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Lung cancer is the leading cause of cancer-related deaths worldwide, yet most currently used in vitro cancer models are confined to traditional 2D cell culture conditions. Recently; however, innovative 3D models such as tumor tissue equivalents (organoids) have been adopted by researchers to recapitulate tissue architecture and physiology in order to improve disease modeling and drug testing. We have hypothesized that 3D lung organoids, incorporating cells directly from patients, will enable personalized disease modeling and tumor cell characterization compared to traditional 2D cultures. Here, we discuss the fabrication of 3D lung cancer organoids using a rare cell source, pleural effusion aspirate. We tracked the phenotypic change that developed in short-term culturing and characterized the cell population within the organoids. We found that isolated patient cells embedded directly into organoids created anatomically relevant structures and displayed lung cancer specific behaviors compared to cultures that first grew in 2D conditions. Additionally, we compared responses of patient cells from pleural effusion aspirates to chemotherapy in 2D and 3D cell culture systems. Our results show that cells in 2D cultures are more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to isolate tumor cells from pleural effusion fluid of lung cancer patients and create organoids that display in vivo like anatomy and drug response. This technology can serve as a more accurate disease model for studying tumor progression and drug development.

Current in vitro three-dimensional (3D) models of liver tissue have been limited by the inability to study the effects of specific extracellular matrix (ECM) components on cell phenotypes. This is in part due to limitations in the availability of chemical modifications appropriate for this purpose. For example, hyaluronic acid (HA), which is a natural ECM component within the liver, lacks key ECM motifs (e.g. arginine–glycine–aspartic acid (RGD) peptides) that support cell adhesion. However, the addition of maleimide (Mal) groups to HA could facilitate the conjugation of ECM biomimetic peptides with thiol-containing end groups. In this study, we characterized a new crosslinkable hydrogel (i.e. HA-Mal) that yielded a simplified ECM-mimicking microenvironment supportive of 3D liver cell culture. We then performed a series of experiments to assess the impact of physical and biochemical signaling in the form of RGD peptide incorporation and transforming growth factor ß (TGF-ß) supplementation, respectively, on hepatic functionality. Hepatic stellate cells (i.e. LX-2) exhibited increased cell–matrix interactions in the form of cell spreading and elongation within HA-Mal matrices containing RGD peptides, enabling physical adhesions, whereas hepatocyte-like cells (HepG2) had reduced albumin and urea production. We further exposed the encapsulated cells to soluble TGF-ß to elicit a fibrosis-like state. In the presence of TGF-ß biochemical signals, LX-2 cells became activated and HepG2 functionality significantly decreased in both RGD-containing and RGD-free hydrogels. Altogether, in this study we have developed a hydrogel biomaterial platform that allows for discrete manipulation of specific ECM motifs within the hydrogel to better understand the roles of cell–matrix interactions on cell phenotype and overall liver functionality.

The field of three-dimensional (3D) bioprinting has advanced rapidly in recent years. Significant reduction in the costs associated with obtaining functional 3D bioprinting hardware platforms is both a cause and a result of these advances. As such, there are more laboratories than ever integrating bioprinting methodologies into their research. However, there is a lack of standards in the field of biofabrication governing any requirements or characteristics to support cross-compatibility with biomaterial bioinks, hardware, and different tissue types. Here we describe a modular extracellular matrix (ECM) inspired bioink comprised of collagen and hyaluronic acid base components that: 1) employ reversible internal hydrogen bonding forces to generate thixotropic materials that dynamically reduce their elastic moduli in response to increased shear stress, thus enabling increased compatibility with printing hardware; and 2) modular addons in the form of chemically-modified fibronectin and laminin that when covalently bound within the bioink support a variety of tissue types, including liver, neural, muscle, pancreatic islet, and adipose tissue. These features aim to accelerate the deployment of such bioinks for tissue engineering of functional constructs in the hands of various end users.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh in vivo environments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

