineered tumor tissue constructs and methods of fabrication and use are described. The preferred “mini-liver tumor” 3-dimensional (3D) cell culture system of engineered liver tumor tissue constructs and methods of fabrication and use are provided. The 3D cell culture system is prepared based on the incorporation of cells of interest obtained from patients or cell-lines in natural (alginate) or synthetic (PEGDA, GelMA, F127, polyacrylamide) hydrogels. The 3D cell scaffold can be heterotypic or homotypic and the cultured cells can include, but are not limited to, tumor cells, immune cells or non-pathological hepatic parenchymal cells and non-parenchymal cells. When used for culturing two or more types of heterotypic cells, the 3D cell scaffold can be used, for example, to study the cell-cell interactions between different types of cells, cell-ECM interaction for mechanistic study and drug screening in a heterotypic cell environment.
The disclosed 3-D liver tumor scaffold uses 3D bio-printing technology to re-create “mini-liver tumor. ” By precisely reconstructing different tumor compartments each embedded with relevant immune cells, the highly clinical mimicking model including the interactive of innate and adaptive tumor immunity components and tumor architecture permit the identification of new HCC related tumor promoting mechanism and personalized drug screening.
Typically, the 3D cell culture system includes a first compartment comprising tumor cells, a second compartment comprising non-tumoral cells, and a third compartment comprising vasculature. In some forms, the tumor cells are of liver cancer, and/or the non-tumoral cells are hepatocytes and/or endothelial cells. In preferred forms, the first and the 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, macrophages are tumor-associated macrophages (TAMs) , and are CD68+CD163+. In other forms, the first and the second compartments include macrophages that are CD68+CD206+. In some forms, T-lymphocytes are CD3+CD4+ T cells and/or CD3+CD8+ T cells.
Preferably, the one or more of the first, second, and third compartments further includes extracellular matrix and/or hydrogel. The 3D cell culture system contains vasculature that allows chemical gradient. Thus, in some forms, the 3D bio-printed scaffold incorporates both normoxic (with vasculature) and hypoxic (without vasculature) compartments to recreate the tumor architecture and complexity observed in patients’ tumor. Typically, the tumor cells, non-tumoral cells, and/or the extracellular matrix are derived from liver tumor tissues of the same subject, for example, one with hepatocellular carcinoma and/or intrahepatic cholangiocarcinoma. In some forms, the hydrogels are natural or synthetic hydrogels such as alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, combinations thereof. To better mimic patient-specific liver conditions such as cirrhosis can be mimicked in the 3D cell culture system by adjusting hydrogel stiffness. Thus, in preferred forms, the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer. Generally, the 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 between 2 and 20, preferably between 3 and 5, microfluidic channels. The inlet and outlet are fluidically connected to the one or more microfluidic channel (s) to mimic the vasculature in the tumor tissue. The 3D cell culture system typically has at least about 10 mm in thickness, for example, a dimension of at least about 10mm x 10mm x 0.5mm.
Methods of fabricating the 3-dimensional (3D) cell culture system are also provided. Generally, the methods include the steps of a) isolating tumor cell, non-tumoral cells, and extracellular matrix (ECM) from a tumor sample; b) mixing with a hydrogel one or more of the tumor cells, non-tumoral cells, and ECM to provide one or more mixtures; and c) 3D bioprinting of the one or more mixtures from step b) to assemble the 3D cell culture system. The tumor sample is typically collected from a subject prior to step a) . in some forms, the tumor sample is a liver biopsy of a subject with liver cancer such as HCC. Methods typically prints 5 or more layers to form the 3D cell culture system. In some forms, methods use hydrogels that are photochemically crosslinked from photosensitive polymers and one or more photo-initiators. In some forms, photosensitive polymers include alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, polycaprolactone triol Methacryloyl (PCLMA) , Hyaluronic acid Methacryloyl (HAMA) , and combinations thereof. In some forms, the photo-initiator includes lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate, lithium acylphosphinate, Irgacure 2959, and camphorquinone. The photochemical crosslinking allows control of the stiffness of the hydrogels and in preferred forms, the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer. Typically, method of fabricating the 3D cell culture system introduces at least one inlet, at least one outlet for microfluidic connection, which are fluidically connected to the microfluidic channel (s) . In preferred forms, the better mimic the vasculature of liver tumor tissue, the 3D cell culture system includes between 2 and 20, preferably between 3 and 5, microfluidic channels. The fabricated 3D cell culture system has at least about 10 mm in thickness, e.g., a dimension of at least about 10mm x 10mm x 0.5mm. In some form, bioprinting uses suspension, extrusion, micro-molding, digital light processing, stereolithography, and combinations thereof. The fabricated 3D cell culture system includes at least one compartment having tumor cells, at least one compartment having non-tumoral cells, and at least one compartment having vasculature.
Methods of using the 3D cell culture system are also described. The application includes mechanistic studies, drug screening, immunotherapy testing, personalized medicine, and/or biomarker discovery. In preferred forms, the cell culture system is well suited for screening of cancer therapy for efficacy. Cancer therapy includes 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 of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists. In some forms, methods include identifying the cancer therapy having the best efficacy in reducing or inhibiting proliferation, migration, invasion, motility, and/or metastatic abilities of the tumor cells in the 3D cell culture system, optionally further include the step of administering the cancer therapy having the best efficacy to the subject in an amount effective to reduce or inhibit tumor growth, tumor burden, and/or increase survival of the subject. In other forms, the 3D cell culture system is suited for mechanistic studies of tumor biology, for example, providing mechanistic information and new therapeutic targets of metastasis in liver cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram showing current platforms available for mechanistic studies and drug screening including 2D monoculture, 3D co-culture on inserts, organ/tumor-on-a-chip, organoids/tumoroid, animal models, and human patients.
Figure 2 is a diagram outlining different components and features of the 3-dimensional scaffold.
Figure 3 is a table comparing features of different platforms including 2D monoculture, 3D co-culture on inserts, organ/tumor-on-a-chip, organoids/tumoroid, animal models, human patient, and tumor in cube (exemplary 3-dimensional scaffold described herein) .
Figures 4A-4C are diagrams outlining and comparing characteristics of liver tumor and the disclosed clinical mimetic model.
Figure 5A is a diagram showing vasculature and multi-cellular interface (e.g., tumor and non-tumor interface and tumor and vasculature interface) manufactured by 3D micro-molding/bioprinting. Figure 5B shows an exemplary 3D scaffold bio-printed using suspension bath and sacrificial extrusion (top) and an exemplary 4D scaffold as described herein e.g., a 4D stimuli-activated programmable platform (bottom) .
Figures 6A and 6B are diagrams showing four exemplary constructs including construct 1 bio-printed by micro-molding; construct 2 bio-printed by suspension, sacrificial extrusion techniques; construct 3 bio-printed by stereolithography; and construct 4 bio-printed by 4D assembled multifunctional building blocks.
Figures 7A-7B are exemplary procedures of manufacturing “tumor in cube” of construct 1 (FIG. 7A) , and “tumor in cube” of construct 2 (FIG. 7B) .
Figure 8A-8F are diagram outlining the features of 3-dimensional scaffold “tumor in cube” including a schematic diagram of running “tumor in cube” (FIG. 8A) ; hypoxic region generated by microfluidics (FIG. 8B) ; long term cell viability (FIG. 8C) ; patient-derived ECM promoting cell proliferation (FIG. 8D) ; capability of cell migration (FIG. 8E) and cell-cell interaction (FIG. 8F) .
Figures 9A and 9B show exemplary methods of simultaneous extraction and maintenance of patient derived ECM and cells. Figure 9A is a diagram outlining the steps of an exemplary method for simultaneously extracting and maintaining patient derived ECM and cells. Figure 9B are exemplary photographs and micrographs of different populations of cells from non-tumor (top) and tumor (bottom) specimens.
Figures 10A-10C show comparisons between the response of 2D construct and an exemplary 3D scaffold (as described herein) to two exemplary drugs commonly used for the treatment of used for hepatocellular carcinoma (HCC) , sorafenib (Figure 10A and 10B) and cisplatin (Figure 10C) .
Figure 11 is a diagram showing an exemplary 3D scaffold (as described herein) as anex vivo model for screening in situ programming of CAR-T cells. As a preclinical model for testing the efficacy and toxicity in situ programming of CAR-T cells, an exemplary 3D scaffold (as described herein) is incorporated with patient’s derived tumor and immune cells for reprogramming as a viral platform (pLV-scFV-GPC3-CD28-41BB-CD3ζ-GFP) .

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions
The term “bioprinting” refers to bioprinting technology based on layer-by-layer printing. Various biomaterials and cell types can be printed simultaneously onto a substrate of cell-compatible biomaterials to build 3D complex constructs 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.
The term “effective amount” or “therapeutically effective amount” means a dosage sufficient to treat, inhibit, or alleviate one or more symptoms of a disease state being treated or to otherwise provide a desired pharmacologic and/or physiologic effect. In some forms, the effective amount refers to the amount which is able to treat one or more symptoms of hepatocellular carcinoma (HCC) , reverse the progression of one or more symptoms of HCC, halt the progression of one or more symptoms of HCC, or prevent the occurrence of one or more symptoms of HCC in a subject to whom the formulation is administered, for example, as compared to a matched subject not receiving the compound. The precise dosage will vary according to a variety of factors such as subject-dependent variables (e.g., injury size/type, age, joint health, immune system health, etc. ) , the disease or disorder, and the treatment being administered. The effective amount can be relative to a control. Such controls are known in the art and discussed herein, and can be, for example the condition of the subject prior to or in the absence of administration of the drug.
The terms “treating” or “preventing” mean to ameliorate, reduce or otherwise stop a disease, disorder or condition from occurring or progressing in an animal which may be predisposed to the disease, disorder and/or condition but has not yet been diagnosed as having it; inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease or condition includes ameliorating at least one symptom of the disease or condition, even if the underlying pathophysiology is not affected, such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. Desirable effects of treatment include decreasing the rate of disease progression, ameliorating, or palliating the disease state, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more symptoms associated with HCC are mitigated or eliminated, including, but are not limited to, reducing and/or inhibiting rate of tumor cell proliferation/growth, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.
The term “pharmaceutically acceptable” or “biocompatible” refers to compositions, polymers and other materials and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. The phrase “pharmaceutically acceptable carrier” refers to pharmaceutically acceptable materials, compositions or vehicles, such as a liquid or solid filler, diluent, solvent or encapsulating material involved in carrying or transporting any subject composition, from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of a subject composition and not injurious to the patient.
The terms “inhibit” or “reduce” in the context of inhibition, mean to reduce or decrease in activity and quantity. This can be a complete inhibition or reduction in activity or quantity, or a partial inhibition or reduction. Inhibition or reduction can be compared to a control or to a standard level. Inhibition can be measured as a %value, e.g., from 1%up to 100%, such as 5%, 10, 25, 50, 75, 80, 85, 90, 95, 99, or 100%. For example, compositions including neutralizing antibodies against S100A10 may inhibit or reduce the activity and/or quantity of S100A10 by about 10%, 20%, 30%, 40%, 50%, 75%, 85%, 90%, 95%, or 99%from the activity and/or quantity of the same S100A10 in subjects that did not receive or were not treated with the compositions. In some forms, the inhibition and reduction are compared according to the level of mRNAs, proteins, cells, tissues, and organs.
The term “contacting” or “culturing ... with” is intended to include incubating the component (s) and the cell/tissue together in vitro (e.g., adding the compound to cells in culture) and the step of “contacting” or “culturing... with” can be conducted in any suitable manner. For example, the cells may be treated in adherent culture, in suspension culture, or in 3D culture; the components can be added temporally substantially simultaneously (e.g., together in a cocktail) or sequentially (e.g., within 1 hour, 1 day or more from an addition of a first component) . The cells can also be contacted with another agent such as a growth factor or other differentiation agent or environments to stabilize the cells, or to differentiate the cells further and include culturing the cells under conditions known in the art.
II. 3-Dimensional Bio-printed Scaffold
3-Dimensional (3D) bio-printed scaffolds prepared to mimic tumor microenvironment including a variety of immune cells, stromal cells, blood vessels, and extracellular matrix, are described. The described 3D bio-printed scaffolds can replicate the tumor immune microenvironment and architecture, offering a model that closely resembles clinical conditions for drug discovery and the investigation of liver disease pathologies.
