CLC number: Q28
On-line Access: 2013-07-04
Received: 2012-08-22
Revision Accepted: 2013-05-28
Crosschecked: 2013-06-19
Cited: 2
Clicked: 4296
Dan Li, Shi-yun Peng, Zhen-wu Zhang, Rui-cheng Feng, Lu Li, Jie Liang, Sheng Tai, Chun-bo Teng. Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion[J]. Journal of Zhejiang University Science B, 2013, 14(7): 596-603.
@article{title="Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion",
author="Dan Li, Shi-yun Peng, Zhen-wu Zhang, Rui-cheng Feng, Lu Li, Jie Liang, Sheng Tai, Chun-bo Teng",
journal="Journal of Zhejiang University Science B",
volume="14",
number="7",
pages="596-603",
year="2013",
publisher="Zhejiang University Press & Springer",
doi="10.1631/jzus.B1200226"
}
%0 Journal Article
%T Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion
%A Dan Li
%A Shi-yun Peng
%A Zhen-wu Zhang
%A Rui-cheng Feng
%A Lu Li
%A Jie Liang
%A Sheng Tai
%A Chun-bo Teng
%J Journal of Zhejiang University SCIENCE B
%V 14
%N 7
%P 596-603
%@ 1673-1581
%D 2013
%I Zhejiang University Press & Springer
%DOI 10.1631/jzus.B1200226
TY - JOUR
T1 - Complete disassociation of adult pancreas into viable single cells through cold trypsin-EDTA digestion
A1 - Dan Li
A1 - Shi-yun Peng
A1 - Zhen-wu Zhang
A1 - Rui-cheng Feng
A1 - Lu Li
A1 - Jie Liang
A1 - Sheng Tai
A1 - Chun-bo Teng
J0 - Journal of Zhejiang University Science B
VL - 14
IS - 7
SP - 596
EP - 603
%@ 1673-1581
Y1 - 2013
PB - Zhejiang University Press & Springer
ER -
DOI - 10.1631/jzus.B1200226
Abstract: The in vitro isolation and analysis of pancreatic stem/progenitor cells are necessary for understanding their properties and function; however, the preparation of high-quality single-cell suspensions from adult pancreas is prerequisite. In this study, we applied a cold trypsin-ethylenediaminetetraacetic acid (EDTA) digestion method to disassociate adult mouse pancreata into single cells. The yield of single cells and the viability of the harvested cells were much higher than those obtained via the two commonly used warm digestion methods. Flow cytometric analysis showed that the ratio of ductal or BCRP1-positive cells in cell suspensions prepared through cold digestion was consistent with that found in vivo. Cell culture tests showed that pancreatic epithelial cells prepared by cold digestion maintained proliferative capacity comparable to those derived from warm collagenase digestion. These results indicate that cold trypsin-EDTA digestion can effectively disassociate an adult mouse pancreas into viable single cells with minimal cell loss, and can be used for the isolation and analysis of pancreatic stem/progenitor cells.
[1]Amsterdam, A., Jamieson, J.D., 1974. Studies on dispersed pancreatic exocrine cells. I. Dissociation technique and morphologic characteristics of separated cells. J. Cell Biol., 63(3):1037-1056.
[2]Baeyens, L., de Breuck, S., Lardon, J., Mfopou, J.K., Rooman, I., Bouwens, L., 2005. In vitro generation of insulin-producing β cells from adult exocrine pancreatic cells. Diabetologia, 48(1):49-57.
[3]Bonner-Weir, S., Toschi, E., Inada, A., Reitz, P., Fonseca, S.Y., Aye, T., Sharma, A., 2004. The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatr. Diabetes, 5(S2):16-22.
[4]Chung, C.H., Hao, E., Piran, R., Keinan, E., Levine, F., 2010. Pancreatic β-cell neogenesis by direct conversion from mature α-cells. Stem Cells, 28(9):1630-1638.
[5]Cole, R.J., Paul, J., 1966. The effects of erythropoietin on haem synthesis in mouse yolk sac and cultured foetal liver cells. J. Embryol. Exp. Morphol., 15(2):245-260.
[6]Donath, M.Y., Halban, P.A., 2004. Decreased β-cell mass in diabetes: significance, mechanisms and therapeutic implications. Diabetologia, 47(3):581-589.
[7]Dono, K., Gotoh, M., Fukuzaki, T., Ohzato, H., Kanai, T., Monden, M., Mori, T., 1992. Low-temperature collagenase digestion: an improved islet isolation method from cold preserved pancreas. Transplant. Proc., 24(4):1511-1512.
