Improved method for rhesus monkey or rat skin preparation and cryosectioning for topical or transdermal skin distribution studies

A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6mm punch biopsies from freshly excised, full-thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap-freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap-frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 mu m cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure.