Histology is the gold standard for analyzing tissue structure and cell morphology. Immunostaining on thin tissue sections enables precise visualization of antigens and proteins. However, for cryosectioning small tissues such as organoids, spheroids, and tumoroids there is a lack of standardized, time- and cost-effective methods, limiting the throughput of analysis. Here, we have adapted to cryosectioning our previously developed HistoBrick approach, in which small tissue arrangement is spatially controlled within arrayed mini-wells. By testing various embedding matrices, we show that an 8% PEGDA and 2.5% gelatine mixture is optimal, providing essential structural support to maintain sample integrity during cryosectioning. This embedding matrix preserves fragile substructures of human retinal organoids, which are particularly susceptible to damage during sample preparation. Using PEGDA-gelatine HistoBricks for the simultaneous embedding of 16 retinal organoids, we analyzed a time course of retinal organoid development. We observed the maintenance of photoreceptors cell bodies up to week 98 in culture, while photoreceptor outer segments were gradually lost. Further, we observed displaced photoreceptors in the region of outer segments. The PEGDA-gelatine HistoBrick is a cost-efficient tool that can be implemented for small tissue studies to increase throughput in experiments such as large-scale screenings or toxicology research.

End-stage liver diseases have an increasing impact worldwide, exacerbated by the shortage of transplantable organs. Recognized as one of the promising solutions, tissue engineering aims at recreating functional tissues and organs in vitro. The integration of bioprinting technologies with biological 3D models, such as multi-cellular spheroids, has enabled the fabrication of tissue constructs that better mimic complex structures and in vivo functionality of organs. However, the lack of methods for large-scale production of homogeneous spheroids has hindered the upscaling of tissue fabrication. In this work, we introduce a fully automated platform, designed for high-throughput sorting of 3D spheroids based on label-free analysis of brightfield images. The compact platform is compatible with standard biosafety cabinets and includes a custom-made microscope and two fluidic systems that optimize single spheroid handling to enhance sorting speed. We use machine learning to classify spheroids based on their bioprinting compatibility. This approach enables complex morphological analysis, including assessing spheroid viability, without relying on invasive fluorescent labels. Furthermore, we demonstrate the efficacy of transfer learning for biological applications, for which acquiring large datasets remains challenging. Utilizing this platform, we efficiently sort mono-cellular and multi-cellular liver spheroids, the latter being used in bioprinting applications, and confirm that the sorting process preserves viability and functionality of the spheroids. By ensuring spheroid homogeneity, our sorting platform paves the way for standardized and scalable tissue fabrication, advancing regenerative medicine applications.

Standardised and high-throughput methods have been developed for the production and experimental handling of some 3D in vitro models. However, adapted analytical tools are still missing for scientists and researchers to fully exploit the potential of complex cellular models in pre-clinical drug testing and precision medicine. Histology is the established, cost-effective and gold standard method for structural and functional tissue analysis. However, standard histological processes are challenging and costly to apply to 3D cell models, as their small size often leads to poor alignment of samples, which lowers analysis throughput. This body of work proposes a new approach: HistoBrick facilitates histological processing of spheroids and organoids by enabling gel embedding of 3D cell models with precise coplanar alignment, parallel to the sectioning plane, thus minimising the loss of sample material. HistoBrick’s features are compatible with automation standards, potentially allowing automated sample transfer from a multi-well plate to the gel device. Moreover, HistoBrick’s technology was validated by demonstrating the alignment of HepG2 cultured spheroids measuring 150–200 µm in diameter with a height precision of ± 80 µm. HistoBrick allows up to 96 samples to be studied across minimal sections, paving the way towards high-throughput micro-histology.

Precise metering in liquid dispensing applications often requires application-specific solutions due to incompatibilities of the sensor and actuator components with the dispensed liquids. Some reoccurring challenges are aggressive liquids that would damage the sensors or tubing, the need for sterile liquids while the pumps or sensors cannot be sterilized, or media that can clog the sensor channels. Two different dispensing systems are here presented where the dispensing flow rate or volume is indirectly measured through a coupled pressure change or airflow, thus avoiding contact between the sensor and liquid. The controlled pressure-driven dispensing (cPDD) system builds an overpressure in the liquid reservoir by pumping air and controls the opening of the liquid output valve based on the internal pressure development. The FlowCap system uses a liquid pump on the outlet, controlled by the measured inflow of air to the reservoir. Both systems are designed for compactness and portability and offer independent operation, as well as control and communication, over a wireless interface.

Self-assembly is a key coordination mechanism for large multi-unit systems and a powerful bottom-up technology for micro/nanofabrication. Controlled self-assembly and dynamic reconfiguration of large ensembles of microscopic particles can effectively bridge these domains to build innovative systems. In this perspective, we present SelfSys, a novel platform for the automated control of the fluidic self-assembly of microparticles. SelfSys centers around a water-filled microfluidic chamber whose agitation modes, induced by a coupled ultrasonic actuator, drive the assembly. Microparticle dynamics is imaged, tracked and analyzed in real-time by an integrated software framework, which in turn algorithmically controls the agitation modes of the microchamber. The closed control loop is fully automated and can direct the stochastic assembly of microparticle clusters of preset dimension. Control issues specific to SelfSys implementation are discussed, and its potential applications presented. The SelfSys platform embodies at microscale the automated self-assembly control paradigm we first demonstrated in an earlier platform.

Integration of valve like structures into microfluidic chips is a key factor for the usability of many microfluidic systems. One approach is utilization of thin PDMS layers as flexible membranes which can be closely packed on the microfluidic chips. These flexible membranes can be deformed by pneumatic actuation, closing or opening the fluidic channel of the corresponding valve. However, PDMS is permeable to gases and this permeability gets non-negligible for common valve layer thicknesses and pressure gradients. This factor restricts the usage of PDMS valves in microfluidic systems, as gas bubbles are strictly prohibited in many applications. Here two methods avoiding the intrusion of gas into the microfluidic channels are introduced and investigated. The first is based on an oil droplet placed in the dead end pneumatic channel of the valve, the second utilizes a parylene coating to make PDMS impermeable to gases. The parylene layer could not completely suppress the gas intrusion into the fluidics of the valve and additionally influenced the sealing capabilities of the valve. The oil droplet, in contrast, suppressed the permeability of the valve completely and provides a straight forward solution. In the experimental setup a novel valve design is introduced and characterized. Sealing capabilities, hydraulic resistance and lifetime are measured, dead and internal volume are calculated to describe the capabilities of thin membrane PDMS valves. These values are of interest if the valves are to be used industrially. The design introduced here is easy to fabricate and requires low alignment accuracy: it uses a Polycarbonate/PDMS sandwich, bonded by a APTES coating. Combining the simplicity of the valve design and the low cost fabrication methods and materials needed, the valve is perfectly suited as a disposable consumable.

In this paper, we present the theoretical background, design, fabrication and characterization of a micromachined chamber assisting the fluidic self-assembly of micro-electro-mechanical systems in a bulk liquid. Exploiting bubble-induced acoustic microstreaming, several structurally-robust driving modes are excited inside the chamber. The modes promote the controlled aggregation and disaggregation of microcomponents relying on strong and reproducible fluid mixing effects achieved even at low Reynolds numbers. The functionality of the microfluidic chamber is demonstrated through the fast and repeatable geometrical pairing and subsequent unpairing of polymeric microcylinders. Relying only on drag and radiation forces and on the natural hydrophobicity of SU-8 in aqueous solutions, assembly yields of approximately 50% are achieved in no longer than ten seconds of agitation. The system can stochastically control the assembly process and significantly reduce the time-to-assembly of building blocks.