Blistering in keratinocyte cultures: a regular phenomenon associated with differentiation.
Blisters have previously been observed in keratinocyte cultures depleted of vitamin A, and in cultures of keratinocytes from patients with epidermolysis bullosa. We have found that blistering may occur in keratinocyte cultures from normal human epidermis, grown under standard conditions, and our aim was to further characterize the mechanism of blister formation. Keratinocytes were seeded at 10(5) cells per 35 mm collagen-coated dish with a 3T3 feeder layer. Blisters were macroscopic, fluid-filled structures which formed irrespective of donor site, or donor age, and were noted on various alternative substrates (collagen, 3T3 + plastic, plastic alone). Blistering commenced around day 12, prior to confluency, and new blisters were formed for up to 5 weeks post-plating. Maximal numbers (up to 70 per dish) were present around days 12 to 20. Cleavage occurred at the cell/collagen interface to form a blister roof composed of 6 to 9 cell layers. The lowest layer appeared metabolically active, but, in contrast to peri-blister regions, lacked hemidesmosomes. The central 2 to 3 layers contained membrane-coating granules and keratohyalin granules while the superficial strata resembled rudimentary corneocytes. Cultures supplemented with 10(-5) M vitamin A formed no blisters, which correlated with suppressed differentiation. Ouabain (10(-7) M) caused blister collapse and a reversible inhibition of new blister formation. We conclude that blisters are a consistent finding in keratinocyte cultures grown under standard conditions. Their formation may be associated with active transport and triggered during differentiation. Further examination of this phenomenon might shed light on whether differentiation itself has an influence on keratinocyte attachment to substrate.
S J
SJ
Chapman
R A
RA
Eady
D000328:Adult; D000367:Age Factors; D001768:Blister / Q000473:pathology* / Q000503:physiopathology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D000078404:Epidermal Cells; D004817:Epidermis / Q000502:physiology / Q000648:ultrastructure; D004820:Epidermolysis Bullosa / Q000473:pathology / Q000503:physiopathology; D006801:Humans; D007633:Keratins; D007811:Lanthanum / Q000494:pharmacology; D008297:Male; D008854:Microscopy, Electron; D010042:Ouabain / Q000494:pharmacology; D014801:Vitamin A / Q000494:pharmacology
D014801:Vitamin A; D010042:Ouabain; D007633:Keratins; D007811:Lanthanum