A Micropatterning Strategy to Study Nuclear Mechanotransduction in Cells

Micropatterning techniques have been widely used in biology, particularly in studies involving cell adhesion and proliferation on different substrates. Cell micropatterning approaches are also increasingly employed as in vitro tools to investigate intracellular mechanotransduction processes. In this report, we examined how modulating cellular shapes on two-dimensional rectangular fibronectin micropatterns of different widths influences nuclear mechanotransduction mediated by emerin, a nuclear envelope protein implicated in Emery–Dreifuss muscular dystrophy (EDMD). Fibronectin microcontact printing was tested onto glass coverslips functionalized with three different silane reagents (hexamethyldisilazane (HMDS), (3-Aminopropyl)triethoxysilane (APTES) and (3-Glycidyloxypropyl)trimethoxysilane (GPTMS)) using a vapor-phase deposition method. We observed that HMDS provides the most reliable printing surface for cell micropatterning, notably because it forms a hydrophobic organosilane monolayer that favors the retainment of surface antifouling agents on the coverslips. We showed that, under specific mechanical cues, emerin-null human skin fibroblasts display a significantly more deformed nucleus than skin fibroblasts expressing wild type emerin, indicating that emerin plays a crucial role in nuclear adaptability to mechanical stresses. We further showed that proper nuclear responses to forces involve a significant relocation of emerin from the inner nuclear envelope towards the outer nuclear envelope and the endoplasmic reticulum membrane network. Cell micropatterning by fibronectin microcontact printing directly on HMDS-treated glass represents a simple approach to apply steady-state biophysical cues to cells and study their specific mechanobiology responses in vitro.

Liver microsystems in vitro for drug response

Engineering approaches were adopted for liver microsystems to recapitulate cell arrangements and culture microenvironments in vivo for sensitive, high-throughput and biomimetic drug screening. This review introduces liver microsystems in vitro for drug hepatotoxicity, drug-drug interactions, metabolic function and enzyme induction, based on cell micropatterning, hydrogel biofabrication and microfluidic perfusion. The engineered microsystems provide varied microenvironments for cell culture that feature cell coculture with non-parenchymal cells, in a heterogeneous extracellular matrix and under controllable perfusion. The engineering methods described include cell micropatterning with soft lithography and dielectrophoresis, hydrogel biofabrication with photolithography, micromolding and 3D bioprinting, and microfluidic perfusion with endothelial-like structures and gradient generators. We discuss the major challenges and trends of liver microsystems to study drug response in vitro.

Geometry and network connectivity govern the mechanics of stress fibers

Actomyosin stress fibers (SFs) play key roles in driving polarized motility and generating traction forces, yet little is known about how tension borne by an individual SF is governed by SF geometry and its connectivity to other cytoskeletal elements. We now address this question by combining single-cell micropatterning with subcellular laser ablation to probe the mechanics of single, geometrically defined SFs. The retraction length of geometrically isolated SFs after cutting depends strongly on SF length, demonstrating that longer SFs dissipate more energy upon incision. Furthermore, when cell geometry and adhesive spacing are fixed, cell-to-cell heterogeneities in SF dissipated elastic energy can be predicted from varying degrees of physical integration with the surrounding network. We apply genetic, pharmacological, and computational approaches to demonstrate a causal and quantitative relationship between SF connectivity and mechanics for patterned cells and show that similar relationships hold for nonpatterned cells allowed to form cell–cell contacts in monolayer culture. Remarkably, dissipation of a single SF within a monolayer induces cytoskeletal rearrangements in cells long distances away. Finally, stimulation of cell migration leads to characteristic changes in network connectivity that promote SF bundling at the cell rear. Our findings demonstrate that SFs influence and are influenced by the networks in which they reside. Such higher order network interactions contribute in unexpected ways to cell mechanics and motility.

Plasma free reversible and irreversible microfluidic bonding

We demonstrate a novel bonding process that allows for facile multilayer microfluidic chip fabrication and is compatible with cell micropatterning.