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CN-121975732-A - Method for co-culturing olfactory organoids and T cells of mice and application

CN121975732ACN 121975732 ACN121975732 ACN 121975732ACN-121975732-A

Abstract

The invention provides a method for co-culturing olfactory organoids and T cells of mice and application thereof. The age-related Olfactory Disorder (OD) organoid model is obtained by co-culturing the mouse olfactory organoids and Th17 cells, and is used for exploring the pathogenesis of the olfactory disorder, screening pathogenic genes and establishing a new method for treating the olfactory disorder. Experimental results show that after coculture with Th17 cells, tuj 1+ and OMP+ cells are significantly reduced, krt 19+ cells are significantly increased and show strong intracellular staining, indicating metaplasia, muc5 ac+ mucus cells and secretion content are increased, and after coculture for 14 days, both young and aged organoids have obvious saccular structures, and aged organoids change more significantly. Importantly, IL-17a neutralizing antibodies effectively block this lineage shift to maintain olfactory differentiation potential, revealing a key role for IL-17a in regulating cell fate decisions. It was shown that by co-culturing olfactory organoids with Th17 cells, the inflammatory microenvironment in age-related OD patients can be mimicked.

Inventors

  • REN WENWEN
  • Liu huanhai
  • XU ZENGYI
  • WANG CHENGLEI
  • ZHA XUDONG
  • XIE YINGQI
  • FU PENGHUI
  • XIONG CHANGHAI

Assignees

  • 中国人民解放军海军军医大学

Dates

Publication Date
20260505
Application Date
20251230

Claims (7)

  1. 1. A method for co-culturing olfactory organoids and T cells of a mouse, comprising the steps of: (1) Olfactory organoids culture Extracting olfactory mucosa from young and old mice, performing enzyme digestion, filtering to obtain single cell suspension, and culturing in organoid medium containing 6% Matrigel to generate organoids containing key olfactory epithelial cell types; (2) Th17 cell induction Preparing single-cell suspension from the mucous membrane barrier part by mechanical disruption, inducing and culturing Th17 cells from the single-cell suspension and detecting the percentage of the Th17 cells; (3) OD organoid construction Adding the separated Th17 cells into a Transwell upper chamber, performing non-contact co-culture with olfactory organoids, culturing mixed cells in a culture medium composed of 20% Th17 differentiation culture medium and 80% organoid culture medium at a ratio of organoids to Th17 cells of 1:40 in each well, removing the Transwell upper chamber after a certain time, and collecting cells of a lower chamber for analysis to obtain OD organoids.
  2. 2. The method of co-culturing a mouse olfactory organoid with T cells of claim 1, wherein: in the step (1), the method for obtaining single-cell suspension by filtering after the olfactory mucosa is digested by enzyme comprises the following steps of digesting the olfactory mucosa in 0.25% trypsin containing EDTA for 15 minutes, and filtering by a 40 mu m filter membrane to obtain single-cell suspension; The organoid medium was composed of DMEM base medium containing 1% Glutamax, 1~10 mM HEPES、10~100 ng/mL Wnt3a、50~300 ng/mL R-Spondin、50~300 ng/mL Noggin、1~20μM/mL Y27632、10~100 ng/mL EGF、1% N2 and 2% B27.
  3. 3. The method of co-culturing a mouse olfactory organoid with T cells of claim 1, wherein: Wherein, in step (2), the mucosal barrier site is selected from the group consisting of spleen; After 5 days of induction culture using the Th17 cell induction kit, the percentage of Th17 cells was measured by flow cytometry to 17.8-28.5 for subsequent related experiments.
  4. 4. The method of co-culturing a mouse olfactory organoid with T cells of claim 1, wherein: wherein, in the step (3), after Th17 cell induction is completed, and after the olfactory organoids are ensured to be in a good growth state on the 4 Th day after passage, co-culture is started; Co-culture of Th17 cells was performed in 24-well plates, with about 500 organoids and 20,000 Th17 cells per well; Th17 differentiation medium uses meitian and CytoBox Th mouse differentiation kit with specification of #130-107-758.
  5. 5. An age-related OD organoid model prepared by the method of any one of claims 1-4.
  6. 6. Use of the age-related OD organoid model of claim 5 for constructing a platform for studying pathogenesis of dysosmia or identification of causative genes.
  7. 7. Use of the age-related OD organoid model of claim 5 for in vitro screening of a medicament for treating age-related dysosmia.