Although not intended to be limiting, exemplary characteristics of the disclosed 3-D bio-printed mimetic model are shown in Figures 4A-4C. The design of the 3D bio-printed scaffold is clinically relevant and adept at 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 e.g., tumor and immune cells from the patient. This is achieved, in one exemplary form, by incorporating patient-specific biomaterials, such as patient-derived extracellular matrix (ECM) , and patient-specific cells, for example, tumor and immune cells from the patient. Additionally, the disclosed 3-D bio-printed scaffold can simulate patient-specific liver conditions, such as cirrhosis, by adjusting the stiffness of the hydrogel, and inflammation, through the deposition of immune cells. Furthermore, the disclosed 3-D bio-printed scaffold is capable of mimicking tumor architecture, achieved by creating multiple compartments and interfaces, including vasculature, tumor, and non-tumor compartments, thereby closely replicating the complexity of the human body.
In an exemplary form, the disclosed 3D bio-printed scaffold is a 3D liver tumor scaffold (also referred to as “Tumor in Cube” or “Mini liver tumor” ) . The 3D liver tumor scaffold utilizes 3D bio-printing technology to create a "mini-liver tumor" which precisely reconstructs different tumor compartments, each embedded with relevant immune cells. This 3D liver tumor scaffold closely mimics clinical scenarios, including the interaction of innate and adaptive tumor immunity components and tumor architecture, and is suitable for the identification of new hepatocellular carcinoma (HCC) related tumor-promoting mechanisms, and personalized drug screening. The benefits of the 3D bio-printed scaffold over current platforms are detailed in Figure 2. A direct comparison between the disclosed 3D bio-printed scaffold and other experimental tools is summarized in Figure 3. Unlike traditional 2D and 3D co-culture methods, the disclosed 3D bio-printed scaffold provides a matrix-rich 3D environment that supports multi-cellular interactions across different interfaces e.g., tumor/non-tumor interfaces, and tumor-vasculature interfaces. Additionally, the nutrient gradient generated within the system, which supports cell growth, is highly biomimetic. In contrast to the lengthy, expensive, and often inefficient in vivo models, the disclosed 3D bio-printed scaffold provides a faster, direct, consistent, and efficient platform for studying cell-cell interactions, dissecting disease mechanisms, identifying new biomarkers, and providing a reliable platform for drug development and personalized medicine (Figure 3) .
In some forms, the disclosed 3D bio-printed scaffolds are capable of regenerating specific cell differentiation and the reorganization of multiple cell clusters, as observed in tumor tissues collected from patients. In one exemplary form, the disclosed 3D bio-printed scaffolds regenerate the tumor heterogeneity that is characteristic of the tumor tissue obtained from patients.
In some forms, the disclosed 3-D bio-printed scaffolds regenerate the complexity of tumors with different functional units such as different cell types, extracellular matrix (ECM) , vasculature system, and multiple chemical factors. In an exemplary form, the disclosed 3-D bio-printed scaffolds mimic physiologically relevant tumor-promoting mechanical forces such as shear stress from the dynamic flow in the vasculature, tension from the solid tumor, and stiffness variation of ECM.
The 3D bio-printed scaffold generally contains multiple compartments. In one form, the 3D bio-printed scaffold contains three compartments: a first compartment containing tumor cells, a second compartment containing non-tumoral cells, and a third compartment containing vasculature. 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. An exemplary 3D bio-printed scaffold is illustrated in Figure 2 (see “Product Design” ) . The embodiment illustrated in Figure 2 has a first compartment containing non-tumor cells e.g., hepatocytes; a second compartment containing tumor cells; and a third compartment containing vasculature e.g., endothelial cells. In some forms, the 3D bio-printed scaffold includes additional compartments, such as a fourth compartment that contains tumor cells from the same patient but at a different stage of disease progression or from a different time point during treatment.
In some forms, the tumor cells and non-tumoral cells are both derived from liver tumor tissues of the same patient. In some forms, the tumor cells, non-tumoral cells, and extracellular matrix are derived from liver tumor tissues of the same subject.
In some forms, the tumor cells are liver cancer cells. In some forms, the non-tumoral cells are hepatocytes and/or endothelial cells. In some forms, the first and the 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.
A. Extracellular Matrix and Biomaterial
The ECM is considered a critical regulator of tissue homeostasis and organ function. It constitutes a complex network of proteins which together construct the overall architecture of an organ. The ECM includes a dynamic assembly of various ECM molecules which activate many intracellular signaling pathways important for modulating cell behavior. The ECM provides structural support, and imparts mechano-elastic properties to tissues, thereby influencing their physical behavior under various physiological conditions, and delivers environmental cues that impact cellular activities such as proliferation, survival, shape, migration, and differentiation of the cells enveloped by the ECM. Further, the ECM maintains cellular equilibrium, thereby affecting cell adhesion, cell proliferation, cell differentiation, cell migration, and establishing cell polarity. The ECM is important for preserving tissue mechanical integrity, facilitating cell signaling pathways, guiding morphogenetic processes, enhancing cell-cell communication, and modulating environmental interactions. The ECM is also involved in developmental processes and regeneration, where its composition and the interaction with cells guide tissue formation and healing. The ECM also acts as a critical barrier and filter, regulating the passage of substances between different tissue compartments.
Alterations in the ECM's composition and a disruption in its regulatory functions are linked to a broad spectrum of diseases. Dysregulation of ECM dynamics can lead to pathological conditions characterized by either excessive ECM deposition, such as in fibrosis, or insufficient ECM, which can compromise tissue integrity. These changes facilitate the development and progression of various diseases, including cardiovascular diseases, which may arise from the disruption of ECM in blood vessels; skin diseases, where ECM alterations affect skin elasticity and repair mechanisms; fibrosis, characterized by excessive ECM accumulation affecting organ function; and cancers, where ECM remodeling facilitates tumor progression and metastasis. The cellular and molecular architecture of the ECM, as well as the ECM’s role in liver pathologies 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) ; Ortiz et al., Chronic Liver Disease, 2: 41-52 (2021) , all of which are incorporated herein by reference in their entireties.
Currently, existing ex vivo cell scaffolds do not include patient-derived ECM proteins, which are necessary for mimicking and studying different liver pathologies. The absence of these tailored ECM proteins in scaffolds means that current models may not fully replicate the unique microenvironmental conditions of liver diseases as they occur in individual patients.
Thus, the first compartment and second compartment of the disclosed 3D bio-printed scaffold contain extracellular matrix (ECM) . Generally, the ECM incorporated into the 3D bio-printed scaffold is derived from the patient. In some forms, the compartment containing the tumor cells also contains ECM derived from tumorous tissue (also referred to as “tumor ECM” ) . In some forms, the first compartment contains tumor cells and tumor ECM. In some forms, the second compartment contains tumor cells and tumor ECM. In some forms, the compartment containing the 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 non-tumor cells and non-tumor ECM. In some forms, the second compartment contains 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 bio-printed scaffold is illustrated in Figure 2 (see “Product Design” ) . The embodiment illustrated in Figure 2 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 vasculature e.g., endothelial cells.
Although the ECM accounts for a relatively small portion of the overall area of the liver, the ECM provides crucial support and structure to the organ, facilitating physical scaffolding for liver cells, and aiding cellular signaling and liver homeostasis as described above. The concept of the "liver matrisome" encompasses the full complement of ECM proteins and associated factors present in the liver; this concept propelled forward by advancements in omics technologies, allowing for a more comprehensive understanding of the ECM's composition and its changes during disease (see Arteel GE, Naba A. JHEP Rep 2: 100115 (2020) ) . The major components of the liver ECM include but are not limited to fibronectin, collagen type IV, and various molecules such as collagen types I, III, laminins, and proteoglycans. Collagen types I and III become particularly abundant during fibrosis. Most of the liver resident cells, including hepatocytes, cholangiocytes, sinusoidal endothelial cells (not epithelial) , and Kupffer cells, contribute to the ECM. They participate both in the secretion of ECM components and in the remodeling processes. Hepatic stellate cells, in particular, play a pivotal role in ECM deposition during fibrosis. Hepatic fibrosis and cirrhosis are canonical examples of ECM dysregulation, characterized by the excessive accumulation of ECM components, notably collagens. 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 hepatic ECM contribute to the pathogenesis of liver fibrosis and cirrhosis.
Thus, the disclosed 3D bio-printed scaffold incorporates ECM proteins derived from patients to accurately replicate and investigate various liver pathologies. In some forms, the 3D bio-printed scaffold contains collagenous proteins, non-collagenous proteins, or both collagenous proteins and non-collagenous proteins.
In some forms, the disclosed 3D bio-printed scaffold contains collagenous proteins. Collagen is a major fibrillar protein present in the ECM that represents approximately 30%of the total protein content in the body; therefore, it constitutes the principal structural protein in mammalian tissues (reviewed in Frantz C, et al., J Cell Sci 123: 4195-4200 (2010) ) .
In some forms, the 3D bio-printed scaffold one or more fibril-forming or fibrillar collagens, fibril-associated collagens with interrupted triple helix (FACITs) , network forming collagens, transmembrane collagens, and/or multiplexins. Fibril-forming or fibrillar collagens include type I, II, III, XI collagen and the more recently discovered type XXIV and XXVII collagen. They are the most abundant and widely distributed collagens in the body. Their role is largely 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) ) . Fibril-associated collagens with interrupted triple helix (FACITs) constitute the largest collagen subclass, including type IX, XII, XIV, XVI, XIX, XX, XXI, and XXII collagen. FACITs do not form fibrils themselves, but they bind the surface of pre-existing collagen fibrils contributing to fibril enlargement. Anchoring fibrils, composed largely of type VII collagen, extend from the basal lamina of epithelial cells, and attach to the lamina reticularis by wrapping around the reticular fiber type III collagen bundles and constituting the basement membrane (reviewed in Ricard-Blum S. Ricard-Blum S. Cold Spring Harb Perspect Biol 3: a004978, 2011) ) . Network-forming collagens contain multiple disruptions in the triple-helical chains, providing flexibility and enabling them to form linear, axial, and lateral associations within protein networks. 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 filamentous networks and binds multiple matricellular proteins. Type VIII and X collagen are very homologous heterotrimeric short-chain molecules that form hexagonal networks; however, they exhibit different localization (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 by proteolysis (reviewed in Ricard-Blum S. and Ruggiero, Pathol Biol (Paris) 53: 430-442, (2005) ) .
In some forms, the 3D bio-printed scaffold contains proteins which constitute the basement membrane (referred to as “basement membrane proteins” ) . The basement membrane is a highly specialized type of ECM that serves as a reservoir of growth factors that direct cellular functions, 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, the 3D bio-printed scaffold contains laminins, nidogen/entactin, heparan sulfate proteoglycans, and the non-fibrillar collagens, like collagen type IV.
In some forms, the 3D bio-printed scaffold contains proteins which constitute the interstitial matrix (IM) (referred to as “interstitial matrix proteins” ) . The interstitial matrix (IM) , is produced by fibroblasts and surrounds cells, making up the bulk of the ECM in the body. In some forms, the 3D bio-printed scaffold contains one or more Types I, III, and/or V collagens. In some forms, the 3D bio-printed scaffold contains one or more of elastin, fibronectin, and tenascin (Karsdal MA, et al. Adv Drug Deliv Rev. 2017; 121: 43-56) .
In some forms, the disclosed 3D bio-printed scaffold incorporates a combination of extracellular matrix (ECM) and bio-active hydrogels. The integration of ECM with bio-active hydrogels in some forms allow for more precise replication of liver tumor microenvironments, thereby facilitating new discoveries in the mechanisms of liver cancer progression and regression. In some forms, the ECM are encapsulated in hydrogels as tissue engineering scaffolds for the 3D cell culture. Hydrogels can be polymerized using light, UV radiation, a redox agent (e.g., sodium thiosulfate in combination with sodium persulfate) , changes in pH or by using some other suitable polymerization initiator such as a divalent cation like calcium.
The polymerizable agent may comprise monomers, macromers, oligomers, polymers, or a mixture thereof. The polymer compositions can consist solely of covalently cross-linkable polymers, or blends of covalently and ionically cross-linkable or hydrophilic polymers.
Suitable hydrophilic polymers include synthetic polymers such as poly (ethylene glycol) , poly (ethylene oxide) , partially or fully hydrolyzed poly (vinyl alcohol) , poly (vinylpyrrolidone) , poly (ethyloxazoline) , poly (ethylene oxide) -co-poly (propylene oxide) block copolymers (poloxamers and meroxapols) , poloxamines, carboxymethyl cellulose, and hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as polypeptides, polysaccharides or carbohydrates such as FICOLL^TM, polysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin or copolymers or blends thereof. “celluloses” includes cellulose and derivatives of the types described above; “dextran” includes dextran and similar derivatives thereof.
