A Drop-on-Demand Bioprinting Approach to Spatially Arrange Multiple Cell Types and Monitor Their Cell-Cell Interactions towards Vascularization Based on Endothelial Cells and Mesenchymal Stem Cells.

Spheroids, organoids, or cell-laden droplets are often used as building blocks for bioprinting, but so far little is known about the spatio-temporal cellular interactions subsequent to printing. We used a drop-on-demand bioprinting approach to study the biological interactions of such building blocks in dimensions of micrometers. Highly-density droplets (approximately 700 cells in 10 nL) of multiple cell types were patterned in a 3D hydrogel matrix with a precision of up to 70 µm. The patterns were used to investigate interactions of endothelial cells (HUVECs) and adipose-derived mesenchymal stem cells (ASCs), which are related to vascularization. We demonstrated that a gap of 200 µm between HUVEC and ASC aggregates led to decreased sprouting of HUVECs towards ASCs and increased growth from ASCs towards HUVECs. For mixed aggregates containing both cell types, cellular interconnections of ASCs with lengths of up to approximately 800 µm and inhibition of HUVEC sprouting were observed. When ASCs were differentiated into smooth muscle cells (dASCs), separate HUVEC aggregates displayed decreased sprouting towards dASCs, whereas no cellular interconnections nor inhibition of HUVEC sprouting were detected for mixed dASCs/HUVEC aggregates. These findings demonstrate that our approach could be applied to investigate cell-cell interactions of different cell types in 3D co-cultures.

Tuning the 3D microenvironment of reprogrammed tubule cells enhances biomimetic modeling of polycystic kidney disease.

Renal tubular cells frequently lose differentiation markers and physiological properties when propagated in conventional cell culture conditions. Embedding cells in 3D microenvironments or controlling their 3D assembly by bioprinting can enhance their physiological properties, which is beneficial for modeling diseases in vitro. A potential cellular source for modeling renal tubular physiology and kidney diseases in vitro are directly reprogrammed induced renal tubular epithelial cells (iRECs). iRECs were cultured in various biomaterials and as bioprinted tubular structures. They showed high compatibility with the embedding substrates and dispensing methods. The morphology of multicellular aggregates was substantially influenced by the 3D microenvironment. Transcriptomic analyses revealed signatures of differentially expressed genes specific to each of the selected biomaterials. Using a new cellular model for autosomal-dominant polycystic kidney disease, Pkd1-/- iRECs showed disrupted morphology in bioprinted tubules and a marked upregulation of the Aldehyde dehydrogenase 1a1 (Aldh1a1). In conclusion, 3D microenvironments strongly influence the morphology and expression profiles of iRECs, help to unmask disease phenotypes, and can be adapted to experimental demands. Combining a direct reprogramming approach with appropriate biomaterials will facilitate construction of biomimetic kidney tubules and disease models at the microscale.

Evaluation of a Novel Thiol-Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells.

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.

Deep Learning-Assisted Nephrotoxicity Testing with Bioprinted Renal Spheroids.

We used arrays of bioprinted renal epithelial cell spheroids for toxicity testing with cisplatin. The concentration-dependent cell death rate was determined using a lactate dehydrogenase assay. Bioprinted spheroids showed enhanced sensitivity to the treatment in comparison to monolayers of the same cell type. The measured dose-response curves revealed an inhibitory concentration of the spheroids of IC 50 = 9 ± 3 µM in contrast to the monolayers with IC 50 = 17 ± 2 µM. Fluorescent labeling of a nephrotoxicity biomarker, kidney injury molecule 1 indicated an accumulation of the molecule in the central lumen of the spheroids. Finally, we tested an approach for an automatic readout of toxicity based on microscopic images with deep learning. Therefore, we created a dataset comprising images of single spheroids, with corresponding labels of the determined cell death rates for training. The algorithm was able to distinguish between three classes of no, mild, and severe treatment effects with a balanced accuracy of 78.7%.

Mechanical properties of polycaprolactone (PCL) scaffolds for hybrid 3D-bioprinting with alginate-gelatin hydrogel.

The generation of artificial human tissue by 3D-bioprinting has expanded significantly as a clinically relevant research topic in recent years. However, to produce a complex and viable tissue, in-depth biological understanding and advanced printing techniques are required with a high number of process parameters. Here, we systematically evaluate the process parameters relevant for a hybrid bioprinting process based on fused-deposition modeling (FDM) of thermoplastic material and microextrusion of a cell-laden hydrogel. First, we investigated the effect of the printing temperature of polycaprolactone (PCL), on the junction strength between individual fused filaments and on the viability of immortalized mesenchymal stem cells (iMSC) in the surrounding alginate-gelatin-hydrogel. It was found that a printing temperature of 140 °C and bonds with an angle of 90° between the filaments provided a good compromise between bonding strength of the filaments and the viability of the surrounding cells. Using these process parameters obtained from individual fused filaments, we then printed cubic test structures with a volume of 10 × 10 × 10 mm3 with different designs of infill patterns. The variations in mechanical strength of these cubes were measured for scaffolds made of PCL-only as well as for hydrogel-filled PCL scaffolds printed by alternating hybrid bioprinting of PCL and hydrogel, layer by layer. The bare scaffolds showed a compressive modulus of up to 6 MPa, close to human hard tissue, that decreased to about 4 MPa when PCL was printed together with hydrogel. The scaffold design suited best for hybrid printing was incubated with cell-laden hydrogel and showed no degradation of its mechanical strength for up to 28 days.

