Separation of Rabbit Intestinal Villus and Crypt Cells Using Improved Cryogenic Chelating Method and Separation Effect Identification

SHEN Xuemei; LI Jing; ZHANG Gang; CUI Hongxiao; LIU Lihui; YAO Junhu; XU Xiurong

doi:10.3969/j.issn.1006-267x.2016.05.020

2016 , Vol. 28 >Issue 5: 1442 - 1449

DOI: https://doi.org/10.3969/j.issn.1006-267x.2016.05.020

EXPERIMENTAL METHODS

Separation of Rabbit Intestinal Villus and Crypt Cells Using Improved Cryogenic Chelating Method and Separation Effect Identification

SHEN Xuemei ,
LI Jing ,
ZHANG Gang ,
CUI Hongxiao ,
LIU Lihui ,
YAO Junhu ,
XU Xiurong

Expand

College of Animal Science and Technology, Northwest Agriculture and Forestry University, Yangling 712100, China

Received date: 2015-12-04

Online published: 2016-05-24

Fold

Abstract

This study aimed to investigate the separation method of rabbit intestinal villus and crypt cells, which can be served as the in vitro model for study on the structure and function of the intestinal epithelium. The experiment used New Zealand white rabbits as test material, and separated their intestinal villus and crypt cells under the conditions of different chelating agent concentrations[5, 10, 15, 20 and 25 mmol/L ethylene diamine tetraacetic acid (EDTA)] and chelating temperatures (4 and 25℃). Then the yield, morphology, cell viability rate and integrity of the separated cells were detected. The results showed as follows:1) under different chelating separation conditions, all of the collected villus and crypt cells remained morphological integrity, and the genomic DNA and total RNA of these villus and crypt cells were intact. 2) The higher chelating temperature and EDTA concentration was, the greater cell enrichment rate was obtained, but the toxic effect on cell was stronger. The enrichment rate of villus and crypt cells in group LT+5 mmol/L EDTA was 7.75% and 1.01%, and that in group RT+25 mmol/L EDTA was 17.89% and 4.99%, respectively; while the relative integrity rate of villus and relative viability rate of crypt cells in the former group were 91.67% and 93.48%, and were 4.25% and 5.17% in the latter group, respectively. 3) By a comprehensive analysis, the best separation effect was observed under the condition of 4℃ and 10 mmol/L EDTA. 4) The relative expression levels of lysozyme and α-defensin genes from crypt enrichment separated under the optimal condition were significantly higher than those of from villus enrichment (P<0.01), and it indicated that cells in a higher purity. In addition, relative expression levels of lysozyme and α-defensin genes from the crypt cell separated under the optimal condition had no significantly change after cultured for 9 h (P>0.05), and it indicated that cells still had a higher vitality after cultured for 9 h. In conclusion, the improved cryogenic chelating method is suitable for separating rabbit intestinal villus and crypt cells effectively.

Key words： crypt; villus; intestine epithelial cell; chelating agents; rabbits

Cite this article

SHEN Xuemei , LI Jing , ZHANG Gang , CUI Hongxiao , LIU Lihui , YAO Junhu , XU Xiurong . Separation of Rabbit Intestinal Villus and Crypt Cells Using Improved Cryogenic Chelating Method and Separation Effect Identification[J]. Chinese Journal of Animal Nutrition, 2016 , 28(5) : 1442 -1449 . DOI: 10.3969/j.issn.1006-267x.2016.05.020

References

[1] PETERSON L W,ARTIS D.Intestinal epithelial cells:regulators of barrier function and immune homeostasis[J].Nature Reviews Immunology,2014,14(3):141-153.

[2] HORITA N,TSUCHIYA K,HAYASHI R,et al.Fluorescent labelling of intestinal epithelial cells reveals independent long-lived intestinal stem cells in a crypt[J].Biochemical and Biophysical Research Communications,2014,454(4):493-499.

