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Permeability of Hydrophilic

Supervisors: Vladan Milovic Professor Per Artursson

SUMMARY

Investigations of the integrity and transport characteristics of 2/4/A1 cells

have been done in this report. The cell line was isolated from rat fetal

intestinal epithelial cells and transfected with thermolabile SV40 large T

antigen.

These cells proliferated at 33 °C, but eliminated the antigen and ceased

proliferating at a non-permissive temperature (39°C). At 39°C 2/4/A1 cells

started to differentiate but simultaneously the cells also underwent massive

cell death.

When cultured at 37°C these cells formed confluent and tight monolayers that

seemed to have paracellular transport characteristics similar to that of the

human intestine. Transmission electron microscopy confirmed the development of

multilayers at 33°C, monolayers at 37°C and defects in the cell layer due to

apoptosis at 39°C.

Different immunostainings of ZO-1, E-cadherin and vinculin confirmed formation

of tight and adherence junctions. Transepithelial resistance reached a plateau

of 25-35 Ohm.cm2, which was similar to the small intestine. In transport studies

2/4/A1 cell line monolayers selectively restricted the permeation of hydrophilic

permeability markers proportional to molecular weight and discriminated more

accurately between the molecules of intermediate molecular weight compared to

Caco-2 cells.

These results indicated that 2/4/A1 cells could be used as a model for

hydrophilic drug absorption.

INTRODUCTION

The small intestine plays a crucial role in the absorption of drugs and

nutrients. Exogenous substances cross a series of barriers during the process

of intestinal absorption: (1) the aqueous boundary/mucus layer, (2) a single

layer of epithelial cells, and (3) the lamina propria, which contains the blood

and lymph vessels that then transport the absorbed drugs to other parts of the

body (Artursson 1991).

The cell monolayer is comprised of two parallel barriers: the cell membrane and

the tight junctions. Most drugs are absorbed by a passive diffusion across the

cell membrane by the transcellular route, or across the tight junctions between

the cells - the paracellular route. Drug transport can also be carrier mediated,

when the drug utilizes transporters located in the cellular membrane.

Transcytosis is another kind of active transport, in which macromolecules can be

transported across the intestinal epithelial cell in endocytosed vesicles.

The hydrophilic and charged drugs are absorbed after passing through the

paracellular route, the water-filled channels between the cells (Artursson

1991). Rates and extent of the paracellular transport are, therefore, highly

influenced by the structure and size of the tight junctions as well as by the

size of the molecules. Only small and hydrophilic drugs can pass between the

cells rapidly and completely; permeation of larger molecules can be limited

proportionally to their size and lipophilicity (Hillgren et al. 1995).

Simple assay methods are needed for drug absorption studies. Excised intestinal

tissue, isolated cells, membrane vesicles and in vivo models have distinct

limitations, which have been previously discussed in detail (Audus et al. 1990;

Artursson 1991; Hillgren et al. 1995). The most suitable method for the study of

drug intestinal transport appeared to be the use of cultured intestinal

epithelial cells. This model has several advantages over conventional drug

absorption models: (a) it is less time-consuming; (b) it enables rapid

evaluation of methods for improving drug absorption; (c) it allows an

opportunity to use human rather than animal tissues; (d) it can minimize

expensive and sometimes controversial animal studies.

Human colorectal carcinoma cell line Caco-2 is nowadays the most widely used and

the best explored model for drug intestinal transport (Hidalgo et al. 1989;

Artursson 1990; Artursson & Karlsson 1991). This cell line displays spontaneous

enterocytic differentiation in culture and forms a polarized monolayer with

apical brush borders and well differentiated tight junctions (Hidalgo, 1989).

Drug transport studies across the Caco-2 cell monolayers showed a satisfactory

correlation with other in vitro absorption models, e.g. rat intestinal segments

(Artursson et al. 1993) and in vivo drug absorption (Lennernäs et al., 1995),

although a considerable variability has been reported, being related to

heterogenity, a number of sub populations, and number of passages (Walter &

Kissel, 1995).

