Development of a primary human Small Intestine-on-a-Chip using biopsy-derived organoids

Abstract

Here we describe a method for fabricating a primary human Small Intestine-on-a-Chip (Intestine Chip) containing epithelial cells isolated from healthy regions of intestinal biopsies. The primary epithelial cells are expanded as 3D organoids, dissociated, and cultured on a porous membrane within a microfluidic device with human intestinal microvascular endothelium cultured in a parallel microchannel under flow and cyclic deformation. In the Intestine Chip, the epithelium forms villi-like projections lined by polarized epithelial cells that undergo multi-lineage differentiation similar to that of intestinal organoids, however, these cells expose their apical surfaces to an open lumen and interface with endothelium. Transcriptomic analysis also indicates that the Intestine Chip more closely mimics whole human duodenum in vivo when compared to the duodenal organoids used to create the chips. Because fluids flowing through the lumen of the Intestine Chip can be collected continuously, sequential analysis of fluid samples can be used to quantify nutrient digestion, mucus secretion and establishment of intestinal barrier function over a period of multiple days in vitro. The Intestine Chip therefore may be useful as a research tool for applications where normal intestinal function is crucial, including studies of metabolism, nutrition, infection, and drug pharmacokinetics, as well as personalized medicine.

Developmentally inspired human ‘organs on chips’

“…Furthermore, because of our deep belief that mechanical forces govern cell and tissue development, it was crucial to replicate organ-relevant physical cues, which in the case of the lung involved surface tension at an air-liquid interface, as well as both fluid flow through the vascular lumen and cyclic mechanical distortion of the tissue-tissue interface due to breathing motions. When we later created a model of the human intestine, we similarly applied cyclic mechanical strain, but at different rates and degrees, to mimic effects of peristalsis-like motions (Kim et al, 2012;Kasendra et al, 2018); when we built a kidney glomerulus chip (Musah et al, 2017), we recreated the deformations this organ unit experiences due to pulsatile blood flow with every beat of the heart. Most importantly, in all of these studies, these mechanical perturbations were absolutely required to drive cell and tissue differentiation, and to robustly mimic human organ-level physiology, as well as pathophysiology.…”

“…For example, one of the key challenges in this field is to obtain highly functional, human, organspecific parenchymal cells. Many adult cell types are available commercially; however, tissue-specific organoids also can be created by isolating stem cells from patient biopsies, and this approach has been leveraged as a cell source to create functional human intestine chips that form highly differentiated villus structures (Kasendra et al, 2018). Induced pluripotent stem cell (iPSC) approaches have also been leveraged to create various types of specialized cells for organ chip studies, including cardiomyocytes, kidney podocytes, brain microvascular endothelial cells, and intestinal enterocytes (Wang et al, 2014(Wang et al, , 2017Musah et al, 2017;Workman et al, 2018), but their clinical relevance is sometimes questioned because most iPSCs generated in vitro remain fetal-like or neonatal in nature.…”

“…When different cell types are placed in the correct microenvironment with appropriate positioning of tissues relative to one another, they spontaneously accumulate ECM along their interface, switch on developmental programs and self-organize, resulting in formation of highly polarized and differentiated epithelial tissue structures that closely resemble those seen in the organs of our bodies, such as mucus and surfactant-producing alveolar epithelium (Huh et al, 2010), ciliated pseudostratified airway epithelium (Benam et al, 2016a), 3D finger-like intestinal villi (Kim et al, 2012;Kasendra et al, 2018;Workman et al, 2018) and reconstitution of podocyte-ECMendothelial contacts of the kidney glomerulus (Musah et al, 2017).…”

“…For example, in the intestine chip, fluid flow was found to be crucial for villus morphogenesis and mucus production (Kim et al, 2012;Kasendra et al, 2018), whereas cyclic peristalsis-like deformations were responsible for suppressing overgrowth of commensal microbes and breakdown of the intestinal barrier . In the lung alveolus chip, absorption of nanoparticulates and associated inflammation were highly sensitive to breathing motions, as was the development of pulmonary edema (Huh et al, 2010;Huh et al, 2012).…”

“…1A,B; Huh et al, 2010). Since this study was published, multi-channel design approaches have been used by multiple research groups to build microfluidic organ-chip models of the human lung small airway, skin, kidney, intestine, placenta, blood-retinal barrier, blood-brain barrier, neurovascular unit and neuromuscular unit, among others (Kim et al, 2012;Achyuta et al, 2013;Abaci et al, 2015;Benam et al, 2016a;Lee et al, 2016;Musah et al, 2017;Yeste et al, 2017;Wang et al, 2017;Workman et al, 2018;Kasendra et al, 2018;Sances et al, 2018; reviewed by Bhatia and Ingber, 2014).…”