Culture environment affects sweat gland cells’ phenotype and their potential to regenerate sweat glands in tissue-engineered skin substitutes

Summary: Abstract Body: Severe burns are challenging to treat, and current tissue-engineered skin substitutes (TESs) lack skin appendages like sweat glands, which contribute to wound healing and are essential for thermoregulation and waste excretion. Our project aims to isolate, culture, and integrate sweat gland cells (SGCs) into TESs to restore functional sweat glands. Briefly, SGCs were isolated from human skin biopsies, cultured on feeder layers (2D) or as spheroids (3D), and integrated into TESs. The expression of skin and sweat gland-specific markers, such as aquaporin 5, keratin 5, and α-smooth muscle actin, was characterized by immunofluorescence and flow cytometry. Our findings revealed that 2D culture influenced the differentiation status of SGCs, promoting cell proliferation and a marker expression profile more characteristic of epidermal keratinocytes. In contrast, cell proliferation was limited in 3D spheroid culture, and the expression of markers associated with the glandular phenotype was preserved. Furthermore, 2D-cultured SGCs incorporated into TESs aggregated but did not, however, re-establish glandular differentiation. Conversely, 3D-cultured SGCs retained glandular marker expression for up to 16 days following incorporation into TESs. In sum, our work highlights the impact of the culture mode on the differentiation fate of SGCs and establishes TESs as an environment conducive to the assembly of SGCs and the preservation of their glandular phenotype. Experiments to characterize SGC differentiation factors are currently underway, with a view to optimizing the culture and facilitating the integration of SGCs into TESs. Ultimately, these results could make it possible to produce the first bilayer skin substitute with functional sweat glands that could be used to improve the treatment of burn victims. Henri De Koninck^{2, 1, 3}, Karel Ferland^{2, 1, 3}, Christian Martel^{2, 3}, Danielle Larouche^{2, 3}, Lucie Germain^{2, 1, 3} 1. Faculty of Medicine, Universite Laval Faculte de Medecine, Québec City, QC, Canada. 2. Centre de recherche en organogénèse expérimentale de l'Université Lava/LOEX, Québec, QC, Canada. 3. Centre de recherche du CHU de Québec-Université Laval Site Hôpital Enfant-Jésus, Québec, QC, Canada. Stem Cell Biology, Tissue Regeneration and Wound Healing