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

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Journal of Food and Nutrition Research, 2017, Vol. 5, No. 3, Available online at Science and Education Publishing DOI: /jfnr Cell Culture Model for
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Journal of Food and Nutrition Research, 2017, Vol. 5, No. 3, Available online at Science and Education Publishing DOI: /jfnr Cell Culture Model for Examining Fat-Soluble Nutrient Absorption In Vitro Alison Kamil, Jeffrey B. Blumberg, C-Y. Oliver Chen * Antioxidants Research Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, 711 Washington St., Boston, MA *Corresponding author: 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: ), cell culture model, micelle, ethanol (PubChem CID: 702) Cite This Article: 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, vol. 5, no. 3 (2017): doi: /jfnr Introduction In vitro cell culture models present a useful means to study mechanisms of intestinal nutrient transport. The human intestinal Caco-2 cell line has been used extensively for studies examining nutrient and drug absorption [1,2,3]. Caco-2 cells originate from a human colonic carcinoma, and after differentiation, exhibit morphological and functional characteristics comparable to those of differentiated epithelial cells lining the mucosa of the small intestine. Differentiation to an enterocyte-like phenotype spontaneously occurs when the cells grow to confluency in conventional monolayer culture conditions [4,5,6]. However, during the early phases of differentiation, these cells express both colonocyte and enterocyte-specific proteins, such as surfactant protein A and α-1 antitrypsin, respectively [7]. As differentiation progresses, morphological and biochemical characteristics of enterocytes develop, including tight junctions, microvilli, enzymes and transport systems, along with the reduction of colonocyte-specific gene expression. Caco-2 cells can be cultured on permeable cell culture support systems (PS) or conventional tissue cultures on treated plastic plates (CONV) for model absorption experiments. While both culture systems are commonly employed, there are important differences between them. Cells cultured in CONV remain in a proliferative stage due to constant cell detachment resulting from the intracellular fluid being transported to the basolateral space of cells [8,9]. PS allows investigation of cell permeability and transepithelial transport because of the complete establishment of functional tight junction assemblies between cells [5]. Further, the apical and basolateral surfaces of cells in the PS face the upper and lower compartments, corresponding to the intestinal lumen side and serosal side, respectively. However, data are absent on whether PS generates more efficient intestinal absorption and more physiologically relevant information than CONV. The absorption of fat-soluble vitamins and carotenoids in vivo is influenced by the vehicle that carries the nutrient to the apical membrane of the enterocytes. During digestion, fat soluble nutrients are released from their food matrix and transferred to mixed micelles or, to a lesser extent, to vesicles such as liposomes [10,11] or associated with proteins such as β-lactoglobulin [12]. Once solubilized, uptake into the intestinal mucosa involves passive diffusion or facilitated transport through cholesterol transporters [13,14,15]. The final step in the absorption assimilates the 161 Journal of Food and Nutrition Research nutrient into chylomicrons and then secretes them into the lymph for systemic distribution. Aside from the major physiological vehicle, i.e., mixed micelles, fat soluble nutrients may also be delivered after they are incorporated into liposomes or water-miscible beadlets [16,17,18,19]. The organic solvents tetrahydrofuran (THF), dimethylsulfoxide (DMSO), and ethanol can be employed as delivery vehicles [20,21,22,23], but can present with serious limitations, such as instability, insolubility, and cytotoxicity. The efficacy of nutrient intestinal absorption using Caco-2 cell monolayers grown in CONV and PS has not been systematically compared. We compared the absorption efficacy of lutein in these 2 systems because of the putative health benefits of lutein and research efforts to optimize its bioavailability [24,25,26]. We also tested 2 delivery vehicles that were employed to solubilize lutein, i.e., a synthetically prepared micelle that models the physiological vehicle within the small intestine and the organic solvent ethanol, which despite its intermediate solubility, is safe and commonly consumed in humans [27]. A better understanding of the properties of CONV vs. PS and the type of delivery vehicle therein can provide practical guidelines regarding in vitro approaches to the study of fat-soluble nutrient absorption. 