Extraction of Primary Human Keratinocytes and Fibroblasts from Adult Foreskin

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Suvara K Wattanapitayakul
Khwandow Kunchana
Linda Chularojmontri
Wattanased Jarisarapurin
Wasan Sedtawong

Abstract

Currently, the extraction of skin keratinocytes and fibroblasts for 2D and 3D cultures is an important tool for research in dermatology, including studies of activity and toxicity properties of agents that are in the process of skin product development. The objective of this study was to extract keratinocytes and fibroblasts from adult foreskin for research purpose. The foreskin samples were obtained from volunteer subjects who underwent foreskin surgery at Lerdsin Hospital. Following DPBS washing step, the skin samples were trimmed off fat and connective tissues. The foreskin was cut into small pieces and digested with dispase. The epidermis and dermis layers were separated and the epidermis layers were then trypsinized to obtain keratinocytes while the dermis was placed onto culture dish to extract fibroblasts. The keratinocytes in passage 1 and 2 grew normally in specific keratinocyte culture media. However, cells at passage 3 showed upward proliferation; thus, prohibiting further culture to increase cell number. On the other hand, fibroblasts proliferated well in normal culture media. In summary, the extraction of keratinocytes from adult foreskin is suitable for short-term cell culture experiment. If higher cell number is desired, pooled samples should be performed in order to obtain adequate cells for 3D skin model construction. In contrast, the extraction and cultivation of primary fibroblasts were easy for general cell culture laboratory.

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Section
Research Articles
Author Biography

Suvara K Wattanapitayakul, Department of Pharmacology Faculty of Medicine Srinakharinwirot University 114 Sukhumvit 23, Wattana Bangkok 10110

Associate Professor Dr.

References

Kim HB, Yoo BS. Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS. Toxicological research2016 Oct;32(4):345-51.

Duval C, Cohen C, Chagnoleau C, Flouret V, Bourreau E, Bernerd F. Key regulatory role of dermal fibroblasts in pigmentation as demonstrated using a reconstructed skin model: impact of photo-aging. PloS one2014;9(12):e114182.

Guerret S, Govignon E, Hartmann DJ, Ronfard V. Long-term remodeling of a bilayered living human skin equivalent (Apligraf) grafted onto nude mice: immunolocalization of human cells and characterization of extracellular matrix. Wound repair and regeneration : official publication of the Wound Healing Society [and] the European Tissue Repair Society2003 Jan-Feb;11(1):35-45.

Black AF, Hudon V, Damour O, Germain L, Auger FA. A novel approach for studying angiogenesis: a human skin equivalent with a capillary-like network. Cell biology and toxicology1999 Apr;15(2):81-90.

Claus S, Fischer J, Megarbane H, Megarbane A, Jobard F, Debret R, et al. A p.C217R mutation in fibulin-5 from cutis laxa patients is associated with incomplete extracellular matrix formation in a skin equivalent model. The Journal of investigative dermatology2008 Jun;128(6):1442-50.

Aasen T, Izpisua Belmonte JC. Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. Nature protocols2010 Feb;5(2):371-82.

Lowenau LJ, Zoschke C, Brodwolf R, Volz P, Hausmann C, Wattanapitayakul S, et al. Increased permeability of reconstructed human epidermis from UVB-irradiated keratinocytes. European journal of pharmaceutics and biopharmaceutics : official journal of Arbeitsgemeinschaft fur Pharmazeutische Verfahrenstechnik eV2017 Jul;116:149-54.

Liu SC, Karasek M. Isolation and growth of adult human epidermal keratinocytes in cell culture. The Journal of investigative dermatology1978 Aug;71(2):157-62.

Soroka Y, Ma'or Z, Leshem Y, Verochovsky L, Neuman R, Bregegere FM, et al. Aged keratinocyte phenotyping: morphology, biochemical markers and effects of Dead Sea minerals. Experimental gerontology2008 Oct;43(10):947-57.

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