Examples of materials that can be used to form a hydrogel include modified alginates. Alginate is a carbohydrate polymer isolated from seaweed, which can be crosslinked to form a hydrogel by exposure to a divalent cation such as calcium. Alginate is ionically crosslinked in the presence of divalent cations, in water, at room temperature, to form a hydrogel matrix. Modified alginate derivatives may be synthesized which have an improved ability to form hydrogels. The use of alginate as the starting material is advantageous because it is available from more than one source and is available in good purity and characterization. The term “modified alginates” refers to chemically modified alginates with modified hydrogel properties. Naturally occurring alginate may be chemically modified to produce alginate polymer derivatives that degrade more quickly.
Polysaccharides that are very viscous liquids or are thixotropic and form a gel over time by the slow evolution of structure, are also useful. For example, hyaluronic acid, which forms an injectable gel with a consistency like a hair gel, may be utilized. Modified hyaluronic acid derivatives are particularly useful. The term “hyaluronic acids” refers to natural and chemically modified hyaluronic acids. Modified hyaluronic acids may be designed and synthesized with preselected chemical modifications to adjust the rate and degree of crosslinking and biodegradation. For example, modified hyaluronic acids may be designed and synthesized which are esterified with a relatively hydrophobic group such as propionic acid or benzylic acid to render the polymer more hydrophobic and gel-forming, or which are grafted with amines to promote electrostatic self-assembly. Modified hyaluronic acids thus may be synthesized which are injectable, in that they flow under stress, but maintain a gel-like structure when not under stress.
Other materials which may be utilized include proteins such as fibrin, collagen, and gelatin. Other polymeric hydrogel precursors include polyethylene oxide-polypropylene glycol block copolymers such as PLURONICS^TM or TETRONICS^TM, which are crosslinked by hydrogen bonding and/or by a temperature change, 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 also may be utilized. For example, a mixture of polyethylene oxide and polyacrylic acid which gels by hydrogen bonding upon mixing may be utilized. In one embodiment, a mixture of a 5%w/w solution of polyacrylic acid with a 5%w/w polyethylene oxide (polyethylene glycol, polyoxyethylene) 100,000 can be combined to form a gel over the course of time, e.g., as quickly as within a few seconds.
Water soluble polymers with charged side groups may be crosslinked by reacting the polymer with an aqueous solution containing ions of the opposite charge, either cations if the polymer has acidic side groups or anions if the polymer has basic side groups. Examples of cations for cross-linking of the polymers with acidic side groups to form a hydrogel are monovalent cations such as sodium, divalent cations such as calcium, and multivalent cations such as copper, calcium, aluminum, magnesium, strontium, barium, and tin, and di-, tri-or tetra-functional organic cations such as alkylammonium salts. Aqueous solutions of the salts of these cations are added to the polymers to form soft, highly swollen hydrogels and membranes. The higher the concentration of cation, or the higher the valence, the greater the degree of cross-linking of the polymer. Additionally, the polymers may be crosslinked enzymatically, e.g., fibrin with thrombin.
Suitable ionically crosslinkable groups include phenols, amines, imines, amides, carboxylic acids, sulfonic acids and phosphate groups. Aliphatic hydroxy groups are not considered to be reactive groups for the chemistry disclosed herein. Negatively charged groups, such as carboxylate, sulfonate 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 carboxylate, sulfonate and phosphate ions. Preferably, the negatively charged ions contain more than one carboxylate, sulfonate, or phosphate group.
The preferred anions for cross-linking of the polymers to form a hydrogel are monovalent, divalent or trivalent anions such as low molecular weight dicarboxylic acids, for example, terepthalic acid, sulfate ions and carbonate ions. Aqueous solutions of the salts of these anions are added to the polymers to form soft, highly swollen hydrogels and membranes, as described with respect to cations.
A variety of polycations can be used to complex and thereby stabilize the polymer hydrogel into a semi-permeable surface membrane. Examples of materials that can be used include polymers having basic reactive groups such as amine or imine groups, having a preferred molecular weight between 3,000 and 100,000, such as polyethylenimine and polylysine. These are commercially available. One polycation is poly (L-lysine) ; examples of synthetic polyamines are: polyethyleneimine, poly (vinylamine) , and poly (allyl amine) . There are also natural polycations such as the polysaccharide, chitosan.
Polyanions that can be used to form a semi-permeable membrane by reaction with basic surface groups on the polymer hydrogel include polymers and copolymers of acrylic acid, methacrylic acid, and other derivatives of acrylic acid, polymers with pendant SO³H groups such as sulfonated polystyrene, and polystyrene with carboxylic acid groups. These polymers can be modified to contain active species polymerizable groups and/or ionically crosslinkable groups. Methods for modifying hydrophilic polymers to include these groups are well known to those of skill in the art.
The polymers may be intrinsically biodegradable but are preferably of low biodegradability (for predictability of dissolution) but of sufficiently low molecular weight to allow excretion. The maximum molecular weight to allow excretion in human beings (or other species in which use is intended) will vary with polymer type but will often be about 20,000 Daltons or below. Usable, but less preferable for general use because of intrinsic biodegradability, are water-soluble natural polymers and synthetic equivalents or derivatives, including polypeptides, polynucleotides, and degradable polysaccharides.
The polymers can be a single block with a molecular weight of at least 600, preferably 2000 or more, and preferably at least 3000. Alternatively, the polymers can include can be two or more water-soluble blocks which are joined by other groups. Such joining groups can include biodegradable linkages, polymerizable linkages, or both. For example, an unsaturated dicarboxylic acid, such as maleic, fumaric, or aconitic acid, can be esterified with hydrophilic polymers containing hydroxy groups, such as polyethylene glycols, or amidated with hydrophilic polymers containing amine groups, such as poloxamines.
Covalently Crosslinkable hydrogel precursors are also useful. For example, a water-soluble polyamine, such as chitosan, can be cross-linked with a water soluble diisothiocyanate, such as polyethylene glycol diisothiocyanate. The isothiocyanates will react with the amines to form a chemically crosslinked gel. Aldehyde reactions with amines, e.g., with polyethylene glycol dialdehyde, also may be utilized. A hydroxylated water-soluble polymer may also be utilized.
Alternatively, polymers may be utilized which include substituents which are crosslinked by a radical reaction upon contact with a radical initiator. For example, polymers including ethylenically unsaturated groups which can be photochemically crosslinked may be utilized. In this embodiment, water soluble macromers that include at least one water soluble region, a biodegradable region, and at least two free radical-polymerizable regions, are provided. The macromers are polymerized by exposure of the polymerizable regions to free radicals generated, for example, by photosensitive chemicals and or light. Examples of these macromers are PEG-oligolactyl-acrylates, wherein the acrylate groups are polymerized using radical initiating systems, such as an eosin dye, or by brief exposure to ultraviolet or visible light. Additionally, water soluble polymers which include cinnamoyl groups which may be photochemically crosslinked may be utilized, as disclosed in Matsuda et al., ASAID Trans., 38: 154-157 (1992) .
In general, the polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions. Methods for the synthesis of 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, N.Y. 1980) . Many polymers, such as poly (acrylic acid) , are commercially available. Naturally occurring and synthetic polymers may 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.
The hydrogel solution is prepared, for example, by mixing 10%weight/volume (w/v) of the polymerizable polymer in sterile phosphate buffered saline (PBS) , which is a suitable solvent, adjusted to a pH of about 7.4. In some embodiments, the polymer is either photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) or photopolymerizable poly (ethylene oxide) diacrylate (PEODA) .
Optionally, various additives can be included in the hydrogel solution such as 100 U/ml of 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, the bioactive additives could include, singly or in combination, growth factors, cell differentiation factors, other cellular mediators, nutrients, antibiotics, anti-inflammatories, and other pharmaceuticals. Although not limiting, some suitable cellular growth factors, depending upon the cell type to be encapsulated in either the hydrogel of the same or adjacent hydrogel layer, include heparin binding growth factor (HBGF) , transforming growth factor (TGFα or TGFβ) , alpha fibroblastic growth factor (FGF) , epidermal growth factor (EGF) , vascular endothelium growth factor (VEGF) , various angiogenic factors, nerve growth factor (NGF) and muscle morphologic growth factor.
In addition, the hydrogel solution optionally includes a suitable non-toxic polymerization initiator, mixed thoroughly to make a final concentration of 0.05%w/v. When PEGDA or PEODA are selected as the polymers, the polymerization initiator is preferably added and selected to be the photoinitiator Igracure 2959 (commercially available from Ciba Specialty Chemicals Corp., Tarrytown, N. Y. ) , although other suitable photoinitiators can be used.
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 (ethyloxazoline) , poly (ethylene oxide) -co-poly (propylene oxide) block copolymers (poloxamers and meroxapols) , poloxamines, carboxymethyl cellulose, and hydroxyalkylated celluloses such as hydroxyethyl cellulose and methylhydroxypropyl cellulose, and natural polymers such as polypeptides, polysaccharides or carbohydrates such aspolysucrose, hyaluronic acid, dextran, heparan sulfate, chondroitin sulfate, heparin, or alginate, and proteins such as gelatin, collagen, albumin, or ovalbumin or copolymers or blends thereof. The term “celluloses” includes cellulose and derivatives of the types described above; “dextran” includes dextran and similar derivatives thereof.
Exemplary photoinitiator is Igracure 2959. Other photoinitiators include HPK, which is commercially available from Polysciences. In addition, various dyes and amine catalysts are known to form an active species when exposed to external radiation. Specifically, light absorption by the dye causes the dye to assume a triplet state, which subsequently reacts with the amine to form the active species that initiates polymerization. Typically, polymerization can be initiated by irradiation with light at a wavelength of between about 200-700 nm, most preferably in the long wavelength ultraviolet range or visible range, 320 nm or higher, and most preferably between about 365 and 514 nm.
Numerous dyes can be used for photopolymerization, and these include erythrosin, phloxime, rose bengal, thonine, camphorquinone, ethyl eosin, eosin, methylene blue, riboflavin, 2, 2-dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone, 2, 2-dimethoxy-2-phenyl acetophenone, other acetophenone derivatives, and camphorquinone. Suitable cocatalysts include amines such as N-methyl diethanolamine, N, N-dimethyl benzylamine, triethanol amine, triethylamine, dibenzyl amine, N-benzylethanolamine, N-isopropyl benzylamine. Triethanolamine is a preferred cocatalyst with one of these dyes. Photopolymerization of these polymer solutions is based on the discovery that combinations of polymers and photoinitiators (in a concentration not toxic to the cells, less than 0.1%by weight, more preferably between 0.05 and 0.01%by weight percent initiator) will crosslink upon exposure to light equivalent to between one and 3 mWatts/cm2.
While photopolymers are preferred for making the hydrogels, because it is convenient to control polymerization using external radiation supplied through a surgical scope, the present invention can be practiced using other polymer materials and polymerization initiators. Examples of other materials which can be used to form a hydrogel include (a) modified alginates, (b) polysaccharides (e.g. gellan cum and carrageenans) which gel by exposure to monovalent cations, (c) polysaccharides (e.g., hyaluronic acid) that are very viscous liquids or are thiotropic and form a gel over time by the slow evolution of structure, and (d) polymeric hydrogel precursors (e.g., polyethylene oxide-polypropylene glycol block copolymers and proteins) .
B. Patient-specific Tissue Stiffness
Beyond the composition of the extracellular matrix (ECM) , the stiffness of tissues is partially determined by the rigidity of the underlying ECM. Studies have demonstrated that variations in tissue stiffness can significantly influence cellular behavior within tissues, particularly in the context of liver diseases (Wong GL, et al., J Hepatol 60: 339-45, 2014; Wells RG, Hepatology 47: 1394-400, 2008) . For instance, focusing on Kupffer cells, which constitute the second largest cell population in the liver, research has underscored the crucial role of substrate stiffness in guiding macrophage activity. This finding is pivotal for creating biomaterials informed by immunological principles, capable of adjusting macrophage responses post-implantation (Sridharan R, et al., Materials Today 18: 313-25, 2015) .
Furthermore, investigations into liver stiffness have explored its potential in predicting the risk of hepatocellular carcinoma (HCC) , given that cirrhosis is a known independent risk factor for HCC. Foucher et al. identified a threshold value of 53.7 kPa for HCC presence, which is significantly higher than the stiffness of healthy liver tissue (2-7 kPa) (Foucher J, et al., Gut 55: 403-408, 2006) . Additional research corroborates that liver stiffness exceeding 8 kPa substantially elevates HCC risk (Jung KS, et al., Hepatology 53 (3) : 885-94, 2010) . Liver stiffness is also increased in patients with other liver conditions, such as viral hepatitis B and C and non-alcoholic steatohepatitis (NASH) (Mueller S, Sandrin L, Hepat Med 2: 49-67, 2010) .
