Microchamber arrays for the identification of individual cells exposed to an X-ray microbeam.

To identify individual cells exposed to a X-ray microbeam in a cell population, we developed a biocompatible microchamber-array chip using UV lithography of photopolymer SU-8. The center-to-center distance between microchambers is 50 mum including a wall of 15 mum height. Using the microchamber-array chip, we performed tracking of individual exposed cells. Sample cells loaded in a microchamber array were selectively irradiated with the X-ray microbeam under microscopic observation. All the irradiated cells were indexed by the array arrangement of the microchambers. For about 24 h of post-irradiation incubation, the irradiated cells were identified successfully by time-lapse observation. In addition, the induction of radiation effects was observed in identified cells using immunofluorescence.

Establishment of immortalized clonal cells derived from periodontal ligament cells by induction of the hTERT gene.

Since the periodontal ligament (PDL) contains a heterogeneous cell population, it is challenging to identify all cell types within the tissue and to determine whether they function alone to produce tissue components or interact with other cell types. Further, it is difficult to isolate and expand single cell clones from PDL cells, as normal cells have a limited life span and are phenotypically unstable. In the present study, we inserted the human telomerase reverse transcriptase (hTERT) gene, which encodes the catalytic subunit of the telomerase holoenzyme, into normal human periodontal ligament (HPL) cells and successfully obtained single cell clones. Expression of the inserted gene and telomerase activity in each of the clones was confirmed. Unlike the original HPL cells, at the end of the study (day 120), clone populations continued to actively double without phenotypic alteration. Osteogenic characteristics were present in some but not all clones. In conclusion, immortalization of HPL cells was successfully accomplished by transduction with the hTERT gene. This is the first report of immortalization of different cell types derived from PDL.