Construction of Multilayered Small Intestine-like Tissue by Reproducing Interstitial Flow

Multilayered Tissue Construction of a Micro-intestinal System Reproduces Mesenchymal Flow

Research Background

In recent years, constructing in vitro models of the human small intestine has made significant progress; however, fully replicating its complex structural and functional characteristics remains challenging. The goal of micro-intestinal tissue models is to create systems applicable in drug metabolism and infectious disease research, but traditional methods have failed to achieve the stratification and maturation of intestinal structures. The research team hypothesized that the mesenchymal flow driven by plasma circulation during embryogenesis might be the key factor in forming these complex structures. To better simulate this process, Professor Sayaka Deguchi and her team employed human pluripotent stem cells (PSCs) to construct a multilayered tissue resembling human fetal intestine by replicating mesenchymal flow in a microfluidic device.

Research Source

This research was jointly led by Professor Sayaka Deguchi and Professor Kazuo Takayama from Kyoto University’s CiRA (Center for iPS Cell Research and Application). The team of authors included researchers from Kyoto University, Ritsumeikan University, and the Japanese Agency for Medical Research and Development, among other institutions. The results were published in the September 5, 2024 issue of the journal “Cell Stem Cell.”

Research Process

Research Design and Methods

This study used PSCs as the research subject, applying microfluidic technology under specific conditions to induce PSCs to differentiate into intestinal epithelium and non-epithelial cells. The main process is as follows:

1. PSCs Differentiation: Initially, differentiated PSCs are injected onto the top of the porous microfluidic device and treated with a series of factors (such as Activin A, FGF2, etc.), prompting them to differentiate into intestinal epithelial and non-epithelial cells.
2. Simulation of Mesenchymal Flow: A slow-flowing culture medium is utilized in the bottom channel of the microfluidic device to simulate mesenchymal flow driven by plasma circulation. Numerical simulations demonstrate that this flow speed is extremely low, exhibiting nearly shear-free flow characteristics, effectively promoting the self-organization of intestinal tissue.
3. Tissue Construction: Between the 5th and 24th day, the culture medium flow in the bottom channel is maintained, during which epithelial cells gradually form a three-dimensional structure resembling intestinal villi, with an aligned mesenchymal layer on the basal side, progressively constructing a multilayered tissue.

Novel Experimental Equipment

The research team designed a novel microfluidic device consisting of two layers of channels, separated by a porous polyethylene terephthalate membrane, segregating the upper cell culture environment from the lower mesenchymal flow to simulate an in vivo mesenchymal flow environment. Additionally, the team optimized the flow rate of the bottom channel through numerical simulations to achieve near-shear-free flow, thereby effectively promoting the organizational self-assembly process of PSCs.

Research Results

Generation of Multilayered Intestinal Tissue

Through experimental analysis, the research team successfully generated tissue structures resembling human fetal intestine, including the stratification of epithelial cells and the mesenchymal layer on the basal side. Specifically:

• Polarity and Stratification of Epithelial Cells: Under flow conditions, the generated epithelial cells acquired cell polarity and formed villi-like structures specific to the small intestine; cell types such as Goblet cells and Paneth cells were present.
• Alignment of Mesenchymal Layer: Compared to static conditions, mesenchymal cells under flow conditions formed aligned structures on the basal side. Immunostaining and mass spectrometry analyses verified the maturity of the mesenchymal layer.
• Distribution of Various Cell Types: Single-cell RNA sequencing analysis further confirmed the presence of various cell types in the generated intestinal tissue, such as smooth muscle and fibroblast cells, with a composition ratio similar to fetal intestinal tissue.

Support from Experimental Data

• Gene and Protein Expression Analysis: qPCR and proteomics analyses showed significantly increased expression levels of intestine-specific markers in the tissue under mesenchymal flow, including Villin, Sucrase-Isomaltase, and other intestinal epithelial markers.
• Permeability Testing: For applications in drug absorption and viral infection, the research team used FITC-labeled dextran to test the permeability of the tissue, showing that tissue in the mesenchymal flow environment exhibited higher barrier function.
• Drug Metabolizing Enzyme Activity: Under flow conditions, cells in the tissue exhibited higher activities of drug metabolizing enzymes (such as CYP, UGT, SULT, etc.), further demonstrating the model’s potential in drug metabolism research.

Application in Viral Infection Model

The research team tested the model’s response to human coronavirus-229E infection. The experiment showed that infection efficiency from the apical side was significantly higher than from the basal side. Under flow conditions, cells exhibited higher expression of ANPEP (coronavirus receptor), primarily concentrated on the apical surface of the epithelium, indicating that the model is closer to the viral infection mechanisms of the human intestine.

Research Conclusion and Value

This micro-intestinal system not only successfully reproduced the three-dimensional structure of multilayered intestinal tissue but also achieved epithelial polarity and functional maturation, becoming a powerful model of the human small intestine. It has broad application prospects in drug metabolism and infectious disease research, suitable for studying intestinal barrier function, drug absorption and metabolism, and the molecular mechanisms of intestinal infections. Additionally, the model’s structure and cell composition are similar to fetal intestinal tissue, providing an important tool for further exploring intestinal development processes.

Research Highlights

1. Innovative Microfluidic Device: By simulating mesenchymal flow, it promotes the maturation and multilayered construction of intestinal tissue, a feat unattainable by existing intestinal models.
2. Multilayered Tissue Structure: The system successfully reproduces the multilayered structure of the intestine, providing a new standard for in vitro intestinal tissue models.
3. Broad Application Potential: Suitable for drug metabolism research, intestinal infection research, and more, aiding in replacing animal models for human intestinal disease research.

Summary and Outlook

The micro-intestinal system developed by Professor Deguchi’s team successfully recreated the multilayered structure of the human intestine, paving a new path for drug metabolism and infection research. In the future, the research team plans to further optimize the system with longer culture durations and different flow conditions to enhance the maturity of intestinal tissue, enabling it to play a bigger role in intestinal development research and human disease modeling.