Fengshan Shen,Hyundoo Hwang,Young Ki Hahn,Je-Kyun Park
This paper describes a new label-free cell separation method using a magnetic repulsion force resulting from the magnetic susceptibility difference between cells and a paramagnetic buffer solution in a microchannel. The difference in the magnetic forces acting on different-sized cells is enhanced by adjusting the magnetic susceptibility of the surrounding medium, which depends on the concentration of paramagnetic salts, such as biocompatible gadolinium diethylenetriamine pentaacetic acid (Gd–DTPA), dissolved therein. As a proof-of-concept demonstration, Gd–DTPA solutions at concentrations of 0–80 mM were applied to separate U937 cells from red blood cells (RBCs) and to distinguish two different-sized polystyrene (PS) beads (8 and 10 μm in diameter). By increasing the Gd–DTPA concentration from 0 to 40 mM, the separation resolution of PS beads was increased from 0.08 to 0.91. Additionally, we successfully achieved label-free separation of U937 cells from RBCs with >90% purity and 1 × 105 cells/h throughput using a 40 mM Gd–DTPA solution.
Cell migration plays an important role in many physiological and biological processes, which is influenced by both physicochemical properties of surrounding matrix and signal gradient generated by neighboring/remote cells. Here we aim to develop a co-culture system of immune cells and smooth muscle cells (SMCs) based on the combination of Transwell and cell-responsive hydrogels. This model can be used to study the cell invasion into hydrogels in dynamic physiological conditions , with better mimicking of the in vivo microenvironment . Methacrylic anhydride-modified hyaluronic acid (MA-HA) macromolecules were crosslinked by matrix metalloproteinases (MMPs) sensitive peptides (MMP SP) to fabricate a cell-degradable hydrogel mimicking dynamic extracellular matrix (ECM). The migration of SMCs into the MMP-sensitive hydrogel was investigated under the existence of U937 cells, a type of macrophage-like cells. The invasion distance of SMCs in the MMP-sensitive hydrogels was much longer than that in the MMP-insensitive ones both in vitro and in vivo . The impact of hydrogel degradability and inductive signal gradient generated by U937 cells on cell invasion was compared, revealing that the degradability plays a major role in regulating cell invasion into the 3D hydrogels. Further mechanism investigation revealed that the expressions of cell migration-related genes and proteins were significantly up-regulated in the MMP-sensitive hydrogels compared to those in the MMP-insensitive hydrogels.
Macrophage foam cell formation triggered by oxLDL is an important event that occurs during the development of atherosclerosis . 2,3,5,4′-Tetrahydroxystilbene-2- O -β- d -glucoside (TSG) exhibits significant anti-atherosclerotic activity. Herein we used U937 cells induced by PMA and oxLDL in vitro to investigate the inhibitory effects of TSG on U937 differentiation and macrophage foam cell formation. TSG pretreatment markedly inhibited cell differentiation induced by PMA, macrophage apoptosis and foam cell formation induced by oxLDL. The inhibition of vimentin expression and cleavage was involved in these inhibitory effects of TSG. The suppression of vimentin by siRNA in U937 significantly inhibited cell differentiation, apoptosis and foam cell formation. Using inhibitors for TGFβR1 and PI3K, we found that vimentin production in U937 cells is regulated by TGFβ/Smad signaling, but not by PI3K-Akt-mTOR signaling. Meanwhile, TSG pretreatment inhibited both the expression of TGFβ1 and the phosphorylation of Smad2 and Smad3, and TSG suppressed the nuclear translocation of Smad4 induced by PMA and oxLDL. Furthermore, TSG attenuated the induced caspase-3 activation and adhesion molecules levels by PMA and oxLDL. PMA and oxLDL increased the co-localization of vimentin with ICAM-1, which was attenuated by pretreatment with TSG. These results suggest that TSG inhibits macrophage foam cell formation through suppressing vimentin expression and cleavage, adhesion molecules expression and vimentin-ICAM-1 co-localization. The interruption of TGFβ/Smad pathway and caspase-3 activation is responsible for the downregulation of TSG on vimentin expression and degradation, respectively.
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