In vitro morphogenesis of mucosal barriers from organoids in synthetic hydrogels

Introduction:: The mucosal barriers of the gut and endometrium are both single-layer epithelia with 3D structures comprising lumen-facing cells joining deep invaginations lined by secretory cells – crypts in the gut and glands in the endometrium – that are rapidly turned over from proliferating stem and progenitor cells. Dynamic models of these mucosal barriers that capture features of the 3D architecture and interactions of the epithelia with underlying stroma and immune cells are desirable for modeling diseases, including Crohn’s disease (gut) and implantation failure (endometrium). The advent of epithelial organoid technology, in which epithelia from biopsies are encapsulated in Matrigel and expanded in a growth factor-rich medium, provides a foundation for tissue engineering approaches to create a true 3D multicellular mucosal barrier with accessible apical and basal compartments. This work follows up on preliminary observations that patient-derived endometrial epithelial organoids in a synthetic hydrogel extracellular matrix (ECM) fuse to form a gland-lumen structure (Gnecco et al, bioRxiv, 2021 and Med, accepted, 2023). Here, we systematically investigate how the biophysical properties of a synthetic extracellular matrix (ECM) hydrogel influence morphogenesis of primary human epithelial organoids into 3D mucosal barriers via a growth and surface emergence process, using the comparative biology of endometrium and intestine. Further, we describe how co-cultures of stroma and organoids in synthetic ECM undergoes morphogenesis to a mucosal barrier in a 3-channel microfluidic mucosal barrier platform.


Materials and Methods:: Patient biopsies of either the intestine or endometrium were processed into human duodenal epithelial organoids (hDOs) and human intestinal stromal cells (hISCs), or to endometrial epithelial organoids (EEOs) and endometrial stromal cells (ESCs), then cultured by standard methods (1,2). A synthetic ECM hydrogel was produced by functionalizing poly(ethylene glycol) (PEG) macromers with peptides designed to either bind to specific integrins or to anchor cell-secreted extracellular matrix proteins (1,2). Microfluidic devices were created by casting PDMS designs and bonding to cover glass-bottom plates. Organoids (with or without stromal cells) were mixed with a liquid hydrogel precursor and loaded into the central channel of the microfluidic devices in a manner that concentrated organoids near the apical hydrogel-media interface. Devices were cultured for up to 14 days, with frequent medium changes and adjustments to media compositions to control growth and migration of organoids and stroma, then fixed and immunostained to identify cell populations and phenotypic markers.






Results, Conclusions, and Discussions:: Organoids show proper apical-basal polarity near the hydrogel surface, and emerge to form two predominant structures: (i) cigar-like projections into the media; and (ii) continuous monolayers spreading across the hydrogel ECM surface. These morphologies depend on cell type and ECM stiffness. Organoid projections that extend into the media reverse apical-basal polarity relative to the embedded portion, visualized with F-actin staining. Softer ECMs favor lumen-like surface monolayer structures, and stiffer ECMs favor projections, with the frequency and length of projections increasing with increasing matrix stiffness. Endometrial cells generate projections only at relatively high stiffnesses (E > 2 kPa), whereas intestinal cells also form projections at physiological stiffnesses (E = 300 Pa), indicating an inherent difference in tissue biomechanics. EdU staining indicates that proliferation localizes to the embedded portion or projections and the leading edge of monolayers. Proliferation also increases with increasing matrix stiffness. With sufficient seeding density, textured monolayer structures grow to cover the entire hydrogel droplet, forming a mucosal barrier.

Inclusion of stromal cells had a significant effect on organoid morphology. In the intestinal cultures, stromal cells promote the formation of lumen-like monolayer structures over projections, towards the ratio seen in a softer hydrogel. This same shift was not observed in the endometrial cells, with stromal cells instead migrating to the hydrogel surface and preventing emergence.

The 3-channel microfluidic device also allows for the investigation of cell migration. Gradients of growth factors were sufficient to drive migration stromal cells towards the higher concentrations in the apical channel, and even to drive invasion of stromal cells into the hydrogel layer from the basal channel. These devices offer a useful platform for further investigation of stromal-epithelial interactions.

PEG hydrogels offer a powerful means to control organoid morphogenesis into mucosal barrier-like structures. These findings support continued work in developing a platform for study of a self-renewing, primary cell-derived mucosal barrier model in a microfluidic device platform with an accessible lumen.


Acknowledgements (Optional): :

References (Optional): : 1. Gnecco, Brown, et al. Med, 2023 (accepted)
2. Hernandez-Gordillo, et al. Biomaterials. 2018.