ARTICLE Synthetic Small Intestinal Scaffolds for Improved Studies of Intestinal Differentiation Cait M. Costello,1 Jia Hongpeng,2 Shahab Shaffiey,2 Jiajie Yu,1 Nina K. Jain,1 David Hackam,2 John C. March1 1

Biological and Environmental Engineering, Cornell University, Ithaca, New York; telephone: þ1-607-254-5471; fax: þ1-607-255-4449; e-mail: [email protected] 2 University of Pittsburgh School of Medicine, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania

ABSTRACT: In vitro intestinal models can provide new insights into small intestinal function, including cellular growth and proliferation mechanisms, drug absorption capabilities, and host-microbial interactions. These models are typically formed with cells cultured on 2D scaffolds or transwell inserts, but it is widely understood that epithelial cells cultured in 3D environments exhibit different phenotypes that are more reflective of native tissue. Our focus was to develop a porous, synthetic 3D tissue scaffold with villous features that could support the culture of epithelial cell types to mimic the natural microenvironment of the small intestine. We demonstrated that our scaffold could support the co-culture of Caco-2 cells with a mucus-producing cell line, HT29-MTX, as well as small intestinal crypts from mice for extended periods. By recreating the surface topography with accurately sized intestinal villi, we enable cellular differentiation along the villous axis in a similar manner to native intestines. In addition, we show that the biochemical microenvironments of the intestine can be further simulated via a combination of apical and basolateral feeding of intestinal cell types cultured on the 3D models. Biotechnol. Bioeng. 2014;111: 1222–1232. ß 2014 Wiley Periodicals, Inc. KEYWORDS: caco-2; egf; cadherin; muc-2; lysozyme; ht29

Introduction Live animal models have proven useful for studying many intestinal disorders and phenomena, including inflammatory Correspondence to: J.C. March. Contract grant sponsor: Cornell Nanoscience Facility Contract grant sponsor: The Hartwell Foundation Contract grant sponsor: Weill Hall Imaging Facilities, Cornell Contract grant sponsor: NIH Contract grant sponsor: DTRA Received 31 August 2013; Revision received 11 November 2013; Accepted 23 December 2013 Accepted manuscript online 4 January 2014; Article first published online 22 January 2014 in Wiley Online Library (http://onlinelibrary.wiley.com/doi/10.1002/bit.25180/abstract). DOI 10.1002/bit.25180

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genomics (Atreya et al., 2000; Uhlig and Powrie, 2009), Crohn’s disease (Darfeuille-Michaud et al., 2004), irritable bowel syndrome (Manning et al., 1978), short-bowel syndrome (Murray, 1999), and gastroenteritis (Moon, 1978) and the use of probiotics to improve barrier function (Madsen et al., 2001; Steidler et al., 2000). However, many intestinal processes are difficult to control using in vivo intestinal models, particularly regarding the behavior of epithelial cells in response to specific environmental cues. Synthetic in vitro intestinal models can potentially enable improved studies of intestinal function in an ethical and well controlled manner, particularly for studies of cellular growth and proliferation (Brown and Phillips, 2007), drug absorption (Yu et al., 2012), and host-microbial interactions (Bernet et al., 1994). Synthetic in vitro intestinal models can enable control over number of different parameters simultaneously, including host cell density, nutrient availability, pH and fluid dynamics. Multiple in vitro models can also be run at the same time, reducing the expense and ethical issues raised by using numerous live animals. Current in vitro intestinal models range from simple 2D systems, such as co-cultured intestinal cells on micro-porous transwell supports (Hidalgo et al., 1989; Hilgers et al., 1990), to more complex 3D models with co-culturing techniques that enable more than one type of intestinal cell (i.e., mucosal epithelia and a submucosal cell type) to be incubated simultaneously with select bacterial populations (Bernardo et al., 2012). It is well known that the 3D physical environment plays a major role in the morphology, biochemistry, and metabolism of mammalian cells (Anselme and Bigerelle, 2011; Bettinger et al., 2009). Attempts have been made to study epithelial intestinal cells in a 3D environment, including using microfabrication techniques to provide supports for cells that contain grooves and ridges (Andersson et al., 2003; Nematollahi et al., 2009), or crypt-like wells (Wang et al., 2009). It has been shown that cells cultured in vitro will present unique cell spreading in response to surface topography, in a process known as contact guidance (Clark et al., 1991), as well as a change in differentiation when compared to flat surfaces. In addition, some groups have made use of microfluidic models ß 2014 Wiley Periodicals, Inc.

