Incubator-independent cell-culture perfusion platform for continuous long-term microelectrode array electrophysiology and time-lapse imaging.

Dirk Saalfrank,Rouhollah Habibey,Shahrzad Latifi,Aurel Vasile Martiniuc,Alois Knoll,Anil Krishna Konduri,Axel Blau,Asiyeh Golabchi,Sven Ingebrandt

doi:10.1098/rsos.150031

Abstract

Most in vitro electrophysiology studies extract information and draw conclusions from representative, temporally limited snapshot experiments. This approach bears the risk of missing decisive moments that may make a difference in our understanding of physiological events. This feasibility study presents a simple benchtop cell-culture perfusion system adapted to commercial microelectrode arrays (MEAs), multichannel electrophysiology equipment and common inverted microscopy stages for simultaneous and uninterrupted extracellular electrophysiology and time-lapse imaging at ambient CO2 levels. The concept relies on a transparent, replica-casted polydimethylsiloxane perfusion cap, gravity- or syringe-pump-driven perfusion and preconditioning of pH-buffered serum-free cell-culture medium to ambient CO2 levels at physiological temperatures. The low-cost microfluidic in vitro enabling platform, which allows us to image cultures immediately after cell plating, is easy to reproduce and is adaptable to the geometries of different cell-culture containers. It permits the continuous and simultaneous multimodal long-term acquisition or manipulation of optical and electrophysiological parameter sets, thereby considerably widening the range of experimental possibilities. Two exemplary proof-of-concept long-term MEA studies on hippocampal networks illustrate system performance. Continuous extracellular recordings over a period of up to 70 days revealed details on both sudden and gradual neural activity changes in maturing cell ensembles with large intra-day fluctuations. Correlated time-lapse imaging unveiled rather static macroscopic network architectures with previously unreported local morphological oscillations on the timescale of minutes.

Highlights

Unlike cells within living organisms, cells in vitro lack a supporting body infrastructure
Perfusion cap templates were designed (Alibre Design) to fit the standard outer diameter (OD) of 24 mm glass or polymer rings that are usually found on commercial microelectrode arrays (MEAs)
Part 3 defined the inner cap diameter, which equalled the OD of the culturing dish; in this case, this was the OD of the Ø 24 mm glass ring

Summary

Introduction

Unlike cells within living organisms, cells in vitro lack a supporting body infrastructure They are not protected by the immune system or other basic system-wide regulatory mechanisms that control the temperature, pH and the turnover of nutrients, metabolites and signalling factors. An incubator-independent and automated perfusion system would stabilize physiological conditions on the experimental set-up and allow for the functional separation of environmental variability from physiological fluctuations. The majority lack an automated medium perfusion function This shortcoming is addressed by a plethora of cell-culture perfusion and microchannel devices in different experimental contexts, as recently reviewed in general [8,9,10,11,12,13,14] and with a special focus on applications in neuroscience [15,16,17]. In combination with a chemically buffered medium and a temperature controller, this platform allows for unsupervised multimodal long-term imaging and extracellular electrophysiology studies at ambient conditions immediately after cell seeding that are unbiased by physical and chemical handling artefacts

Methods

Results

Discussion

Conclusion