Biofabrication of 3D breast cancer models for dissecting the cytotoxic response of human T cells expressing engineered MAIT cell receptors.

Immunotherapy has revolutionized cancer treatment with the advent of advanced cell engineering techniques aimed at targeted therapy with reduced systemic toxicity. However, understanding the underlying immune-cancer interactions require development of advanced three-dimensional (3D) models of human tissues. In this study, we fabricated 3D tumor models with increasing complexity to study the cytotoxic responses of CD8+T cells, genetically engineered to express mucosal-associated invariant T (MAIT) cell receptors, towards MDA-MB-231 breast cancer cells. Homotypic MDA-MB-231 and heterotypic MDA-MB-231/human dermal fibroblast tumor spheroids were primed with precursor MAIT cell ligand 5-amino-6-D-ribitylaminouracil (5-ARU). Engineered T cells effectively eliminated tumors after a 3 d culture period, demonstrating that the engineered T cell receptor recognized major histocompatibility complex class I-related (MR1) protein expressing tumor cells in the presence of 5-ARU. Tumor cell killing efficiency of engineered T cells were also assessed by encapsulating these cells in fibrin, mimicking a tumor extracellular matrix microenvironment. Expression of proinflammatory cytokines such as interferon gamma, interleukin-13, CCL-3 indicated immune cell activation in all tumor models, post immunotherapy. Further, in corroborating the cytotoxic activity, we found that granzymes A and B were also upregulated, in homotypic as well as heterotypic tumors. Finally, a 3D bioprinted tumor model was employed to study the effect of localization of T cells with respect to tumors. T cells bioprinted proximal to the tumor had reduced invasion index and increased cytokine secretion, which indicated a paracrine mode of immune-cancer interaction. Development of 3D tumor-T cell platforms may enable studying the complex immune-cancer interactions and engineering MAIT cells for cell-based cancer immunotherapies.

Chemotherapeutics and CAR-T Cell-Based Immunotherapeutics Screening on a 3D Bioprinted Vascularized Breast Tumor Model.

Despite substantial advancements in development of cancer treatments, lack of standardized and physiologically-relevant in vitro testing platforms limit the early screening of anticancer agents. A major barrier is the complex interplay between the tumor microenvironment and immune response. To tackle this, a dynamic-flow based 3D bioprinted multi-scale vascularized breast tumor model, responding to chemo and immunotherapeutics is developed. Heterotypic tumors are precisely bioprinted at pre-defined distances from a perfused vasculature, exhibit tumor angiogenesis and cancer cell invasion into the perfused vasculature. Bioprinted tumors treated with varying dosages of doxorubicin for 72 h portray a dose-dependent drug response behavior. More importantly, a cell based immune therapy approach is explored by perfusing HER2-targeting chimeric antigen receptor (CAR) modified CD8+ T cells for 24 or 72 h. Extensive CAR-T cell recruitment to the endothelium, substantial T cell activation and infiltration to the tumor site, resulted in up to ≈70% reduction in tumor volumes. The presented platform paves the way for a robust, precisely fabricated, and physiologically-relevant tumor model for future translation of anti-cancer therapies to personalized medicine.

Studying Tumor Angiogenesis and Cancer Invasion in a Three-Dimensional Vascularized Breast Cancer Micro-Environment.

