Akademik Veri Yönetim Sistemi
26.ulusal elektron mikroskopi kongresi, Eskişehir, Türkiye, 20 - 23 Eylül 2023, sa.15, ss.93
Human skin equivalents (HSEs), being developed as alternatives to animal models that are physiologically
different from humans, have been used in studies related to wound healing, photoaging, skin
development, cancer, toxicology screening. However, a HSE that fully mimics the natural tissue has not
been constructed yet. One of the most important problems in the development of HSE is the selection of
the medium to be used for co-culturing more than one cell type. In this study, in order to optimize the coculture medium, human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs)
were embedded into the methacrylated gelatin (GelMA) hydrogel to serve as dermis, and human
keratinocytes (HKs) were seeded onto the dermis as the epidermis of the HSE.
The GelMA was produced via a chemical reaction between gelatin and methacrylic anhydride. HDFs
(6x106 cells/mL, passages 2-5) and HUVECs (3x106 cells/mL, passages 2-5) were coated with atelocollagen
(0.3 mg/mL) for 20 min at 37°C, to prevent excess water loss from the cells during the photocrosslinking.
The cells were subsequently resuspended in GelMA prehydrogel solution (8%w/v), and exposed to UV A
for 22 min. HUVECs (6.3x104 cells/cm2) were seeded on this cell-laden hydrogel and incubated in DMEMlow glucose(DMEM-LG):EGM-2 (1:1v/v) medium containing 5% human plasma (HP) for 26 days. After
incubation, HKs (6.3x105 cells/cm2) were seeded onto the HUVEC layer on the hydogel. The hydrogels
were incubated at four different media containing 5% HP (Cascade medium [CM], CM: DMEM-LG [1:1v/v],
CM: EGM-2 [1:1 v/v], CM: DMEM-LG: EGM-2 [1:1:1v/v/v]) for 14 days. The cells were immunostained with
anti-vimentin for HDFs, anti-CD31 for HUVECs, anti-cytokeratin5 for HKs, and finally counterstained with
DAPI. HSEs were examined using the confocal microscope (Zeiss LMS700).
Confocal micrographs showed that the best medium supporting formation of a complete epidermal layer
was CM:DMEM-LG:EGM-2 [1:1:1 v/v/v]. However, the epidermal cells with elongated morphology implied
that HKs dedifferentiated and underwent the epithelial-mesenchymal transition in this medium. In dermal
layer, this medium supported fibroblast growth and the formation of vessel-like structures.
It can be concluded that CM:DMEM-LG:EGM-2 [1:1:1v/v/v] medium can be used to develop especially a
dermis containing vessel-like structures.
Anahtar Kelimeler: Human skin equivalent, Human dermal fibroblast, Human keratinocyte, Human
umbilical vein endothelial cell, confocal microscopy