[Analysis of success rate of organoid construction of nasopharyngeal carcinoma by first-day suspension method].

Objective: To investigate the efficacy of the first-day suspension method for improving the success rate of construction of nasopharyngeal carcinoma-patient derived organoids (NPC-PDO). Methods: The tumor samples of 14 nasopharyngeal carcinoma(NPC) patients, i.e.,13 males and 1 female, with a mean age of 43.0±12.0 years old, were collected from the Affiliated Tumor Hospital of Guangxi Medical University and the First Affiliated Hospital of Guangxi Medical University from January 2022 to July 2022. The tumor samples of 3 patients were digested into single cell suspension and divided into 2 groups, for comparing the efficacy of NPC-PDO construction by the direct inoculation method and the first-day suspension method. The remaining 11 patients were randomized to receive either the direct inoculation method or the first-day suspension method for NPC-PDO construction. The diameter and the number of spheres of NPC-PDO constructed by the two methods were compared by optical microscope; the 3D cell viability detection kit was used to compare the cell viability; the survival rates were compared by trypan blue staining; the success rates of the two construction methods were compared; the number of cases which could be successfully passaged for more than 5 generations and were consistent with the original tissue by pathological examination was counted; and the dynamic changes of cells in suspension overnight were observed by live cell workstation. The independent sample t-test was applied to compare the measurement data of the two groups, and the chi-square test was used to compare the classification data. Results: Compared with the direct inoculation, the diameter and the number of spheres of NPC-PDO constructed by the first-day suspension method were increased, with a higher cell activity, and the success rate of construction was obviously improved (80.0% vs 16.7%, χ2=4.41, P<0.05). In the suspension state, some of the cells aggregated and increased their ability to proliferate. Conclusion: The first-day suspension method can improve the success rate of NPC-PDO construction, especially for those whose original tumor sample size is small.目的： 验证首日悬浮法对于提高鼻咽癌类器官（nasopharyngeal carcinoma-patient derived organoids，NPC-PDO）构建成功率的有效性。方法： 收集2022年1月至7月广西医科大学附属肿瘤医院和广西医科大学第一附属医院初诊的14例鼻咽癌（nasopharyngeal carcinoma，NPC）患者样本，男性患者13例，女性1例，年龄（43.0±12.0）岁。取其中3例患者的肿瘤样本消化成单细胞悬液后平均分成2组，同时采用直接接种法和首日悬浮法对比NPC-PDO的构建效果；取余下11例患者样本随机采用直接接种法或首日悬浮法进行NPC-PDO的构建。通过光学显微镜对比两种方法构建的NPC-PDO直径大小和成球数量；采用3D细胞活力检测试剂盒对比细胞活力；采用台盼蓝染色计算细胞存活率；对可成功传代5代以上且经病理学检测与来源组织一致的病例数进行统计；采用活细胞工作站观察悬浮过夜状态下细胞的动态变化情况。采用独立样本t检验对2组计量资料进行比较，采用卡方检验对分类资料进行比较。结果： 相比于直接接种法，首日悬浮法构建的NPC-PDO直径和成球数都增加，且保持更高的细胞活性，同时，构建成功率明显提高（80.0%比16.7%，χ2=4.41，P<0.05），差异均有统计学意义。悬浮状态下部分细胞发生聚集现象并提高了增殖能力。结论： 首日悬浮法可以在样本量较少时提高NPC-PDO的培养成功率。.