Paper-based open microfluidic platform for protein electrophoresis and immunoprobing.

Protein electrophoresis and immunoblotting are indispensable analytical tools for the characterization of proteins and posttranslational modifications in complex sample matrices. Owing to the lack of automation, commonly employed slab-gel systems suffer from high time demand, significant sample/antibody consumption, and limited reproducibility. To overcome these limitations, we developed a paper-based open microfluidic platform for electrophoretic protein separation and subsequent transfer to protein-binding membranes for immunoprobing. Electrophoresis microstructures were digitally printed into cellulose acetate membranes that provide mechanical stability while maintaining full accessibility of the microstructures for consecutive immunological analysis. As a proof-of-concept, we demonstrate separation of fluorescently labeled marker proteins in a wide molecular weight range (15-120 kDa) within only 15 min, reducing the time demand for the entire workflow (from sample preparation to immunoassay) to approximately one hour. Sample consumption was reduced 10- to 150-fold compared to slab-gel systems, owing to system miniaturization. Moreover, we successfully applied the paper-based approach to complex samples such as crude bacterial cell extracts. We envisage that this platform will find its use in protein analysis workflows for scarce and precious samples, providing a unique opportunity to extract profound immunological information from limited sample amounts in a fast fashion with minimal hands-on time.

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.

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.

Open microfluidic gel electrophoresis: Rapid and low cost separation and analysis of DNA at the nanoliter scale.

Gel electrophoresis is one of the most applied and standardized tools for separation and analysis of macromolecules and their fragments in academic research and in industry. In this work we present a novel approach for conducting on-demand electrophoretic separations of DNA molecules in open microfluidic (OM) systems on planar polymer substrates. The approach combines advantages of slab gel, capillary- and chip-based methods offering low consumable costs (<0.1$) circumventing cost-intensive microfluidic chip fabrication, short process times (5 min per analysis) and high sensitivity (4 ng/µL dsDNA) combined with reasonable resolution (17 bases). The open microfluidic separation system comprises two opposing reservoirs of 2-4 µL in volume, a semi-contact written gel line acting as separation channel interconnecting the reservoirs and sample injected into the line via non-contact droplet dispensing and thus enabling the precise control of the injection plug and sample concentration. Evaporation is prevented by covering aqueous structures with PCR-grade mineral oil while maintaining surface temperature at 15°C. The liquid gel line exhibits a semi-circular cross section of adaptable width (∼200-600 µm) and height (∼30-80 µm) as well as a typical length of 15-55 mm. Layout of such liquid structures is adaptable on-demand not requiring time consuming and repetitive fabrication steps. The approach was successfully demonstrated by the separation of a standard label-free DNA ladder (100-1000 bp) at 100 V/cm via in-line staining and laser induced fluorescent end-point detection using an automated prototype.

Technologies for Single-Cell Isolation.

The handling of single cells is of great importance in applications such as cell line development or single-cell analysis, e.g., for cancer research or for emerging diagnostic methods. This review provides an overview of technologies that are currently used or in development to isolate single cells for subsequent single-cell analysis. Data from a dedicated online market survey conducted to identify the most relevant technologies, presented here for the first time, shows that FACS (fluorescence activated cell sorting) respectively Flow cytometry (33% usage), laser microdissection (17%), manual cell picking (17%), random seeding/dilution (15%), and microfluidics/lab-on-a-chip devices (12%) are currently the most frequently used technologies. These most prominent technologies are described in detail and key performance factors are discussed. The survey data indicates a further increasing interest in single-cell isolation tools for the coming years. Additionally, a worldwide patent search was performed to screen for emerging technologies that might become relevant in the future. In total 179 patents were found, out of which 25 were evaluated by screening the title and abstract to be relevant to the field.

Particle Detection in Free-Falling Nanoliter Droplets.

Sorting and dispensing distinct numbers of cellular aggregates enables the creation of three-dimensional (3D) in vitro models that replicate in vivo tissues, such as tumor tissue, with realistic metabolic properties. One method for creating these models involves utilizing Drop-on-Demand (DoD) dispensing of individual Multicellular Spheroids (MCSs) according to material jetting processes. In the DoD approach, a droplet dispenser ejects droplets containing these MCSs. For the reliable printing of tissue models, the exact number of dispensed MCSs must be determined. Current systems are designed to detect MCSs in the nozzle region prior to the dispensing process. However, due to surface effects, in some cases the spheroids that are detected adhere to the nozzle and are not dispensed with the droplet as expected. In contrast, detection that is carried out only after the droplet has been ejected is not affected by this issue. This work presents a system that can detect micrometer-sized synthetic or biological particles within free-falling droplets with a volume of about 30 nanoliters. Different illumination modalities and detection algorithms were tested. For a glare point projection-based approach, detection accuracies of an average of 95% were achieved for polymer particles and MCF-7 spheroids with diameters above 75 µm. For smaller particles the detection accuracy was still in the range of 70%. An approach with diffuse white light illumination demonstrated an improvement for the detection of small opaque particles. Accuracies up to 96% were achieved using this concept. This makes the two demonstrated methods suitable for improving the accuracy and quality control of particle detection in droplets for Drop-on-Demand techniques and for bioprinting.

