Phenotyping of Human Pulp Cells After Cryopreservation

This study aimed to compare cell viability, morphology, cell yield, and stem cell-associated marker expression of cryopreserved human dental pulp cells (HDPCs) across different passages following thawing and serial in vitro expansion. HDPCs were isolated from three healthy donors, cryopreserved at -80°C for 3 months, and subsequently evaluated at different passages. Non-cryopreserved passage 0 (P0) cells were used as the baseline control. Cell viability was assessed using the MTT assay, cell morphology and complementary cell counting by scanning electron microscopy (SEM), and phenotypic marker expression (CD44, CD146, STRO-1, and CD45) by flow cytometry. P6 and P10 showed significantly higher cell viability than P0 and P12 (p < 0.05), whereas SEM-based cell counting showed a higher number of cells at P6 than at P0, P3, and P12 (p < 0.05). The percentage of CD146-positive cells decreased from 32.1% and 32.3% at P0 and P3, respectively, to 4.9% and 5.2% at P10 and P12 (p < 0.05). A similar passage-dependent reduction was observed for STRO-1 expression. Overall, HDPCs exhibited passage-dependent changes following cryopreservation, thawing, and serial expansion. Intermediate passages, particularly P6, showed favourable cell viability and yield, whereas earlier passages better preserved progenitor-associated phenotypic markers. These findings highlight the importance of passage selection when cryopreserved HDPCs are expanded for subsequent experimental applications.