Microfluidics and multielectrode array-compatible organotypic slice culture method

Abstract

Organotypic brain slice cultures are used for a variety of molecular, electrophysiological, and imaging studies. However, the existing culture methods are difficult or expensive to apply in studies requiring long-term recordings with multielectrode arrays (MEAs). In this work, a novel method to maintain organotypic cultures of rodent hippocampus for several weeks on standard MEAs in an unmodified tissue culture incubator is described. Polydimethylsiloxane (Sylgard) mini-wells were used to stabilize organotypic cultures on glass and MEA surfaces. Hippocampus slices were successfully maintained within PDMS mini-wells for multiple weeks, with preserved pyramidal layer organization, connectivity, and activity. MEAs were used to record the development of spontaneous activity in an organotypic cultures for 4 weeks. This method is compatible with integration of microchannels into the culture substrate. Microchannels were incorporated into the mini-wells and applied to the guidance of axons originating within the slice, paving the way for studies of axonal sprouting using organotypic slices.

Figure 1

Left: fabrication sequence for creating PDMS mini-wells. Top right: schematic representation of the hippocampus slice in a PDMS mini-well. Bottom right, micrograph of an organotypic hippocampus slice in a mini-well, 7 DIV

Figure 2

A: Nissl-stained 20 μm cryosection of DIV 7 hippocampus slice. B: Evoked activity, arrow shows the stimulus artifact, left: stimulus electrode in DG, recording electrode in CA3, right: stimulus electrode in CA3, recording electrode in CA1. C: spontaneous activity in CA1, D: short and long spontaneous bursts in CA1. Recordings in B-D were carried out with glass electrodes.

Figure 3

A: Hippocampus slice with neurite extension at 3 DIV, cultured on a polylysine-coated surface. B: calcein staining of neurite outgrowth from the slice (bright region on the left), 7 DIV, C: calcein staining of microchannel-guided axons, 7 DIV, D: axons extended by a hippocampus slice enter the microchannels, 10 DIV, E: axons span the length of the microchannels and emerge on the other side of the PDMS mini-well, 10 DIV.

Figure 4

Left: hippocampus organotypic slice (20 DIV) on the MEA, Right: spontaneous population bursts recorded at different time points with the same recording electrode.