2. Materials and Methods 2.1. Chemicals and Materials β-cryptoxanthin (BC, 97%), 1-oleoyl-rac-glycerol [monoolein (MO), 99%], 2-oleoyl-1-palmitoyl-sn-glycero- 3-phosphocholine (PC, 99%), 1-palmitoyl-sn-glycero-3- phospho-choline [lysolecithin, (LC), 99%], sodium glycodeoxycholate (GDC, 97%), sodium taurodeoxycholate hydrate (TDC, 97%), taurocholic acid sodium salt hydrate (TC, 95%), sodium oleate (OA, 99%) and bovine serum albumin (97%) were purchased from Sigma-Aldrich (St. Louis, MO). Advanced Dulbecco s Modified Eagle Medium (DMEM), 200 mm L-glutamine, penicillinstreptomycin (10,000 U/mL) were purchased from Gibco, Life Technologies Inc. (Grand Island, NY). Hyclone phosphate buffer saline (PBS) without Ca 2+ or Mg 2+, Pierce RIPA buffer, and Pierce bicinchoninic acid (BCA) protein assay were purchased from Thermo Fischer Scientific (Rockford, IL). Multi-well tissue culture treated plates, Transwell permeable supports, cell culture treated flasks, and sterile filters (0.22 µm pore size) were purchased from Corning Life Sciences (Tewksbury, MA). Caco-2 cells, fetal bovine serum, and trypsin-edta (0.25%) were purchased from ATCC (Manassas, VA). All other solvents were HPLC grade and purchased from Sigma-Aldrich (St. Louis, MO). Purified lutein (70-75%) was a gift from Kemin Industries Inc. (Des Moines, IO) Preparation of Lutein Unmodified pure lutein in ethanol was added directly to serum-free medium, vortexed, and sonicated at room temperature for 2 min. Micelles containing lutein were prepared according to Chitchumroonchokchai et al. (2004) [28]. Briefly, MO, PC, and LC in chloroform (500, 200, and 200 µm, respectively), OA (1500 µm) in methanol, and lutein in ethanol were added to a conical glass tube and dried under N 2 gas at room temperature. Filtered sterilized serum-free medium containing 800, 450, and 750 µm GDC, TDC, and TC, respectively, were added to reconstitute the dry residue, and the resulting mixture was sonicated for 30 min at room temperature under red light to minimize photo-oxidation. In the time course experiment, lutein concentration at 4 and 14.6 µg/ml for ethanol and micelles, respectively, was employed to evaluate the effect of the test vehicle and culture system on its absorption. The concentration of 14.6 µg/ml of lutein (~5 mg/d) was selected for micelles based on the consideration that it would be an adequate dose to quantify the magnitude of the absorption. Further, this concentration was calculated based on the assumption of a mean dietary intake of 1 mg/d lutein [29] and concurrent delivery of 2-3 L of water to the small intestine. Lutein at 4 μg/ml (~1.3 mg/d) was selected for testing with ethanol to assure lutein solubility and cell viability. Although the solubility of lutein in ethanol has been shown to be 300 μg/ml [27], we were unable to successfully prepare lutein in ethanol at 4 μg/ml. Further, the viability of Caco-2 cells in ethanol decreases when exposed to ethanol concentrations 7% [30]. In the dose-response experiment, the lutein concentration in the medium for the ethanol arm was 0.35, 1.18, and 4 µg/ml, equivalent to a low to average daily lutein intake (~0.11, 0.39, and 1.3 mg in 3 L intestinal water). The lutein concentration in the medium for the micelle arm was 0.35, 1.18, 4, and 14.6 μg/ml, equivalent to a low to high average daily lutein intake of ~0.11, 0.39, 1.3, and 5 mg in 3 L intestinal water Cell Culture Caco-2 cells were maintained in advanced DMEM supplemented with 10% fetal bovine serum and 1% L-glutamine and the absence of antibiotics in a humidified incubator (Thermo Scientific Series 7000, Cambridge, MA) at 37 C and 5% CO 2. Cells between the 12 th and 27 th passages were used for all experiments. To examine the uptake of lutein in CONV, cells grown in the medium in the absence of antibiotics were seeded at a density of 7 x 10 4 cells/well on 24-well tissue culture treated plates (1.9 cm 2 growth area/well). Cells were grown for 48 h to attain ~80% confluence. At the beginning of each experiment, serum-containing media was removed and replaced with 1 ml of lutein enriched serum-free media. Since cells do not adhere tightly to the plates, washing of the cells with PBS to remove any residual serum was avoided. Serum-free media was utilized to remove any variability in