| GSM8304052 channel 1 treatment |
PSCs were cultured on Matrigel-coated 6-well plates, fed every other day with mTeSR Plus (Stemcell Technologies, 100-0276), and chemically passaged using ReLeSR (Stemcell Technologies, 100-0483). The brain organoid differentiation was modified from the cortical organoid protocol described in Trujillo et al. and Fitzgerald et al.. A 24-well AggreWell 800 plate (Stemcell Technologies, 34811) was prepared by adding Anti-Adherence Rinsing Solution (Stemcell Technologies, 07010) to each well, centrifuging the plate at 1300 xg for 5 minutes, and rinsing each well with D-PBS. On Day 1, PSCs were dissociated into single cells Accutase (Stemcell Technologies, 07920) in D-PBS at a 1:1 ratio for 10 minutes at 37 °C. The cells were then centrifuged for 3 minutes at 150 xg and the pellet was resuspended in mTeSR Plus supplemented + 10 μM SB431542 (SB; Stemgent, 04-0010-base) + 1 μM Dorsomorphin (Dorso; R&D Systems, 3093) + 5 μM ROCKi). 2.5×106cells were transferred to each well of the prepared AggreWell 800 plate, and the plate was centrifuged at 1300 xg for 5 minutes. The AggreWell plate was incubated at 37 °C overnight. On day 2, embryonic bodies were transferred from each used well of the AggreWell plate to each well of a 6-well plate in fresh mTeSR Plus + 10 μM SB + 1 μM Dorso and kept in suspension on an orbital shaker (90 rpm rotation) for the duration of the brain organoid differentiation. Media was changed daily through day 6. From days 7-11, media was changed every other day with M1 media [Neurobasal Medium (Life Technologies, 21103049) + 1% GlutaMAX (ThermoFisher Scientific, 35050061) + 1% Gem21 NeuroPlex (Gemini Bio, 400-160-010) + 1% N2 NeuroPlex (Gemini Bio, 400-163-005) + 1% NEAA (ThermoFisher Scientific, 11140050) + 1% PS (ThermoFisher Scientific, 15140122) + 10 μM SB + 1 μM Dorso] and embryoid bodies were split between new wells approximately once during this period to prevent overcrowding. From days 12-18, media was changed every day with M2 media [Neurobasal Medium + 1% GlutaMAX + 1% Gem21 NeuroPlex + 1% NEAA + 1% PS] + 20 ng/mL FGF2 (Peprotech, 100-25). From days 19-25, media was changed every other day with M2 + 20 ng/mL FGF2 + 20 ng/mL EGF (PeproTech, AF-100-15). From days 26-29, half media changes were performed with M3 media [M2 medium + 10 ng/mL BDNF (PeproTech, 450-02) + 10 ng/mL GDNF (PeproTech, 450-10) + 10 ng/mL NT-3 (PeproTech, 450-03) + 200 μM L-ascorbic acid (Sigma-Aldrich, A4403) + 1 mM dibutyryl-cAMP (Stemcell Technologies, 100-0244)] + 20 ng/mL FGF2. From days 30-35, media was changed every 3-4 days with M3 media. From days 36-42, media was changed every 3-4 days with M2.5 media [M3 medium with half the concentration of factors]. From day 43 on, brain organoids were maintained in M2 media, media changes every 3-4 days. |
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| GSM8304052 channel 1 growth |
CytoView MEA 48 plates (Axion Biosystems, M768-tMEA-48B) were first sterilized by incubating the wells with 1% Terg-a-zyme (Sigma-Aldrich, 7273287) in sterile DI water for 2 hours. Terg-a-zyme was removed from the wells and the wells rinsed with sterile DI water. The wells were sterilized a second time by incubating 70% ethanol (Sigma-Aldrich, E7023) in sterile DI water for 30 minutes. Ethanol was removed from the wells and the wells rinsed with sterile DI water. M2 media was added to the wells and incubated for at least 48 hours at 37 °C to pre-condition the surface. After pre-conditioning, wells were rinsed with sterile DI water, air-dried, and a solution 0.07% PEI (Sigma-Aldrich, P3143) in 1X borate buffer (ThermoFisher Scientific, 28341) sterilized through a 0.22 μm filter (Millipore Sigma, SCGP00525) was added to the wells and incubated at 37 °C for 1 hour. Then the wells were rinsed with sterile DI water, air-dried, and coated with 0.4 mg/mL laminin (Life Technologies, 23017015) in D-PBS by pipetting a small volume directly onto the electrodes, without allowing the solution to spread to the edges of the well. The plates were then incubated at 37 °C for 1 hour. M2 media was added to the wells, followed by 2-4 organoids between 60-90 days of differentiation and then 100 μg of sterilized, Matrigel-coated nanofibers were added to each well. Micropipette tips were used the move the organoids onto the electrodes, taking care not to scratch the wells’ surfaces. The plates were then incubated at 37 °C for 5 days to allow organoids to attach. Half media changes were performed every 4-5 days, taking care not to disturb the brain organoids as networks were forming. MEA recordings were performed on the Meastro Pro (Axion Biosystems) approximately 2 weeks after seeding the brain organoids on MEA plates, every 4-5 days (the day after media change) for 8 weeks. 1 hour of daily light stimulation was produced using the Lumos (Axion Biosystems) with the following parameters: both blue and green lights at 100% intensity, 1-2 Hz pulse frequency 1 ms pulse rise, 10 ms pulse duration, 5 ms pulse off. Light frequency was increased from 1 to 2 Hz in 0.3 Hz intervals over the 8 weeks of light training. Control plates were never exposed to light stimulation and a single 5 minute recording was made on recording day for these plates. |
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