DOI: 10.6084/m9.figshare.27233032.v1
title: A single-cell 3D dynamic volume control system for chondrocytes

description: In articular cartilage, zone-specific cellular morphology is a typical characteristic of cartilage tissue, which is related with chondrocyte function, inflammation and osteoarthritis (OA). Chondrocyte hypertrophic phenotype is a criticle physiological process which indicates a hallmark of chondrocyte terminal differentiation and bone formation. Thus, developing a <i>in vitro</i> cell culture system for dynamic regulation of single chondrocyte volume at a three-dimensional (3D) level is particularly necessary for understanding how physical cues of matrix microenvironment regulate chondrocyte fate and the degeneration of articular cartilage. Here, based on the soft lithography techniques, we have constructed well-defined single-cell 3D dynamic volume control system to recapitulate the physiological matrix microenvironment of single chondrocyte niche. The results of finite element analysis indicated that the stress and strain distribution in the cell culture region is homogeneous during the stretching process. Additionally, 3D dynamic volume expansion and compression of single cells in physiological or hyperphysiological can be realized in this cell culture system. Our device for single-cell 3D dynamic culture provides a microphysiological culture system for chondrocytes to explore the mechanisms of cartilage hypertrophy, as well as develops a new paradigm for functional cartilage tissue engineering and regenerative medicine. SolidWorks software was used to design various components of 3D dynamic volume control system. Leveraging lithography microfabrication techniques, the polydimethylsiloxane (PDMS) 3D niches were fabricated with controlled geometry and volume on PDMS stretching substrate. Finite element analysis softwares (Abaqus and Comsol) were used to analyze the strain distribution of PDMS stretching substrate and single 3D niche. The elastic moduli of PDMS stretching substrate were measured using an Instron 5544. Furthermore, immunofluorescence staining was performed to assess the morphology and cytoskeletal reorganization of chondrocyte encapsulated in PDMS 3D niches. ImageJ software was used to quantitatively analyze immunofluorescence images. The design idea is derived from the concept that chondrocyte function is strongly related to their cartilage zone-specific volume change defined by extracellular matrix. Breaking through traditional 2D and 3D cell culture techniques of cartilage chondrocytes, we recapitulate the characteristics of <i>in vivo</i> 3D chondrocyte niche. We engineered a unique single-cell 3D dynamic volume control system which consists of a dynamic actuating apparatus and a PDMS 3D niches. The cell culture system can realize 3D dynamic volume expansion and compression of single chondrocytes in physiological or hyperphysiological strain ranges. The cell culture system provides the added value of 3D single cell niches to mechanically regulate chondrocyte fate and promote cartilage tissue regeneration. The cell culture system can be used for fundamental research on pathological mechanisms of osteoarthritis, as well as contributing to regenerative medicine.