In preferred forms, the 3D bio-printed scaffold is engineered to precisely emulate the stiffness characteristic of a patient's liver, thereby facilitating the replication of biophysical parameters found in clinical liver specimens for advanced studies in cellular behavior. This customization allows for the exploration of cellular responses under conditions that closely mirror the physiological environment, offering invaluable insights into the mechanisms governing cellular interactions within the liver tissue.
Furthermore, the 3D bio-printed scaffold is designed to encompass stiffness profiles that represent various stages of carcinogenesis, including fibrosis, cirrhosis, and steatosis. This approach enables a nuanced examination of the progression of liver disease, facilitating a deeper understanding of the transitions between these stages and their impact on cellular dynamics and pathology.
In some forms, the hydrogel used in the disclosed 3D bio-printed scaffolds has a stiffness ranging from about 2kPa to about 75kPa. This range is deliberately chosen to cover a broad spectrum of liver pathologies, ensuring that the model can be tailored to simulate the specific conditions of interest with high fidelity. In some forms, the hydrogel used in the disclosed 3D bio-printed models has a stiffness from about 5kPA to about 75kPa, from about 20kPA to about 60kPa, or from about 30kPA to about 50kPa. In some forms, the hydrogel used in the disclosed 3D bio-printed models has a stiffness of about 2kPa, about 5kPa, about 10kPa, about 15kPa, about 20kPa, about 25kPa, about 30kPa, about 35kPa, about 40kPa, about 45kPa, about 50kPa, about 55kPa, about 60kPa, about 65kPa, about 70kPa, or about 75kPa.
In some forms, the hydrogel used in the disclosed 3D bio-printed scaffolds have varying stiffness depending on the compartment of the hydrogel. For example, in some forms, the compartment containing tumoral cells have a stiffness from about 20kPa to about 75kPa to recapitulate the stiffness of the diseases liver tissues extracted from the patient. In some forms, the compartment containing non-tumoral cells have a stiffness from about 2kPa to about 19kPa to recapitulate the stiffness of healthy tissues extracted from the patient.
C. Cell composition for immune microenvironment of tumor cells
Tumor cells stimulate significant molecular, cellular, and physical changes within their host tissues to support tumor growth and progression. Numerous risk factors for liver cancer have immunological bases, e.g., infections with hepatitis viruses and conditions like non-alcoholic steatohepatitis (NASH) , which lead to persistent and skewed immunological changes as the tumor develops and progresses. The tumor immune microenvironment (TIME) plays a role in tumor immune surveillance and the mechanisms of immune evasion; thus, being important for the development of new insights into checkpoint inhibitor therapies (Tang T, et al., Signal Transduction and Targeted Therapy 6: 72 (2021) ) . A single-cell sequencing study involving over 100 hepatocellular carcinoma (HCC) patients identified five distinct immune subtypes, each characterized by unique immune cell and extracellular matrix compositions (Xue, R., Zhang, Q., Cao, Q. et al., Nature 612, 141-147 (2022) ) . This heterogeneity in immune profiles leads to diverse somatic mutations and transcriptomic patterns in tumor cells, significantly influencing the effectiveness of immunotherapies. Despite the critical role of immune cell diversity and TIME in understanding liver tumors, current models have not yet integrated or demonstrated this spectrum of immunological variability in liver cancer research.
The disclosed 3D-bioprinted scaffold is designed to preserve the unique immunological profiles of individual patients. This approach enables a more accurate representation of the liver cancer tumor immune microenvironment (TIME) within the scaffold, thereby enhancing personalized drug screening efforts. Such precision in mimicking patient-specific immunological characteristics in the scaffold allows for more tailored evaluations of therapeutic responses. As illustrated in Figures 4A and 4B, different immune cell types including macrophages, dendritic cells, B regulatory cells, T regulatory cells, epithelial progenitor cells individually contribute to tumor development, progression and recurrence before and after treatment (Yeung OWH, Lo C-M, Ling C-C, 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., J Hepatol 65: 944-52 (2016) ; Shao Y, Lo CM, Ling CC, et al., Cancer Letters 355: 264-72 (2014) ; and Ling C-C, Ng KTP, Shao Y, et al., Journal of Hepatology 60: 103-9 (2014) ) .
In some forms, the disclosed 3D bio-printed scaffold includes tumor cells, cancer-associated fibroblast, endothelial cells, immune cells etc. In some forms, the immune cells include innate 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 bio-printed scaffold includes malignant liver cells, healthy hepatocytes and non-parenchymal cells including macrophages, lymphocytes, and endothelial cells. In some exemplary forms, the disclosed 3D bio-printed scaffold includes macrophages and T lymphocytes that are abundant in hepatocellular carcinoma. In further forms, the disclosed 3D bio-printed scaffold is suited for the study of the interactions of adaptive and innate immunity.
In one exemplary embodiment, the disclosed 3D bio-printed scaffold preserves the unique immunological characteristics of an individual patient, enabling accurate replication of the tumor microenvironment specific to liver cancer. In additional embodiments, this 3D bio-printed scaffold is tailored for enhanced personalized drug screening, offering improved compatibility and effectiveness in therapeutic testing.
D. Vasculature in the Tumor Compartment
Tumors are not only aggregates of malignant cells but also well-organized complex vasculature. Primary tumors are divided into the tumor core, tumor stroma, and invasion margin based on tumor compartments. The vasculature in tumors is abnormal, which impairs blood flow and limits the delivery of oxygen, nutrients, and therapeutics, including antibodies and immune cells. Apart from nutrient supplement, vasculature also allows molecular gradients of waste metabolic products.
Vasculature (also referred to as a vascular system) includes a structure composed of vascular endothelial cells and its supporting cells. Vascular systems not only maintain tissues but also play an important role in the maturation process of tissues. In some forms, vascular structures supply the tissues with oxygen and nutrients that are necessary for their survival. Even before blood flows into the tissue, recapitulating three-dimensional tissue structures with blood vessels and cell polarity is important for the differentiation, proliferation, and maintenance of cells.
Thus, the 3D and 4D bio-printed scaffolds contain vasculature or a vascular system. The vascular system or vasculature includes a population of vascular cells. In one preferred form, the vascular cells of the 3D and 4D bio-printed scaffolds are derived from the patient’s own vascular tissues. In other forms, the vascular cells of the 3D and 4D bio-printed scaffolds can be derived from totipotent or pluripotent cells (such as induced pluripotent stem cells and embryonic stem cells) by induction of differentiation. The vascular cells included in the bio-printed scaffolds are generally vascular endothelial cells. The vascular endothelial cells include cells constituting vascular endothelium or cells capable of differentiating into such cells e.g., vascular endothelial progenitor cells and vascular endothelial stem cells. Whether a cell is vascular endothelial cell or not can be determined by checking to see if they express marker proteins such as TIE2, VEGFR-1, VEGFR-2, VEGFR-3 and CD31. If any one or more of the above-listed marker proteins are expressed, the cell can safely be regarded as a vascular endothelial cell. Further, markers for vascular endothelial progenitor cells include but are not limited to c-kit, and Sca-1. If these markers are expressed, the cell of interest can be confirmed as a vascular endothelial progenitor cell (Fang, et al., PLOS Biology, 2012; 10 (10) : e1001407) .
As chemical forces influence the TME and directly affect cancer growth, one critical factor in this scenario is oxygen. Oxygen deficiency mainly occurs when the oxygen demand at a tumor exceeds the supply being provided from the adjacent vessel system, known as hypoxia. Most healthy organs reside in 3-6%oxygen while conditions lower than 3%oxygen are described as hypoxia. Hypoxia occurs widely in malignant tumors and is known to cause tumor progression through multiple mechanisms. The response typically includes abnormal growth of the vascular system in angiogenesis and EMT of cancer cells, which eventually leads to metastasis.
With the vasculature, a gradient of oxygen can be generated in the 3D bio-scaffold with hypoxic region. The resulting hypoxia (low oxygen concentration) and low pH can in turn 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 the intratumor regions of HCC patients. Abnormal microvasculature and unrestrained proliferation of HCC cells lead to oxygen deficiency. Hypoxia is involved in multiple biological processes of HCC and promotes tumor aggressiveness, chemoresistance and immunotherapy resistance (Cramer T, Vaupel P. Journal of Hepatology 76: 975-80 (2022) .
Thus, in some forms, the 3D bio-printed scaffold incorporates one or more normoxic (with vasculature) compartments to recreate the tumor architecture and complexity observed in patients’ tumor. In some forms, the 3D bio-printed scaffold incorporates one or more hypoxic (without vasculature) compartments to recreate the tumor architecture and complexity observed in patients’ tumor.
In tumor tissue, chronic hypoxia occurs when the tumor expands beyond 70 μm from the pre-existing nutritive blood vessels, which prevents an adequate delivery of oxygen (Emami Nejad, et al., Cancer Cell Int 21, 62 (2021) ) . In some forms, regions with different oxygen and nutrient gradients are generated by their distance from the vasculature within the 3D bio-printed scaffold. In some forms, the distance between the tumor cells and vasculature with oxygen and nutrient supply is about 0.5 μm to about 400 μm simultaneously resulting in normoxic and hypoxic cells as illustrated in Figure 8B. In some forms, the distance between the tumor cells and the vasculature is about 0.5 μm, about 50 μm, about 100 μm, about 150 μm, about 200 μm, about 250 μm, about 300 μm, about 350 μm, or about 400 μm. In some forms, the distance between non-tumor cells and vasculature is about 0.5 μm to about 1400μm. In some forms, the distance between non-tumor cells and the vasculature is about 0.5 μm, about 50 μm, about 100 μm, about 200 μm, about 300 μm, about 500 μm, about 700 μm, about 900 μm, about 1000 μm, about 1100 μm, about 1200 μm, about 1300 μm, or abour1400μm. The average size of a single tumor or non-tumor cell is about 5 μm to about 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.
E. Geometry of Bio-printed Scaffolds
The bio-printed scaffolds can have either a 3-dimensional configuration i.e., a 3D bio-printed scaffold, or a 4-dimensional configuration i.e., a 4D bio-printed scaffold.
1. 3D Bio-printed Scaffolds
The disclosed 3D scaffold is made of bio-printed, living, three-dimensional constructs that simulate liver tumor tissues. The 3D scaffold contains one or more bio-printed compartments, including at least one compartment containing tumor cells and macrophages, a second compartment containing non-tumoral cells such as hepatocytes and T-lymphocytes, and a third compartment containing vasculature.
In preferred forms, the 3D scaffold is fabricated with microfluidic channels and dynamic flow systems that mimics physiological systems such as blood flow. In some forms, the disclosed 3D bio-printed scaffold includes at least one inlet and one outlet for microfluidic connection. The construct is incorporated with multi-branch microfluidic channels to increase their contact area with tumor and non-tumor regions for effective material exchanges. To simulate the different diameters of blood vessel in the vasculature system, the width of the microfluidic channels in the construct are ranged from about 0.5 mm to about 1.0 mm. In some forms, 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 forms, to bio mimic blood vessels, all of the microfluidic channels are coated with endothelial cells (see Figure 5) .
In other forms, the 3D scaffold enables control of the chemical microenvironment to provide physiologically relevant approaches such as generation of well-controlled chemical gradients across 3D ECMs and multi-cell systems in ECMs with spatiotemporal distributions successfully mimicking in vivo conditions.
In some forms, the disclosed 3D bio-printed scaffold can be constructed with any one of three different designs as shown in Figure 6, each containing inlet and outlet for microfluidic connection. Each layer of the engineered liver tissue includes multiple cells in the X, Y, and Z axes, and is at least about 10 mm in thickness. The hydrogel matrix can be produced in one or more layers. In some forms, the disclosed 3D bio-printed scaffold includes 5 or more layers. In preferred forms, the dimension of the construct is at least 10mm x 10mm x 0.5mm. Suitable range of dimensions of the constructs are length (about 20 to about 40mm) , width (about 20 to about 40mm) and height (about 10 to about 30mm) .
2. 4D Bio-printed Scaffolds
In general, the in vivo physiological system of the human body represents a complex dynamic environment, with the microenvironmental conditions (such as the temperature, pH, enzymes, and potentials) fluctuating in real time across different tissues (see Kavand, et al., Adv. Mater. 2022, 34, 2107876) . To satisfy the real-time demands of this dynamic microenvironment of tissues and organs, an innovative paradigm of 4D bioprinting (3D bioprinting + time) has emerged, aimed at generating smart scaffolds for tissue engineering (S. Miao, et al., Sci. Rep. 2016, 6, 27226) . In recent years, the concept of 4D printing has evolved, allowing the shape, property, or functionality of a 3D printed object 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) .
