In this report, we present multiparameter deformability cytometry (m-DC), in which we explore a large set of parameters describing the physical phenotypes of pluripotent cells and their derivatives. m-DC utilizes microfluidic inertial focusing and hydrodynamic stretching of single cells in conjunction with high-speed video recording to realize high-throughput characterization of over 20 different cell motion and morphology-derived parameters. Parameters extracted from videos include size, deformability, deformation kinetics, and morphology. We train support vector machines that provide evidence that these additional physical measurements improve classification of induced pluripotent stem cells, mesenchymal stem cells, neural stem cells, and their derivatives compared to size and deformability alone. In addition, we utilize visual interactive stochastic neighbor embedding to visually map the high-dimensional physical phenotypic spaces occupied by these stem cells and their progeny and the pathways traversed during differentiation. This report demonstrates the potential of m-DC for improving understanding of physical differences that arise as cells differentiate and identifying cell subpopulations in a label-free manner. Ultimately, such approaches could broaden our understanding of subtle changes in cell phenotypes and their roles in human biology.

Standard tissue culture of adherent cells is known to poorly replicate physiology and often entails suspending cells in solution for analysis and sorting, which modulates protein expression and eliminates intercellular connections. To allow adherent culture and processing in flow, we present 3D-shaped hydrogel cell microcarriers, which are designed with a recessed nook in a first dimension to provide a tunable shear-stress shelter for cell growth, and a dumbbell shape in an orthogonal direction to allow for self-alignment in a confined flow, important for processing in flow and imaging flow cytometry. We designed a method to rapidly design, using the genetic algorithm, and manufacture the microcarriers at scale using a transient liquid molding optofluidic approach. The ability to precisely engineer the microcarriers solves fundamental challenges with shear-stress-induced cell damage during liquid-handling, and is poised to enable adherent cell culture, in-flow analysis, and sorting in a single format.

All cell types generate mechanical forces in the contexts of their single-cell or tissue-level physiological roles. Since aberrant force-generating phenotypes directly lead to diseases, cellular force-generation mechanisms are high-value targets for new therapies. We report a scalable microtechnology to embed single-cell force sensors into elastomers that seamlessly integrates with the multi-well plate format to leverage laboratory automation workflows and achieves ~100-fold improvements in throughput for single-cell force measurements. We perform highly-parallelized time-course studies investigating airway biology and show that airway smooth muscle cells isolated from fatally asthmatic patients exhibit innately greater, and more rapid force generation in response to agonist than non-diseased cells. By also simultaneously tracing agonist-induced calcium flux and contractility in the same single cells, we reveal that calcium level is ultimately a poor quantitative predictor of cellular force generation. Finally, our flexible bio-functionalization approach uniquely enabled quantification of phagocytic forces in 1,000s of individual human macrophages and revealed that initiation of this force is a digital rather than a proportional response to the proper immunogen.

Vesicle transport is a major underlying mechanism of cell communication. Inhibiting vesicle transport in brain cells results in blockage of neuronal signals, even in intact neuronal networks. Modulating intracellular vesicle transport can have a huge impact on the development of new neurotherapeutic concepts, but only if we can specifically interfere with intracellular transport patterns. Here, we propose to modulate motion of intracellular lipid vesicles in rat cortical neurons based on exogenously bioconjugated and cell internalized superparamagnetic iron oxide nanoparticles (SPIONs) within microengineered magnetic gradients on-chip. Upon application of 6–126 pN on intracellular vesicles in neuronal cells, we explored how the magnetic force stimulus impacts the motion pattern of vesicles at various intracellular locations without modulating the entire cell morphology. Altering vesicle dynamics was quantified using, mean square displacement, a caging diameter and the total traveled distance. We observed a de-acceleration of intercellular vesicle motility, while applying nanomagnetic forces to cultured neurons with SPIONs, which can be explained by a decrease in motility due to opposing magnetic force direction. Ultimately, using nanomagnetic forces inside neurons may permit us to stop the mis-sorting of intracellular organelles, proteins and cell signals, which have been associated with cellular dysfunction. Furthermore, nanomagnetic force applications will allow us to wirelessly guide axons and dendrites by exogenously using permanent magnetic field gradients.