Recent advances in biofabrication technologies, such as cell culture systems, and biomaterials have led to the development of three-dimensional (3D) cell culture platforms, such as tumor organoids. Tumor organoids are more physiologically accurate to the in vivo system, which they are intended to model, compared with traditional 2D cancer cell culture systems. Tumor organoids can mimic pathological and physical characteristics of tumors, as well as maintain genetic stability of the cancer cells. Furthermore, tumor organoids have advantage over animal models, being made from human cells and easily controlled in the laboratory to attain the desired tissue characteristics. In this section, we describe general tumor organoid technologies, the importance of the tumor microenvironment in model culture systems, and the use of tumor organoids in drug development and precision medicine. Organoid technologies continue to develop rapidly for applications in academic, clinical, and pharmaceutical settings.

The current drug development pipeline takes approximately fifteen years and $2.6 billion to get a new drug to market. Typically, drugs are tested on two-dimensional (2D) cell cultures and animal models to estimate their efficacy before reaching human trials. However, these models are often not representative of the human body. The 2D culture changes the morphology and physiology of cells, and animal models often have a vastly different anatomy and physiology than humans. The use of bioengineered human cell-based organoids may increase the probability of success during human trials by providing human-specific preclinical data. They could also be deployed for personalized medicine diagnostics to optimize therapies in diseases such as cancer. However, one limitation in employing organoids in drug screening has been the difficulty in creating large numbers of homogeneous organoids in form factors compatible with high-throughput screening (e.g., 96- and 384-well plates). Bioprinting can be used to scale up deposition of such organoids and tissue constructs. Unfortunately, it has been challenging to 3D print hydrogel bioinks into small-sized wells due to well&ndash, bioink interactions that can result in bioinks spreading out and wetting the well surface instead of maintaining a spherical form. Here, we demonstrate an immersion printing technique to bioprint tissue organoids in 96-well plates to increase the throughput of 3D drug screening. A hydrogel bioink comprised of hyaluronic acid and collagen is bioprinted into a viscous gelatin bath, which blocks the bioink from interacting with the well walls and provides support to maintain a spherical form. This method was validated using several cancerous cell lines, and then applied to patient-derived glioblastoma (GBM) and sarcoma biospecimens for drug screening.

INTRODUCTION. We have hypothesized that biofabrication of appendiceal tumor organoids allows for a more personalized clinical approach and facilitates research in a rare disease. METHODS. Appendiceal cancer specimens obtained during cytoreduction with hyperthermic intraperitoneal chemotherapy procedures (CRS/HIPEC) were dissociated and incorporated into an extracellular matrix-based hydrogel system as three-dimensional (3D), patient-specific tumor organoids. Cells were not sorted, preserving tumor heterogeneity, including stroma and immune cell components. Following establishment of organoid sets, chemotherapy drugs were screened in parallel. Live/dead staining and quantitative metabolism assays recorded which chemotherapies were most effective in killing cancer cells for a specific patient. Maintenance of cancer phenotypes were confirmed by using immunohistochemistry. RESULTS. Biospecimens from 12 patients were applied for organoid development between November 2016 and May 2018. Successful establishment rate of viable organoid sets was 75% (9/12). Average time from organoid development to chemotherapy testing was 7 days. These tumors included three high-grade appendiceal (HGA) and nine low-grade appendiceal (LGA) primaries obtained from sites of peritoneal metastasis. All tumor organoids were tested with chemotherapeutic agents exhibited responses that were either similar to the patient response or within the variability of the expected clinical response. More specifically, HGA tumor organoids derived from different patients demonstrated variable chemotherapy tumor-killing responses, whereas LGA organoids tested with the same regimens showed no response to chemotherapy. One LGA set of organoids was immune-enhanced with cells from a patient-matched lymph node to demonstrate feasibility of a symbiotic 3D reconstruction of a patient matched tumor and immune system component. CONCLUSIONS. Development of 3D appendiceal tumor organoids is feasible even in low cellularity LGA tumors, allowing for individual patient tumors to remain viable for research and personalized drug screening.

