Prolonged Cryopreservation of Rat Submandibular Salivary-Glands Progenitor Cells

Introduction: Head and neck cancer patients undergo irradiation-therapy (IR) leading to salivary-glands (SGs) dysfunction. Recent studies support the hypothesis that adult SGs contain epithelial stem-cells such as integrin-α6β1 expressing cells (SGIE), which can be isolated, multiply in culture and implanted into the irradiated SGs to regenerate their function. One challenge implementing this modality is keeping the isolated SGIE in an undifferentiated state, and in relatively low passage numbers throughout the IR timeframe. To achieve such conditions, a cell cryo-preservation in liquid-nitrogen (LNCP) followed by defrosting and re-cultivation before transplantation is needed. Hence, our objective was to test the effects of LNCP on the progenitor characteristics of SGIE, as well as on their in-vitro differentiation ability.

Materials & Methods: Rat SGIEs were secluded by Percoll-gradient-technique followed by MACS-methodology, cultured for several passages on collagen type-1 and cryopreserved for up to three years. Thereafter cells were defrosted, re-cultivated, examined for integrin-α6β1 expression, grown on Matrigel® extra-cellular-matrix and collagen type-1 and examined for the expression of cell differentiation-markers.

Results: SGIE could be cryopreserved for up to three years without losing integrin-α6β1 expression. Furthermore, growing on Matrigel® SGIE produced both acinar and duct like structures. Immuno-staining of collagen-1 and Matrigel® SGIE cultures showed the expression of P63 and integrin-α6β1 stem-cells-markers and Cystatin-C and α-amylase SGs differentiation-markers.

Conclusions: The cryopreservation method described here maintains SGIE functionality for prolonged times, thus it may have a role in establish a therapeutic procedure for stem-cells-based auto-transplantation treatment to IR-derived dysfunction of SGs.