Description

Method for co-culturing olfactory organoids and T cells of mice and application Technical Field The invention belongs to the technical field of organoid construction, and provides a method for co-culturing olfactory organoids and T cells of mice and application thereof. Background Olfaction is an important sensory function of mammals, deeply affecting human physiology, cognition and emotion. Olfactory Dysfunction (OD) refers to deterioration or loss of olfactory function, an early indicator of neurodegenerative disease, a hallmark symptom of viral infection, and a common manifestation of aging. As the population ages rapidly, the incidence of olfactory dysfunction increases year by year, affecting the elderly up to 50%. This situation can seriously impair the quality of life and may even endanger life, especially the elderly, because they lose the ability to detect warning odors such as gas leaks, smoke or spoiled foods. Impaired Olfactory Epithelium (OE) regeneration is a key factor in age-related OD prevalence. However, the pathogenesis and key regulatory mechanisms of age-induced defects in OE regeneration remain largely unknown. Olfactory epithelium is a pseudo-stratified epithelial structure consisting mainly of Horizontal Basal Cells (HBC), spherical basal cells (GBC), supporting cells (SUS), olfactory Sensory Neurons (OSN), and the like. The olfactory function is mediated primarily by sensory neurons, which detect odors and transmit signals to the olfactory bulb and the advanced brain regions. Under steady state conditions, mitotically active GBCs continuously generate new neurons throughout life, while quiescent HBCs attached to the basal layer do not participate in maintaining normal tissue turnover. When severely damaged, HBC is activated and converted to GBC, which then differentiates into sensory neurons and non-sensory support cells, HBC can also directly produce support cells, thereby completing epithelial regeneration. However, in the inflammatory aging state, accumulation of immune cells releases more inflammatory factors, resulting in an imbalance in the inflammatory microenvironment. This imbalance impedes basal cell activation and differentiation, leading to reduced neuronal turnover and, in turn, to dysolfaction. In the process of inflammatory aging, T cells play a key role in tissue renewal, homeostasis and repair, and a variety of signaling pathways and inflammatory factors are involved in tissue aging, such as IL-6, tnfα, ifnα, etc. Th17 cells are a key subset of T helper cells, characterized by secretion of interleukin-17, and play a unique "double sword" role in the immune system. Th17 cells are mainly present in mucosal barrier sites such as intestinal tract, lung, spleen, skin, etc., and are used for defending extracellular bacteria and fungi, maintaining epithelial barrier integrity, recruiting neutrophils, etc. When Th17 cells are overreacted or deregulated, severe inflammatory and autoimmune diseases can result, and the Th17 pathway has become the "golden target" for development of autoimmune disease drugs due to their clear pathogenic effects. Since the pathogenesis of dysosmia is unknown, no targeted treatment method exists at present, and common means comprise drug treatment based on corticosteroid hormone, olfactory training and surgical treatment. Disease models for research of the pathogenesis of dysolfactory disorders remain lacking, and are mainly dependent on animal models, including Lipopolysaccharide (LPS) -induced dysolfactory disorder models, methylindole-induced olfactory injury models, neuronal transduction defects, and inflammatory factor-related transgenic mice. The in vitro model of the dysosmia disease is not reported yet, if an effective cell model is established to replace an animal model, the animal consumption can be greatly reduced to improve animal welfare, and the research progress of the pathogenesis of the dysosmia can be accelerated by the characteristics of low cost, easy maintenance and convenient experimental operation. Organoids are three-dimensional structures developed from stem cells that accurately reflect tissue structure and cellular composition. Organoids may be derived from two types of stem cells, including pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells), and organ-specific adult stem cells. With both of the above cells, scientists have successfully established organoids of a variety of vital organs such as brain, stomach, intestine, lung, liver and kidney, usually from healthy or diseased tissue, as a good model for studying tissue homeostasis and regeneration and disease occurrence mechanisms. The olfactory epithelium has tissue specific stem cells, and is an ideal tissue source for in vitro cultivation of organoids. The organoids can maintain genetic stability while being stably passaged, and the development of the technology opens up a new way for testing and developing preclinical treatmen