Journal of Food and Nutrition Research
ISSN (Print): 2333-1119 ISSN (Online): 2333-1240 Website: http://www.sciepub.com/journal/jfnr Editor-in-chief: Prabhat Kumar Mandal
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Journal of Food and Nutrition Research. 2017, 5(3), 160-167
DOI: 10.12691/jfnr-5-3-4
Open AccessArticle

Cell Culture Model for Examining Fat-Soluble Nutrient Absorption In Vitro

Alison Kamil1, Jeffrey B. Blumberg1 and C-Y. Oliver Chen1,

1Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, 711 Washington St., Boston, MA 02111

Pub. Date: March 04, 2017

Cite this paper:
Alison Kamil, Jeffrey B. Blumberg and C-Y. Oliver Chen. Cell Culture Model for Examining Fat-Soluble Nutrient Absorption In Vitro. Journal of Food and Nutrition Research. 2017; 5(3):160-167. doi: 10.12691/jfnr-5-3-4

Abstract

Permeable support systems (PS) are employed in in vitro nutrient absorption studies but data are absent on their efficacy compared to conventional cell culture models (CONV). The in vivo absorption of fat soluble nutrients is influenced by its delivery vehicle, yet a fundamental understanding of the influence of the vehicle on cells in culture is lacking. We compared the efficacy of lutein absorption in Caco-2 cells cultured with CONV and PS, and examined the role of micelles, the physiological vehicle within the small intestine. After plating for 2 and 21 d to attain confluence and differentiation in CONV and PS, respectively, cells were treated with lutein in micelles or ethanol. After incubation, lutein in cell lysate, as well as apical and basolateral mediums, were quantified by HPLC-UV. After 24 h, cellular lutein in CONV was ≥460 and 8% greater in ethanol and micelle, respectively, than in PS. However, the intracellular AUC over time was only different for ethanol (P ≤ 0.05). In PS, 0.15% of micellized lutein was secreted into the basolateral medium in contrast to 0.016% of lutein in ethanol. The absorption of lutein (uptake + secretion), independent of the vehicle, in CONV increased in a linear manner with dose (0.35 to 4 or to 14.6 µg/mL for ethanol or micelle, respectively), while that in PS peaked at 1.18 μg/mL. Caco-2 cells cultured in PS grow to display the phenotype and function of small intestine enterocytes and suggest this in vitro platform generates information closest to the natural physiology of the absorptive process. However, although the CONV has the physiology of colonic tissue, it appears to display a greater efficacy for lutein uptake by Caco-2 cells and so can provide a more rapid, preliminary method for nutrient absorption studies.

Keywords:
Caco-2 cells lutein (PubChem CID: 5281243) cell culture model micelle ethanol (PubChem CID: 702)

Creative CommonsThis work is licensed under a Creative Commons Attribution 4.0 International License. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

References:

[1]  Delie, F., and Rubas, W. “A human colonic cell line sharing similarities with enterocytes as a model to examine oral absorption: advantages and limitations of the Caco-2 model,” Critical Reviews in Therapeutic Drug Carrier Systems, 14 (3), 221-286, 1997.
 
[2]  Le Ferrec, E., Chesne, C., Artusson, P., Brayden, D., Fabre, G., Gires, P., Guillou, F., Rousset, M., Rubas, W., and Scarino, M.L. “In vitro models of the intestinal barrier. The report and recommendations of ECVAM Workshop 46. European Centre for the Validation of Alternative methods,” Alternatives to Laboratory Animals, 29 (6), 649-668, Nov.-Dec. 2001.
 
[3]  Sambruy, Y., Ferruzza, S., Ranaldi, G., and De Angelis, I. “Intestinal cell culture models: applications in toxicology and pharmacology,” Cell Biology and Toxicology, 17 (4-5), 301-317, 2001.
 
[4]  Ferruzza, S., Rossi, C., Scarino, M.L., and Sambuy, Y. “A protocol for differentiation of human intestinal Caco-2 cells in asymmetric serum-containing medium,” Toxicology In Vitro, 26 (8), 1252-1255, Dec. 2012.
 
