A Novel Bioreactor of Caco-2 Cell Cultured on Polyethersulfone Hollow Fiber Membranes For

Applied Microbiology and Biotechnology

Hollow Fiber Culture Accelerates Differentiation of Caco-2 Cells

Xudong Deng1, Guoliang Zhang2, Chong Shen1, Jian Yin1, Qin Meng1,*

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

*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

Materials and Methods

Materials. Cimetidine, ketoconazole, midazolam, verapamil, dexamethasone, testosterone, ibuprofen, omeprazole and Triton X-100 were purchased from Sigma-Aldrich (St. Louis, MO,

USA). Methotrexate was purchased from Bio Basic Inc. (Markham Ontario, Canada).

Clozapine was kindly offered by The First Affiliated Hospital of Zhejiang (Hangzhou, China).

Propranolol, amiloride and warfarin were generously donated by Mr. Wang Yanming and

Miss. Chen Qiuxia (College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,

China).

Chemical analysis. The concentration of each drug was analyzed by a HPLC system (ProStar

210 Series, Varian, Walnut Creek, CA) equipped with a reverse phase column (ChromSpher

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

Table S1.

Results

Drug transport across Caco-2 cell layers

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.

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.

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

(r2>0.87) was observed in both cultures (Figure S1). Table S1

The operation conditions of mobile phase and wavelength for HPLC detection.

Compound Mobile phase (1 mL/min) Wavelength (nm) Acetaminophen Water: methanol (50: 50) 254

Amiloride Water: methanol: acetic acid (60: 40: 1) 286

Cimetidine Water: methanol: triethylamine: phosphate (80: 20: 0.1: 0.06) 219

Clozapine Water: methanol: acetonitrile: acetic acid: n-butylamine (44: 28: 254 28: 1.9: 0.4) Dexamethasone Water: methanol (25: 75) 240

Erythromycin 0.02 mol/L KH2PO4: acetonitrile (45: 55) 215

Ibuprofen 0.04 mol/L KH2PO4: methanol (30:70) 263

Ketoconazole 0.02 mol/L KH2PO4: methanol (25: 75) 230

Methotrexate 0.05 mol/L sodium acetate: acetonitrile: methanol (87: 6.5: 6.5) 260

Midazolam Water: acetonitrile: acetic acid: triethylamine (60: 40: 0.03:0.08) 225

Propranolol 0.02 mol/L KH2PO4: methanol (50: 50) 290

Testosterone 0.02 mol/L KH2PO4: methanol: triethylamine (50: 50: 0.1) 254

Verapamil 0.1 mol/L sodium acetate (pH=5.8): methanol (42: 58) 278

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

(day 10) and in Transwell (day 21). (n = 3 for each point)

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

Figure S1. Cross-section of human small intestine showing one circular fold projecting into intestinal lumen which formed a circular intervalvular space. Figure S2

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.

References:

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