IADR Abstract Archives
Influence of Isolation Methodologies on Biological Properties of Rabbit Dental Pulp Stem Cells for Regenerative Dentistry Applications
Objectives: To evaluate the effects of digestion and explant cultures on the biological properties of rabbit dental pulp stem cells (DPSCs).
Methods: Upper and lower incisors were extracted from a 2.2kg male rabbit. The pulp tissue was extracted and two methods were used to isolate cells; explant and digestion. Enzymatic digestion was done using 3mg/ml collagenase NB4 and 4mg/ml dispase. Cells were cultured in DMEM F-12, 10% fetal bovine serum (FBS), 100 units/ml penicillin-100µg/ml streptomycin, 50µg/ml L-ascorbic acid and 1% L-glutamine. Cells from passages 2-4, isolated by both methods, underwent colony forming assays (CFU), morphological characterization, population doubling levels (PDLs) and population doubling times (PDTs). Additionally, the effect of different concentrations of FBS (Serum-free, 2%, 10%) on cell migration was evaluated using wound scratch assays.
Results: DPSCs isolated using explant cultures depicted lower CFU capacity, lower PDTs and higher total number of PDLs compared to those cultured using digestion. Migration of DPSCs from explant displayed significantly different rates depending on the concentration of FBS used (p<0.05). However, DPSCs from digestion cultures did not display significant migration differences among the different FBS conditions.
Conclusions: DPSCs from both digestion and explant cultures retained their proliferation, colony forming and migration capacities throughout passages. However, cells from explant cultures appeared to exhibit a more uncommitted and responsive state. Furthermore, explant cultured cells retain their native microenvironment during the critical initial days of culture. These benefits combined may render the explant technique potentially more appealing for versatile clinical applications of DPSCs, especially since this technique is more time and cost efficient.
Methods: Upper and lower incisors were extracted from a 2.2kg male rabbit. The pulp tissue was extracted and two methods were used to isolate cells; explant and digestion. Enzymatic digestion was done using 3mg/ml collagenase NB4 and 4mg/ml dispase. Cells were cultured in DMEM F-12, 10% fetal bovine serum (FBS), 100 units/ml penicillin-100µg/ml streptomycin, 50µg/ml L-ascorbic acid and 1% L-glutamine. Cells from passages 2-4, isolated by both methods, underwent colony forming assays (CFU), morphological characterization, population doubling levels (PDLs) and population doubling times (PDTs). Additionally, the effect of different concentrations of FBS (Serum-free, 2%, 10%) on cell migration was evaluated using wound scratch assays.
Results: DPSCs isolated using explant cultures depicted lower CFU capacity, lower PDTs and higher total number of PDLs compared to those cultured using digestion. Migration of DPSCs from explant displayed significantly different rates depending on the concentration of FBS used (p<0.05). However, DPSCs from digestion cultures did not display significant migration differences among the different FBS conditions.
Conclusions: DPSCs from both digestion and explant cultures retained their proliferation, colony forming and migration capacities throughout passages. However, cells from explant cultures appeared to exhibit a more uncommitted and responsive state. Furthermore, explant cultured cells retain their native microenvironment during the critical initial days of culture. These benefits combined may render the explant technique potentially more appealing for versatile clinical applications of DPSCs, especially since this technique is more time and cost efficient.