Effects of growth factor supplementation on the proliferation of cryopreserved canine amniotic membrane stem cells

Amniotic membrane-derived mesenchymal stem cells (AM-MSCs) possess significant proliferative and multilineage differentiation potential. However, prolonged in vitro culture and cryopreservation can negatively impact cell viability and expansion capacity. This study investigates the effects of growth factor supplementation on the proliferation of advanced-passage canine AM-MSCs (cAM-MSCs) before and after cryopreservation. cAM-MSCs were isolated from late-gestation canine fetuses, cultured in vitro, and supplemented with different growth factors: fibroblast growth factor 2 (FGF-2), fibroblast growth factor 4 (FGF-4), platelet-derived growth factor-ββ (PDGF-ββ), vascular endothelial growth factor-β (VEGF-β), and transforming growth factor-β1 (TGF-β1), with a control group receiving no supplementation. Growth curves were analyzed before and after cryopreservation, and gene expression of SCAPER (mitotic potential) and TP53 (apoptosis) was quantified using qPCR. As result, supplementation with FGF-2, FGF-4, and PDGF-ββ significantly enhanced cAM-MSC proliferation without altering the expression of proliferation-associated genes. These findings suggest that growth factor supplementation may be a viable strategy to improve cell viability in long-term culture and post-thaw recovery.