Biology subjects

Romanov, V.

Publications and source records attributed to Romanov, V..

4 recordsLinked to original sources

bioRxiv2025

Robotic Handling Preserves Induced Pluripotent Stem Cell Derived Vascular Smooth Muscle Cells Differentiation using a Weekend-free, Automatable, Low-variability, Low-cost (WALL) Protocol

Vascular disease modeling and tissue engineering require scalable, efficient, and reproducible methods for differentiating cells, however no studies have validated whether automation, essential for scale-up, preserves differentiation quality in cells. Here, we developed a differentiation protocol for induced pluripotent stem cell (iPSC)-derived vascular smooth muscle cells (iVSMCs) that is applicable to both manual, and automated robotic culture. iPSC lines generated from male and female donors across various ages were used to optimize the generation of iVSMCs. iVSMCs had consistent morphology and protein expression was largely similar to primary VSMCs, including expression of myosin heavy chain-11 (MYH11), -smooth muscle actin (-SMA), transgelin (TAGLN) and calponin (CNN1). Functionally, these iVSMCs could respond to the vasoconstrictor carbachol. Coupling this protocol with an automated Hamilton liquid-handling robotics system allowed the generation of iVSMCs in large quantities, morphologically similar to manually differentiated iVSMCs. Comparative proteomic analysis confirmed protein expression did not differ between automated and manual differentiation methods. Automation markedly reduced manual labor and facilitated increased production without sacrificing cell quality. This study demonstrates the feasibility of automating iVSMC differentiation, marking a significant step towards scalable VSMC manufacturing for three-dimensional applications, and for the ever-growing demands of organoid and tissue engineering applications. Statement of significanceThe rapidly expanding fields of tissue engineering and high-throughput disease modeling require large quantities of viable cells, making automation essential. Robotic liquid handling systems are increasingly employed for generating large quantities of induced pluripotent stem cell (iPSC)-derived cells, requiring the development of scalable protocols. No studies have yet evaluated whether the use of robotic liquid handling alters the differentiation of cells compared to traditional manual handling. Here, we report a scalable differentiation protocol that robustly generates iPSC-derived VSMCs (iVSMCs) from a cohort of female and male donors of varying age (n=5 lines). We demonstrate for the first time that iVSMCs, differentiated through an automated robotic pipeline, had no significant differences in morphological or proteomic phenotype compared to manual differentiation. The ability to automate this protocol facilitates robust and reproducible differentiation of large quantities of iVSMC, setting a precedent for the application of automation in generating iPSC-derived cells. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=57 SRC="FIGDIR/small/690512v1_ufig1.gif" ALT="Figure 1000"> View larger version (18K): org.highwire.dtl.DTLVardef@673d61org.highwire.dtl.DTLVardef@1eead89org.highwire.dtl.DTLVardef@c3089eorg.highwire.dtl.DTLVardef@6af2cd_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2020

Biomechanical characterization of endothelial cells exposed to shear stress using acoustic force spectroscopy

Characterizing mechanical properties of cells is important for understanding many cellular processes, such as cell movement, shape, and growth, as well as adaptation to changing environments. In this study, we explore mechanical properties of endothelial cells that form the biological barrier lining blood vessels, whose dysfunction leads to development of many cardiovascular disorders. Stiffness and contractile prestress of living endothelial cells were determined by Acoustic Force Spectroscopy (AFS) focusing on the displacement of functionalized microspheres located at the cell-cell periphery. The specific configuration of the acoustic microfluidic channel allowed us to develop a long-term dynamic culture protocol exposing cells to laminar flow, reaching shear stresses in the physiological range (i.e. 8 dyne cm-2) within 48 hours of barrier function maturation. A staircase-like sequence of increasing force steps, ranging from 186 pN to 3.5 nN, was applied in a single measurement revealing a force-dependent apparent stiffness in the kPa range. Moreover, our results show that different degrees of stiffening, defining the elastic behavior of the cell under different experimental conditions, i.e. static and dynamic, are caused by different levels of contractile prestress in the cytoskeleton, and are modulated by shear stress-mediated junction development and stabilization at cell borders. These results demonstrate that the AFS is capable of fast and high-throughput force measurements of adherent cells under conditions mimicking their native microenvironment, and thus revealing the shear stress dependence of mechanical properties of neighbouring endothelial cells.

bioengineering↗

bioRxiv2020

An acoustic platform for single-cell, high-throughput measurements of the viscoelastic properties of cells

Cellular processes including adhesion, migration and differentiation are governed by the distinct mechanical properties of each cell. Importantly, the mechanical properties of individual cells can vary depending on local physical and biochemical cues in a time-dependent manner resulting in significant inter-cell heterogeneity. While several different methods have been developed to interrogate the mechanical properties of single cells, throughput to capture this heterogeneity remains an issue. While new high-throughput techniques are slowly emerging, they are primarily aimed at characterizing cells in suspension, whereas high-throughput measurements of adherent cells have proven to be more challenging. Here, we demonstrate single-cell, high-throughput characterization of adherent cells using acoustic force spectroscopy. We demonstrate that cells undergo marked changes in viscoelasticity as a function of temperature, the measurements of which are facilitated by a closed microfluidic culturing environment that can rapidly change temperature between 21 {degrees}C and 37 {degrees}C. In addition, we show quantitative differences in cells exposed to different pharmacological treatments specifically targeting the membrane-cytoskeleton interface. Further, we utilize the high-throughput format of the AFS to rapidly probe, in excess of 1000 cells, three different cell-lines expressing different levels of a mechanosensitive protein, Piezo1, demonstrating the ability to differentiate between cells based on protein expression levels.

bioengineering↗

bioRxiv2019

A tunable microfluidic device enables cargo encapsulation by cell- or organelle-sized lipid vesicles comprising asymmetric lipid monolayers

Cellular membranes play host to a wide variety of morphologically and chemically complex processes. Although model membranes, like liposomes, are already widely used to reconstitute and study these processes, better tools are needed for making model bilayers that faithfully mimic cellular membranes. Existing methods for fabricating cell-sized (m) or organelle-sized (tens to hundreds of nm) lipid vesicles have distinctly different requirements. Of particular note for biology, it remains challenging for any technique to efficiently encapsulate fragile cargo molecules or to generate liposomes with stable, asymmetric lipid-leaflets within the bilayer. Here we describe a tunable microfluidic device and protocol for fabricating liposomes with desired diameters ranging from ~10 m to ~100 nm. Lipid vesicle size is templated by the simple inclusion of a polycarbonate filter within the microfluidic system and tuned with flow rate. We show that the vesicles made with our device are stable, unilamellar, lipid asymmetric, and capable of supporting transmembrane protein assembly, peripheral membrane protein binding, as well as soluble cargo encapsulation (including designer nanocages for biotechnology applications). These fabricated vesicles provide a new platform for studying the biophysically rich processes found within lipid-lipid and lipid-protein systems typically associated with cellular membranes.

bioengineering↗