The understanding that systemic context and tissue crosstalk are essential keys for bridging the gap between in vitro models and in vivo conditions led to a growing effort in the last decade to develop advanced multi-organ-on-a-chip devices. However, many of the proposed devices have failed to implement the means to allow for conditions tailored to each organ individually, a crucial aspect in cell functionality. Here, we present two 3D-print-based fabrication methods for a generic multi-organ-on-a-chip device: One with a PDMS microfluidic core unit and one based on 3D-printed units. The device was designed for culturing different tissues in separate compartments by integrating individual pairs of inlets and outlets, thus enabling tissue-specific perfusion rates that facilitate the generation of individual tissue-adapted perfusion profiles. The device allowed tissue crosstalk using microchannel configuration and permeable membranes used as barriers between individual cell culture compartments. Computational fluid dynamics (CFD) simulation confirmed the capability to generate significant differences in shear stress between the two individual culture compartments, each with a selective shear force. In addition, we provide preliminary findings that indicate the feasibility for biological compatibility for cell culture and long-term incubation in 3D-printed wells. Finally, we offer a cost-effective, accessible protocol enabling the design and fabrication of advanced multi-organ-on-a-chip devices.

This CloneSeq protocol combines clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing to resolve the limited sensitivity of single-cell approaches. CloneSeq can reveal rare subpopulations and support cellular stemness. CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging clonal enhanced sensitivity. Important considerations include the hydrogel composition, adaptation of 3D cultured clones to the inDrops system, and inherent adhesive properties of the cells. CloneSeq is only validated for cell lines so far.

Real-time knowledge of contaminants in water is an essential component of any potable water security system. Latex immunoassay is the basic technique in rapid identification of pathogens. We used acoustic radiation to accelerate the latex immunoassay and to bring about separation between free and bound antigen (rotavirus SA-11) in less than one minute. The ultrasonic cylindrical standing-wave device of NDT Instruments, UltraAssay 101, creates areas of maximum and minimum potential energy (nodes). Acoustic radiation forces acting on the pathogens drive them directly to the central node. In this manner, antibody-antigen complexes accumulate in the nodes in 40-60 sec. The nonbound antibodies are washed out of the separation area by the buffer flow. The forces responsible for separation of particles in the UltraAssay 101 depend on the frequency of the standing-wave resonator and on the density, compressibility and size of the immune complexes. Additionally, UltraAssay 101 is able to directly monitor water salinity, turbidity and specific gravity.

Cell cycle and differentiation decisions are tightly linked; however, the underlying principles that drive these decisions are not fully understood. Here, we combined cell-cycle reporter system and single-cell RNA-seq profiling to study the transcriptomes of mouse embryonic stem cells (ESCs) in the context of cell cycle states and differentiation. By applying retinoic acid, a multi-linage differentiation assay, on G1 and G2/M pre-sorted ESCs, we show that only G2/M ESCs were capable of differentiating into extraembryonic endoderm cells (XENs), whereas cells in the G1 phase predominantly produce Epiblast Stem Cells. We identified ESRRB, a key pluripotency factor that is upregulated during G2/M phase, as a central driver of XEN differentiation. Furthermore, enhancer chromatin states based on WT and Esrrb -KO ESCs show association of ESRRB with XEN poised enhancers. Cells engineered to overexpress Esrrb during G1 allow ESCs to produce XENs, while ESRRB-KO ESCs lost their potential to differentiate into XEN. In addition, Embryonic bodies (EBs) are not affected by deletion of ESRRB but trigger apoptosis upon attempts to apply direct XEN differentiation. Taken together, this study reveals an important functional link between Esrrb and cell-cycle states during the exit from pluripotency. Finally, the experimental scheme of single cell RNA-seq in the context of cell cycle can be further expanded into other cellular systems to better understand differentiation decisions and cancer models.