original article Wien Klin Wochenschr [Suppl] DOI 10.1007/s00508-015-0771-1

HUIEC, Human intestinal epithelial cell line with differentiated properties: process of isolation and characterisation Lidija Gradisnik · Martin Trapecar · Marjan Slak Rupnik · Tomaz Velnar

Received: 30 November 2014 / Accepted: 16 February 2015 © Springer-Verlag Wien 2015

Summary  The intestinal epithelium is composed of diverse cell types, most abundant being the enterocytes. Among other functions, they maintain the intestinal barrier and play a critical role in the absorption of nutrients, drugs and toxins. This study describes the development and characterization of human intestinal epithelial cells (HUIEC), a spontaneously arising cell line established by selective trypsinization and cloning of the intestinal epithelium, resulting in a uniform population of highly epithelial cells with a strong growth potential. Keywords  Intestinal epithelial cells · Cell isolation · Cell line · Intestinal epithelium · Cell model

Introduction Cell models are becoming increasingly important for in vitro study of physiological and pathophysiological processes. An ideal cell model most closely mimics the in vivo conditions and consists of one or more cell lines. As there is no universal cell line to fit these models, isolation of variT. Velnar, MD, MSc () Department of Neurosurgery, University Medical Centre Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia e-mail: [email protected] L. Gradisnik · M. Trapecar Laboratory for Cell Cultures, Faculty of Medicine, University of Maribor, Maribor, Slovenia M. S. Rupnik Institute of Physiology, Faculty of Medicine, University of Maribor, Maribor, Slovenia T. Velnar, MD, MSc Chair of Surgery, Faculty of Medicine Ljubljana, University of Ljubljana, Maribor, Slovenia

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ous new types of cell lines from tissues is essential. Under specific conditions, these isolated cells may be maintained outside the body, in in vitro environment [1, 2]. The development of tissue and cell cultures dates back to the early 20th century when the pioneers of cell cultures, Harrison and Carrel, developed methods for cell isolation, their maintenance and study of cell physiology [3, 4]. The first isolation techniques have included the method of explantation, where cells migrate from a tissue sample, resulting in a tissue culture [5, 6]. Nowadays, isolation techniques of various tissues of plant, animal and human origin have become routine activities of research laboratories [6, 7]. Among many isolated cell lines, the intestinal epithelial cells and their functional cell models are often used. They are becoming an indispensable research tool in medicine and pharmacy to study absorption, cell transport, drug bioavailability and for nutritional sciences, such as studies of allergic effects of nutrients and their components, probiotics and intestinal interactions between hosts and pathogens [2, 8–12]. Additionally, the experimentation on cell cultures is becoming widely recognized due to the declining trend in animal tests and consequently lower costs [13, 14]. The intestinal epithelium is composed of diverse cell types. The most abundant are enterocytes [15, 16]. The main function is the absorption of nutrients, drugs and toxins. These cells have specific structural markers, like cytokeratin 18 and vimentin and enzymatic markers, on the basis of which they can be distinguished from other cells during isolation [17]. Among the markers characteristic of intestinal epithelial cells are also the brush border enzymes maltase-glucoamylase, sucrose-isomaltase, alkaline phosphatase and fatty acid binding protein, all involved in the digestion of carbohydrates, fats and apolipoprotein B [17, 18]. Specific ligands or specific antibodies for these markers can be used to determine the cell types in the tissue sample or culture [16, 19].

HUIEC, Human intestinal epithelial cell line with differentiated properties: process of isolation and characterisation  

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Many cell types from different parts of the gastrointestinal tract have been isolated [15, 20–23]. Less frequently, cultures of intestinal stem cells of animal and human origin, duodenal endocrine cells, cryptic intestinal cells and differentiated villus enterocytes have been described [6, 18, 24–26]. Their isolation is challenging due to a short viability of the epithelial intestinal cells in vitro, mainly owing to a lack of attachment to the substrate, which is crucial for cell survival [28]. Most research on the intestinal epithelium so far has been made on experimental animals and on human colon cancer cells. The usefulness and limitations of animal cells and transformed cell lines are well known and research carried out on these cells cannot be directly transferred to humans [2, 29]. The purpose of this study was to describe the isolation and characterisation of adult human ileal intestinal epithelial cells (HUIEC) in order to establish an untransformed cell line for future investigations.

