Isolation and culture of bone marrow mesenchymal stem cells by red blood cell lysis method
- VernacularTitle:红细胞裂解法分离及培养大鼠骨髓间充质干细胞
- Author:
Jinping DENG
;
Zhongquan DAI
;
Shou LIU
;
Jielin NIE
;
Yinghui LI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2007;11(3):579-582,封3
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are few in bone marrow, and they are easily mingled with other cells, especially red blood cells. Therefore, intervention needs to be depleted in the process of isolation of red blood cells so as to obtain highlypurified BMSCs as many as possible.OBJECTIVE: To isolate and culture BMSCs of rats with red blood cell lysis method, and perform biological identification.DESIGN: Observation and controlled trial.SETTING: Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts.MATERIALS: Fifty 30-day-old male SD rats, weighing about 100 g, of SPF degree, were purchased from Beijing Experimental Animal Center (License No. SCXK (Jing) 2002-0003) and involved in this trial. DAB condensed chromogen (hydrogen dioxide included, Zhongshan Company), rabbit anti-ret polyclonal antibody (Boster Co.,Ltd., Wuhan), fetal calf serum (PAA, Austria) and LG-DMEM medium (Sigma Company, USA) were used in this trial.METHODS: This trial was carried out in the Laboratory of Aerospace Cell Molecular Biology, Scientific Research Training Center for Chinese Astronauts during September 2004 to September 2005. The rats were sacrificed by dislocation to expose bone marrow cavity. Cell suspension was collected. BMSCs were isolated and cultured primarily by whole bonemarrow culture method. ① Red blood cells lysis test: A, B, C and D 4 tubes were chosen and filled with 0.5 mL bonemarrow rinse solution which was filtered and fully beat upon. Then, red blood cell lysis buffer of 2 mL, ammonium chloride of 2 mL, phosphate buffer normal saline of 2 mL and 0.04 volume fraction acetic acid of 0.5 mL were correspondingly added into the 4 tubes. In each tube, absorbance and hemoglobin concentration were measured and cell growth was observed. ② Observation of growth curve, doubling time and surface marker molecule expression of BMSCs: Based on the formula, population doubling time (TD) =t[lg2/(lgNt-lgN0)] (NO and Nt represented the cell number after inoculation and t hours after culture ,respectively), cell population doubling time was calculated and traced and growth curve of the 2nd, 4th and 6th generations of BMSCs were analyzed; The proliferation of BMSCs was measured by methylene blue staining method; Surface marker molecule expression of BMSCs was detected with immunocytochemical staining.MAIN OUTCOME MEASURES: ①Observation of isolation and culture of bone marrowmesenchymal stems of rats. ②Effect of different methods on the lysis of red blood cells and the growth of BMSCs. ③ The growth curve and cell doubling time of the 2nd, 4th and 6th generations of cells. ④Surface marker molecule expression of BMSCs of rats.RESULTS: ① Results of isolation and culture of BMSCs of rats: After 48-hour primary culture, most of the cells had adhered to the wall, and 72 hours later, division growth of the adherent cells presented. Seven to eight days later, cell colonies formed obviously, and then increased quickly, expanded incessantly and fused with each other. On 14 to 16 days, cell clones grew densely. Immediately generative cells presented ball-shape, subsided and adhered the wall verysoon. Some few round cells suspended. Adherent cells distributed evenly and proliferated quickly within 3 to 5 days. Although cell morphology of generative cells did not change after passage, cell proliferation was speeded up obviously and cells covered the bottom of the whole bottom on about 6 days. ② Effect of different methods on red blood cell lysis and the growth of BMSCs: hemoglobin concentration in the red blood cell lysate-treated group, ammonium chloride-treated group and 4％ acetic acid-treated group was significantly higher than that in the phosphate buffer normal saline-treated group, with significant difference (P ＜ 0.01). ③ Observation of growth curve of different generations of BMSCs: The growth curves of the 2nd, 4th and 6th generations of the cells were basically the same: latent period about 1 to 2 days, then logarithmic growth phase, peak on the 5th day and finally plateau phase (about on the 5th to 7th days). The latent period of the 6th generation of BMSCs was not obvious and the population doubling time of BMSCs was about 34 hours. ④ldentification of immunophenotype of different generations of BMSCs: Both CD44 and CD106 staining of each generation of cells were positive, presenting brown granule sediments, and CD34 staining was negative.CONCLUSION: Inoculation of red blood cells lysate-treated bone marrow rinse solution can boost the adherent rate of BMSCs, and does not influence its post-adherent growth, so it is a feasible separation method. Cell surface marker staining confirms that thecells isolated in this study are BMSCs.