[5]  Sambruy, Y., De Angelis, I., Ranaldi, G., Scarino, M.L., Stammati, A., and Zucco, F. “The Caco-2 cell line as a model of the intestinal barrier: influence of cell and culture-related factors on Caco-2 cell functional characteristics,” Cell Biology and Toxicology, 21 (1), 1-26, Jan. 2005.
 
[6]  Reisher, S.R., Hughes, T.E., Ordovas, J.M., Schaefer, E.J., and Feinstein, S.I. “Increased expression of apolipoprotein genes accompanies differentiation in the intestinal cell line Caco-2,” Proceedings of the National Academy of Sciences USA, 90 (12), 5757-5761, Jun. 1993.
 
[7]  Engle, M.J., Goetz, G.S., and Alpers, D.H. “Caco-2 cells express a combination of colonocyte and enterocyte phenotypes,” Journal of Cell Physiology, 174 (3), 362-369, Mar. 1998.
 
[8]  Perdikis, D.A., and Basson, M.N. “Basal nutrition promotes human intestinal epithelial (Caco-2) proliferation, brush border enzyme activity, and motility,” Critical Care Medicine, 25 (1), 159-165, Jan. 1997.
 
[9]  Cereijido, M., Robbins, E.S., Dolan, W.J., Rotunno, C.A., and Sabatini, D.D. “Polarized monolayers formed by epithelial cells on a permeable and translucent support,” Journal of Cell Biology, 77 (3), 853-880, June 1978.
 
[10]  Borel, P. “Factors affecting intestinal absorption of highly lipophilic food microconstituents (fat-soluble vitamins, carotenoids and phytosterols),” Clinical Chemistry and Laboratory Medicine, 41 (8), 979-994, Aug. 2003.
 
[11]  Noy, N., Kelleher, D.J., and Scotto, A.W. “Interactions of retinol with lipid bilayers: studies with vesicles of different radii,” Journal of Lipid Research, 36 (2), 375-382, Feb. 1995.
 
[12]  Pérez, M.D., and Calvo, M. “Interaction of beta-lactoglobulin with retinol and fatty acids and its role as a possible biological function for this protein: a review,” Journal of Dairy Sciences, 78 (5), 978-988, May 1995.
 
[13]  Reboul, E., and Borel, P. “Proteins involved in uptake, intracellular transport and basolateral secretion of fat-soluble vitamins and carotenoids by mammalian enterocytes,” Progress in Lipid Research, 50 (4), 388-402, Oct. 2011.
 
[14]  Narushima, K., Takada, T., Yamanashi, Y., and Suzuki, H. “Niemann-pick C1-like 1 mediates alpha-tocopherol transport,” Molecular Pharmacology, 74 (1), 42-49, Jul. 2008.
 
[15]  Reboul, E. “Absorption of vitamin A and carotenoids by the enterocyte: focus on transport proteins,” Nutrients, 5(9), 3563-3581, Sep. 2013.
 
[16]  Garrett, D.A., Failla, M.L., and Sarama, R.J. “Development of an in vitro digestion method to assess carotenoid bioavailability from meals,” Journal of Agricultural and Food Chemistry, 47 (10), 4301-4309, Oct. 1999.
 
[17]  Reboul, E., Thap, S., Perrot, E., Amiot, M.J., Lairon, D., and Borel, P. “Effect of the main dietary antioxidants (carotenoids, gamma-tocopherol, polyphenols, and vitamin C) on alpha-tocopherol absorption,” European Journal of Clinical Nutrition, 61 (10), 1167-1173, Oct. 2007.
 
[18]  Shahrzad, S., Cadenas, E., Sevanian, A., Packer, L. “Impact of water-dispersible beadlets as a vehicle for the delivery of carotenoids to cultured cells,” Biofactors, 16 (3-4), 83-91, 2002.
 
[19]  Lancrajan, I., Diehl, H.A., Socaciu, C., Engelke, M., and Zorn-Kruppa, M. “Carotenoid incorporation into natural membranes from artificial carriers: liposomes and beta-cyclodextrins,” Chemistry and Physics of Lipids, 112 (1), 1-10, Jul. 2001.
 
