Investigation of human CNS disease and drug effects has been hampered by the lack of a system that enables single cell analysis on live adult patient brain cells. We developed a culturing system, based on a papain-aided procedure, for resected adult human brain tissue removed during neurosurgery. We performed single-cell transcriptomics on over 300 cells permitting identification of oligodendrocytes, microglia, neurons, endothelial cells, and astrocytes after 3 weeks in culture. Using deep sequencing, we detected over 12,000 expressed genes including hundreds of cell-type enriched mRNAs, lncRNAs and pri-miRNAs. We describe cell-type and patient specific transcriptional hierarchies. Single-cell transcriptomics on cultured live adult patient derived cells is a prime example of the promise of personalized precision medicine. As these cells derive from subjects ranging in age into their sixties, this system permits human aging studies previously possible only in rodent systems.

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Skin has a highly regulated pH environment of around pH 7.2 but with an acid barrier mantle of around pH 5.5. Trauma, inflammation, and infection are all thought to disrupt this pH environment but the lack of a non-invasive technique to measure pH within discrete locations within skin has hindered investigating what role pH plays in wound healing. In this study, a confocal Raman microspectroscopy method was used for measuring pH in a 3D tissue engineered model of human skin (TE-skin) and evaluated for its ability to detect changes in pH in response to wounding, inflammation and bacterial infection. The state of protonation of phosphate groups within the TE-skin was used to indicate pH in a non-destructive manner exploring depths of skin from the stratum corneum to 600 microns into the dermis. Deliberate wounding or inflammation (induced by IL-17) resulted in a loss of the acid mantle. Detailed scanning of TE-skin infected with Staphylococcus aureus or Pseudomonas aeruginosa revealed heterogeneous pH microenvironments ranging in size from 10 Â 10 to 50 Â 100 microns and ranging from pH 5 to 9. These microenvironments were not detected if an average pH for the TEskin model was used.

Many cancer treatments rely on inhibition of epidermal growth factor (EGF)-induced cellular responses. Evaluating drug effects on such responses becomes critical to the development of new cancer therapeutics. In this report, we have employed a label-free acoustic sensor, the quartz crystal microbalance with dissipation monitoring (QCM-D), to track the EGF-induced response of mutant MCF10A cells under various inhibitory conditions. We have identified a complex cell de-adhesion process, which can be distinctly altered by inhibitors of signaling pathways and cytoskeleton formation in a dose-dependent manner. The dose dependencies of the inhibitors provide IC50 values which are in strong agreement with the values reported in the literature, demonstrating the sensitivity and reliability of the QCM-D as a screening tool. Using immunofluorescence imaging, we have also verified the quantitative relationship between the ΔD-response (change in energy dissipation factor) and the level of focal adhesions quantified with the areal density of immunostained vinculin under those inhibitory conditions. Such a correlation suggests that the dynamic restructuring of focal adhesions can be assessed based on the time-dependent change in ΔD-response. Overall, this report has shown that the QCM-D has the potential to become an effective sensing platform for screening therapeutic agents that target signaling and cytoskeletal proteins.

Epidermal growth factor receptor (EGFR) plays a major role in cell migration and invasion and is considered to be the primary source of activation of various malignant tumors. To gain insight into how elevated levels of EGFR influence cellular function, particularly cell motility, we used a quartz crystal microbalance with dissipation monitoring (QCM-D) to examine restructuring of focal adhesions in MCF-10A cells induced by epidermal growth factor. Engineered cells that overexpress epidermal growth factor receptor (EGFR) exhibited a very different kinetic profile from wildtype MCF-10A cells that have a lower level of EGFR with a higher rate for the initial disassembly of focal adhesion and a much lower rate for the later reassembly of focal adhesions. It is conceivable that these effects exhibited by EGFR-overexpressing cells may promote the initiation and maintenance of a more favorable adhesion state for cell migration. This study has demonstrated the capability of the dissipation monitoring function of the QCM-D to quantitatively assess kinetic aspects of cellular processes with a high temporal resolution and sensitivity.