Microfluidic preparation of anchored cell membrane sheets for in vitro analyses and manipulation of the cytoplasmic face

Abstract

Molecular networks on the cytoplasmic faces of cellular plasma membranes are critical research topics in biological sciences and medicinal chemistry. However, the selective permeability of the cell membrane restricts the researchers from accessing to the intact intracellular factors on the membrane from the outside. Here, a microfluidic method to prepare cell membrane sheets was developed as a promising tool for direct examination of the cytoplasmic faces of cell membranes. Mammalian cells immobilized on a poly(ethylene glycol)-lipid coated substrate were rapidly and efficiently fractured, with the sheer stress of laminar flow in microchannels, resulting in isolation of the bottom cell membrane sheets with exposed intact cytoplasmic faces. On these faces of the cell membrane sheets, both ligand-induced phosphorylation of receptor tyrosine kinases and selective enzymatic modification of a G-protein coupling receptor were directly observed. Thus, the present cell membrane sheet should serve as a unique platform for studies providing new insights into juxta-membrane molecular networks and drug discovery.

Unroofing site-specific α-synuclein–lipid interactions at the plasma membrane

“…After photobleaching, there was negligible fluorescence intensity recovery observed for regions labeled with CT-B, confirming that these are rigid structures. In contrast, fluorescence intensity fully recovered for regions stained with DiD after 10 s, consistent with a fluid environment as previously reported for unroofed cells (43). This platform retains native-like properties of the inner leaflet of the plasma membrane, composed of fluid and ordered lipids.…”

Single Cell Array Enclosed with a Photodegradable Hydrogel in Microwells for Image-Based Cell Classification and Selective Photorelease of Cells

“…Glass coverslips (20 × 20 × 5 mm, each with an injection hole of 1 mm in diameter) were precoated with agarose. In the experiments for cell enrichment and culture, a microfluidic device was prepared as reported previously . The microwell glass slide was combined with a microchannel slide (ibidi GmbH, Munich, Germany) reversibly using a set of steel clips.…”