to culture intestinal epithelial cells in three dimensions. For example, the NASA developed Rotating Wall Vessels (RWV) have enabled prolonged 3D culture of both mammalian cell types and bacterial populations (Barrila et al., 2010; Goodwin et al., 1993; Skardal et al., 2010). These devices are optimized to produce laminar flow to enable the growth of, for example, intestinal organoids in suspension culture in conjunction with bacteria to simulate an enteric infection in a fluidic setting (Barrila et al., 2010; Bentrup et al., 2006; Nickerson et al., 2007). Similarly, on-chip peristaltic devices have been created that enable separation of basolateral and apical membranes for more accurate intestinal transport and absorption studies, as well as co-culture with bacteria (Kim et al., 2012). However, the main disadvantage with these reactors/and or on-chip models is that the topography of the intestine has thus far been poorly recreated, despite it being known that the physical environment plays a major role in the morphology, biochemistry, and metabolism of epithelial cells and bacteria, meaning that current cell culture conditions are not accurately reflecting the native environment. In addition to having motility, the upper GI tract is populated by finger-like villi of up to a millimeter in length. These villi affect intestinal fluid dynamics, pressure and wall stiffness (Chen et al., 2008) in a way that has thus far not been replicated in current in vitro models. Our early studies have shown that simply by recreating the topograpy of the small intestine with the same shape, size, and distribution of human intestinal villi, Caco-2 monolayers present differentiation morphology along the crypt-villus axis and TEER values more highly correlated with that of in vivo intestines than a 2D (flat) model (Yu et al., 2012). However, we have also shown that despite enabling the recreation of surface topography, hydrogels such as collagen can form a barrier to the diffusion of some drugs, and they also are relatively shortlived (i.e., unable to sustain long term-cell culture without loss of villus integrity). A common alternative for the creation of scaffolds and supports for tissue culture in a 3D environment is the use of biodegradable polymers (such as poly-lactic-glycolic acid and poly-lactic-acid). Previous researchers have shown that these polymers can be made porous to facilitate cell culture in a variety of ways, including particulate leaching (Beatty et al., 2002; Guan and Davies, 2004; Lin et al., 2002; Mattioli-Belmonte et al., 2008) and thermally induced solvent extraction (Ho et al., 2004; Li et al., 2004; Pavia et al., 2013). These polymers have also been shown to have improved resistance to degradation, retaining structural integrity for up to 3 months in the case of poly-lactic-glycolic acid (Cao et al., 2006; Lu et al., 2000). We have devised an improved biomimetic in vitro model of the intestine, using porous poly-lactic-glycolic acid (PLGA), which not only presents accurate villus geometry similar to real intestine, but also addresses some of the problems faced with using hydrogels, including ease of fabrication and degradability. These scaffolds can be used to support the co-culture and differentiation of the immortal epithelial cell lines Caco-2

and HT29-MTX, and for the study of biomolecule transport through basolateral feeding when integrated into a custom tissue-culture insert. Previous researchers have shown that intestinal crypts can be extracted from mice, seeded onto biodegradable materials or cell-free intestine supports and implanted into animal intestines (Choi and Vacanti, 1997; Day, 2006; Grikscheit et al., 2003, 2004; Spurrier and Grikscheit, 2013); however, there has not yet been a realistic 3D model available to study primary intestinal crypt differentiation and proliferation directly in vitro without implantation. In addition to immortal cell lines, we have therefore also cultured intestinal crypt cells from mice onto the villus scaffolds.

Materials and Methods Cell Lines Caco-2 cells (ATCC, Manassas, VA) passage 18–25, and the mucus producing cell line HT29-MTX (Lesuffleur et al., 1990), passage 30–35, were expanded and maintained in co-culture media [DMEM with 10% Fetal Bovine Serum (FBS), 1 anti-anti and 40 mM GlutaMAXTM] (all from Invitrogen, Long Island, NY). Cells were maintained in a 37 C incubator with 5% CO2 with regular media change every 2 days. In addition, small intestinal crypts were extracted from wild type c57 small intestine following a procedure described previously (Sato et al., 2009). Briefly, the small intestine was dissected into small pieces, followed by suspension in PBS with antibiotics (0.1% gentamicin, 0.2% amphotericin B, from Invitrogen) and 4 mM EDTA. After centrifugation for 45 min at 200g, the supernatant was discarded and cells were re-suspended vigorously in ice-cold PBS with antibiotics. Cells were spun at 200g for 5 min and the supernatant above the crypt layer discarded. This procedure was repeated three times, leaving a thin layer of enriched crypts, which were removed and re-suspended in crypt culture media-DMEM/F12 with 20% FBS, 1% pen/ strep, 1% L-glutamine, 0.1% gentamycin, 0.2% amphotericin B, and 5% 1 M HEPES (all from Invitrogen). All animals used in these experiments were managed in accordance with University of Pittsburgh IACUC-approved protocols. Fabrication of Porous PLGA Intestinal Scaffolds An overview of the procedure for construction of porous PLGA scaffolds is depicted in Figure 1. Laser ablation was used to create a template array of 500 mm deep, high aspect ratio holes on a Polymethyl methacrylate (PMMA) template, with Polydimethylsiloxane (PDMS; Dow Corning, MI) used to fabricate (approximately 1 cm2) replicas with a full villous array as described previously (Sung et al., 2011). Molten agarose (3% in water, from Sigma, St Louis, MO) was poured over the PDMS scaffolds and cooled at room temperature to form hydrogel replicas of the initial PMMA molds. These can be produced more rapidly and in higher quantities than

