Enterocyte Culture on a Hybrid Transwell-Inserted Gut-on-a-Chip for Liquid–Liquid and Air–Liquid Interface Conditions

Charoensri, Tidaporn; Kulthong, Kornphimol; Pormrungruang, Pareesa; Wongwanakul, Ratjika; Kunyanee, Chanikarn; Aueviriyavit, Sasitorn; Rahong, Sakon

doi:10.1021/acsomega.5c03215

Enterocyte Culture on a Hybrid Transwell-Inserted Gut-on-a-Chip for Liquid–Liquid and Air–Liquid Interface Conditions

Author(s)

Charoensri, Tidaporn

Kulthong, Kornphimol

Pormrungruang, Pareesa

Wongwanakul, Ratjika

Kunyanee, Chanikarn

Aueviriyavit, Sasitorn

Rahong, Sakon

Date Issued

November 18, 2025

Type

Article

DOI

10.1021/acsomega.5c03215

Abstract

Intestinal models have been studied extensively using in vitro microfluidic platforms, often called gut-on-a-chip devices, which replicate the physiological environment and functions of the human intestine. The role of fabrication techniques in developing these microfluidic systems is increasingly significant. Photolithography has traditionally been used for microchannel preparation, oxygen-plasma surface treatment, and conventional bonding methods that attach microchannels to surfaces. However, photolithography is limited to perfectly flat surfaces, is costly, requires specialized equipment, and must be conducted in controlled environments free from contaminants, making the process slow and labor-intensive. Consequently, there is a need for a fabrication method that enables rapid prototyping and is easy to use, cost-effective, and suitable for mass production. In this study, a hybrid chip was developed using an uncured poly(dimethylsiloxane) (PDMS) bonding technique, which provides strong adhesion. This hybrid chip incorporates a transwell insert and a PDMS fluid channel, which can be used to culture human intestinal cells (Caco-2 cells) at a flow rate of 30 μL/h over 5 days without removing the fluid channel. Furthermore, the hybrid chip enables the cultivation of Caco-2 cells under both the liquid–liquid interface (LLI) and air–liquid interface (ALI) conditions. Evaluations of cell viability, morphology, and gene expression of Caco-2 cells grown on the hybrid chip demonstrated improved cell viability, clear expression of ZO-1, and a significant increase in MUC2 expression in both LLI and ALI cultures, along with GLUT2 expression in the ALI culture, indicating that this fabricated hybrid chip is a viable in vitro fluid flow system for culturing Caco-2 cells.

Citation

ACS Omega, 10(45), 53865-53873, 2025