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.

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.

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.

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.

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.

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%.