Abstract. Currently, culture and growth keratinocytes are important stages in achieving a reliable and reproducible skin tissue. In the present study, two different methods, enzymatic and explant methods, for keratinocytes isolation from human foreskin were compared. Foreskins were cut into 2-3 mm pieces and placed in trypsin at 4˚C overnight for separation of the epidermis from the dermis. Subsequently, these samples were divided into two groups: i) Keratinocytes separated from the epidermis by trypsin and ii) by the explant method. These keratinocytes were divided into two groups: i) With no feeder layer and ii) onto a type I collagen scaffold. The cells were evaluated using immunocytochemistry and 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining. In the enzymatic treatment, after 7-10 days no attached cells were found in the cell culture dishes. In the explant method, keratinocytes were separated after ~24 h, attached rapidly and formed big colonies into a collagen scaffold. In the absence of a feeder layer, small colonies were developed with rapid loss of proliferation within 2-3 days. Keratinocytes showed positive immunoreactivity for the pan-cytokeratin marker and keratinocytes' nuclei were clearly observed. This method could be applied and developed as a component of skin substitutes to treat burns and wounds and also in laboratory testing.
IntroductionThe skin is the largest organ in the body that is divided into two anatomically distinct regions, the dermis and epidermis. The normal structure and function of this organ is dependent on the intact epidermis anchored to its vascular, elastic dermis (1,2). Fibroblasts are the most prevalent cell types in the dermis, which produce different growth factors that induce proliferation of keratinocytes in vivo and in vitro (3). The principal cell type of the epidermis is the keratinocyte (1,4), which is a small epithelial cell, located at the top of the epidermal basal membrane and characterized by a low division rate (5,6).Thus far, various enzymatic methods for dermal-epidermal separation have been applied (6-8). For instance, trypsin separates suprabasal hemidesmosomes that causes the basal layer cells to attach to the dermal layer (6). Thermolysin is another enzyme that selectively separates desmosomes (5,9) and is able to separate the epidermis at the basal membrane zone level (5,10).Although the dissociation method of keratinocytes in primary culture is well-established, attempts to acquire purified adult stem-cell like⁄progenitor keratinocytes from whole human skin are still ongoing. In particular, different techniques are currently being applied to achieve high purity or homogeneous primary cultures enriched in keratinocyte progenitor⁄precursor cells. These include filtration, density gradient centrifugation and fluorescence-activated cell sorting using cell surface antibodies, as well as differential adhesion to enrich the cells that rapidly attach to particular substrates (11).A previous study showed that when keratinocytes/precursor cells...
