Establishment of Immortal Multipotent Rat Salivary Progenitor Cell Line Toward Salivary Gland Regeneration

Abstract

Adult salivary gland stem cells are promising candidates for cell therapy and tissue regeneration in cases of irreversible damage to salivary glands in head and neck cancer patients undergoing irradiation therapy. At present, the major restriction in handling such cells is their relatively limited life span during in vitro cultivation, resulting in an inadequate experimental platform to explore the salivary gland-originated stem cells as candidates for future clinical application in therapy. We established a spontaneous immortal integrin α6β1-expressing cell line of adult salivary progenitor cells from rats (rat salivary clone [RSC]) and investigated their ability to sustain cellular properties. This line was able to propagate for more than 400 doublings without loss of differentiation potential. RSC could differentiate in vitro to both acinar- and ductal-like structures and could be further manipulated upon culturing on a 3D scaffolds with different media supplements. Moreover, RSC expressed salivary-specific mRNAs and proteins as well as epithelial stem cell markers, and upon differentiation process their expression was changed. These results suggest RSC as a good model for further studies exploring cellular senescence, differentiation, and in vitro tissue engineering features as a crucial step toward reengineering irradiation-impaired salivary glands.

Long-Term Cryopreservation Model of Rat Salivary Gland Stem Cells for Future Therapy in Irradiated Head and Neck Cancer Patients

“…2,4,10 Cultivation of integrin a6b1-positive (SGIE) cells and murine prostate cancer PC3 cells SGIE culture. The isolated cells were inoculated into 25-cm 2 Falcon flasks preseeded with 2 · 10 5 lethally irradiated (50 Gy) NIH 3T3 mouse fibroblasts serving as a feeder layer, as previously described, or into flasks precoated with 10 mg/cm 2 of rat collagen type I (Sigma).…”

“…10 Briefly, growth factor reduced Matrigel or basement membrane Matrigel (200 mL; Becton Dickinson) was added to 24-well tissue culture dishes and incubated for at least 30 min at 37°C to polymerize. SGIE were plated at a density of 1.5 · 10 5 cells/well and were cultured in a 37°C, 5% CO 2 incubator for 3 days.…”

“…Immunofluorescence assay was performed as we previously described 10 with the following modifications: The antibodies against amylase (2 mg/mL, mouse monoclonal IgG; Santa Cruz Biotechnology), p63 (200 mg/mL, rabbit polyclonal IgG; Santa Cruz Biotechnology), cystatin C (1 mg/ mL, rabbit polyclonal IgG; Chemicon), integrin-a6b1 (mouse monoclonal IgG; Chemicon), c-KIT (CD117, 2 mg/mL; Santa Cruz), and CK17 (Rabbit Polyclonal; Epitomics) were diluted 1:100 with 1.25% FCS in PBS and incubated overnight at 4°C. Alexa 488-and Alexa 647-conjugated secondary antibodies against rabbit and mouse IgG respectively (Molecular Probes) were added to samples at a dilution of 1:500 and incubated for 90 min at room temperature.…”

“…Extraction of total RNA and synthesis of complementary DNA were performed using TaqMan Cells to CT kit (Applied Biosystems) according to the manufacturer's instructions, as previously described. 10 To extract RNA from 3D cultures, 24-well Matrigel-cultured cells were incubated with 500 mL dispase solution (Becton Dickinson) for 2 h in a 37°C, 5% CO 2 incubator. The depolarized mixture of cells, Matrigel and dispase was collected into 15-mL tubes (contents of six to nine wells per tube) and centrifuged at 4°C (5 min, 1300 g).…”

“…Cultures of nCP and 1-3 years CP SGIE were further estimated for division number assays (Fig. 2b, c; passages [10][11][12][13]. No differences were found when comparing the averages of both the division numbers within each passage (Fig.…”