Mesenchymal stem cell chondrogenic differentiation medium

Normal Human Articular Chondrocytes (NHAC, LONZA Catalog #: CC-2550) were cultured in CGM™ Chondrocyte Growth Medium (LONZA), while human adipose tissue-derived mesenchymal stem cells (hMSC-AT, PromoCell) were cultured in supplemented Mesenchymal Stem Cells Growth Medium 2 (PromoCell); both in a humidified 5% CO₂ atmosphere, at 37 °C in tissue culture flasks (Falcon®). The culture medium was changed every three days and cells were passaged with TrypLE (Gibco) at 80–85% confluency. Additionally, hMSC-AT were cultured in Mesenchymal Stem Cell Chondrogenic Differentiation Medium (PromoCell) as a reference for gene expression analysis.

Chondrogenic differentiation was based on the spheroid model. For spheroid generation, the cells were seeded on a Nunc 96-well Round Bottom Microwell Plate (Thermo Scientific, Waltham, MA, USA) with 300,000 cells per well. The plates were incubated at 5% CO₂ and 37 °C for 48 h, after which the cells had assembled into spheroids suitable for subsequent studies. After spheroid formation, the Mesenchymal Stem Cell Chondrogenic Differentiation Medium (PromoCell, Heidelberg, Germany) was added to half of the wells; whereas, in the other half, a standard culture medium was utilized for negative controls. The culture was conducted for 21 days, with a change of medium every 72 h. The results of the differentiation were evaluated with Alcian Blue (Sigma-Aldrich, Saint Louis, MO, USA) staining for aggrecan detection. Spheroids were washed gently with PBS and fixed with Saccomanno Fixative solution for 3 h at room temperature. Subsequently, the spheroids were washed twice with distilled water and stained with Alcian Blue, previously filtered with the use of a 0.22 µm syringe filter (Millex, Merck, Germany), for 45 min. The spheroids were washed three times with a destaining solution. The results of the staining were observed using an inverted phase-contrast microscope (Olympus IX70, Olympus, Tokyo, Japan).

The BioX printer (Cellink) with temperature-control, pressure extrusion printhead was used, with printhead temperature set at 25 °C, and printbed temperature set at 10 °C.
After bioprinting, the constructs were crosslinked with sterile 200 mM CaCl₂ (Sigma-Aldrich) dissolved in 4.6% (w/v) D-mannitol (Sigma-Aldrich) for 10 min at room temperature.
The constructs were cultured in Mesenchymal Stem Cell Growth Medium 2 (Promocell), and the Mesenchymal Stem Cell Chondrogenic Differentiation Medium (Promocell) in the case of cells used as differentiation control. The constructs were cultured in standard conditions (37 °C, 5% CO₂) and the medium was changed every 3 days.

As described before, mMSCs were differentiated into adipogenic, osteogenic, and chondrogenic cells [21 (link)]. In brief, adipogenic and osteogenic differentiation was induced with mesenchymal stem cell adipogenic differentiation medium 2 (PromoCell) and osteogenic differentiation medium (PromoCell), respectively, by culturing 5 × 10⁴ cells on round glass slides in 6-well plates. After 14 days, the cells were fixed and stained with oil red O (adipogenic differentiation) and alizarine red (osteogenic differentiation) [21 (link)]. Chondrogenic differentiation was induced using micro mass body cultivation with chondrogenic differentiation medium. After centrifugation and incubation, mesenchymal stem cell chondrogenic differentiation medium (PromoCell) was added at 48 h and replaced every 3 days with fresh medium. After 21 days, the pellet was collected and put in paraffin wax, cut, and stained with Alcian blue.

hDPSCs (1 × 10³ cells) seeded in spheroid-forming culture plates (PrimeSurface; Sumitomo Bakelite, Tokyo, Japan) were cultured in a chondrogenic differentiation medium (Mesenchymal Stem Cell Chondrogenic Differentiation Medium; PromoCell, Heidelberg, Germany) for 25 days. Alcian Blue (Fujifilm Wako Pure Chemical) staining was performed on the frozen sections (5 μm thickness).