Metastatic breast cancer is one of the deadliest forms of malignancy, primarily driven by its characteristic micro-environment comprising cancer cells interacting with stromal components. These interactions induce genetic and metabolic alterations creating a conducive environment for tumor growth. In this study, a physiologically relevant 3D vascularized breast cancer micro-environment is developed comprising of metastatic MDA-MB-231 cells and human umbilical vein endothelial cells loaded in human dermal fibroblasts laden fibrin, representing the tumor stroma. The matrix, as well as stromal cell density, impacts the transcriptional profile of genes involved in tumor angiogenesis and cancer invasion, which are hallmarks of cancer. Cancer-specific canonical pathways and activated upstream regulators are also identified by the differential gene expression signatures of these composite cultures. Additionally, a tumor-associated vascular bed of capillaries is established exhibiting dilated vessel diameters, representative of in vivo tumor physiology. Further, employing aspiration-assisted bioprinting, cancer-endothelial crosstalk, in the form of collective angiogenesis of tumor spheroids bioprinted at close proximity, is identified. Overall, this bottom-up approach of tumor micro-environment fabrication provides an insight into the potential of in vitro tumor models and enables the identification of novel therapeutic targets as a preclinical drug screening platform.

Fabrication of PDMS microfluidic devices using nanoclay-reinforced Pluronic F-127 as a sacrificial ink.

Three-dimensional (3D) printing technology is increasingly being employed in biochemical as well as clinical applications and more importantly in fabrication of microfluidic devices. However, the microfluidic community mainly relies on photolithography for fabrication of a defined mask, which is both tedious and expensive requiring clean room settings as well as limited to the generation of two-dimensional (2D) features. In this work, we 3D printed nanoclay-reinforced Pluronic ink as a sacrificial material, which exhibited shear thinning behavior and superior printability allowing the fabrication of unsupported or overhanging templates of channels with uniform diameter and circular cross-sections. To highlight the potential and effectiveness of the presented approach, we fabricated a human blood vessel-on-a-chip model with curved as well as straight channels. These channels were then lined with Human Umbilical Vein Endothelial cells (HUVECs) and subjected to a dynamic culture for 10 days to explore the effect of shear stress on HUVEC morphology based on the location of HUVECs in the devices. Overall, we presented a highly affordable, useful, and practical approach in fabrication of closed microfluidic channels in PDMS based devices, which holds great potential for numerous applications, such as but not limited to tissue/organ-on-chip, microfluidics, point-of-care devices and drug screening platforms.

Aspiration-assisted bioprinting of co-cultured osteogenic spheroids for bone tissue engineering.

Conventional top-down approaches in tissue engineering involving cell seeding on scaffolds have been widely used in bone engineering applications. However, scaffold-based bone tissue constructs have had limited clinical translation due to constrains in supporting scaffolds, minimal flexibility in tuning scaffold degradation, and low achievable cell seeding density as compared with native bone tissue. Here, we demonstrate a pragmatic and scalable bottom-up method, inspired from embryonic developmental biology, to build three-dimensional (3D) scaffold-free constructs using spheroids as building blocks. Human umbilical vein endothelial cells (HUVECs) were introduced to human mesenchymal stem cells (hMSCs) (hMSC/HUVEC) and spheroids were fabricated by an aggregate culture system. Bone tissue was generated by induction of osteogenic differentiation in hMSC/HUVEC spheroids for 10 d, with enhanced osteogenic differentiation and cell viability in the core of the spheroids compared to hMSC-only spheroids. Aspiration-assisted bioprinting (AAB) is a new bioprinting technique which allows precise positioning of spheroids (11% with respect to the spheroid diameter) by employing aspiration to lift individual spheroids and bioprint them onto a hydrogel. AAB facilitated bioprinting of scaffold-free bone tissue constructs using the pre-differentiated hMSC/HUVEC spheroids. These constructs demonstrated negligible changes in their shape for two days after bioprinting owing to the reduced proliferative potential of differentiated stem cells. Bioprinted bone tissues showed interconnectivity with actin-filament formation and high expression of osteogenic and endothelial-specific gene factors. This study thus presents a viable approach for 3D bioprinting of complex-shaped geometries using spheroids as building blocks, which can be used for various applications including but not limited to, tissue engineering, organ-on-a-chip and microfluidic devices, drug screening and, disease modeling.

3D bioprinting for reconstituting the cancer microenvironment.