Materials and methods Origin of cell line The HUIEC cell line was derived from the small intestine of a human donor. Ethical approvals were obtained. Stored in cold phosphate buffer for 3 hours prior to plating, a part of the terminal ileum resection specimen was collected. Since the small intestine is fundamentally contaminated with intestinal bacterial flora, the sample was first washed in phosphate buffer containing 100 U/ ml penicillin and 1 mg/ml streptomycin (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). The tissue was cut into small pieces to achieve coarse mechanical decomposition. Pieces of tissue were incubated for 30 min at 37 °C with dispersal solution containing 0.25 % (w/v) trypsin (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany). After trypsin inactivation, centrifugation at 2400 rpm for 30 min followed, separating coarse incompletely digested fragments of tissue from the cell containing sediment. The cell sediment was then washed twice with the cell culture medium Advanced DMEM (Life Technologies Ltd, Paisley, UK) and centrifuged for 15  min at 1400  rpm. The sediment was again resuspended in 10 ml of cell culture medium containing penicillin (100 IU/ml), streptomycin (1 mg/ml), L-glutamine (2mM), and 5 % fetal bovine serum (FBS) (Life Technologies Ltd, Paisley, UK) and plated in two T25 tissue culture flasks. Mixed primary cell cultures were incubated for five days at 37 °C in 5 % CO2 and the medium was replaced twice a week.

Routine conditions for cell culture and passage: the cultivation and grafting The cells were routinely cultured in T25 flasks and incubated at 37 °C in controlled atmosphere with 5 % CO2. A mixed cell culture was growing in flasks and it was split

with trypsin when confluent in ratio 1:3. This was followed by centrifugation at 1000 rpm for 5 min. The cell sediment was resuspended in 9 ml of fresh medium. The cell suspension was transferred to T25 flasks and every 3  ml of cell suspension was supplemented with 7  ml of cell culture medium. The cultures were then incubated and growth was monitored. In this way, the cell culture of the first passage was obtained.

Cell cloning and establishment of permanent cell line Following the isolation, the primary cell culture consisted of heterogeneous cell types. The aim was to establish a permanent intestinal epithelial cell line. Therefore, after trypsinization the cells were inoculated in Petri dishes in such low concentrations that after seven to ten days separate colonies have formed from each of the attached cell. It was noted immediately that this epithelial cell culture had a distinct potential for growth. The cells were extensively pigmented and large areas of polygonal cells were present in the mixed culture. The HUIEC cell line was purified by the selective trypsinization of the confluent culture and by removal of weekly adherent and morphologically different cells. The remaining adherent cells were allowed to grow until confluent and the selective trypsinization was then repeated. Under the microscope, the morphology of colonies was observed and those colonies that formed epithelial cells were identified. The medium was discarded, the surface gently washed with phosphate buffer solution and trypsin-impregnated discs were placed on the identified colonies. After five to ten minutes, the cells at the edges of the disks became round and the disks were transferred to 24-well microtitre plates with the wells filled with 1 ml of cell medium and 10 % FBS. With the discs, the attached cells were also transferred. The plate was incubated as described. After 48 hours, the cells attached and the discs were removed again. After ten days, the culture was confluent. For further subcultivation, the cells from those wells were selected, morphologically corresponding to epithelial cells. This was performed several times and a highly epithelial, uniform intestinal epithelial cell culture was obtained.

