Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains
Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains
复制标题
使用 WST-1 和膜完整性染色评估软骨细胞原位存活
作者：
N. Jomha;J. Elliott;G. Law;L. Mcgann
评估软骨细胞原位记录软骨保存技术的有效性已被证明是非常困难的。本研究旨在确定WST-1在冷却至-10 °C（无冰形成）后原位对猪软骨细胞的有效性，并与膜完整性染色剂（MIS）进行比较。在处死后24小时内，从性成熟猪中收获骨软骨钉（直径10 mm），并随机分为三组：（1）未处理对照组，（2）在-10 °C下储存1天（在冷冻保护剂溶液中以防止结冰），（3）在-10 °C下储存7天（在冷冻保护剂溶液中）。在70 μm切片上使用荧光MISs（Syto 13和溴化乙锭）。将代表性图像数字化，并将绿色和红色像素数确定完整细胞的回收百分比。使用分光光度计在450 nm处使用每个样品20个70 μm厚的切片测定线粒体活性（WST-1），以获得可靠的读数。在生长培养基中孵育0、3、24、48、72和144 h后，对所有样品进行了膜完整性和代谢活性的重复测量。WST-1始终高估细胞回收率，结果大于新鲜对照。在低温储存7天后，WST-1测量显示线粒体活性降低，到48小时恢复。当考虑到细胞密度，将“绝对”细胞回收率与原始对照进行比较时，MIS是最准确的。总之，WST-1可以随时间跟踪软骨细胞的代谢活性，但在48小时孵育后通过MIS确定的“绝对”细胞恢复可能是原位活软骨细胞数量的最准确测定。
Evaluating chondrocytes in situ to document the effectiveness of cartilage preservation techniques has proven exceedingly difficult. This study was conducted to determine the effectiveness of WST-1 on porcine chondrocytes in situ after cooling to −10°C (without ice formation) compared to membrane integrity stains (MIS). Osteochondral dowels (10 mm in diameter) were harvested from sexually mature pigs within 24 h of sacrifice and randomized into three groups: (1) untreated control, (2) one day storage at −10°C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at −10°C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 μm slices. Representative images were digitized and green and red pixel numbers determined the percent recovery of intact cells. Mitochondrial activity (WST-1) was determined using 20 slices of 70 μm thickness per sample to obtain reliable readings using a spectrophotometer at 450 nm. All samples underwent repeated measures of membrane integrity and metabolic activity obtained after 0, 3, 24, 48, 72, and 144 h incubation in growth media. WST-1 consistently overestimated cell recovery with results greater than fresh controls. After hypothermic storage for 7 days, the WST-1 measurement demonstrated decreased mitochondrial activity that recovered by 48 h. MIS was most accurate when “absolute” cell recovery was compared to original controls, taking into account cell density. In conclusion, WST-1 can track metabolic activity of chondrocytes in situ over time but “absolute” cell recovery determined by MISs after 48 h incubation may be the most accurate determination of the number of live chondrocytes in situ.
DOI：
10.2106/00004623-200311000-00008
发表时间：
2003-11-01
影响因子：
5.3
作者：
Williams, SK;Amiel, D;Bugbee, WD
通讯作者：
Bugbee, WD