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Figure 1. Schematic representation of porous PLGA intestinal scaffold formation. Laser ablation is used to create an array of 500 mm deep holes in a PMMA (A). PDMS replicas fabricated to produce a PDMS intestinal scaffold template (B and C). Agarose replicas are made to enable improved detachment of the final PLGA scaffolds (D and E). High PLGA/low porogen solution under vacuum to forms initial villous layer (F) with additional polymer layer (low PLGA/high porogen) added to increase the porosity. Total thickness of the base measures approximately 500 mm (G). Scaffolds are then frozen at 20 C, immersed in pre-cooled ethanol to extract the solvent, then incubated in warm water to dissolve the porogen. PMMA molds, and enable improved detachment of the final PLGA scaffolds. Porous PLGA scaffolds were then fabricated using a modified version of a porogen leaching/thermally induced phase separation technique (Yang et al., 2008). PLGA (100 mg/mL in chloroform, from Lactel Absorbable Polymers, Birmingham, AL) was mixed with a porogen (sodium bicarbonate, 400 mg/mL) that had been pre-minced to a fine powder. The PLGA/porogen solution was homogenized with a hand held homogenizer (OmniTech International, Midland, MI) for 2 min to further reduce porogen size. The agarose molds were then coated with 100 mL of PLGA/ porogen solution and placed under vacuum for 30 s to draw the solution into the array holes. Following PLGA casting, the scaffolds were frozen at 20 C overnight, and then immersed in pre-cooled ethanol for a further 12 h to extract the chloroform. The scaffolds were then covered in warm distilled water for 24 h to dissolve the porogen, and sterilized with 70% ethanol for 24 h prior to use. Prior to cell seeding, the PLGA scaffolds were placed into a custom designed insert kit from previously reported methods (Yu et al., 2012), and then soaked overnight in co-culture media which was added to the basolateral and apical sides. Cell Seeding Onto Porous PLGA Intestinal Scaffolds Caco-2 and HT29-MTX cells were removed from culture flasks with 0.25% (v/v) trypsin, 0.02% EDTA solution in PBS and seeded onto the PLGA scaffolds with a cell concentration of 1  106 cells/mL (with a Caco-2/HT29-MTX ratio of 3:1). Media was added to both the basolateral and apical compartments after a 30-min cell attachment period and replaced every 2 days. For studies of basolateral stimulation, media containing epidermal growth factor (EGF; 100 ng/mL,

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from Invitrogen) was added to the basolateral compartments, with EGF negative media in the apical compartments. Control experiments were performed on cells cultured on standard 0.4 mm pore size 12 mm transwell inserts (Corning Inc., Lowell, MA). Cells were cultured for up to 28 days. PLGA scaffolds were prepared for crypt seeding by coating the scaffold surface with MatrigelTM (BD Biosciences, San Jose, CA), diluted 1 to 10 in crypt cell media, followed by polymerization at 37 C for 45 min. Intestinal crypt suspensions were then seeded onto the scaffolds for 3 h at 37 C. Unattached crypts were then removed by washing with media, and then samples were incubated with crypt culture media containing added growth factors (500 ng/mL Rspondin, 200 ng/mL WNT3a, 200 ng/mL Noggin, and 50 ng/mL EGF, all from Invitrogen) to enable crypt cell differentiation (Sato et al., 2009). Crypts were cultured for 7 days on the scaffolds. Scanning Electron Microscopy (SEM) and Determination of Porosity Cell-free porous PLGA scaffolds were dried under vacuum overnight prior to imaging. The dried samples were then mounted on aluminum stubs and sputter coated with a gold/ palladium alloy. Pore sizes in the scaffolds were determined by examining with a Leica 440 SEM. Scaffold porosity was measured using the liquid displacement method adopted by Shi et al. (2002). Immunofluorescence and Confocal Imaging Intestinal cell-coated PLGA scaffolds were fixed with 4% paraformaldehyde overnight at 4 C, and then either imaged