Caco-2 cells however, form monolayers that resemble colonic rather than small

intestinal epithelial cells. Due to its well-formed tight junctions, Caco-2 cell

monolayers have a transepithelial electrical resistance of 260 Ohm.cm2 which is

similar to the transepithelial electrical resistance of the colon rather than of

the small intestine (Hillgren et al. 1995). Therefore, there is a need to

investigate drug intestinal transport in a model which has apparent transport

characteristics corresponding to the human intestine, and several studies have

been attempted to characterize a cell line that can be used for this purpose.

A novel intestinal epithelial cell line (2/4/A1) is derived from the rat fetal

intestinal epithelial cells conditionally immortalized with thermolabile SV40

large T antigen, pzipSVtsa58 (Paul et al. 1993). According to the original

report, these cells form more leaky monolayers, with paracellular transport

characteristics similar to that of the human intestine. When cultured at 32°C

these cells continually proliferate and display few markers of intestinal

differentiation. However, after being switched to a non-permissive temperature

(39°C), these cells cease proliferating and exhibit a more markedly

differentiated phenotype. They form a polarized monolayer covered with a few

microvilli; tight junctions are also present (Paul et al. 1993; Hochman,

personal communication).

The 2/4/A1 cell line has been preliminary investigated in this laboratory. It

appeared that cells grown at 39°C underwent massive apoptotic cell death

simultaneously with differentiation, and that those grown at permissive

temperature continued proliferating and form multilayers. However, when grown at

an intermediate temperature (37°C), the cells underwent apoptosis to a lesser

extent, but maintained their proliferative capacity sufficiently to form tight

and continuous monolayers.

The aim of this study was to investigate permeability of paracellular marker

molecules across the 2/4/A1 cell line monolayers and to look at the

characteristics of the cell line.

MATERIALS AND METHODS

Cell culture

2/4/A1 cells were expanded in flasks at 33°C, in RPMI 1640 medium supplemented

with 2% fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 200 mg/ml geneticin, 1

mg/ml BSA, 2 mg/ml dexamethasone, 20 ng/ml EGF, 50 ng/ml IGF-I, 10 mg/ml insulin,

10 mg/ml transferrin and 10 ng/ml selenic acid (ITS premixTM, Collaborative

Research), with 5-6% CO2 and 95% humidity.

The cells were seeded on Transwell polycarbonate filter inserts (Ø 6.5 mm)

coated with ECL extracellular matrix (entactin-collagen IV-laminin; Promega,

Madison, Wisconsin, USA), at a density of 100,000 cm2 in a serum-free RPMI 1640

medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 200 mg/ml geneticin, 1

mg/ml BSA, 2 mg/ml dexamethasone, 20 ng/ml EGF, 10 mg/ml insulin, 10 mg/ml

transferrin and 10 ng/ml selenic acid.

Transport studies

Paracellular markers of different size and molecular weight labelled with 14C or

fluorescein were used: mannitol (MW 182), fluorescein (MW 376), lucifer yellow

(MW 450), polyethylene-glycol 4000 (MW 4000), and dextran (MW 50,000). The

experiments were performed at 37°C in Hank's Balanced Salt Solution pH 7.2 under

"sink conditions". When PEG 4000 was used unlabelled PEG 4000 was also added to

the donor solution to limit possible drug metabolism. The labelled marker

molecules, 250 ml, were added to the apical side of the monolayer and after 20,

40, 60 and 80 minutes the inserts were moved to new wells and 500 ml samples

taken from the basolateral solution. Prior to the experiments samples of 50 ml

were taken from the apical solutions for measurements of the initial

concentration (C0). All solutions were preheated to 37°C, and a heating plate

was used when the wells were moved. Transport was measured over time (days 1-10)

and compared with the values obtained from Caco-2 monolayers used as standard.

The radioactivity of the samples was determined...