Cell characterisation Immunocytochemistry was used in order to characterize the cell culture for the presence of specific structural and enzymatic epithelial markers: (I) cytokeratin 18, (II) intestinal alkaline phosphatase, (III) vimentin and (IV) FABP or intestinal fatty acid binding protein. The cells were grown on four chamber glass slides until confluence, as described previously. The medium was then discarded and the monolayer in each chamber washed with cold PBS. The cells were fixed with CellFix (BD Biosciences, Heidelberg, Germany) for 15  min and then

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original article

permeabilised with 0.1 % polyethylene glycol (SigmaAldrich Chemie GmbH, Schnelldorf, Germany) in PBS, supplemented with 2 % FBS. For the investigation of structural molecules, the cells were stained simultaneously with the conjugated antibodies anti-cytokeratin 18 FITC, anti-vimentin DyLight 594 and the nucleus visualized with DRAQ5 according to the manufacturer’s protocols (Abcam, Cambridge, UK). Further key enzymes were labelled with goat derived primary antibodies to intestinal alkaline phosphatase and rabbit derived antiintestinal FABP, visualized with the secondary antibodies anti-rabbit PE-Cy5 and anti-goat TexasRed (Abcam, Cambridge, UK). Optical images were acquired using a Leica TCS SP5 II broadband confocal microscope (Leica Microsystems, Mannheim, Germany). Fluorescence was excited with lasers at 488 nm for FITC (cytokeratin 18), 594 nm for DyLight 594 and TexasRed (vimentin, alkaline phosphatase) and at 633 nm for DRAQ5 (nucleus) as well as PE-Cy5 (FABP). Emission was collected with detectors set in the range from 495–515 nm, 570–625 nm and 705– 730 nm. Images were taken from the absolute centre of each chamber.

Results HUIEC is a rapidly growing, spontaneously evolved intestinal epithelial cell line that was purified by selective trypisnization and cloning of the intestinal epithelium. This process involves the removal of other cell types from the mixed cell population, which was isolated from the tissue sample. In the initial stage of plating, various cell types were present in the suspension to obtain the primary culture of intestinal cells (Fig.  1). After several repetitions of trypsinization and cloning, the culture developed a uniform epithelial morphology. HUIEC cells had a distinctive epithelial appearance (Fig. 2). The characteristic shape was polygonal to round

Fig. 1 Primary intestinal cell culture after attachment with various cell types visible. Selective trypsinization and cloning followed. Bar = 100 μm

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Fig. 2  HUIEC cell culture in full confluence. Cells have distinct borders and adopted cobblestone appearance. Bar = 100 μm

and triangular with pseudopodia, with dotted, finely granular, pigmented cytoplasm and a large central round and hypercromatic nucleus. In some places nucleoli were clearly visible. The shape of the cells varied during the attachment process and growth from round or oval to polygonal, which was characterized during growth in confluent culture. After four to eight hours, most of the cells were attached to the substrate and shape alterations from round to polygonal were visible at that time. Cells were growing well and were easy to maintain in culture, which had a cobblestone appearance with defined cell borders, a feature of the epithelial cells growing in culture. The average time to confluent culture formation was seven to ten days. Then, the growth stopped due to the contact inhibition, a characteristic of primary epithelial cells. A survival of 95 % was observed when cells were thawed and reseeded. To further characterize the epithelial function of the cultured HUIEC cells, we recorded the extent of tight junction formation and transepithelial resistance of HUIEC two weeks after plating. Cells were grown in monolayers on 12 well Transwel plates with microporous inserts and fed with a defined medium. A resistance greater than 120Ω/cm was measured, which remained stable during growth in culture. The ability of the epithelium to polarise and to form transepithelial resistance confirms the in vivo-like functionality of the culture. During cell characterisation, the presence of structural proteins cytokeratin 18 and vimentin was confirmed as well as the expression of the enterocyte brush border enzymes intestinal alkaline phosphatase and FABP was confirmed. All cells were positive for the presence of the specific epithelial cytoskeletal protein cytokeratin 18 and for the mesenchymal stem cell marker vimentin (Fig. 3). This implies that the cells were at a crossover stage, during the progression from mesenchymal transformation into an epithelial phenotype in vitro. Immunocytochemistry revealed the production by cells of key brush border enzymes intestinal alkaline phosphatase and FABP in all cultured cells (Fig.  4). The presence of cell mark-

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Fig. 3 Confocal micrography of HUIEC after immunohistochemistry demonstrating presence of structural proteins cytokeratin 18 (red) and vimentin (green). Bar = 25 μm

ers strongly suggests the epithelial nature of the isolated culture.

