<p>Applied Microbiology and Biotechnology</p><p>Hollow Fiber Culture Accelerates Differentiation of Caco-2 Cells </p><p>Xudong Deng1, Guoliang Zhang2, Chong Shen1, Jian Yin1, Qin Meng1,*</p><p>1 Department of Chemical and Biological Engineering, Zhejiang University, Zhejiang 310027, P.R. China 2 Institute of Biological and Environmental Engineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310012, P.R. China</p><p>*Corresponding author: Qin Meng Department of Chemical and Biological Engineering, Zhejiang University, 38 Zheda Road, Hangzhou, Zhejiang, 310027, PR China. E-mail address: [email protected] Tel: 86-571-87953193 Fax: 86-571-87951227 Supporting information</p><p>Materials and Methods</p><p>Materials. Cimetidine, ketoconazole, midazolam, verapamil, dexamethasone, testosterone, ibuprofen, omeprazole and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO,</p><p>USA). Methotrexate was purchased from Bio Basic Inc. (Markham Ontario, Canada).</p><p>Clozapine was kindly offered by The First Affiliated Hospital of Zhejiang (Hangzhou, China).</p><p>Propranolol, amiloride and warfarin were generously donated by Mr. Wang Yanming and</p><p>Miss. Chen Qiuxia (College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,</p><p>China). </p><p>Chemical analysis. The concentration of each drug was analyzed by a HPLC system (ProStar</p><p>210 Series, Varian, Walnut Creek, CA) equipped with a reverse phase column (ChromSpher</p><p>C18, 150 mm × 4.6 mm, 5μm particle size, Varian, Walnut Creek, CA) except for inulin. The mobile phases and the wavelengths monitored for isolated absorbance peaks were listed in</p><p>Table S1. </p><p>Results</p><p>Drug transport across Caco-2 cell layers</p><p>Sixteen drugs, showing no or low adsorption (less than 10%) by PES membrane, were selected for prediction of intestinal absorption. Among these drugs, fourteen were transported mostly via passive pathways and two were transported by efflux transporters.</p><p>The Papp of each drug across the cell layer from apical to basolateral (A-B) was evaluated in hollow fiber culture at day 10 and Transwell culture at day 21 (Table S2). Majority of drugs presented equivalent Papp between two cultures.</p><p>The A-B permeability of all the sixteen drugs in each culture was plotted against the oral absorption percentage in humans using semilogarithmic coordinates. A good linearity</p><p>(r2>0.87) was observed in both cultures (Figure S1). Table S1</p><p>The operation conditions of mobile phase and wavelength for HPLC detection.</p><p>Compound Mobile phase (1 mL/min) Wavelength (nm) Acetaminophen Water: methanol (50: 50) 254</p><p>Amiloride Water: methanol: acetic acid (60: 40: 1) 286</p><p>Cimetidine Water: methanol: triethylamine: phosphate (80: 20: 0.1: 0.06) 219</p><p>Clozapine Water: methanol: acetonitrile: acetic acid: n-butylamine (44: 28: 254 28: 1.9: 0.4) Dexamethasone Water: methanol (25: 75) 240</p><p>Erythromycin 0.02 mol/L KH2PO4: acetonitrile (45: 55) 215</p><p>Ibuprofen 0.04 mol/L KH2PO4: methanol (30:70) 263</p><p>Ketoconazole 0.02 mol/L KH2PO4: methanol (25: 75) 230</p><p>Methotrexate 0.05 mol/L sodium acetate: acetonitrile: methanol (87: 6.5: 6.5) 260</p><p>Midazolam Water: acetonitrile: acetic acid: triethylamine (60: 40: 0.03:0.08) 225</p><p>Propranolol 0.02 mol/L KH2PO4: methanol (50: 50) 290</p><p>Testosterone 0.02 mol/L KH2PO4: methanol: triethylamine (50: 50: 0.1) 254</p><p>Verapamil 0.1 mol/L sodium acetate (pH=5.8): methanol (42: 58) 278</p><p>Warfarin Water: acetonitrile: acetic acid (60: 40: 1) 308 Table S2 The apical to basolateral permeability of 13 drugs across Caco-2 cell layers in PES hollow fiber</p><p>(day 10) and in Transwell (day 21). (n = 3 for each point)</p><p>Compound Papp in hollow fiber culture Papp in Transwell FA (%) (× 10-6 cm/s) (× 10-6 cm/s) Acetaminophen 16.43 ± 3.56 21.90 ± 1.40 95a Amiloride 3.81 ± 0.29 5.31 ± 0.51 50b Clozapine 19.66 ± 1.74 27.54 ± 4.74 100b Dexamethasone 34.29 ± 6.01 29.03 ± 1.78 100c Ibuprofen 20.19 ± 3.81 35.90 ± 4.21 100c Inulin 1.36 ± 0.17 0.92 ± 0.26 5a Ketoconazole 12.13 ± 0.26 16.31 ± 1.13 76b Methotrexate 0.98 ± 0.05 1.21 ± 0.38 20b Midazolam 32.33 ± 3.82 29.22 ± 2.62 95d Omeprazole 24.14 ± 2.07 27.76 ± 1.58 80b Propranolol 25.33 ± 2.16 37.30 ± 4.50 90b Testosterone 22.99 ± 9.81 40.71 ± 8.25 100c Verapamil 18.98 ± 1.91 20.72 ± 2.12 95b Warfarin 19.71 ± 0.08 20.23 ± 1.35 98b FA, fraction absorbed in human intestinal; aData taken from (Marino et al. 2005) bData taken from (Skolnik et al. 2010) cData taken from (Yee 1997) dData taken from (Li et al. 2007) Figure S1</p><p>Figure S1. Cross-section of human small intestine showing one circular fold projecting into intestinal lumen which formed a circular intervalvular space. Figure S2</p><p>Figure S2. Prediction of the oral adsorption of 16 drugs. Oral adsorption fraction of 16 drugs in human and their permeability values across Caco-2 cell layers cultured in PES hollow fibers at day 10 (A) as well as in Transwell at day 21 (B). Linear fit was assessed in semilogarithmic coordinates.</p><p>References: </p><p>Li C, Liu T, Cui X, Uss AS, Cheng KC. 2007. Development of in vitro pharmacokinetic screens using Caco- 2, human hepatocyte, and Caco-2/human hepatocyte hybrid systems for the prediction of oral bioavailability in humans. Journal of biomolecular screening 12(8):1084-91. Marino AM, Yarde M, Patel H, Chong S, Balimane PV. 2005. Validation of the 96 well Caco-2 cell culture model for high throughput permeability assessment of discovery compounds. Int J Pharm 297(1-2):235-41. 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