Serial passaging has a profound effect on primary cells. Since serially passaged cells show signs of cellular aging, serial passaging is used as an in vitro model of aging. To relate the effect of in vitro aging more to in vivo aging, we generated human skin equivalents (HSEs). We investigated if HSEs generated with late passage fibroblasts show characteristics of aged skin when compared with HSEs generated with early passage fibroblasts. Late passage fibroblasts had enlarged cell bodies and were more often positive for myofibroblast marker α-smooth muscle actin, senescence associated β-galactosidase and p16 compared with early passage fibroblasts. Skin equivalents generated with late passage fibroblasts had a thinner dermis, which could partly be explained by increased matrix metalloproteinase-1 secretion. In equivalents generated with late passage fibroblasts epidermal expression of keratin 6 was increased, and of keratin 10 slightly decreased. However, epidermal proliferation, epidermal thickness and basement membrane formation were not affected. In conclusion, compared with HSEs generated with early passage fibroblasts, HSEs generated with late passage fibroblasts showed changes in the dermis, but no or minimal changes in the basement membrane and the epidermis.
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