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.

We present a disposable, normally closed, non-contact dispensing valve for the sub-µL range. The miniaturized solenoid valve (diameter: 8 mm, height: 27.25 mm) is compatible to standard Luer-Lock interfaces. A highly dynamic actuation principle enables opening times down to 1 ms. The dispensing performance was evaluated for water (η = 1.03 mPas) and a 66% (w/w) glycerol/water solution (η = 16.98 mPas), at pressures varying from 200 to 800 mbar. The experimentally determined minimal dispensing volume was 163 nL (CV 1.6%) for water and 123 nL (CV 4.5%) for 66% (w/w) glycerol/water. The low-cost polymer valve enables high precision dispensing of liquid volumes down to the lower end of the sub-µL range comparable to high-end non-disposable micro-dispensing valves.

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.

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.

Additive manufacturing (AM), so‐called 3D printing, has reshaped manufacturing technologies with attractive alternatives to the traditional centralized, subtractive‐based mass production. Further functionalities like multimaterial AM (MMAM) are essential for mainstream manufacturing. With MMAM of electrical traces, the traditional printed circuit board can be transformed to freeform 3D electronics using the entire volumetric space. Although research groups attempt MMAM for freeform 3D electronics, achieving easy integration of the electrical traces with high conductance into dielectric substrates in a streamlined and simplified process remains challenging. Herein, an in‐house developed printer, named Synkròtima, is demonstrated for one‐stop printing of 3D dielectric parts with fully embedded and structured conductive features via drop‐on‐demand printing of molten metal for the first time. Printed traces show 12 times lower electrical resistance of at 230 μm linewidth than the lines from conductive inks. Printed traces with 100 μm dot spacing reveal exemplary shape fidelity with a standard deviation of only 7 μm. Quantitative estimation of adhesion of metal traces to dielectric reveals a mean shear‐off force of 7.20 N which is half of what is obtained for reflow soldered electronics. Demonstrators with embedded functional electrical circuits, directly interconnected components, and 3D out‐of‐plane metal structures are printed via Synkròtima.

We present a miniaturized, disposable, normally-closed electromagnetic dispensing valve for the microliter range to process 96-well plates. The novel injection-molded valve is designed to fit into a 9 mm grid to realize an eight channel dispensing head, enabling the serial processing of well plates row-by-row. The presented dispensing valve design originates from a miniaturization study of a previously developed functional model. The outer diameter of the valve, including all actuating components, was reduced by 8 mm to an overall diameter of 8.5 mm without compromising the performance. Additionally, the actuation current of the valve could be reduced to 5 A. The valve is characterized for a volume range between 840 nL and 5.3 μL. The performance of the injection molded valve is competitive to commercially available dispensing valves, featuring the advantages of low fabrication costs, disposability, low mounting size, easy handling, and super silent actuation.