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

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2062
Author(s):  
Marcus Lindner ◽  
Anna Laporte ◽  
Stephan Block ◽  
Laura Elomaa ◽  
Marie Weinhart
Keyword(s):  
Cell Model ◽  
Culture Conditions ◽  
Solid Surfaces ◽  
Shear Stresses ◽  
Strong Impact ◽  
Intestinal Epithelial ◽  
Mucus Layer ◽  
3 Dimensional ◽  
Compositional Changes

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.

scholarly journals Physiological shear stress enhances differentiation and mucus-formation of intestinal epithelial cells in vitro.

2020 ◽  
Author(s):  
Marcus Lindner ◽  
Anna Laporte ◽  
Stephan Block ◽  
Laura Elomaa ◽  
Marie Weinhart
Keyword(s):  
Shear Stress ◽  
Culture Conditions ◽  
Shear Stresses ◽  
Intestinal Epithelial ◽  
Culture Chamber ◽  
3 Dimensional ◽  
Static Culture ◽  
The Impact

The gastrointestinal (GI) mucus layer plays a pivotal role in tissue homoeostasis and functionality of the gut. However, due to the shortage of affordable, realistic in vitro mucus models, studies with deeper insights into its structure and characteristics are rare. To obtain an improved mucus model, we developed a reusable culture chamber facilitating the application of physiologically relevant GI shear stresses (0.002-0.08 dyn/cm) to cells in a bioreactor system. Differentiation of a confluent monolayer of human mucus-producing epithelial HT29-MTX cells was monitored under dynamic and static culture conditions. Cells under flow remained highly proliferative and analysis via confocal microscopy revealed superior reorganization into 3-dimensional villi-like structures compared to static culture (up to 120 vs. 80 µm in height). Additionally, the median mucus thickness was significantly increased under dynamic conditions compared to static culture (41±14 vs. 29±14 µm) with a simultaneous drastic reduction of culture time from three to two weeks for sufficient maturation into goblet-like cells. We demonstrated the impact of culture conditions on the differentiation of HT29-MTX cells, revealing outstanding in vivo like reorganization of cells and the production of thick adherent mucus networks when cultured under physiological shear stress using our newly designed culture chamber.

scholarly journals Transcriptome comparisons of in vitro intestinal epithelia grown under static and microfluidic gut-on-chip conditions with in vivo human epithelia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kornphimol Kulthong ◽  
Guido J. E. J. Hooiveld ◽  
Loes Duivenvoorde ◽  
Ignacio Miro Estruch ◽  
Victor Marin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Culture Conditions ◽  
Gene Set Enrichment Analysis ◽  
Shear Stresses ◽  
Static Culture ◽  
Dynamic Culture ◽  
Intestinal Segments ◽  
On Chip

AbstractGut-on-chip devices enable exposure of cells to a continuous flow of culture medium, inducing shear stresses and could thus better recapitulate the in vivo human intestinal environment in an in vitro epithelial model compared to static culture methods. We aimed to study if dynamic culture conditions affect the gene expression of Caco-2 cells cultured statically or dynamically in a gut-on-chip device and how these gene expression patterns compared to that of intestinal segments in vivo. For this we applied whole genome transcriptomics. Dynamic culture conditions led to a total of 5927 differentially expressed genes (3280 upregulated and 2647 downregulated genes) compared to static culture conditions. Gene set enrichment analysis revealed upregulated pathways associated with the immune system, signal transduction and cell growth and death, and downregulated pathways associated with drug metabolism, compound digestion and absorption under dynamic culture conditions. Comparison of the in vitro gene expression data with transcriptome profiles of human in vivo duodenum, jejunum, ileum and colon tissue samples showed similarities in gene expression profiles with intestinal segments. It is concluded that both the static and the dynamic gut-on-chip model are suitable to study human intestinal epithelial responses as an alternative for animal models.

The Promise of Patient-Derived Colon Organoids to Model Ulcerative Colitis

2021 ◽  
Author(s):  
Babajide A Ojo ◽  
Kelli L VanDussen ◽  
Michael J Rosen
Keyword(s):  
Ulcerative Colitis ◽  
Self Organization ◽  
Great Promise ◽  
Preclinical Model ◽  
Intestinal Epithelial ◽  
Epigenetic Changes ◽  
Nutrient Delivery ◽  
3 Dimensional ◽  
Tissue Systems

Abstract Physiologic, molecular, and genetic findings all point to impaired intestinal epithelial function as a key element in the multifactorial pathogenesis of ulcerative colitis (UC). The lack of epithelial-directed therapies is a conspicuous weakness of our UC therapeutic armamentarium. However, a critical barrier to new drug discovery is the lack of preclinical human models of UC. Patient tissue–derived colon epithelial organoids (colonoids) are primary epithelial stem cell–derived in vitro structures capable of self-organization and self-renewal that hold great promise as a human preclinical model for UC drug development. Several single and multi-tissue systems for colonoid culture have been developed, including 3-dimensional colonoids grown in a gelatinous extracellular matrix, 2-dimensional polarized monolayers, and colonoids on a chip that model luminal and blood flow and nutrient delivery. A small number of pioneering studies suggest that colonoids derived from UC patients retain some disease-related transcriptional and epigenetic changes, but they also raise questions regarding the persistence of inflammatory transcriptional programs in culture over time. Additional research is needed to fully characterize the extent to which and under what conditions colonoids accurately model disease-associated epithelial molecular and functional aberrations. With further advancement and standardization of colonoid culture methodology, colonoids will likely become an important tool for realizing precision medicine in UC.