performance [4]. The range of the time course (0-48 h) was selected based on the data from pilot experiments of cell uptake, which showed that the maximal uptake was not reached by 24 h but plateaued by 48 h (results not shown). Lutein in the medium was found to be stable for at least 48 h. At the end of the selected incubation times, the medium was collected and cells were then washed twice with PBS containing 2 mg/ml bovine serum albumin to remove any residual lutein. Subsequently, the cells in the wells were incubated with 300 μl RIPA buffer for 5 min on ice for lysis and removal. All samples were collected into 2 ml Journal of Food and Nutrition Research 162 Eppendorf tubes, flushed with N 2 gas, wrapped in parafilm, and stored at -80 C until analysis. All experiments and analyses were conducted under red light. To examine the uptake and secretion of lutein in PS, cells grown in the medium supplemented with 1% antibiotics (penicillin-streptomycin) were seeded at a density of 5 x 10 4 cells/well on collagen-coated Transwell permeable filters (12 well plate, 0.9 cm 2 growth area, 3 µm pore size). Cells were grown for 21 d (medium changed every 2-3 d) to attain complete differentiation and monolayer integrity [4]. Alkaline phosphatase activity, an index of differentiation, was measured spectrophometrically in cell lysate, according to the manufacturer s instructions (Sigma-Aldrich, St. Louis, MO). The cell lysate was collected using 210 μl 0.16% digitonin in 2 mm EDTA solution at 37 C and then utilized to measure protein content to reflect cell count. Trans-epithelial electrical resistance (TEER), reflective of monolayer integrity, was measured using a voltohmmeter equipped with a chopstick electrode (EVOHM2, World Precision Instruments, Sarasota, FL). At the beginning of each PS experiment, the apical side of Caco-2 cells was washed with PBS twice and replaced with 0.5 ml lutein enriched serum-free media. The basolateral compartment was filled with 1.5 ml serumfree media. The range of the time course (0-48 h) was selected based on the data of chylomicron production and secretion to the basolateral compartment in a pilot experiment. We noted a 50% increase in apo-lipoprotein- B (apo-b) production between 6 and 16 h, but a steady state was not apparent at h (data not shown). Apo-B was quantified using an ELISA kit (Antiobodies- Online.com, Product # ABIN612664) after chylomicron isolation using a gradient ultracentrifugation [31]. At the end of selected incubation times, both apical and basolateral media were collected. Cells remaining on the permeable filters were washed twice with PBS containing 2 mg/ml bovine serum albumin, incubated with 350 μl above and 600 μl below with 0.25% trypsin-edta solution for 30 min at 37 C to detach cells, and then lysed with 300 μl RIPA buffer for 5 min on ice. All samples were collected into Eppendorf tubes, flushed with N 2 gas, wrapped in parafilm, and stored at -80 C until analysis. All experiments and analysis were conducted under red light. To determine the proportion of free intracellular lutein in cell lysate, which was not attached to cell membranes, additional cell samples were collected at T max (identified in kinetic experiments) and combined in duplicate (all treatments for CONV and lutein in micelle for PS) or quadruplet (lutein in ethanol for PS) to ensure adequate quantification Lutein Extraction and Analysis Apical and basolateral media and cell lysates (sample size of 3-4 wells) were thawed and briefly vortexed. Cell lysates were sonicated for 30 sec at room temperature. To determine the proportion of intracellular lutein, cell lysate samples were further centrifuged at 14,000 rpm for 30 min to collect cell pellets and supernatant. Lutein and β-cryptoxanthin (internal standard) in the samples were quantified according to the method of Kamil et al. [32]. Briefly, samples were extracted sequentially with FOLCH solution (chloroform/methanol: 2/1) and hexane. The organic layer was transferred, combined, and dried under N 2 gas, reconstituted in 100 µl acetone, and then analyzed by reverse phase HPLC-UV at a flow rate of 1 ml/min at 20 C on a ProntoSIL C30 column (4.6 x 150 nm, 3.0 µm, MAC-MOD Analytical, Inc., Chadds Ford, PA). Lutein and β-cryptoxanthin were monitored at 443 and 450 nm, respectively. Their concentrations were calculated using standard curves constructed with authenticated standards with concentrations ranging from 1 to 825 ng on column. The limit of detection and quantification for lutein was 0.63 and 1.0 ng on column, respectively. The recovery rate for the