Normal human ectocervical and colorectal tissues were used. Polarized explant cultures were set-up as previously described [29] (link), [30] (link). Briefly, the explant was placed with the luminal side up in a transwell. The edges around the explant were sealed with Matrigel™ (BD Biosciences, San Jose, CA). The explants were maintained with the luminal surface at the air-liquid interface. The lamina propria was immersed in medium for ectocervical explants or resting on medium-soaked gelfoam for colorectal explants. Cultures were maintained at 37°C in a 5% CO2 atmosphere.
The explants were prepared on day of surgery in duplicate. To ensure even spread of the gels and to allow it to be mixed with HIV-1 for the efficacy testing (below), a 1∶5 dilution of tenofovir or vehicle control gels was applied to the apical side of the explants for 18 h. As controls, explants were untreated or a 1∶5 dilution of 3% N9 gel was applied apically. The next day, explants were washed and viability was evaluated using the MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] assay and histology [29] (link), [30] (link).
The explants were prepared on day of surgery in duplicate. To ensure even spread of the gels and to allow it to be mixed with HIV-1 for the efficacy testing (below), a 1∶5 dilution of tenofovir or vehicle control gels was applied to the apical side of the explants for 18 h. As controls, explants were untreated or a 1∶5 dilution of 3% N9 gel was applied apically. The next day, explants were washed and viability was evaluated using the MTT [1-(4,5-dimethylthiazol-2-yl)-3,5-diphenylformazan] assay and histology [29] (link), [30] (link).
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