Toward a 3D Printed Perfusable Islet Embedding Structure: Technical Notes and Preliminary Results.

To date, islet transplantation to treat type 1 diabetes mellitus remains unsuccessful in long-term follow-up, mainly due to failed engraftment and reconstruction of the islet niche. Alternative approaches, such as islet embedding structures (IESs) based on 3D printing have been developed. However, most of them have been implanted subcutaneously and only a few are intended for direct integration into the vascular system through anastomosis. In this study, we 3D printed a proof-of-concept IES using gelatin methacrylate biocompatible ink. This structure consisted of a branched vascular system surrounding both sides of a central cavity dedicated to islets of Langerhans. Furthermore, we designed a bioreactor optimized for these biological structures. This bioreactor allows seeding and perfusion experiments under sterile and physiological conditions. Preliminary experiments aimed to analyze if the vascular channel could successfully be seeded with mature endothelial cells and the central cavity with rat islets. Subsequently, the structures were used for a humanized model seeding human endothelial progenitor cells (huEPC) within the vascular architecture and human islets co-cultured with huEPC within the central cavity. The constructs were tested for hemocompatibility, suture strength, and anastomosability. The 3D printed IES appeared to be hemocompatible and anastomosable using an alternative cuff anastomosis in a simple ex vivo perfusion model. While rat islets alone could not successfully be embedded within the 3D printed structure for 3 days, human islets co-cultivated with huEPC successfully engrafted within the same time. This result emphasizes the importance of co-culture, nursing cells, and islet niche. In conclusion, we constructed a proof-of-concept 3D printed islet embedding device consisting of a vascular channel that is hemocompatible and perspectively anastomosable to clinical scale blood vessels. However, there are numerous limitations in this model that need to be overcome to transfer this technology to the bedside.

Biofabrication of synthetic human liver tissue with advanced programmable functions.

Advances in cellular engineering, as well as gene, and cell therapy, may be used to produce human tissues with programmable genetically enhanced functions designed to model and/or treat specific diseases. Fabrication of synthetic human liver tissue with these programmable functions has not been described. By generating human iPSCs with target gene expression controlled by a guide RNA-directed CRISPR-Cas9 synergistic-activation-mediator, we produced synthetic human liver tissues with programmable functions. Such iPSCs were guide-RNA-treated to enhance expression of the clinically relevant CYP3A4 and UGT1A1 genes, and after hepatocyte-directed differentiation, cells demonstrated enhanced functions compared to those found in primary human hepatocytes. We then generated human liver tissue with these synthetic human iPSC-derived hepatocytes (iHeps) and other non-parenchymal cells demonstrating advanced programmable functions. Fabrication of synthetic human liver tissue with modifiable functional genetic programs may be a useful tool for drug discovery, investigating biology, and potentially creating bioengineered organs with specialized functions.

Blends of gelatin and hyaluronic acid stratified by stereolithographic bioprinting approximate cartilaginous matrix gradients.

Stereolithographic bioprinting holds great promise in the quest for creating artificial, biomimetic cartilage-like tissue. To introduce a more biomimetic approach, we examined blending and stratifying methacrylated hyaluronic acid (HAMA) and methacrylated gelatin (GelMA) bioinks to mimic the zonal structure of articular cartilage. Bioinks were suspended with porcine chondrocytes before being printed in a digital light processing approach. Homogenous constructs made from hybrid bioinks of varying polymer ratios as well as stratified constructs combining different bioink blends were cultivated over 14 days and analyzed by histochemical staining for proteoglycans/collagen type II, cartilage marker expression analysis, and for cellular viability. The stiffness of blended bioinks increased gradually with HAMA content, from 2.41 ± 0.58 kPa (5% GelMA, 0% HAMA) to 8.84 ± 0.11 kPa (0% GelMA, 2% HAMA). Cell-laden constructs maintained vital chondrocytes and supported the formation of proteoglycans and collagen type II. Higher concentrations of GelMA resulted in increased formation of cartilaginous matrix proteins and a more premature phenotype. However, decreased matrix production in central areas of constructs was observed in higher GelMA content constructs. Biomimetically stratified constructs retained their gradient-like structure even after ECM formation, and exclusively exhibited a significant increase in COL2A1 gene expression (+178%). Concluding, we showed the feasibility of blending and stratifying photopolymerizable, natural biopolymers by SLA bioprinting to modulate chondrocyte attributes and to create zonally segmented ECM structures, contributing to improved modeling of cartilaginous tissue for regenerative therapies or in vitro models.

A Reproducible Bioprinted 3D Tumor Model Serves as a Preselection Tool for CAR T Cell Therapy Optimization.

