Hydrogel Micropost Arrays with Single Post Tunability to Study Cell Volume and Mechanotransduction

Abstract

with both physical signals and constraints. Mesenchymal stromal cells (MSCs) have served as an important model cell type to understand how physical properties of the ECM impact cell size and functions because they are highly mechanosensitive. On 2D substrates, matrix elasticity is known to mediate MSC volume by water flux. [2] Matrix elasticity also activates nuclear translocation of Yes-associated protein (YAP) in MSCs, [3] which is known to regulate cell volume. [4] In this context, the ability of cells to sense and respond to matrix elasticity requires force generation by actomyosin contractility, [5] which subsequently impacts various biological processes, including cell division, [6] differentiation, [7,8] migration, [9] and paracrine signaling. [10] In 3D substrates, degradable, [11] fast stress-relaxing [12] or microscale thin [13] hydrogels increase cell volume expansion, cell tension, and osteogenic differentiation of MSCs, while spatial confinement restricts these processes. Nuclear localization of YAP is enhanced in 3D hydrogels when MSCs are allowed to spread or increase in cell volume as facilitated by stress relaxation, degradation or less crosslinking. [14-16] While these studies have revealed important insights into how the ECM mediates cell volume and mechanotransduction, the ECM varies considerably in biophysical properties at the microscale as revealed by mechanical force mapping of tissues. [17] This aspect of cell-matrix interactions is currently difficult to study using bulk engineered hydrogels, which lack control over microscale properties. Combination of instrumentation and biomaterial design offers promising strategies to control the placement of matrix signals independently of their mechanics at microscale resolution. While electrospinning has been used to decouple polymer network architecture and mechanics by creating fibrous materials as a means to study mechanosensing of cells, [18] it is presently difficult to control where matrix fibers are placed. Microfabrication by stereolithography has the potential to precisely specify matrix mechanical properties at the microscale. So far, most studies have focused on 2D interaction of cells with micropatterned substrates as a means to study the effect of adhesion area, [19] geometry, [20-22] and surface topography [23] on MSC functions. Recently, micropatterning was used to study The extracellular matrix varies considerably in mechanical properties at the microscale. It remains unclear how cells respond to these properties, in part, due to lack of tools to create precisely defined microenvironments in a discrete manner. Here, freeform stereolithography is leveraged to control the placement and elastic modulus of individual hydrogel microposts that serve as discrete matrix signals to interface with cells. Mesenchymal stromal cells (MSCs) located in the interstitial spaces between microposts above a base layer are analyzed. Cell volume is higher when MSCs interact with more microposts. MSCs show higher strain energy when they interact simultan...

Tension sensing by FAK governs nuclear mechanotransduction, endothelial transcriptome and fate

“…We assessed if FAK was required to soften EC to maintain lung homeostasis. We used atomic force microscopy (AFM) to quantify intracellular tension, i.e., Young's modulus (E) in control inactivated versus FAK depleted EC (Discher et al, 2005;Wong et al, 2020). We found that inactivated EC generates physiologically relevant intracellular EC tension ⁓8 KPa (Fig.…”

Automatic Multi-functional Integration Program (AMFIP) towards all-optical mechano-electrophysiology interrogation

“…The intended purposes of AMFIP to study dynamic, multi-faceted, and long-term biological questions are achieved by demonstrating the previously unknown relationship between YAP dynamics and cell mechanics. We think AMFIP is a reliable platform with proven advantages over existing alternatives and validated long-term utility that will benefit the scientific community in multidisciplinary biomedical research [34,37,54,[64][65][66][67][68][69][70][71]. The charge-free source code of AMFIP is available to the public on GitHub (link: https://github.com/njheadshotz/AMFIP), and we hope researchers will develop and expand more new functions based on AMFIP to enhance their specific automatic operations in research.…”

Cell–Matrix Interactions Regulate Functional Extracellular Vesicle Secretion from Mesenchymal Stromal Cells

“…To covalently bond cross-linked PEG-DA hydrogel to a thin glass surface, acrylate groups were attached to glass coverslips by silanization as described . A solution of 3% v / v 3-(trimethoxysilyl)­propyl acrylate (TCI Chemicals) and 5% v / v glacial acetic acid (Fisher Scientific) was prepared in methanol.…”