as a whole (3D) scaffold, or sliced by cryo-sectioning prior to immunofluorescence and confocal microscopy. For scaffold slices, cells were cryo-protected by immersion in 30% sucrose in PBS for 30 min, before embedding in OCT solution and snap freezing. Vertical slices from the tips of the villi to the base, 12 mm thick, were cut using a cryostat microtome (Microm 505e, Gorilla Scientific, Silver Spring, MD). Fixed samples were blocked with normal donkey serum (5% in PBS with 0.3% Triton x-100, from Sigma) for 1 h then washed three times with PBS. Caco-2/HT29-MTX were stained for alkaline phosphatase through overnight incubation with goat primary antibody (Santa Cruz Biotech, Dallas, TX) at a dilution of 1:200, then immersion in Alexa Fluor1 555 donkey anti-goat secondary antibodies (Invitrogen) at a dilution of 1:500. Mouse crypts were stained for MUC-2 and lysozyme through overnight incubation with rabbit primary antibodies (Santa Cruz Biotech), with a 1:200 dilution. Alexa Fluor1 488 donkey anti-rabbit and Alexa Fluor1 555 donkey anti-goat secondary antibodies (Invitrogen) were used for immunofluorescence at a dilution of 1:500. Both whole scaffold and sliced scaffolds were then stained with TOPRO1-3 for (nucleic acid) and/or Alexa Fluor1 488 Phalloidin (for actin), and WGA-Texas Red1 (for mucus) for 30 min (all from Invitrogen). Samples were scanned using a Leica SP2 confocal microscope (Leica Microsystems, Buffalo Grove, IL) with Z-series capability. Three dimensional rendering images and sections were assembled with Volocity 5.0 software (Perkin Elmer, Waltham, MA) and ImageJ. Transepithelial Electrical Resistance (TEER) To measure TEER values of the Caco-2/HT29-MTX coculture monolayers, the media was aspirated from the inserts, replaced with fresh DMEM both basolaterally and apically, and incubated at 37 C for 15 min. TEER was measured with an EVOM2 Epithelial Voltohmmeter with STX3 electrodes (World Precision Instruments, Sarasota, FL). Electrodes were placed on the apical and basolateral sides of the insert kits, and the resistance was corrected for surface area (0.5 mm) and expressed as Ohm cm2. The intrinsic resistance (scaffold) was subtracted from the total resistance (scaffold and cells) to give the monolayer resistance, and values were compared to cell monolayers grown on standard transwell inserts.

405 nm and converted to concentration in reference to a standard curve of p-NP in Tris Buffer. Results of the alkaline phosphatase analysis were expressed as U ¼ nmol of p-NP/ min at 37 C, and normalized to mg of total protein using a BCA assay kit (Thermo Scientific, Rockford, IL). Enzyme-Linked Lectin Assay (ELLA) for Mucus Quantification Mucus production was assessed in HT29-MTX cells cultured on scaffolds with and without EGF, with standard transwell inserts as controls. Mucus was removed from the scaffold surface in a method described previously (Wikman et al., 1993). Briefly, spent media was aspirated from basolateral and apical compartments, and the scaffolds were washed with shaking three times for 10 min with 2 mL (FBS-free) DMEM. The media was collected and the procedure repeated twice. Total collected mucus was then assessed using a wheat germ agglutinin peroxidase ELLA adapted from previous researchers. (Maierhofer et al., 2007; Zoghbi et al., 2006). Microtiter plates (96 well) were incubated overnight at 4 C with 200 mL of the mucus samples diluted in sodium carbonate buffer (0.5 M, pH 9). The plate was washed with PBS Tween (1% Tween-20 in PBS, pH 7) and blocked with 250 mL PBS Tween BSA (0.2 g BSA in 100 mL PBS Tween) for 1 h at 37 C. After washing with PBS Tween, plates were incubated with 100 mL WGAperoxidase (5 mg/mL, Sigma) for 1 h at 37 C. Plates were then incubated with 100 mL O-phenylenediamine (Sigma) for 5 min and stopped with 25 mL 3 M sulfuric acid. Absorbance was read at 492 nm and converted to concentration in reference to a standard curve of porcine mucin in sodium carbonate buffer (0.5 M). Results of the ELLA were normalized to nanogram of mucin per 100,000 cells. Statistical Analysis TEER and all assays were performed in triplicate, and data are presented as mean  SD. Statistical differences were determined by using a Student’s unpaired t-test, with P values of less than 0.01 considered significant.

