ABSTARCT: The aim of this study was to phenotype determination of dermal-epidermal organotypics (DEpOs) made with a hypotetraploid human keratinocyte cell line known as HaCaT. Three matrices were used, polyethylene terephthalate, human fıbrin or type I and type III rat collagen mixture. Methods: Population doubling time and the mitogenic effect of several media were assessed by formazan production assay. Microanatomical features were evaluated by bright-field/phase-contrast microscopy and hematoxylin and eosin (H&E) staining. Keratinization development markers (i.e. Ki67, K14, K10, filaggrin and loricrin) were determined by indirect immunohistochemistry. Free fatty acids (FFAs) fraction was estimated by gas chromatography with fire ionization detector (GC-FID). Barrier function was estimated by transepithelial electrical resistance (TEER) as well as permeability to Lucifer yellow (LY) measuring. DNA content of HaCaT was determined by propidium iodide (PI) staining and flow cytometry. Findings: Used HaCaT need 16.92±2.03 hours to divide in used medium. Irrespective to matrix type, histology and immunohistochemistry DEpOs showed a poor stratified epidermis (3-5 layers). Flattened cell shape in all layers, no enucleation and a poor spatiotemporal expression of differentiation markers was obtained. All observed features were in accordance with parakeratotic keratinization. Poor FFA detected profile and low TEER values showed absence of a significantly barrier function which correlates with parakeratosis and results of permeability assay. HaCaT DNA content analysis by flow cytometry revealed the presence of at least two different populations. High genetic alterations events might provoke a decreased sensitivity of this cell line to differentiation factors (e.g. air-liquid raising, insulin, adenine, etc.), as was reported previously.