Scaffold-free 3D cellulose acetate membrane-based cultures form large cartilaginous constructs

Scaffold-free three-dimensional (3D) cultures provide clinical potential in cartilage regeneration. The purpose of this study was to characterize a scaffold-free 3D membrane-based culture system, in which human articular chondrocytes were cultivated on a cellulose acetate membrane filter, and compare it to pellet and monolayer cultures. Chondrocytes were expanded in monolayer culture for up to 5 passages, transferred to membrane-based or pellet cultures and harvested after 7 or 21 days. The chondrogenic potential was assessed by histology (toluidine blue, safranin-O), immunohistochemistry for collagen type II and quantitative analysis of collagen type II α(1) (COL2A1). Membrane-based cultures (P1) formed flexible disc-like constructs (diameter 4000 µm, thickness 150 µm) with a large smooth surface after 7 days. Positive safranin-O and collagen type II staining was found in membrane-based and pellet cultures at P1-3. Expression of COL2A1 after 7 days was increased in both culture systems compared to monolayer culture up to P3, whereas cells from monolayer > P3 did not redifferentiate. The best results for COL2A1 expression were obtained from membrane-based cultures at P1. After 21 days the membrane-based cultures did not express COL2A1. We concluded that membrane-based and pellet cultures showed the ability to promote redifferentiation of chondrocytes expanded in monolayer culture. The number of cell passages had an impact on the chondrogenic potential of cells. Membrane-based cultures provided the highest COL2A1 expression and a large, smooth and cartilage-like surface. As these are appropriate features for clinical applications, we assume that membrane-based cultures might be of use in cartilage regeneration if they displayed similar results in vivo.