Living skin equivalent (LSE) was used to identify nonvascular aspects of freeze-thaw injury to human skin cells. Pairs of transwell cell culture inserts, containing LSE, were washed twice for 30 min in maintenance media at 37 degrees C in a CO2 incubator and placed in two wells of a six-well assay tray (containing assay media). One specimen was maintained at room temperature. The other was cooled at 1 degrees C/min to -15 degrees C and rapidly rewarmed. Both were incubated for 24 h on fresh maintenance media at 37 degrees C in a CO2 incubator. This process was repeated producing 12 per group. Prostaglandin E2 (PGE2), interleukin-1 alpha (IL-1 alpha), and K+ were measured (mean +/- SE) in the assay medium, after the rewarming interval, and in the maintenance media after the 24-h incubation. Specimens were then processed for electron microscopy. After the rewarming interval, PGE2 (164.0 +/- 23.2 pg/0.1 ml), IL-1 alpha (24.3 +/- 2.2 pg/0.1 ml), and K+ (6.47 +/- 0.41 mM) released from frozen LSE were significantly increased compared to controls (PGE2 = 22.2 +/- 4.6 pg/0.1 ml, IL-1 alpha = 5.7 +/- 1.0 pg/0.1 ml, K+ = 4.32 +/- 0.02 mM). After the 24-h incubation, PGE2 and IL-1 alpha released by frozen LSE (PGE2 = 1126 +/- 208 pg/0.1 ml; IL-1 alpha = 80.6 +/- 6.8 pg/0.1 ml) remained significantly higher than controls (PGE2 = 229.0 +/- 45 pg/0.1 ml; IL-1 alpha = 4.9 +/- 0.7 pg/0.1 ml). At this time, K+ leakage from frozen LSE (4.39 +/- 0.03 mM) had returned to a normal range (control = 4.65 +/- 0.02 mM). Keratinocytes and, to a lesser extent, fibroblasts showed ultrastructural freeze-thaw damage.(ABSTRACT TRUNCATED AT 250 WORDS)