Sains Malaysiana 51(5)(2022): 1425-1436

http://doi.org/10.17576/jsm-2022-5105-13

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

(Pencirian Keratinosit, Fibroblas dan Melanosit Dipencilkan daripada Kulit Manusia menggunakan Penanda Gen)

PAU-RING WONG¹, NUR FATIN NABILAH MOHD SAHARDI¹, JEN-KIT TAN¹, KIEN-HUI CHUA², WAN ZURINAH WAN NGAH¹ & SUZANA MAKPOL^1,*

¹Department of Biochemistry, Faculty of Medicine, Level 17, Preclinical Building, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

²Department of Physiology, Faculty of Medicine, Level 17, Preclinical Building, Universiti Kebangsaan Malaysia, Kuala Lumpur Campus, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Kuala Lumpur, Federal Territory, Malaysia

Received: 24 July 2021/Accepted: 16 September 2021

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.

Keywords: Epidermis; fibroblasts; gene markers; keratinocytes; melanocytes

Abstrak

Sel yang dipencilkan daripada kulit mempunyai kegunaan meluas dalam kajian patogenesis penyakit berkaitan dengan kulit serta pembinaan setara kulit 3D. Kajian ini bertujuan untuk memencilkan sel keratinosit, fibroblas dan melanosit daripada kulit manusia serta mencirikan ketulenan jenis sel tersebut. Sel keratinosit, fibroblas dan melanosit telah dipencilkan dengan cara pentripsinan pembezaan dan pemilihan media. Sementara itu, ketulenan jenis sel telah dicirikan melalui ekspresi penanda gen. Pengekspresan penanda gen melibatkan pengekstrakan RNA, mereka bentuk primer, tindak balas rantai polimerase (qPCR) kuantitatif dan pewarnaan imunositokimia. Hasil yang diperoleh menunjukkan fibroblas di dalam ko-kultur sel keratinosit-fibroblas yang dipencilkan daripada lapisan dermis boleh diasingkan daripada keratinosit melalui proses pentripsinan pantas dan dikulturkan di dalam medium Eagle Pengubahsuaian Dulbecco, manakala sel keratinosit yang berbaki dikulturkan di dalam medium Epilife. Sel melanosit di dalam ko-kultur melanosit-keratinosit yang dipencilkan daripada lapisan epidermis boleh dipilih dengan menukarkan medium Epilife kepada medium M254. Keputusan penanda gen mencadangkan bahawa gen sitokeratin 14 (CK14) adalah penanda gen yang sesuai untuk sel keratinosit dan gen elastin (ELN) sesuai digunakan untuk sel fibroblas. Sementara itu, gen tirosin (TYR) dan tirosinase berkaitan protein 1 dan 2 (TYRP1 dan TYRP2) sesuai digunakan untuk sel melanosit. Secara kesimpulannya sel keratinosit, fibroblas dan melanosit boleh dipencilkan daripada sumber kulit manusia yang sama melalui proses pentripsinan pembezaan dan teknik pemilihan media; serta dicirikan melalui penanda gen yang sesuai. Hasil kajian ini boleh membantu dalam memencilkan jenis sel kulit tulen untuk pelbagai kegunaan dalam perubatan regeneratif dan kajian kesitotoksikan.

Kata kunci: Epidermis; fibroblas; keratinosit; melanosit; penanda gen

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^*Corresponding author; email: suzanamakpol@ppukm.ukm.edu.my