Abstract
BackgroundHuman dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We recently reported that a xenogeneic serum-free culture medium (XFM) is preferable to fetal bovine serum-containing culture medium for ex vivo expansion of DPSCs; however, we observed that, upon reaching overconfluence, XFM cells developed a multilayered structure and frequently underwent apoptotic death, resulting in reduced cell yield. Therefore, we focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs.MethodsWe selected type I collagen (COL) as the optimal coating substrate for the cultureware and compared DPSCs cultured on COL in XFM (COL-XFM cells) to the conventional XFM cultures (XFM cells).ResultsOur results demonstrated that COL coating facilitated significantly higher rates of cell isolation and growth; upon reaching overconfluence, cell survival and sustained proliferative potential resulted in two-fold yield compared to the XFM cells. Surprisingly, after subculturing the overconfluent COL-XFM cultures, the cells retained stem cell behavior including stable cell growth, multidifferentiation potential, stem cell phenotype, and chromosomal stability, which was achieved through HIF-1α-dependent production and uniform distribution of collagen type I and its interactions with integrins α2β1 and α11β1 at overconfluency. In contrast, cells undergoing apoptotic death within overconfluent XFM cultures had disorganized mitochondria with membrane depolarization.ConclusionThe use of COL as a coating substrate promises safe and reliable handling of DPSCs in XFM culture, allowing translational stem cell medicine to achieve stable isolation, expansion, and banking of donor-derived stem cells.
Highlights
Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine
After a 4-day incubation period, DPSCs grew in culture plates coated with COL and fibronectin, while the cells did not adhere in the plates coated with laminin and poly-D-lysine (Additional file 1: Figure S1)
We observed that the time period required to show cell division of adherent cells in COL-xenogeneic serum-free culture medium (XFM) primary culture was significantly shorter compared to the XFM primary culture (4.0 ± 1.0 vs 7.8 ± 1.3 days; n = 4; P = 0.0073)
Summary
Human dental pulp stem cells (DPSCs) are a readily accessible and promising cell source for regenerative medicine. We focused on optimization of the XFM culture system to avoid the undesirable death of DPSCs. Populations of dental pulp-derived stromal cells contain multipotent stem cells with a high proliferative capacity and potent differentiation potential into a wide range of cell types, such as osteo/odontogenic cells [1,2,3,4] and Mochizuki et al Stem Cell Research & Therapy (2020) 11:267 routine tooth extraction; DPSCs present a safe and minimally invasive MSC source for therapeutic applications and cell bank storage for future use [19,20,21,22]. It is widely known that the use of xenogeneic serum, i.e., fetal bovine serum (FBS), is necessary for MSC culture to isolate and expand donor-derived stem/stromal cells, including DPSCs [23, 24]. Development and application of serum-free MSC culture methodology will ensure safe and reliable cellbased therapies for regenerative medicine [39, 40]
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