Microtissues (such as organoids or spheroids) are useful to model healthy or diseased human tissues and organs, with application in drug testing, tissue engineering, and personalized medicine. To characterize their morphology and phenotype, microtissues are often monitored using histology and high‐resolution imaging. While this technique is well‐established and standard for macroscopic tissue sections, it remains challenging to execute and has low throughput in the context of these sub‐millimeter structures, mainly due to the fact that their size makes them difficult to locate within the embedding medium. To enhance the efficiency of microtissue histology, an acoustofluidic device is developed to arrange microtissues in a coplanar manner within HistoGel, a widely used embedding medium. Prepatterning microtissues within HistoGel streamlines downstream processing by positioning the centers of mass of the microtissues at a predefined plane in the sample, simplifying sectioning, labeling, and imaging. The method is validated using differently sized HepG2 microtissues and tumor microtissues from osteosarcoma cells (Saos‐2, MG‐63, HOS), demonstrating that this method can be applied to different microtissue sizes and types, without modifying established postprocessing techniques. Our approach substantially enhances the microtissue histology workflow and broadens possibilities for microtissue analysis.

High-throughput transcriptomics is of increasing fundamental biological and clinical interest. The generation of molecular data from large collections of samples, such as biobanks and drug libraries, is boosting the development of new biomarkers and treatments. Focusing on gene expression, the transcriptomic market exploits the benefits of next-generation sequencing (NGS), leveraging RNA sequencing (RNA-seq) as standard for measuring genome-wide gene expression in biological samples. The cumbersome sample preparation, including RNA extraction, conversion to cDNA and amplification, prevents high-throughput translation of RNA-seq technologies. Bulk RNA barcoding and sequencing (BRB-seq) addresses this limitation by enabling sample preparation in multi-well plate format. Sample multiplexing combined with early pooling into a single tube reduces reagents consumption and manual steps. Enabling simultaneous pooling of all samples from the multi-well plate into one tube, our technology relies on smart labware: a pooling lid comprising fluidic features and small pins to transport the liquid, adapted to standard 96-well plates. Operated with standard fluidic tubes and pump, the system enables over 90% recovery of liquid in a single step in less than a minute. Large scale manufacturing of the lid is demonstrated with the transition from a milled polycarbonate/steel prototype into an injection molded polystyrene lid. The pooling lid demonstrated its value in supporting high-throughput barcode-based sequencing by pooling 96 different DNA barcodes directly from a standard 96-well plate, followed by processing within the single sample pool. This new pooling technology shows great potential to address medium throughput needs in the BRB-seq workflow, thereby addressing the challenge of large-scale and cost-efficient sample preparation for RNA-seq. Graphical abstract

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.

Cardiomyocyte alignment in myocardium tissue plays a significant role in the physiological, electrical, and mechanical functions of the myocardium. It remains, however, difficult to align cardiac cells in a 3D in vitro heart model. This paper proposes a simple method to align cells using microfabricated Polydimethylsiloxane (PDMS) grooves with large dimensions (of up to 350 µm in width), similar to the dimensions of trabeculae carneae, the smallest functional unit of the myocardium. Two cell groups were used in this work; first, H9c2 cells in combination with Nor10 cells for proof of concept, and second, neonatal cardiac cells to investigate the functionality of the 3D model. This model compared the patterned and nonpatterned 3D constructs, as well as the 2D cell cultures, with and without patterns. In addition to alignment, we assessed the functionality of our proposed 3D model by comparing beating rates between aligned and non-aligned structures. In order to assess the practicality of the model, the 3D aligned structures should be demonstrated to be detachable and alignable. This evaluation is crucial to the use of this 3D functional model in future studies related to drug screening, building blocks for tissue engineering, and as a heart-on-chip by integrating microfluidics.

