Complex, multi-scale small intestinal topography replicated in cellular growth substrates fabricated via chemical vapor deposition of Parylene C

Abstract

Native small intestine possesses distinct multi-scale structures (e.g., crypts, villi) not included in traditional 2D intestinal culture models for drug delivery and regenerative medicine. The known impact of structure on cell function motivates exploration of the influence of intestinal topography on the phenotype of cultured epithelial cells, but the irregular, macro- to submicron-scale features of native intestine are challenging to precisely replicate in cellular growth substrates. Herein, we utilized chemical vapor deposition of Parylene C on decellularized porcine small intestine to create polymeric intestinal replicas containing biomimetic irregular, multi-scale structures. These replicas were used as molds for polydimethylsiloxane (PDMS) growth substrates with macro to submicron intestinal topographical features. Resultant PDMS replicas exhibit multiscale resolution including macro- to micro-scale folds, crypt and villus structures, and submicron-scale features of the underlying basement membrane. After 10 d of human epithelial colorectal cell culture on PDMS substrates, the inclusion of biomimetic topographical features enhanced alkaline phosphatase expression 2.3-fold compared to flat controls, suggesting biomimetic topography is important in induced epithelial differentiation. This work presents a facile, inexpensive method for precisely replicating complex hierarchal features of native tissue, towards a new model for regenerative medicine and drug delivery for intestinal disorders and diseases.

Figure 1.

Stepwise process overview for utilizing native small intestinal tissue as a substrate for chemical vapor deposition of a Parylene C mold. The Parylene mold is used to fabricate biomimetic PDMS replicas used as cellular growth substrates.

Figure 2.

SEM images show varying topographical features of osmium fixed native small intestinal tissue, including (a) macroscopic folds (*) and (b) microscopic villi (*) and crypts (arrows). Bar = 1 mm in (a) and 500 μM in (b).

Figure 3.

Macroscale photograph of representative osmium fixed native small intestinal tissue (left), CVD positive (top view) Parylene replica (center), and final positive PDMS growth substrate (right). Sputter coated Parylene and PDMS are shown for increased contrast due to transparency of the materials. Bar = 3 mm.

Figure 4.

Topography of native intestinal tissue, including complex villus structures (A,C), is mimicked in vapor deposited positive Parylene molds (B,D). Top view shown, *denote villus structures, black arrow denotes pores. Bar = 1mm in (A,B) and 500 μM in (C,D).

Figure 5.

Small intestinal multi-scale features shown on native tissue (A–C) are imprinted into negative Parylene molds (D–F) used for solution casting of positive PDMS replicas (G–I) as biomimetic growth substrates. A,D,G bar = 1 mm; B,E,H bar = 500 μM; C,F,I bar = 200 μM, * denotes villus structure, arrow denotes crypt.

Figure 6.

Representative fluorescent images of Caco-2 monolayers cultured for 10 days on flat PDMS substrates (a) and biomimetic PDMS replicas (b). Optical cross sections of growth substrates are shown as Y-orthogonal views of the flat (c) and replica (d) substrate surfaces. Blue = Hoechst nuclei, green = filamentous actin cytoskeleton. Bars = 50 um. Confocal maximum intensity projections shown.

Figure 7.

Alkaline phosphatase (ALP) expression of Caco-2 cells is enhanced on topographical replicas of native small intestine (right) compared to flat controls (left). Cells were cultured for 10 days prior to measuring ALP, n = 3, * = p < 0.05