Optimized dissociation protocol for isolating human glioma stem cells from tumorspheres via fluorescence-activated cell sorting.
Fluorescence-activated cell sorting (FACS) based on the surface marker CD133 is the most common method for isolating glioma stem cells (GSCs) from heterogeneous glioma cell populations. Optimization of this method will have profound implications for the future of GSC research. Five commonly used digestion reagents, Liberase-TL, trypsin, TrypLE, Accutase, and non-enzymatic cell dissociation solution (NECDS), were used to dissociate glioma tumorspheres derived from two primary glioma specimens (091214 and 090116) and the cell lines U87 and T98G. The dissociation time, cell viability, retention of CD133, and stemness capacity were assessed. The results showed that single cells derived from the Liberase-TL (200 µg/ml) group exhibited high viability and less damage to the antigen CD133. However, the efficiency of NECDS for dissociating the tumorspheres into single cells was fairly low. Meanwhile, the use of this digestion reagent resulted in obvious cellular and antigenic impairments. Taken together, Liberase-TL (200 µg/ml) is an ideal reagent for isolating GSCs from tumorspheres. In contrast, the use of NECDS for such a protocol should be carefully considered.
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China.
Donglai
D
Lv
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China.
Qing-Hua
QH
Ma
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China.
Jiang-Jie
JJ
Duan
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China.
Hai-Bo
HB
Wu
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China.
Xi-Long
XL
Zhao
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China. Electronic address: yushicang@163.com.
Shi-Cang
SC
Yu
Institute of Pathology and Southwest Cancer Center, Southwest Hospital, Third Military Medical University, Chongqing 400037, China; Key Laboratory of Tumor Immunology and Pathology of Ministry of Education, Chongqing 400037, China. Electronic address: bianxiuwu@263.net.
Xiu-Wu
XW
Bian
D000071916:AC133 Antigen / Q000378:metabolism*; D000818:Animals; D014408:Biomarkers, Tumor / Q000378:metabolism*; D045744:Cell Line, Tumor; D000066673:Cell Self Renewal; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D017364:Collagenases / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005910:Glioma / Q000378:metabolism* / Q000473:pathology; D006801:Humans; D008819:Mice, Nude; D039902:Multipotent Stem Cells / Q000378:metabolism* / Q000473:pathology; D014411:Neoplastic Stem Cells / Q000378:metabolism* / Q000473:pathology; D010447:Peptide Hydrolases / Q000378:metabolism; D010641:Phenotype; D018874:Spheroids, Cellular; D013820:Thermolysin / Q000378:metabolism*; D013997:Time Factors; D014357:Trypsin / Q000378:metabolism; D047368:Tumor Burden
D000071916:AC133 Antigen; D014408:Biomarkers, Tumor; C000606023:PROM1 protein, human; C575294:accutase; D010447:Peptide Hydrolases; D014357:Trypsin; D017364:Collagenases; C106635:Liberase; D013820:Thermolysin
CD133; Cell dissociation; Flow cytometry; Glioma; Stem cell