Bulge-Free and Homogeneous Metal Line Jet Printing with StarJet Technology.

The technology to jet print metal lines with precise shape fidelity on diverse substrates is gaining higher interest across multiple research fields. It finds applications in additively manufactured flexible electronics, environmentally friendly and sustainable electronics, sensor devices for medical applications, and fabricating electrodes for solar cells. This paper provides an experimental investigation to deepen insights into the non-contact printing of solder lines using StarJet technology, eliminating the need for surface activation, substrate heating, curing, or post-processing. Moreover, it employs bulk metal instead of conventional inks or pastes, leading to cost-effective production and enhanced conductivity. The effect of molten metal temperature, substrate temperature, standoff distance, and printing velocity was investigated on polymer foils (i.e., PET sheets). Robust printing parameters were derived to print uniform, bulge-free, bulk metal lines suitable for additive manufacturing applications. The applicability of the derived parameters was extended to 3D-printed PLA, TPU, PA-GF, and PETG substrates having a much higher surface roughness. Additionally, a high aspect ratio of approx. 16:1 wall structure has been demonstrated by printing multiple metal lines on top of each other. While challenges persist, this study contributes to advancing additively manufactured electronic devices, highlighting the capabilities of StarJet metal jet-printing technology.

Automated Nanodroplet Dispensing for Large-Scale Spheroid Generation via Hanging Drop and Parallelized Lossless Spheroid Harvesting.

Creating model systems that replicate in vivo tissues is crucial for understanding complex biological pathways like drug response and disease progression. Three-dimensional (3D) in vitro models, especially multicellular spheroids (MCSs), offer valuable insights into physiological processes. However, generating MCSs at scale with consistent properties and efficiently recovering them pose challenges. We introduce a workflow that automates large-scale spheroid production and enables parallel harvesting into individual wells of a microtiter plate. Our method, based on the hanging-drop technique, utilizes a non-contact dispenser for dispensing nanoliter droplets of a uniformly mixed-cell suspension. The setup allows for extended processing times of up to 45 min without compromising spheroid quality. As a proof of concept, we achieved a 99.3% spheroid generation efficiency and maintained highly consistent spheroid sizes, with a coefficient of variance below 8% for MCF7 spheroids. Our centrifugation-based drop transfer for spheroid harvesting achieved a sample recovery of 100%. We successfully transferred HT29 spheroids from hanging drops to individual wells preloaded with collagen matrices, where they continued to proliferate. This high-throughput workflow opens new possibilities for prolonged spheroid cultivation, advanced downstream assays, and increased hands-off time in complex 3D cell culture protocols.

Hybrid Printing of Conductive Traces from Bulk Metal for Digital Signals in Intelligent Devices.

In this article, we explore multi-material additive manufacturing (MMAM) for conductive trace printing using molten metal microdroplets on polymer substrates to enhance digital signal transmission. Investigating microdroplet spread informs design rules for adjacent trace printing. We studied the effects of print distance on trace morphology and resolution, noting that printing distance showed almost no change in the printed trace pitch. Crosstalk interference between adjacent signal traces was analyzed across frequencies and validated both experimentally and through simulation; no crosstalk was visible for printed traces at input frequencies below 600 kHz. Moreover, we demonstrate printed trace reliability against thermal shock, whereby no discontinuation in conductive traces was observed. Our findings establish design guidelines for MMAM electronics, advancing digital signal transmission capabilities.

Towards Automation in 3D Cell Culture: Selective and Gentle High-Throughput Handling of Spheroids and Organoids via Novel Pick-Flow-Drop Principle.

3D cell culture is becoming increasingly important for mimicking physiological tissue structures in areas such as drug discovery and personalized medicine. To enable reproducibility on a large scale, automation technologies for standardized handling are still a challenge. Here, a novel method for fully automated size classification and handling of cell aggregates like spheroids and organoids is presented. Using microfluidic flow generated by a piezoelectric droplet generator, aggregates are aspirated from a reservoir on one side of a thin capillary and deposited on the other side, encapsulated in free-flying nanoliter droplets to a target. The platform has aggregate aspiration and plating efficiencies of 98.1% and 98.4%, respectively, at a processing throughput of up to 21 aggregates per minute. Cytocompatibility of the method is thoroughly assessed with MCF7, LNCaP, A549 spheroids and colon organoids, revealing no adverse effects on cell aggregates as shear stress is reduced compared to manual pipetting. Further, generic size-selective handling of heterogeneous organoid samples, single-aggregate-dispensing efficiencies of up to 100% and the successful embedding of spheroids or organoids in a hydrogel with subsequent proliferation is demonstrated. This platform is a powerful tool for standardized 3D in vitro research.