[20]  O’Sullivan, S.M., Woods, J.A., and O'Brien, N.M. “Use of Tween 40 and Tween 80 to deliver a mixture of phytochemicals to human colonic adenocarcinoma cell (CaCo-2) monolayers,” British Journal of Nutrition, 91 (5), 757-764, May 2004.
 
[21]  Zhang, P., and Omaye, S.T. “Antioxidant and prooxidant roles for beta-carotene, alpha-tocopherol and ascorbic acid in human lung cells,” Toxicology In Vitro, 15 (1), 13-24, Feb. 2001.
 
[22]  Liu, C.S., Glahn, R.P., and Liu, R.H. “Assessment of carotenoid bioavailability of whole foods using a Caco-2 cell culture model coupled with an in vitro digestion,” Journal of Agricultural and Food Chemistry, 52 (13), 4330-4337, Jun. 2004.
 
[23]  El-Metwally, T.H., and Adrian, T.E. “Optimization of treatment conditions for studying the anticancer effects of retinoids using pancreatic adenocarcinoma as a model,” Biochemical and Biophysical Research Communications, 257 (2), 596-603, Apr. 1999.
 
[24]  Tapiero, H., Townsend, D.M., and Tew, K.D. “The role of carotenoids in the prevention of human pathologies,” Biomedicine and Pharmacotherapy, 58 (2), 100-110, Mar. 2004.
 
[25]  Krinsky, N.I., and Johnson, E.J. “Carotenoid actions and their relation to health and disease,” Molecular Aspects of Medicine, 26 (6), 459-516, Dec. 2005.
 
[26]  Johnson, E.J. “The role of carotenoids in human health,” Nutrition in Clinical Care, 5 (2), 56-65, Mar.-Apr. 2002.
 
[27]  Craft, N.E., and Soares, J.H. “Relative solubility, stability, and absorptivity of lutein and β-carotene in organic solvents,” Journal of Agricultural and Food Chemistry, 40 (3), 431-434, Mar. 1992.
 
[28]  Chitchumroonchokchai, C., Schwartz, S.J., and Failla, M.L. “Assessment of lutein bioavailability from meals and a supplement using simulated digestion and caco-2 human intestinal cells,” Journal of Nutrition, 134 (9), 2280-2286, Sept. 2004.
 
[29]  Monsen, E.R. “Dietary reference intakes for the antioxidant nutrients: vitamin C, vitamin E, selenium, and carotenoids,” Journal of American Dietetic Association, 100 (6), 637-640, Jun. 2000.
 
[30]  Park, S.C., Lim, J.Y., Jeen, Y.T., Keum, B., Seo, Y.S., Kim, Y.S., Lee, S.J., Lee, H.S., Chun, H.J., Um, S.H., Kim, C.D., Ryu, H.S., Sul, D., and Oh, E. “Ethanol-induced DNA damage and repair-related molecules in human intestinal epithelial Caco-2 cells,” Molecular Medicine Reports, 5(4), 1027-1032, Apr. 2012.
 
[31]  Luchoomun, J., and Hussain, M.M. “Assembly and secretion of chylomicrons by differentiated Caco-2 cells. Nascent triglycerides and preformed phospholipids are preferentially used for lipoprotein assembly,” Journal of Biological Chemistry, 274 (28), 19565-19572, Jul. 1999.
 
[32]  Kamil, A., Smith D.E., Blumberg, J.B., Astete, C., Chen, C.Y. “Bioavailability and biodistribution of nanodelivered lutein,” Food Chemistry, 192, 915-923, Feb. 2016.
 
[33]  Nielsen, I.L., Chee, W.S., Poulsen, L., Offord-Cavin, E., Rasmussen, S.E., Frederiksen, H., Enslen, M., Barron, D., Horcajada, M.N., and Williamson, G. “Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial,” Journal of Nutrition, 136 (2), 404-408, Feb. 2006.
 
[34]  Yi, J., Lam, T.I., Yokoyama, W., Cheng, L.W., and Zhog, F. “Cellular uptake of beta-carotene from protein stabilized solid lipid nano-particles prepared by homogenization-evaporation method,” Journal of Agricultural and Food Chemistry, 62 (5), 1096-1104, Feb. 2014.
 