Chimeric antigen receptor (CAR) T cell performance against solid tumors in mouse models and clinical trials is often less effective than predicted by CAR construct selection in two-dimensional (2D) cocultures. Three-dimensional (3D) solid tumor architecture is likely to be crucial for CAR T cell efficacy. We used a three-dimensional (3D) bioprinting approach for large-scale generation of highly reproducible 3D human tumor models for the test case, neuroblastoma, and compared these to 2D cocultures for evaluation of CAR T cells targeting the L1 cell adhesion molecule, L1CAM. CAR T cells infiltrated the model, and both CAR T and tumor cells were viable for long-term experiments and could be isolated as single-cell suspensions for whole-cell assays quantifying CAR T cell activation, effector function and tumor cell cytotoxicity. L1CAM-specific CAR T cell activation by neuroblastoma cells was stronger in the 3D model than in 2D cocultures, but neuroblastoma cell lysis was lower. The bioprinted 3D neuroblastoma model is highly reproducible and allows detection and quantification of CAR T cell tumor infiltration, representing a superior in vitro analysis tool for preclinical CAR T cell characterization likely to better select CAR T cells for in vivo performance than 2D cocultures.

3D bioprinting of tissue-specific osteoblasts and endothelial cells to model the human jawbone.

Jawbone differs from other bones in many aspects, including its developmental origin and the occurrence of jawbone-specific diseases like MRONJ (medication-related osteonecrosis of the jaw). Although there is a strong need, adequate in vitro models of this unique environment are sparse to date. While previous approaches are reliant e.g. on scaffolds or spheroid culture, 3D bioprinting enables free-form fabrication of complex living tissue structures. In the present work, production of human jawbone models was realised via projection-based stereolithography. Constructs were bioprinted containing primary jawbone-derived osteoblasts and vasculature-like channel structures optionally harbouring primary endothelial cells. After 28 days of cultivation in growth medium or osteogenic medium, expression of cell type-specific markers was confirmed on both the RNA and protein level, while prints maintained their overall structure. Survival of endothelial cells in the printed channels, co-cultured with osteoblasts in medium without supplementation of endothelial growth factors, was demonstrated. Constructs showed not only mineralisation, being one of the characteristics of osteoblasts, but also hinted at differentiation to an osteocyte phenotype. These results indicate the successful biofabrication of an in vitro model of the human jawbone, which presents key features of this special bone entity and hence appears promising for application in jawbone-specific research.

Simplified Bioprinting-Based 3D Cell Culture Infection Models for Virus Detection.

Studies of virus-host interactions in vitro may be hindered by biological characteristics of conventional monolayer cell cultures that differ from in vivo infection. Three-dimensional (3D) cell cultures show more in vivo-like characteristics and may represent a promising alternative for characterisation of infections. In this study, we established easy-to-handle cell culture platforms based on bioprinted 3D matrices for virus detection and characterisation. Different cell types were cultivated on these matrices and characterised for tissue-like growth characteristics regarding cell morphology and polarisation. Cells developed an in vivo-like morphology and long-term cultivation was possible on the matrices. Cell cultures were infected with viruses which differed in host range, tissue tropism, cytopathogenicity, and genomic organisation and virus morphology. Infections were characterised on molecular and imaging level. The transparent matrix substance allowed easy optical monitoring of cells and infection even via live-cell microscopy. In conclusion, we established an enhanced, standardised, easy-to-handle bioprinted 3D-cell culture system. The infection models are suitable for sensitive monitoring and characterisation of virus-host interactions and replication of different viruses under physiologically relevant conditions. Individual cell culture models can further be combined to a multicellular array. This generates a potent diagnostic tool for propagation and characterisation of viruses from diagnostic samples.

The microfollicle: a model of the human hair follicle for in vitro studies.

Access to complex in vitro models that recapitulate the unique markers and cell-cell interactions of the hair follicle is rather limited. Creation of scalable, affordable, and relevant in vitro systems which can provide predictive screens of cosmetic ingredients and therapeutic actives for hair health would be highly valued. In this study, we explore the features of the microfollicle, a human hair follicle organoid model based on the spatio-temporally defined co-culture of primary cells. The microfollicle provides a 3D differentiation platform for outer root sheath keratinocytes, dermal papilla fibroblasts, and melanocytes, via epidermal-mesenchymal-neuroectodermal cross-talk. For assay applications, microfollicle cultures were adapted to 96-well plates suitable for medium-throughput testing up to 21 days, and characterized for their spatial and lineage markers. The microfollicles showed hair-specific keratin expression in both early and late stages of cultivation. The gene expression profile of microfollicles was also compared with human clinical biopsy samples in response to the benchmark hair-growth compound, minoxidil. The gene expression changes in microfollicles showed up to 75% overlap with the corresponding gene expression signature observed in the clinical study. Based on our results, the cultivation of the microfollicle appears to be a practical tool for generating testable insights for hair follicle development and offers a complex model for pre-clinical substance testing.

