Type 1 collagenase and dispase

The study was conducted in accordance with the Declaration of Helsinki and applicable local regulatory requirements and laws. All procedures were approved by the Ethics Committee of the University of Valencia (Spain) and all donors provided informed consent. The hDPSCs were isolated as previously described [25 (link)]. Briefly, dental pulp was gently removed from human third molars under aseptic conditions using cow horn forceps with a small excavator, and immersed in culture tubes filled with cell culture medium. The specimens were then divided into small pieces using a bistoury blade, immersed in Hank’s balanced salt solution (HBSS), and incubated for 2 h at 37 °C in an atmosphere of 5% CO₂ and 95% air. The supernatant was removed, 0.1% type I collagenase and dispase (Sigma-Aldrich, Madrid, Spain) in filter-sterilized HBSS were added to the pellet, and the mixture was incubated for 15 min, followed by centrifugation at 1500 rpm for 10 min. The supernatant was removed and the cells were plated in 25-cm² flasks in alpha Minimum Essential Medium (αMEM) culture medium (Sigma-Aldrich) containing 10% fetal calf serum (FCS) (Sigma-Aldrich), penicillin/streptomycin, amphotericin B, 2 mM of L-glutamine, and 100 µM of ascorbic acid (Sigma-Aldrich). The medium was replaced every 3–4 days. Once the cells reached 90% confluence, flow cytometry was performed.

DPSCs were cultivated and identified according to our previous developed protocol[56 (link)]. Briefly, impacted third molars from healthy individuals (15–25 years old) were collected from the Department of Stomatology, Renmin Hospital of Wuhan University. The study was approved by the Ethics Committee of Renmin Hospital of Wuhan University and informed consent was obtained from all donors. After cleaning and disinfecting the tooth surface with alcohol, the teeth were dissected aseptically, and the pulp tissue was isolated and rinsed in PBS. The dental pulp tissue was cut into small pieces of 1mm³, and digested with type I collagenase and dispase (Sigma, Germany) at 37 °C for 1 h with shaking once every 10 min. After termination of digestion, the cell pellet was resuspended with α-modified Eagle’s medium (α-MEM, Gibco, USA) containing 20% fetal bovine serum (FBS, Gibco, USA), 100 µg/mL streptomycin, and 100 U/mL penicillin (Gibco, USA) and incubated in T-25 culture flasks in a standard culture environment (37 °C, 5% CO₂). The culture medium was changed every 3 days. Cells from passages 3 to 5 were used for experiments.