Cancer is often a complicated and dynamic disease, which makes determining the optimal treatment for a given patient a difficult endeavor. Moreover, even within a particular cancer type, different patients often have varying responses to the same therapies. Bioengineered tumor model systems specific to patients would allow preemptive screening of personalized therapies, facilitating identification of the most effective treatments prior to administration in the patients. Here, we provide an overview of organoid technology, and how these bioengineered tumor models can be harnessed for patient-centric personalized oncology. Organoid models have ranged from simple cell spheroids to more complex tumor-on-a-chip systems. The earliest of these models were comprised of easy to culture cell lines, but recent advances in 3D cell culture approaches have facilitated generation of human primary cell-based organoids. Importantly, recent efforts have been made to employ tumor biospecimens from human patients to create personalized tumor models for patient-specific predictive drug screening. Bioengineering and tissue engineering technologies have advanced significantly in recent years, culminating in the capability to biofabricate tissue and tumor organoids derived from individual human patients. In the near future, we anticipate such models being implemented in parallel with clinical practice as patient-oriented screening tools, thereby improving the success rates of oncology.

Variability in patient response to anti-cancer drugs is currently addressed by relating genetic mutations to chemotherapy through precision medicine. However, practical benefits of precision medicine to therapy design are less clear. Even after identification of mutations, oncologists are often left with several drug options, and for some patients there is no definitive treatment solution. There is a need for model systems to help predict personalized responses to chemotherapeutics. We have microengineered 3D tumor organoids directly from fresh tumor biopsies to provide patient-specific models with which treatment optimization can be performed before initiation of therapy. We demonstrate the initial implementation of this platform using tumor biospecimens surgically removed from two mesothelioma patients. First, we show the ability to biofabricate and maintain viable 3D tumor constructs within a tumor-on-a-chip microfluidic device. Second, we demonstrate that results of on-chip chemotherapy screening mimic those observed in subjects themselves. Finally, we demonstrate mutation-specific drug testing by considering the results of precision medicine genetic screening and confirming the effectiveness of the non-standard compound 3-deazaneplanocin A for an identified mutation. This patient-derived tumor organoid strategy is adaptable to a wide variety of cancers and may provide a framework with which to improve efforts in precision medicine oncology. Wake Forest Baptist Medical Center Clinical and Translational Science Institute Open Pilot Program; Wake Forest University School of Medicine; Comprehensive Cancer Center at Wake Forest Baptist Medical Center's NCI Cancer Center Support Grant [P30CA012197]; Defense Threat Reduction Agency (DTRA) under Space and Naval Warfare Systems Center Pacific (SSC PACIFIC) [N6601-13-C-2027] AS acknowledges funds from the Wake Forest Baptist Medical Center Clinical and Translational Science Institute Open Pilot Program. ARH acknowledges start-up funds from Wake Forest University School of Medicine. AS and ARH acknowledge services from the Wake Forest Cellular Imaging Shared Resource supported by the Comprehensive Cancer Center at Wake Forest Baptist Medical Center's NCI Cancer Center Support Grant P30CA012197. AS and ARH acknowledge funding by the Defense Threat Reduction Agency (DTRA) under Space and Naval Warfare Systems Center Pacific (SSC PACIFIC) Contract No. N6601-13-C-2027. The publication of this material does not constitute approval by the government of the findings or conclusions herein. The authors gratefully acknowledge Dr. Frank Marini for access to the confocal microscopy equipment employed in the studies.

