RRC ID 43636
Author Oze H, Hirao M, Ebina K, Shi K, Kawato Y, Kaneshiro S, Yoshikawa H, Hashimoto J.
Title Impact of medium volume and oxygen concentration in the incubator on pericellular oxygen concentration and differentiation of murine chondrogenic cell culture.
Journal In Vitro Cell Dev Biol Anim
Abstract Previous studies have demonstrated that oxygen environment is an important determinate factor of cell phenotypes and differentiation, although factors which affect pericellular oxygen concentration (POC) in murine chondrogenic cell culture remain unidentified. Oxygen concentrations in vivo were measured in rabbit musculoskeletal tissues, which were by far hypoxic compared to 20% O(2) (ranging from 2.29 ± 1.16 to 4.36 ± 0.51%). Oxygen concentrations in murine chondrogenic cell (C3H10T1/2) culture medium were monitored in different oxygen concentrations (20% or 5%) in the incubator and in different medium volumes (3,700 or 7,400 μl) within 25-cm(2) flasks. Chondrogenic differentiation was assessed by glycosaminoglycan production with quantitative evaluation of Alcian blue staining in 12-well culture dishes. Expression of chondrogenic genes, aggrecan, and type II collagen α1, was examined by quantitative real-time polymerase chain reaction. Oxygen concentrations in medium decreased accordingly with the depth from medium surface, and POC at Day 6 was 18.99 ± 0.81% in 3,700-μl medium (1,480-μm depth) and 13.26 ± 0.23% in 7,400-μl medium (2,960-μm depth) at 20% O(2) in the incubator, which was 4.96 ± 0.08% (1,480-μm depth) and 2.83 ± 0.42% (2,960-μm depth) at 5% O(2), respectively. The differences of POC compared by medium volume were statistically significant (p = 0.0003 at 20% and p = 0.001 at 5%). Glycosaminoglycan production and aggrecan gene expression were most promoted when cultured in moderately low POC, 1,000 μl (2,960-μm depth) at 20% O(2) and 500 μl (1,480-μm depth) at 5% O(2) in 12-well culture dishes. We demonstrate that medium volume and oxygen concentration in the incubator affect not only POC but also chondrogenic differentiation.
Volume 48(2)
Pages 123-30
Published 2012-2-1
DOI 10.1007/s11626-011-9479-3
PMID 22259015
MeSH Aggrecans / biosynthesis Animals Cell Culture Techniques* Cell Differentiation Cell Hypoxia Cell Line Cell Proliferation Chondrocytes / cytology Chondrocytes / metabolism* Chondrogenesis* Collagen Type II / biosynthesis Culture Media* Female Glycosaminoglycans / biosynthesis Mice Mice, Inbred C3H Oxygen* Rabbits
IF 1.665
Times Cited 13
Human and Animal Cells 10T1/2(RCB0247)