Assessment of tissue viability following electroosmotic push-pull perfusion from organotypic hippocampal slice cultures.

We have developed a novel sampling technique that allows both introduction and removal of fluid from the extracellular space of living tissue. This method is based on the fluidics of push-pull perfusion but flow is driven by electroosmosis. We have applied this method to organotypic hippocampal cultures. A source capillary is inserted into the tissue and a collection capillary is in contact with the tissue surface through a thin layer of fluid. A voltage is applied across the proximal ends of source and collection capillary. In the applied field, fluid will move from source, into the tissue, and then be collected. In this process, damage to cells may occur. To understand better what sampling conditions influence damage most, we tested various sampling geometries and applied voltages, quantifying damage 16-24 h later using propidium iodide as a cell death marker. We found that damage correlates with both voltage drop and power dissipated in the tissue, but that voltage drop is a better indicator of damage when comparing models in which capillary arrangement and length are different.

Electroosmotic push-pull perfusion: description and application to qualitative analysis of the hydrolysis of exogenous galanin in organotypic hippocampal slice cultures.

We demonstrate here a method that perfuses a small region of an organotypic hippocampal culture with a solution containing an enzyme substrate, a neuropeptide. Perfusate containing hydrolysis products is continually collected and subsequently analyzed for the products of the enzymatic degradation of the peptide substrate. The driving force for perfusion is an electric field. The fused silica capillaries used as "push" and "pull" or "source" and "collection" capillaries have a ζ-potential that is negative and greater in magnitude than the tissue's ζ-potential. Thus, depending on the magnitudes of particular dimensions, the electroosmotic flow in the capillaries augments the fluid velocity in the tissue. The flow rate is not directly measured; however, we determine it using a finite-element approach. We have determined the collection efficiency of the system using an all d-amino acid internal standard. The flow rates are low, in the nL/min range, and adjustable by controlling the current or voltage in the system. The collection efficiency of the d-amino acid peptide internal standard is variable, increasing with increased current and thus electroosmotic flow rate. The collection efficiency can be rationalized in the context of a Peclet number. Electroosmotic push-pull perfusion of the neuropeptide galanin (gal1-29) through the extracellular space of an organotypic hippocampal culture results in its hydrolysis by ectopeptidase reactions occurring in the extracellular space. The products of hydrolysis were identified by MALDI-MS. Experiments at two levels of current (8-12 µA and 19-40 µA) show that the probability of seeing hydrolysis products (apparently from aminopeptidases) is greater in the Cornu Ammonis area 3 (CA3) than in the Cornu Ammonis area 1 (CA1) in the higher current experiments. In the lower current experiments, shorter peptide products of aminopeptidases (gal13-29 to gal20-19) are seen with greater frequency in CA3 than in CA1 but there is no statistically significant difference for longer peptides (gal3-29 to gal12-29).

A simple method for measuring organotypic tissue slice culture thickness.

This paper presents a simple method to measure tissue slice thicknesses using an ohmmeter. The circuit described here is composed of a metal probe, an ohmmeter, a counter electrode, culture medium or physiological buffer, and tissue slice. The probe and the electrode are on opposite interfaces of an organotypic hippocampal slice culture. The circuit closes when the metal probe makes contact with the surface of the tissue slice. The probe position is recorded and compared to its position when it makes contact with the insert membrane on which the tissue grows, thus yielding a thickness measurement. The method does not reduce the viability of slice cultures. Thicknesses of the slice cultures were measured under a number of culturing protocols. An initial drop in thickness occurred between 0 and 4 days in culture. Thicknesses are rather constant thereafter. The type of culture medium and the initial thickness of the tissue explant influence the thickness. Slice thicknesses were compared to a known technique by using optical measurements of slice cross-sections to obtain thicknesses. In contrast to this known technique, the proposed method does not sacrifice the slice culture for measurement purposes. The proposed measurement technique described is straightforward and rapid, about 1 min per culture.