Comparison of gene expression and activation of transcription factors in organoid-derived monolayer intestinal epithelial cells and organoids

2021 ◽  
Author(s):  
Yu Takahashi ◽  
Yu Inoue ◽  
Keitaro Kuze ◽  
Shintaro Sato ◽  
Makoto Shimizu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cells ◽  
Gene Expression Regulation ◽  
Intestinal Epithelial Cells ◽  
Three Dimensional ◽  
Culture Conditions ◽  
Dependent Manner ◽  
Intestinal Epithelial

Abstract Intestinal organoids better represent in vivo intestinal properties than conventionally used established cell lines in vitro. However, they are maintained in three-dimensional culture conditions that may be accompanied by handling complexities. We characterized the properties of human organoid-derived two-dimensionally cultured intestinal epithelial cells (IECs) compared with those of their parental organoids. We found that the expression of several intestinal markers and functional genes were indistinguishable between monolayer IECs and organoids. We further confirmed that their specific ligands equally activate intestinal ligand-activated transcriptional regulators in a dose-dependent manner. The results suggest that culture conditions do not significantly influence the fundamental properties of monolayer IECs originating from organoids, at least from the perspective of gene expression regulation. This will enable their use as novel biological tools to investigate the physiological functions of the human intestine.

scholarly journals Tissue Specific Differentiation of Human Chondrocytes Depends on Cell Microenvironment and Serum Selection

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 934 ◽  
Author(s):  
Ecke ◽  
Lutter ◽  
Scholka ◽  
Hansch ◽  
Becker ◽  
...  
Keyword(s):  
Articular Chondrocytes ◽  
Culture Conditions ◽  
Serum Supplement ◽  
3 Dimensional ◽  
The Matrix ◽  
Differentiation Degree ◽  
Overlay Technique ◽  
Fetal Bovine ◽  
Cell Culture Conditions

Therapeutic options to cure osteoarthritis (OA) are not yet available, although cell-based therapies for the treatment of traumatic defects of cartilage have already been developed using, e.g., articular chondrocytes. In order to adapt cell-based therapies to treat OA, appropriate cell culture conditions are necessary. Chondrocytes require a 3-dimensional (3D) environment for redifferentiation after 2-dimensional (2D) expansion. Fetal bovine serum (FBS) is commonly used as a medium supplement, although the usage of a xenogeneic serum could mask the intrinsic behavior of human cells in vitro. The aim of this study was to compare human articular chondrocytes cultivated as monolayers (2D) and the development of microtissues (3D) in the presence of FBS with those cultivated with human serum (HS). Evaluation of the expression of various markers via immunocytochemistry on monolayer cells revealed a higher dedifferentiation degree of chondrocytes cultivated with HS. Scaffold-free microtissues were generated using the agar overlay technique, and their differentiation level was evaluated via histochemistry and immunohistochemistry. Microtissues cultivated in the medium with FBS showed a higher redifferentiation level. This was evidenced by bigger microtissues and a more cartilage-like composition of the matrix with not any/less positivity for cartilage-specific markers in HS versus moderate-to-high positivity in FBS-cultured microtissues. The present study showed that the differentiation degree of chondrocytes depends both on the microenvironment of the cells and the serum type with FBS achieving the best results. However, HS should be preferred for the engineering of cartilage-like microtissues, as it rather enables a "human-based" situation in vitro. Hence, cultivation conditions might be further optimized to gain an even more adequate and donor-independent redifferentiation of chondrocytes in microtissues, e.g., designing a suitable chemically-defined serum supplement.

Biological activity of the non-microbial fraction of kefir: antagonism against intestinal pathogens

2017 ◽  
Vol 84 (3) ◽  
pp. 339-345 ◽  
Author(s):  
Carolina Iraporda ◽  
Mário Abatemarco Júnior ◽  
Elisabeth Neumann ◽  
Álvaro Cantini Nunes ◽  
Jacques R Nicoli ◽  
...  
Keyword(s):  
Acetic Acid ◽  
Biological Activity ◽  
Cell Model ◽  
Acetic Acid Bacteria ◽  
Fermented Milk ◽  
In Vitro Assays ◽  
Intestinal Epithelial ◽  
Milk Fermentation ◽  
Intestinal Pathogens

