DOI: 10.5281/zenodo.12555054
title: Longitudinal development of 2D neuronal iPSC and primary neuronal cultures within NEUREKA project

description: Dataset Description

Several cell culture experiments were performed to evaluate and assess performance of novel electrode geometries and characteristics as described here: http://neureka.gr

iPSC culture experiment with HD-MEA chips performed at MaxWell Biosystems

In this experiment, iPS cells have been tested on HD-MEA chips, following the standard MaxOne Chips Neuronal Cell Plating Protocol.





Name of the experiment: MEX-1552

Date of the plating: 05.10.2023

Cell Type: Human-derived iPSC Excitatory Glutaneurons & Astrocytes

Cell Plating Protocol Details: Based on MaxWell Biosystem MaxOne Neuronal Cell Plating Protocol (https://www.mxwbio.com/resources/product-literature/neuronal-cell-culture-protocol/). In brief, both HD-NEA and HD-MEA chips have been treated with Terg-a-zyme solution for 2h at room temperature (RT), then sterilized in 70% EtOH for 30 min inside the biological safety cabinet. To prepare the surface for cell attachment, all chips were incubated in Complete BrainPhys medium for 48h in the cell culture incubator (37°C, 95% RH, 5% CO2).

Coatings: PEI 0.07% (Primary Surface Coating) + Laminin 20 ug/mL (Secondary Surface Coating).

Cell concentration: 385.000 cells/well

Plating modality: whole area (50 uL of cell suspension).

Consumables used: MaxOne Chips with HD-MEA (Standard)

Replicates: 5

Replicates IDs: 22553, 22637, 22565, 22706, 22654

Recordings Parameters:



Activity Scan Assay



Electrode configuration: Sparse 7

Recording window time: 30s/configuration



Network Activity Assay (based on Activity Scan Assay)



Scanning option: Neuronal Units (based on spike amplitude) for all the replicates for both conditions

Recording window time: 300 seconds





Analysis Parameters:



Activity Scan Assay:



Firing Rate Threshold (Hz): 0.1

Amplitude Threshold (uV): 20

ISI Threshold (ms): 200



Network Activity Assay:



Smoothing Window Size: 0.3 seconds

Burst Detection Threshold: 1.6

Minimum Peak Distance: 5 seconds

Start-stop threshold: 0.3






Primary neuronal culture experiment with HD-MEA chips performed at MaxWell Biosystems

In this experiment, primary cortical neuronal cultures have been tested on HD-MEA MaxOne Chips, following the standard MaxOne Chips Neuronal Cell Plating Protocol.



Name of the experiment: MEX-1556

Date of the plating: 22.11.2023

Cell Type: E18 Rat cortical primary neurons

Cell Plating Protocol Details: from MaxWell Biosystem MaxOne Neuronal Cell Plating Protocol (https://www.mxwbio.com/resources/product-literature/neuronal-cell-culture-protocol/). In brief, HD-MEA chips have been treated with Terg-a-zyme solution for 2h at room temperature (RT), then sterilized in 70% EtOH for 30 min inside the biological safety cabinet. To prepare the surface for cell attachment, all chips were incubated in Complete Neurobasal Medium for 48h in the cell culture incubator (37°C, 95% RH, 5% CO2).

Coatings: PEI 0.05% (Primary Surface Coating) + Laminin 20 ug/mL (Secondary Surface Coating).

Cell concentration: 100.000 cells/well

Plating modality: whole area (50 uL of cell suspension).

Consumables used: MaxOne Chips with HD-MEA (Standard, as control) + MaxOne Chips with HD-NEA.

Replicates: 3

Replicates IDs: 22531, 22722, 22615



Recordings Parameters:



Activity Scan Assay





Electrode configuration: Sparse 7




Recording window time: 30s/configuration






Network Activity Assay (based on Activity Scan)



Scanning option: Neuronal Units (based on spike amplitude) for all the replicates for both conditions

Recording window time: 300 seconds





Analysis Parameters:



Activity Scan Assay:



Firing Rate Threshold: 0.1 Hz

Amplitude Threshold: 20 µV

ISI Threshold: 200 ms



Network Activity Assay:



Smoothing Window Size: 0.05 seconds

Burst Detection Threshold: 1.6

Minimum Peak Distance: 5 seconds

Start-stop threshold: 0.3






Immunostaining Assay Details

Cells were fixed at the end of the culture (DIV 48) in 4% paraformaldehyde for 20 min at RT and then washed for 3 times with PBS 1X. Immunocytochemistry was performed immediately after the fixation to assess cells morphology, localization on the array, and neuronal health status. More specifically, cells were permeabilized and blocked using a PBS 10X solution containing 10% normal donkey serum (NDS), 1% bovine serum albumin (BSA) and 0.5% Triton X. Permeabilization facilitated antigen access to the cell, while blocking prevented non-specific binding of antibodies to the tissue. Primary and secondary antibodies were diluted in a PBS solution containing 3% NDS, 1% BSA and 0.5% Triton X. Cells were first incubated for 2h at room temperature with Primary Antibodies solution. The primary antibodies used were anti S-100 β for Astrocytes and anti β-Tubulin III for Neurons. Afterwards, the cells were washed three times with PBS and incubated for 1.5h at room temperature with the Secondary Antibodies solution, containing 4′,6-diamidino-2-phenylindole (DAPI) to mark cell nuclei. Subsequently, cells were washed three more times with PBS and immediately imaged by using an upright epifluorescence microscope.

Immunostaining Images Details: the directories are named after the corresponding Chip ID. For example, the directory “HD-MEA Chip 22565” contains all  pictures related to chip ID 22565. Inside the chip directory, there are two subdirectories: one with the raw data images (in .tiff format) and one with images analyzed with the ImageJ Software (in .png format).

TIFF images folder: contains the images corresponding to each single fluorescence channel (red for neurons, green for astrocytes, blue for cell nuclei). The pictures have been taken from two different areas of the chips: center of the array (“array”) and from outside of the array (“amplifier”). An example is: 22565_blue_amplifier_5x. In this case:



22565: ChipID.

blue: fluorescence channel. In this case referring to the one showing the DAPI, marking the cell nuclei.

amplifier: portion of the chip represented in the picture.

5x: objective magnification.


PNG images folder: contains the images analyzed with the ImageJ software. There are three images corresponding to each fluorescence channel, one picture representing the overlapping of the three fluorescence channels (“merge”) and the one showing the overview of the three single channels along with the merge (“montage”).