Figure 1. Isolation of cryopreserved periodontal ligament stem cells. (A) Periodontal ligament stem cells recovered from 6-month-cryopreserved periodontal ligament were capable of forming single-cell clusters after being plated at low density and cultured with regular culture medium for 10 days as described in METHODS. The number of single colonies derived from cryopreserved periodontal ligament (CP) was significantly decreased (*p < 0.05) in comparison with that derived from the fresh non-frozen periodontal ligament (P) when the same number (5000) of cells was plated (CP = 4.7 ± 0.6, P = 24.8 ± 1.5, mean ± SD) (N = 3). (B) The proliferation rates were assessed by bromodeoxyuridine (BrdU) incorporation for 12 hrs. Cryopreserved periodontal ligament stem cells (CP) maintain a high level of proliferation rate, similar to that of the regular periodontal ligament stem cells (P), showing that there is no significant difference between the regular periodontal ligament stem cells and cryopreserved periodontal ligament stem cells (73.8 ± 3.7, 67.4 ± 10.3, respectively, mean ± SD) (N = 3). (C) H&E staining of non-frozen human periodontal ligament tissue. (D,E) H&E staining of periodontal ligament cryopreserved for 6 mos. Most areas of periodontal ligament tissue showed normal histological structures in H&E staining. However, some nuclear anisokaryosis was found in frozen periodontal ligament (E, arrow), indicating that the cryopreservation can cause some tissue damage (scale bar, 200 µm for C-E). (F-M) Cryopreserved periodontal ligament stem cells expressed STRO-1, one of the early progenitor markers for mesenchymal stem cells. The cryopreserved periodontal ligament stem cells may co-express STRO-1 with bone sialoprotein (BSP) and TGFβ receptor type I (TGFβRI), as shown on the merged Figs. Some cryopreserved periodontal ligament stem cells may express STRO-1 and BSP separately (scale bar, 50 µm for F–M).