Discussion HUIEC is one of several isolated intestinal cell lines. Similar cells may be isolated from various sources, including human, and from the gut of other vertebrates and invertebrates [30–32]. In the literature, there are many descriptions of intestinal cells with a rich experimental portfolio [33, 34]. The drawback of animal cell models is that the results cannot be directly transferred to humans. Compared to animal intestinal cell isolation, there are fewer reports about the isolation of intestinal epithelial cells of human origin [19, 24, 35]. Despite this lower number of human cultures, these cells are widely used for studies of

human physiology and metabolic processes that would otherwise not be possible in vivo [36, 37]. The isolation of these cells is demanding because in vitro epithelial intestinal cells die rapidly, mainly due to the lack of attachment to the substrate, which is crucial for their survival [28]. To our knowledge this is the first report about isolation of adult human untransformed intestinal epithelial cell line from ileum. There are; however, descriptions of epithelial intestinal cells isolated from foetal tissues [37, 38]. Intestinal epithelial cells may be isolated from different parts of the intestine. We decided for the terminal ileum resection specimens due to accessibility. The intestinal mucosa of this part has abundant villi, which means a high concentration of cells per unit area [19]. Isolation procedures are simpler as yield at higher concentration of cells is better. Since the aim was the isolation of untransformed cultures of human intestinal epithelial cells, it was necessary to obtain a healthy part of the small intestine. Intestinal tissue affected by cancer growth or chronic inflammatory processes is not suitable for isolation of these types of cells, as their characteristics are completely different from the native line [6, 39]. During the isolation, it was necessary to develop an effective technique for the maintenance of cell culture, which is often challenging and complicated, so it may take long to establish an efficient and reliable line of cell culture [39, 40]. As stable cell culture without oncogenic transformation or use of viral proteins, which are commonly used in the establishment of permanent transformed cell lines, this allows the cells to maintain the characteristics of the tissue from which they originate [6]. Despite the ideal characteristics in contrast to transformed cell lines, the untransformed cell culture can dedifferentiate and the cells lose their phenotypic characteristics after a certain number of passages, usually 20–40, depending on the cell type. This can be somewhat corrected by special conditions of cultivation and selective cell media [2, 6]. HUIEC retained the characteristics of the tissue from which they were isolated. This is typically characteristic for cells up to 20 passages, making HIUEC a good model for research.

Fig. 4  Confocal micrographies of HUIEC after immunohistochemistry. The cells positive for the enzyme intestinal alkaline phosphatase are stained green. The nucleus is blue a Positive signal for FABP (red) and the nucleus (blue) b Bar = 25 μm

