Osteogenesis differentiation medium

To assess the differentiation capacity of ASC towards osteoblasts, ASC (passage 1) were seeded in 12-well culture dishes at 10,000 cells/cm². The cells were then cultured in monolayer in Osteogenesis Differentiation Medium (Gibco). Control cells were cultured in normal culture medium. Both differentiation and control media were changed twice a week. Differentiation was stopped at 12 days by fixating the cells with 4 % formaldehyde at RT for 10 min. Differentiation was demonstrated by staining alkaline phosphatase (ALP). For this, cells were incubated in 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium, 0.1 M magnesium chloride solution for 10 min, whereafter a solution containing 0.2 M TRIS-hydrochloride (pH 10), 0.2 M calcium chloride, 0.1 M magnesium chloride solution and nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) was added (1:100, Roche Diagnostics, Mannheim, Germany) for 15 min. This stains ALP blue. Microscopic images were taken using a Zeiss microscope.

For differentiation into keratocytes, 3 × 10⁵ cells were spun down for pellet preparation and cultured in the Keratocyte Differentiation Medium consisting of Advanced DMEM with 10 ng/ml fibroblast growth factor 2 and 0.5 mM ascorbic acid; Thermo Fisher Scientific) for 21 days^{44 (link)}. For differentiation into corneal endothelial cells, single cells were seeded at the density of 1 × 10⁴ cells per cm² in 24-well plate coated with collagen IV in MESCM + 5% FBS. After cell adhesion in 24 h, the medium was switched to Corneal Endothelial Differentiation Medium consisting of low-glucose DMEM with 10% FBS and 50 µg/ml gentamicin, and 1.25 µg/ml amphotericin B) for 28 days⁴⁵ . For differentiation into adipocytes or osteocytes, single cells were seeded at the density of 1 × 10⁴ cells per cm² in 24-well plate coated with Matrigel in MESCM + 5% FBS. After cell adhesion in 24 h, the medium was switched to the Adipogenesis Differentiation Medium (Invitrogen) or Osteogenesis Differentiation Medium (Invitrogen), respectively, for 21 days^{18 (link)}. For differentiation into chondrocytes, 3 × 10⁵ cells were prepared for pellets and cultured in Chondrogenesis Differentiation Medium (Life Line) for 28 days^{18 (link)}. All the above media were changed every 3 days.

MSCs were grown to 90% confluency and cultured for 28 days in StemPro Adipogenesis or Osteogenesis differentiation medium (Thermo Fisher, USA). Adipogenic and osteogenic terminal differentiation was monitored by Oil Red O (Sigma Aldrich, USA) or Alizarine Red S (Sigma Aldrich, USA) staining, respectively.

For adipogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 4 × 10⁴ cells/well and cultured in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution until 80% confluency. The medium was then replaced with 1 mL of adipogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. Adipogenesis was analyzed by Oil Red O staining after 21 days.
For osteogenic differentiation, passage 2 cADSC were seeded on 12-well plates at a concentration 2 × 10⁵ cells/well and incubated in DMEM supplemented with 10% FBS and 1% antibiotic-antimycotic solution for 24 h. The medium was then replaced with 1 mL of osteogenesis differentiation medium (Thermo Fisher Scientific) and changed every 3 days. For osteogenic analysis, mineral deposits were analyzed quantitatively by Alizarin Red staining after 21 days.
For chondrogenic differentiation, 2 × 10⁵ passage 2 cADSC were suspended in 1 mL of chondrogenic differentiation medium (STEMCELL Technologies, Vancouver, BC, Canada) and 0.5 mL was aliquoted into a 15 mL conical tube and then centrifuged at 300× g for 5 min. The lid was then loosened and the cells were incubated for 3 days, after which 0.5 mL of medium was added and replaced every 3 days. Chondrogenic differentiation was evaluated by Alcian Blue staining after 21 days.