悬浮培养构建源于胃癌患者的类器官及鉴定

Establishment and identification of organoid derived from patients with gastric cancer based on suspension culture

目的探索悬浮培养构建源于胃癌患者类器官的可行性。方法选取2021年7–12月期间于兰州大学第一医院行手术切除的3例胃癌患者的新鲜胃癌组织,将新鲜组织经混合消化酶消化后制成细胞悬液,将此细胞悬液接种于超低吸附板中悬浮培养类器官,待类器官生长到一定大小后进行传代、冻存,然后在倒置显微镜下观察胃癌类器官的形成过程,再通过苏木精-伊红和免疫组织化学染色观察类器官与原发肿瘤的一致性。结果本研究中有1例患者成功培养出可传代、冻存的类器官。在倒置显微镜下见类器官初始时(培养至第5天时)呈球形结构,至第10天时见类器官逐渐呈短棒状结构,至第15天时出现分枝样改变,至第20天时形成不规则的腺管样结构。苏木精-伊红染色结果显示类器官与原发胃癌组织的结构高度相似,免疫组织化学染色结果显示在类器官及它对应的原发胃癌组织中低分子量细胞角蛋白、p53、Ki67表达情况基本相同,即在类器官及原发胃癌组织中低分子量细胞角蛋白、p53均呈阳性表达,Ki67均呈高比例(阳性率约为70%)表达。结论本研究初步研究结果提示,采用悬浮培养可以构建出在组织和细胞层面与原发胃癌组织基本一致的类器官。

Objective To explore the feasibility of organoid culture derived from the patients with gastric cancer by suspension culture.Methods The fresh gastric cancer tissues of the 3 patients with gastric cancer were selected,which were digested with mixed enzymes and then made into cell suspensions,and were inoculated into ultra-low attachment plates to culture organoid by suspension.When the organoid growth was dense,the passage and freezing were carried out.The formation process of organoid was observed under the inverted microscope(IM).Further the consistency between the organoid and primary gastric cancer tissue was evaluated by hematoxylin-eosin(HE)and immunohistochemical(IHC)staining.Results In this study,an organoid that could be passaged and frozen was successfully established in one patient.The results under the IM showed that the organoid was initially spherical in shape(cultured on day 5),then gradually became short rod-shaped on day 10,showed a branching like change on day 15,and formed irregular glandular tubular structures on day 20.The structures between the organoids and primary gastric cancer tissues were highly similar by HE staining.The IHC staining results showed that the expressions of low molecular weight cytokeratin(CK-LMW),p53,and Ki67 in the organoid and its corresponding primary gastric cancer tissue were basically the same.That was,the CK-LMW and p53 expressions were positive in the organoid and primary gastric cancer tissue,and the Ki67 was highly expressed(with a positive rate of approximately 70%).Conclusion Based on the preliminary research results of this study,it suggests that the suspension culture can be used to establish organoid derived from patients with gastric cancer,which is in accordance with primary tumor tissue at the tissue and cellular levels.