Nalgene cryo 1 c freezing container

Whole blood was collected from 8 subjects (SA = 5; HC = 3) into BD Vacutainer® Sodium Heparin Tubes (BD Biosciences, Franklin Lakes, NJ). PBMCs were isolated using Lymphoprep and SepMate™-15(IVD) (STEMCELL Technologies, Vancouver, Canada) according to the manufacturer’s protocol. Isolated PBMCs were aliquoted and stored in cryotube vials (Thermo Scientific, Waltham, MA) with freezing medium (80% FBS, 20% DMSO) at a concentration of 5 × 10⁶ cells/mL. Cells were stored overnight in Nalgene™ Cryo 1 °C Freezing Container (Thermo Fisher, Rochester, NY) at − 80 °C, then transferred into liquid nitrogen for long-term storage. Figure 1 (created with Biorender.com) summarizes the study’s experimental and analytical workflow.

The CD146⁺ DPSCs (P3) in residual T75 flasks were harvested and re‐suspended with freezing medium, consisting of 10% dimethyl sulphoxide (DMSO, Sigma‐Aldrich, Steinheim, Germany) and 90% FBS. 1 × 10⁶ cells were aliquoted into cryovials (Corning, Lowell, MA) and stored in the Nalgene^™ Cryo 1°C Freezing Container (Thermo Fisher Scientific, USA), then kept in −80°C Freezer overnight for gradient cooling enabling a cooling rate at −1°C/min. Finally, they were transferred into liquid nitrogen.
In 3 months, CD146⁺ DPSCs were thawed in water bath at 37°C and seeded in 96‐well plates (2 × 10³ cells per well), marked as P4. Complete culture medium was used as the control medium (CM). 5 ng/mL bFGF was chosen as the minimal concentration in this experiment according to the literature, which showed highly proliferative response from bone marrow mesenchymal stem cells (BMMSCs).³¹ Moreover, a study had been reported that 100 ng/mL bFGF had obvious impact on the proliferation of dental pulp cells.³² Thus, in experiment groups, recovered DPSCs (P4) were immediately supplemented with 5, 10, 20, 50, 80 and 100 ng/mL bFGF. Fresh medium with bFGF was changed every 2 days.