Myocardium consists of cardiac cells that interact with their environment through physical, biochemical, and electrical stimulations. The physiology, function, and metabolism of cardiac tissue are affected by this dynamic structure. Within the myocardium, cardiomyocytes’ orientations are parallel, creating a dominant orientation. Additionally, local alignments of fibers, along with a helical organization, become evident at the macroscopic level. For the successful development of a reliable in vitro cardiac model, evaluation of cardiac cells’ behavior in a dynamic microenvironment, as well as their spatial architecture, is mandatory. In this study, we hypothesize that complex interactions between long-term contraction boundary conditions and cyclic mechanical stimulation may provide a physiological mechanism to generate off-axis alignments in the preferred mechanical stretch direction. This off-axis alignment can be engineered in vitro and, most importantly, mirrors the helical arrangements observed in vivo. For this purpose, uniaxial mechanical stretching of dECM-fibrin hydrogels was performed on pre-aligned 3D cultures of cardiac cells. In view of the potential development of helical structures similar to those in native hearts, the possibility of generating oblique alignments ranging between 0° and 90° was explored. Indeed, our investigations of cell alignment in 3D, employing both mechanical stimulation and groove constraint, provide a reliable mechanism for the generation of helicoidal structures in the myocardium. By combining cyclic stretch and geometric alignment in grooves, an intermediate angle toward favored direction can be achieved experimentally: while cyclic stretch produces a perpendicular orientation, geometric alignment is associated with a parallel one. In our 2D and 3D culture conditions, nonlinear cellular addition of the strains and strain avoidance concept reliably predicted the preferred cellular alignment. The 3D dECM-fibrin model system in this study shows that cyclical stretching supports cell survival and development. Using mechanical stimulation of pre-aligned heart cells, maturation markers are augmented in neonatal cardiomyocytes, while the beating culture period is prolonged, indicating an improved model function. We propose a simplified theoretical model based on numerical simulation and nonlinear strain avoidance by cells to explain oblique alignment angles. Thus, this work lays a possible rational basis for understanding and engineering oblique cellular alignments, such as the helicoidal layout of the heart, using approaches that simultaneously enhance maturation and function.

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.

Complex three-dimensional (3D) in vitro models are emerging as a key technology to support research areas in personalised medicine, such as drug development and regenerative medicine. Tools for manipulation and positioning of microtissues play a crucial role in the microtissue life cycle from production to end-point analysis. The ability to precisely locate microtissues can improve the efficiency and reliability of processes and investigations by reducing experimental time and by providing more controlled parameters. To achieve this goal, standardisation of the techniques is of primary importance. Compared to microtissue production, the field of microtissue manipulation and positioning is still in its infancy but is gaining increasing attention in the last few years. Techniques to position microtissues have been classified into four main categories: hydrodynamic techniques, bioprinting, substrate modification, and non-contact active forces. In this paper, we provide a comprehensive review of the different tools for the manipulation and positioning of microtissues that have been reported to date. The working mechanism of each technique is described, and its merits and limitations are discussed. We conclude by evaluating the potential of the different approaches to support progress in personalised medicine.

Despite increasing interest in situ monitoring of proteolytic activity in chronic wound is not possible and information can only be obtained by sampling wound exudate. In this context, we developed an evanescent wave (EW) fiber-optic sensor to quantify protease activity directly in the wound bed. Detection is based on the degradation of thin gelatin films deposited on the fiber core by dip-coating, which serve as a substrate for proteases. After staining with a chlorophyllin copper sodium salt biocompatible dye, EW absorption occurs proportionally to the dye concentration, which is detected by the variation in light transmission intensity. The sensor response varies proportionally to enzymatic activity, showing sensitivity against MMP-2 and MMP-9 down to 2 μg/mL and 10 μg/mL, respectively. In addition, it is sensitive to film thickness and crosslink density, thus allowing tuning of the sensitivity and lifetime. Designed to be totally biocompatible and low cost, this miniature sensor has potential for use as a point-of-care disposable device in a clinical environment to assist physicians with quantitative information about the wound healing process.