[3] BEDFORD A,CHEN T,HUYNH E,et al.Epidermal growth factor containing culture supernatant enhances intestine development of early-weaned pigs in vivo:potential mechanisms involved[J].Journal of Biotechnology,2015,196-197:9-19.
[4] GRABINGER T,LUKS L,KOSTADINOVA F,et al.Ex vivo culture of intestinal crypt organoids as a model system for assessing cell death induction in intestinal epithelial cells and enteropathy[J].Cell Death & Disease,2014,5(5):e1228.
[5] VINCENT A,KAZMIERCZAK C,DUCHÊNE B,et al.Cryosectioning the intestinal crypt-villus axis:an ex vivo method to study the dynamics of epigenetic modifications from stem cells to differentiated cells[J].Stem Cell Research,2015,14(1):105-113.

[6] BERDASCO M,ESTELLER M.DNA methylation in stem cell renewal and multipotency[J].Stem Cell Research and Therapy,2011,2(5):42.
[7] CLEVERS H.The intestinal crypt,a prototype stem cell compartment[J].Cell,2013,154(2):274-284.

[8] TANCOS Z,NEMES C,POLGAR Z,et al.Generation of rabbit pluripotent stem cell lines[J].Theriogenology,2012,78(8):1774-1786.

[9] BEVINS C L,SALZMAN N H.Paneth cells,antimicrobial peptides and maintenance of intestinal homeostasis[J].Nature Reviews Microbiology,2011,9(5):356-368.

[10] SALZMAN N H.Paneth cell defensins and the regulation of the microbiome:detente at mucosal surfaces[J].Gut Microbes,2010,1(6):401-406.

[11] EVANS G S,FLINT N,SOMERS A S,et al.The development of a method for the preparation of rat intestinal epithelial cell primary cultures[J].Journal of Cell Science,1992,101(Pt 1):219-231.
[12] POTHIER P,HUGON J S.Characterization of isolated villus and crypt cells from the small intestine of the adult mouse[J].Cell and Tissue Research,1980,211(3):405-418.
[13] BJERKNES M,CHENG H.Methods for the isolation of intact epithelium from the mouse intestine[J].The Anatomical Record,1981,199(4):565-574.

[14] AYABE T,SATCHELL D P,WILSON C L,et al.Secretion of microbicidal α-defensins by intestinal Paneth cells in response to bacteria[J].Nature Immunology,2000,1(2):113-118.

[15] FLINT N,COVE F L,EVANS G S.A low-temperature method for the isolation of small-intestinal epithelium along the crypt-villus axis[J].Biochemical Journal,1991,280(2):331-334.

[16] FULLER M K,FAULK D M,SUNDARAM N,et al.Intestinal crypts reproducibly expand in culture[J].Journal of Surgical Research,2012,178(1):48-54.

[17] 宁宇,王锋超,刘登群,等.在体分离小鼠小肠隐窝绒毛上皮细胞的生化完整性研究[J].现代生物医学进展,2009,9(14):2610-2612.
[18] 高博,杨晓农,于学辉,等.家兔GAPDH基因实时荧光定量RT-PCR方法的建立[J].中国畜牧兽医,2010,37(1):69-73.
[19] GROSSMANN J,WALTHER K,ARTINGER M,et al.Progress on isolation and short-term ex-vivo culture of highly purified non-apoptotic human intestinal epithelial cells (IEC)[J].European Journal of Cell Biology,2003,82(5):262-270.

[20] CANO-GAUCI D F,LUALDI J C,OUELLETTE A J,et al.In vitro cDNA amplification from individual intestinal crypts:a novel approach to the study of differential gene expression along the crypt-villus axis[J].Experimental Cell Research,1993,208(2):344-349.

[21] CHOUGULE P,HERLENIUS G,HERNANDEZ N M,et al.Isolation and characterization of human primary enterocytes from small intestine using a novel method[J].Scandinavian Journal of Gastroenterology,2012,47(11):1334-1343.

[22] TAUC H M,TASDOGAN A,PANDUR P.Isolating intestinal stem cells from adult Drosophila midguts by FACS to study stem cell behavior during aging[J].Journal of Visualized Experiments,2014(94):52223.

Options

Outlines

模态框（Modal）标题

Abstract

Cite this article

References