Thus, bio-printed scaffolds having a four-dimensional (4D) configuration are also disclosed. In some forms, the 4D bio-printed scaffolds incorporate various types of stimuli, such as humidity, temperature, pH, light, and magnetic fields.
4D printing technology has been used to create 4D programmable building blocks that respond to stimuli, offering an innovative approach to constructing architectural structures with both exceptional structural and mechanical features. The disclosed 4D bio-printed scaffolds enables precise control over environmental conditions within a culture system, allowing for the accurate observation of interactions between various cell types in vitro. The system is composed of porous scaffolds integrated with a stimuli-responsive platform, designed to mimic the architecture of a hepatic plate.
By leveraging advancements in 3D printing and intelligent biomaterials, the 4D bio-printed scaffolds more accurately position tumor cells, immune cells, stromal cells, and vascular cells in alignment with their natural physiological roles. The 4D bio-printed scaffolds includes microchannels that serve as potential endothelialized vasculatures and fluid conduits, as well as 4D printed building blocks made from stimuli-responsive hydrogels that act as sources for biological signal release. The modules are designed to be stackable and interlockable, akin to traditional building blocks, which provides customizable and flexible options for creating multiple functional geometries. This feature is particularly beneficial for reconstructing patient-specific tumor structures, offering a new level of controllability and adaptability in medical modeling and therapeutic applications.
Biomaterials that can be incorporated into the 4D bio-printed scaffold include but are not limited to shape memory polymers (SMPs) and liquid crystal elastomers. In some forms, the biomaterial incorporated into the 4D bio-printed scaffold is an alloy such as a shape memory alloy (SMA) . Shape memory alloys (SMAs) undergo phase transition (between austenite and martensite) when subjected to mechanical stress or temperature variations. In particular, SMAs “remember” their initial form and return to it once conditions normalize. Exemplary SMAs that can be used include copper-aluminum-nickel alloy and nickel-titanium (NiTi) alloys.
In some forms, the biomaterial incorporated into the 4D bio-printed scaffold is ceramic. An exemplary ceramic that can be used is “ceramic ink” made of elastomeric poly (dimethylsiloxane) matrix nanocomposites that can be printed, deformed, and then transformed into silicon oxycarbide (SiOC) -based ceramic nanomaterials (G. Liu, Y. Zhao, G. Wu, J. Lu, Sci. Adv. 2018, 4: eaat0641) .
In some forms, the biomaterial incorporated into the 4D bio-printed scaffold is a polymer e.g., shape memory polymer (SMP) . SMPs represent the most widely used smart materials in 4D printing (Zhou, et al., Macromol. Rapid Commun., 2021, 42, 2100176) . SMPs can maintain a temporary shape and recover their initial shape upon exposure to an external stimulus (Kuang, et al., Adv. Funct. Mater. 2019, 29, 1805290) . When the temperature exceeds the transition temperature, SMPs can be temporarily deformed. Upon cooling below this threshold and unloading, the SMP retains its deformed shape. This process can be repeated to achieve reversible deformations and has been applied in many TE instances.
In some forms, the SMP is a hydrogel as described above for 3D printing. Hydrogels are 3D polymer networks permeated with water. Their unique properties, such as wetness, responsiveness, softness, biocompatibility, and bioactivity, make them promising materials for tissue engineering. Furthermore, hydrogels exhibit stimuli-responsive behavior, which renders them promising materials for 4D bioprinting. The capacity of hydrogel structures to dynamically change and adapt is crucial for tissue engineering (TE) applications. The hydrogel structures can exhibit shapeshifting capabilities, respond to specific physiological cues, and even undergo controlled degradation as they integrate with the surrounding biological environment. For example, Gladman and co-workers presented a biomimetic 4D printing of a hydrogel (Gladman, et al. Nat. Mater. 2016, 15, 413) . Cellulose fibrils were oriented along the printing direction by shear forces during the bioprinting process. Anisotropic swelling behavior was observed when the hydrogel was immersed in water, resulting in programmable shape changes and complex 3D morphologies. In another example, Ding et al. developed a cell-laden bioink for 4D bioprinting (Ding, et al., Adv. Mater. 2022, 34, 2109394) . The printed cell-loaded construct exhibited high cell viability and programmable deformation under external stimulation. These exemplary 4D living cell bioprinting techniques can be leveraged to prepare the 4D bio-printed scaffolds as disclosed herein. 4D Bio-printed materials and methods of making are further described in Chen et al., Advanced Materials, 2307686 (2023) .
III. Methods of Making
The composition of the tumor microenvironment varies between tumor types, but hallmark features include immune cells, stromal cells, blood vessels, and extracellular matrix. To fabricate the highly clinically relevant model, one or more methods to incorporate the clinical components in the bioprinting process are applied.
Methods of preparing a 3D in vitro cell culture mimicking liver cancer and the associated TME in a subject are provided. In some embodiments, one or more of the following steps of biomaterial preparation and 3D bioprinting techniques are used:
(a) Simultaneously isolating and reserving patients derived cells and ECM;
(b) Patients derived cells maintenance for bioprinting;
(c) Micro-molding combined with 3D multi-materials inject exclusion and suspension bioprinting techniques in manufacturing “Tumor in cube” or “Mini liver tumor. ”
A. Simultaneously isolating and reserving patients derived cells and EC
Due to its clinical nature, ECM is tissue-derived biomaterial that can be used as a bioactive component for tissue engineering applications (Kim YS, Majid M, Melchiorri AJ, Mikos AG. Bioengineering &translational medicine 4: 83-95 (2018) ) . The versatility of ECM allows it to be the excellent candidate for various applications, from a whole tissue scaffold to a digested solution that could be used as a bioink for 3D printing.
Current methods in isolating tissue ECM yield low reproductivity with high variation. Utilization of ECM in tissue engineering applications is still in development and a large portion of the current work still focuses on exploring the effects of different decellularization methods. More importantly, all of the current protocols are established to extract either ECM or cells from clinical specimens. Thus, in preferred forms, the disclosed methods preserve these two key elements simultaneously to recreate and bio-print the TIME in vitro.
In an exemplary method of extraction, fresh patients’ liver tissue including tumor and non-tumor removed from surgical operation, measured about 1-3 cm in diameter is first washed with 1xHBSS containing 1xpenicillin-streptomycin to remove blood and potential contaminant. It is then cut into 1-2 mm diameter pieces by sterile surgical blade scissors. To simultaneously preserve the extracellular matrix compartment of HCC tissues, primary cells are first isolated with the combination of 1mM EDTA and other components with mechanical disruption in a 37℃ orbital shaker at 150 rpm for 30 mins. Supernatants containing various cell types are then harvested and isolated with newly developed protocol. For primary hepatocytes and HCC cells enrichment, the supernatant is centrifuged at 50 g for 5 min followed by cell pellet suspension. For the enrichment of smaller hepatic non-parenchymal cells (NPC) -fractiona) liver endothelial cells, centrifuge 300 g for 5 min followed by overlaying in two-layer (25%/50%) density gradient to removal dead cells and then purified by FACS sorting of CD31+; b) macrophages and Kupffer cells, first centrifuge 300 g for 5 min followed by centrifuge 650 g for 7 min and FACS enrichment; c) Immune cells, Ficoll enrichment of PBMC in patient derived blood and followed by FACS sorting of desired immune populations.
To extract extracellular matrix from HCC tissues, 1%Triton X-100 and other components are added to the cell-deprived cubes to deplete any remaining cells. The cubes are then incubated in 37℃ orbital shaker at 300 rpm overnight. Supernatants are discarded, and the remaining cubes are then freeze-dried by Thermo ModulyoD Freeze Dryer for 24 hours to obtain lyophilized powder of tissue-derived ECM.
1. Source of Liver Tissues
Early-stage liver cancer can be treated curatively by local ablation, surgical resection, or liver transplantation. Treatment selection depends on tumor characteristics, the severity of underlying liver dysfunction, age, other medical comorbidities, and available medical resources and local expertise. Catheter-based locoregional treatment is used in patients with intermediate-stage cancer. Kinase and immune checkpoint inhibitors have been shown to be effective treatment options in patients with advanced-stage HCC.
In some forms, the subject is undergoing a conventional treatment for liver cancer. For example, in some forms, the additional therapy or procedure is surgery, transplant surgery, a radiation therapy, or chemotherapy.
In some forms, the subject is undergoing an immunotherapy such inhibition of checkpoint proteins such as components of the PD-1/PD-L1 axis or CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists) , adoptive T cell therapy, and/or a cancer vaccine.
In some forms, the samples are collected during surgical removal of tumors. In other forms, the samples are metastasized liver cancer.
In some forms, the cancer may have developed a resistance to the previously administered chemotherapeutic agent (s) . Therefore, in some forms, the subject from where the biomaterials for the 3D bio-printed scaffold are derived is one in which the cancer being treated is resistant or insensitive to one or more conventional chemotherapeutic agents prior to sample collection.
i. Hepatocellular Carcinoma
In some forms, the liver tissues are surgically removed from a patient with hepatocellular carcinoma (HCC) . HCC is the most common primary liver malignancy and is a leading cause of cancer-related death worldwide.
Chronic liver disease and cirrhosis remain the most important risk factors for the development of HCC of which viral hepatitis and excessive alcohol intake are the leading risk factors worldwide.
Chronic viral hepatitis can lead to cirrhosis and/or HCC. Hepatitis B and C are the most common causes of chronic hepatitis in the world. Hepatitis B virus (HBV) is a double-stranded, circular DNA molecule with eight genotypes (Ato 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 via contaminated blood transfusions, intravenous injections, and sexual contact. Vertical transmission from mother to fetus is the leading cause for HBV infection worldwide. Five percent of the world’s population is infected with hepatitis B.
Several epidemiological studies have demonstrated significant hepatocarcinogenicity with chronic HBV infection. 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 has been associated with a higher risk of HCC than genotypes A, B, and D. Active infection with HBV carries an independent risk of HCC with HBV DNA levels >105/mL viral copies associated with a 2.5-3 times increased risk of developing HCC in 8-10 years follow-up. Hepatitis B surface antigen (HBsAg) is not the only hematological marker that carries a significant risk for development of HCC. Patients with positive hepatitis B core antibody (anti-HBc) who are HBsAg-negative also remain at risk for development of HCC. The hepatocarcinogenicity of HBV can be significantly reduced with antiviral treatment for hepatitis B. Suppression of the virus can result in a significant 5-year reduction of the incidence of HCC from 13.7% (controls) to 3.7%, with the greatest reduction occurring in cirrhotic patients. 10 The use of HBV vaccination has resulted in significant declines in the incidence of HCC from HBV. The East Asian neonatal vaccination program is estimated to result in a 70%-85%decrease in the incidence of hepatitis B-related HCC. Despite perinatal immunization, 5%-10%of infants remain at risk of acquiring hepatitis B infection. The use of nucleoside analogs in treating chronic hepatitis B mothers in their third trimester of pregnancy has demonstrated superiority to vaccination alone in preventing neonatal transmission.
Hepatitis C virus (HCV) is a small, single-stranded RNA virus, which exhibits high genetic variability. There are six different genotypes of HCV isolated. Genotypes I, II, and III are predominant in the Western countries and the Far East, while type IV is predominant in the Middle East. The highest rates of chronic hepatitis C infection occur in Egypt (18%) , with lower rates occur in Europe (0.5%-2.5%) , the United States (1.8%) , and Canada (0.8%) . 16 Once infected with HCV, 80%of patients progress to chronic hepatitis, with ～20%developing cirrhosis. In hepatitis C, the development of HCC occurs almost exclusively in the liver with established cirrhosis; however, in the HALT-C trial, 8%of HCC occurred in patients with only advanced fibrosis. Dual infection with HBV and HCV in a cirrhotic patient increases the risk of HCC with an odds ratio (OR) of 165 compared to 17 for hepatitis C and 23 for hepatitis B alone. A synergistic effect with alcohol increases the incidence of HCC between 1.7-and 2.9-fold when compared to HCV-HCC alone. The risk of HCC is reduced significantly in patients who obtained a sustained viral response after treatment of HCV with a 54%reduction in all-cause mortality. While advances in medications recently have made treating HCV easier, vaccinations against the virus remain elusive.
