Enzymatic Dissociation of hCS-FF and hCS/hSS

For the enzymatic dissociation of hCS-FF for culture in monolayer and
immunocytochemistry (ani-GFAP), up to 3 spheroids were incubated with 200
μl of Accutase (Innovative Cell Technologies) for 20 min at 37 °C,
washed with neural medium and gently triturated using a P200 pipette. Cells were
plated on glass coverslips (15 mm, Warner Instruments) coated with
poly-L-ornithine and laminin (Sigma-Aldrich) at a density of around 250,000
cells per coverslip in neural medium supplemented with BDNF, NT3 and, for the
first 24 hours, 10 μM Y-27632.
To dissociate hCS and hSS for single-cell profiling, we used a
previously published protocol^{7 (link)}.
Briefly, 3–10 spheroids were chopped using a #10 blade and then incubated
in 40 U/ml papain enzyme solution containing 0.46% D(+)-Glucose (Sigma-Aldrich),
26 mM NaHCO₃ (Sigma-Aldrich), 0.5 mM EDTA (Sigma-Aldrich) in EBSS
(1X, Sigma-Aldrich) at 37 °C in 5% CO₂ for 70 minutes. The
digested spheroids were then washed and carefully triturated with a protease
inhibitor stock solution containing 0.46% D(+)-Glucose (Sigma-Aldrich), 26 mM
NaHCO₃ (Sigma-Aldrich), 5 mg trypsin inhibitor (Sigma-Aldrich) in
EBSS (1X, Sigma-Aldrich). After centrifugation (200 × g for 4 min), the
pellet was resuspended in 0.2% bovine serum albumin (BSA) diluted in PBS and
supplemented with 10 μ M Y-27632, and the cells were used for the
single-cell RNA sequencing.