(684c) Replicating the Topography of the Intestinal Basement Membrane

The rapidly growing market for oral drug delivery is forecast to reach $52.1 billion in revenues by 2010, with an annual growth rate of 10 percent.[1] With tens of thousands of new oral pharmaceuticals being produced each year, there is a need for efficient and effective analysis of ability to pass through the intestinal membrane and become bioactive in the body. Currently, the method utilized is to culture intestinal cells on flat membranes and test the drug's transport through the resulting cell monolayer. Unfortunately, these tests are not always predictive; one approach to address this issue is to alter the substrate properties to influence cell behavior. We are exploring the influence of substrate topography, as flat substrates are not indicative of the intestinal micro architecture. The intestine has a unique complex micro geometry consisting of finger-like villi and deep wells or crypts; position relative to this topography is known to affect cellular movement and protein expression.[2] Therefore, development of a cell culture substrate that replicates the intestinal basement membrane topography at the micro and, potentially, nano scale could lead to engineered intestinal tissues that behave more similarly to tissues in vivo and hence, give more accurate drug absorption prediction data.

First, experiments were run to find a suitable substrate for attachment and growth of intestinal epithelial cells that would enable modification of microtopography. Investigation of cell attachment on chemical vapor deposited (CVD) cross-linked pHEMA, collagen, PDMS (alone and coated with fibronectin), and glass was performed. Cells adhered to high cross-linked pHEMA just as well as to collagen, polystyrene and PDMS coated with fibronectin and better than to glass and PDMS alone. Based on these results,, it was determined to explore two materials for replication of native intestinal topography: CVD cross-linked pHEMA, because it offers the advantage of enabling replication of the complex geometry of the intestinal basement membrane at the micro and sub-micron scale,[3] and collagen, due to its high cell attachment and the fact that it is a key ingredient of the intestinal basement membrane.

Poly dimethyl siloxane (PDMS) was selected for creation of a mold of the complex structure of the intestinal basement membrane due to its strong mechanical properties and established ability to replicate micron and sub-micron scale structures.[4] Poly dimethyl siloxane (PDMS) was used in a 10:1 base to cross-linking agent ratio and was placed over a fixed and dried segment of porcine intestine. After the PDMS was cured for 2 hours at 70C, it was placed in bleach to remove the intestinal tissue to produce the biomimetic replica. Next, two different substrates were used, collagen and CVD pHEMA, to produce a biocompatible substrate from the PDMS replica for cell culture studies. Future work will explore the use of both biocompatible replicas in cell culture experiments and compare the resulting cultures to those on flat substrates of the same materials, to investigate if the topography changes the cellular behavior.

[1] Gotensparre, S. ?Oral drug delivery sector tipped for explosive growth? In-Pharma Technologists, Jan. 2007.

[2] Cigna, N., C. Nicoleeti, A. Durand, J. C. Chaix, T. Giardina, J. Perrier, Cell Biology International 2007, 31, 966-973.

[3] Pfluger, C., R. Carrier, B. Sun, K. Ziemer, D. Burkey, Macromolecular Rapid Communications, 2009, 30, 126 ? 132.

[4] Pins, G., M. Toner, J. R. Morgan, The FASEB Journal 2000, 14, 593-602.