Page 357 - IJB-9-2

International Journal of Bioprinting Laser bioprinting of hiPSC-derived neural stem cells and neurons

permeabilized with 0.3% Triton X-100/PBS. After blocking Table 1. Grading system for rating the neuronal activity
with 2% bovine serum albumin/PBS solution at 37°C for visualized by calcium imaging
1 hour, the cell samples were incubated with primary Spontaneous activity Without bursting With bursting
antibodies (see Table S1 in Supplementary File for details) events events
diluted in 0.3% Triton X-100/PBS overnight at 4°C. After No 0 N/A
several washing steps, the cell samples were incubated
with a fluorescence-conjugated secondary antibody at an Scarcely 2 N/A
appropriate dilution (Table S1) for 1 hour at 37 °C. Cell Little 3 N/A
nuclei were stained with Hoechst 33342. Some 4 8
Substantial 8 12
2.4.3. Western blot analysis Widespread 12 16
NSCs, neurons and astrocytes were harvested at the Abundant 16 20
indicated time points from three independent experiments. Abundant and intensive 20 24
Total protein was isolated from the cells with RIPA lysis
buffer containing protease and phosphatase inhibitors
(25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% Nonidet 2.5.2. Rating of neuronal activity visualized by
P-40, 0.1% sodium dodecyl sulfate [SDS], and 1% sodium calcium imaging
deoxycholate). The protein concentrations of the cell lysates Approximately 500 videos were taken from printed
were determined using a BCA protein assay (Carl Roth, samples with calcium imaging to investigate neuronal
Karlsruhe, Germany). Ten micrograms of the total protein activity. Some of them were assessed in detail by statistical
extract were loaded per lane on a 10% polyacrylamide data analysis method described below. Additionally, the
resolving gel and separated by SDS-PAGE (sodium activity visualized in all videos was rated between 0 and 24
dodecyl sulfate–polyacrylamide gel electrophoresis). (points) with the grading of Table 1. Thereby, intermediary
After electrophoresis, proteins were transferred onto ratings were also given.
polyvinylidene fluoride (PVDF) membranes (Carl Roth) 2.6. Calcium imaging data analysis
by wet blotting using 20% methanol in Tris-glycine Fluorescence recordings were analyzed with the custom-
buffer. The membranes were blocked for 1 hour in a 5% made software NETCAL (www.itscnetcal.com) . First,
[49]
nonfat milk solution in TBS with 0.1% Tween™ 20 before all frames of the fluorescence recording were averaged to
incubation with primary antibodies (Table S1) overnight generate a highly contrasted gray scale image where bright
at 4°C. After washing, the membranes were incubated with spots are active neurons. Regions of interest (ROIs) were
a horseradish peroxidase-conjugated secondary antibody then automatically identified as those circular-like spots
(Table S1) for 2 hours at room temperature. The blots were with at least 5 µm in diameter. The fluorescence trace
developed with an enhanced chemiluminescence reagent F (t) for each ROI was then extracted and normalized as
i
SuperSignal West Femto Substrate (Thermo Scientific). %DFF = 100 (F (t)-F ) F , where F is the fluorescence
-1
i_0
i_0
i
i_0
The band densities of the immunoblots were quantified by trace of the neuron at rest. Sharp increases in the
applying the ImageJ software. The relative expressions levels fluorescence trace revealed activity. The train of spikes’
of neuronal proteins were normalized to the housekeeping onset times for each ROI was extracted using the
protein levels (β-actin) and are presented as mean values Schmitt trigger method, which accepts a sharp change
from three independent experiments. in fluorescence as a spike when the fluorescence stays
elevated for at least 100 ms between a lower and a higher
2.5. Calcium imaging threshold . The two thresholds are necessary to prevent
[50]
2.5.1. Culture preparation and visualization that camera noise or other artifacts are identified as
Neuronal cell cultures were incubated with the calcium- spikes. The complete set of spiking events extended to
sensitive dye Fluo-8 AM (10 µM Biomol, Hamburg, all the neurons in the network provided the raster plots,
Germany) in a neuronal differentiation medium for and from which all statistical analyses were carried out.
40 minutes at 37°C in a 5% CO environment. Cell We associated all ROIs as neurons. Typically, a recording
2
cultures were then washed twice with prewarmed external contained about 2,000 active neurons. Glial cells showed
medium (EM) consisting of 10 mM Hepes, 130 mM NaCl, smooth fluorescence calcium transients that were
4 mM KCl, 1 mM MgCl 2 mM CaCl , 45 mM sucrose, automatically filtered out during data analysis.
2,
2
and 10 mM glucose. Spontaneous calcium activity was
acquired upon an AxioImager A1.m microscope equipped 2.6.1. Average neuronal activity
with an AxioCm ICc1 camera and AxioVision Rel. 4.8 The average neuronal activity quantified the neurons’
software. spontaneous activity and was computed by the number of

Volume 9 Issue 2 (2023) 349 https://doi.org/10.18063/ijb.v9i2.672