Constructing an in vitro model of ulcerative colitis in mice based on organoid technology

Abstract

BACKGROUND: The pathogenesis of ulcerative colitis is highly complex, necessitating the development of models that more closely mimic human physiological and pathological responses to study the mechanisms underlying its onset and progression. OBJECTIVE: To establish a mouse ulcerative colitis organoid model. METHODS: Colon organoids of C57BL/6J mice were extracted, cultured and passaged in vitro. Colon organoids from mice after three generations of passage were taken and incubated in lipopolysaccharide at varying concentrations [0 (control), 150, 175, 200, 225, 250, 275, 300, 325, and 350 μg/mL] to induce inflammation for 24 hours. The morphology of mouse colon organoids was observed under a microscope, and changes in proliferation viability were assessed using the cell counting kit-8 assay. After 24 hours of incubation with 0, 225, 250, 275 μg/mL lipopolysaccharide, the levels of tumor necrosis factor α, interleukin-6, interleukin-9, and interleukin-10 were measured by ELISA. After 24 hours of incubation with 0 and 275 μg/mL lipopolysaccharide, the expression of occludin and zonula occludens-1 was detected by immunofluorescence staining, and the mRNA expression of tumor necrosis factor α, interleukin-6, interleukin-9, occludin, and zonula occludens-1 was detected by q-PCR. RESULTS AND CONCLUSION: (1) Under the microscope, colon organoids in the 150-275 μg/mL lipopolysaccharide group showed varying degrees of swelling, while those in the 300-350 μg/mL lipopolysaccharide group had inhibited growth and swelling. CCK-8 assay showed that 150-350 μg/mL lipopolysaccharide reduced the proliferation viability of mouse colon organoids, with 225-350 μg/mL having a more pronounced effect. Based on cell morphology and proliferation viability results, 225, 250, and 275 μg/mL lipopolysaccharide were selected for ELISA. (2) Compared with the control group, the levels of interleukin-6 and tumor necrosis factor α were increased in the 225, 250, and 275 μg/mL lipopolysaccharide groups (P < 0.05), and the level of interleukin-9 was increased in the 275 μg/mL lipopolysaccharide group (P < 0.05). (3) Immunofluorescence staining showed that compared with the control group, the expression of occludin and zonula occludens-1 was decreased in the 275 μg/mL lipopolysaccharide group. q-PCR detection showed that compared with the control group, the mRNA expression of interleukin-6 and tumor necrosis factor α was increased (P < 0.05), occludin mRNA expression was decreased (P < 0.05), and there was no significant difference in the expression of interleukin-9 and zonula occludens-1 (P > 0.05). (4) These results indicate that an in vitro mouse ulcerative colitis model based on organoids was successfully constructed, providing a powerful tool for studying the mechanisms of ulcerative colitis and screening effective drugs.

1. Introduction

Ulcerative colitis is a chronic non-specific inflammatory bowel disease characterized by inflammation of the colorectal mucosa and submucosa. Its incidence has been increasing annually, and it is difficult to cure, prone to relapse, and has a risk of canceration. Therefore, a better understanding of the pathogenesis of ulcerative colitis is needed to overcome this challenge. Epidemiological studies suggest that the interaction of environmental and genetic factors increases susceptibility to ulcerative colitis, with epithelial barrier defects, immune dysregulation, and dysbiosis being important components in initiating and maintaining inflammation. In the early stage of ulcerative colitis, pathogenic microorganisms invade the intestinal mucosa and activate cellular immune responses, producing a large number of pro-inflammatory cytokines, leading to persistent inflammation of the intestinal mucosa. The intestinal mucosa is the main defense mechanism against environmental, physiological, and immune stimuli, but during active ulcerative colitis, pro-inflammatory cytokines can directly or indirectly disrupt the integrity of the intestinal mucosal barrier, aggravating the disease. Although there are many treatments for ulcerative colitis, some patients still require total proctocolectomy, which often leads to postoperative complications.

To develop safer and more reliable treatment options for ulcerative colitis, there is a need for a platform that can effectively study the disease and screen drugs. Colon organoid culture technology derived from tissue stem cells can not only highly recapitulate the structure and organization of the intestinal epithelium, effectively simulating the microenvironment of multiple cell interactions, but also maintain morphological characteristics and genomic stability, enabling efficient and precise drug screening. Traditional methods for studying ulcerative colitis mainly rely on two-dimensional cell culture and animal experiments. Two-dimensional cell culture is commonly used for rapid high-throughput screening and basic cell biology research, but it cannot replicate the rich in vivo environment and complex conditions, leading to results that may not match in vivo situations. Animal models, on the other hand, have limitations in terms of species differences and ethical concerns. Therefore, organoid models offer a promising alternative that bridges the gap between traditional cell culture and animal models.