Alcohol consumption remains an important risk factor for the development of HCC. The relationship between alcohol and liver disease correlates with the amount of alcohol consumed over a lifetime, with heavy alcohol use rather than social drinking being the main risk of HCC. The prevalence rate of alcohol abuse in the United States is five times higher than that of hepatitis C. Alcohol abuse accounts for 40%-50%of all HCC cases in Europe. Studies in Europe reported an increase in the relative risk of developing liver disease above 7-13 drinks per week in women and 14-27 drinks per week in men. In the United States, studies showed that the risk of liver cancer is increased two-to fourfold among persons drinking more than 60 g/d of ethanol. A meta-analysis of 19 prospective studies showed that consumption of three or more drinks per day resulted in a 16%increase risk of liver cancer and consumption of six or more drinks per day resulted in a 22%increase risk.
Sixty percent of patients older than 50 years with diabetes or obesity are thought to have NASH with advanced fibrosis. Chronic medical conditions such as diabetes mellitus and obesity increase the risk of HCC. Diabetes mellitus directly affects the liver because of the essential role the liver plays 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 between a 1.8-and 4-fold increased risks of HCC. It is well-known that obesity is associated with many hepatobiliary diseases, including nonalcoholic fatty liver disease (NAFLD) , steatosis, and cryptogenic cirrhosis all of which can lead to the development of HCC.
In some forms, the cells and ECM are extracted from clinical specimens including patients with HCC and intrahepatic cholangiocarcinoma.
B. Patient-derived cells maintenance for bioprinting
Compared to cell-lines or stem cell-derived cells, patient-derived cells (PDC) including tumor and immune cells generated from patient specimens directly reflect patient tumor characteristics and clinical responses (Kim S-Y, Lee JY, Kim DH, et al., Scientific Reports 9: 19909 (2019) ) . It is also considered to be the key object for successful personalized medicine development. The practical challenges for primary culture of cells involve limited availability of tumor specimens, restoration of tumor microenvironment for signal stimulation, outgrowth of stromal cells, and tumor cell senescence.
Both innate and adaptive immune components are commonly found in patient tumor microenvironment in tumor tissues including macrophages, lymphocytes and tumor cells. All the cells are derived from one or more of the following sources: adult human liver tissue; established liver-derived and monocytic 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 of isolated primary cells are immortalized with genes modification.
In preferred forms, the disclosed method involves the extraction of different populations of cells from tumor specimens and maintain their viability in bioprinting scaffold for further mechanistic and drug screening study.
C. 3D bioprinting of patient-derived ECM and cells
Currently bioprinting of specific tissues and organs of high cellular heterogeneity and complex structural organization still poses a significant challenge, mainly due to the construct of vascularization for adequate tissue oxygenation, nutrient delivery, and removal of waste (Bertassoni LE. Advanced Materials 34: 2101321 (2022) ) .
The disclosed method involves the procedure of combining bioprinting techniques including extrusion, micro-molding, digital light processing (DLP) with sacrificial cell-laden hydrogel to construct and combine different tumor compartments with multiple cells-materials interaction interfaces (tumor/non-tumor, tumor/vasculature, non-tumor/vasculature) for recreating the complexity of tumor architecture in HCC. An exemplary 3D bio-printed scaffold includes vasculature and multi-cellular interface (e.g., tumor and non-tumor interface and tumor and vasculature interface) (Figure 5) .
Based on bioprinting technology, bioinks can create cell aggregates including different types of cells, including cancer-associated fibroblasts, immune cells, and endothelial cells, to form vascular networks. In addition, bioprinting is able to create a biomimetic microenvironment for the heterogeneous distribution of biologically relevant proteins and growth factors, which are important to control tumor cell signaling, proliferation, and migration. Bioprinting technology has the ability to directly print/pattern cells in microfluidic devices, modeling vasculature and biological barriers. Vascularization is very important to maintain tissue activities and can be used to separate different tissue compartments. The vasculature of tumors is very different from the blood vessels that supply healthy tissues, especially in terms of heterogeneity, permeability, multidirectional blood flow, and unordered distribution throughout the tumor.
In preferred forms, bioprinting technology is used to fabricate mini-liver 3D cell culture system to mimic the heterogeneous microenvironment and complex 3D microstructures of the tumor.
1. Fabrication of patient derived ECM hydrogel
Lyophilized patient-derived ECM can be dissolved hydrogel. In one form, lyophilized patient-derived ECM is dissolved in PEGDA, alginate-gelatin and Gelatin methacryloyl (GelMA) hydrogel to generate 0.1-0.5%solution, and patient-derived cells are then ladened in the hydrogen before bioprinting.
2. 3D bio-printing of hydrogel compartments
All of the designs include three compartments, tumor, tumor stroma and vasculature. The first two compartments are 3D bio-printed by the bioink 1 and bioink 2 with designed path. The hydrogel matrix can also include a first cell type and a second cell type embedded therein. In certain aspects, the hydrogel compartment can include a first cell type embedded therein.
Photosensitive polymers which can be used include, by example but not limitation, poly (ethylene glycol) diacrylate (PEDGA) , gelatin methacrylate (GelMA) , polyacrylamide (PMA) , and alginate gelatin. Photosensitive polymers can also be modified by the conjugation of peptides such as collagen and fibronectin. Biomaterials that highly mimics liver tumor microenvironment and vessel specific ECM such as alginate, gelatin, collagen IV, and fibronectin will be used as the biomaterial for mimicking HCC tissue. Apart from patients’ ECM, such natural extra-ECM proteins are reported to be highly presented in both liver healthy and tumor tissue. For mimicking blood vessels, GelMA, and PMA, can be applied as the biomaterial as it is recently to assemble many properties of blood and lymph vessels. To mimic the tumor microenvironment, different cell types are ladened with ECM mixed hydrogels, hepatocytes in non-tumor compartment, HCC, immune cells in tumor compartment and endothelial cells in vasculature compartment.
The 3D construct includes inlet and outlet for connecting the microfluidic device to supply cells, nutrients and remove waste. Hypoxic can be induced in compartment 1 and 2 in area without vasculature.
3. Hydrogel matrix
After bio-printing, the photosensitive hydrogel solution is casted over the printed hydrogel matrix. The photosensitive hydrogel solution is composed of photosensitive polymer and photo-initiator, and various cells. Such photosensitive polymers can include, by way of example but not limitation, Gelma, PEGDA, polycaprolactone triol Methacryloyl (PCLMA) , Hyaluronic acid Methacryloyl (HAMA) . Such photo-initiators can include, by way of example but not limitation, lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate, lithium acylphosphinate, Irgacure 2959, and camphorquinone which is typically used with either ethyl 4-N, N-dimethylaminobenzoate or TEA and the photosensitizer isopropyl thioxanthone. 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 the bule light. In some forms, the hydrogel solution is 0.5-10%hydrogel. In some forms, the concentration of photoinitiator is 0.05-0.5%. In another embodiment, the cell density is 0-5.0 x10⁶ cells/mL hydrogel solution. In some forms, the curing time is 10-100s.
4. 3D bio-printing methods of hydrogels
The protocol of fabricating the 3D bio-printed scaffold with two designs is summarized in (Figure 7A-7B) . In one form, the bioinks are printed by Bioscaffold BS5.3 3D bio-printer by GeSim, Germany. 410 μm diameter nozzle is selected to realize the smooth printing. To obtain perfect aligned structures, the printing speed is optimized at 8 mm·s-1. The printing pressures are optimized as 0.08 -0.095 and 0.09-0.1 MPa for PEGDA and PAM bioinks, respectively. The spacing distance is optimized at 800 μm. After printing, the samples are irradiated under 365 nm UV-lamp (350 mW·cm-2 /2 min) to polymerize and crosslink the hydrogel. Then, the printed structures are placed in water overnight to completely remove the uncross-linked monomer and reach to the equilibrium shapes at the swelled state.
In some forms, bioprinting techniques used including extrusion, micro-molding, digital light processing (DLP) with sacrificial cell-laden hydrogel to construct, bioprinting in suspension, any combinations thereof. Bioprinting combines different tumor compartments for recreating the complete and complexity of tumor architecture in liver tumor.
In some forms, 3D bio-printed scaffolds are bio-printed by stereolithography.
5. Stiffness determination of clinical specimens and 3D bio-printed scaffold
In preferred forms, the 3D bio-printed scaffold is prepared to mimic patients liver stiffness to replicate the biophysical parameters of clinical specimens for studying cell behavior. In other forms, ECM stiffness gradient is generated, for example, by controlling the extent of photo-polymerization.
Measurement on fresh liver tissues and bio-printed scaffold are carried out on samples embedded in 4%low melting point agarose and cut into 50 μm sections using a Leica VT1000S vibratome (Buffalo Grove, IL) . Measurements on frozen liver tissues are performed on livers embedded in OCT compound (Sakura, Torrance, CA) , snap frozen via direct immersion into liquid nitrogen, and cut into 50μm sections on a Leica CM1900-13 cryostat. Measurements are conducted using a MFP3D-BIO inverted optical atomic force microscope (AFM) (Asylum Research, Santa Barbara, CA) mounted on a Nikon TE200-U inverted fluorescence microscope (Melville, NY) . Silicon nitride cantilevers (k = 0.06 N/m) modified with a 5μm diameter borosilicate glass spherical tip (Novascan Tech, Ames, IA) are used for indentation. For each session, cantilevers are calibrated using the thermal oscillation method. AFM force maps are performed on 90μm×90μm fields. Gradients are generated post-acquisition. Each experimental group included at least 3 different mice, with 3 sections from each mouse, and 2 gradients (one orthogonal and one parallel to the fibrosis) generated per section. Data analyses were done using the Hertz model in Igor Pro v. 6.22A (WaveMetrics, Lake Oswego, OR) and a Poisson’s ratio of 0.5.
To replicate the biophysical characteristics of clinical specimens to the 3D bio-printed scaffold, the value of stiffness of patient tumor and non-tumor tissue are first measured by AFM. Based on the results, the 3D scaffold is then constructed by variation of %of hydrogel (4a ofFigure 4) .
6. Perfusion with microfluidics
In some embodiments, after bio-fabrication, the hydrogel is connected with microfluidic system for delivery of nutrients, chemicals, proteins and cells in the culture model accompanied with time dependent diffusion (Figure 8A) . Continuous perfusion of the model with culture medium using microfluidics prolong the cell survivability and culture duration. Due to the diffusible nature of hydrogel used in the model, nutrient gradients can be generated and hence the hypoxic region similar to the phenomenon observed in tumor core in patients (Figure 8B) . Importantly, novel immunotherapeutic drug screening, such as pembrolizumab (anti-PD1) , monalizumab (anti-NKG2a) and ipilimumab (anti -CTLA-4) can also be feasible to be tested in the system. Microfluidic with flow rate range from 0 to 50μL/min can be used in the construct for delivery of nutrients, chemicals, proteins and cells.
7. Cell characteristics and viability
In preferred forms, a high cell viability is achieved in hydrogels.
In some forms, after bio-fabrication, all of the hydrogel ladened cells are cultured under standard conditions for 14 days with nutrients supplement by microfluidics. Confocal microscopy confirmed the support of the invention as a long-term 3D tumor culture system (Figure 8C) . In addition, the presence of patient-derived ECM is essential in stimulating the proliferation of the tumor cells (Figure 8D) .
8. Microstructure examination of 3D bio-printed scaffold by scanning electronic microscopy
Standard SEM microscopy on lyophilization of swollen hydrogels can be applied. The hydrogel samples are dried by lyophilization (sample 2 mm × 2 mm × 2 mm first frozen in a refrigerator at -30 ℃ for three hours and then freeze-dried for 12 hours at -90 ℃ at 0.1 mbar using lyophilizer Gregor Instrument) . Then, the sample is attached by conductive adhesive tape to an aluminum specimen holder and covered with a thin platinum layer (vacuum sputter coater SCD 050; Leica, Austria) . Then the samples can be inserted in the SEM microscope and observed in high vacuum using a secondary electron detector at accelerating voltage 10 kV (Figure 8E) . The presence of pore permit cell interactions and biological functions e.g., cell migration, phagocytosis etc. (Figure 8F) .
D. 4D printed stimuli-activated programmable building blocks fabrication
The preparation of 4D bio-printed scaffolds includes creating a 3D structure with integrated vascular networks. An exemplary technique for incorporating these networks involves using an aqueous fugitive ink made of Pluronic F127 for printing the vasculature, which can then be easily removed under gentle conditions. This triblock copolymer is selected for its dual benefits: it facilitates the printing of synthetic microvascular networks due to its printability and, crucially, exhibits biological inertness across a variety of cell types during the brief period needed for scaffold fabrication.
A key characteristic of Pluronic F127 is its ability to undergo thermally reversible gelation. Specifically, the fugitive ink transitions to a liquid state with reduced elasticity at temperatures below approximately 4℃. This property significantly aids in the ink's removal from the printed scaffold, allowing for the creation of precise internal voids or channels that mimic vascular structures. This method enhances the fidelity and functionality of the 3D bio-printed scaffolds, enabling the development of more complex and biologically relevant tissue models.
