Anionic polymers amplify electrokinetic perfusion through extracellular matrices.

Electrical stimulation (ES) promotes healing of chronic epidermal wounds and delays degeneration of articular cartilage. Despite electrotherapeutic treatment of these non-excitable tissues, the mechanisms by which ES promotes repair are unknown. We hypothesize that a beneficial role of ES is dependent on electrokinetic perfusion in the extracellular space and that it mimics the effects of interstitial flow. In vivo, the extracellular space contains mixtures of extracellular proteins and negatively charged glycosaminoglycans and proteoglycans surrounding cells. While these anionic macromolecules promote water retention and increase mechanical support under compression, in the presence of ES they should also enhance electro-osmotic flow (EOF) to a greater extent than proteins alone. To test this hypothesis, we compare EOF rates between artificial matrices of gelatin (denatured collagen) with matrices of gelatin mixed with anionic polymers to mimic endogenous charged macromolecules. We report that addition of anionic polymers amplifies EOF and that a matrix comprised of 0.5% polyacrylate and 1.5% gelatin generates EOF with similar rates to those reported in cartilage. The enhanced EOF reduces mortality of cells at lower applied voltage compared to gelatin matrices alone. We also use modeling to describe the range of thermal changes that occur during these electrokinetic experiments and during electrokinetic perfusion of soft tissues. We conclude that the negative charge density of native extracellular matrices promotes electrokinetic perfusion during electrical therapies in soft tissues and may promote survival of artificial tissues and organs prior to vascularization and during transplantation.

Electrokinetic Perfusion Through Three-Dimensional Culture Reduces Cell Mortality.

Cell proliferation and survival are dependent on mass transfer. In vivo, fluid flow promotes mass transfer through the vasculature and interstitial space, providing a continuous supply of nutrients and removal of cellular waste products. In the absence of sufficient flow, mass transfer is limited by diffusion and poses significant challenges to cell survival during tissue engineering, tissue transplantation, and treatment of degenerative diseases. Artificial perfusion may overcome these challenges. In this work, we compare the efficacy of pressure driven perfusion (PDP) with electrokinetic perfusion (EKP) toward reducing cell mortality in three-dimensional cultures of Matrigel extracellular matrix. We characterize electro-osmotic flow through Matrigel to identify conditions that generate similar interstitial flow rates to those induced by pressure. We also compare changes in cell mortality induced by continuous or pulsed EKP. We report that continuous EKP significantly reduced mortality throughout the perfusion channels more consistently than PDP at similar flow rates, and pulsed EKP decreased mortality just as effectively as continuous EKP. We conclude that EKP has significant advantages over PDP for promoting tissue survival before neovascularization and angiogenesis. Impact statement Interstitial flow helps promote mass transfer and cell survival in tissues and organs. This study generated interstitial flow using pressure driven perfusion (PDP) or electrokinetic perfusion (EKP) to promote cell viability in three-dimensional cultures. EKP through charged extracellular matrices possesses significant advantages over PDP and may promote cell survival during tissue engineering, transplantations, and treatment of degenerative diseases.

Electromigration of cell surface macromolecules in DC electric fields during cell polarization and galvanotaxis.

DC electric fields (EFs) can often induce cellular polarity, and direct migration of cells toward one of the electrical poles. The mechanism(s) by which cells sense weak EFs is not established. We present here a molecular flux model to describe electromigration of plasma membrane macromolecules and compare its predictions to electromigration of a lipid-anchored surface protein, tdTomato-GPI, under different experimental conditions. Gradients of tdTomato-GPI are assembled based on its electrophoretic and electro-osmotic mobilities and collapsed by its own diffusion. The flux model predicts greatest cathodal accumulation for tdTomato-GPI under slightly acidic conditions, and weak cathodal accumulation under alkaline conditions. Predictions by the flux model align closely with measurements of the electromigration of tdTomato-GPI except at pH 6, the only condition examined in which the protein exhibits a net positive surface charge. We use the model to predict the time course and relative steady state concentration difference for asymmetric accumulation of other surface macromolecules based on their physical properties. We also describe a method for identifying the physical properties of the plasma membrane proteins in zebrafish keratocytes, in order to predict likely candidates for the electric field receptor in this model migratory system that exhibits cathodal galvanotaxis, and to predict the asymmetric distribution of proteins in other cell types. We provide a physical basis for predicting the dynamics of electromigration for numerous cell surface macromolecules and provide evidence for supporting the role of electromigration in directing cell polarity, migration and growth in response to weak EFs.

Advances in Electrochemistry for Monitoring Cellular Chemical Flux.

The transport of molecules and inorganic ions across the plasma membrane results in chemical fluxes that reflect cellular function in healthy and diseased states. Measurement of these chemical fluxes enables the characterization of protein function and transporter stoichiometry, characterization of the viability of single cells and embryos prior to implantation, and screening of pharmaceutical agents. Electrochemical sensors are sensitive and noninvasive tools for measuring chemical fluxes immediately outside the cells in the boundary layer, that are capable of monitoring a diverse range of transported analytes including inorganic ions, gases, neurotransmitters, hormones, and pharmaceutical agents. Used on their own or in combination with other methods, these sensors continue to expand our understanding of the function of rare cells and small tissues. Advances in sensor construction and detection strategies continue to improve sensitivity under physiological conditions, diversify analyte detection, and increase throughput. These advances will be discussed in the context of addressing technical challenges to measuring in the boundary layer of cells and measuring the resultant changes to the chemical concentration in the bulk media.

Reversing the direction of galvanotaxis with controlled increases in boundary layer viscosity.

Weak external electric fields (EFs) polarize cellular structure and direct most migrating cells (galvanotaxis) toward the cathode, making it a useful tool during tissue engineering and for healing epidermal wounds. However, the biophysical mechanisms for sensing weak EFs remain elusive. We have reinvestigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells, by reducing it with neutral, viscous polymers. We report that increasing viscosity with low molecular weight polymers decreases cathodal migration and promotes anodal migration in a concentration dependent manner. In contrast, increased viscosity with high molecular weight polymers does not affect directionality. We explain the contradictory results in terms of porosity and hydraulic permeability between the polymers rather than in terms of bulk viscosity. These results provide the first evidence for controlled reversal of galvanotaxis using viscous agents and position the field closer to identifying the putative electric field receptor, a fundamental, outside-in signaling receptor that controls cellular polarity for different cell types.