High density micromass cultures of a human chondrocyte cell line: A reliable assay system to reveal the modulatory functions of pharmacological agents

Abstract

Osteoarthritis is a highly prevalent and disabling disease for which we do not have a cure. The identification of suitable molecular targets is hindered by the lack of standardized, reproducible and convenient screening assays. Following extensive comparisons of a number of chondrocytic cell lines, culture conditions, and readouts, we have optimized an assay utilizing C-28/I2, a chondrocytic cell line cultured in high-density micromasses. Utilizing molecules with known effects on cartilage (e.g. IL-1β, TGFβ1, BMP-2), we have exploited this improved protocol to (i) evoke responses characteristic of primary chondrocytes; (ii) assess the pharmacodynamics of gene over-expression using non-viral expression vectors; (iii) establish the response profiles of known pharmacological treatments; and (iv) investigate their mechanisms of action. These data indicate that we have established a medium-throughput methodology for studying chondrocyte-specific cellular and molecular responses (from gene expression to rapid quantitative measurement of sulfated glycosaminoglycans by Alcian blue staining) that may enable the discovery of novel therapeutics for pharmacological modulation of chondrocyte activation in osteoarthritis.

IFT80 Is Required for Fracture Healing Through Controlling the Regulation of TGF-β Signaling in Chondrocyte Differentiation and Function

“…For micromass cultures, the protocol was described by De Bari and colleagues (34) and modified by Greco and colleagues. (35) Briefly, micromasses were obtained by pipetting 20 μL of primary chondrocytes into individual wells of 24-well plates. Following a 3-hour attachment period without medium, the growth medium was gently added.…”

Regulation of multiple target genes by miR-1 and miR-206 is pivotal for C2C12 myoblast differentiation

“…C28/I2 cells were cultured and induced to chondrogenic differentiation as described previously (Greco et al, 2011). Alcian Blue staining was performed as previously described (Swingler et al, 2012) and C28/I2 cells served as positive control.…”

Preparation and characterization of directed, one‐day‐self‐assembled millimeter‐size spheroids of adipose‐derived mesenchymal stem cells

“…In vivo , cells express tissue‐specific characteristics in a three‐dimensional (3D) microenvironment called “niche.” Once the cells are separated from their niche and cultured in vitro , they dedifferentiate and lose several‐tissue specific characteristics . Thus, 3D cell aggregates such as multicellular spheroids, mammospheres, or macromass have been utilized as niche‐mimicking models by using suspension culture technologies based on static systems using nonadhesive cell culture dishes and dynamic systems that incorporate spinner flasks with stirred devices or microgravity bioreactors . These culture systems have been used as the models for tumor growth, hepatic metabolism, and for drug screening and enabled cultivation of induced pluripotent stem cells, embryonic stem cells, and somatic stem cells …”