Development of a Robust Manufacturing Route for Molnupiravir, an Antiviral for the Treatment of COVID-19.

Herein is described the development of a large-scale manufacturing process for molnupiravir, an orally dosed antiviral that was recently demonstrated to be efficacious for the treatment of patients with COVID-19. The yield, robustness, and efficiency of each of the five steps were improved, ultimately culminating in a 1.6-fold improvement in overall yield and a dramatic increase in the overall throughput compared to the baseline process.

Endothelial cell dynamics during anastomosis in vitro.

Vascular anastomosis - the fusion of vessels from two distinct branches of the vascular system - represents a critical step in vascular growth under both healthy and pathological conditions, in vivo, and presents an important target for engineering of vascularized tissues, in vitro. Recent works in animal models have advanced our understanding of the molecular and cellular players in vascular anastomosis, but questions remain related to cellular dynamics and control of this process, in vitro. In this study, we exploited a three-dimensional (3-D) culture platform to examine the dynamics of endothelial cell (EC) during and after vascular anastomosis by allowing angiogenesis and vasculogenesis to proceed in parallel. We show that anastomosis occurs between sprouts formed by angiogenesis from an endothelium and tubes formed by vasculogenesis in the bulk of a 3-D matrix. This fusion leads to highly connected vessels that span from the surface of the matrix into the bulk in a manner that depends on cell density and identity. Further, we observe and analyze intermixing of endothelial cells of distinct origin (surface versus bulk) within the vessels structures that are formed; we provide evidence that the cells migrate along pre-existing vessels segments as part of this intermixing process. We conclude that anastomosis can occur between vessels emerging by angiogenesis and vasculogenesis and that this process may play an important role in contexts such as wound healing.

A contact line pinning based microfluidic platform for modelling physiological flows.

This work introduces a contact line pinning based microfluidic platform for the generation of interstitial and intramural flows within a three dimensional (3D) microenvironment for cellular behaviour studies. A contact line pinning method was used to confine a natively derived biomatrix, collagen, in microfluidic channels without walls. By patterning collagen in designated wall-less channels, we demonstrated and validated the intramural flows through a microfluidic channel bounded by a monolayer of endothelial cells (mimic of a vascular vessel), as well as slow interstitial flows within a cell laden collagen matrix using the same microfluidic platform. The contact line pinning method ensured the generation of an engineered endothelial tube with straight walls, and spatially uniform interstitial fluid flows through the cell embedded 3D collagen matrix. Using this device, we demonstrated that the breast tumour cells' (MDA-MB-231 cell line) morphology and motility were modulated by the interstitial flows, and the motility of a sub-population of the cells was enhanced by the presence of the flow. The presented microfluidic platform provides a basic framework for studies of cellular behaviour including cell transmigration, growth, and adhesion under well controlled interstitial and intramural flows, and within a physiologically realistic 3D co-culture setting.

Formation of microvascular networks in vitro.

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (µVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.

In vitro microvessels for the study of angiogenesis and thrombosis.

Microvascular networks support metabolic activity and define microenvironmental conditions within tissues in health and pathology. Recapitulation of functional microvascular structures in vitro could provide a platform for the study of complex vascular phenomena, including angiogenesis and thrombosis. We have engineered living microvascular networks in three-dimensional tissue scaffolds and demonstrated their biofunctionality in vitro. We describe the lithographic technique used to form endothelialized microfluidic vessels within a native collagen matrix; we characterize the morphology, mass transfer processes, and long-term stability of the endothelium; we elucidate the angiogenic activities of the endothelia and differential interactions with perivascular cells seeded in the collagen bulk; and we demonstrate the nonthrombotic nature of the vascular endothelium and its transition to a prothrombotic state during an inflammatory response. The success of these microvascular networks in recapitulating these phenomena points to the broad potential of this platform for the study of cardiovascular biology and pathophysiology.