Mean Cumulative Population Doubling (CPD) Data and Mean Dead Cell Fraction (DCF) Data from Serial Culture of 8 Human Oral Alveolar Bone-Derived Mesenchymal Tissue Cell (MTC) Preparations

Oral alveolar bone MTC preparations were isolated for 8 dental patients after obtaining their informed consent. After two passages in cell culture, the MTC preparations were cryopreserved. Thawed samples for each donor were used to initiate triplicate serial cultures with an input of 100,000 viable cells (determined by trypan dye-exclusion) per well of 6-well culture plates. Approximately every 3 days, the adherent cells were trypsinized and harvested; and 1/20 of the harvested cells were transferred to begin the next culture in the series. The remaining harvested cells were stained with trypan blue dye and counted with triplicate sampling to determine the mean total number of dead cells per culture and the mean total number of live cells per culture. The mean total cell numbers were used to calculate the mean CPD for each patient donor’s triplicate cultures, which are reported. The mean dead cell numbers and the mean total cell numbers were used to calculate the mean DCF for each donor’s triplicate cultures, which are reported. The culture medium for all procedures was: MEMα supplemented with 15% fetal bovine serum (FBS) along with 1% antibiotic antimycotic (Gibco), 1% L-glutamine (Gibco), and 1% L-ascorbic acid 2-phosphastase (Gibco).The reported mean CPD data and mean DCF data were input into Asymmetrex®’s TORTOISE Test® kinetic stem cell (KSC) counting software (https://asymmetrex.com/tortoise-test/) to determine the mesenchymal stem cell-specific fraction (SCF) initially and at each serial passage.