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.

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.

In-line measurements of the physical and thermodynamic properties of single and multicomponent liquids.

Microfluidic devices are becoming increasingly important in various fields of pharmacy, flow chemistry and healthcare. In the embedded microchannel, the flow rates, the dynamic viscosity of the transported liquids and the fluid dynamic properties play an important role. Various functional auxiliary components of microfluidic devices such as flow restrictors, valves and flow meters need to be characterised with liquids used in several microfluidic applications. However, calibration with water does not always reflect the behaviour of the liquids used in the different applications. Therefore, several National Metrology Institutes (NMI) have developed micro-pipe viscometers for traceable inline measurement of the dynamic viscosity of liquids used in flow applications as part of the EMPIR 18HLT08 MeDDII project. These micro-pipe viscometers allow the calibration of any flow device at different flow rates and the calibration of the dynamic viscosity of the liquid or liquid mixture used under actual flow conditions. The validation of the micro-pipe viscometers has been performed either with traceable reference oils or with different liquids typically administered in hospitals, such as saline and/or glucose solutions or even glycerol-water mixtures for higher dynamic viscosities. Furthermore, measurement results of a commercially available device and a technology demonstrator for the inline measurement of dynamic viscosity and density are presented in this paper.

Holographic PIV/PTV for nano flow rates-A study in the 70 to 200 nL/min range.

Accurately measuring flow rates is a key requirement in many medical applications such as infusion and drug delivery systems. A major drawback of current systems is the low resolution of the sensors in the low flow rate regime. In this article, we present a method based on Holographic PIV/PTV that has been used for the first time to measure flow rates in the range of a few nL/min. Our method requires a very simple setup that combines lensless holography with particle velocimetry. For flow rates in the 70 to 200 nL/min range, the highest uncertainty was 5.6% (coverage factor k=2). This is an open-source project; the CAD designs and software source code are available at https://github.com/gui-miotto/holovel.

Calibration methods for flow rates down to 5 nL/min and validation methodology.

Improving the accuracy and enabling traceable measurements of volume, flow, and pressure in existing drug delivery devices and in-line sensors operating at very low flow rates is essential in several fields of activities and specially in medical applications. This can only be achieved through the development of new calibrationmethods and by expanding the existing metrological infrastructure to perform micro-flow and nano-flow measurements. In this paper, we will investigate new traceable techniques for measuring flow rate, from 5 nL/min to 1,500 nL/min and present the results of an inter-comparison between nine laboratories for the calibration of two different flow meters and a syringe pump.

Bioprinting-based automated deposition of single cancer cell spheroids into oxygen sensor microelectrode wells.

Three-dimensional (3D) cell agglomerates, such as microtissues, organoids, and spheroids, become increasingly relevant in biomedicine. They can provide in vitro models that recapitulate functions of the original tissue in the body and have applications in cancer research. For example, they are widely used in organ-on-chip systems. Microsensors can provide essential real-time information on cell metabolism as well as the reliability and quality of culture conditions. The combination of sensors and 3D cell cultures, especially single spheroids, is challenging in terms of reproducible formation, manipulation, and access to spheroids, precise positioning near sensors, and high cell-to-volume ratios to obtain meaningful biosignals in the most parallel approach possible. To overcome this challenge, we combined state-of-the-art bioprinting techniques to automatically print tumour spheroids directly into microwells of a chip-based electrochemical oxygen sensor array. We demonstrated highly accurate and reproducible spheroid formation (diameter of approx. 200 µm) and a spheroid deposition precision of 25 µm within a volume of 22 nl per droplet. Microstructures and hydrogel-coated microwells allowed the placement of single MCF-7 breast cancer spheroids close to the sensor electrodes. The microelectrode wells were sealed for oxygen measurements within a 55 nl volume for fast concentration changes. Accurate and stable amperometric oxygen sensor performance was demonstrated from atmospheric to anoxic regions. Cellular respiration rates from single tumour spheroids in the range of 450-850 fmol min-1 were determined, and alterations of cell metabolism upon drug exposure were shown. Our results uniquely combine bioprinting with 3D cell culture monitoring and demonstrate the much-needed effort for facilitation, parallelization, sensor integration, and drug delivery in 3D cell culture and organ-on-chip experiments. The workflow has a high degree of automation and potential for scalability. In order to achieve greater flexibility in the automation of spheroid formation and trapping, we employed a method based on drop-on-demand liquid handling systems, instead of the typical on-chip approach commonly used in microfluidics. Its relevance ranges from fundamental metabolic research over standardization of cell culture experiments and toxicological studies, to personalized medicine, e.g. patient-specific chemotherapy.

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.

Large scale production and controlled deposition of single HUVEC spheroids for bioprinting applications.

We present (1) a fast and automated method for large scale production of HUVEC spheroids based on the hanging drop method and (2) a novel method for well-controlled lateral deposition of single spheroids by drop-on-demand printing. Large scale spheroid production is achieved via printing 1536 droplets of HUVEC cell suspension having a volume of 1 µl each within 3 min at a pitch of 2.3 mm within an array of 48 × 32 droplets onto a flat substrate. Printing efficiencies between 97.9% and 100% and plating efficiencies between 87.3% and 100% were achieved. Harvested spheroids (consisting of approx. 250 HUVECs each) appear uniform in size and shape. After incubation and harvesting, the spheroids are deposited individually in user-defined patterns onto hydrogels using an automated drop-on-demand dispenser setup. Controlled by an image detection algorithm focusing the dispenser nozzle, droplets containing exactly one spheroid are printed onto a substrate, while all other droplets are discarded. Using this approach an array of 6 × 3 HUVEC spheroids with intermediate distances of 500 µm embedded in fibrin was generated. Successful progress of spheroid sprouting and merging of neighboring sprouts was observed during the first 72 h of incubation indicating a good viability of the deposited spheroids.