[Establishment of the isolation and culture system for monocloning human pancreatic stem cell]

The present study aimed to establish an isolation and culture system for the monoclonal human pancreatic stem cells and monoclonal human pancreatic stem cell line. Some factors which would influence the proliferation of the monoclonal human pancreatic stem cells were assessed. Pancreatic tissues, taken from abortive fetuses by sterile procedures, were dissected into 1 mm3 segments and digested with 0.1% type IV collagenase. The isolated cells were grown in media containing low glucose Dulbecco's Modified Eagles's Medium supplemented with 10% fetal bovine serum (FBS), 3.7 g/L sodium pyruvate, 0.08 g/L penicillin and 0.1 g/L streptomycin. These cells were further digested with 0.25 g/L trypsin and 0.4 g/L EDTA for propagation. The monoclonal human pancreatic stem cells were selected by clone-ring, and further proliferated after addition of 10 ng/mL epidermal growth factor (EGF) in culture media. The cell chromosome set was determined by karyotype analysis. The growth curve was made by the 3-(4, 5)-dimethylthiahiazo (-z-yl)-3, 5-di-phenytetrazoliumromide (MTT) method. The results showed that pancreatic tissues were digested to many single cells and cell clusters with collagenase. Adherently cultured, primary epidermal-like pancreatic stem cells grew clonally. After several times of dissociation and propagation, pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the monoclonal human pancreatic stem cells were obtained. Continuously propagated, a monoclonal human pancreatic stem cell line which was derived from a male abortive fetus of 4 month-old had been passed through 50 generations. Karyotype analysis demonstrated that the chromosome set of the monoclonal human pancreatic stem cell line was normal diploid. Growth curve revealed that monoclonal human pancreatic stem cells grew slowly in initial 1-4 days. Then, they entered the logarithmic growth period in next 5-6 days. Their proliferation was speeded up by supplementation with 15% FBS in culture media, which was even more quickly after addition of the 15 ng/mL EGF or 10 ng/mL insulin growth factor II (IGF-II). The result identified that the monoclonal human pancreatic stem cell line could be obtained by the cell isolation and culture system.