Results and Discussion Characterization of Porous PLGA Scaffolds

Alkaline Phosphatase Assay Alkaline phosphatase activity was assessed in Caco-2/HT29MTX co-cultures in a method adapted from previous researchers (Ferruzza et al., 2012). Media was aspirated from the inserts, and scaffolds were incubated with pNitrophenyl phosphate (1 mg/mL, Sigma) in 0.2 M Tris buffer for 30 min at 37 C on a 500 rpm shaker (MTS 2/4 digital shaker, IKA Works, Wilmington, NC). Scaffolds were put on ice, and 100 mL of the yellow soluble end product pNitrophenol (p-NP) was collected and added to 50 mL NaOH in a 96-well-plate to stop the reaction. Absorbance was read at

In our previous studies we demonstrated that villous architecture can be produced on collagen scaffolds, and that cells grown on these scaffolds appear to differentiate in a manner consistent with in vivo differentiation. Here, we investigated a new scaffold comprised of PLGA, a material that has been shown to allow for improved stiffness over collagen [1–2 GPa (Leung et al., 2008) compared to 0.001–0.8 GPa (Piechocka et al., 2011)], enabling the fabrication of an intestinal model that is more structurally robust and easily manipulated. Three dimensional scaffolds with an array of intestinal villi were constructed from PLGA via a combination

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Figure 2. SEM images of PLGA scaffolds showing non-porous PLGA scaffolds (A) and PLGA scaffolds made porous by porogen leaching and solvent extraction (B) Cell-free resistance of PLGA scaffolds made porous through a combination of solvent extraction and porogen leaching, or porogen leaching in isolation (C).

of laser ablation of PMMA and replica molding techniques (Fig. 2A). Each individual scaffold measured 1 cm3 with a full array of villi with physiologically realistic geometries. The villi measured 500 mm in height by 200 mm at the base, and had high aspect ratio shapes and smooth curvature at the tips. Our PLGA scaffolds can also be rendered porous to enable epithelial cell attachment and proliferation, which will provide a test bed to examine the effects of transport through the epithelial cell barrier from the luminal side and cytokine/signal stimulation that can be selected to mimic submucosal transport to the epithelia from the rest of the body. The SEM image in Figure 2B shows that the scaffolds can be made porous without loss of villous shape and structural integrity. Pores were homogenous and range between 5 and 10 mm, which we estimated would be the correct size to allow large molecule and potentially bacterial translocation without epithelial cell invasion into the pores. Use of particulate leaching is a widely adopted method for creating porous scaffolds with relative ease for mammalian cell attachment (Beatty et al., 2002; Beckstead et al., 2005; Brown and Phillips, 2007; Lin et al., 2002; Mattioli-Belmonte et al., 2008; Shi et al., 2002); however, we found through preliminary experimentation that this process alone did not form adequate connective porosity in our scaffolds, partly due to the intricate nature of our villous surface topography. In our method, we also make use of thermally induced solvent extraction, which involves a reduction in temperature of the solvent solution to enable phase separation of the polymer solution, and has been shown to induce greater pore connectivity (Ho et al., 2004; Li et al., 2004; Molladavoodi et al., 2013; Yang et al., 2008), as well as reducing cellular penetration into the polymer and improving polymer durability (Cao et al., 2006). We have shown that combining porogen leaching and solvent extraction significantly alters the cell-free scaffold resistance (across the apical-basolateral sides) compared to porogen leaching in isolation (Fig. 2C), suggesting that solvent leaching improves connectivity between the individual pores in the villi and in the base of the scaffold. Adequate

