Strongly Metastable Assemblies of Particles at Liquid Interfaces

The self-assembly of floating particles is a widely observed phenomenon. Ideally, rafts of identical floating spheres or cylinders should assemble in a closed-packed fashion. However, rafts are observed to exhibit large and various defects, and we show that the conjunction of lateral liquid bridges between particles and contact angle hysteresis freezes the rotation of particles around their neighbors, a mechanism that generates imperfect rafts. Conversely, we demonstrate how this capillary bond can be exploited to sculpt 2D aggregates far from equilibrium that are persistent

Strongly Metastable Assemblies of Particles at Liquid Interfaces

The self-assembly of floating particles is a widely observed phenomenon. Ideally, rafts of identical floating spheres or cylinders should assemble in a closed-packed fashion. However, rafts are observed to exhibit large and various defects, and we show that the conjunction of lateral liquid bridges between particles and contact angle hysteresis freezes the rotation of particles around their neighbors, a mechanism that generates imperfect rafts. Conversely, we demonstrate how this capillary bond can be exploited to sculpt 2D aggregates far from equilibrium that are persistent

Stepwise addition of DMSO.

<p><b>(A)</b> Comparison of the mortality rate of cells between stepwise and single-step addition. <b>(B)</b> Bleb index in the stepwise addition method. HeLa cells were treated with 20% DMSO for 30 minutes, and the solution was removed quickly and changed to 40% DMSO for 30 minutes. It was then changed to 60% DMSO for 30 minutes and, finally, to 80% DMSO. The inverted fluorescence microscope was used to observe dead cells labeled by PI and Hoechst. For <b>(A)</b>, the number of cells used was approximately 500 and the experiment was repeated 5 times. For <b>(B)</b>, the number of cells used was approximately 40. **p<0.01 was considered statistically significant.</p

Cell blebs and cytoskeleton under different DMSO concentrations.

<p><b>(A)</b> The bleb and cytoskeleton were observed by an inverted fluorescence microscope (membrane: red; cytoskeleton: green). <b>(B)</b> The bleb and cytoskeleton were observed by a confocal microscope (cytoskeleton: green; nucleus: blue). <b>(C)</b> The fluid flows in the formation of blebs under a hypoosmotic condition (0.1×PBS) and a hyperosmotic condition (25% DMSO in PBS). The experiments were repeated 3 times.</p

The autophagy induced by the addition of CPAs.

<p><b>(A)</b> GFP-LC3/HeLa cells were treated with various concentrations of DMSO, and GFP green fluorescence dots appeared in cells. <b>(B)</b> LC3 conversion was determined by western blot in HeLa cells treated with different concentrations of DMSO. <b>(C)</b> Effect of DMSO on the autophagy rate. <b>(D)</b> GFP-LC3 /HeLa cells were inhibited by 3-MA, and then stimulated by 30% DMSO. Shrinkage of cell nuclei is a hallmark of apoptosis. <b>(E)</b> Autophagy reduced the apoptosis in the presence of 30% DMSO. **p<0.01 was considered statistically significant. The experiments were repeated 5 times. The number of cells used was approximately 500.</p

Effect of the concentration of CPAs: (A) number of cell blebs; (B) total area of cell blebs; (C) bleb index; (D) mortality rate of cells; (E) schematic of A_lip and A_cyto.

<p>HeLa cells were treated with a series of solutions containing different amounts of DMSO or glycerol, as well as the fluorochromes Hoechst and PI. After 30 minutes, when cells were stable, an inverted fluorescence microscope was used to observe cell death. For <b>(A)</b>, <b>(B)</b> and <b>(C)</b>, the cell number was approximately 40. For <b>(D)</b>, the cell number was approximately 500 and the experiment was repeated 5 times. For <b>(E)</b>, the red boundary denotes the lipid bilayer and the green boundary denotes the cortical cytoskeleton.</p

Cell blebs induced by the addition of CPAs.

<p>Various concentrations of <b>(A)</b> DMSO and <b>(B)</b> glycerol were applied to HeLa cells for 30 minutes. The development of cell blebs during the first 3 minutes was observed as the initial state and after 30 minutes as the stable state. Initiate: 3 minutes, and Stabilized: 30 minutes. The experiments were repeated 3 times.</p

Life cycle of a dynamic bleb.

<p><b>(A)</b> the inflation and retraction of one bleb (black arrows); <b>(B)</b> the actin microfilament reorganization during the bleb inflation and retraction; <b>(C)</b> the comparison of the inflation and retraction time between DMSO and glycerol. For <b>(A)</b> and <b>(B)</b>, the experiments were repeated 3 times. For <b>(C)</b>, the number of cells used was approximately 20.</p

Marrow VME for the study of thrombopoiesis <i>in vitro</i>.

<p><b>A</b>. Z-stack projection of confocal fluorescence imaging of megakaryocytes co-cultured within a 3D microvascular system. Green: CD41, red: CD31, blue: nuclei. <b>B</b>. Enlarged view, z-projection of confocal fluorescence images (left panel) and orthogonal views (right two panels) of locations at dotted lines 1 and 2, showing megakaryocytes interacting with the vessel wall (stars) and in the lumen and on the abluminal vessel wall (arrowheads). Green: CD41, red: CD31, blue: nuclei. <b>C</b>. (i) Zoomed view of megakaryocyte indicated in A (arrowhead) showing CD41a+ (green) and nucleus staining (blue) (ii) 3D reconstruction of the nucleus lobes from the megakaryocyte in i. <b>D</b>. A TEM image showing a megakaryocyte with four nucleus lobes close to a vessel. <b>E</b>. Megakaryocyte lobe counts near and far from the vessel wall shows more mature megakaryocytes are located closer to the vessel wall.</p

Microvasculature-directed thrombopoiesis in a 3D <i>in vitro</i> marrow microenvironment

<div><p>Vasculature is an interface between the circulation and the hematopoietic tissue providing the means for hundreds of billions of blood cells to enter the circulation every day in a regulated fashion. The precise mechanisms that control the interactions of hematopoietic cells with the vessel wall are largely undefined. Here, we report on the development of an <i>in vitro</i> 3D human marrow vascular microenvironment (VME) to study hematopoietic trafficking and the release of blood cells, specifically platelets. We show that mature megakaryocytes from aspirated marrow as well as megakaryocytes differentiated in culture from CD34+ cells can be embedded in a collagen matrix containing engineered microvessels to create a thrombopoietic VME. These megakaryocytes continue to mature, penetrate the vessel wall, and release platelets into the vessel lumen. This process can be blocked with the addition of antibodies specific for CXCR4, indicating that CXCR4 is required for megakaryocyte migration, though whether it is sufficient is unclear. The 3D marrow VME system shows considerable potential for mechanistic studies defining the role of marrow vasculature in thrombopoiesis. Through a stepwise addition or removal of individual marrow components, this model provides potential to define key pathways responsible for the release of platelets and other blood cells.</p></div