Accuracy of Three Techniques to Determine Cell Viability in 3D Tissues or Scaffolds
Accuracy of Three Techniques to Determine Cell Viability in 3D Tissues or Scaffolds
复制标题
DOI：
10.1089/ten.tec.2008.0313
复制
发表时间：
2008-12-01
影响因子：
3
通讯作者：
Ito, Keita
中科院分区：
文献类型：
--
作者：
Gantenbein-Ritter, Benjamin;Potier, Esther;Ito, Keita
几种不同的测定方法通常用于评估组织内细胞或三维载体的存活率，但其准确性和可靠性尚未得到评估。在这里，我们比较了三种测定细胞活力（CV）的方法：(i)低温切片乳酸脱氢酶（LDH）染色，（ii）钙黄蛋白AM/乙二酸同二聚体-1 （CaAM/EthH）染色，以及（iii）载体消化和台盼蓝（TB）测定。活细胞群和死细胞群从牛软骨细胞中产生，并结合产生大约0%，25%,50%，75%和100% CV混合物。在将细胞植入纤维蛋白载体之前，用TB试验（MIX）测量CV比。然后用三种方法测定CV （n = 5/method）。使用自定义编写的宏处理LDH-和CaAM/ ethh染色图像，TB方法使用血细胞计手工计数。采用绝对误差和类内相关性（ICC）进行准确性和可靠性评价。所有方法估计的CV值都接近MIX值。TB法最准确（ICC=0.99），其次是CaAM/EthH法（ICC=0.98）和LDH法（ICC=0.97）。对于活细胞和死细胞的绝对定量，TB和LDH方法表现良好（ICC=0.75-0.96），而CaAM/EthH方法严重高估了细胞数量（活细胞，ICC=0.30；死细胞，ICC=0.30）。虽然TB是最准确的，但LDH和CaAM/EthH提供了关于细胞形状和细胞在组织或支架中的空间分布的有价值的信息。
Several different assays are commonly used to evaluate survival of cells inside tissues or three-dimensional carriers, but their accuracy and reliability have not been evaluated. Here, we compare three methods for cell viability (CV) determination: (i) lactate dehydrogenase (LDH) staining on cryosections, (ii) calcein AM/ethidium homodimer-1 (CaAM/EthH) staining, and (iii) carrier digestion and trypan blue (TB) assay. Living and dead cell populations were generated from bovine chondrocytes and combined to produce approximately 0%, 25%, 50%, 75%, and 100% CV mixtures. CV ratios were measured with TB assay (MIX) before seeding cells into fibrin carriers. CV was then determined using the three methods (n = 5/method). Custom-written macros were used to process LDH- and CaAM/EthH-stained images, and hand counting with hemocytometer was used for the TB method. Absolute error and intraclass correlation (ICC) were used for accuracy and reliability evaluation. All methods estimated CV values close to MIX values. TB method was the most accurate (ICC=0.99) followed by CaAM/EthH (ICC=0.98) and LDH (ICC=0.97). As for absolute quantification of living and dead cells, TB and LDH methods performed well (ICC=0.75-0.96), whereas CaAM/EthH largely overestimated cell numbers (living, ICC=0.30; dead, ICC=0.30). Although TB was the most accurate, LDH and CaAM/EthH provide valuable information on cell shape and spatial distribution of cells in tissue or a scaffold.