In vitro serial passage culture of mouse pancreatic stem cells under the condition of embryonic fibroblast feeder layers

Yanhui CEN

ABSTRACT

BACKGROUND:

Pancreatic stem cells exhibit the capacities to division and high differentiation and can be successfully isolated and cultured in vitro. But how to perform effective in vitro proliferation remains to be solved.

OBJECTIVE:

To in vitro subculture mouse pancreatic stem cells under the condition of embryonic fibroblast feeder layers. DESIGN, TIME AND

SETTING:

A cytological, in vitro observation was performed at the Room for Cell Sterile Culture, School of Basic Medical Sciences, Guangxi Traditional Chinese Medical University between July 2007 and January 2008. MATERIALS Twenty newborn Kunming mice of specific pathogen free and of either gender, as well as some 14-day-pregnant Kunming mice, were purchased from Laboratory Animal Center, Guangxi Traditional Chinese Medical University.

METHODS:

The pancreas were obtained from newborn Kunming mice and digested with V type collagenase. When tissue blocks that were not completely digested settled naturally, the cell suspension on the upper layer was collected and centrifuged. After supernatant removal and addition of serum-free low glucose dulbecco's modified eagle's medium containing keratinocyte growth factors and basic fibroblast growth factors, cell suspension was cultured in polylysine-treated 24-well plate. Mouse embryo was taken from 14-day-pregnant Kunming mouse. After head and internal organs removal, the body and four limbs were isolated and cultured for obtaining fibroblasts by trypsin digestion. Following density adjustment, cells were inoculated into a 24-well plate at a density of 5×108/L. After 3 passages, mitomycin C treatment was conducted to prepare embryonic fibroblast feeder layer. Pancreatic stem cells primarily cultured for 5 days were inoculated into feeder layer cells-containing 24-well plate at a density of 30 cells per square centimeter. When cells spread about 80% of the whole bottom, passage could be continued. MAIN OUTCOME

MEASURES:

Cell morphology was observed under an inverted microscope. After 48 hours of primary culture, cells were dyed with alkaline phosphatase(ALP), and Nestin expression was identified by immunohistochemistry. ALP, Nestin, pancreatic duodenal homeobox-1(PDX-1) detections were performed at 2, 3, 4, and 5 days after passage.

RESULTS:

Among the cells derived from pancreas by primary culture, some large, round cells with single nucleus could be found, which exhibited strong endochylema refraction and high karyoplasmic ratio, grew adhering to the wall, were immunoreactive for ALP and Nestin, and simultaneously showed strong division and proliferation capacity. Under the condition of mouse embryonic fibroblast feeder layer, pancreatic stem cells could be serially passaged to the third generation. Each generation of cells still maintained their properties including large, round appearance with single nucleus, high karyoplasmic ratio, and proliferative capability. At each time point after passage, the cells were positive for ALP and Nestin staining but negative for PDX-1 staining, and maintained undifferentiated state.

CONCLUSION:

After mitomycin treatment, mouse embryonic fibroblast feeder layer can secrete the factors that promote the growth of pancreatic stem cells but inhibit the self-differentiation. After in vitro serial passages, the third generation of pancreatic stem cells can maintain their properties including good growth characteristics, high proliferation capacity, and undifferentiated state.