小鼠胃类器官培养中真菌污染的鉴定与控制

Identification and control of fungal contamination in mouse gastric organoids culture

目的探讨小鼠胃类器官培养过程中污染真菌的种类及其控制措施。方法利用小鼠胃成体干细胞培养胃类器官;分离小鼠胃类器官培养过程中产生的污染物,革兰染色观察镜下形态;将污染物分离培养,并进行真菌ITS基因测序分析;梯度浓度试验探究可抑制污染物的药物及最适浓度。结果小鼠胃类器官培养过程中的污染物为出芽短梗霉菌;500μg/L的两性霉素B可有效抑制污染的出芽短梗霉菌;控制真菌污染后成功构建小鼠胃类器官,第9 d时检测类器官仍具有活性。结论出芽短梗霉菌可引起小鼠胃类器官培养过程中的真菌污染,通过添加两性霉素B控制真菌污染可成功构建小鼠胃类器官。

Objective To explore the specific causes and solutions of common pollution in mouse gastric organoids culture.Methods Gastric organoids were cultured from mouse gastric stem cells.The sediment that obtained by centrifugation was stained with gram to observe the morphology under a microscope.The pollutants were isolated and cultured and the FUNGI were sequenced by ITS.The selection experiment of action concentration was conducted to explore the drugs and the optimal concentration that could inhibit the pollutants.Results In the process of mouse gastric organoids culture,the contaminant was Aureobasidium pullulans,and they were spore brachium.Amphotericin B at 500μg/L could effectively inhibit the contamination of A.pullulans.After controlling the fungal contamination,the gastric organoid of mice were successfully constructed,and the gastric organoids were still active on the ninth day.Conclusion A.pullulans can cause fungal contamination in the process of gastric organoid culture in mice.Then mice gastric organoids were successfully constructed by adding amphotericin B to control fungal contamination..