Isolation and Characterization of Stem Cells from the Submandibular and Parotid Salivary Glands of Albino Rats

Abstract

Salivary function in mammals may be defective for various reasons, such as aging, Sjogren’s syndrome or due to radiation therapy in head and neck cancer patients. Recently, tissue-specific stem cell therapy has attracted public attention as a next-generation therapeutic reagent. The aim of this work is to isolate stem cells from the Submandibular and Parotid salivary glands of albino rats for 1- Identification and characterization. 2- Assessment of proliferation rate. 3- Cryopreservation. 4- Culturing of stem cells in cell culture inserts. Fifteen adult male albino rats were used in this study. They were sacrificed by IV administration of anesthetic overdose (sodium thiopental 3ml).The submandibular (SM) and Parotid salivary glands were dissected and prepared for tissue culture. Isolation, culturing and passaging of the salivary gland stem cells (SGSC) were the same for both glands .The dissected salivary glands were weighed and chopped into a homogenous pulp. Steps proceeded by washing , filtering, plating, suspending in media, subculturing and passaging after reaching confluence and cell counting using the Haemocytometer. Immunophenotypic analysis by flow cytometry was carried out. Proliferation rate of Submandibular and parotid salivary glands’ stem cells was assessed and the stem cells from both groups were tested for colony formation capability. Culture of cells using cell culture insert (preparation of multicellular layers): was performed for both types of salivary gland stem cells and histological confirmation of multicellular layer formation was done. Cryopreservation was performed for both groups followed by Cell thawing. Counting of viable cells after thawing was performed. Finally, samples were prepared for Examination by SEM. The results from the current research showed that Stem cells from both glands were successfully isolated enzymatically. the initial culture contained cell population of round- shaped cells and the fibroblast like cells. In both groups and over the culture period cells initially assumed a rounded appearance and were initially dispersed in the media. Most cells retained their rounded morphology and then Cells started to form processes in order to adhere to the plastic flask, other cells were spindle and star shapes. Increase in number of stellate and spindle-shaped cells followed. Changing the media after a week caused rounded non adherent cells to be removed leaving the adherent cells. Spindle shaped cells, triangular shaped and spindle shaped. Increase in number of stellate or spindle in shape then occurred. By the third week, flasks showed confluency of 70% and colony formation. II- Characterization of Submandibular and Parotid gland stem cells using Flow cytometry analysis: Salivary gland stem cells from both groups were characterized by Flow cytometry with two markers after culture in medi