Cellstart substrate

The human NSCs used in this study were derived from NIH-approved H9 human embryonic stem cells (Thermo Fisher Scientific, MA, USA). The cells were handled following a kit’s protocol. The complete medium used to maintain the human NSCs consisted of 1 × KnockOut D-MEM/F12, 2 mM GlutaMAX-I supplement, 20 ng/ml FGF-β, 20 ng/ml EGF and 2% StemPro neural supplement (all from Thermo Fisher Scientific, MA, USA). Adherent human NSCs were thawed and subcultured in plates precoated with a matrix consisting of CELLStart substrate and D-PBS containing calcium and magnesium (both from Thermo Fisher Scientific, MA, USA). The cells were incubated at 37 °C with 5% CO_2, and the medium was replaced with new complete medium every 2 days. When the NSCs were approximately 90% confluent, they were ready to be passaged. The spent medium was removed from the cells, and the cellular surface was rinsed with 1 × D-PBS. To detach the cells, they were treated with Accutase and incubated at 37 °C in a 5% CO₂ incubator for 1–2 min, after which complete medium was added to stop the dissociation reaction. The cells were gently pipetted several times, transferred to a 15 ml tube, and centrifuged at 200 × g for 5 min. The supernatant was aspirated, and the cells were resuspended in complete medium and seeded in precoated plates for maintenance.

The expanded cells were cultured in growth media supplemented with different sera according to the study groups: 15% fetal bovine serum (FBS) (Gibco), 15% human serum (HS) (Sigma–Aldrich), and 15% synthetic serum (SS) (StemPro™ MSC SFM, Gibco). For the SS group, cell culture plates were precoated with CELLstart™ Substrate (Gibco) at a concentration of 1:200 and 10 mL per 75 cm² for 1 h before cell seeding following the manufacturer’s instructions. Culture media were changed every 2–3 days.
To generate cell batches, the expanded cells from each donor at passage 1 at 80% confluence were collected and frozen. Afterward, the frozen cells from three donors were thawed and pooled as one batch and expanded in the designated sera into passages 3–5 for the investigations. Cells were seeded at 5 × 10³ cells/cm² for expansion and investigations. Growth medium comprised DMEM/F-12 (Corning, Corning, NY, USA), 1% penicillin, 0.1% fungizone (Gibco), and 15% serum (FBS, HS, or SS). The osteogenic medium was growth media supplemented with 50 mM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone (Sigma-Aldrich) [40 (link)].

Cryopreserved BMSCs were thawed and plated at a cell density of 3000 cells·cm⁻², on T-75 flasks with low glucose (1 g·L⁻¹) Dulbecco’s Modified Eagle Medium [DMEM, (Gibco, Thermo Fisher Scientific, New York, NY, USA) supplemented with 5% v/v human platelet lysate (hPL) UltraGRO™-PURE (AventaCell, Atlanta, GA, USA) and Antibiotic-Antimycotic (1×) (Gibco, Thermo Fisher Scientific) (DMEM/HPL). Alternatively, cells were plated at the same cell density in CELLstart™ substrate (Gibco, Thermo Fisher Scientific) pre-coated T-75 flasks with StemPro™ MSC SFM XenoFree medium (Gibco, Thermo Fisher Scientific) supplemented with Glutamax (1×) (Gibco, Thermo Fisher Scientific) and Antibiotic-Antimycotic (1%) (StemPro). Cells cultured in DMEM-HPL were grown in VWBR, whereas cells grown in SP were cultured in StemPro. At 70% cell confluence, MSCs were harvested with 1× TrypLE™ Select Enzyme solution (Gibco, Thermo Fisher Scientific) for 5 min at 37 °C. Cell number and viability were determined using the Trypan Blue (Gibco, Thermo Fisher Scientific) exclusion method.

Human iPSC lines were maintained in StemProTM hESC SFM media (A1000701, Gibco) supplemented with 20 ng/mL human basic FGF (R&D) on either CELLstart Substrate (A1014201, Gibco) or Vitronectin (A31804, Gibco) coated wells. Media was changed daily. iPSCs were passaged when wells reached approximately 70%–80% confluency with a StemPro EZPassage Disposable Stem Cell Passaging Tool (23181010, Gibco).