Scalable fabrication of renal spheroids and nephron-like tubules by bioprinting and controlled self-assembly of epithelial cells.

Scalable fabrication concepts of 3D kidney tissue models are required to enable their application in pharmaceutical high-throughput screenings. Yet the reconstruction of complex tissue structures remains technologically challenging. We present a novel concept reducing the fabrication demands, by using controlled cellular self-assembly to achieve higher tissue complexities from significantly simplified construct designs. We used drop-on-demand bioprinting to fabricate locally confined patterns of renal epithelial cells embedded in a hydrogel matrix. These patterns provide defined local cell densities (cell count variance <11%) with high viability (92 ± 2%). Based on these patterns, controlled self-assembly leads to the formation of renal spheroids and nephron-like tubules with a predefined size and spatial localization. With this, we fabricated scalable arrays of hollow epithelial spheroids. The spheroid sizes correlated with the initial cell count per unit and could be stepwise adjusted, ranging from Ø = 84, 104, 120-131µm in diameter (size variance <9%). Furthermore, we fabricated scalable line-shaped patterns, which self-assembled to hollow cellular tubules (Ø = 105 ± 22µm). These showed a continuous lumen with prescribed orientation, lined by an epithelial monolayer with tight junctions. Additionally, upregulated expression of kidney-specific functional genes compared to 2D cell monolayers indicated increased tissue functionality, as revealed by mRNA sequencing. Furthermore, our concept enabled the fabrication of hybrid tubules, which consisted of arranged subsections of different cell types, combining murine and human epithelial cells. Finally, we integrated the self-assembled fabrication into a microfluidic chip and achieved fluidic access to the lumen at the terminal sites of the tubules. With this, we realized flow conditions with a wall shear stress of 0.05 ± 0.02 dyne cm-2driven by hydrostatic pressure for scalable dynamic culture towards a nephron-on-chip model.

Open-source hybrid 3D-bioprinter for simultaneous printing of thermoplastics and hydrogels.

3D-bioprinting is a promising technology applicable in areas such as regenerative medicine or in vitro organ model development. Various 3D-bioprinting technologies and systems have been developed and are partly commercially available. Here, we present the construction and characterization of an open-source low-cost 3D-bioprinter that allows the alternated microextrusion of hydrogel and fused deposition modeling (FDM) of thermoplastic filaments. The presented 3D-bioprinter is based on a conventional Prusa i3 MK3 printer and features two independent printheads: the original FDM-head and a syringe-based microextrusion printhead for soft materials. Modifications were designed modularly to fit various syringe formats or heating elements to the device. The bioprinter is the first hybrid DIY 3D-bioprinter that allows switching between materials as often as required during a print run to produce complex multi-material constructs with arbitrary patterns in each layer. For validation of the printer, two designs suitable for relevant bioprinting applications were realized. First, a porous plastic construct filled with hydrogel was printed, serving as a mechanically stable bone replacement tissue model. Second, a plastic chamber, which might be used in organ-on-a-chip applications, was printed with an extruded silicone sealing that enables the liquid-tight attachment of glass slides to the top and bottom of the chamber.

In vivo evaluation of bioprinted prevascularized bone tissue.

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.

Bioprinting of high cell-density constructs leads to controlled lumen formation with self-assembly of endothelial cells.

Active nutrient supply and waste product removal are key requirements for the fabrication of long-term viable and functional tissue constructs of considerable size. This work aims to contribute to the fabrication of artificial perfusable networks with a bioprinting process, based on drop-on-demand (DoD) printing of primary endothelial cell (EC) suspension bioink (25 × 106 ± 3 × 106 cells/ml). The process results in prescribed lumen between two hydrogel layers, allowing its integration in common layering based bioprinting processes. Low volume bioink droplets (appr. 10 nl) as building blocks were deposited between two fibrin or Collagen I layers to realize shapeable, cell-rich aggregates. Unattainable with manual positioning, DoD printing allowed precise fabrication of various designs, such as spheroidal-, line-shaped, and Y-branch cellular structures, with a mean lateral extension of 285 ± 81 µm. For basic characterization, the cell suspension building blocks were systematically compared with preformed spheroids of the same cell type, passage, and number. Post printing investigations of initially loose cell arrangements showed self-assembly and formation of central lumen with a mean cross-sectional area of Ølumen = 6,400 µm2 at Day 3, lined by a single layer of CD31 positive ECs, as evaluated by confocal microscopy. Originating from this main lumen smaller, undirected side branches (Øbranches = 740 µm2 ) were formed by sprouting cells, inducing a first step towards a simplistic hierarchically organized network. These lumen could prospectively help for tissue construct perfusion in vitro or, potentially, as niche for angiogenesis of host vascularization in implants.