The prefrontal cortex tissues were digested according to a previously described method90 (link) with some minor modifications. Briefly, the prefrontal cortex tissues from human PCW9 and macaque E51 fetal brains were transferred into a new sterile 3.5-cm dish with 100 µL fresh sterile ice-cold HBSS++ buffer. After discarding the HBSS++ buffer, the tissues were sliced into sizes less than 1 mm3 using a sterile surgical blade. Immediately, the minced prefrontal cortex tissues were digested in a 15-mL tube with 2 mL of 20-unit/mL papain (Worthington, Lakewood, NJ, USA, Cat. No. LK003178) and 200 µL 200-Knuitz-units/mL DNase I (Worthington, Cat. No. LK003172), both of which were dissolved in HBSS with Ca2+ and Mg2+ (Thermo Fisher Scientific, Cat. No. 14025092). The tube was then rotated 30 rounds per minute (rpm) at 30 °C for 30 minutes (min).
After incubation, the tube was centrifuged at 150 × g for 1 min at room temperature (RT), and the supernatant was removed as much as possible with a P1000 pipette. The tissue/cell pellet was suspended using 2 mL RT Neurobasal++ medium, containing Neurobasal medium (Thermo Fisher Scientific, Cat. No. 21103049) supplemented with 1× B27 (Thermo Fisher Scientific, Cat. No. 17504044) and 1× GlutaMAX (Thermo Fisher Scientific, Cat. No. 35050-061). Single cells were released by triturating the tissue/pellet by pipetting up and down ten times with 1-mL LoBind tip (Thermo Fisher Scientific, Cat. No. TFLR1121000-Q) in 45 s without introducing bubbles. The tube was settled for 1 min, and the supernatant containing the released single cells was transferred to a new 15-mL tube without disturbing the remaining tissues at the tube bottom. Another 2 mL Neurobasal++ was added into the tube with the remaining tissues. The trituration, as described above, was repeated two more times to achieve maximum single-cell release. The supernatant containing the released single cells was combined and centrifuged at 800 × g for 5 min at RT. After discarding the supernatant fraction, 6 mL complete medium, which was comprised of Neurobasal medium supplemented with 1× B27, 1× GlutaMAX, 1× penicillin/streptomycin, and 5 ng/mL human FGF2 (Novoprotein, Wuhan, China, Cat. No. GMP-C046), was added to resuspend the pelleted cells. An aliquot of 10 µL cell suspension was used to assess the cell viability. Usually, the isolated cells showed a cell viability of more than 98%.
Next, the isolated single cells were seeded in the 6-well plate wells that were pre-coated with 0.1 mg/mL Poly-D-Lys (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. P6407) dissolved in sterile miliQ water. Each well was seeded with 1.2 million cells and added with 2 mL of complete medium. For culturing the primary neuronal cells that were used for immunostaining, ~10,000 cells were seeded into each well of the 24-well plate with a 12-mm coverslip (Electron Microscopy Science, Hatfield, PA, USA, Cat. No. 72196-12) pre-coated with Poly-D-Lys. The seeded cells were maintained in a 37 °C incubator with 5% CO2 by half medium exchange every 2 days since day 5 after seeding cells. The primary neuronal cells were collected on day 20 and day 15 for human and macaque cells, respectively. Those primary neuronal cells were applied for subsequential experiments.
After incubation, the tube was centrifuged at 150 × g for 1 min at room temperature (RT), and the supernatant was removed as much as possible with a P1000 pipette. The tissue/cell pellet was suspended using 2 mL RT Neurobasal++ medium, containing Neurobasal medium (Thermo Fisher Scientific, Cat. No. 21103049) supplemented with 1× B27 (Thermo Fisher Scientific, Cat. No. 17504044) and 1× GlutaMAX (Thermo Fisher Scientific, Cat. No. 35050-061). Single cells were released by triturating the tissue/pellet by pipetting up and down ten times with 1-mL LoBind tip (Thermo Fisher Scientific, Cat. No. TFLR1121000-Q) in 45 s without introducing bubbles. The tube was settled for 1 min, and the supernatant containing the released single cells was transferred to a new 15-mL tube without disturbing the remaining tissues at the tube bottom. Another 2 mL Neurobasal++ was added into the tube with the remaining tissues. The trituration, as described above, was repeated two more times to achieve maximum single-cell release. The supernatant containing the released single cells was combined and centrifuged at 800 × g for 5 min at RT. After discarding the supernatant fraction, 6 mL complete medium, which was comprised of Neurobasal medium supplemented with 1× B27, 1× GlutaMAX, 1× penicillin/streptomycin, and 5 ng/mL human FGF2 (Novoprotein, Wuhan, China, Cat. No. GMP-C046), was added to resuspend the pelleted cells. An aliquot of 10 µL cell suspension was used to assess the cell viability. Usually, the isolated cells showed a cell viability of more than 98%.
Next, the isolated single cells were seeded in the 6-well plate wells that were pre-coated with 0.1 mg/mL Poly-D-Lys (Sigma-Aldrich, St. Louis, MO, USA, Cat. No. P6407) dissolved in sterile miliQ water. Each well was seeded with 1.2 million cells and added with 2 mL of complete medium. For culturing the primary neuronal cells that were used for immunostaining, ~10,000 cells were seeded into each well of the 24-well plate with a 12-mm coverslip (Electron Microscopy Science, Hatfield, PA, USA, Cat. No. 72196-12) pre-coated with Poly-D-Lys. The seeded cells were maintained in a 37 °C incubator with 5% CO2 by half medium exchange every 2 days since day 5 after seeding cells. The primary neuronal cells were collected on day 20 and day 15 for human and macaque cells, respectively. Those primary neuronal cells were applied for subsequential experiments.
