Open AccessA Reliable Protocol for the Isolation of Viable, Chondrogenically Differentiated Human Mesenchymal Stem Cells from High-Density Pellet Cultures
Author(s) -
Mujib Ullah,
Houda Hamouda,
Stefan Stich,
Michael Sittinger,
Jochen Ringe
Publication year - 2012
Publication title -
bioresearch open access
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.457
H-Index - 26
eISSN - 2164-7860
pISSN - 2164-7844
DOI - 10.1089/biores.2012.0279
Subject(s) - mesenchymal stem cell , collagenase , chondrogenesis , cartilage , extracellular matrix , stem cell , chemistry , microbiology and biotechnology , type ii collagen , chondrocyte , biology , biochemistry , anatomy , enzyme
Administration of chondrogenically differentiated mesenchymal stem cells (MSC) is discussed as a promising approach for the regenerative treatment of injured or diseased cartilage. The high-density pellet culture is the standard culture for chondrogenic differentiation, but cells in pellets secrete extracellular matrix (ECM) that they become entrapped in. Protocols for cell isolation from pellets often result in cell damage and dedifferentiation towards less differentiated MSC. Therefore, our aim was to develop a reliable protocol for the isolation of viable, chondrogenically differentiated MSC from high-density pellet cultures. Human bone marrow MSC were chondrogenically stimulated with transforming growth factor-β3, and the cartilaginous structure of the pellets was verified by alcian blue staining of cartilage proteoglycans, antibody staining of cartilage collagen type II, and quantitative real-time reverse-transcription polymerase chain reaction of the marker genes COL2A1 and SOX9. Trypsin and collagenases II and P were tested alone or in combination, and for different concentrations and times, to find a protocol for optimized pellet digestion. Whereas trypsin was not able to release viable cells, 90-min digestion with 300 U of collagenase II, 20 U of collagenase P, and 2 mM CaCl2 worked quite well and resulted in about 2.5×10(5) cells/pellet. The protocol was further optimized for the separation of released cells and ECM from each other. Cells were alcian blue and collagen type II positive and expressed COL2A1 and SOX9, verifying a chondrogenic character. However, they had different morphological shapes. The ECM was also uniformly alcian blue and collagen type II positive but showed different organizational and structural forms. To conclude, our protocol allows the reliable isolation of a defined number of viable, chondrogenically differentiated MSC from high-density pellet cultures. Such cells, as well as the ECM components, are of interest as research tools and for cartilage tissue engineering.