机械法与机械-酶消化法制备大鼠膈肌组织单细胞悬液的比较

Preparing single cell suspension of rat diaphragm tissues: mechanical grinding versus mechanical-enzymatic digestion

背景:流式细胞术作为目前先进的细胞分析技术,其研究基础是单细胞悬液,但目前尚无有关膈肌组织单细胞悬液制备方法的相关报道。目的:探索应用机械法与机械-酶消化法分别制备大鼠膈肌单细胞悬液的可行性并比较2种方法获得单细胞的数量和活性。方法:以SD大鼠的新鲜膈肌组织为标本,在机械法的基础上,选用胰蛋白酶、胶原酶Ⅰ、胶原酶Ⅱ、胶原酶Ⅳ及其不同组合进行消化制备膈肌单细胞悬液,观察细胞形态并经锥虫蓝染色后测定细胞活性,对活细胞、失活细胞和细胞团块进行计数并计算出细胞存活率和单细胞悬液浓度,结果进行统计学比较分析。结果与结论:①机械-酶消化法所制得的单细胞悬液较机械研磨法细胞分散度好、形态完整、边界清楚、杂质及细胞碎片较少、背景较干净;②单纯机械研磨法制备的单细胞悬液活细胞数较低,失活细胞数较高,细胞存活率最低,团块较多;③在机械法的基础上,加入胰蛋白酶、胶原酶Ⅰ和胶原酶Ⅳ3种等体积混合酶所得单细胞悬液的活细胞数及悬液浓度最高,每0.1 g膈肌组织可获得(1.0-2.0)×106个细胞,细胞存活率较高,与单纯机械法获得单细胞悬液比较,在活细胞、失活细胞、细胞团块、细胞存活率及悬液浓度方面差异均有显著性意义(P<0.05);其次是加入胰蛋白酶和胶原酶Ⅳ这2种等体积混合酶消化法,所得单细胞悬液浓度、细胞成活率及细胞团块方面也均较为理想;④结果表明,机械法和机械-酶消化法均能成功制备出大鼠膈肌单细胞悬液,机械-酶消化法优于单纯机械法,其中加入胰蛋白酶、胶原酶Ⅰ和胶原酶Ⅳ3种等体积混合酶的机械-酶消化法所得单细胞悬液效果最佳,是较优选的膈肌单细胞悬液制作方法。

BACKGROUND: Flow cytometry is currently the advanced cell analysis technique with single cell suspension as a research basis, but there is no report on the preparation method of single cell suspension of diaphragm tissue. OBJECTIVE: To explore the feasibility of preparing single cell suspension of rat diaphragm tissue by mechanical grinding method and mechanical-enzymatic digestion method and to compare cell number and viability of the cells obtained using different methods. METHODS: The fresh diaphragm tissues of Sprague-Dawley rats were harvested. Based on the mechanical method, trypsin, collagenase I, collagenase II, collagenase IV and their different combinations were used to digest and prepare the single cell suspension of diaphragm tissues. Cell morphology was observed;cell number and viability were determined by trypan blue staining. The living cells, inactivated cells, and cell aggregates were counted, and cell survival rate and concentration of the single cell suspension were calculated. Statistical analyses were performed thereafter. RESULTS AND CONCLUSION: (1) The single cell suspension with better cell dispersion, more complete morphology, clearer boundary, fewer impurities and cell debris, and cleaner background were obtained by mechanical-enzymatic digestion compared with mechanical grinding method. (2) The single cell suspension prepared by simple mechanical grinding method has low number of living cells, high number of inactivated cells, low survival rate and many cell aggregates. (3) The number of living cells and concentration of single cell suspension obtained by same-volume addition of trypsin, collagenase I and collagenase IV mixed enzymes based on the mechanical grinding method were the highest, with (1.0-2.0)×10 6 cells per 0.1 g diaphragm tissue. There were significant differences between mechanical-enzymatic digestion and mechanical grinding method in terms of living cells, inactivated cells, cell aggregates, cell survival rate and suspension concentration (P < 0.05). Moreover, the single cell suspension prepared by the same-volume addition of trypsin and collagenase IV had higher suspension concentration, higher cell survival rate, and less inactivated cells and cell aggregates. To conclude, the single cell suspension of diaphragm tissues could be prepared successful by both mechanical and mechanical-enzyme digestion methods. Mechanical-enzyme digestion is superior to simple mechanical grinding method, with the best single cell suspension after same-volume addition of trypsin, collagenase I and collagenase IV. This is the preferred method for preparation of single cell suspension of the diaphragm tissue.