Yet, the culture time needed for the complete functional maturation of individual neurons and networks is unpredictable. The progress of spontaneous electrophysiological activity and pharmacological responses was examined for more than one year in culture using multi-electrode arrays (MEAs). About 20–30 weeks were needed for the full maturation of spontaneous firing, evoked responses, and modulation of activity by glutamatergic and GABAergic receptor antagonists/agonists. During this phase, neural networks also exhibited epileptiform synchronized burst firing (SBF) as a result of pro-convulsants and SBF suppression using clinical anti-epilepsy drugs.
The outcomes exhibit the feasibility of long-term MEA measurements from hiPSC-derived neuronal networks in vitro for drug screening and mechanistic analyses. However, developmental variations in pharmacological and electrophysiological properties represent the need for the international standardization of culture and evaluation procedures.
Material and
Methods
Human iPSC-Derived Cerebral Cortical Neurons
(Axol Bioscience Inc.)(1)
·
Using astrocyte co-culture method, (2) long-term culture of
hiPSC-derived neurons were carried out for more than 300 days.
Multi-Electrode
Array System (Alpha MED Scientific Inc.)
·
The
long-term electrophysiological characteristics and drug effects of
hiPSC-derived neurons were assessed using a planar MEA measurement system
(Alpha MED Scientific Inc., Japan). The MEA chips consist of 64 electrodes
(MED-P515A) with high S/N ratio and low impedance.
·
Spike
analyses were carried out using MATLAB and Mobius software (Alpha MED
Scientific).
Result 1: Development of
Spontaneous Firing During Long-Term Culture
Figure 2. (A) Changes in
spontaneous firing pattern in the same long-term culture at 7, 14, 29, and 34
weeks in vitro (WIV). (a) Typical spontaneous firing patterns. (b) Raster plots
of the array-wide spike detection rate (AWSDR, spikes/second). Bin size is 1
ms. (c) Corresponding raster plots for all 64 electrodes over 5 minutes. (B)
Electrode grids color-coded to indicate mean spontaneous firing frequency from
same culture at 2, 6, 14, 20, 26, and 34 WIV. Red indicates electrodes with
higher firing frequencies. Scale bar in Hz (maximum: 28 Hz). (C) Time course of
the average firing frequency per channel from 2 to 34 WIV. Firing frequency (±
standard deviation) was calculated as the average of all 64 electrodes from
each of the three MEA dishes. (D) Development of spontaneous synchronized burst
firings (SBFs) during long-term culture. The number of SBFs per minute (average
for 15 minutes) from 13 to 34 WIV.
Continue full article:
.png)
.jpg)
.gif)