[35]  Sy, C., Gleize, B., Dangles, O., Landrier, J.F., Veyrat, C.C., Borel, P. “Effects of physicochemical properties of carotenoids on their bioaccessibility, intestinal cell uptake, and blood and tissue concentrations,” Molecular Nutrition and Food Research, 56 (9), 1385-1397, Sep. 2012.
 
[36]  O'Sullivan, L., Ryan, L., and O'Brien, N. “Comparison of the uptake and secretion of carotene and xanthophyll carotenoids by Caco-2 intestinal cells,” British Journal of Nutrition, 98 (1), 38-44, Jul. 2007.
 
[37]  During, A., Hussain, M., Morel, D., and Harrison, E. “Carotenoid uptake and secretion by CaCo-2 cells: beta-carotene isomer selectivity and carotenoid interactions,” Journal of Lipid Research, 43 (7), 1086-1095, Jul. 2002.
 
[38]  Traber, M.G., Kayden, H.J., and Rindler, M.J. “Polarized secretion of newly synthesized lipoproteins by the Caco-2 human intestinal cell line,” Journal of Lipid Research, 28 (11), 1350-1363, Nov. 1987.
 
[39]  Franssen-van Hal, N.L., Bunschoten, J.E., Venema, D.P., Hollman, P.C., Riss, G., and Keijer, J. “Human intestinal and lung cell lines exposed to beta-carotene show a large variation in intracellular levels of beta-carotene and its metabolites,” Archives of Biochemistry and Biophysics, 439 (1), 32-41, Jul. 2005.
 
[40]  Dashti, N., Smith, E.A., and Alaupovic, P. “Increased production of apolipoprotein B and its lipoproteins by oleic acid in Caco-2 cells,” Journal of Lipid Research, 31 (1), 113-123, Jan. 1990.
 
[41]  Wagner, R.D., Krul, E.S., Moberly, J.B., Alpers, D.H., and Schonfeld, G. “Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells,” American Journal of Physiology, 263 (2 Pt1), E374-E382, Aug. 1992.
 
[42]  Hilgendorf, C., Spahn-Langguth, H., Regårdh, C.G., Lipka, E., Amidon, G.L., and Langguth, P. “Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport,” Journal of Pharmaceutical Sciences, 89 (1), 63-75, Jan. 2000.
 
[43]  Mahler, G.J., Shuler, M.L., and Glahn, R.P. “Characterization of Caco-2 and HT29-MTX cocultures in an in vitro digestion/cell culture model used to predict iron bioavailability,” Journal of Nutritional Biochemistry, 20 (7), 494-502, Jul. 2009.
 
[44]  Nollevaux, G., Devillé, C., El Moualij, B., Zorzi, W., Deloyer, P., Schneider, Y.J., Peulen, O., and Dandrifosse, G. “Development of a serum-free co-culture of human intestinal epithelium cell-lines (Caco-2/HT29-5M21),” BMC Cell Biology, 7, 20-31, May 2006.
 
[45]  des Rieux, A., Ragnarsson, E.G., Gullberg, E., Préat, V., Schneider, Y.J., and Artursson, P. “Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium,” European Journal of Pharmaceutical Sciences, 25 (4-5), 455-465, Jul.-Aug. 2005.
 
[46]  Kadiyala, I., Loo, Y., Roy, K., Rice, J., and Leong, K.W. “Transport of chitosan-DNA nanoparticles in human intestinal M-cell model versus normal intestinal enterocytes,” European Journal of Pharmaceutical Sciences, 39 (1-3), 103-109, Jan. 2010.
 
[47]  Gullberg, E., Leonard, M., Karlsson, J., Hopkins, A.M., Brayden, D., Baird, A.W., and Artursson, P. “Expression of specific markers and particle transport in a new human intestinal M-cell model,” Biochemical and Biophysical Research Communications, 279 (3), 808-813, Dec. 2000.
 
[48]  Antunes, F., Andrade, F., Araújo, F., Ferreira, D., and Sarmento, B. “Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs,” European Journal of Pharmaceutics and Biopharmaceutics, 83 (3), 427-435, Apr. 2013.