Early hMSC morphology and proliferation on model polyelectrolyte multilayers.

Polyelectrolyte multilayers (PEMs) are a category of materials commonly used as coatings on surfaces that interact with cells. The properties of PEMs have been established to be controlled by not only polyelectrolyte choice, but by the identity of the initially applied (bottom) layer. In this work, 5-bilayer PEMs consisting of poly(diallyldimethylammonium chloride) (PDADMAC) and poly(sodium 4-styrenesulfonate) (PSS) were coated on gold-sputtered quartz substrates with different first layer materials. A final poly-l-lysine (PLL) layer was added to all PEMs to provide identical top layers conducive to cell growth. As in previous work, initial layer selection affected PEM roughness. All coated surfaces, including the PLL-only control, showed increased dispersive surface energy but decreased polar surface energy, as compared to uncoated surfaces. When human mesenchymal stem cells (hMSCs) were cultured on these surfaces, analysis through lateral cell imaging for the first 90 min and fluorescent staining after 1 day showed improved attachment on surfaces with a PDADMAC bottom layer. However, after 4 days, a higher cell density was observed on the PLL-only and uncoated control surfaces, indicating that the PEMs negatively affected hMSC proliferation. Both the long and short time period results did not correlate to any of the roughness and surface energy trends, indicating more complex interactions between the cells and the surface relating to charge distribution and functional group density.

Correlation of Cell Surface Biomarker Expression Levels with Adhesion Contact Angle Measured by Lateral Microscopy.

Immunophenotyping is typically achieved using flow cytometry, but any influence a biomarker may have on adhesion or surface recognition cannot be determined concurrently. In this manuscript, we demonstrate the utility of lateral microscopy for correlating cell surface biomarker expression levels with quantitative descriptions of cell morphology. With our imaging system, we observed single cells from two T cell lines and two B cell lines adhere to antibody-coated substrates and quantified this adhesion using contact angle measurements. We found that SUP-T1 and CEM CD4+ cells, both of which express similar levels of CD4, experienced average changes in contact angle that were not statistically different from one another on surfaces coated in anti-CD4. However, MAVER-1 and BJAB K20 cells, both of which express different levels of CD20, underwent average changes in contact angle that were significantly different from one another on surfaces coated in anti-CD20. Our results indicate that changes in cell contact angles on antibody-coated substrates reflect the expression levels of corresponding antigens on the surfaces of cells as determined by flow cytometry. Our lateral microscopy approach offers a more reproducible and quantitative alternative to evaluate adhesion compared to commonly used wash assays and can be extended to many additional immunophenotyping applications to identify cells of interest within heterogeneous populations.

Thioether-stapled macrocyclic inhibitors of the EH domain of EHD1.

Recycling of receptors from the endosomal recycling compartment to the plasma membrane is a critical cellular process, and recycling is particularly important for maintaining invasiveness in solid tumors. In this work, we continue our efforts to inhibit EHD1, a critical adaptor protein involved in receptor recycling. We applied a diversity-oriented macrocyclization approach to produce cyclic peptides with varied conformations, but that each contain a motif that binds to the EH domain of EHD1. Screening these uncovered several new inhibitors for EHD1's EH domain, the most potent of which bound with a Kd of 3.1µM. Several of the most potent inhibitors were tested in a cellular assay that measures extent of vesicle recycling. Inhibiting EHD1 could potentially slow cancer invasiveness and metastasis, and these cyclic peptides represent the most potent inhibitors of EHD1 to date.

Beyond Wicking: Expanding the Role of Patterned Paper as the Foundation for an Analytical Platform.

While a number of assays for soluble analytes have been developed using paper-based microfluidic devices, the detection and analysis of blood cells has remained an outstanding challenge. In this Feature, we discuss how the properties of paper determine the performance of paper-based microfluidic devices and permit the design of cellular assays, which can ultimately impact disparities in healthcare that exist in limited-resource settings.

Lateral Microscope Enables the Direct Observation of Cellular Interfaces and Quantification of Changes in Cell Morphology during Adhesion.

The ability to observe cell adhesion processes in real-time remains a grand challenge in basic biology and medicine. Toward this goal, we have developed a lateral optical microscope that allows direct observation of cell-substrate interactions in real-time on any substrate-transparent, opaque, or coated-without requiring labels or specialized optical components. We demonstrate the use of our lateral microscope by quantifying dynamic changes in cell morphology during the first 90 min of adhesion to various materials. Specifically, we determined the rates of change in contact angle of HeLa, 3T3, HEK293, and MDA-MB-231 cells on five different substrates: glass, collagen-coated glass, Nylon, PTFE, and collagen-alginate hydrogels. We used these rates of change to compare adhesion of different cell lines on each surface, and to rank the adhesion-promoting capacities of the five surfaces for each cell line. For HeLa, 3T3, and HEK293 cells, we observed maximal rates of change in contact angle (0.058 min-1) on collagen-coated glass substrates. All five cell lines exhibited minimal rates of change (0.006 min-1) on PTFE. Lateral microscopy also revealed a unique morphology among MDA-MB-231 breast cancer cells during initial adhesion, which we quantified using measurements of changes in cell height. Our lateral microscope will not only enable more comprehensive, quantitative studies of cell adhesion to inform the development of biomaterials but will ultimately assist in advancing our understanding of many important biological processes and discovering new behaviors related to cell adhesion.

Combining Step Gradients and Linear Gradients in Density.

Combining aqueous multiphase systems (AMPS) and magnetic levitation (MagLev) provides a method to produce hybrid gradients in apparent density. AMPS­solutions of different polymers, salts, or surfactants that spontaneously separate into immiscible but predominantly aqueous phases­offer thermodynamically stable steps in density that can be tuned by the concentration of solutes. MagLev­the levitation of diamagnetic objects in a paramagnetic fluid within a magnetic field gradient­can be arranged to provide a near-linear gradient in effective density where the height of a levitating object above the surface of the magnet corresponds to its density; the strength of the gradient in effective density can be tuned by the choice of paramagnetic salt and its concentrations and by the strength and gradient in the magnetic field. Including paramagnetic salts (e.g., MnSO4 or MnCl2) in AMPS, and placing them in a magnetic field gradient, enables their use as media for MagLev. The potential to create large steps in density with AMPS allows separations of objects across a range of densities. The gradients produced by MagLev provide resolution over a continuous range of densities. By combining these approaches, mixtures of objects with large differences in density can be separated and analyzed simultaneously. Using MagLev to add an effective gradient in density also enables tuning the range of densities captured at an interface of an AMPS by simply changing the position of the container in the magnetic field. Further, by creating AMPS in which phases have different concentrations of paramagnetic ions, the phases can provide different resolutions in density. These results suggest that combining steps in density with gradients in density can enable new classes of separations based on density.