Cell Surface Modification-Mediated Primary Intestinal Epithelial Cell Culture Platforms for Assessing Host-Microbiota Interactions

Panida Sittipo; Laurensia Danis Anggradita; Hyunbum Kim; Chanyoung Lee; Nathaniel S Hwang; Yun Kyung Lee; Yongsung Hwang

doi:10.34133/bmr.0004

Cell Surface Modification-Mediated Primary Intestinal Epithelial Cell Culture Platforms for Assessing Host-Microbiota Interactions

Biomater Res. 2024 Jan 25:28:0004. doi: 10.34133/bmr.0004. eCollection 2024.

Authors

Panida Sittipo¹, Laurensia Danis Anggradita^{1

2}, Hyunbum Kim^{1

3}, Chanyoung Lee^{1

2}, Nathaniel S Hwang^{3

4

5}, Yun Kyung Lee^{1

2}, Yongsung Hwang^{1

2}

Affiliations

¹ Soonchunhyang Institute of Medi-bio Science (SIMS), Soonchunhyang University, Cheonan-si, Chungnam-do 31151, Republic of Korea.
² Department of Integrated Biomedical Science, Soonchunhyang University, Asan-si, Chungnam-do 31538, Republic of Korea.
³ School of Chemical and Biological Engineering, Institute of Chemical Processes, Seoul National University, Seoul 08826, Republic of Korea.
⁴ Bio-MAX/N-Bio Institute, Institute of Bio-Engineering, Seoul National University, Seoul 08826, Republic of Korea.
⁵ Institute of Engineering Research, Seoul National University, Seoul 08826, Republic of Korea.

Abstract

Background: Intestinal epithelial cells (IECs) play a crucial role in regulating the symbiotic relationship between the host and the gut microbiota, thereby allowing them to modulate barrier function, mucus production, and aberrant inflammation. Despite their importance, establishing an effective ex vivo culture method for supporting the prolonged survival and function of primary IECs remains challenging. Here, we aim to develop a novel strategy to support the long-term survival and function of primary IECs in response to gut microbiota by employing mild reduction of disulfides on the IEC surface proteins with tris(2-carboxyethyl)phosphine. Methods: Recognizing the crucial role of fibroblast-IEC crosstalk, we employed a cell surface modification strategy, establishing layer-to-layer contacts between fibroblasts and IECs. This involved combining negatively charged chondroitin sulfate on cell surfaces with a positively charged chitosan thin film between cells, enabling direct intercellular transfer. Validation included assessments of cell viability, efficiency of dye transfer, and IEC function upon lipopolysaccharide (LPS) treatment. Results: Our findings revealed that the layer-by-layer co-culture platform effectively facilitates the transfer of small molecules through gap junctions, providing vital support for the viability and function of primary IECs from both the small intestine and colon for up to 5 days, as evident by the expression of E-cadherin and Villin. Upon LPS treatment, these IECs exhibited a down-regulation of Villin and tight junction genes, such as E-cadherin and Zonula Occludens-1, when compared to their nontreated counterparts. Furthermore, the transcription level of Lysozyme exhibited an increase, while Mucin 2 showed a decrease in response to LPS, indicating responsiveness to bacterial molecules. Conclusions: Our study provides a layer-by-layer-based co-culture platform to support the prolonged survival of primary IECs and their features, which is important for understanding IEC function in response to the gut microbiota.