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Development and characterization of 2-dimensional culture for buffalo intestinal cells

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Abstract

Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant’s primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17 µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10 days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.

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Abbreviations

IEC:

Intestinal epithelial cells

FBS:

Fetal bovine serum

PBS:

Phosphate buffered saline

DTT:

Dithiothreitol

ZO1:

Zonula occludens

FABP2:

Fatty acid binding protein 2

IP:

Intestinal peptidase

2D:

Two-dimensional

RNA:

Ribonucleic acid

HBSS:

Hank’s Balanced Salt Solution

DMEM:

Dulbecco’s Modified Eagle’s Medium

ITS:

Insulin Transferrin Selenium

cDNA:

complementary deoxyribonucleic acid

dNTP:

Deoxynucleotide triphosphate

qRT-PCR:

quantitative real time polymerase chain reaction

eTEM:

Experiments that showed typical epithelial morphology

euTEM:

Experiments that did not show typical epithelial morphology

RPLP:

Ribosomal Protein Lateral Stalk Subunit P1

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Acknowledgements

The authors are thankful to the Director, ICAR-NDRI for providing infrastructure to carry out the present study. This research work was financially supported by Department of Biotechnology, Ministry of Science and Technology, India (Grant No. 102/IFD/SAN/3670/2014-15).

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Authors and Affiliations

  1. Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry Division, ICAR-National Dairy Research Institute, Karnal, Haryana, 132001, India

    Nidhi Chaudhary, Himanshu Agrawal, Mamta Pandey, Suneel Onteru & Dheer Singh

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  1. Nidhi Chaudhary
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  2. Himanshu Agrawal
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  3. Mamta Pandey
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  4. Suneel Onteru
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  5. Dheer Singh
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Correspondence to Dheer Singh.

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Chaudhary, N., Agrawal, H., Pandey, M. et al. Development and characterization of 2-dimensional culture for buffalo intestinal cells. Cytotechnology 70, 361–373 (2018). https://doi.org/10.1007/s10616-017-0151-y

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