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

Abstract

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.

A one-step procedure to probe the viscoelastic properties of cells by Atomic Force Microscopy

“…3b-III ), despite their similar morphology. As discussed above, the absence of T low for the patterned PDAC p53 R172H cells is associated with their limited capability of reorganising their cytoskeleton and restricted movement 36 , 37 . The presence of T low in PDAC p53 fl/fl cells shows that individual cells in a cluster were not limited by their neighbouring cells, and maintain similar levels of restructuring activities as the freely moving PDAC p53 R172H cells.…”

“…Therefore, we first evaluated the influence of cell shape on their viscoelastic properties. The micropatterning technique was used as a simple physical means to regulate cell shape as well as restrict cell movement 36 – 40 . Mutant PDAC p53 R172H cells were cultured on a micropatterned substrate with an array of 20 μm adhesive circles, giving rise to round cell shapes on the patterns (Fig.…”

“…It is worth noting, because of geometrical restrictions, cells on patterns were prevented from changing shape and have limited adhesion sites on the adhesive patterns, whereas their counterparts on unpatterned substrates could reorganise their cytoskeleton and move freely 36 , 37 . This indicates that cells on patterns have limited capability of reorganising their cytoskeleton, dynamically restructuring F-actin networks and modulating interactions with substrates.…”

Engineering a Cell Home for Stem Cell Homing and Accommodation

“…However, if the aim is the generation of tissue‐ and organ‐like structures for reconstructive purpose, higher complexity levels of biomaterial systems (such as in vitro 3D culture models and in situ biosensing tools) presenting physicochemical and biological cues in a temporospatial manner are absolutely justified . We expect that the future 3D tools and systems will be capable of inducing tissue‐specific cell differentiation, modulating cell geometry and monitoring cellular responses, among others; generate new insights into fundamental cellular behavior information; and lead to more and more biomedical applications ranging from 3D cell culture and production, stem cell therapy, and tissue engineering to drug discovery and development …”

The Good the Bad and the Ugly of Glycosaminoglycans in Tissue Engineering Applications

“…Histological analysis demonstrated that these scaffolds alone (i.e., cell-free) were able to initiate the formation of well-structured osteochondral tissue. Collagen I gels in combination with 2D micro patterning and single cell culture has also been used as a platform to investigate conditions that control chondrocyte de-differentiation [375]. This approach also offers a method from which niches for targeted differentiation of hMSCs could be investigated.…”