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pore interconnectivity is necessary to prevent separation of the basolateral membranes from culture media, as tight junction formation in post-confluent cultures can limit the diffusion of nutrients from the apical side. In addition, we expected that gradients of nutrients and oxygen would change as cell coverage changes. In early culture, cells residing near the crypt region at the base of the villi would be exposed equally to both basolateral and apical feeding, however once migration up the villi occurs, these gradients will change, as cells on the tips of the villi will be further away from growth factors/nutrients in the basolateral membrane, just like in vivo. Cell Morphology on Scaffolds Is Consistent With In Vivo Morphology Porous villous scaffolds were able to support the adhesion and proliferation of co-cultured Caco-2 and HT29-MTX (Fig. 3). We chose a ratio of 3:1 for the Caco-2/HT29-MTX to make the epithelial cell composition more indicative of the native intestine. Cells migrate up from the base of the scaffold to the villi in a directional movement, much like in the real intestine where epithelial cells move from the crypts to the tips of the villi. We show directional movement of the cells as they proliferate half way up the villi at day 4 (Fig. 3A), and completely cover the scaffolds after 7 days of culture (Fig. 3B and C). We also showed through confocal microscopy in Figure 4A–C that epithelial cell morphology on PLGA scaffolds over the course of the co-culture becomes more columnar and polarized after 21 days of culture, and that tips of the villi have more well-differentiated cells than the bases in a manner consistent with epithelial differentiation in vivo. Spontaneous differentiation in intestinal enterocytes is accompanied with an increase in expression of brush-border hydrolases, which are therefore commonly used as markers of cellular differentiation in intestinal models (Matsumoto et al., 1990; Pinto et al., 1983). On our PLGA scaffolds, alkaline phosphatase enzymes became visible via immunofluorescence on the brush

Figure 3. Confocal microscopy of a PLGA scaffold co-cultured with Caco-2 and HT29-MTX with staining for nuclei (blue) and actin (green). Twenty times magnification shows partial coverage of villi by cells after 4 days (A), with full coverage of cells over a section of scaffold from the base to the tips after 7 days (B) and 3D rendering shows full coverage of cells on an individual villus measuring 500 mm (C).

Figure 4. Confocal imaging of whole 3D scaffolds with staining for nuclei (blue), actin (green). Z stack Images show change in cell morphology after 7 days (A), 14 days (B), and 21 days co-culture (C). Cryo-sectioned scaffold slices, with staining for alkaline phosphatase (red), show an increase in enzyme expression between 7, 14 and 21 days (D–F, respectively).

border after 14 days of culture along the whole length of the villi (Fig. 4D and F). Effect of Villous Shape and Basolateral Stimulation on Cell Differentiation We have previously shown that cells can differentiate in a manner similar to in vivo conditions when growing on high

aspect ratio scaffolds rather than on flat surfaces (Yu et al., 2012). However, scaffolds from our previously published work were composed of collagen and were resistant to transport of hydrophobic molecules. We wanted to determine the performance of our new PLGA scaffolds with respect to transport of signal peptides that may be presented basolaterally to cells in vivo. In order to determine the combined effects of basolateral stimulation and villous

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shape on cellular differentiation, we grew cells on our new villous scaffolds and stimulated them with epidermal growth factor (EGF) and compared their growth to cells grown on traditional well inserts with the same basolateral stimulation. We chose EGF as previous studies have shown that it plays a role in the regulation of intestinal epithelial proliferation and differentiation (Barnard et al., 1995; Basson et al., 1992). In the developed intestine, concentrations of EGF are primarily sourced from duodenal Brunner’s glands, with the majority of epithelial EGF receptors located basolaterally (Scheving et al., 1989). EGF is believed to aid in intestinal regeneration after injury (Wright et al., 1990). We measured the effect of EGF and villous shape on cell growth and differentiation through changes in tight junction integrity using TEER, and brush border enzyme activity through alkaline phosphatase over a 28-day growth period. Figure 5A displays the TEER values of co-cultures on PLGA scaffolds with and without basolateral stimulation with EGF, compared to transwell insert controls. Although TEER continuously increased from day 4 to day 28, the TEER of co-culture monolayers grown on villous PLGA scaffolds was significantly lower than that of cells grown on transwell inserts throughout the study. It has been shown that tight junctions increase in leakiness from the tip of the villi to the crypts in normal juvenile intestines (Schulzke et al., 1998), and as in our previously reported data we have shown that our scaffolds enable similar differentiation along the length of the crypt-villus axis (Sung et al., 2011; Yu et al., 2012). It is well known that Caco-2 permeability data on transwells often has poor correlation to in vivo studies, and even when cocultured with HT29-MTX results are still 5–30-fold lower compared to in situ rat perfusion studies (Walter et al., 1996). Our data supports the hypothesis that the poor correlation between traditional in vitro intestinal models and in vivo data may be at least partially due to missing villous geometry. In

