Capturing and Quantifying Particle Transcytosis with Microphysiological Intestine‐on‐Chip Models

Abstract

gens, and exclusion of microbiota and exogenous hazards including bacteria. The ability of intestinal epithelial cells to actively transcytose nano-and micronsized particles has long been established, but the mechanisms and extent to which this process occurs in healthy intestinal tissues have been the object of significant discussion. [1] Specialized epithelial Microfold (M) cells located in the gut associated lymphoid tissue (GALT) have high particle transcytotic capabilities. [2] In addition to their role in shaping intestinal immune homeostasis, M cells have been postulated to play a key role in particle transport. However, they represent <1% of the cellular population in the human small intestine and while M cells may contribute to particle transport across the intestinal epithelium, several studies have shown that they are not essential to particle transport. [3][4][5] Conversely, there is strong evidence that nano-and micro-particles are efficiently transported across enterocytes in villi within the small intestine. [1,[6][7][8] For example, Reineke et al. conclusively demonstrated in a rat model that nonphagocytic processes are key to the absorption of nano-and even micro-particles in the small intestine and, consequently, to their systemic bioavailability. [1] These studies refute the view that enterocyte transcytosis is a neglectable process in the small Understanding the intestinal transport of particles is critical in several fields ranging from optimizing drug delivery systems to capturing health risks from the increased presence of nano-and micro-sized particles in human environment. While Caco-2 cell monolayers grown on permeable supports are the traditional in vitro model used to probe intestinal absorption of dissolved molecules, they fail to recapitulate the transcytotic activity of polarized enterocytes. Here, an intestine-on-chip model is combined with in silico modeling to demonstrate that the rate of particle transcytosis is ≈350× higher across Caco-2 cell monolayers exposed to fluid shear stress compared to Caco-2 cells in standard "static" configuration. This relates to profound phenotypical alterations and highly polarized state of cells grown under mechanical stimulation and it is shown that transcytosis in the microphysiological model is energy-dependent and involves both clathrin and macropinocytosis mediated endocytic pathways. Finally, it is demonstrated that the increased rate of transcytosis through cells exposed to flow is explained by a higher rate of internal particle transport (i.e., vesicular cellular trafficking and basolateral exocytosis), rather than a change in apical uptake (i.e., binding and endocytosis). Taken together, the findings have important implications for addressing research questions concerning intestinal transport of engineered and environmental particles.

Stimuli‐Responsive Nanocarriers for Transcytosis‐Based Cancer Drug Delivery

“…[176] Inhibitors such as Filipin, [27,195] Nystatin, [196,197] Genistein, [198,199] and Indomethacin [200] are frequently employed to obstruct caveolae-mediated endocytosis, whereas Chloroquine, [201,202] Chlorpromazine, [198] and Dynasore [197,203] are typically used against clathrin-mediated endocytosis. Similarly, macropinocytosis is often inhibited by agents like Wortmannin, [204] Colchicine, [205,206] Imipramine, [207] 5-(N-Ethyl-N-isopropyl) amiloride (EIPA), [203,208] and Cytochalasin D. [197,209] For instance, in the research conducted by Fan, [210] endocytosis inhibition experiments indicated that NPs were internalized via caveolae-and macropinocytosis-related pathways, as evidenced by the reduced cellular uptake in the presence of their respective inhibitors. Conversely, in Liu's study, [211] the use of various endocytosis inhibitors revealed that, except for nocodazole, which disrupts microtubule dynamics and blocks endocytic vesicle trafficking, all other inhibitors were ineffective in preventing cellular uptake of NPs.…”

A pumpless and tubeless microfluidic device enables extendedin vitrodevelopment ofCryptosporidium parvum

“…The present study proposes the use of a user-friendly gut-on-chip to extend the current applications of the HCT-8 cell line for the study of Cryptosporidium biology, while maintaining ease of use and low costs. The control of fluid flow, and consequent application of fluid shear stress to transformed cells cultured within microfluidic devices, has been frequently documented to change the phenotype of these cells to reflect more physiologically relevant structure and function (27)(28)(29)(30). To foster implementation in settings with limited microfluidic experience and facilities, a previously reported pumpless and tubeless microfluidic device was used.…”

Bio–Microfabrication of 2D and 3D Biomimetic Gut-on-a-Chip

“…To address these challenges, specialized biomaterial-based microfabrication processes for three-dimensional structures need to be developed. With continuous progress in bio–microfabrication techniques, biomaterial-based microfabrication technology for a gut-on-a-chip has implemented a great complexity of gut tissues in vitro to mimic physiological three-dimensional structures with effective gut functions [ 47 , 48 , 49 ]. The three-dimensional structure in the gut-on-a-chip represents many intestine villi and microvilli on an enormous surface.…”