Effects of ethanol washing and storage duration on primary culture of stem cells from human exfoliated deciduous teeth

Abstract

Purpose: Since the oral environment harbors various microorganisms, the removal of contaminants during the primary culture process of stem cells from human exfoliated deciduous teeth (SHEDs) is very important. We investigated optimal methods for primary culture of SHEDs with minimal contamination rates.

Materials and methods: Three different storage conditions for deciduous teeth were utilized:1) storing teeth in Hank's Balanced Salt Solution (HBSS) with 3% penicillin and streptomycin (P/S), 2) storing teeth in HBSS with 3% antibiotics and antimycotics (A-A), and 3) storing teeth in HBSS with A-A, and additional washing with 70% ethanol just before primary culture of dental pulp. In addition, the storage time from the extraction of teeth to the primary culture was measured.

Results: The contamination rates were about 70% for HBSS with P/S, 40% for HBSS with A-A, and less than 10% for HBSS with A-A and additional washing with 70% ethanol. When the primary culture was conducted within 12 h after teeth extraction, the contamination rate was the lowest in all conditions. Furthermore, when the teeth were delivered in HBSS with A-A and an additional 70% ethanol washing was performed, the contamination rate was 0% until 48 h after teeth extraction. Ethanol washing had little effect on the cellular characteristics and stemness of SHEDs, including their morphology, growth rate, expression of surface markers, and differentiation potential.

Conclusions: We suggested that both delivering teeth in HBSS with A-A and additional 70% ethanol washing are critical considerations for the successful culture of SHEDs without contamination.

Keywords: Antibiotics; Contamination; Ethanol; Primary culture; Stem cells from human exfoliated deciduous teeth.

Graphical abstract

Fig. 1

Characterization of SHEDs. SHEDs were isolated from the dental pulp of deciduous teeth and cultured until in vitro passage 9 (P9). Primary culture of SHEDs was conducted in three conditions; Condition#1 (C#1) stored in HBSS with P/S (n = 64), Condition#2 (C#2) stored in HBSS with A-A (n = 29), or Condition#3 (C#3) stored in HBSS with A-A and washed by 70% ethanol (70% EtOH) (n = 172). (A) SHEDs at P6 showed typical MSC-like morphology irrespective of conditions. These images were one of representatives of SHEDs at each condition. (B) Growth curves were illustrated by average ± standard deviation calculated from three lines of SHEDs of each condition. There were no significant differences (P > 0.05, two-way ANOVA with repeated measures) in growth among the three conditions until P9. (C) To characterize immunophenotypes of SHEDs at P6, FACS analysis was performed (n = 3 for each condition). Results of one of three representatives of SHEDs for each condition were illustrated. SHEDs were positive for MSC-specific markers (CD44, CD73, CD90, CD105, and CD166), but negative for hematopoietic or endothelial cell markers (CD11b, CD14, CD19, CD34, CD45, and HLA-DR). (D) There were no differences in positive-cell percentages among the three conditions (P > 0.05, one-way ANOVA). (E) To confirm differentiation potential of SHEDs, adipogenic or osteogenic differentiation were performed at P6 (n = 3 for each condition). Lipid vacuoles and calcium deposits were stained with Oli red O and Alizarin Red, respectively. Undifferentiated SHEDs and Wharton's Jelly-derived MSCs (data not shown) were used as negative and positive controls, respectively. Results of one of three representatives of SHEDs for each condition were illustrated. (F) When the red signals were quantitatively analyzed, there were no differences among the three conditions (P > 0.05, one-way ANOVA).

Fig. 2

Contamination rate of primary culture of SHEDs. Contamination rates were analyzed according to the three conditions and storage time from extracting teeth to primary culture. Contamination by oral microorganisms was visually determined. (A) Condition #1 (C#1). (B) Condition #2 (C#2). (C) condition 3 (C#3). (D) Contamination rates of C#1 and C#2 were compared with that of C#3. P values were calculated by Fisher's exact test. (E) When primary culture was performed within 48 h after tooth extraction in C#3 (n = 81), contamination rate was significantly lower than that of 93 C#3 cases in which primary culture were done after 48 h after tooth extraction (P = 0.0002, Fisher's exact test).