Probing Single-Cell Macrophage Polarization and Heterogeneity Using Thermo-Reversible Hydrogels in Droplet-Based Microfluidics.

Human immune cells intrinsically exist as heterogenous populations. To understand cellular heterogeneity, both cell culture and analysis should be executed with single-cell resolution to eliminate juxtacrine and paracrine interactions, as these can lead to a homogenized cell response, obscuring unique cellular behavior. Droplet microfluidics has emerged as a potent tool to culture and stimulate single cells at high throughput. However, when studying adherent cells at single-cell level, it is imperative to provide a substrate for the cells to adhere to, as suspension culture conditions can negatively affect biological function and behavior. Therefore, we combined a droplet-based microfluidic platform with a thermo-reversible polyisocyanide (PIC) hydrogel, which allowed for robust droplet formation at low temperatures, whilst ensuring catalyzer-free droplet gelation and easy cell recovery after culture for downstream analysis. With this approach, we probed the heterogeneity of highly adherent human macrophages under both pro-inflammatory M1 and anti-inflammatory M2 polarization conditions. We showed that co-encapsulation of multiple cells enhanced cell polarization compared to single cells, indicating that cellular communication is a potent driver of macrophage polarization. Additionally, we highlight that culturing single macrophages in PIC hydrogel droplets displayed higher cell viability and enhanced M2 polarization compared to single macrophages cultured in suspension. Remarkably, combining phenotypical and functional analysis on single cultured macrophages revealed a subset of cells in a persistent M1 state, which were undetectable in conventional bulk cultures. Taken together, combining droplet-based microfluidics with hydrogels is a versatile and powerful tool to study the biological function of adherent cell types at single-cell resolution with high throughput.

Critical behaviour in the nonlinear elastic response of hydrogels.

In this paper we study the elastic response of synthetic hydrogels to an applied shear stress. The hydrogels studied here have previously been shown to mimic the behaviour of biopolymer networks when they are sufficiently far above the gel point. We show that near the gel point they exhibit an elastic response that is consistent with the predicted critical behaviour of networks near or below the isostatic point of marginal stability. This point separates rigid and floppy states, distinguished by the presence or absence of finite linear elastic moduli. Recent theoretical work has also focused on the response of such networks to finite or large deformations, both near and below the isostatic point. Despite this interest, experimental evidence for the existence of criticality in such networks has been lacking. Using computer simulations, we identify critical signatures in the mechanical response of sub-isostatic networks as a function of applied shear stress. We also present experimental evidence consistent with these predictions. Furthermore, our results show the existence of two distinct critical regimes, one of which arises from the nonlinear stretch response of semi-flexible polymers.

Unusual temperature dependence of elastic constants of an ambient-temperature discotic nematic liquid crystal.

We report the first experimental studies on the temperature dependence of viscoelastic properties of a room temperature discotic nematic liquid crystal. The splay elastic constant is greater than the bend elastic constant and both show unusual temperature and order parameter dependence. The rotational viscosity is remarkably larger than conventional calamitic liquid crystals. We provide a simple physical explanation based on the columnar short-range order to account for the the unusual temperature dependence of the elastic constants.

Solid-state NMR characterization of tri-ethyleneglycol grafted polyisocyanopeptides.

In aqueous media, ethylene glycol substituted polyisocyanopeptides (PICPs) change their state (undergo a sol-to-gel transition) as a response to temperature. This makes them promising materials for various biomedical applications, for instance, for controlled drug release and non-damaging wound dressing. To utilize PICP in biomedical applications, understanding of the origin of the gelation process is needed, but this is experimentally difficult because of the notoriously low gelator concentration in combination with the slow polymer dynamics in the sample. This paper describes a detailed characterization of the dried state of PICPs by solid-state NMR measurements. Both the (13) C and the (1) H NMR resonances were assigned using a combination of 1D cross-polarization magic angle spinning, 2D (13) C-(1) H heteronuclear correlation spectra and (1) H-(1) H single quantum-double quantum experiments. In addition, the chemical groups involved in dipolar interaction with each other were used to discuss the dynamics and spatial conformation of the polymer. In contrast to other PICP polymers, two resonances for the backbone carbon are observed, which are present in equal amounts. The possible origin of these resonances is discussed in the last section of this work. The data obtained during the current studies will be further used in elucidating mechanisms of the bundling and gelation. A comprehensive picture will make it possible to tailor polymer properties to meet specific needs in different applications. Copyright © 2015 John Wiley & Sons, Ltd.

Phase structure and molecular dynamics of liquid-crystalline side-on organosiloxane tetrapodes.

X-ray diffraction and proton NMR relaxation measurements were carried out on two liquid-crystalline organosiloxane tetrapodes with side-on mesogenic groups, exhibiting nematic and smectic- C phases, and on a monomeric analog. Packing models for the mesophases exhibited by these systems are proposed on the basis of x-ray diffraction data. As a consequence of microsegregation, the aromatic cores are packed in between two sublayers formed by a mixture of interdigitated aliphatic and siloxane chains. The mixed sublayers are characteristic for the tetrapodes with side-on mesogenic groups presented in this work and have not been observed in tetrapodes with terminally attached mesogens. The tilt angle in the smectic- C phase is found very large, i.e., approximately 61 degrees -62 degrees . Notably, smectic- C clusters are present also in the whole temperature range of the nematic phase. NMR relaxometry yields T(1)-1 dispersions clearly different from those of conventional calamitics. The influence of molecular tendency to form interdigitated structures is evidenced by frequency-dependent relaxation rate in the isotropic phase-indicating the presence of ordered clusters far above the phase transition-and by the diminished role of molecular self-diffusion in ordered phases. Nematiclike director fluctuations are the dominating relaxation mechanism whereas the translational displacements are strongly hindered by the interdigitation of dendrimer arms.

Structure and molecular dynamics of the mesophases exhibited by an organosiloxane tetrapode with strong polar terminal groups.

The polymorphism of a new organosiloxane tetrapode compound with cyano terminal polar groups was characterized by means of polarizing optical microscopy and x-ray diffraction. The compound exhibits smectic- A and smectic- C phases with a partial bilayer arrangement due to a certain degree of head-to-head association of the mesogenic units through their cyano end groups. On the basis of x-ray diffraction results, evidencing the microsegregation of polyphilic molecules, packing models for the smectic- A and smectic- C phases are proposed. A high degree of smectic positional order and a relatively low value of the tilt angle in the smectic- C phase are indicated. Molecular dynamics of the studied compound was investigated by means of proton NMR relaxometry. The frequency dispersions of the spin-lattice relaxation time (T1) show that the relaxation is induced by three rotational modes of individual dendrimer arms with frequencies between 10;{6} and 10;{9}Hz . In the smectic phases, the effect of individual rotations is overwhelmed by a well expressed contribution of layer undulations at Larmor frequencies below approximately 10MHz . The appearance of this relaxation mechanism over the frequency range of three decades is so far unique in the case of thermotropic liquid crystals. The analysis of the layer undulations contribution supports the microsegregation model of the smectic phases by revealing a slowing-down of translational diffusion and the lack of interactions among the sublayers formed by the mesogenic groups.