Isolation And Cultivation Of Human Submandibular Gland Stem/Progenitor Cells

Objectives:In this study,we isolated and cultured stem/progenitor cells(hSGPs) from human fetus submandibular gland,and then analyzed its surface markers and biological characters by using immunofluorescence,flow cytometry,growth curve and karyotype.These cells might be differentiated into functional cells of liver,pancreas or salivary gland by further research.Methods:①Human fetus submandibular gland cells(hSMGs) were isolated and cultured by mechanical method and enzyme digestion.②Colonies of epithelium-like cells were harvested by selectively cultured of hSMGs in vitro.We purified the cells by limited dilution and one of the cells was designated hSGPs.③The growth curves were drawn by cell counting.④We performed immunofluorescence staining for CK19, laminin and analyzed cell-surface markers CD29,Thy-1(CD90.1),c-Kit(CD117) by flow cytometry(FCM).⑤Karyotype was analyzed in long-term cultured hSGPs.Results:①The hSMGs were obtained by mechanical method and enzyme digestion. The hSMGs exhibit triangle,irregular polygonal and a small number of acinar-like cells. There were three cell clusters formed when hSMGs were cultured 12 days in vitro.②After been cultured for 9 days,one colony of epithelium-like cells formed. Morphologically the cells showed the same shape with a larger proportion of nuclear plasma.These cells exhibit cobblestone-like appearance when selectively cultured in vitro,and then we purified the cells by limited dilution.One of the cells was designated hSGPs.③Growth curve were drawn with passages 6 hSGPs.For the f'trst day,the population of hSGPs already proliferated,the second day,it entered into the log phase till the 7th day,then the capacity of cell proliferation significantly weakened,but still maintain a slow upward trend,did not show platform phase.From the growth curve,we concluded that the population doubling time was 36 hours,and the lag time was 33.6 hours.④hSGPs expressed intracellular laminin and cytokeratinl9 in immunofluorescence. Flow cytometric analysis of hSGPs,the percentage of cells positive for Thy-1(CD90.1),CD29,c-Kit(CD117) were 99.69%,98.83%,67.27%respectively.⑤Karyotype by G-band manifested that chromosome number of hSGPs cell line was 2n=46 with no abnormality in number,morphology and structure.Conclusion:hSGPs we harvested had the characteristics of tissue stem cells that with better adherence,powerful proliferation,strong colony ability.The SGP-1 cell also maintained its diploid in long-time culture.