@misc{10481/91097,
year = {2023},
month = {9},
url = {https://hdl.handle.net/10481/91097},
abstract = {For the development of advanced therapies, the use of primary cells instead of cell lines is
preferred. The manufacture of human tissue-engineered skin substitutes requires efficient isolation
and culture protocols allowing a massive expansion of the cells in culture from an initial specimen of a
minimal size. This study compared two skin cell isolation protocols, routinely applied in two clinical
laboratories. Epithelial (keratinocytes) and dermal (fibroblasts) cells were isolated and cultured
from three human skin biopsies (N = 3). The two-step digestion protocol (LOEX-Protocol) firstly
used thermolysin to enzymatically disrupt the dermal–epidermal junction while, for the one-step
digestion protocol (UPCIT-Protocol), mechanical detachment with scissors was applied. Then, the
epidermal and dermal layers were digested, respectively, to achieve cell isolation. The cell size,
viability, yield and growth were analyzed over five passages (P). The colony-forming efficiency (CFE)
and Keratin 19 (K19) expression of epithelial cells were also assessed after P0 and P1. Regarding the
dermal cells, no significant differences were observed in the tested parameters of isolation and culture.
However, for the epithelial cells, viability was higher (93% vs. 85%) and the number of cells extracted
per cm2 of skin was 3.4 times higher using the LOEX-Protocol compared to the UPCIT-Protocol.
No significant difference was observed for any parameter once the keratinocytes were cultured from
P1 to P4. The CFE and K19 expression decreased from P0 to P1 in both protocols, probably due to the
culture process. This study shows that both protocols enable the efficient isolation of skin dermal and
epithelial cells and subsequent culture to produce grafts destined for the treatment of patients.},
organization = {Instituto de Salud Carlos III (Spain) through the
project PI17/02083 (co-funded by the European Regional Development Fund “A way to make
Europe”)},
organization = {Regional Government of Andalusia (Spain) (PIGE-0242-2019)},
organization = {Canadian Institutes
for Health Research (CIHR) (FDN-143213 and IC-132948)},
organization = {Fondation des Pompiers du Québec
pour les Grands Brûlés (FPQGB)},
organization = {Quebec Network for Cell, Tissue and Gene Therapy—
ThéCell (a thematic network supported by the Fonds de recherche du Québec—Santé (FRQS))},
organization = {Tier 1 Canadian Research Chair in Stem Cells and Tissue Engineering and a
Research Chair on Tissue-Engineered Organs and Translational Medicine from the Fondation de l’Université Laval},
organization = {Postdoctoral fellowship from FRQS
(Canada)},
organization = {Doctoral fellowship from FRQS
(Canada)},
organization = {Predoctoral fellowship (BOE 05/01/2018) funded by the Instituto de Salud Carlos III (Spain) (co-funded by the European Social Fund “Investing in your future”) with the dossier number FI18/00269},
organization = {Mobility aid (BOE 26/12/2019) funded by the Instituto de Salud Carlos III (Spain) (co-funded by the European Social Fund “Investing in your future”) with the dossier number MV20/00043},
publisher = {MDPI},
keywords = {Skin cell isolation},
keywords = {CFE},
keywords = {Fibroblast},
title = {Comparison of Two Human Skin Cell Isolation Protocols and Their Influence on Keratinocyte and Fibroblast Culture},
doi = {10.3390/ijms241914712},
author = {Sierra Sánchez, Álvaro and Barbier, Martin A. and Magne, Brice and Larouche, Danielle and Arias Santiago, Salvador Antonio and Germain, Lucie},
}