addition, we found that culture time on PLGA scaffolds was maintained for longer than on transwell inserts seeded with the same concentration of cells, whereas after 28 days the cocultures on transwells started to disintegrate due to cell overgrowth and multilayer formation; co-cultures grown on scaffolds were intact (Fig. 5). Transwells that were treated with EGF yielded TEER readings that were significantly higher than untreated transwells after 4 days, but thereafter there was no significant difference between the two. In contrast, EGF did not increase the TEER compared to untreated PLGA scaffold co-cultures until after 14 days. As with TEER values, alkaline phosphatase activity increased continuously over the 28-day in all samples, and activity from cells cultured on PLGA scaffolds became significantly higher than that of transwells after 21 days (Fig. 5B). However, the difference in activity was never significant in cells grown on transwell inserts, suggesting they reach a maximal differentiation state early in the culture period. Changes in alkaline phosphatase activity in relation to surface topography have also been found by other groups. For example, Wang et al. (2010) found that cells growing in artificial crypts actually displayed a reduction in alkaline phosphatase activity in Caco-2 compared to the same cells growing on flat surfaces. In conjunction with our data on villous structures, this highlights the significance of surface topography in variations of intestinal cell differentiation. In addition, our results are also markedly more comparable to in vivo data. Fan et al. (2001) studied the distribution of alkaline phosphatase in 14-day-old neonatal rats, and found that there was a linear increase in digestive enzymes along the cryptvillus axis, with an approximate 8.8-fold increase from the crypts to the tips, with activity increasing with cell maturity/ differentiation (Fan et al., 2001). We hypothesize that cocultures on transwell inserts reach a maximal level of maturity earlier in the culture period due to the lack of 3D

Figure 5. TEER values (A) and alkaline phosphatase activity (B) of co-cultures on PLGA scaffolds and transwell inserts with and without EGF stimulation basolaterally. Statistics were performed using a paired t-test (P < 0.01).

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architecture, and that this is maintained until cells slough away, which is supported by the fact that the increase in alkaline phosphatase activity was never significantly different across time points for cultures grown on transwells. We also show in Figure 5B that EGF significantly increases alkaline phosphatase activity in co-cultures on PLGA scaffolds. Alkaline phosphatase activity in cells grown on PLGA scaffolds stimulated basolaterally with EGF was not only statistically significant from the transwell inserts across all time points, but more than doubled between 14 and 28 days compared to PLGA scaffolds without EGF stimulation. Importantly, we also observed that EGF had little effect on alkaline phosphatase activity in transwell insert cultures, which suggests they reach a maximal activity rate earlier in the culture period. This corresponds to other findings in the literature, with no significant difference in alkaline phosphatase activity on transwell inserts with a subclone of Caco-2, even with EGF levels as high as 200 ng/mL (Cross and Quaroni, 1991). Effect of Villous Shape and Basolateral Stimulation on Mucus Production by HT29-MTX Confocal imaging and ELLA were used to determine mucus production during HT29-MTX differentiation. After 21 days full coverage of mucus was found along the villi (Fig. 6A), with the highest intensity near the differentiated cells at the tips and middle (Fig. 6B and C) compared to the bottom (Fig. 6D). Differences in mucus production between cocultures on PLGA scaffolds and transwell inserts, as well as the effect of basolateral stimulation by EGF, are shown in Figure 6E. After 21 days the mucus production by HT29MTX growing on the 3D PLGA scaffolds was statistically

higher than on the flat transwells, however the addition of EGF dramatically increased mucus production, showing a peak on the PLGA scaffolds after 7 days. In contrast, EGF did not have a significant effect on mucus production on the transwells at any of the time points. Mucus production in vivo has been found to be increased through parenteral stimulation by EGF, including studies on newborn rabbits that showed an increase in the number of mucus producing goblet cells in the intestine (Okuyama et al., 1998), and in the human gastric mucosa (Kelly and Hunter, 1990). EGF stimulation has also been shown to increase the thickness of the mucus layer on the villi tips in both healthy premature rats, and premature rats suffering from necrotizing enterocolitis, restoring the protective barrier function to almost normal levels (Clark et al., 2006). Although it was not statistically significant, mucus production overall in the transwell co-cultures appeared to decrease between 7 and 21 days, and at 21 days mucus production was lower in the EGF positive compared to EGF negative controls. A possible explanation could be that the rapid growth of the HT29-MTX cells on the transwell inserts (cultures were confluent by 7 days and lifted by 21 days) led to more rapid decline and cessation of mucus production. PLGA Villous Scaffolds Can Support the Culture and Differentiation of Small Intestinal Crypts From Mice Investigation of the small intestine in vitro has typically been restricted in the past to just immortalized cancer cell lines; however, recent advances in cell culture techniques have enabled primary cell lines to be cultured in vitro for a number of weeks. Intestinal stem cells isolated during crypt

Figure 6. Confocal imaging of scaffold slice (A) and 3D z stacks of scaffolds with staining for nuclei (blue), and mucus (red). Images were taken after 21 days. Three dimensional scanning shows the tip (B), middle (C) and bottom of an individual villus (D). Mucus production over 21 days was also assessed using an ELLA with wheat germ agglutinin to bind and quantify mucus. Results are expressed as ng/mL mucus in reference to a standard curve, and normalized to cell density (mucus production per 100,000 cells (P < 0.01).

