Tunnel Culture System to Examine Cell-Cell Interaction on Microelectrode Array
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1
ETH Zurich, Department of Biosystems Science and Engineering, Switzerland
Motivation: Synaptic, dendritic, and axonal signals are the mechanism for central nervous system communication over long and short distances. Synaptic and dendritic signal have been extensively studied, however, electrophysiological investigation of axons remains a challenge. In an effort to advance the common neurobiological tool set, our lab previously developed a high-density microelectrode array (MEA) based on CMOS technology, featuring 26,400 recording sites. Such high resolution allows efficient investigation of electrical conduction with multiple measurement sites along a single axon. When culturing neurons, axons tend to grow in an undefined pattern and electrical signals must be followed precisely to identify the location of each neuron. Additionally, when axons overlap it is difficult to identify which soma is communicating through each axon. Tunnel systems have been designed previously that reduce the noise from surrounding culture conditions, however they tend to provide structures that allow multiple axons to grow in the same area [1, 2].
The goal of this project is to develop a system to culture multiple cell types on a MEA to examine the effects of cell-cell interaction on signal conduction. To achieve this goal, we have developed microfluidic tunnel systems and cultured neurons in this device on coverslips. This in vitro platform will allow researchers to examine multi-cell diseases, test pharmaceutical effects on neurons, and measure real-time changes in neuronal activity.
Materials and Methods:
Design of the MEA1k was published previously [3]. The CMOS array includes 26,400 electrodes, with 1024 simultaneous recording channels. The active array is on a 3.85 x 2.10mm area and each electrode measures 9.3 x 5.45um with a pitch of 17.5um. All electrodes, amplification, signal conditioning, analog-to-digital conversion, and simulation buffers are housed on the chip.
To create the PDMS structures, SU-8 molds were created using standard soft-lithography techniques. First, masks were designed in AutoCAD to introduce tunnels and culture well structures with care to create tunnel spacing not as a multiple of the MEA pitch. SU-8 3005 (MicroChem USA) was spin-coated on a 4-inch silicon wafer for 30 seconds at 6000rpm for 4um thickness. The SU-8 was soft baked at 95°C before being exposed to UV on a mask aligner (Suss MicroTec AG, Germany) with a chrome mask. Post-exposure bake and development were completed before spin-coating the next layers. Center channels to introduce secondary cell types were created with SU-8 100 spin-coated at 1600rpm for 30 seconds to create 200um tall structures, the wafers were soft-baked, and exposed to UV before spin-coating the second layer of SU-8 at 100rpm for 1 minute to create a total thickness of ~500um for the tall culture wells. The wafer was soft baked, exposed, hard baked, and developed upside down for 40 minutes.
PDMS (Sylgard 184, Dow Corning) was poured onto the wafers and allowed to rest for one hour to facilitate redistribution of the PDMS over all structures. PDMS on the wafer was degassed and cured at 80°C for one hour. Any remaining PDMS films over the tall structures were removed. The PDMS structures were cut to create individual devices, rinsed with isopropanol, and blown dry with nitrogen gas. PDMS structures were placed on the coverslips with tweezers and pressed gently to ensure contact with the glass.
All animal work was conducted with permission from the Basel-City Cantonal Veterinary Authority under license 2358. Primary cortical neurons were isolated from Wistar rat embryos (E18-19) in accordance to Swiss federal laws on animal welfare. Timed pregnant rats (Charles River, France), were anesthetized using isoflurane and immediately euthanized by decapitation. Embryos were removed and euthanized by decapitation. Cortices were isolated in Hanks Balanced Salt Solution and dissociated chemically in 0.25% trypsin for 20 minutes. Tissue was moved to serum-containing medium and mechanically dissociated by trituration. Neurons were cultured in Neurobasal medium (Invitrogen) supplemented with horse serum (Hyclone), GlutaMAX (Invitrogen), and B27 (Invitrogen) for 48 hours before medium was replaced with Dulbecco's Modified Eagle Medium (Invitrogen) supplemented with horse serum, GlutaMAX, and sodium pyruvate (Invitrogen). Medium was changed once per week and cells were cultured in humidified incubator with 5% CO2 at 37°C.
After two weeks, cells were fixed with 4% paraformaldehyde and stained to visualize neurons. Cells were incubated with primary antibody anti-b-III tubulin (abcam) overnight, rinsed in PBS, then incubated at room temperature for one hour in secondary antibody (abcam) and Hoechst before being rinsed again in PBS prior to mounting the coverslip with tunnels onto a concave slide for imaging. Images were obtained using a Leica confocal microscope.
Results: Culture wells with tunnels connecting them were developed with 4um tall channels, 200um tall middle wells, and 500um tall culture wells open to culture medium. Neurons were successfully cultured in these devices and axons grew into the tunnels.
Discussion: These culture platforms can be used to introduce many different cell types and create a controlled network. Neurons can be cultured on one side of the tunnels with oligodendrocytes, Schwann cells, astrocytes, or other cell types introduced in the center channels and wells after axons are present. These systems can be used to examine neuronal response to in vitro disease models, pharmaceutical intervention, and cell invasion models.
Conclusion: These culture platforms will be introduced to the MEA1k system to examine neuronal responses to different conditions.
References:
[1] Lewandowska MK, Bakkum DJ, Rompani SB, Hierlemann A. Recording Large Extracellular Spikes in Microchannels along Many Axonal Sites from Individual Neurons. Plos One. 2015;10.
[2] FitzGerald JJ, Lacour SP, McMahon SB, Fawcett JW. Microchannels as axonal amplifiers. Ieee T Bio-Med Eng. 2008;55:1136-46.
[3] Ballini M, Muller J, Livi P, Chen YH, Frey U, Stettler A, et al. A 1024-Channel CMOS Microelectrode Array With 26,400 Electrodes for Recording and Stimulation of Electrogenic Cells In Vitro. Ieee J Solid-St Circ. 2014;49:2705-19.
Acknowledgements
Financial support through the ERC Advanced Grant 267351 "NeuroCMOS", Whitaker Foundation Scholars Program (SAG). Olivier Frey and Jan Muller assisted with training.
Keywords:
Neural Network,
cell-cell interaction,
Directed axonal growth,
axonal sign,
microelectrode array
Conference:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays, Reutlingen, Germany, 28 Jun - 1 Jul, 2016.
Presentation Type:
Poster Presentation
Topic:
MEA Meeting 2016
Citation:
Geissler
S and
Hierlemann
A
(2016). Tunnel Culture System to Examine Cell-Cell Interaction on Microelectrode Array.
Front. Neurosci.
Conference Abstract:
MEA Meeting 2016 |
10th International Meeting on Substrate-Integrated Electrode Arrays.
doi: 10.3389/conf.fnins.2016.93.00003
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Received:
22 Jun 2016;
Published Online:
24 Jun 2016.
*
Correspondence:
Dr. Sydney Geissler, ETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland, sydney.geissler@bsse.ethz.ch