Taveras, C. | bioRxiv Science

bioRxiv2024

Parallelized Droplet Vitrification Enables Single-Run Vitrification of the Whole Rat Liver Hepatocyte Yield

Drug discovery pipelines rely on the availability of isolated primary hepatocytes for investigating potential hepatotoxicity prior to clinical application. These hepatocytes are typically isolated from livers rejected for transplantation and subsequently cryopreserved for later usage. The gold-standard cryopreservation technique, slow-freezing, is a labor-intensive process, with significant post-storage viability loss. In this work, we introduce parallelized droplet vitrification, a technique for high-volumetric, rapid vitrification of suspended cells. We show the utility of this technique through the single-run vitrification of the whole-rate liver hepatocyte yield, resulting in a 1600% increase in single-batch vitrification and a 500% increase in droplet generation rate compared to previous droplet vitrification approaches. Additionally, we showed that these implementations maintained improved post-preservation outcomes in primary rat hepatocytes.

bioengineering↗

bioRxiv2022

Rab8-, Rab11-, and Rab35-dependent mechanisms coordinating lumen and cilia formation during Left-Right Organizer development

An essential process during Danio rerios left-right organizer (Kupffers Vesicle, KV) development is for the majority of developing KV cells to form a motile cilium that extend into the KV lumen. Left-right beating of motile cilia within the KV lumen directs fluid flow to establishment the embryos left-right axis. However, when KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation has not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumenal cavity. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 m2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

cell biology↗