Engineered tissue modules can be fabricated using a layer-by-layer technique that involves the sequential printing of multiple bioinks. In an exemplary method, the perimeter of each module is outlined with PDMS (Polydimethylsiloxane) ink, establishing a defined boundary for the subsequent bioink deposition. Following the digestion of tumor tissues, isolated Hepatocellular Carcinoma (HCC) cells are suspended and adjusted to a specific concentration. These cells are then combined with a bioink composed of Gelatin Methacryloyl (GelMA) and sodium alginate.
The HCC cell-laden constructs can next be stabilized by immersing them in a 100 mM solution of calcium chloride (CaCl₂) for one minute. This process enhances the structural integrity of the constructs, leveraging the ability of calcium ions (Ca²⁺) to facilitate cross-linking within the sodium alginate matrix. Subsequently, a fugitive ink based on Pluronic F127 is patterned onto a glass substrate to define the vasculature channels.
Upon completion of the printing process, the vasculature and cell-containing scaffold are encapsulated within a layer of pure GelMA bioink. This bioink is preheated to 37℃ and then exposed to 405 nm blue light for at least 60 seconds, initiating cross-linking within the GelMA matrix to solidify the structure.
To finalize the fabrication, the entire assembly is cooled to 4℃. This cooling step liquifies the Pluronic F127 ink, allowing it to be evacuated under a moderate vacuum. The removal of the fugitive ink reveals the meticulously designed channels within the encapsulated structure, emulating interconnected vascular networks. This intricate procedure ensures the creation of robust, biofunctional engineered tissue modules with potential applications in tissue engineering and regenerative medicine.
IV. Methods of Use
Methods of using a 3D in vitro cell culture mimicking liver cancer and the associated TME in a subject are provided. The disclosed 3D bio-printed culture systems mimic the complex physiological architecture and provide integrated features including 3D scaffolding, multicellular compartments (s) , and a vasculature system to simulate dynamic flow in vivo. The disclosed 3D bio-printed culture platform also enables precise spatiotemporal control, and are this well suited for functional investigations of metastatic mechanisms and drug responses.
With its clinical relevance and biomimicry features, the 3D bio-printed culture system is suitable for use in vitro assays such as personalized drug screening, drug discovery, drug testing, preclinical research, three-dimensional biology studies, and cell-based screening for liver cancer.
With vasculature network and nutrients supply, the system is sustainable for at least two weeks hence the system is very suitable for multiple purposes including immuno-and chemo-drug screening, novel therapeutic drug testing and research purposes.
Although not intended to be limiting, the 3D and 4D bio-printed culture systems are useful for (i) studying tumor and immune cell interactions for the discovery of new therapeutic targets; (ii) testing drug safety and efficacy for precision medicine in patients in need thereof; and (iii) potentially the first tailor made and patient specific drug testing platform.
A. Mechanistic Study of Tumor Biology
One major objective in cancer research is to understand tumor biology related to metastasis mechanisms and TME, which could support the development of anti-cancer drugs and treatment approaches. This is important as it is appreciated that factors such as mechanical force, shear stress, chemotaxis, and hypoxia influence tumor progression, invasion, metastasis, and lastly drug metabolism.
In the microfluidic systems of the 3D bio-printed scaffold, the fabricated microchannels can be used to precisely control the interstitial fluidic flow or wall shear stress, pressure, etc. while networks can be made to closely resemble small capillaries in size and branching by integrating with various microelements.
Metastasis is still the leading cause of mortality for most cancer patients and is increasingly the most studied topic in cancer research (Hanahan D and Weinberg RA, Cell, 2011, 144, 646-674. ) . Cancer models, including in vitro cell culture and animal models, have contributed tremendously to developing diagnostics and treatments for cancer over the past several 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) . In vitro models mostly utilize 2D platforms which are easy to use but do have limitations due to a lack of mimicking the physiological in vivo environment (Tibbitt MW and Anseth KS, Biotechnology and Bioengineering, 2009, 103, 655-663) . There have been advances toward 3D systems in cancer research as scientists strive to make the systems.
The disclosed 3D bio-printed scaffold systems are more physiologically relevant compared to 2D platforms and other 3D platforms. It incorporates both normoxic (with vasculature) and hypoxic (without vasculature) compartments to recreate the tumor architecture and complexity observed in patients’ tumor. In preferred embodiments, the methods of using the 3D bio-printed scaffold provide mechanistic information of metastasis in liver cancer.
Solid TME produces many environmental stress factors, such as acidic pH, nutrient depletion, hypoxia, or accumulated waste product that blunt the action of immune response and suppress NK and T cells’a ntitumor activity. The microfluidic platforms of the 3D bio-printed scaffold are helpful to mimic and understand the cancer metastasis process and the mutual interactions between tumor cells and immune cells. In preferred forms, the disclosed microfluidic-based 3D bio-printed scaffold systems can reveal cell-cell interactions such as interactions between macrophages, tumor cells, and/or mesothelial cells. In some forms, tumor cell adhesion is studies under controlled static and shear conditions.
Cancer cells need to undergo extravasation, a process by which cancer cells move to secondary tissues or organs through blood vessels to form metastases. The endothelial barrier that cancer cells need to extravasate to reach the metastatic site is a major regulator of metastasis. Cancer cells adhere to blood vessels, transmigrate through the endothelium, and finally escape the circulation to invade into the secondary site. Therefore, in some forms, the 3D bio-printed scaffold is designed to investigate the role of surface adhesion molecules and endothelium permeability in cancer extravasation.
Studying angiogenesis or vasculogenesis is still a great challenge. The 3D bio-printed scaffold includes perfused microvessels that can represent endothelial cell lining. Thus, in some forms, the 3D bio-printed scaffold is use for studying angiogenesis. Angiogenesis is a procedure by which the growth of new blood vessels is formed from the pre-existing blood vessels.
B. New Therapeutic Targets
There is a lack of effective treatments available for liver cancer. The systems and methods are useful to investigate mechanistically cancer biology in vitro under controlled conditions.
The disclosed 3D bio-printed scaffold systems include immune cells and are therefore understand the process of immunosuppression and thus, the development of new immunotherapies by bridging the gap between in vitro and animal models.
In some forms, the methods can identify new therapeutic targets in liver cancer. In preferred forms, the methods enable validation of the new therapeutic target (s) on the 3D bio-printed scaffold. In further forms, two or more therapeutic targets are targeted simultaneously for enhanced efficacy.
C. Drug Efficacy for Precision Medicine in Individual Patients
The systems and methods are useful to investigate efficacy of one or more existing therapies including chemotherapeutic agents and immunotherapeutic agents to identify therapies likely to be most effective in decreasing or inhibiting the proliferation and/or viability of the cancer cells within the patient from where the biomaterials of the 3D bio-printed scaffold are derived from. For example, as demonstrated in Figures 10A-10C, the drug responses of two exemplary drug sorafenib and cisplatin used for HCC treatment were validated using the disclosed 3D bio-printed scaffolds (Figures 10A-10C) . With the multiple compartment design, the cytotoxic effects of different drugs with different dosages in regions with different cell populations including healthy hepatocytes were simultaneously assessed with live and dead assay (Figure 10A) . Compared to the 2D culture, the drug response rate with two dosages in the 3D construct showed similar clinical results (Figure 10B and 10C) . In another example, the 3D bio-printed scaffold was tested for efficacy and toxicities testing in situ programming of CAR-T cells, the invention is incorporated with patient’s derived tumor and immune cells for the reprogramming with viral platform (pLV-scFV-GPC3-CD28-41BB-CD3ζ-GFP) (Figure 11) . The transforming and anti-tumor efficacy of the treatment can be assessed by live-dead assay and used as preclinical reference for further clinical application. Thus, the disclosed 3D bio-printed scaffolds are suitable use in in vitro assays, drug discovery, drug testing, preclinical research, three-dimensional biology studies; and cell-based screening for liver and bile duct cancer.
In some forms, the disclosed methods are suitable for guiding treatment strategies in subjects identified as having HCC. Methods can assist in risk stratification and effective management of patients with HCC.
In other forms, the methods stratify cancer patients into responders and non-responders to current therapies. In some forms, the patients are non-responders to one or more existing therapies.
Exemplary conventional cancer therapeutics to be screened for efficacy on the 3D bio-printed scaffold include chemotherapeutic agents, cytokines, and chemokines. Most chemotherapeutic drugs can be divided 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. Additional therapeutics include monoclonal antibodies and the tyrosine kinase inhibitors e.g., imatinib mesylate (or) , which directly targets a molecular abnormality in certain types of cancer (chronic myelogenous leukemia, gastrointestinal stromal tumors) .
In some forms, one or more existing therapies to be screened for efficacy on the 3D bio-printed scaffold is an immunotherapy such inhibition of checkpoint proteins such as components of the PD-1/PD-L1 axis or CD28-CTLA-4 axis using one or more immune checkpoint modulators (e.g., PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists) , adoptive T cell therapy, and/or a cancer vaccine. Exemplary immune checkpoint modulators used in immunotherapy include Pembrolizumab (anti-PD1 mAb) , Durvalumab (anti-PDL1 mAb) , PDR001 (anti-PD1 mAb) , Atezolizumab (anti-PDL1 mAb) , Nivolumab (anti-PD1 mAb) , Tremelimumab (anti-CTLA4 mAb) , Avelumab (anti-PDL1 mAb) , and RG7876 (CD40 agonist mAb) .
In preferred forms, the methods further include a step of administering an effective amount of the therapeutic agent (s) identified as effective for the patient using the 3D bio-printed scaffold to reduce or inhibit proliferation, migration, invasion, motility, and/or metastatic abilities of the cancer cells in the patient. In further preferred forms, the therapeutic agent (s) are identified as effective for the patient using the 3D bio-printed scaffold are effective to reduce or inhibit tumor growth, tumor burden, and/or increase survival of the subject.
D. Patient-Specific Drug Screening &Testing Platform
The systems and methods are useful to investigate the activity or applicability of one or more test compounds to treat or alleviate or prevent one or more symptoms of liver cancer. The methods can effectively screen for new therapeutic agents that decrease or inhibit the proliferation and/or viability of the cancer cells within the 3D bio-printed scaffold compared to untreated control cancer cells in a separate 3D bio-printed scaffold prepared in a similar way with cells and biomaterials derived from the same patient.
In some forms, the methods include contacting cancer cells with a potential therapeutic agent and select the therapeutic agent as a suitable new drug candidate if it decreases or inhibits the proliferation and/or viability of the cancer cells by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more than 100%, compared to untreated control cancer cells.
In preferred forms, the methods further include a step of administering the new drug identified as effective on the 3D bio-printed scaffold to the patient where the cells and biomaterials of the 3D bio-printed scaffold are derived from in an amount effective to reduce or inhibit proliferation, migration, invasion, motility, and/or metastatic abilities of the cancer cells. In further preferred forms, the new drug identified as effective on the 3D bio-printed scaffold are effective to reduce or inhibit tumor growth, tumor burden, and/or increase survival of the subject.
E. Clinically Relevant Platform for Diagnostics
As described above, liver cancer is characterized by a complex landscape of cellular heterogeneity and 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 treatments (Atkins et al., Br J Cancer 123: 1496-501 (2020) ) . These findings underscore the importance of developing drug screening models that can accurately replicate a patient's specific TIME to predict therapeutic outcomes effectively.
To address this need, the described 3D bio-printed scaffold emerges as a tailored in vitro diagnostic tool, offering a high-fidelity platform for precision medicine in liver cancer. This scaffold enables the classification of immune subtypes and the simulation of the individual tumor immune environments, facilitating precision medicine approaches. It is specifically designed to assess the efficacy and safety of various treatment regimes, including traditional therapies, targeted molecular therapies, and immunotherapies.
The construction of the 3D bio-printed scaffold involves the use of cell-laden and Extracellular Matrix (ECM) component-embedded bioinks. These bioinks are crafted through advanced bioprinting technology, based on a detailed patient-specific TIME atlas derived from proteomics, RNA sequencing, and spatial transcriptomics data. By incorporating this comprehensive molecular and spatial information, the scaffold provides a precise, patient-specific platform for evaluating therapeutic responses, paving the way for more personalized and effective liver cancer treatment strategies.
The disclosed compositions and methods can be further understood through the following numbered paragraphs.
1. A 3-dimensional (3D) cell culture system comprising a first compartment comprising tumor cells, a second compartment comprising non-tumoral cells, and a third compartment comprising vasculature.
