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Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.

Kanagasabapathi TT, Ciliberti D, Martinoia S, Wadman WJ, Decré MM - Front Neuroeng (2011)

Bottom Line: Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated.The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining.In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques.

View Article: PubMed Central - PubMed

Affiliation: Minimally Invasive Healthcare Department, Philips Research Laboratories Eindhoven, Netherlands.

ABSTRACT
We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

No MeSH data available.


Related in: MedlinePlus

(A) Schematic layout of the dual-compartment device; (B) Planar MEA substrate with dual-compartment PDMS device; (C) Spontaneous activity of a culture on DIV 14 (the electrode number along the y-axis run from 11 through 88 with the first digit representing column and the second digit its row respectively. Each dot represents an action potential recorded by one of the MEA channels); (D) Typical cortical cell culture in a compartment on DIV 4.
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Figure 1: (A) Schematic layout of the dual-compartment device; (B) Planar MEA substrate with dual-compartment PDMS device; (C) Spontaneous activity of a culture on DIV 14 (the electrode number along the y-axis run from 11 through 88 with the first digit representing column and the second digit its row respectively. Each dot represents an action potential recorded by one of the MEA channels); (D) Typical cortical cell culture in a compartment on DIV 4.

Mentions: The 3-mm-thick polydimethylsiloxane (PDMS) devices used for this study have two microfluidic compartments of 100 μm height, 1.5 mm width, and 8 mm length interconnected with microchannels of 10 μm width, 3 μm height, and 150 μm length spaced at regular intervals of 60 μm (Figure 1A; Taylor et al., 2003). The small size of microchannels prevents migration of cells between compartments while allowing only neurites to pass through (Taylor et al., 2003). Conventional soft lithographic techniques as pioneered by Whitesides and his collaborators (McDonald et al., 2000; McDonald and Whitesides, 2002) were utilized in the fabrication of the device. PDMS stamps were replicated using Si master molds and the fabrication techniques were described in an earlier work (Kanagasabapathi et al., 2009). Four 6-mm-diameter reservoir holes were laser cut into the fabricated device. The fabricated PDMS stamps were rinsed thoroughly in an ultrasonic bath, stored in Milli-Q water for 24 h and decontaminated in 70% ethanol. Each PDMS device was then sterilized in a dry oven at 120°C for 20 min. To selectively hydrophilize the microcompartments and the microchannels, the PDMS devices were placed on cover-slips with the microchannels facing downward for oxygen–plasma treatment (Owen and Smith, 1994; Katzenberg, 2005; Bodas and Khan-Malek, 2007). This treatment renders the microfluidic compartments and the microchannels hydrophilic, while preserving hydrophobicity of the contact surface, thereby preventing leakage. The PDMS devices were then reversibly bonded onto a commercially available planar microelectrode array (Multichannel Systems, Germany) with 60 electrodes of 30 μm diameter and 200 μm spacing placed in an 8 × 8 array (no corner electrodes) as shown in Figure 1B. While bonding, PDMS device was aligned on the MEA to include 30 electrodes each per compartment. Prior to the placement of the PDMS devices, MEAs were sterilized in a vacuum oven, coated overnight with a solution of Polyethylenimine (PEI; Sigma-Aldrich, The Netherlands) at a concentration of 40 μg/ml and rinsed thoroughly in sterile water (GIBCO, Invitrogen, CA, USA) on the day of culture.


Dual-compartment neurofluidic system for electrophysiological measurements in physically segregated and functionally connected neuronal cell culture.

