Frozen Dental Pulp Cells Keep Their Strength at Just the Right Passage

Human dental pulp cells, the resident regenerative population tucked inside the soft core of every tooth, have long been viewed as one of the most accessible sources of mesenchymal stem cells for tissue engineering. Harvested routinely from extracted wisdom teeth and exfoliated baby teeth, they proliferate vigorously, differentiate along multiple lineages, and carry immunomodulatory properties that make them attractive far beyond dentistry, with potential roles in bone, cartilage, neural, and vascular repair. But a fundamental practical question has shadowed the field: what happens to these cells when they are frozen, stored, thawed, and then grown in the laboratory for weeks on end? A new study published in the Journal of Cellular and Molecular Medicine offers one of the most detailed passage-by-passage answers to date, and its findings carry real consequences for anyone banking dental pulp cells for research or therapy.

Researchers at the Federal University of Uberlândia in Brazil isolated human dental pulp cells from three third molars, each contributed by a different donor to capture inter-individual genetic variability. The pulp tissue was washed, dissected, and cultured as explants in DMEM/F-12 medium supplemented with fetal bovine serum, antibiotics, L-glutamine, and non-essential amino acids. Within three to four days, fibroblast-like cells began migrating from the tissue pieces, and the explants were repeatedly transferred to fresh dishes to keep the cultures expanding. Once sufficient numbers of cells were available, the team froze them using a freezing medium composed of 80 percent DMEM, 10 percent fetal bovine serum, and 10 percent dimethyl sulfoxide, a cryoprotectant that limits the formation of damaging intracellular ice crystals.

The freezing protocol itself was deliberately simple and accessible. Rather than relying on expensive controlled-rate freezers, the researchers placed cryovials in a standard minus 20 degrees Celsius freezer for twenty minutes to encourage gradual cooling, then transferred them to an ultra-low temperature freezer held at minus 80 degrees Celsius, where the cells remained for three months. This stepwise approach was designed to minimize thermal and osmotic stress during the transition to a frozen state. Storage at minus 80 degrees Celsius has previously been shown to preserve the viability and key biological traits of dental pulp stem cells for six to twelve months, and the new work extends that evidence by tracking how thawed cells behave across many subsequent passages rather than at a single snapshot in time.

After rapid thawing in a 37 degree water bath, the cells were reseeded and expanded, then evaluated at passages 0, 3, 6, 10, and 12 using three complementary methods: an MTT assay for metabolic viability, flow cytometry for surface marker expression, and scanning electron microscopy for morphology and cell attachment. Passage 0, collected before freezing, served as the non-cryopreserved reference point. Because the same donor-derived cultures were followed across passages, the team used repeated-measures statistics, applying the Friedman test for overall differences and Wilcoxon signed-rank comparisons with Holm correction for pairwise contrasts, with significance set at p less than 0.05.

The viability results revealed a striking sweet spot in the middle of the expansion timeline. Metabolic activity peaked at passages 6 and 10, with passage 6 showing significantly higher viability than both the pre-freeze baseline at passage 0 and the late passage 12. Passage 10 followed the same pattern, significantly outperforming passages 0 and 12. Scanning electron microscopy told a matching story: the number of cells adhered to the substrate was significantly greater at passage 6 than at passages 0, 3, and 12, and microscopy across all groups confirmed that the cells retained their characteristic fibroblast-like shapes with odontoblastic extensions, remaining well attached to the glass substrate throughout.

Flow cytometry, however, uncovered a subtler and arguably more important trend. The staining panel targeted CD45 as a negative, hematopoietic marker and CD44, CD146, and STRO-1 as stem cell-associated markers. CD44 expression remained statistically stable across all passages, suggesting that the broad mesenchymal identity of the cells was never lost. But the more lineage-restricted progenitor markers told a different story. CD146 positivity, which sat at roughly 32 percent at passages 0 and 3, collapsed to around 5 percent by passages 10 and 12, a highly significant decline. STRO-1 showed a parallel drop, with significantly fewer positive cells at the later passages compared with the earlier ones.

