Towards the development of biomimetic in vitro models of intestinal epithelium derived from intestinal organoids

Abstract

Intestinal epithelium is highly specialized tissue organized into crypt-villus units relevant for their effective barrier function and nutrient absorption. In the crypt units reside the proliferative intestinal stem cells (ISCs) that divide and differentiate while migrating along the villi to generate the epithelium. The proliferation, migration and differentiation of ISCs is governed by the tightly controlled spatio-chemical gradients of ISC niche factors; bone morphogenic protein (BMP), wingless/Int (Wnt) and epidermal growth factor (EGF) pathway modulators. In vitro models of the intestinal epithelium, for the most part, based on culturing of intestinal stem cells/crypts in 3D cultures forming structures called organoids. These structures faithfully recapture diverse cell populations and their multicellular organization of native intestinal epithelium. However, 3D closed geometry of intestinal organoids prevents access to the apical region of the epithelium, making them unsuitable for conventional functionality assays. Experimental modeling of intestinal epithelial biology and physiology are limited due to the lack in vitro platforms that recapitulate these key aspects of the small intestinal epithelium: its distinct cell populations, 3D architecture and the gradients of ISC niche biochemical factors along the crypt-villus axis. Here, we describe development of in vitro models of intestinal epithelium obtained from intestinal organoid-derived crypts. First, we present a method that takes the advantage of substrate stiffness to dictate the formation of monolayers with accessible lumen rather than 3D organoids with a closed geometry. The 2D intestinal epithelium model has in vivo-like crypt-villus cellular organization with all major epithelial cell types and show physiologically relevant tissue barrier function. Then, we describe the development of a more complex model of intestinal epithelium by incorporating a 3D villus-like basement membrane substitute fabricated on hydrogels. For that, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are chosen due to their highly tunable chemical, and mechanical properties, porosity and photocrosslinkable nature allowing easy microstructuring. The formation of 3D bullet-like complex shapes was achieved by photolithography-based crosslinking of PEGDA, a simple, cost-effective approach. The bioactive functionalization of otherwise inert PEGDA for cell adhesion, was achieved by copolymerizing it with acrylic acid and a variety of cell adhesion proteins can be covalently anchored to the 3D villus-like hydrogels. We establish the optimal conditions for the growth of intestinal organoid-derived epithelial monolayers and demonstrated that organoid-derived intestinal epithelial cells successfully formed epithelial monolayers on collagen type I functionalized 3D villus-like PEGDA-acrylic acid hydrogels. Finally, we describe methods to create spatiotemporal gradients of biochemical ISC niche factors on 3D villus-like hydrogels and demonstrate that these gradients can be used to compartmentalize the differentiated epithelial cells. The spatio-chemical gradients of ISC niche biochemical factors on PEGDA hydrogels with proper porosity were successfully generated based on the free diffusion of the factors from a source to a sink chamber in a custom-made microfluidic device allocating the hydrogel and visualized with light-sheet fluorescence microscopy. In silico models were developed to simulate the spatio-chemical gradients formed within the hydrogels. The 3D villus-like PEGDA hydrogels were fabricated on porous membranes and successfully adapted to Transwell® inserts that permitted access to both sides of the hydrogel and the generation of spatio-chemical gradients. The gradients generated in this fashion can be used to compartmentalize the differentiated epithelial cells more towards the tips of the villus-like microstructures. The 3D villus-like platform improves the current models in providing cells with physiologically representative topographical and mechanical cues and biochemical gradients. Due to its utility, this platform might find uncountable applications. It can be used for the understanding of the basic biology of the intestinal epithelium. In addition, it can be used to culture human intestinal stem cells allowing for the screening of novel therapies and disease modeling.

El epitelio intestinal es un tejido altamente especializado, organizado en unidades de criptas y vellosidades que son relevantes para sus eficaces funciones de barrera y absorción de nutrientes. En las unidades de criptas residen las células madre intestinales (ISC) proliferativas que se dividen y diferencian mientras migran a lo largo de las vellosidades, las cuales generan el epitelio maduro. En el epitelio maduro, las ISC y las células proliferativas se localizan en las criptas y las células absorbentes y secretoras diferenciadas en las vellosidades. La proliferación, migración y diferenciación de las ISC se rigen por los gradientes químicos espaciales altamente controlados de los factores de nicho de la ISC; Moduladores de la vía de bone morphogenic protein (BMP), wingless/Int (Wnt) y epidermal growth factor (EGF). El modelado experimental de la biología y la fisiología del epitelio intestinal está limitado debido a la falta de plataformas in vitro que recapitulan estos aspectos clave del epitelio del intestino delgado: sus distintas poblaciones celulares, la arquitectura 3D y los gradientes de factores bioquímicos de nicho ISC a lo largo del eje cripta-vellosidad. Aquí, describimos el desarrollo de modelos in vitro de epitelio intestinal obtenidos de criptas derivadas de organoides intestinales. En primer lugar, presentamos un método para obtener monocapas epiteliales intestinales 2D con lumen accesible y función de barrera fisiológica. A continuación, describimos el desarrollo de andamios biomiméticos 3D similares a vellosidades en hidrogeles de diacrilato de polietilenglicol (PEGDA) utilizando un enfoque fotolitográfico simple y rentable. Demostramos que nuestra plataforma de vellosidades sintéticas apoya la formación de monocapas epiteliales de células epiteliales intestinales derivadas de organoides. Finalmente, describimos métodos para crear gradientes espaciotemporales de factores nicho bioquímicos ISC en hidrogeles 3D similares a vellosidades y demostramos que estos gradientes se pueden usar para compartimentar las células epiteliales diferenciadas. La plataforma 3D que recrea las vellosidades intestinalesmejora los modelos actuales al proporcionar a las células las señales topográficas y mecánicas y los gradientes bioquímicos fisiológicamente representativos. Debido a su utilidad, esta plataforma puede encontrar innumerables aplicaciones. Puede ser utilizada para la comprensión de la biología básica del epitelio intestinal. Además, se puede utilizar para cultivar células madre intestinales humanas que permitan la detección de nuevas terapias y el modelado de enfermedades.