We develop a process to surface pattern PDMS with ferromagnetic structures of varying sizes (micron to mm) and thicknesses (> 70 micron). We utilize their flexibility and magnetic reach to confer dynamic, additive properties to a variety of substrates such as coverslips and eppendorf tubes. We find these substrates can generate additional modes of magnetic droplet manipulation, and can tunably steer magnetic-cell organization.

Carcinomas contain tight junctions that can limit the penetration and therefore therapeutic efficacy of anticancer agents, especially those delivered by nano-carrier systems. The junction opener (JO) protein is a virus-derived protein that can transiently open intercellular junctions in epithelial tumors by cleaving the junction protein desmoglein-2 (DSG2). Co-administration of JO was previously shown to significantly increase the efficacy of various monoclonal antibodies and chemotherapy drugs in murine tumor models by allowing for increased intratumoral penetration of the drugs. To investigate the size-dependent effect of JO on nanocarriers, we used PEGylated gold nanoparticles (AuNPs) of two different sizes as model drugs and investigated their biodistribution following JO protein treatment. By inductively coupled plasma mass spectrometry (ICP-MS), JO was found to significantly increase bulk tumor accumulation of AuNPs of 35nm but not 120nm particles in both medium (200-300mm) and large (500-600mm) tumors. Image analysis of tumor sections corroborates this JO-mediated increase in tumor accumulation of AuNPs. Quantitative intratumoral distribution analyses show that most nanoparticles were found within 100μm of the vasculature, and that the penetration profiles of AuNPs are not significantly affected by JO treatment at the 6h timepoint.

The fractures of the orbital floor are common after craniofacial trauma. Repair with resorbable plates is a viable reconstructive option; however, there are few reports in the literature. This study describes our experience using copolymer polylactic and polyglycolic acid (PLLA/PGA) orbital reconstruction plates (LactoSorb, Lorenz Surgical, Jacksonville, FL) in 29 cases of the orbital floor fracture repair. We conducted a retrospective review of 29 orbital floor fractures at a single institution repaired through transconjunctival, preseptal dissection using PLLA/PGA plates fashioned to repair the orbital floor defect. Associated fractures included zygomaticomaxillary, LeFort, and nasoethmoid fractures. There were six patients with complications. Four patients had transient diplopia with complete resolution of symptoms within 1 year. One patient had diplopia postoperatively, but was later lost to follow-up. Two patients have had persistent enophthalmos since 1 year. In each of these cases, the floor fracture was coincident with significant panfacial or neurotrauma. We did not encounter any adverse inflammatory reactions to the implant material itself. The study concluded that orbital floor fracture repair with resorbable plates is safe, relatively easy to perform, and in the majority of cases was effective without complications. In the presence of severe orbital trauma, more rigid implant materials may be appropriate.

Microalgal biofuels and biomass have ecofriendly advantages as feedstocks. Improved understanding and utilization of microalgae require large-scale analysis of the morphological and metabolic heterogeneity within populations. Here, with Euglena gracilis as a model microalgal species, we evaluate how fluorescence- and brightfield-derived-image-based descriptors vary during environmental stress at the single-cell level. This is achieved with a new multiparameter fluorescence-imaging cytometric technique that allows the assaying of thousands of cells per experiment. We track morphological changes, including the intensity and distribution of intracellular lipid droplets, and pigment autofluorescence. The combined fluorescence-morphological analysis identifies new metrics not accessible with traditional flow cytometry, including the lipid-to-cell-area ratio (LCAR), which shows promise as an indicator of oil productivity per biomass. Single-cell metrics of lipid productivity were highly correlated ( R > 0.90, p < 0.005) with bulk oil extraction. Such chemomorphological atlases of algal species can help optimize growth conditions and selection approaches for large-scale biomass production.