Physiological Shear Stress Enhances Differentiation, Mucus-Formation and Structural 3D Organization of Intestinal Epithelial Cells In Vitro

Abstract

Gastrointestinal (GI) mucus plays a pivotal role in the tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro GI models with a physiologically relevant mucus layer, studies with deeper insights into structural and compositional changes upon chemical or physical manipulation of the system are rare. To obtain an improved mucus-containing cell model, we developed easy-to-use, reusable culture chambers that facilitated the application of GI shear stresses (0.002–0.08 dyn∙cm−2) to cells on solid surfaces or membranes of cell culture inserts in bioreactor systems, thus making them readily accessible for subsequent analyses, e.g., by confocal microscopy or transepithelial electrical resistance (TEER) measurement. The human mucus-producing epithelial HT29-MTX cell-line exhibited superior reorganization into 3-dimensional villi-like structures with highly proliferative tips under dynamic culture conditions when compared to static culture (up to 180 vs. 80 µm in height). Additionally, the median mucus layer thickness was significantly increased under flow (50 ± 24 vs. 29 ± 14 µm (static)), with a simultaneous accelerated maturation of the cells into a goblet-like phenotype. We demonstrated the strong impact of culture conditions on the differentiation and reorganization of HT29-MTX cells. The results comprise valuable advances towards the improvement of existing GI and mucus models or the development of novel systems using our newly designed culture chambers.

Flow-induced glycocalyx formation and cell alignment of HUVECs compared to iPSC-derived ECs for tissue engineering applications

“…To comparatively analyze the effect of shear stress applied to the apical surface of HUVEC or iPSC-EC monolayers on cellular orientation as well as glycocalyx composition and thickness a previously established reusable slide cultivation chamber was deployed for the culture of cells under flow on conventional microscopy slides ( Figure 1A ) ( Lindner et al, 2021 ). This chamber can be implemented into a simple circuit further comprising a peristaltic pump and a medium reservoir simultaneously working as a bubble trap ( Figure 1B ).…”

“…In this study we investigated the influence of physiological shear stress on cellular alignment and glycocalyx formation of HUVECs compared to iPSC-ECs. In order to apply homogenous Frontiers in Cell and Developmental Biology frontiersin.org laminar flow to the cellular monolayers our recently reported slide chamber system (Lindner et al, 2021) was used, which can reproducibly be operated in a simple flow circuit without the need for a complex bioreactor system (Figures 1A-D).…”

“…The final pump speed was gradually increased in three steps with an interval of 45 min to initially allow cells to adapt to the shear stress. CFD simulations were performed as previously published (Lindner et al, 2021), confirming laminar flow and homogenous surface shear stress (Figures 1C,D).…”

“…Cells were cultured under fluid flow conditions in parallel plate flow chambers as described before ( Lindner et al, 2021 ). In brief, culture chambers fabricated from polycarbonate which can accommodate a standard microscopy slide are used to apply a homogenous shear stress onto the cell surface ( Figure 1A ).…”

“…The chamber is included into a simple circuit comprising a bubble trap/medium reservoir and a peristaltic pump is used to generate a constant fluid flow ( Figure 1B ). With a channel height of 0.15 mm and a volume flow rate of 3.4 ml min −1 , the applied shear stress is approximately 6.6 dyn cm −2 ( Lindner et al, 2021 ). The final pump speed was gradually increased in three steps with an interval of 45 min to initially allow cells to adapt to the shear stress.…”