Cellular methods

Several in vitro techniques have been used for assessing intestinal permeability and are favoured as they are less labour and cost effective in comparison to the in vivo methods. Each in vitro method has distinct advantages however are not representative of the in vivo

physiological environment. Hence extrapolation of data to the in vivo situation might require additional data.

Everted Gut Sac technique: 2.2.4.1.

The everted gut sac technique of the rat small intestine has been used as early as the 1950s to study the transport of sugars and amino acids from the mucosal to the serosal side159, 160_{. More recently has been used to quantify the paracellular transport of hydrophilic} molecules161_{. Mannitol flux (as a paracellular marker) showed an apparent permeability (P}

app) of 1.5 x 10-5 _{to 1.7 x 10}-5 _{cm s}-1_{, a value similar to that reported with low molecular weight} hydrophilic drugs in human perfusion studies162_{. Also, molecules that traverse the epithelial} barrier via the transcellular route have been accurately quantified using the everted gut sac technique162_{. The oxygenated tissue culture media and specific preparation ensures that the} tissue is viable for up to 2 hr and is suited to measure absorption at different sites across the intestine and into epithelial cells163_.

Thiry Vella loop 2.2.4.2.

The Thiry-Vella loop model has been used to study the physiology of epithelial transport, digestion and absorption and to determine regional differences in the absorption of

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a molecule from the intestine or stomach. In the model, a loop of bowel with intact nerve and blood supply and lymphatic drainage is removed from continuity with the remainder of the intestine and the ends of the loop are brought to the skin surface of the abdomen by the creation of two stomas164, 165_{. For jejunal loops the small bowel is divided at 10 and 30 cm distal}

to the ligament of Treitz, leaving the mesentry intact. The two ends of the 2 cm segment are then brought out the left side of the abdominal wall and sutured to the abdominal skin166_{. For}

ileal studies a segment of distal ileum 10 – 30 cm proximal to the cecum is used166_{. The} perfusate is then introduced into the exteriorized loop and at regular time intervals the loop is emptied and the amount of substrate remaining in the solution determined167_.

Ussing Chamber: 2.2.4.3.

The use of the Ussing Chamber to assess intestinal permeability is well recognized. The unique feature of this approach is that electrical resistance of the membrane can be measured during the course of the experiment, with the short circuit current and resistance across the membrane routinely used as indicators of intestinal tissue viability47, 168_{. The technique allows} the electrical resistance of the membrane to be measured from changes in current and corresponds to the integrity of the tissue. The short circuit current is used as an indicator of active ion transport taking place across the intestinal epithelium47_.

Basically, the chamber consists of two halves that are mounted together containing the tissue specimen with the apical side isolated from the basolateral side. The active ion transport produces a potential difference across the epithelium and the generated voltage difference is measured using two voltage electrodes that are placed as near as possible to the tissue. The spontaneous voltage is cancelled out by injecting a counter current using two other current

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electrodes that are placed away from the epithelium. This current externally injected is called the short-circuit current and is the exact measure of net ion transport taking place across the epithelium169_.

The Ussing Chamber technique is adopted to study regional differences in the absorption of molecules as the investigator is able to mount the tissue from specific regions of the gut170_{. A comparison of interspecies data is also therefore possible. However as with other} in vitro techniques physiological conditions cannot be maintained, i.e. blood and nerve supply, rapid loss of tissue viability, changes in morphology and functionality of transporter proteins during the process of surgery and mounting168_{. It is therefore critical that fresh and viable} resections from surgery are obtained and more importantly tissue viability is maintained during the experiment in the chamber. Other recommendations to ensure better results include that the excised segment be instantly rinsed to remove debris, solutions be well oxygenated, nutrients be added to the buffer, and low temperatures be maintained during preparation171, 172_.

Additionally careful handling of the excised segment is required so that the tissue is not stretched during preparation to maintain the tip to crypt axis structure172_.

Cell based models: 2.2.4.4.

A variety of cell monolayer models that mimic the in vivo intestinal epithelium in humans have been developed. However unlike enterocytes, tumor cells grow rapidly into confluent monolayers and differentiate to provide an ideal system for transport studies168_{. Cell} monolayers are grown on filter support in a multiple well format and permeability is measured by the movement of molecules from one side of the cell monolayer to the other. The

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adenocarcinoma cell lines HT-29 and Caco2 are most commonly used as they display a number of properties characteristic of undifferentiated intestinal cells.

Caco2 cells are considered by some to be a gold standard technique173_{. The cells are} derived from colorectal adenocarcinomas differentiate spontaneously into polarized intestinal cells possessing an apical brush border and TJ between adjacent cells and express hydrolases and transporters174_{. Caco2 despite their colonic origin, express the majority of morphological} and functional characteristics of small intestinal absorptive epithelial cells, including phase I and phase II enzymes, detected either by measurement of their activities toward specific substrates, or by immunological techniques175_.

Whilst these cultures are used to study the rate of absorption through the absorptive epithelial cells the major criticism is that the model does not assess transport across the mucus layer, the lamina propria and/or the muscularis mucosa. Additionally the size of pores in the monolayer, estimated to be ~3.7 ± 0.1 Å, is smaller than that in the mucosa of the human small intestine and hence express much 'tighter' TJs176_{. Accordingly the epithelial resistance}

across the cell is much greater at about 234 ohms._cm2 _{whilst that in the typical small intestine is} estimated to be in the range of 25 - 40 ohms._cm2 176_{. Differences in culture conditions and} composition of cell sub populations of Caco2 cells, derived from different laboratories, result in varying epithelial resistance values177_{. These differences have been thought to affect the} permeation of molecules through the ‘larger’ pores in the monolayer, i.e. the paracellular route174, 176_{. This has been demonstrated using mannitol as a paracellular marker whose} permeability can vary as much as a 100 fold depending on the source of Caco2 cells178_.

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