Kanagasabapathi TT, Ciliberti D, Martinoia S, Wadman WJ, Decré MM - Front Neuroeng (2011)

(A) Schematic layout of the dual-compartment device; (B) Planar MEA substrate with dual-compartment PDMS device; (C) Spontaneous activity of a culture on DIV 14 (the electrode number along the y-axis run from 11 through 88 with the first digit representing column and the second digit its row respectively. Each dot represents an action potential recorded by one of the MEA channels); (D) Typical cortical cell culture in a compartment on DIV 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198030&req=5

Figure 1: (A) Schematic layout of the dual-compartment device; (B) Planar MEA substrate with dual-compartment PDMS device; (C) Spontaneous activity of a culture on DIV 14 (the electrode number along the y-axis run from 11 through 88 with the first digit representing column and the second digit its row respectively. Each dot represents an action potential recorded by one of the MEA channels); (D) Typical cortical cell culture in a compartment on DIV 4.
Mentions: The 3-mm-thick polydimethylsiloxane (PDMS) devices used for this study have two microfluidic compartments of 100 μm height, 1.5 mm width, and 8 mm length interconnected with microchannels of 10 μm width, 3 μm height, and 150 μm length spaced at regular intervals of 60 μm (Figure 1A; Taylor et al., 2003). The small size of microchannels prevents migration of cells between compartments while allowing only neurites to pass through (Taylor et al., 2003). Conventional soft lithographic techniques as pioneered by Whitesides and his collaborators (McDonald et al., 2000; McDonald and Whitesides, 2002) were utilized in the fabrication of the device. PDMS stamps were replicated using Si master molds and the fabrication techniques were described in an earlier work (Kanagasabapathi et al., 2009). Four 6-mm-diameter reservoir holes were laser cut into the fabricated device. The fabricated PDMS stamps were rinsed thoroughly in an ultrasonic bath, stored in Milli-Q water for 24 h and decontaminated in 70% ethanol. Each PDMS device was then sterilized in a dry oven at 120°C for 20 min. To selectively hydrophilize the microcompartments and the microchannels, the PDMS devices were placed on cover-slips with the microchannels facing downward for oxygen–plasma treatment (Owen and Smith, 1994; Katzenberg, 2005; Bodas and Khan-Malek, 2007). This treatment renders the microfluidic compartments and the microchannels hydrophilic, while preserving hydrophobicity of the contact surface, thereby preventing leakage. The PDMS devices were then reversibly bonded onto a commercially available planar microelectrode array (Multichannel Systems, Germany) with 60 electrodes of 30 μm diameter and 200 μm spacing placed in an 8 × 8 array (no corner electrodes) as shown in Figure 1B. While bonding, PDMS device was aligned on the MEA to include 30 electrodes each per compartment. Prior to the placement of the PDMS devices, MEAs were sterilized in a vacuum oven, coated overnight with a solution of Polyethylenimine (PEI; Sigma-Aldrich, The Netherlands) at a concentration of 40 μg/ml and rinsed thoroughly in sterile water (GIBCO, Invitrogen, CA, USA) on the day of culture.

Bottom Line: Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated.The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining.In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques.

View Article: PubMed Central - PubMed

Affiliation: Minimally Invasive Healthcare Department, Philips Research Laboratories Eindhoven, Netherlands.

ABSTRACT
We developed a dual-compartment neurofluidic system with inter-connecting microchannels to connect neurons from their respective compartments, placed on a planar microelectrode arrays. The design and development of the compartmented microfluidic device for neuronal cell culture, protocol for sustaining long-term cultures, and neurite growth through microchannels in such a closed compartment device are presented. Using electrophysiological measurements of spontaneous network activity in the compartments and selective pharmacological manipulation of cells in one compartment, the biological origin of network activity and the fluidic isolation between the compartments are demonstrated. The connectivity between neuronal populations via the microchannels and the crossing-over of neurites are verified using transfection experiments and immunofluorescence staining. In addition to the neurite cross-over to the adjacent compartment, functional connectivity between cells in both the compartments is verified using cross-correlation (CC) based techniques. Bidirectional signal propagation between the compartments is demonstrated using functional connectivity maps. CC analysis and connectivity maps demonstrate that the two neuronal populations are not only functionally connected within each compartment but also with each other and a well connected functional network was formed between the compartments despite the physical barrier introduced by the microchannels.

No MeSH data available.


Related in: MedlinePlus