Isolation and Characterization of Primary Human Periodontal Ligament Fibroblasts

Primary human PDLFs were isolated from the periodontal connective tissue of extracted human teeth of healthy donors that were free of decay and periodontal disease and were extracted for medical reasons. Tissue samples were cultivated in six-well cell culture plates (353046, Corning GmbH, Kaiserslautern, Germany) (37 °C, 5% CO₂, 100% H₂O) in complete media (DMEM High Glucose, D5671, Merck KGaA, Darmstadt, Germany) with 10% FBS (P30-3302, PAN-Biotech GmbH, Aidenbach, Germany); 1% antibiotic/antimycotic (A5955, Merck KGaA, Darmstadt, Germany); 1% L-glutamine (G7513, Merck KGaA, Darmstadt, Germany) and 100 µM ascorbic acid (A8960, Merck KGaA, Darmstadt, Germany) until proliferative outgrowth of adherently growing fibroblasts was observed. Cells were characterised by human PDLF-specific marker genes and a spindle-shaped morphology, as reported previously [49 (link),56 (link)]. For the experiments, human PDLFs from the third to fifth passage were pooled from six patients (male: 3; female: 3; age: 17–27 years).