The cancer microenvironment is known for its complexity, both in its content as well as its dynamic nature, which is difficult to study using two-dimensional (2D) cell culture models. Several advances in tissue engineering have allowed more physiologically relevant three-dimensional (3D) in vitro cancer models, such as spheroid cultures, biopolymer scaffolds, and cancer-on-a-chip devices. Although these models serve as powerful tools for dissecting the roles of various biochemical and biophysical cues in carcinoma initiation and progression, they lack the ability to control the organization of multiple cell types in a complex dynamic 3D architecture. By virtue of its ability to precisely define perfusable networks and position of various cell types in a high-throughput manner, 3D bioprinting has the potential to more closely recapitulate the cancer microenvironment, relative to current methods. In this review, we discuss the applications of 3D bioprinting in mimicking cancer microenvironment, their use in immunotherapy as prescreening tools, and overview of current bioprinted cancer models.

3D Bioprinting of Carbohydrazide-Modified Gelatin into Microparticle-Suspended Oxidized Alginate for the Fabrication of Complex-Shaped Tissue Constructs.

Extrusion-based bioprinting of hydrogels in a granular secondary gel enables the fabrication of cell-laden three-dimensional (3D) constructs in an anatomically accurate manner, which is challenging using conventional extrusion-based bioprinting processes. In this study, carbohydrazide-modified gelatin (Gel-CDH) was synthesized and deposited into a new multifunctional support bath consisting of gelatin microparticles suspended in an oxidized alginate (OAlg) solution. During extrusion, Gel-CDH and OAlg were rapidly cross-linked because of the Schiff base formation between aldehyde groups of OAlg and amino groups of Gel-CDH, which has not been demonstrated in the domain of 3D bioprinting before. Rheological results indicated that hydrogels with lower OAlg to Gel-CDH ratios possessed superior mechanical rigidity. Different 3D geometrically intricate constructs were successfully created upon the determination of optimal bioprinting parameters. Human mesenchymal stem cells and human umbilical vein endothelial cells were also bioprinted at physiologically relevant cell densities. The presented study has offered a novel strategy for bioprinting of natural polymer-based hydrogels into 3D complex-shaped biomimetic constructs, which eliminated the need for cytotoxic supplements as external cross-linkers or additional cross-linking processes, therefore expanding the availability of bioinks.

Aspiration-assisted bioprinting for precise positioning of biologics.

Three-dimensional (3D) bioprinting is an appealing approach for building tissues; however, bioprinting of mini-tissue blocks (i.e., spheroids) with precise control on their positioning in 3D space has been a major obstacle. Here, we unveil "aspiration-assisted bioprinting (AAB)," which enables picking and bioprinting biologics in 3D through harnessing the power of aspiration forces, and when coupled with microvalve bioprinting, it facilitated different biofabrication schemes including scaffold-based or scaffold-free bioprinting at an unprecedented placement precision, ~11% with respect to the spheroid size. We studied the underlying physical mechanism of AAB to understand interactions between aspirated viscoelastic spheroids and physical governing forces during aspiration and bioprinting. We bioprinted a wide range of biologics with dimensions in an order-of-magnitude range including tissue spheroids (80 to 600 µm), tissue strands (~800 µm), or single cells (electrocytes, ~400 µm), and as applications, we illustrated the patterning of angiogenic sprouting spheroids and self-assembly of osteogenic spheroids.

Extrusion-based printing of sacrificial Carbopol ink for fabrication of microfluidic devices.