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Typical characteristics of HUIEC were observed during growth. Similar features were described in other intestinal epithelial lines [19, 40]. Cells were grown in flasks in a single layer and after the formation of confluent culture the growth stopped. Cells did not accumulate in domes or grew in several layers. This indicates that the contact inhibition was maintained, which is not unique to the cells HUIEC, but also for other untransformed cells and is not seen in cancer lines [19, 24, 41]. HUIEC exhibit a high proliferative index. This enables a rapid formation of confluent culture. Due to fast growth, the cultures require continuous monitoring of the growth conditions as other rapidly growing cells [6, 19]. On the other hand, a number of growth factors with autocrine and paracrine effects on the adjacent cells are secreted during cell growth [1]. Since these substances exhibit affects on the entire culture, it is recommended that one fifth of the volume of the condition media be returned to the flasks for normal growth. This principle was observed during the maintenance of HUIEC and proved to be useful. The phenotypical and functional characterization of the cells was performed with immunocytochemistry and the search for the presence of key epithelial markers. In order to distinguish cells from epithelial origin from potentially contaminating fibroblasts, the cells were labelled with antibodies directed against the intermediary type II filaments cytokeratin 18, specifically expressed in epithelia and vimentin, an intermediary type III filament [42]. The ability of cells to express small intestinal organotypic brush border enzymes has also been investigated with the antibodies against intestinal alkaline phosphatase and FABP which has been recognised as a marker of gut maturity [43–46]. Positive signals from the labelled probes provide the evidence of epithelial lineage designation. According to immunocytochemical analysis, all cells expressed the epithelial cytoskeleton component cytokeratin 18 and were double positive also for vimentin. Although in vivo expression of vimentin decreases during epithelial differentiation, Kaeffer et al. found that epithelial cells which undergo mesenchymal transformation in vitro, re-express vimentin [45]. Moreover, the presence of vimentin correlates with the maintenance of epithelial cell function and those of specific intestinal enzymes [42]. It is thus not surprising that the double positive cells have all been shown to express high levels of the enzymes intestinal alkaline phosphatase and FABP that provide the evidence of gut-like epithelial function.

Conclusions HUIEC is a stable and fast-growing untransformed, adult human epithelial intestinal cell line, which can be used for further experiments with functional cell models. Isolation of other cell types is needed for functional cell models in biochemistry, medicine, pharmacology, food

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technology, and as a credible replacement for experimental animals. These are just small steps, which in future will bring more information about the development and usefulness of cell cultures. Aknowledgements We would like to thank Ms Avrelija Cencic, PhD, from the Faculty of Medicine, University of Maribor for the support during the isolation process and the Department of Abdominal Surgery, University Medical Centre Ljubljana for tissue samples. Conflict of interest  The authors declare that there are no actual or potential conflicts of interest in relation to this article.

References   1. Alberts B, Bray D, Johnson A, Lewis J, Raff J, Roberts K, et al. Essential cell biology. New York: Garland; 1997.  2. Freshney RI. Culture of animal cells. A manual of basic technique. New York: Willey-Liss; 2000.   3. Carell A. On the permanent life of tissues outside of the organisms. J Exp Med. 1912;15(4):516–28.   4. Harrison RG. Observations on the living developing nerve fiber. Proc Soc Exp Biol Med. 1907;4:140–3.   5. Draper JS, Moored HD, Ruban LN, Gokhale PJ, Andrews PW. Culture and characterization of human embryonic stem cells. Stem Cells Dev. 2004;13(4):325–36.   6. Freshney RI. Culture of cells for tissue engineering. Basic principles of cell culture. New York: Willey; 2006. pp. 3–22.  7. Fresheny RI. Tumour cells disaggregated in collagenase. Lancet. 1972;2(7775):488–9.   8. Hoffmann W. Self—renewal of the gastric epithelium from stem and progenitor cells. Front Biosci. 2013;5:720–31.   9. Jirillo E, Jirillo F, Magrone T. Healthy effects exerted by prebiotics, probiotics and symbiotics with special reference to their impact on the immune system. Int J Vitam Nutr Res. 2012;82(3):200–8. 10. Macfarlane S, Macfarlane GT, Cummings JH. Review article: prebiotics in the gastrointestinal tract. Aliment Pharmacol Ther. 2006;24(5):701–14. 11. Robles Alonso V, Guarner F. Linking the gut microbiota to human health. Br J Nutr. 2013;109(2):21–6. 12. Viladomiu M, Hontecillas R, Yuan L, Lu P, BassaganyaRiera J. Nutritional protective mechanisms against gut inflammation. J Nutr Biochem. 2013;24(5):929–39. 13. De Vreese M, Schrezenmeir J. Probiotics, prebiotics and synbiotics. Adv Biochem Eng Biotechnol. 2008;111:1–66. 14. EFSA GMO Panel Working Group on Animal Feeding Trials. Safety and nutritional assessment of GM plants and derived food and feed: the role of animal feeding trials. Food Chem Toxicol. 2008;46(1):2–70. 15. Carrasco A, Mane J, Santaolalla R, Pedrosa E, Mallolas J, Loren V, et al. Comparison of lymphocyte isolation methods for endoscopic biopsy specimens from the colonic mucosa. J Immunol Methods. 2013;389(1–2):29–37. 16. Thopmson JN. Small bowel disease and intestinal obstruction. Clinical Surgery. Toronto: Elsevier; 2005. p. 377–92. 17. Clevers H. The intestinal crypt, a prototype stem cell compartment. Cell. 2013;154(2):274–84.

