Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains

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 degrees 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 degrees C (in cryoprotectant solution to prevent ice formation), and (3) seven day storage at -10 degrees C (in cryoprotectant solution). Fluorescent MISs (Syto 13 and ethidium bromide) were used on 70 microm 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 microm 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.