Overview

Gut barrieroids are advanced 3D models of the human intestine closely mimic the structure and function of the gut barrier. These organoids are created from stem cells and replicate key features of the intestinal lining, including epithelial cells, tight junctions, and mucus production. The gut barrier plays a crucial role in maintaining gut health by regulating nutrient absorption and preventing harmful substances from entering the bloodstream. Using organ-on-a-chip technology is more beneficial for animal welfare as it reduces the amount of animal use. Additionally, it allows for a more intuitive observation of changes in the physicochemical properties of tissues under in vitro culture conditions.

To verify and simulate the intestinal permeation efficiency of the FGF21-LMWP fusion protein, and as a preparation for animal experiments, we designed an intestinal barrier organ-on-a-chip. Unlike traditional transwell culture plates, its significant advantage is that it allows for the seeding of cells on both sides of the semi-permeable membrane. Moreover, different cell types can be cultured in different chambers to simulate various locations within the intestine, better mimicking the 3D structural barrier of the intestinal mucosa.

Design

Device design

Our chip is composed of three chambers as a set (Figure 2). The upper layer consists of two seeding units, A and B, which function as separate culture units when 100 μm of culture medium is added. When 500 μm of culture medium is added, they can be connected as a single unit. The upper layer is separated from the lower C culture chamber by a 0.4-micron semi-permeable membrane. The lower layer is an independent culture unit that includes columnar areas corresponding to seeding units A and B, with liquid reservoirs D on both sides for convenient medium replacement.

Usage Method

Step 1: Seeding in the C Chamber

First, coat the porous membrane with type I collagen at 37°C for two hours. Invert the chip and seed fibroblasts to allow them to settle on the porous membrane for approximately 2 to 4 hours. After that, place the chip upright and culture it on a shaking platform for 24 hours.

Step 2: Seeding in the A and B Chambers

Seed IEC cells (derived from small intestine crypts, polarized monolayer with long, dense microvilli) in the A chamber and HT29 cells (derived from human colon, capable of forming tight junctions) in the B chamber. After independently culturing both chambers for 48 hours, add enough culture medium to connect the two chambers.

Step 3: Adding FGF21-LMWP Fusion Protein

After verifying the successful construction of the intestinal mucosal barrier in the connected chambers A and B under a microscope, we added the purified FGF21-LMWP fusion protein, which had been sterilized by filtering through a 0.22-micron cell filter. One hour later, the concentration of FGF21 in chamber C was measured using ELISA.

Figure 1- Figure 1: Schematic Diagram of a Single set

Figure 2: Cross-sectional view of a Single set

Figure 3: Traditional Transwell Migration Assay

Figure 4: Chip Overall Diagram