A novel culture system for modulating single cell geometry in 3D.

Dedifferentiation of chondrocytes during in vitro expansion remains an unsolved challenge for repairing serious articular cartilage defects. In this study, a novel culture system was developed to modulate single cell geometry in 3D and investigate its effects on the chondrocyte phenotype. The approach uses 2D micropatterns followed by in situ hydrogel formation to constrain single cell shape and spreading. This enables independent control of cell geometry and extracellular matrix. Using collagen I matrix, we demonstrated the formation of a biomimetic collagenous "basket" enveloping individual chondrocytes cells. By quantitatively monitoring the production by single cells of chondrogenic matrix (e.g. collagen II and aggrecan) during 21-day cultures, we found that if the cell's volume decreases, then so does its cell resistance to dedifferentiation (even if the cells remain spherical). Conversely, if the volume of spherical cells remains constant (after an initial decrease), then not only do the cells retain their differentiated status, but previously de-differentiated redifferentiate and regain a chondrocyte phenotype. The approach described here can be readily applied to pluripotent cells, offering a versatile platform in the search for niches toward either self-renewal or targeted differentiation.