Kefir is a fermented milk obtained by the activity of kefir grains which are composed of lactic and acetic acid bacteria, and yeasts. Many beneficial health effects have been associated with kefir consumption such as stimulation of the immune system and inhibition of pathogenic microorganisms. The biological activity of kefir may be attributed to the presence of a complex microbiota as well as the microbial metabolites that are released during fermentation. The aim of this work was to characterise the non-microbial fraction of kefir and to study its antagonism againstEscherichia coli,Salmonellaspp. andBacillus cereus.During milk fermentation there was a production of organic acids, mainly lactic and acetic acid, with a consequent decrease in pH and lactose content. The non-microbial fraction of kefir added to nutrient broth at concentrations above 75% v/v induced a complete inhibition of pathogenic growth that could be ascribed to the presence of un-dissociated lactic acid. In vitro assays using an intestinal epithelial cell model indicated that pre-incubation of cells with the non-microbial fraction of kefir did not modify the association/invasion ofSalmonellawhereas pre-incubation ofSalmonellawith this fraction under conditions that did not affect their viability significantly decreased the pathogen's ability to invade epithelial cells. Lactate exerted a protective effect againstSalmonellain a mouse model, demonstrating the relevance of metabolites present in the non-microbial fraction of kefir produced during milk fermentation.

Fabrication of a cell-adhesive microwell array for 3-dimensional in vitro cell model

2015 ◽  
Vol 5 (2) ◽  
pp. 140-146 ◽  
Author(s):  
Jihwang Park ◽  
Michael Müller ◽  
Jungtae Kim ◽  
Helmut Seidel
Keyword(s):  
Cell Model ◽  
3 Dimensional ◽  
Microwell Array ◽  
Cell Adhesive ◽  
A Cell

scholarly journals Human Co-culture Model of Neurons and Astrocytes to Test Acute Cytotoxicity of Neurotoxic Compounds

2017 ◽  
Vol 36 (6) ◽  
pp. 463-477 ◽  
Author(s):  
Uliana De Simone ◽  
Francesca Caloni ◽  
Laura Gribaldo ◽  
Teresa Coccini
Keyword(s):  
Cell Line ◽  
Cell Morphology ◽  
Cell Model ◽  
Culture Conditions ◽  
Test Method ◽  
Alternative Methods ◽  
Mitochondrial Activity ◽  
Neuroprotective Effects ◽  
Toxicological Studies

Alternative methods and their use in planning and conducting toxicology experiments have become essential for modern toxicologists, thus reducing or replacing living animals. Although in vitro human co-culture models allow the establishment of biologically relevant cell–cell interactions that recapitulate the tissue microenvironment and better mimic its physiology, the number of publications is limited specifically addressing this scientific area and utilizing this test method which could provide an additional valuable model in toxicological studies. In the present study, an in vitro model based on central nervous system (CNS) cell co-cultures was implemented using a transwell system combining human neuronal cells (SH-SY5Y cell line) and glial cells, namely astrocytes (D384 cell line), to investigate neuroprotection of D384 on SH-SY5Y and vice versa. The model was applied to test acute (24-48 hours) cytotoxicity of 3 different neurotoxicants: (1) methyl mercury (1-2.5 μM), (2) Fe3O4 nanoparticles (1-100 μg/mL), and (3) methylglyoxal (0.5-1 mM). Data were compared to mono-cultures evaluating the mitochondrial function and cell morphology. The results clearly showed that all compounds tested affected the mitochondrial activity and cell morphology in both mono-culture and co-culture conditions. However, astrocytes, when cultured together with neurons, diminish the neurotoxicant-induced cytotoxic effects that occurred in neurons cultured alone, and astrocytes become more resistant in the presence of neurons. This human CNS co-culture system seems a suitable cell model to feed high-throughput acute screening platforms and to evaluate both human neuronal and astrocytic toxicity and neuroprotective effects of new and emerging materials (eg, nanomaterials) and new products with improved sensitivity due to the functional neuron–astrocyte metabolic interactions.

scholarly journals Exploring Effects of Chitosan Oligosaccharides on the DSS-Induced Intestinal Barrier Impairment In Vitro and In Vivo

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2199
Author(s):  
Yujie Wang ◽  
Rong Wen ◽  
Dongdong Liu ◽  
Chen Zhang ◽  
Zhuo A. Wang ◽  
...  
Keyword(s):  
Tight Junction ◽  
Structural Characteristics ◽  
Intestinal Barrier ◽  
Intestinal Barrier Function ◽  
Chitosan Oligosaccharide ◽  
Intestinal Epithelial ◽  
Mucus Layer ◽  
Intestinal Mucus ◽  
Epithelial Tight Junction

Intestinal barrier dysfunction is an essential pathological change in inflammatory bowel disease (IBD). The mucus layer and the intestinal epithelial tight junction act together to maintain barrier integrity. Studies showed that chitosan oligosaccharide (COS) had a positive effect on gut health, effectively protecting the intestinal barrier in IBD. However, these studies usually focused on its impact on the intestinal epithelial tight junction. The influence of COS on the intestinal mucus layer is still poorly understood. In this study, we explored the effect of COS on intestinal mucus in vitro using human colonic mucus-secreted HT-29 cells. COS relieved DSS (dextran sulfate sodium)-induced mucus defects. Additionally, the structural characteristics of COS greatly influenced this activity. Finally, we evaluated the protective effect of COS on intestinal barrier function in mice with DSS-induced colitis. The results indicated that COS could manipulate intestinal mucus production, which likely contributed to its intestinal protective effects.

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