Abstract
BackgroundCurrently, ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. However, because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells.MethodsDental pulp stem cells were derived from wisdom teeth donated by eight healthy volunteers and cultured in xenogeneic serum-free culture medium (XFM) or xenogeneic serum-containing culture medium (SCM). Cells were subjected to morphological, proliferation, karyotype, differentiation, marker expression, cryopreservation, and cytotoxic susceptibility analyses in vitro, as well as transplantation in vivo.ResultsIn primary culture, XFM cells showed lower adhesion and slightly different morphology, although the single-cell size was similar to that of SCM cells. XFM cells exhibited typical mesenchymal stem cell (MSC) characteristics in vitro and in vivo, including marker gene/protein expression, trilineage differentiation potential, and hard, osteo-dentin tissue formation. Additionally, XFM cells maintained a normal karyotype in vitro and nontumorigenic potential in vivo; however, XFM cells were more susceptible to H2O2 and ultraviolet cytotoxic stimuli. XFM cells formed a multilayered structure showing excessive cell death/division in contrast to the monolayered structure of SCM cells when reaching overconfluence. Proliferation was disrupted in overconfluent XFM cells, and these cells could not be subcultured. Dimethyl sulfoxide-free cryopreserved XFM cells yielded similar results in all of the experiments.ConclusionsThis study is the first reporting successful isolation and expansion of an MSC population from donor-derived tissue (dental pulp) under xenogeneic serum-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments.
Highlights
Ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation
We thoroughly investigated the isolation and expansion of Dental pulp stem cell (DPSC) under FBS-free culture conditions, as well as the application of cryopreservation, using a research strategy based on clinically oriented in-vitro and in-vivo experiments
We demonstrated in-vitro stem cell properties of xenogeneic serum-free culture medium (XFM) and serum-containing culture medium (SCM) cells, including active cell growth (Fig. 2a), a normal karyotype (Fig. 2b), global gene/protein expression associated with the mesenchymal stem cell (MSC) phenotype (Fig. 2c, d), and trilineage differentiation potential, such as osteogenic, adipogenic, and chondrogenic lineages (Fig. 2e–g)
Summary
Ex-vivo handling of stem cells, including transport after harvest and therapeutic preparation, is generally done in culture media containing fetal bovine serum (FBS), which promotes cell attachment, proliferation, and differentiation. Because of safety concerns associated with the use of FBS, including potential transmission of zoonotic agents and transplant rejection because of the incorporation of foreign proteins into the stem cells, there is a need for xenogeneic serum-free culture media for clinical handling of stem cells. Mesenchymal stem cells (MSCs) isolated from dental pulp tissue of extracted teeth, which are generally discarded after dental treatments, represent autologous stem cell candidates for future cell therapies. Clinical studies reported the therapeutic use of DPSCs for bone augmentation in tooth-extraction defects [20] and for dental pulp regeneration in cariesaffected, pulpectomized teeth [21]. DPSCs are currently regarded as a valuable, promising MSC type among somatic stem cells with clinical potential
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