harvesting and seeding of human umbilical vein endothelial cells (huvecs) for human intestine-on-chip model

Enhanced Intestine-on-Chip Platform for Immunological Insight

Organ-on-Chip (OoC) systems represent an emerging technique of 3D cell culture that is capable of bridging the gap between conventional 2D cell culture and animal models. OoC platforms typically consist of one or more compartments containing tissue-specific cells grown on a wide range of scaffolds such as membranes or hydrogels1. The models are capable of mimicking one or more defined organotypic functions. Pumps enable continuous microfluidic perfusion of cell culture medium for removal of cellular waste products, supply with nutrition and growth factors for improved cellular differentiation, and recreating essential in vivo conditions. With the integration of immune cells, OoC systems can mimic human immune response in vitro2. To date, a wide range of organs and functional units have been presented1. These systems include models of the vasculature3, lung4, liver2,5, and intestine6 that can be facilitated for drug testing5,7 and infection studies6,8.
We here present a human intestine-on-chip model integrating human epithelial cells forming an organotypic 3D topography of villus-like and crypt-like structures combined with an endothelial lining and tissue-resident macrophages. The model is cultured in a microfluidically perfused biochip in the format of a microscopic slide.&#160;Each biochip consists of two separate microfluidic cavities. Each cavity is divided by a porous polyethylene terephthalate (PET) membrane into an upper and lower chamber. The membrane itself also serves as the scaffold for the cells to grow on each side. The pores of the membrane enable cellular crosstalk and cell migration between cell layers. Each chamber can be accessed by two female luer lock-sized ports. Optionally, an additional mini-luer lock-sized port can provide access to the upper or lower chamber (Figure 1).
The OoC platform offers a number of readouts that can be obtained from a single experiment. The intestine-on-chip is tailored towards combining perfused 3D cell culture, effluent analysis, and fluorescence microscopy to assess cell marker expression, metabolization rates, immune response, microbial colonization and infection, and barrier function3,6,8. The model includes tissue-resident immune cells and direct contact of living microorganisms with the host tissue, which is a benefit compared to other published models9. Further, epithelial cells self-organize into three-dimensional structures that provide a physiologically relevant interface for the colonization with a living microbiota6.

Author Spotlight: Developing Immunocompetent Organ-on-Chip Models for Infectious Disease Research

Immunocompetent Intestine-on-Chip Model for Analyzing Gut Mucosal Immune Responses

Our research focuses on the development and use of immunocompetent organ-on-chip platforms. By creating these physiologically relevant, humanized in vitro models, we are able to study the complex host-pathogen interaction. The knowledge we create can be used to manipulate and identify molecular targets for the therapy of infectious diseases.One key challenge is maintaining a living microbiota under homeostatic conditions alongside host tissue in vitro. This includes replicating the organotypic 3D topography of crypt and villus structures and their microbial colonization. Intestine-on-chip platforms address this by using flow to shape the organotypic structures and prevent bacterial overgrowth.This ensures stable culture conditions. Compared to other available intestine-on-chip platforms, our model enables the detailed study of the human immune response to pathogens due to the inclusion of primary tissue-resident immune cells. Additionally, the model size enables multiple readouts in one single experiment, reducing the overall cost and number of experiments.Our laboratory is focusing on using induced pluripotent stem cells to develop autologous systems that enable personalized medicine.

Harvesting and Seeding of Human Umbilical Vein Endothelial Cells (HUVECs) for Human Intestine-on-Chip Model

harvesting-seeding-human-umbilical-vein-endothelial-cells-huvecs-for

Research

JoVE Journal

Immunology and Infection

120 vues.  Jena University Hospital. Pour commencer, préparez une dilution de 1 à 100 d’une solution mère de collagène dans du DPBS avec du magnésium et du calcium. Ajoutez 350 microlitres de la solution mère diluée dans la chambre correspondante et incubez pendant cinq minutes à température ambiante. Rincez toutes les chambres deux fois en utilisant 350 microlitres de DPBS avec du magnésium et du calcium pour éliminer le collagène et l’acide acétique restants.Retirez les bouchons d’un site et basculez-...

Vidéo: Prélèvement et ensemencement de cellules endothéliales de veine ombilicale humaine (HUVEC) pour le modèle d’intestin humain sur puce

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent model includes Human Umbilical Vein Endothelial Cells (HUVEC), Caco-2 intestinal epithelial cells, tissue-resident macrophages, and dendritic cells, which self-organize within the tissue, mirroring characteristics of the human intestinal mucosa. A unique aspect of this platform is its capacity to integrate circulating human primary immune cells, enhancing physiological relevance. The model is designed to investigate the intestinal immune system&#39;s response to bacterial and fungal colonization and infection. Due to its enlarged cavity size, the model offers diverse functional readouts such as permeation assays, cytokine release, and immune cell infiltration, and is compatible with immunofluorescence measurement of 3D structures formed by the epithelial cell layer. It hereby provides comprehensive insights into cell differentiation and function. The intestine-on-chip platform has demonstrated its potential in elucidating complex interactions between surrogates of a living microbiota and human host tissue within a microphysiological perfused biochip platform.

Our detailed protocol outlines the creation and use of the advanced intestine-on-chip model, which simulates human intestinal mucosa with 3D structures and various cell types, enabling in-depth analysis of immune responses and cellular functions in response to microbial colonization.

An advanced intestine-on-chip model recreating epithelial 3D organotypic villus-like and crypt-like structures has been developed. The immunocompetent ...