nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome.

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity or impact on performance vs 1-plex signals, with sensitivities as low as 0.1pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating ~1.5M protein datapoints in under a week, a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We also validated the nELISA's use in phenotypic screening, and propose its application to drug discovery.

Functional Effects of Delivering Human Mesenchymal Stem Cell-Seeded Biological Sutures to an Infarcted Heart.

Stem cell therapy has the potential to improve cardiac function after myocardial infarction (MI); however, existing methods to deliver cells to the myocardium, including intramyocardial injection, suffer from low engraftment rates. In this study, we used a rat model of acute MI to assess the effects of human mesenchymal stem cell (hMSC)-seeded fibrin biological sutures on cardiac function at 1 week after implant. Biological sutures were seeded with quantum dot (Qdot)-loaded hMSCs for 24 h before implantation. At 1 week postinfarct, the heart was imaged to assess mechanical function in the infarct region. Regional parameters assessed were regional stroke work (RSW) and systolic area of contraction (SAC) and global parameters derived from the pressure waveform. MI (n = 6) significantly decreased RSW (0.026 ± 0.011) and SAC (0.022 ± 0.015) when compared with sham operation (RSW: 0.141 ± 0.009; SAC: 0.166 ± 0.005, n = 6) (p < 0.05). The delivery of unseeded biological sutures to the infarcted hearts did not change regional mechanical function compared with the infarcted hearts (RSW: 0.032 ± 0.004, SAC: 0.037 ± 0.008, n = 6). The delivery of hMSC-seeded sutures exerted a trend toward increase of regional mechanical function compared with the infarcted heart (RSW: 0.057 ± 0.011; SAC: 0.051 ± 0.014, n = 6). Global function showed no significant differences between any group (p > 0.05); however, there was a trend toward improved function with the addition of either unseeded or seeded biological suture. Histology demonstrated that Qdot-loaded hMSCs remained present in the infarcted myocardium after 1 week. Analysis of serial sections of Masson's trichrome staining revealed that the greatest infarct size was in the infarct group (7.0% ± 2.2%), where unseeded (3.8% ± 0.6%) and hMSC-seeded (3.7% ± 0.8%) suture groups maintained similar infarct sizes. Furthermore, the remaining suture area was significantly decreased in the unseeded group compared with that in the hMSC-seeded group (p < 0.05). This study demonstrated that hMSC-seeded biological sutures are a method to deliver cells to the infarcted myocardium and have treatment potential.

Aprotinin extends mechanical integrity time of cell-seeded fibrin sutures.

Cell therapy has the potential to treat different pathologies, including myocardial infarctions (heart attacks), although cell engraftment remains elusive with most delivery methods. Biological sutures composed of fibrin have been shown to effectively deliver human mesenchymal stem cell (MSC) to infarcted hearts. However, human MSCs rapidly degrade fibrin making cell seeding and delivery time sensitive. To delay the degradation process, we propose using Aprotinin, a proteolytic enzyme inhibitor that has been shown to slow fibrinolysis. Human MSCs seeded on fibrin sutures and incubated with Aprotinin demonstrated similar cell viability, examined using a LIVE/DEAD stain, to controls. No differences in proliferation, as determined by Ki-67 presence, were observed. Human MSCs incubated in Aprotinin differentiated into adipocytes, osteocytes, and chondrocytes, confirming multipotency. The number of cells adhered to fibrin sutures increased through Aprotinin supplementation at 2, 3, and 5 day time points. Uniaxial tensile testing was used to examine the effect of Aprotinin on suture integrity. Sutures exposed to Aprotinin had higher ultimate tensile strength and modulus when compared to sutures exposed to standard growth media. Fibrin sutures incubated in Aprotinin had larger diameters and less fibrin degradation products compared to the controls, confirming decreased fibrinolysis. These data suggest that Aprotinin can reduce degradation of fibrin sutures without significant effects on MSC function, providing a novel method for extending the implantation window and increasing the number of cells delivered via fibrin sutures. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2271-2279, 2016.

Aneurysm permeability following coil embolization: packing density and coil distribution.

BACKGROUND: Rates of durable aneurysm occlusion following coil embolization vary widely, and a better understanding of coil mass mechanics is desired. The goal of this study is to evaluate the impact of packing density and coil uniformity on aneurysm permeability. METHODS: Aneurysm models were coiled using either Guglielmi detachable coils or Target coils. The permeability was assessed by taking the ratio of microspheres passing through the coil mass to those in the working fluid. Aneurysms containing coil masses were sectioned for image analysis to determine surface area fraction and coil uniformity. RESULTS: All aneurysms were coiled to a packing density of at least 27%. Packing density, surface area fraction of the dome and neck, and uniformity of the dome were significantly correlated (p<0.05). Hence, multivariate principal components-based partial least squares regression models were used to predict permeability. Similar loading vectors were obtained for packing and uniformity measures. Coil mass permeability was modeled better with the inclusion of packing and uniformity measures of the dome (r(2)=0.73) than with packing density alone (r(2)=0.45). The analysis indicates the importance of including a uniformity measure for coil distribution in the dome along with packing measures. CONCLUSIONS: A densely packed aneurysm with a high degree of coil mass uniformity will reduce permeability.