A microengineered collagen scaffold for generating a polarized crypt-villus architecture of human small intestinal epithelium

Abstract

The human small intestinal epithelium possesses a distinct crypt-villus architecture and tissue polarity in which proliferative cells reside inside crypts while differentiated cells are localized to the villi. Indirect evidence has shown that the processes of differentiation and migration are driven in part by biochemical gradients of factors that specify the polarity of these cellular compartments; however, direct evidence for gradient-driven patterning of this in vivo architecture has been hampered by limitations of the in vitro systems available. Enteroid cultures are a powerful in vitro system; nevertheless, these spheroidal structures fail to replicate the architecture and lineage compartmentalization found in vivo, and are not easily subjected to gradients of growth factors. In the current work, we report the development of a micropatterned collagen scaffold with suitable extracellular matrix and stiffness to generate an in vitro self-renewing human small intestinal epithelium that replicates key features of the in vivo small intestine: a crypt-villus architecture with appropriate cell-lineage compartmentalization and an open and accessible luminal surface. Chemical gradients applied to the crypt-villus axis promoted the creation of a stem/progenitor-cell zone and supported cell migration along the crypt-villus axis. This new approach combining microengineered scaffolds, biophysical cues and chemical gradients to control the intestinal epithelium ex vivo can serve as a physiologically relevant mimic of the human small intestinal epithelium, and is broadly applicable to model other tissues that rely on gradients for physiological function.

Introduction

The small intestine is a highly specialized organ that is the major site for digestion and nutrient absorption as well as serving critical roles in oral drug uptake and metabolism, while also being a target of many pathogens and toxins [1]. The lining of the small intestine is a monolayer composed primarily of absorptive enterocytes that are arranged along a microscale topography [2] composed of tightly spaced invaginations (crypts) that are contiguous with larger projections into the intestinal lumen (villi). Stem cells reside in the crypts and proliferate to give rise to differentiated cells that migrate up the crypt-villus axis and cover the villi projections [3]. The microenvironment of the crypt-villus axis, particularly the presence of chemical gradients of growth and morphogens, is believed to play an important role in patterning the polarity of proliferative and differentiated compartments and regulation of proper stem/progenitor cell differentiation of the small intestinal epithelium [4]. It has also been recognized that metabolic factors generated by the gut microbiome form gradients from the lumen to crypt that likely impact development, health and disease of the intestine as well as the organism as a whole [5], [6], [7], [8]. Despite a wealth of indirect evidence, the detailed role of various chemical factors and their concentrations within the crypt-villus microenvironment remains poorly understood [9]. Experimental modeling of intestinal epithelial biology and physiology are limited due to the lack of ex vivo platforms that mimic key features of the small intestinal epithelium such as distinct stem/progenitor and differentiated cell compartments, forward and reverse gradients of mitogens/morphogens and nutrients, and ordered cellular migration along the crypt-villus axis [10].

Prior to the current age of organoid (or enteroid) technologies [11], ex vivo small intestine models were relegated to the use of colon cancer cell lines as no reliable small intestinal transformed cell line exits. With the goal of developing a miniaturized in vitro assay platform having the potential to replicate small intestinal form and function, a number of microdevices have been developed, almost all of which incorporate the Caco-2 cancer cell line as the tissue mimic [2], [10], [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24], [25], [26]. These devices use a variety of techniques to create a topological representation of the microenvironment. Microscale 3D scaffolds and surface coatings employing extracellular matrices (ECM) have been shown to influence growth properties, differentiation, gene expression and absorptive functions of Caco-2 cells cultured on these devices [2], [12], [13], [19], [21]. To better replicate biomimetic surface properties, a number of different materials have been incorporated into the devices as substrates for cell culture, including hydrogels, PDMS, silk and decellularized porcine intestine [12], [17], [20], [22]. The introduction of dynamic properties such as fluid-flow-induced shear stress and cyclic mechanical strain have also been shown to impact the physiological properties of the Caco-2 cells within a microenvironment [14], [15], [18], [23], [24], [25]. Co-culture of the Caco-2 cells with other cell types impacts the differentiation and function of these tumor cells [20], [26]. However, it is well-recognized that tissue-cultured cells derived from colon tumors fail to recapitulate normal small intestine cell lineages and function [27], [28]. Cancer cell lines harbor other features including accelerated accumulation of genetic mutations, loss of normal physiologic responses, and adaptations to tissue-cultured growth, e.g. the ability to grow in the absence of an ECM, that render them imperfect compared to primary tissue cultures [27], [28], [29], [30]. Despite these drawbacks, use of cancer cells dominates microdevice development since native intestinal tissue quickly undergoes differentiation and/or apoptosis when cultured on non-biological surfaces [31], [32], [33].

Fortuitously, innovations in intestinal stem-cell culture methods have made it possible to use primary, non-transformed cells to create specialized tissues in vitro [34], [35]. With these methods, primary animal and human intestinal stem cells can be used to create multi-cellular structures known as enteroids that mimic many of the aspects of the small intestinal epithelium [11], [36]. The potential for intestinal enteroid technology is enormous as evidenced by the burgeoning number of reports and reviews on the topic [36], [37], [38]. Nevertheless, the cystic, spherical architecture of enteroids presents severe limitations in many applications. Current culture systems yield an enteroid with an enclosed lumen buried within a thick mass of Matrigel (a hydrogel) [39]. The luminal aspect of the enteroid is thus not easily accessed by drugs, toxins, probiotics, microbiota and other agents. Furthermore, culture of cells embedded within a hydrogel presents additional problems for assay development including the need for compounds to diffuse through the hydrogel to reach the enteroid, which may lead to artifacts during time sensitive assays, and the potential for dosage errors due to adsorption of the compound into the hydrogel. Thus, there is a pressing need for engineered culture systems to address the enteroid limitations by providing an accessible luminal surface for ready assay performance yet preserving the stem and differentiated cells present in the enteroids.

The combination of advances in the culture of primary stem cells and their differentiated progeny with cutting-edge microengineered systems now makes it possible to develop functional human intestinal epithelium on lab-on-chip devices [40], [41]. Critical to this endeavor are identification of a biomimetic material with appropriate ECMs and stiffness to support both stem and differentiated cells and to construct scaffolding of the proper 3D topography for crypts and villi. When this is combined with tightly controlled environments enabled by microscale devices, sustained, biochemical gradients should be capable of directing a polarized cellular architecture. In this manuscript, we describe our work to identify a cross-linked collagen hydrogel that permits stem cell maintenance and proliferation as well as formation of the differentiated lineages of the small intestine. This hydrogel was then micromolded on a porous substrate in a 3D topography that recapitulated the crypt/villus architecture of the small intestinal epithelium. This human 3D tissue was then subjected to chemical gradients applied to the basal-luminal axis to create stem/proliferative and differentiated cells zones, formation of appropriate differentiated lineages and cell movement along the crypt-villus axis analogous to that seen in vivo.