Stochastic electrotransport selectively enhances the transport of highly electromobile molecules

Nondestructive chemical processing of porous samples such as fixed biological tissues typically relies on molecular diffusion. Diffusion into a porous structure is a slow process that significantly delays completion of chemical processing. Here, we present a novel electrokinetic method termed stochastic electrotransport for rapid nondestructive processing of porous samples. This method uses a rotational electric field to selectively disperse highly electromobile molecules throughout a porous sample without displacing the low-electromobility molecules that constitute the sample. Using computational models, we show that stochastic electrotransport can rapidly disperse electromobile molecules in a porous medium. We apply this method to completely clear mouse organs within 1–3 days and to stain them with nuclear dyes, proteins, and antibodies within 1 day. Our results demonstrate the potential of stochastic electrotransport to process large and dense tissue samples that were previously infeasible in time when relying on diffusion.Simons Foundation. Postdoctoral FellowshipLife Sciences Research FoundationBurroughs Wellcome Fund (Career Awards at the Scientific Interface)Searle Scholars ProgramMichael J. Fox Foundation for Parkinson's ResearchUnited States. Defense Advanced Research Projects AgencyJPB FoundationNational Institutes of Health (U.S.)National Institutes of Health (U.S.) (Grant 1-U01-NS090473-01

In situ expansion of engineered human liver tissue in a mouse model of chronic liver disease

Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. Control of both tissue architecture and scale is a fundamental translational roadblock in tissue engineering. An experimental framework that enables investigation into how architecture and scaling may be coupled is needed. We fabricated a structurally organized engineered tissue unit that expanded in response to regenerative cues after implantation into mice with liver injury. Specifically, we found that tissues containing patterned human primary hepatocytes, endothelial cells, and stromal cells in a degradable hydrogel expanded more than 50-fold over the course of 11 weeks in mice with injured livers. There was a concomitant increase in graft function as indicated by the production of multiple human liver proteins. Histologically, we observed the emergence of characteristic liver stereotypical microstructures mediated by coordinated growth of hepatocytes in close juxtaposition with a perfused vasculature. We demonstrated the utility of this system for probing the impact of multicellular geometric architecture on tissue expansion in response to liver injury. This approach is a hybrid strategy that harnesses both biology and engineering to more efficiently deploy a limited cell mass after implantation.NIH (Grants R01EB008396, R01DK85713, EB00262, and U24DK059635, P30-CA14051)National Institute of Environmental Health Sciences (Grant P30-ES002109)NIGMS Training (Grant T32GM007753

Protection of tissue physicochemical properties using polyfunctional crosslinkers

Understanding complex biological systems requires the system-wide characterization of both molecular and cellular features. Existing methods for spatial mapping of biomolecules in intact tissues suffer from information loss caused by degradation and tissue damage. We report a tissue transformation strategy named stabilization under harsh conditions via intramolecular epoxide linkages to prevent degradation (SHIELD), which uses a flexible polyepoxide to form controlled intra-and intermolecular cross-link with biomolecules. SHIELD preserves protein fluorescence and antigenicity, transcripts and tissue architecture under a wide range of harsh conditions. We applied SHIELD to interrogate system-level wiring, synaptic architecture, and molecular features of virally labeled neurons and their targets in mouse at single-cell resolution. We also demonstrated rapid three-dimensional phenotyping of core needle biopsies and human brain cells. SHIELD enables rapid, multiscale, integrated molecular phenotyping of both animal and clinical tissues.NIH (Grant 1-DP2-ES027992)NIMH (Grants R01MH107742, R01MH108594, U01MH114829