Culture of Pancreatic Duct-Derived Stem Cells of Rats in The Three-Dimension and Two-dimension Cell Culture_CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Authors：

CHEN Xuchun , CHENG Ying , YANG Lei , LI Hong , LIU Yongfeng

Department of Organ Transplantation Surgery and General Surgery, The First Affiliated Hospital of China Medical University, Shenyang 110001, Liaoning Province,China;

Corresponding author：

Keywords：

Three-dimension cell culture; Two-dimension cell culture; Pancreatic duct-derived stem cell

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Objective 　To identify pancreatic duct-derived stem cells （PDSCs） from the pancreatic duct of rats, and culture in three-dimension cell culture system or two-dimension cell culture, with characterization on their morphology and phenotype.
Methods 　Adult male Wistar rats underwent perfusion with collagenaseⅤvia the pancreatic duct, the pancreas was surgically procured, digested, followed by discontinued density gradient centrifuge to isolate islets from acinar and ductal tissue. The acinar and ductal tissues were cultivated in serum-containing medium in the three-dimension or two-dimension cell culture seperatedly to obtain adherent cells, as PDSCs, which were expanded by consecutive passages. The cell junction, cell cycle, and cell apoptosis rate between the two-dimension and three-dimision cell culture were compared by means of microscope and flow cytometry.
Results 　PDSCs of rats showed direct adherence culture in three-dimension and two-dimision cell culture systerm. PDSCs grew in many layers in three-dimension cell culture system on 2 days after culture, and possessed capacity of high proliferation on 7 days. Cells were adherent to the bottom of flask on 5 days after culture in two-dimision cell culture. Cell junctions were more obvious in three-dimension cell culture system. Compared with the two-dimision cell culture, the cells of G1 phase increased〔（56.46±1.17） % vs.（37.33±1.21）%〕, and the cells of G2 phase decreased〔（14.62±0.15） % vs. （32.40±1.21）%〕in three-dimension cell culture system （P＜0.01）. The early period apoptosis rate was lower in three-dimension cells culture system than that in two-dimision cell culture 〔（1.48±0.39）% vs. （5.24±1.33）%, P＜0.05〕. The late period apoptosis rate was no statistically significant difference between the three-dimension and two-dimension cell culture （P＞0.05）.
Conclusions 　PDSCs of rats could be obtained in the three-dimension cell culture system. There is cell junction in three-dimension culture mode, which shows a significant enhancement on intercellular interaction, and the biological characters are different between the three-dimension and two-dimension cell culture.

Citation： CHEN Xuchun,CHENG Ying,YANG Lei,LI Hong,LIU Yongfeng,.. Culture of Pancreatic Duct-Derived Stem Cells of Rats in The Three-Dimension and Two-dimension Cell Culture. CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY, 2012, 19(7): 722-726. doi: Copy

