Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

Abstract

OBJECTIVES:Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days.METHODS:Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee.RESULTS:SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected.CONCLUSION:We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

Synovial fluid niche promoted differentiation of dental follicle mesenchymal stem cells toward chondrogenesis in rheumatoid arthritis

“…The third passage cells were trypsinized and placed into the 96 U-bottom well-plates each containing 5x10[ 5 ] cells in a 20-µL volume of DMEM supplemented with 10% FBS, and 1% penicillin/streptomycin (100 U/mL), as described previously. [ 13 ] The cells were, then, cultured for the initial 2 h at 37°C in a 5% CO 2 and 95% humidified chamber to obtain micromass cultures. After 2 h, 100 µL of DMEM, chondrogenesis differentiation medium (StemPro™; Thermo Fisher Scientific Inc., MA, USA) or with SF samples (1:1 v/v) or only SF samples were added to the micromass culture wells.…”

Cell spheroids as a versatile research platform: formation mechanisms, high throughput production, characterization and applications

“…In recent decades, in turn, the wide use of the term 'tissue spheroid' to designate the spheroid intended for research in the area of tissue engineering [39] is observed. Also noted is the term 'micromass' or 'high density micromass' , often used to describe high cell density 3D spherical cell cultures [40]. Regarding more complex spherical models, the term 'organoid' has been used, which involve various cell types and aim to mimic structures similar to mini-organs capable of executing a few more advanced roles such as hormone synthesis [3,41,42].…”