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preparation have the ability to divide and differentiate into the four intestinal cell types, including paneth cells and goblet cells, and have been shown to generate self-renewing enteroids that display many features of native intestinal epithelium (Kaeffer, 2002; Ouellette, 1997; Sato et al., 2009, 2011; Simon-Assman et al., 2007; Spence et al., 2011). We show here through confocal microscopy that our villous scaffolds can be used to culture crypt cells from small mouse intestine that accurately reflect the spatial arrangement in the small intestine, including paneth cells near the base, and goblet cells which have migrated up the crypt-villus axis. Confocal imaging was used to show the differentiation of small intestinal crypts from mice after 7 days in culture along the crypt-villous axis. It can be seen in Figure 7 that as well as enterocytes, the primary crypt cells differentiated in vitro into paneth cells expressing lysozyme (Fig. 7A), which remained at the base of the scaffold, and goblet cells expressing MUC-2 (Fig. 7B), which were proliferative and migrated up the villi. Since both secretory cell types have been shown to play a role in bacterial attachment and communication with the host epithelium both in vivo (Ayabe et al., 2000, 2002; Salzman et al., 2003) and in vitro (Bernet et al., 1994; Lin et al., 2004), we hypothesize that our in vitro model could provide a platform for more accurate studies of both commensal and pathogenic bacterial distribution in along the crypt-villus axis in a 3D system, particularly with respect to the localization of mucus and antibacterial secretions.

Conclusions An improved method for the construction of biomimetic synthetic intestines with accurately sized villous surface

topography was demonstrated. We showed that scaffolds can be produced with relative ease and can be rendered porous for both epithelial cell attachment and to allow biomolecules such as growth factors to diffuse through the scaffold to stimulate the cells basolaterally in addition to typical apical side feeding. In addition, we showed that compared to traditional 2D transwell inserts, culturing epithelial cells onto these scaffolds leads to epithelial cell morphology and differentiation that is more indicative of native intestinal tissue, as shown by image analysis, quantification of differentiation markers and mucus production by goblet cells. Stimulation by EGF does not cause a significant change in TEER at the different time points on 2D cultures over 21 days because the cells form confluent, differentiated monolayers very quickly, and tight junctions are already formed regardless of growth factor stimulation. In contrast, there are both undifferentiated and differentiated cell types on the 3D PLGA scaffold after 21 days, just like in vivo. This means that cells in our system can be affected by basolateral stimulation by growth factors such as EGF, in the same manner that native intestinal cells in vivo are basolaterally stimulated by either mesenchyme or blood derived growth factors. We also showed that our model enabled differentiation of primary crypt cells from mice in a spatial distribution consistent with that seen in vivo, and we suggest that this model can provide a platform for other researchers to conduct improved studies on intestinal processes, including absorption through the epithelial layer and cell–cell communication studies with host cells and/or bacterial populations.

Figure 7. Confocal microscopy of a PLGA scaffold co-cultured with mouse small intestinal crypts for 5 days with staining for nuclei (blue), e-cadherin (green) lysozyme and MUC-2 (red). 10 magnification shows full coverage of villi by crypt cells that have differentiated into enterocytes, paneth and goblet cells. Arrows point to paneth cells stained for lysozyme that remain at the base (A) and goblet cells stained for MUC-2, which have migrated up the villi (B).

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We are grateful to Dr. Longying Dong, Immunopathology R & D Lab, Department of Biomedical Sciences, Cornell, for use and support with the cryostat facilities. We thank the fine mechanics workshop, Chemical Engineering, Cornell for constructing the tissue culture inserts. We thank Carol Bayles for support with confocal microscopy at the Weill Hall Imaging Facilities, Cornell. The HT29-MTX cells were a kind gift from Dr. Thécla Lesuffleur (INSERM U560, Lille France). Also, we acknowledge The Hartwell Foundation (Collaborative Award to D.J.H. and J.C.M.) and NIH (DP2-New Innovator to J.C.M.) for financial support, and the Cornell Nanoscience Facility for access to the SEM and Versalaser.

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