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1. Shapiro AM， Ricordi C， Hering BJ， et al. International trial of the Edmonton protocol for islet transplantation [J]. N Engl J Med， 2006，355(13)：1318-1330.
2. Dorrell C， Grompe MT， Pan FC， et al. Isolation of mouse pancreatic alpha， beta， duct and acinar populations with cell surface markers [J]. Mol Cell Endocrinol， 2011， 339(1-2)：144-150.
3. Li XY， Zhan XR， Lu C， et al. Mechanisms of hepatocyte growth factor-mediated signaling in differentiation of pancreatic ductal epithelial cells into insulin-producing cells [J]. Biochem Biophys Res Commun， 2010， 398(3)：389-394.
4. Pittenger MF， Martin BJ. Mesenchymal stem cells and their potential as cardiac therapeutics [J]. Circ Res， 2004， 95(1)：9-20.
5. Taylor-Fishwick DA， Pittenger GL. Harnessing the pancreatic stem cell [J]. Endocrinol Metab Clin North Am， 2010, 39(4)：763-776.
6. Seo MJ， Suh SY， Bae YC， et al. Differentiation of human adipose stromal cells into hepatic lineage in vitro and in vivo [J].Biochem Biophys Res Commun， 2005， 328(1)：258-264.
7. Peck AB， Yin L， Ramiya V. Animal models to study adult stem cell-derived, in vitro-generated islet implantation [J]. ILAR J，2004， 45(3)：259-267.
8. 黄孝伦. 胰岛细胞移植治疗糖尿病的历史、现状与未来 [J].中国普外基础与临床杂志， 2012， 19(2)： 125-128.
9. 黄子星，刘洋洋，宋彬. 胰岛移植的分子影像示踪与监测 [J].中国普外基础与临床杂志， 2012， 19(2)：140-145.[ 10] Hori Y， Rulifson IC， Tsai BC， et al. Growth inhibitors promote differentiation of insulin-producing tissue from embryonic stem cells [J]. Proc Natl Acad Sci USA, 2002, 99(25)：16105-16110.
10. Bonner-Weir S， Weir GC. New sources of pancreatic beta-cells [J]. Nat Biotechnol， 2005， 23(7)：857-861.
11. Grapin-Botton A. Ductal cells of the pancreas [J]. Int J Biochem Cell Biol， 2005， 37(3)：504-510.
12. Lin HT， Chiou SH， Kao CL， et al. Characterization of pancreatic stemcells derived from adult human pancreas ducts by fluorescence activated cell sorting [J]. World J Gastroenterol，2006， 12(28)：4529-4535.
13. Zulewski H， Abraha EJ， Gerlach MJ， et al. Multipotential Nestin-positive stem cells isolated from adult pancreatic islets differentiates ex vivo into pancreatic endocrine, exocrine, and hepatic phenotypes [J]. Diabetes， 2001， 50(3)：521-533.
14. Behrous D， Laertis I， Bruce M， et al. Human islet-derived precursor cells are mesenchymal stromal cells that differentiate and mature to hormone-expressing cells in vivo [J]. Stem Cells，2007， 25(12)：3215-3222.
15. Petropavlovskaia M， Rosenberg L. Identification and characterization of small cells in the adult pancreas： potential progenitor cells? [J]. Cell Tissue Res， 2002， 310(1)：51-58.
16. Seeberger KL, Dufour JM， Shapiro AM， et al. Expansion of mesenchymal stem cells from human pancreatic ductal epithelium [J]. Lab Invest， 2006， 86(2)：141-153.
17. Ramiya V， Maraist M， Arfors KE， et al. Reversal of insulindependent diabetes using islets generated in vitro from pancreatic stem cells [J]. Nat Med， 2000， 6(3)：278-282.
18. Chase LG， Ulloa-Montoya F， Kidder BL， et al. Islet-derived fibroblast-like cells are not derived via epithelial-mesenchymal transition from Pdx-1 or insulin-positive cells [J]. Diabetes，2007， 56(1)：3-7.
19. Giselbrecht S， Gottwald E， Truckenmueller R， et al. Microfabrication of chip-sized scaffolds for three-dimensional cell cultivation [J]. J Vis Exp， 2008， 12(5)：691-699.
20. 夏豪，李庚山，李建军，等. 三维培养内皮细胞形成血管样结构的实验研究 [J]. 微循环学杂志， 2000， 10(2)：5-7.
21. 徐强，胡亚，廖泉，等. 胰腺癌细胞在二维和三维培养系统中的生长特性研究 [J]. 中国普外基础与临床杂志， 2011，18(1)：33-38.
22. 施森，何延政，刘勇，等. 外周血来源内皮祖细胞三维血管新生模型的建立及其特性分析 [J]. 中国普外基础与临床杂志，2009， 16(2)：119-123.
23. 王萍，孙立，林森森，等. 内皮细胞三维培养芽生模型的构建 [J]. 中国药科大学学报， 2012， 43(1)：60-63.

CHINESE JOURNAL OF BASES AND CLINICS IN GENERAL SURGERY

Culture of Pancreatic Duct-Derived Stem Cells of Rats in The Three-Dimension and Two-dimension Cell Culture

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