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.

On the reproducibility of extrusion-based bioprinting: round robin study on standardization in the field.

The outcome of three-dimensional (3D) bioprinting heavily depends, amongst others, on the interaction between the developed bioink, the printing process, and the printing equipment. However, if this interplay is ensured, bioprinting promises unmatched possibilities in the health care area. To pave the way for comparing newly developed biomaterials, clinical studies, and medical applications (i.e. printed organs, patient-specific tissues), there is a great need for standardization of manufacturing methods in order to enable technology transfers. Despite the importance of such standardization, there is currently a tremendous lack of empirical data that examines the reproducibility and robustness of production in more than one location at a time. In this work, we present data derived from a round robin test for extrusion-based 3D printing performance comprising 12 different academic laboratories throughout Germany and analyze the respective prints using automated image analysis (IA) in three independent academic groups. The fabrication of objects from polymer solutions was standardized as much as currently possible to allow studying the comparability of results from different laboratories. This study has led to the conclusion that current standardization conditions still leave room for the intervention of operators due to missing automation of the equipment. This affects significantly the reproducibility and comparability of bioprinting experiments in multiple laboratories. Nevertheless, automated IA proved to be a suitable methodology for quality assurance as three independently developed workflows achieved similar results. Moreover, the extracted data describing geometric features showed how the function of printers affects the quality of the printed object. A significant step toward standardization of the process was made as an infrastructure for distribution of material and methods, as well as for data transfer and storage was successfully established.

Completely superhydrophobic PDMS surfaces for microfluidics.

This study presents a straightforward two-step fabrication process of durable, completely superhydrophobic microchannels in PDMS. First, a composite material of PDMS/PTFE particles is prepared and used to replicate a master microstructure. Superhydrophobic surfaces are formed by subsequent plasma treatment, in which the PDMS is isotropically etched and PTFE particles are excavated. We compare the advancing and receding contact angles of intrinsic PDMS samples and composite PTFE/PDMS samples (1 wt %, 8 wt %, and 15 wt % PTFE particle concentration) and demonstrate that both the horizontal and vertical surfaces are indeed superhydrophobic. The best superhydrophobicity is observed for samples with a PTFE particle concentration of 15 wt %, which have advancing and receding contact angles of 159° ± 4° and 158° ± 3°, respectively.

Numerical investigations on electric field characteristics with respect to capacitive detection of free-flying droplets.

In this paper a multi-disciplinary simulation of a capacitive droplet sensor based on an open plate capacitor as transducing element is presented. The numerical simulations are based on the finite volume method (FVM), including calculations of an electric field which changes according to the presence of a liquid droplet. The volume of fluid (VOF) method is applied for the simulation of the ejection process of a liquid droplet out of a dispenser nozzle. The simulations were realised using the computational fluid dynamic (CFD) software CFD ACE+. The investigated capacitive sensing principle enables to determine the volume of a micro droplet passing the sensor capacitor due to the induced change in capacity. It could be found that single droplets in the considered volume range of 5 nL < V(drop) < 100 nL lead to a linear change of the capacity up to ΔQ < 30 fC. The sensitivity of the focused capacitor geometry was evaluated to be S(i) = 0.3 fC/nL. The simulation results are validated by experiments which exhibit good agreement.

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.

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.

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.

Characterization of CRISPR/Cas9 RANKL knockout mesenchymal stem cell clones based on single-cell printing technology and Emulsion Coupling assay as a low-cellularity workflow for single-cell cloning.

The homogeneity of the genetically modified single-cells is a necessity for many applications such as cell line development, gene therapy, and tissue engineering and in particular for regenerative medical applications. The lack of tools to effectively isolate and characterize CRISPR/Cas9 engineered cells is considered as a significant bottleneck in these applications. Especially the incompatibility of protein detection technologies to confirm protein expression changes without a preconditional large-scale clonal expansion creates a gridlock in many applications. To ameliorate the characterization of engineered cells, we propose an improved workflow, including single-cell printing/isolation technology based on fluorescent properties with high yield, a genomic edit screen (Surveyor assay), mRNA RT-PCR assessing altered gene expression, and a versatile protein detection tool called emulsion-coupling to deliver a high-content, unified single-cell workflow. The workflow was exemplified by engineering and functionally validating RANKL knockout immortalized mesenchymal stem cells showing bone formation capacity of these cells. The resulting workflow is economical, without the requirement of large-scale clonal expansions of the cells with overall cloning efficiency above 30% of CRISPR/Cas9 edited cells. Nevertheless, as the single-cell clones are comprehensively characterized at an early, highly parallel phase of the development of cells including DNA, RNA, and protein levels, the workflow delivers a higher number of successfully edited cells for further characterization, lowering the chance of late failures in the development process.

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.