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original article 18. Galand G. Brush border membrane sucrase-isomaltase, maltase-glucoamylase and trehalase in mammals. Comparative development, effects of glucocorticoids, molecular mechanisms, and phylogenetic implications. Comp Biochem Physiol B. 1989;9(1):1–11. 19. Beaulieu JF, Menard D. Isolation, characterization, and culture of normal human intestinal crypt and villus cells. Methods Mol Biol. 2012;806:157–73. 20. Leonard F, Collnot EM, Lehr CM. A three—dimensional coculture of enterocytes, monocytes and dendritic cells to model inflamed intestinal mucosa in vitro. Mol Pharm. 2010;7(6):2103–19. 21. Monfatur-Solis D, Klein JR. An improved method for isolating intraepithelial lymphocytes (IELs) from the murine small intestine with consistently high purity. J Immunol Methods. 2006;308(1–2):251–4. 22. Naschberger E, Schellerer VS, Rau TT, Croner RS, Sturzl M. Isolation of endothelial cells from human tumors. Methods Mol Biol. 2011;731:209–18. 23. Schellerer VS, Croner RS, Weinlander K, Hohenberger W, Sturzl M, Naschberger E. Endothelial cells of human colorectal cancer and healthy colon reveal phenotypic differences in culture. Lab Invest. 2007;87(11):1159–70. 24. Booth C, O‘Shea JA. Isolation and culture of intestinal epithelial cells. Culture of epithelial cells. New York: Wiley – Lyss; 2002. p. 303–35. 25. Petto C, Lesko S, Gabel G, Bottner M, Wedel T, Kacza J, et al. Establishment and characterization of porcine colonic epithelial cells grown in primary culture. Cells Tissues Organs. 2011;194(6):457–68. 26. Traber PG, Gumucio DL, Wang W. Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt—villus axis. Am J Physiol. 1991;260(6):895–903. 27. Wang F, Scoville D, He XC, Mahe MM, Box A, Perry J. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony-formation assay. Gastroenterology. 2013;145(2):383–95. 28. Grossmann J, Maxon JM, Whitacre CM, Orosz DE, Berger NA, Fiocchi C, et al. New isolation technique to study apoptosis in human intestinal epithelial cells. Am J Pathol. 1998;153(1):53–62. 29. Manconi F, Markham R, Fraser IS. Culturing endothelial cells of microvascular origin. Methods Cell Sci. 2000;22:89–99. 30. Magness ST, Puthoff ST, Crissey MA, Dunn MA, Henning SJ, Houchen C. A multicenter study to standardize reporting and analyses of fluorescence—activated cell—sorted murine intestinal epithelial cells. Am J Physiol Gastrointest Liver Physiol. 2013;305(8):542–51. 31. Soret R, Coquenlorge S, Cossais F, Meurette G, RolliDerkinderen M, Neunlist M. Characterization of human, mouse, and rat cultures of enteric glial cells and their effect on intestinal epithelial cells. Neurogastroenterol Motil. 2013;25(11):755–64. 32. Wang F, Scoville D, He XC, Mahe M, Box A, Perry J, et al. Isolation and characterization of intestinal stem cells based on surface marker combinations and colony—formation assay. Gastroenterology. 2013;145(2):383–95.

