Penicillin streptomycin glutamine 100 solution

hMSCs were isolated from human bone marrow aspirates obtained from routine orthopedic surgical procedures involving exposure of the iliac crest, after ethical approval (Ethikkommission beider Basel, Ref.78/07) and informed donor consent. Briefly, marrow aspirate (20 mL volume) was harvested from healthy young donor using a bone marrow biopsy needle inserted through the cortical bone and immediately transferred into plastic tubes containing 15,000 IU heparin. After diluting the marrow aspirate with phosphate buffered saline (PBS) at a ratio of 1:4, nucleated cells were counted and seeded at a density of 3.10⁶ cells/cm² in complete medium supplemented with 5 ng/mL of fibroblast growth factor-2 (FGF-2, R&D Systems) and cultured in a humidified 37 °C/5% CO₂ incubator. hMSCs were selected on the basis of adhesion and proliferation on the plastic substrate one week after seeding and cultured with complete medium, consisting of α-minimum essential Medium (αMEM) with 10% fetal bovine serum, 1% HEPES (1 M), 1% Sodium pyruvate (100 mM) and 1% of Penicillin–Streptomycin–Glutamine (100×) solution (all from Gibco). The cytofluorimetric profile of the expanded cells corresponds to what was previously reported [7 (link)].

After 7 days of primary 2D conventional monolayer culture (first expansion phase), NPCs were isolated from tissue culture dishes by trypsinization, washed, and seeded back into tissue culture dishes coated with fibronectin (#F2006, Sigma) at a density of 10 × 10³ cells/cm². Thereafter, 3D NP cell alginate beads were digested in alginate dissociation buffer, composed of NaCl at 150 mM (#71379, Sigma) and Na₃Citrate.2H₂O at 55 mM (#71406, Sigma) dissolved in Milli‐Q‐Water and adjusted at pH = 6.8, at 37°C for 15 minutes. After washing, NPCs were seeded into a tissue culture dish coated with fibronectin (#F2006, Sigma) at a density of 10 × 10³ cells/cm² with LG‐DMEM with 10% fetal bovine serum, 1% HEPES (1 M), 1% sodium pyruvate (100 mM), and 1% of penicillin‐streptomycin‐glutamine (100×) solution (all from Gibco) and 10 ng/mL of FGF‐2 (R&D Systems) until confluence. After reaching confluence, cells were directly used for the differentiation assays. Microscopic pictures were taken for each condition at the end of the first and second phase of expansion using the Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan).

For the 3D culture in the first expansion phase, NP cells isolated from NP tissue were washed with PBS and centrifuged at 1500 rpm for 3 minutes. Cells were mixed and encapsulated in 1.2% alginate at a density of 4 × 10⁶ cells/mL using a syringe (22 G needle) by forming approximately 30 μL droplets in a 102 mM CaCl₂ salt solution.²⁰ The NP cell alginate beads (six beads per well of 12‐well plate) were maintained in LG‐DMEM with 10% fetal bovine serum, 1% HEPES (1 M), 1% sodium pyruvate (100 mM), and 1% of penicillin‐streptomycin‐glutamine (100×) solution (all from Gibco) and 100 μM l‐ascorbic acid‐2‐phosphate (Sigma‐Aldrich) under a 5% CO₂ and 5% oxygen atmosphere at 37°C, for 7 days. The medium was replaced twice a week.