2. The 3D cell culture system of paragraph 1, wherein the tumor cells are of liver cancer.
3. The 3D cell culture system of paragraph 1 or 2, wherein the non-tumoral cells are hepatocytes and/or endothelial cells.
4. The 3D cell culture system of any one of paragraphs 1-3, wherein the first and the second compartments further comprise one or more types of immune cells.
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.
6. The 3D cell culture system of paragraph 5, wherein the macrophages are tumor-associated macrophages (TAMs) , and wherein the macrophages are CD68+CD163+.
7. The 3D cell culture system of paragraph 5, wherein the macrophages are CD68+CD206+.
8. The 3D cell culture system of paragraph 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 paragraphs 1-8, wherein one or more of the first, second, and third compartments further comprise extracellular matrix.
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 comprise hydrogel.
11. The 3D cell culture system of any one of paragraphs 1-10, wherein the system comprises oxygen gradient resulting in one or more normoxic regions and one or more hypoxic regions.
12. The 3D cell culture system of any one of paragraphs 1-11, wherein the tumor cells and non-tumoral cells are both derived from liver tumor tissues of the same subject.
13. The 3D cell culture system of any one of paragraphs 9-12, wherein the tumor cells, non-tumoral cells, and extracellular matrix are derived from liver tumor tissues of the same subject.
14. The 3D cell culture system of paragraph 12 or 13, wherein the subject has hepatocellular carcinoma and/or intrahepatic cholangiocarcinoma.
15. The 3D cell culture system of any one of paragraphs 10-14, wherein the hydrogels are natural or synthetic hydrogels selected from the group consisting of alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, combinations thereof.
16. The 3D cell culture system of any one of paragraphs 10-15, wherein the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer.
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.
18. The 3D cell culture system of any one of paragraphs 1-17, wherein the vasculature of the 3D cell culture system comprises at least one microfluidic channel.
19. The 3D cell culture system of any one of paragraphs 1-18, wherein the vasculature of the 3D cell culture system comprises between 2 and 20, preferably between 3 and 5, microfluidic channels.
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 fluidically connected to the microfluidic channel (s) .
21. The 3D cell culture system of any one of paragraphs 1-20, wherein the 3D cell culture system has at least about 10 mm in thickness.
22. The 3D cell culture system of any one of paragraphs 1-21, wherein the 3D cell culture system has a dimension of at least about 10mm x 10mm x 0.5mm.
23. A method of preparing a 3-dimensional (3D) cell culture system comprising
(a) isolating tumor cell, non-tumoral cells, and extracellular matrix (ECM) from a tumor sample;
(b) mixing with a hydrogel one or more of the tumor cell, non-tumoral cells, and ECM to provide one or more mixtures; and
(c) 3D bioprinting of the one or more mixtures from step (b) to assemble the 3D cell culture system.
24. The method of paragraph 23, further comparing the step of collect the tumor sample from a subject prior to step (a) .
25. The method of paragraph 23 or 24, wherein the tumor sample is a liver biopsy of a subject with liver cancer.
26. The method of any one of paragraphs 23-25, wherein the 3D bioprinting prints 5 or more layers.
27. The method of any one of paragraphs 23-26, wherein the hydrogels are natural or synthetic hydrogels.
28. The method of any one of paragraphs 23-27, wherein the hydrogels are photochemically crosslinked from photosensitive polymers and one or more photo-initiators.
29. The method of paragraph 28, wherein the photosensitive polymers selected from the group consisting of alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, polycaprolactone triol Methacryloyl (PCLMA) , Hyaluronic acid Methacryloyl (HAMA) , and combinations thereof.
30. The method of paragraph 28 or 29, wherein the photo-initiator 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 paragraphs 28-30, wherein the stiffness of the hydrogels is controlled by the extent of the photochemical crosslinking.
32. The method of any one of paragraphs 23-31, wherein the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer.
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.
34. The method of any one of paragraphs 23-33, wherein the 3D cell culture system comprises at least one microfluidic channel.
35. The method of any one of paragraphs 23-34, wherein the 3D cell culture system comprises between 2 and 20, preferably between 3 and 5, microfluidic channels.
36. The method of any one of paragraphs 33-35, wherein the at least one inlet and the at least one outlet are fluidically connected to the microfluidic channel (s) .
37. The method of any one of paragraphs 33-36, wherein the 3D cell culture system has at least about 10 mm in thickness.
38. The method of any one of paragraphs 33-37, wherein the 3D cell culture system has a dimension of at least about 10mm x 10mm x 0.5mm.
39. The method of any one of paragraphs 33-38, wherein the bioprinting comprises suspension, extrusion, micro-molding, digital light processing, stereolithography, and combinations thereof.
40. The method of any one of paragraphs 33-39, wherein the 3D cell culture system comprises at least one compartment comprising tumor cells, at least one compartment comprising non-tumoral cells, and at least one compartment comprising vasculature.
41. A method of using the 3D cell culture system of any one of paragraphs 1-22 for mechanistic studies, drug screening, immunotherapy testing, personalized medicine, and/or biomarker discovery.
42. The method of paragraph 41, wherein the 3D cell culture system is used for screening of cancer therapy for efficacy.
43. The method of paragraph 41 and 42, wherein the cancer therapy comprises conventional chemotherapeutic agents.
44. The method of paragraph 43, wherein the conventional chemotherapeutic agents are one or more selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents.
45. The method of paragraph 41 and 42, wherein the cancer therapy comprises immunotherapeutic agents.
46. The method of paragraph 45, wherein the immunotherapeutic agents are 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 paragraphs 42-46, further comprising identifying the cancer therapy having the best efficacy in reducing or inhibiting proliferation, migration, invasion, motility, and/or metastatic abilities of the tumor cells in the 3D cell culture system.
48. The method of paragraph 47, further comprising administering the cancer therapy having the best efficacy to the subject in an amount effective to reduce or inhibit tumor growth, tumor burden, and/or increase survival of the subject.
49. The method of paragraph 41, wherein the 3D cell culture system is used for mechanistic studies of tumor biology.
50. The method of paragraph 49, wherein the 3D cell culture system provides mechanistic information and new therapeutic targets of metastasis in liver cancer.

Claims

A 3-dimensional (3D) cell culture system comprising a first compartment comprising tumor cells, a second compartment comprising non-tumoral cells, and a third compartment comprising vasculature.
The 3D cell culture system of claim 1, wherein the tumor cells are of liver cancer.
The 3D cell culture system of claim 1 or 2, wherein the non-tumoral cells are hepatocytes and/or endothelial cells.
The 3D cell culture system of any one of claims 1-3, wherein the first and the second compartments further comprise one or more types of immune cells.
The 3D cell culture system of 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.
The 3D cell culture system of claim 5, wherein the macrophages are tumor-associated macrophages (TAMs) , and wherein the macrophages are CD68+CD163+.
The 3D cell culture system of claim 5, wherein the macrophages are CD68+CD206+.
The 3D cell culture system of claims 5, wherein the T-lymphocytes are CD3+CD4+ T cells and/or CD3+CD8+ T cells.
The 3D cell culture system of any one of claims 1-8, wherein one or more of the first, second, and third compartments further comprise extracellular matrix.
The 3D cell culture system of any one of claims 1-9, wherein one or more of the first, second, and third compartments further comprise hydrogel.
The 3D cell culture system of any one of claims 1-10, wherein the system comprises oxygen gradient resulting in one or more normoxic regions and one or more hypoxic regions.
The 3D cell culture system of any one of claims 1-11, wherein the tumor cells and non-tumoral cells are both derived from liver tumor tissues of the same subject.
The 3D cell culture system of any one of claims 9-12, wherein the tumor cells, non-tumoral cells, and extracellular matrix are derived from liver tumor tissues of the same subject.
The 3D cell culture system of claim 12 or 13, wherein the subject has hepatocellular carcinoma and/or intrahepatic cholangiocarcinoma.
The 3D cell culture system of any one of claims 10-14, wherein the hydrogels are natural or synthetic hydrogels selected from the group consisting of alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, combinations thereof.
The 3D cell culture system of any one of claims 10-15, wherein the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer.
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.
The 3D cell culture system of any one of claims 1-17, wherein the vasculature of the 3D cell culture system comprises at least one microfluidic channel.
The 3D cell culture system of any one of claims 1-18, wherein the vasculature of the 3D cell culture system comprises between 2 and 20, preferably between 3 and 5, microfluidic channels.
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 fluidically connected to the microfluidic channel (s) .
The 3D cell culture system of any one of claims 1-20, wherein the 3D cell culture system has at least about 10 mm in thickness.
The 3D cell culture system of any one of claims 1-21, wherein the 3D cell culture system has a dimension of at least about 10mm x 10mm x 0.5mm.
A method of preparing a 3-dimensional (3D) cell culture system comprising

(a) isolating tumor cell, non-tumoral cells, and extracellular matrix (ECM) from a tumor sample;

(b) mixing with a hydrogel one or more of the tumor cell, non-tumoral cells, and ECM to provide one or more mixtures; and

(c) 3D bioprinting of the one or more mixtures from step (b) to assemble the 3D cell culture system.
The method of claim 23, further comparing the step of collect the tumor sample from a subject prior to step (a) .
The method of claim 23 or 24, wherein the tumor sample is a liver biopsy of a subject with liver cancer.
The method of any one of claims 23-25, wherein the 3D bioprinting prints 5 or more layers.
The method of any one of claims 23-26, wherein the hydrogels are natural or synthetic hydrogels.
The method of any one of claims 23-27, wherein the hydrogels are photochemically crosslinked from photosensitive polymers and one or more photo-initiators.
The method of claim 28, wherein the photosensitive polymers selected from the group consisting of alginate, gelatin, Polyethylene Glycol Diacrylate (PEGDA) , Gelatin methacryloyl (GelMA) , F127, polyacrylamide, polycaprolactone triol Methacryloyl (PCLMA) , Hyaluronic acid Methacryloyl (HAMA) , and combinations thereof.
The method of claim 28 or 29, wherein the photo-initiator is selected from the group consisting of lithium phenyl-2, 4, 6-trimethylbenzoylphosphinate, lithium acylphosphinate, Irgacure 2959, and camphorquinone.
The method of any one of claims 28-30, wherein the stiffness of the hydrogels is controlled by the extent of the photochemical crosslinking.
The method of any one of claims 23-31, wherein the hydrogels have a stiffness similar to that of the liver tissue of the subject having liver cancer.
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.
The method of any one of claims 23-33, wherein the 3D cell culture system comprises at least one microfluidic channel.
The method of any one of claims 23-34, wherein the 3D cell culture system comprises between 2 and 20, preferably between 3 and 5, microfluidic channels.
The method of any one of claims 33-35, wherein the at least one inlet and the at least one outlet are fluidically connected to the microfluidic channel (s) .
The method of any one of claims 33-36, wherein the 3D cell culture system has at least about 10 mm in thickness.
The method of any one of claims 33-37, wherein the 3D cell culture system has a dimension of at least about 10mm x 10mm x 0.5mm.
The method of any one of claims 33-38, wherein the bioprinting comprises suspension, extrusion, micro-molding, digital light processing, stereolithography, and combinations thereof.
The method of any one of claims 33-39, wherein the 3D cell culture system comprises at least one compartment comprising tumor cells, at least one compartment comprising non-tumoral cells, and at least one compartment comprising vasculature.
A method of using the 3D cell culture system of any one of claims 1-22 for mechanistic studies, drug screening, immunotherapy testing, personalized medicine, and/or biomarker discovery.
The method of claim 41, wherein the 3D cell culture system is used for screening of cancer therapy for efficacy.
The method of claim 41 or 42, wherein the cancer therapy comprises conventional chemotherapeutic agents.
The method of claim 43, wherein the conventional chemotherapeutic agents are one or more selected from the group consisting of alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other antitumor agents.
The method of claim 41 or 42, wherein the cancer therapy comprises immunotherapeutic agents.
The method of claim 45, wherein the immunotherapeutic agents are one or more immune checkpoint modulators selected from the group consisting of PD-1 antagonists, PD-1 ligand antagonists, and CTLA4 antagonists.
The method of any one of claims 42-46, further comprising identifying the cancer therapy having the best efficacy in reducing or inhibiting proliferation, migration, invasion, motility, and/or metastatic abilities of the tumor cells in the 3D cell culture system.
The method of claim 47, further comprising administering the cancer therapy having the best efficacy to the subject in an amount effective to reduce or inhibit tumor growth, tumor burden, and/or increase survival of the subject.
The method of claim 41, wherein the 3D cell culture system is used for mechanistic studies of tumor biology.
The method of claim 49, wherein the 3D cell culture system provides mechanistic information and new therapeutic targets of metastasis in liver cancer.