Amir Bein

LPS Induces Hyper‐Permeability of Intestinal Epithelial Cells

Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder leading to high morbidity and mortality rates. A growing body of evidence demonstrate the key role of the Toll like receptor 4 (TLR4) in NEC. This membranal receptor recognizes lipopolysaccharides (LPS) from the bacterial wall and triggers an inflammatory response. The aim of the present study was to elucidate the effect of LPS on paracellular permeability known to be severely affected in NEC. IEC‐18 cells were treated with LPS and the effects on morphology, paracellular permeability and their associated gene and protein expressions were measured. Our results show that LPS down regulated the expression of occludin and ZO‐1 mRNAs while up regulating Cdkn1a. In addition LPS caused a significant increase in paracellular permeability and epithelial barrier damage. Finally ZO‐1 protein was found to be spatially disarrayed in the intercellular junctions in response to LPS. We conclude that LPS adversely affected the functionality of the intestinal epithelial barrier suggesting a new mechanism by which bacterial infection may contribute to the development of NEC. J. Cell. Physiol. 232: 381–390, 2017.

Surface plasmon-based infrared spectroscopy for cell biosensing

Cell morphology is often used as a valuable indicator of the physical condition and general status of living cells. We demonstrate a noninvasive method for morphological characterization of adherent cells. We measure infrared reflectivity spectrum at oblique angle from living cells cultured on thin Au film, and utilize the unique properties of the confined infrared waves (i.e., surface plasmon and guided modes) traveling inside the cell layer. The propagation of these waves strongly depends on cell morphology and connectivity. By tracking the resonant wavelength and attenuation of the surface plasmon and guided modes we measure the kinetics of various cellular processes such as (i) cell attachment and spreading on different substrata, (ii) modulation of the outer cell membrane with chlorpromazine, and (iii) formation of intercellular junctions associated with progressive cell polarization. Our method enables monitoring of submicron variations in cell layer morphology in real-time, and in the label-free manner.

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

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.

Microfluidic Organ-on-a-Chip Models of Human Intestine

Microfluidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physiology and pathophysiology. In this article, we review this field and describe how microfluidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine.

Surface plasmon resonance-based infrared biosensor for cell studies with simultaneous control.

Abstract. We report a label-free infrared surface plasmon biosensor with a double-chamber flow cell for continuous monitoring of morphological changes in cell culture exposed to various stimuli. In this technique, the monolayer of cultured cells is divided into two halves by a barrier, allowing the treatment of one half while the other serves as control. We demonstrate the advantages of this setup in test experiments that track kinetics of the IEC-18 cell layer response to variations in extracellular Ca2+ concentration. The sensitivity of the presented method was found to be an order of magnitude higher compared to the single-chamber biosensor.

Intestinal tight junctions are severely altered in NEC preterm neonates

BACKGROUND & AIMS Necrotizing Enterocolitis (NEC) is a severe inflammatory disorder of the intestine endangering the health and survival of preterm infants. It is well established that the gut barrier is severely damaged in NEC patients, nonetheless an in depth investigation of modifications at the transcriptional and translational levels of tight junction genes and proteins during NEC are still missing. The aim of this study was to investigate changes in the expression of tight junctions and other associated proteins during NEC and determine their correlation to the disease severity. METHODS We examined intestinal specimens from six NEC patients and compared them with six control specimens from patients that underwent surgeries for reasons other than NEC. The expression of genes was analyzed by real time PCR and protein expression by immunohistochemistry. RESULTS The tight junction genes ZO-1, occludin, cingulin and claudin-4 were significantly down regulated in NEC. Furthermore TLR4, BAX and SIRT1 genes were found to be significantly down regulated while HIF-1A showed a trend of up regulation in NEC patients. These changes were found to correlate with the severity of the disease. Additionally we demonstrated in an ex-vivo model that hypoxic conditions initiated a destructive process of the epithelial barrier. We also showed that the expression of the tight junction proteins ZO-1 and occludin were significantly down regulated in NEC specimens. CONCLUSIONS The expression of tight junction proteins and their encoding genes are significantly altered in NEC. We surmise that SIRT1 and HIF-1A may play a role in controlling these effects.

TIMP-1 inhibition of occludin degradation in Caco-2 intestinal cells: a potential protective role in necrotizing enterocolitis

Background:Necrotizing enterocolitis (NEC), a common intestinal disease affecting premature infants, is a major cause of morbidity and mortality. Previous reports indicate an upregulation of intestinal matrix metalloproteinases (MMPs) activity that may play key roles on the higher permeability of the intestinal barrier, typical to NEC. Recently, TIMP-1, a natural inhibitor of MMP’s, was found to be over expressed in preterm human breast milk (HBM). Previous studies have shown that infants fed with HBM have a significant reduction in the incidence of NEC. The aim of the present study was to investigate the possible role that TIMP-1 may play on the maintenance of tight junctions and therefore the gut barrier integrity.Methods:Timp-1-treated Caco-2 intestinal cells were tested for MMP-2 enzymatic activity and cell junction integrity.Results:TIMP-1 inhibited MMP-2 activity, which induced a significant increase in the expression of occludin but not of claudin-4. TIMP-1 did not affect apoptosis.Conclusion:One of the putative mechanisms associated with HBM protection against NEC is mediated by TIMP-1, which downregulates MMP-2 activity, inhibits the degradation of occluding, and preserves tight junctions and gut barrier integrity.

Directional freezing for the cryopreservation of adherent mammalian cells on a substrate

Successfully cryopreserving cells adhered to a substrate would facilitate the growth of a vital confluent cell culture after thawing while dramatically shortening the post-thaw culturing time. Herein we propose a controlled slow cooling method combining initial directional freezing followed by gradual cooling down to -80°C for robust preservation of cell monolayers adherent to a substrate. Using computer controlled cryostages we examined the effect of cooling rates and dimethylsulfoxide (DMSO) concentration on cell survival and established an optimal cryopreservation protocol. Experimental results show the highest post-thawing viability for directional ice growth at a speed of 30 μm/sec (equivalent to freezing rate of 3.8°C/min), followed by gradual cooling of the sample with decreasing rate of 0.5°C/min. Efficient cryopreservation of three widely used epithelial cell lines: IEC-18, HeLa, and Caco-2, provides proof-of-concept support for this new freezing protocol applied to adherent cells. This method is highly reproducible, significantly increases the post-thaw cell viability and can be readily applied for cryopreservation of cellular cultures in microfluidic devices.