Characterization of Keratinocytes, Fibroblasts and Melanocytes Isolated from Human Skin using Gene Markers

Abstract

Cells isolated from skin have wide applications in studies of the pathogenesis of skin-related diseases and the construction of 3D skin equivalents. This study aimed to isolate keratinocytes, fibroblasts, and melanocytes from human foreskin and characterize the purity of the cell types. Keratinocytes, fibroblasts, and melanocytes from human foreskin were isolated by differential trypsinization and media selection. The purity of the cell types was characterized based on the expression of gene markers. The assessment of gene marker expression involved RNA extraction, primer design, quantitative polymerase chain reaction (qPCR) and immunocytochemical staining. Our results showed that in cocultures of keratinocytes and fibroblasts isolated from the dermis, fibroblasts could be separated from keratinocytes by quick trypsinization and culture in Dulbecco’s modified Eagle’s medium. The remaining keratinocytes are cultured in Epilife medium. Melanocytes in cocultures of melanocytes and keratinocytes isolated from the epidermis could be selected by changing Epilife medium to M254 medium. Gene marker results suggested that cytokeratin 14 (CK14) is a suitable marker for keratinocytes, elastin (ELN) is a suitable marker for fibroblasts, and tyrosinase (TYR) and tyrosinase-related proteins 1 and 2 (TYRP1 and TYRP2) are suitable markers for melanocytes. In conclusion, keratinocytes, fibroblasts, and melanocytes can be isolated from the same human foreskin sample by differential trypsinization and media selection techniques and characterized by suitable gene markers. This finding will aid in the isolation of pure skin cell types for various applications in regenerative medicine and toxicity studies.

In Vitro Cell Proliferation and Migration Properties of Oral Mucosal Fibroblasts: A Comparative Study on the Effects of Cord Blood- and Peripheral Blood-Platelet Lysate

“…After 7–10 days, the cells were cultured until 70–80% confluence and underwent quick trypsinization to harvest the pure fibroblasts and leave the epithelial cells in the culture flask. Quick trypsinization was performed with incubated the cells through 2 mL 0.05% trypsin-EDTA (Gibco-BRL, USA) at 37 °C for 5 min [ 49 ]. Passage 2 HOMF was harvested and used for cell characterization via cell morphology observation and the mRNA expression of vimentin (fibroblast marker).…”

Contour Analysis of Three-Dimensional Peri-Implant Mucosal Model as an Endpoint Analysis of Photofunctionalization Effects on Implant Abutment Materials

“…On this account, the ascertainment of the specific type of cells that remain attached to various abutment surfaces after conducting the pull test would be the focus to further explore the effect of photofunctionalization on abutment surfaces, and the nature of the soft tissue attachment should be conducted. To accomplish this, the method involves conducting double immunocytochemistry staining for both human gingival fibroblast and oral epithelium in one specimen; for example, vimentin or α-SMA can be used to stain human gingival fibroblasts, and pancytokeratin or cytokeratin 14 for epithelial cells and gene markers [ 52 , 53 ]. In the same manner, double immunohistochemical staining can also be performed on the histological ground section of the soft tissue–implant interface in addition to the hematoxylin and eosin stain.…”

Effects of Platelet Lysate Gels Derived from Different Blood Sources on Oral Mucosal Wound Healing: An In Vitro Study

“…The culture medium was replenished every 2–3 days until the cells reached 70–80% confluency. After that, the cells were treated with 2 mL of 0.05% trypsin-EDTA (Gibco-BRL, USA) at 37 °C for 5 min to detach the HOMFs [ 43 ]. The epithelial cells remained in the culture flask because they required a longer time to detach from the surface of flask.…”