Cryopreservation of amniotic fluid stem cells derived from Zobor rabbit

Abstract

The aims of our preliminary study were to evaluate the viability and to investigate the occurrence of early and late apoptosis of fresh, frozen-thawed and thawed-cultured rabbit amniotic fluid stem cells (AFSCs). Rabbit AFSCs were isolated from young (4-6 months old) Zobor rabbit females (n = 3), cultured in endothelial basal medium (EBM-2) to passage 3 (P3) and frozen. One month after cryopreservation AFSCs were thawed and cultured for 72 hours. We identified the apoptotic (Annexin-V - AnV; Yo-Pro-1 - YoP) and dead (propidium iodide - PI) AFSCs using flow cytometry and evaluated the viability of fresh (F-P3), frozen-thawed (F-T) and thawed-cultured (72 hours of post-thaw in vitro culture – T-72h) AFSCs. To examine the phenotype of P3 AFSCs we performed an antibody-based staining of surface markers CD29, CD44 and CD45. We have found decreased (P < 0.05) viability in F-T cells (77.5 ± 2.2 % and 75.2 ± 0.7 % for AnV- /PI- and YoP- /PI- , respectively), compared to F-P3 (92.5 ± 2.2 and 89.45 ± 1.3 for AnV- /PI- and YoP- /PI- , respectively). However, viability of T-72h cells was similar to F-P3 (90.9 ± 1.5 % and 84.6 ± 1.7 % for AnV- /PI- and YoP- /PI, respectively). Our AFSCs were both CD44+ (95.98 ± 1.51 %) and CD29+ (92.33 ± 4.46) positive but CD45- (0.73 ± 0.55 %) negative, according to defined MSCs phenotype.