There was also a modest but statistically significant rise in CD45, the hematopoietic marker that mesenchymal stem cells should not express. At passages 3, 6, and 10, CD45 positivity remained vanishingly small, between 0.1 and 0.8 percent, but at passage 12 it climbed to a median of 4.85 percent, differing significantly from passages 0, 3, and 6. The authors suggest this may reflect stress-induced phenotypic modulation or the emergence of minor non-mesenchymal subpopulations during extended culture and post-thaw recovery, rather than a wholesale loss of mesenchymal identity. Notably, cells at passage 3 behaved comparably before and after cryopreservation, hinting that early-passage cells possess a greater biological resilience to the freezing and thawing process.

Perhaps the most consequential insight of the study is a disconnect between what the eye can see and what the molecules reveal. Scanning electron microscopy found no dramatic ultrastructural differences across passages, yet flow cytometry documented clear immunophenotypic drift. In other words, molecular instability can emerge well before any visible change in cell shape or attachment, which means that morphology alone is an unreliable gauge of whether banked cells remain fit for purpose. This echoes earlier reports that functional deterioration can occur at advanced passages even when conventional mesenchymal markers remain detectable, and that CD146, which is tied to perivascular stem cell niches, clonogenic potential, and immunoregulatory capacity, serves as a sensitive barometer of stem cell potency.

The findings align with a growing body of literature on passage-dependent decline in dental pulp-derived cells. Previous comparisons of early, extended, and late passages have documented marked reductions in proliferation, with late-passage cells growing 50 to 70 percent more slowly than early ones, alongside membrane damage, pre-apoptotic changes, and a transition from spindle-shaped cells to enlarged, polygonal, heterogeneous forms. The new study adds an important twist by showing that these changes unfold in cells that have been frozen and thawed, suggesting that the combined burden of serial expansion and cryostorage-related stress, rather than freezing alone, shapes the final cellular phenotype. The authors caution, however, that because passage-matched non-cryopreserved controls were not included, the relative contributions of freezing and passaging cannot be fully separated, and SEM-based cell counting should be read as a complementary measure rather than a direct proliferation assay.

For the rapidly growing field of dental stem cell banking, the practical message is clear and actionable. Cryopreservation at minus 80 degrees Celsius with 10 percent dimethyl sulfoxide preserves the fundamental mesenchymal character of dental pulp cells over three months, and intermediate passages, particularly 6 and 10, deliver the best combination of viability, yield, and intact morphology after thawing. Yet the earliest passages, 3 and 6, hold the richest reservoir of stemness-associated markers, meaning that the optimal window depends on whether the priority is maximum cell number or maximum progenitor content. As the authors conclude, extended storage periods, functional differentiation assays, and larger sample sizes will be needed to confirm long-term stability, but the study provides a much-needed roadmap for standardizing how frozen dental pulp cells are expanded, characterized, and ultimately deployed in regenerative medicine.

Subject of Research: Effects of cryopreservation and passage number on the viability, morphology, and stem cell marker expression of human dental pulp cells

Article Title: Phenotyping of Human Pulp Cells After Cryopreservation

Article References: dos Santos, C. M. M. L., da Silva, W. H. T., Guedes, F. R., de Paula, M. C., Morse, L., Battaglino, R., Silva, M. J. B., & Turrioni, A. P. (2026). Phenotyping of Human Pulp Cells After Cryopreservation. Journal of Cellular and Molecular Medicine, 30(18), Article e71362. https://doi.org/10.1111/jcmm.71362

Image Credits: AI Generated

DOI: 10.1111/jcmm.71362

Keywords: dental pulp stem cells, cryopreservation, dimethyl sulfoxide, flow cytometry, CD146, STRO-1, CD45, passage number, cell banking, regenerative medicine, scanning electron microscopy, mesenchymal stem cells