Lung cancer is the leading cause of cancer-related death worldwide yet in vitro disease models have been limited to traditional 2D culture utilizing cancer cell lines. In contrast, recently developed 3D models (organoids) have been adopted by researchers to improve the physiological relevance of laboratory study. We have hypothesized that 3D hydrogel-based models will allow for improved disease replication and characterization over standard 2D culture using cells taken directly from patients. Here, we have leveraged the use of 3D hydrogel-based models to create lung cancer organoids using a unique cell source, pleural effusion aspirate, from multiple lung cancer patients. With these 3D models, we have characterized the cell populations comprising the pleural effusion aspirate and have tracked phenotypic changes that develop during short-term in vitro culture. We found that isolated, patient cells placed directly into organoids created anatomically relevant structures and exhibited lung cancer specific behaviors. On the other hand, cells first grown in plastic dishes and then cultured in 3D did not create similar structures. Further, we have been able to compare chemotherapeutic response of patient cells between 2D and 3D cell culture systems. Our results show that cells in 2D culture were more sensitive to treatment when compared with 3D organoids. Collectively, we have been able to utilize tumor cells from pleural effusion fluid of lung cancer patients to create organoids that display in vivo like anatomy and drug response and thus could serve as more accurate disease models for study of tumor progression and drug development.

INTRODUCTION. We hypothesized that engineering a combined lymph node/melanoma organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. METHODS. Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. RESULTS. Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor organoids between September 2017 and June 2018. Successful establishment of viable organoid sets was 90% (9/10), although organoid yield varied with biospecimen size. Average time from organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. CONCLUSION. Development of 3D mixed immune-enhanced tumor/node organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.

Current 3D printing of tissue is restricted by the use of biomaterials that do not recapitulate the native properties of the extracellular matrix (ECM). These restrictions have thus far prevented optimization of composition and structure of the in vivo tissue microenvironment. The artificial nature of currently used biomaterials affects cellular phenotype and function of the bioprinted tissues, and results in inaccurate modeling of disease and drug metabolism significantly. Collagen type I is the major structural component in the ECM, and is widely used as a 3D hydrogel, but is less applicable for 3D bioprinting due to low viscosity and slow polymerization. We have hypothesized that a combination of hyaluronic acid with collagen I yields a bioink with the properties required for extrusion bioprinting, while supporting native cell-matrix interactions and preservation of the native microenvironment properties. To test this hypothesis, we tested the viscoelastic properties of three bioink formulations -2:1, 3:1, and 4:1 collagen type I to hyaluronic acid, and examined cellular behavior in order to determine an optimal formulation that allows for bioprinting while supporting biological activity. We then employed this formulation to bioprint 3D liver tissue constructs containing primary human hepatocytes and liver stellate cells and tested the effects of acetaminophen, a common liver toxicant. Our results have shown that the combination of methacrylated collagen type I and thiolated hyaluronic acid yield a simple, printable bioink that allows for modulation that was directly related to stromal cell elongation. Further, the bioink adequately allowed for implementation as a support hydrogel for hepatocytes which were able to remain viable over two weeks and responded to drug treatment appropriately.

Over the past decade, advances in biomedical and tissue engineering technologies, such as cell culture techniques, biomaterials, and biofabrication, have driven increasingly widespread use of 3D cell culture platforms, and subsequently, the use of organoids in a variety of research endeavors. Given the 3D nature of these organoid systems, and the frequent inclusion of extracellular matrix (ECM) components, these constructs typically have more physiologically accurate cell-cell and cell-matrix interactions than traditional 2D cell cultures. As a result, 3D organoids have the capability to serve as superior model systems than their 2D counterparts. Moreover, as organoids can be biofabricated from highly functional human cells, they have certain advantages over animal models, being human in nature, and more easily manipulated in the laboratory. In this review, we describe such organoid technologies, and their deployment in drug development and precision medicine efforts. Organoid technologies are being rapidly developed for these applications and now represent a wide variety of tissue types and diseases. There is emerging evidence that organoids arepoised for widespread adoption not only in academia, but also the pharmaceutical industry and clinical diagnostic applications, positioning them as indispensable tools in medicine.