[8]Dono, K., Gotoh, M., Monden, M., Kanai, T., Fukuzaki, T., Mori, T., 1994. Low temperature collagenase digestion for islet isolation from 48-hour cold-preserved rat pancreas. Transplantation, 57(1):22-26.
[9]Dor, Y., Brown, J., Martinez, O.I., Melton, D.A., 2004. Adult pancreatic β-cells are formed by self-duplication rather than stem-cell differentiation. Nature, 429(6987):41-46.
[10]Githens, S., 1988. The pancreatic duct cell: proliferative capabilities, specific characteristics, metaplasia, isolation, and culture. J. Pediatr. Gastroenterol. Nutr., 7(4):486-506.
[11]Gross, J., Harper, E., Harris, E.D., McCroskery, P.A., Highberger, J.H., Corbett, C., Kang, A.H., 1974. Animal collagenases: specificity of action, and structures of the substrate cleavage site. Biochem. Biophys. Res. Commun., 61(2):605-612.
[12]Hao, E., Tyrberg, B., Itkin-Ansari, P., Lakey, J.R., Geron, I., Monosov, E.Z., Barcova, M., Mercola, M., Levine, F., 2006. β-cell differentiation from nonendocrine epithelial cells of the adult human pancreas. Nat. Med., 12(3):310-316.
[13]Inada, A., Nienaber, C., Katsuta, H., Fujitani, Y., Levine, J., Morita, R., Sharma, A., Bonner-Weir, S., 2008. Carbonic anhydrase II-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth. PNAS, 105(50):19915-19919.
[14]Ji, B., Gaiser, S., Chen, X., Ernst, S.A., Logsdon, C.D., 2009. Intracellular trypsin induces pancreatic acinar cell death but not NF-κB activation. J. Biol. Chem., 284(26):17488-17498.
[15]Kayali, A.G., van Gunst, K., Campbell, I.L., Stotland, A., Kritzik, M., Liu, G., Flodstrom-Tullberg, M., Zhang, Y.Q., Sarvetnick, N., 2003. The stromal cell-derived factor-1α/CXCR4 ligand-receptor axis is critical for progenitor survival and migration in the pancreas. J. Cell Biol., 163(4):859-869.
[16]Kikugawa, R., Katsuta, H., Akashi, T., Yatoh, S., Weir, G.C., Sharma, A., Bonner-Weir, S., 2009. Differentiation of COPAS-sorted non-endocrine pancreatic cells into insulin-positive cells in the mouse. Diabetologia, 52(4):645-652.
[17]McKeehan, W.L., 1977. The effect of temperature during trypsin treatment on viability and multiplication potential of single normal human and chicken fibroblasts. Cell Biol. Int. Rep., 1(4):335-343.
[18]Oliver, C., 1980. Isolation and maintenance of differentiated exocrine gland acinar cells in vitro. In Vitro, 16(4):297-305.
[19]Poliakova, L., Pirone, A., Farese, A., MacVittie, T., Farney, A., 2004. Presence of nonhematopoietic side population cells in the adult human and nonhuman primate pancreas. Transplant. Proc., 36(4):1166-1168.
[20]Rhodes, C.J., 2005. Type 2 diabetes—a matter of β-cell life and death? Science, 307(5708):380-384.
[21]Seglen, P.O., 1976. Preparation of isolated rat liver cells. Methods Cell Biol., 13:29-83.
[22]Shapiro, A.M., Lakey, J.R., Ryan, E.A., Korbutt, G.S., Toth, E., Warnock, G.L., Kneteman, N.M., Rajotte, R.V., 2000. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med., 343(4):230-238.
[23]Stock, P.G., Bluestone, J.A., 2004. β-cell replacement for type I diabetes. Annu. Rev. Med., 55(1):133-156.
[24]Suzuki, A., Nakauchi, H., Taniguchi, H., 2004. Prospective isolation of multipotent pancreatic progenitors using flow-cytometric cell sorting. Diabetes, 53(8):2143-2152.
[25]Xu, X., D'Hoker, J., Stange, G., Bonne, S., de Leu, N., Xiao, X., van de Casteele, M., Mellitzer, G., Ling, Z., Pipeleers, D., et al., 2008. β cells can be generated from endogenous progenitors in injured adult mouse pancreas. Cell, 132(2):197-207.
[26]Zhou, S., Schuetz, J.D., Bunting, K.D., Colapietro, A.M., Sampath, J., Morris, J.J., Lagutina, I., Grosveld, G.C., Osawa, M., Nakauchi, H., et al., 2001. The ABC transporter Bcrp1/ABCG2 is expressed in a wide variety of stem cells and is a molecular determinant of the side-population phenotype. Nat. Med., 7(9):1028-1034.
Open peer comments: Debate/Discuss/Question/Opinion
<1>