internal standard, calculated from 57 samples, was 71.8 ± 19.4%. The protein content of cell lysates was determined using a BCA protein assay kit (Thermo Fischer Scientific, Rockford, IL) and was used to reflect cell count Statistics All data are expressed as mean ± SD (n = 3-4 wells). The peak concentration of cell lysate and plateauing basolateral medium (C max ) and the time to reach C max (T max ) were determined. The area under the curve (AUC) of apical, cell lysate, and basolateral concentration vs. time curve (0-48 h) were calculated from random complete time course curves using the linear trapezoidal integrations [33]. The differences between culture models (CONV vs PS) were analyzed using Student s t-test while the differences between doses in the culturing systems were analyzed by one-way ANOVA, followed by post hoc Tukey-Kramer honestly significant difference (HSD) test. Pearson s correlation test (r value) was performed to analyze the correlation between parameters. Simple linear regression test (r 2 value) was employed to analyze the dose-response of absorption. Three-way ANOVA was performed to test the statistical significance of dose, delivery vehicle, and culturing system. Differences with P 0.05 were considered significant. The JMP IN 4 statistical software package (SAS Institute, Cary, NC) was used to perform all statistical analyses. 3. Results 3.1. Delivery Vehicle The time effect on the cellular uptake of lutein and its pharmacokinetic parameters in CONV is presented in Figure 1 and Table 1. The cellular uptake of lutein in ethanol reached C max at 39 ± 6 h in a non-linear fashion (Table 1). The uptake of lutein increased gradually to 24 h followed by a spike and plateau at 36 h (Figure 1A). Concurrently, there was a 27% increase in lutein concentration in the apical medium from 0-4 h, followed by a decrease and then leveling off as the cell uptake reached its C max. Lutein concentrations in the cell lysate were not correlated with those in the apical medium. The uptake of lutein in micelles into the cells followed a similar trend to that of the ethanol vehicle (Figure 1B). The uptake increased up until 24 h reaching C max at 42 ± 7h (Table 1). Lutein in the apical medium decreased as the 163 Journal of Food and Nutrition Research uptake by cells increased and then reached a steady state as cell uptake reached its C max. Unlike lutein in ethanol, there was a negative correlation between the concentrations in the cell lysate and in the apical medium (r = -0.54, P = ). The time effect on the cell uptake and secretion of lutein and its pharmacokinetic parameters in PS are presented in Figure 2 and Table 1. The cellular uptake of lutein in ethanol reached C max at 12 ± 10.6 h (Table 1). Concurrently, lutein concentration in the apical medium decreased by 24% over 8 h without further change to 48 h (Figure 2A). The secretion of lutein in ethanol increased after lutein in the cell lysate reached C max at 0.016% of dose without further change to 48 h. Although lutein concentrations in the basolateral medium were inversely correlated over time with that in the apical medium (r = -0.81, P ), no correlation was observed between lutein in the cell lysate and the other 2 compartments. The uptake of lutein in micelle into cells reached C max at 8 h followed by a subsequent decrease towards its baseline concentration at 2 h (Figure 2B). Concurrently, lutein concentration in the apical medium decreased over time as the cell lysate reached C max, followed by a decrease through 48 h. Lutein concentrations in the cell lysate were inversely correlated with that in the apical medium (r = -0.82, P = 0.001). The secretion of lutein in micelle increased in a linear manner starting at 4 h and reached a plateau at 0.15% of the dose after 36 h (Figure 2B). Similarly, lutein concentrations in the cell lysate as well as in the apical medium were correlated with that in the basolateral medium (r = 0.91, P = and r = -0.97, P , respectively). Table 1. Pharmacokinetics of cell lysate of Caco-2 cells cultured on CONV and PS and treated with lutein in ethanol or micelles 1,2 C max (μg/mg protein) T max (h) AUC (h*ng/mg protein) Vehicles CONV PS CONV PS CONV PS Ethanol 2.8 ± ± 0.3* 39 ± 6 12 ± 10.6* 41 ± ± 6.7* Micelle 13.2 ± ± 1.5* 42 ± 7 8* 178 ± ± Values are expressed as mean ± SD (n = 3 and 4 wells for PS and CONV, respectively) 2 Percentage C max free in cell indicates proportion of C max within cytosol and not attached to cell membranes *Means within the same the vehicles in t
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