Yuhan Chang1, Pang-Hsin Hsieh, Chuck C-K Chao. 1. Department of Orthopaedic Surgery, Chang Gung Memorial Hospital, Taipei, Chang Gung University College of Medicine, Taoyuan, Taiwan; Department of Biochemistry and Molecular Biology; Graduate Institute of Clinical Medicine; Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Abstract
BACKGROUND: Percoll and Ficoll density gradient media are widely used in the isolation of human mesenchymal stem cells (hMSCs) for research. However, the efficacy of the two methods in the isolation of hMSCs and their differentiation potentiality have not been evaluated. In this study, the ability of the two methods to isolate MSCs from human bone marrow was compared. METHODS: Colony forming unit-fibroblasts (CFU-Fs) are considered colonies of stem and progenitor cells. Thus it was proposed that MSCs be represented by CFU-Fs. Therefore, we compared the relative efficiencies of the two media, Ficoll and Percoll, in isolating colony forming unit-fibroblasts with alkaline phosphatase activity (CFU-F/ALP(+)) and the percentages of CD166(+)/CD34(-), CD90(+)/CD34(-), SH3(+)/CD34(-) and CD105(+)/CD34(-) cells in all nucleated cells from bone marrow (BM). RESULTS: A significantly higher number of nucleated cells could be isolated with Ficollthan with Percoll. The percentages of cells which were CD166(+)/CD34(-), CD90(+)/CD34(-), SH3(+)/CD34(-) and CD105(+)/CD34(-) were significantly higher for the Ficoll group. The colony-forming efficiency from Ficoll isolates (119 +/- 69 CFU-F/ALP(+) per dish) was also higher than that from Percoll isolates (46 +/- 35 CFU-F/ALP(+) per dish) (p < 0.01). However, the average colony size, percentage of CFU-Fs with ALP(+) and the differentiation abilities of CFU-Fs were not significantly different between groups. CONCLUSIONS: Ficoll and Percoll are both suitable but the Ficoll methodology is superior to that of Percoll in the preparation of hMSCs.
BACKGROUND: Percoll and Ficoll density gradient media are widely used in the isolation of human mesenchymal stem cells (hMSCs) for research. However, the efficacy of the two methods in the isolation of hMSCs and their differentiation potentiality have not been evaluated. In this study, the ability of the two methods to isolate MSCs from human bone marrow was compared. METHODS: Colony forming unit-fibroblasts (CFU-Fs) are considered colonies of stem and progenitor cells. Thus it was proposed that MSCs be represented by CFU-Fs. Therefore, we compared the relative efficiencies of the two media, Ficoll and Percoll, in isolating colony forming unit-fibroblasts with alkaline phosphatase activity (CFU-F/ALP(+)) and the percentages of CD166(+)/CD34(-), CD90(+)/CD34(-), SH3(+)/CD34(-) and CD105(+)/CD34(-) cells in all nucleated cells from bone marrow (BM). RESULTS: A significantly higher number of nucleated cells could be isolated with Ficollthan with Percoll. The percentages of cells which were CD166(+)/CD34(-), CD90(+)/CD34(-), SH3(+)/CD34(-) and CD105(+)/CD34(-) were significantly higher for the Ficoll group. The colony-forming efficiency from Ficoll isolates (119 +/- 69 CFU-F/ALP(+) per dish) was also higher than that from Percoll isolates (46 +/- 35 CFU-F/ALP(+) per dish) (p < 0.01). However, the average colony size, percentage of CFU-Fs with ALP(+) and the differentiation abilities of CFU-Fs were not significantly different between groups. CONCLUSIONS: Ficoll and Percoll are both suitable but the Ficoll methodology is superior to that of Percoll in the preparation of hMSCs.