A Novel Serum-Free 2D and 3D Culture System Comprising Two Small Molecules for Culturing Mouse Lacrimal Gland Epithelial Cells

Baihui Zeng; Shurong Wang; Cheng Li

doi:10.3791/70438

Method Article

A Novel Serum-Free 2D and 3D Culture System Comprising Two Small Molecules for Culturing Mouse Lacrimal Gland Epithelial Cells

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DOI:

10.3791/70438

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February 10th, 2026

Baihui Zeng¹^,² , Shurong Wang¹ , Cheng Li²^,³^,⁴

¹Department of Ophthalmology Optometry Center, China-Japan Union Hospital of Jilin University, ²Eye Institute & Affiliated Xiamen Eye Center, School of Medicine, Xiamen University, ³Huaxia Eye Hospital of Quanzhou, ⁴Shen Zhen Research Institute of Xiamen University

Summary

A novel serum-free culture system using the small molecule combination 2C (Y27632 and SB431542) is introduced to efficiently expand lacrimal gland epithelial cells (LGECs) in both 2D and 3D cultures, enabling precise control of their proliferation and differentiation.

Abstract

Lacrimal gland (LG) dysfunction is a major cause of aqueous-deficient dry eye disease, and cell-based and tissue engineering therapies show significant potential for this condition. However, the limited availability of sufficient seed cells cultured under serum-free conditions has hindered their widespread application. In this study, we developed a novel serum-free culture system using two small molecules, Y27632 and SB431542 (2C), to efficiently expand LG epithelial cells (LGECs). For the 2D primary culture of LGECs, LGs were isolated from 6-8 weeks old mice and enzymatically digested using Dispase II and Collagenase A. The resulting cell suspension was seeded into culture dishes at a density of 7,500 cells/cm² and cultured with 2C for 12-14 days. When the cell confluence reached 80-90%, subculture was initiated at a 1:2 ratio. For 3D culture, 10,000 P1 LGECs were resuspended in 10 µL of 2C and 10 µL of matrix gel, then seeded in the center of a 24-well plate. After a 30 min solidification period at 37 °C, 600 µL of 2C was added for continued culture. For differentiation, the 3D spheroids were cultured with 2C for 7 days, followed by removal of 2C and continued culture for an additional 7 days. LGECs cultured with 2C exhibited high proliferation, with elevated expression of stemness and proliferation markers (Ki67, K14, P63, K5, K15 [P = 0.0011, 0.0002, 0.0012, 0.0003, 0.0014, respectively]), and maintained morphology and proliferative capacity even after ten passages. Furthermore, under 3D conditions, LGECs formed spheroids with stem/progenitor characteristics, which further differentiated into microglandular structures containing multiple LG cell types (AQP5, K19, α-SMA-positive) and mature secretory functions after the removal of 2C. This approach is expected to provide a stable source of seed cells for tissue engineering and offers a new in vitro model to study LG physiology.

Introduction

The lacrimal gland (LG) is essential for producing the aqueous layer of the tear film, which is crucial for maintaining ocular surface homeostasis¹. Dysfunction of the LG, caused by injury or inflammation, results in aqueous-deficient dry eye disease (ADDE). This condition can lead to severe ocular surface inflammation, chronic corneal disease, and, in severe cases, permanent vision loss². Current treatments for ADDE primarily manage symptoms, without addressing the underlying glandular dysfunction, which limits their long-term efficacy¹. The development of targeted therapies for LG dysfunction pr....