Skin is the largest organ in vertebrates that is of great importance and performs as a protective barrier against the external world. It serves various functions such as protecting against external insults, fluid homeostasis, self-healing, and sensory detection. Skin disorders impose major financial and social burden. Therefore, the regeneration potential of the skin is an important area of research in tissue engineering (TE). This potential is due to the stem cells; they play a vital role in the skin regeneration process, since one of the main characteristics of the stem cells is their ability to differentiate into the organ specific specialized cells. Numerous factors such as growth factors, topography, etc. are involved in stem cell differentiation. In the current study, primary human keratinocytes were isolated from the foreskin samples and cultured. Then, the substrate was developed using PDMS (polydimethylsiloxane) silicon following keratinocyte fixation. This substrate can be used in further stem cell culture and differentiation studies.