Current technologies for manufacturing of microfluidic devices include soft-lithography, wet and dry etching, thermoforming, micro-machining and three-dimensional (3D) printing. Among them, soft-lithography has been the mostly preferred one in medical and pharmaceutical fields due to its ability to generate polydimethylsiloxane (PDMS) devices with resin biocompatibility, throughput and transparency for imaging. It is a multi-step process requiring the preparation of a silicon wafer pattern, which is fabricated using photolithography according to a defined mask. Photolithography is a costly, complicated and time-consuming process requiring a clean-room environment, and the technology is not readily accessible in most of the developing countries. In addition, generated patterns on photolithography-made silicon wafers do not allow building 3D intricate shapes and silicon direct bonding is thus utilized for closed fluid channels and complex 3D structures. 3D Printing of PDMS has recently gained significant interest due to its ability to define complex 3D shapes directly from user-defined designs. In this work, we investigated Carbopol as a sacrificial gel in order to create microfluidic channels in PDMS devices. Our study demonstrated that Carbopol ink possessed a shear-thinning behavior and enabled the extrusion-based printing of channel templates, which were overlaid with PDMS to create microfluidic devices upon curing of PDMS and removal of the sacrificial Carbopol ink. To demonstrate the effectiveness of the fabricated devices, channels were lined up with human umbilical vein endothelial cells (HUVECs) and human bone marrow endothelial cells (BMECs) in separate devices, where both HUVECs and BMECs demonstrated the formation of endothelium with highly aligned cells in the direction of fluid flow. Overall, we here present a highly affordable and practical approach in fabrication of PDMS devices with closed fluid channels, which have great potential in a myriad of applications from cancer treatments to infectious disease diagnostics to artificial organs.

Challenges in Bio-fabrication of Organoid Cultures.

Three-dimensional (3D) organoids have shown advantages in cell culture over traditional two-dimensional (2D) culture, and have great potential in various applications of tissue engineering. However, there are limitations in current organoid fabrication technologies, such as uncontrolled size, poor reproductively, and inadequate complexity of organoids. In this chapter, we present the existing techniques and discuss the major challenges for 3D organoid biofabrication. Future perspectives on organoid bioprinting are also discussed, where bioprinting technologies are expected to make a major contribution in organoid fabrication, such as realizing mass production and constructing complex heterotypic tissues, and thus further advance the translational application of organoids in tissue engineering and regenerative medicine as well drug testing and pharmaceutics.

Sprouting angiogenesis in engineered pseudo islets.

Despite the recent achievements in cell-based therapies for curing type-1 diabetes (T1D), capillarization in beta (ß)-cell clusters is still a major roadblock as it is essential for long-term viability and function of ß-cells in vivo. In this research, we report sprouting angiogenesis in engineered pseudo islets (EPIs) made of mouse insulinoma ßTC3 cells and rat heart microvascular endothelial cells (RHMVECs). Upon culturing in three-dimensional (3D) constructs under angiogenic conditions, EPIs sprouted extensive capillaries into the surrounding matrix. Ultra-morphological analysis through histological sections also revealed presence of capillarization within EPIs. EPIs cultured in 3D constructs maintained their viability and functionality over time while non-vascularized EPIs, without the presence of RHMVECs, could not retain their viability nor functionality. Here we demonstrate angiogenesis in engineered islets, where patient specific stem cell-derived human beta cells can be combined with microvascular endothelial cells in the near future for long-term graft survival in T1D patients.

3D Printing of PDMS Improves Its Mechanical and Cell Adhesion Properties.

Despite extensive use of polydimethylsiloxane (PDMS) in medical applications, such as lab-on-a-chip or tissue/organ-on-a-chip devices, point-of-care devices, and biological machines, the manufacturing of PDMS devices is limited to soft-lithography and its derivatives, which prohibits the fabrication of geometrically complex shapes. With the recent advances in three-dimensional (3D) printing, use of PDMS for fabrication of such complex shapes has gained considerable interest. This research presents a detailed investigation on printability of PDMS elastomers over three concentrations for mechanical and cell adhesion studies. The results demonstrate that 3D printing of PDMS improved the mechanical properties of fabricated samples up to three fold compared to that of cast ones because of the decreased porosity of bubble entrapment. Most importantly, 3D printing facilitates the adhesion of breast cancer cells, whereas cast samples do not allow cellular adhesion without the use of additional coatings such as extracellular matrix proteins. Cells are able to adhere and grow in the grooves along the printed filaments demonstrating that 3D printed devices can be engineered with superior cell adhesion qualities compared to traditionally manufactured PDMS devices.