Despite having yielded extensive breakthroughs in cancer research, traditional 2D cell cultures have limitations in studying cancer progression and metastasis and screening therapeutic candidates. 3D systems can allow cells to grow, migrate, and interact with each other and the surrounding matrix, resulting in more realistic constructs. Furthermore, interactions between host tissue and developing tumors influence the susceptibility of tumors to drug treatments. The past decade has seen a rapid advancement of the application of 3D cellular systems to cancer research. These 3D tumor models, or tumor organoids, occupy a range of distinct form factors, each with their own strengths and weaknesses, and appropriateness for particular applications. In this chapter we highlight the major categories of tumor organoids and the methods by which they are biofabricated, aiming to provide the reader with an overview of the types of tumor organoids currently employed in cancer research applications.

Typical in vitro barrier and co-culture models rely upon thick semi-permeable polymeric membranes that physically separate two compartments. Polymeric track-etched membranes, while permeable to small molecules, are far from physiological with respect to physical interactions with co-cultured cells and are not compatible with high-resolution imaging due to light scattering and autofluorescence. Here we report on an optically transparent ultrathin membrane with porosity exceeding 20%. We optimize deposition and annealing conditions to create a tensile and robust porous silicon dioxide membrane that is comparable in thickness to the vascular basement membrane (100-300 nm). We demonstrate that human umbilical vein endothelial cells (HUVECs) spread and proliferate on these membranes similarly to control substrates. Additionally, HUVECs are able to transfer cytoplasmic cargo to adipose-derived stem cells when they are co-cultured on opposite sides of the membrane, demonstrating its thickness supports physiologically relevant cellular interactions. Lastly, we confirm that these porous glass membranes are compatible with lift-off processes yielding membrane sheets with an active area of many square centimeters. We believe that these membranes will enable new in vitro barrier and co-culture models while offering dramatically improved visualization compared to conventional alternatives.

Efforts to create physiologically relevant microenvironments for cell differentiation have led to the creation of three-dimensional (3D) support matrices with varying physical and chemical properties. In an effort to simplify the complexity of these matrices, while maintaining their unique permeable nature, we investigated the culture and differentiation of adipose-derived stem cells (ADSCs) on porous membranes. Membranes offer many of the benefits of a 3D matrix, but simplify cell seeding, imaging, analysis and post-differentiation recovery. After inducing the differentiation of ADSCs into endothelial cells (ECs), the cells cultured on porous substrates produce more branch points and greater tube length in angiogenesis assays compared to the cells cultured on non-porous controls. While we confirm that ADSCs can be induced with vascular endothelial growth factor to express endothelial adhesion molecule CD31 (PECAM-1), only when co-cultured across a membrane with human umbilical vein endothelial cells (HUVECs), do a subset of ADSCs show appropriate CD31 distribution along cell boundaries. Others have recently described that mesenchymal stem cells derive from perivascular cells including pericytes, which are known to wrap circumferentially around microvessels. We used ultrathin porous membranes to permit limited physical interactions between polarized HUVECs and ADSCs. In this arrangement, we found that the majority of ADSCs aligned perpendicular to the polarized HUVECs even though contact between the cell types was limited by pores less than 500 nm in diameter. Together, this ADSC pericyte behavior in combination with the ability to differentiate into ECs shows the potential versatility of ADSCs in the engineering of vascular networks.

High-throughput technologies have become essential in many fields of pharmaceutical and biological development and production. Such technologies were initially developed with compatibility with liquid handling-based cell culture techniques to produce large-scale 2D cell culture experiments for the compound analysis of candidate drug compounds. Over the past two decades, tools for creating 3D cell cultures, organoids, and other 3D in vitro models, such as cell supportive biomaterials and 3D bioprinting, have rapidly advanced. Concurrently, a significant body of evidence has accumulated which speaks to the many benefits that 3D model systems have over traditional 2D cell cultures. Specifically, 3D cellular models better mimic aspects such as diffusion kinetics, cell-cell interactions, cell-matrix interactions, inclusion of stroma, and other features native to in vivo tissue and as such have become an integral part of academic research. However, most high throughput assays were not developed to specifically support 3D systems. Here, we describe the need for improved compatibility and relevant advances toward deployment and adoption of high throughput 3D models to improve disease modeling, drug efficacy testing, and precision medicine applications.