Inspired by the human placenta: a novel 3D bioprinted membrane system to create barrier models.

Barrier organ models need a scaffold structure to create a two compartment culture. Technical filter membranes used most often as scaffolds may impact cell behaviour and present a barrier themselves, ultimately limiting transferability of test results. In this work we present an alternative for technical filter membrane systems: a 3D bioprinted biological membrane in 24 well format. The biological membrane, based on extracellular matrix (ECM), is highly permeable and presents a natural 3D environment for cell culture. Inspired by the human placenta we established a coculture of a trophoblast-derived cell line (BeWo b30), together with primary placental fibroblasts within the biological membrane (simulating villous stroma) and primary human placental endothelial cells-representing three cellular components of the human placental villus. All cell types maintained their cell type specific marker expression after two weeks of coculture on the biological membrane. In permeability assays the trophoblast layer developed a barrier on the biological membrane, which was even more pronounced when cocultured with fibroblasts. In this work we present a filter membrane free scaffold, we characterize its properties and assess its suitability for cell culture and barrier models. Further we show a novel placenta inspired model in a complex bioprinted coculture. In the absence of an artificial filter membrane, we demonstrate barrier architecture and functionality.

Vascular bioprinting with enzymatically degradable bioinks via multi-material projection-based stereolithography.

Introduction of cavities and channels into 3D bioprinted constructs is a prerequisite for recreating physiological tissue architectures and integrating vasculature. Projection-based stereolithography inherently offers high printing speed with high spatial resolution, but so far has been incapable of fabricating complex native tissue architectures with cellular and biomaterial diversity. The use of sacrificial photoinks, i.e. photopolymerisable biomaterials that can be removed after printing, theoretically allows for the creation of any construct geometry via a multi-material printing process. However, the realisation of this strategy has been challenging because of difficult technical implementation and a lack of performant biomaterials. In this work, we use our projection-based, multi-material stereolithographic bioprinter and an enzymatically degradable sacrificial photoink to overcome the current hurdles. Multiple, hyaluronic acid-based photoinks were screened for printability, mechanical properties and digestibility through hyaluronidase. A formulation meeting all major requirements, i.e. desirable printing properties, generation of sufficiently strong hydrogels, as well as fast digestion rate, was identified. Biocompatibility of the material system was confirmed by embedding of human umbilical vein endothelial cells with followed enzymatic release. As a proof-of-concept, we bioprinted vascular models containing perfusable, endothelial cell-lined channels that remained stable for 28 days in culture. Our work establishes digestible sacrificial biomaterials as a new material strategy for 3D bioprinting of complex tissue models.

Photopolymerizable gelatin and hyaluronic acid for stereolithographic 3D bioprinting of tissue-engineered cartilage.

To create artificial cartilage in vitro, mimicking the function of native extracellular matrix (ECM) and morphological cartilage-like shape is essential. The interplay of cell patterning and matrix concentration has high impact on the phenotype and viability of the printed cells. To advance the capabilities of cartilage bioprinting, we investigated different ECMs to create an in vitro chondrocyte niche. Therefore, we used methacrylated gelatin (GelMA) and methacrylated hyaluronic acid (HAMA) in a stereolithographic bioprinting approach. Both materials have been shown to support cartilage ECM formation and recovery of chondrocyte phenotype. We used these materials as bioinks to create cartilage models with varying chondrocyte densities. The models maintained shape, viability, and homogenous cell distribution over 14 days in culture. Chondrogenic differentiation was demonstrated by cartilage-typical proteoglycan and type II collagen deposition and gene expression (COL2A1, ACAN) after 14 days of culture. The differentiation pattern was influenced by cell density. A high cell density print (25 × 106 cells/mL) led to enhanced cartilage-typical zonal segmentation compared to cultures with lower cell density (5 × 106 cells/mL). Compared to HAMA, GelMA resulted in a higher expression of COL1A1, typical for a more premature chondrocyte phenotype. Both bioinks are feasible for printing in vitro cartilage with varying differentiation patterns and ECM organization depending on starting cell density and chosen bioink. The presented technique could find application in the creation of cartilage models and in the treatment of articular cartilage defects using autologous material and adjusting the bioprinted constructs size and shape to the patient. © 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B:2649-2657, 2019.