GFP+ Motor Neurons: Development and in-vitro Functional Assessment on Microelectrode Arrays

Anthony NicoliniApplications Scientist

Axion BioSystemsAruna BiomedicalNeuromics (ArunA

Strategic Selling Partner)June 10, 2015

Agenda

• Background on Microelectrode Arrays– Trends in Research– Types of Data

• Working with ArunA mMNGFP+ Motorneurons– Density Optimization– Longevity in Culture– Characterization of Network Development– Stabilization of Network Phenotype

• What this means

Stem Cells as Tools and Disease Models

Tox & Animal Reduction

Phenotypic Drug Screening

*

Model complex systems in vitro* Swinney 2011

Converging Trends in Research

MEAs: Grids of Extracellular Electrodes

The microelectrode array can track individual neural action potentials.

The Maestro Advantage

• Label-free, non-invasive system enabling native cellular activity monitoring over minutes, hours, or days

• Interchangeable plate formats allow a range of assay throughput from 12- to 96-wells without hardware changes

• Industry-leading 768 electrodes provide access to network-level information and ensure high quality, reliable data

• High-speed data sampling across all electrodes (12.5 kHz) guarantees accurate temporal resolution of fast-acting electrophysiological events

Different Levels of Data

Working with mMNGFP+ Motorneurons on MEAs• Surface Coating

– PEI-laminin for adhesion and uniform monolayer development

• Dotting– Constrains cells to the array– Requires fewer cells per well

• Media changes every 2-3 days• Variations on Cell Density

Microelectrode Array

Ground Electrodes

60,000 Cells/Well

Motorneurons on 12-well MEAs

20,000 Cells/Well

Microelectrode

Neuron Cell Body

Neurites

• Cell in close contact with microelectrodes• Voltage measurement of field potentials• Neural spike detection via thresholding

Neural Spike Detection

Monitoring Culture Development

0 5 10 15 20 25 30 35 40 45 500

2

4

6

8

10

12

14

20,000 Cells/Well

0 5 10 15 20 25 30 35 40 45 500

2

4

6

8

10

12

14

40,000Cells/Well

0 5 10 15 20 25 30 35 40 45 500

2

4

6

8

10

12

14

60,000 Cells/Well

0 5 10 15 20 25 30 35 40 45 500

2

4

6

8

10

12

14

80,000 Cells/Well

Mean Firing Rate (MFR)

Hz

Days in vitro Days in vitro Days in vitro Days in vitro

N=6 for each cell density

Neural Network Connectivity

Beyond MFR

• Neural networks show synchronized bursts across channels

Neural Network Connectivity

• Visually track using live raster plots

Synchrony Height of Baseline

80,000 Cells/WellIsolated Network Bursts

Burst

Regular Network Bursting

Burst

Network Modulation by Bicuculline

Incr. Synchrony

Monitoring Culture Network Development

Synchrony

0 5 10 15 20 25 30 35 40 45 500

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30 35 40 45 500

0.2

0.4

0.6

0.8

1

0 5 10 15 20 25 30 35 40 45 500

0.2

0.4

0.6

0.8

1

Days in vitro Days in vitro Days in vitro Days in vitro

Kre

uz

Spik

e D

ista

nce

0 5 10 15 20 25 30 35 40 45 500

0.2

0.4

0.6

0.8

1

20,000 Cells/Well

40,000 Cells/Well

60,000 Cells/Well

80,000 Cells/Well

N=6 for each cell density

What does this mean?

• Drug and Toxicology Screening– New drug screening– Environmental toxicology

• Future Disease Models– Motorneuron diseases– Neuromuscular junction diseases

Applications

• Advantages of mMNGFP+ Motorneurons on MEAs• Cells become active rapidly• Consistent across wells and conditions

• Readily usable for various levels of throughput

Thank you

ArunA Biomedical

Axion BioSystems Applications Team

Questions?