Transplantation of Bioprinted Tissues and Organs: Technical and Clinical Challenges and Future Perspectives.

: Three-dimensional (3D) bioprinting is a revolutionary technology in building living tissues and organs with precise anatomic control and cellular composition. Despite the great progress in bioprinting research, there has yet to be any clinical translation due to current limitations in building human-scale constructs, which are vascularized and readily implantable. In this article, we review the current limitations and challenges in 3D bioprinting, including in situ techniques, which are one of several clinical translational models to facilitate the application of this technology from bench to bedside. A detailed discussion is made on the technical barriers in the fabrication of scalable constructs that are vascularized, autologous, functional, implantable, cost-effective, and ethically feasible. Clinical considerations for implantable bioprinted tissues are further expounded toward the correction of end-stage organ dysfunction and composite tissue deficits.

Competent processing techniques for scaffolds in tissue engineering.

Engineering a functional tissue ex vivo requires a synchronized effort towards developing technologies for ECM mimicking scaffold and cultivating tissue-specific cells in an integrated and controlled manner. Cell-interactive scaffolds in three dimensions (3D), designed and processed appropriately with an apt biomaterial to yield optimal porosity and mechanical strength is the key in tissue engineering (TE). In order to accomplish these facets in a 3D scaffold, multiple techniques and processes have been explored by researchers all over the world. New techniques offering reasonable flexibility to use blends of different materials for integrated tissue-specific mechanical strength and biocompatibility have an edge over conventional methods. They may allow a combinatorial approach with a mix of materials while incorporating multiple processing techniques for successful creation of tissue-specific ECM mimics. In this review, we analyze the material requirement from different TE perspectives, while discussing pros and cons of advanced fabrication techniques for scale-up manufacturing.

The bioink: A comprehensive review on bioprintable materials.

This paper discusses "bioink", bioprintable materials used in three dimensional (3D) bioprinting processes, where cells and other biologics are deposited in a spatially controlled pattern to fabricate living tissues and organs. It presents the first comprehensive review of existing bioink types including hydrogels, cell aggregates, microcarriers and decellularized matrix components used in extrusion-, droplet- and laser-based bioprinting processes. A detailed comparison of these bioink materials is conducted in terms of supporting bioprinting modalities and bioprintability, cell viability and proliferation, biomimicry, resolution, affordability, scalability, practicality, mechanical and structural integrity, bioprinting and post-bioprinting maturation times, tissue fusion and formation post-implantation, degradation characteristics, commercial availability, immune-compatibility, and application areas. The paper then discusses current limitations of bioink materials and presents the future prospects to the reader.

A comprehensive review on droplet-based bioprinting: Past, present and future.

Droplet-based bioprinting (DBB) offers greater advantages due to its simplicity and agility with precise control on deposition of biologics including cells, growth factors, genes, drugs and biomaterials, and has been a prominent technology in the bioprinting community. Due to its immense versatility, DBB technology has been adopted by various application areas, including but not limited to, tissue engineering and regenerative medicine, transplantation and clinics, pharmaceutics and high-throughput screening, and cancer research. Despite the great benefits, the technology currently faces several challenges such as a narrow range of available bioink materials, bioprinting-induced cell damage at substantial levels, limited mechanical and structural integrity of bioprinted constructs, and restrictions on the size of constructs due to lack of vascularization and porosity. This paper presents a first-time review of DBB and comprehensively covers the existing DBB modalities including inkjet, electrohydrodynamic, acoustic, and micro-valve bioprinting. The recent notable studies are highlighted, the relevant bioink biomaterials and bioprinters are expounded, the application areas are presented, and the future prospects are provided to the reader.