Optimizing a serum-free/xeno-free culture medium for culturing and promoting the proliferation of human dental pulp stem cells

Abstract

Background: Dental pulp stem cells (DPSCs) hold great promise for utilization in tissue repair and regenerative medicine. Routinely, culture media used for culturing stem cells are supplemented with animal serum for promoting growth and successful maintenance of stem cells. However, there is a growing demand for optimizing a well-defined culture media that could safely increase the efficacy and reproducibility of the cultured cells. In this study, we aimed at optimizing a serum-free/xeno-free culture medium.

Methods: A cocktail of various supplements intended to enrich DPSCs proliferation in defined concentrations was designed. It consisted of recombinant human basic fibroblast growth factor (hbFGF), insulin transferrin selenium (ITS), ascorbic acid (vitamin C), Beta mercaptoethanol and cholesterol. The effect of this optimized media on the proliferation of DPSCs was assessed by MTT assay and flow cytometric analysis (FACS) of early apoptotic marker annexin V. Expression of stemness-related genes (OCT4, SOX and NANOG) was assessed by qRT-PCR.

Results: Proliferation results by MTT illustrated a significant increase in the proliferation rate of DPSCs cultured in the proposed media. FACS analysis of annexin V expression was nil. Expression of OCT4, SOX and NANOG genes was also up-regulated.

Conclusions: The proposed combination of supplements utilized in the proposed culture media successfully increased the proliferation potential of DPSCs in addition to enhancing the stemness properties. Thus, it can be considered a promising and safe substitute to traditional animal derived supplements like fetal bovine serum (FBS).

Keywords: Culture medium; dental pulp stem cells (DPSCs); serum free; xeno free.

Figure 1

DPSCs cultured successfully in control group. (A) Photomicrograph illustrating morphological appearance of DPSCs cultured in control group. On day 3, spindle-shaped cells started to appear forming a colony. Cells started to increase in number by days 5 & 7 while assuming various morphological appearances and reaching confluence by day 10 (scale bar 100 µm). (B) Representative plots of flow-cytometry for CD90 and CD105 in DPSCs cultured in basal culture medium supplemented with serum, showing positive expression mesenchymal cell surface markers, and negative expression of hematopoietic cell surface markers CD34. DPSCs, dental pulp stem cells.

Figure 2

DPSCs cultured successfully in serum-free/xeno-free (SF/XF) culture medium. (A) Photomicrograph illustrating morphological appearance of DPSCs cultured in SF/XF medium on days 3, 5 and 7. Similar morphological appearances to control group was illustrated (scale bar 100 µm). (B) Representative plots of flow-cytometry for CD90 and CD105 in DPSCs cultured in SF/XF medium, showing positive expression mesenchymal cell surface markers, and negative expression of hematopoietic cell surface markers CD34. DPSCs, dental pulp stem cells.

Figure 3

DPSCs cultured in serum-free/xeno-free (SF/XF) culture medium demonstrate enhanced cell viability and proliferation capacity. (A) Cell count of cultured DPSCs after the first and second week of culture. (B) MTT assay of DPSCs on days 3, 7 and 14. Proliferation of DPSCs was significantly higher on days 7 and 14. Statistical significance was analysed with paired Student t-test. Results are illustrated as mean ± SEM of triple independent experiments and were expressed in P values. Results were considered significant at P≤0.05. (C) Representative plots of flow-cytometry for apoptotic cell surface markers annexin V in DPSCs cultured in SF/XF medium, showing negative expression. DPSCs, dental pulp stem cells; SEM, standard error of the mean.

Figure 4

DPSCs cultured in serum-free/xeno-free (SF/XF) culture medium demonstrate stemness characteristics. (A) Quantitative RT-PCR for mRNA levels of stemness specific genes; OCT4, SOX and NANOG, showing up-regulation of those genes in DPSCs grown in SF/XF medium. Results are presented in fold change in relation to control. Results are presented as mean ± SEM of three independent experiments (n=3). *, P<0.05. (B) Adipogenic differentiation of DPSCs was evaluated by Oil Red O staining (right) and revealed accumulated lipid droplets (red) in differentiated cells. Chondrogenic differentiation ability of DPSCs in monolayers was evaluated by Alcian blue staining (left) (scale bar 200 µm).