33. Schierack P, Nordhoff M, Pollmann M, Weyrauch KD, Amasheh S, Lodemann U. Characterization of a porcine intestinal epithelial cell line for in vitro studies of microbial pathogenesis in swine. Histochem Cell Biol. 2006;125(3):293–305. 34. Von Furstenberg RJ, Gulati AS, Baxi A, Doherty JM, Stappenbeck TS, Gracz AD, et al. Sorting mouse jejunal epithelial cells with CD24 yields a population with characteristics of intestinal stem cells. Am J Physiol Gastrointes Liver Physiol. 2011;300(3):409–17. 35. Kanauchi O, Andoh A, Mitsuyama K. Effects of the modulation of microbiota on gastrointestinal immune system and bowel function. J Agric Food Chem. 2013;61(42):9977–83. 36. Liu Z, Zhankg P, Zhou Y, Qin H, Shen T. Culture of human intestinal epithelial cell using the dissociating enzyme thermolysin and endothelin-3. Braz J Med Biol Res. 2010;42(5):451–9. 37. Pageot LP, Perreault N, Basora N, Francoeur C, Magny P, Beaulieu JF. Human cell models to study small intestinal functions: recapitulation of the crypt—villus axis. Microsc Res Tech. 2000;49(4):394–06. 38. Levin TG, Powell AE, Davies PS, Silk AD, Dismuke AD, Anderson EC, et al. Characterization of the intestinal cancer stem cell marker CD166 in the human and mouse gastrointestinal tract. Gastroenterology. 2010;139(6):2072–82. 39. Welhelm A, Jahns F, Boecker S, Mothes H, Greulich KO, Glei M. Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells. Toxicol In Vitro. 2012;26(1):133–41. 40. Jung P, Sato T, Merlos-Suarez A, Barriga FM. Isolation and in vitro expansion of human colonic stem cells. Nat Med. 2011;17(10):1225–7. 41. Baudin B, Bruneel A, Bosselut N, Vaubordolle M. A protocol for isolation and culture of human umbilical vein endothelial cells. Nat Protoc. 2007;2(3):481–5. 42. Rusu D, Loret S, Peulen O, Mainil J, Dandrifosse G. Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures. BMC Cell Biol. 2005;6:42. 43. Shin J, Carr A, Corner GA, Togel L, Davalos-Salas M, Tran H, et al. The intestinal epithelial cell differentiation marker alpi is selectively induced by hdac inhibitors in colon cancer cells in a klf5-dependent manner. J Biol Chem. 2014. http://www.jbc.org/content/early/2014/07/18/jbc. M114.557546.long. 44. Reisinger KW, Elst M, Derikx JP, Nikkels PG, de Vries B, Adriaanse MP, et al. Intestinal fatty acid binding protein (I-FABP): a possible marker for gut maturation. Pediatr Res. 2014. doi: 10.1038/pr.2014.89. 45. Kaeffer B, Bottreau E, Velge P, Pardon P. Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid. Eur J Cell Biol. 1993;62(1):152–62. 46. Cencic A, Lefevre F, Komiotis D, Manta S, Botic T. 1-(6-O-Acetyl-3,4-Dideoxy-3-Fluoro-β-D-Glycero-Hex-3enopyranosyl-2-Ulose)-N4-Benzoyl Cytosine is a potent inhibitor of pseudorabies virus replication in infected cells. Acta Medico-biotechnica. 2009;2(2):41–7.

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HUIEC, Human intestinal epithelial cell line with differentiated properties: process of isolation and characterisation.

The intestinal epithelium is composed of diverse cell types, most abundant being the enterocytes. Among other functions, they maintain the intestinal ...
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