Evaluation of chondrocyte survival in situ using WST-1 and membrane integrity stains
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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.
KeywordsChondrocytes Cryopreservation Metabolic function Membrane integrity stains Articular cartilage
analysis of variance
chondrocyte growth medium
membrane integrity stain
phosphate buffered saline
statistical package for the social sciences
The authors would like to acknowledge Sturgeon Valley Pork Ltd for supplying the pig joints. The authors acknowledge Dr. Lauren Beaupre for statistical analysis. JAW Elliott holds a Canada Research Chair in Interfacial Thermodynamics. This research was funded by the Canadian Institutes of Health Research.
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