Wang K, Yu Y, Han R, Wang X, Zhao Y, Tang H, Li G
Department of Otolaryngology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Jun 20;42(6):868-877. doi: 10.12122/j.issn.1673-4254.2022.06.10.
To establish a culture system for human nasal mucosal organoids with controllable differentiation to reproduce the structure and function of the source tissue through staged expansion-differentiation culture.
Fresh samples of surgically resected middle turbinate and nasal polyp tissues were collected, from which the nasal mucosa epithelial cells were isolated by enzymatic digestion and filtration for continuous culture at the air-liquid interface for expansion (EO group) or staged culture for expansion and differentiation (DO group). Immunohistochemical staining was used to characterize the structure, cellular composition and ciliary function of nasal mucosal organoids in the two groups. The secretion function of the differentiated nasal mucosal organoids in DO group was evaluated using PAS staining.
Both of the two organoid culture systems yielded vacuolar or solid spherical 3D organoids, and their diameters increased progressively with time. On day 16 of culture, more vacuolar organoids occurred in DO group, while more solid spherical organoids were seen in EO group, and the proportion of vacuoles was significantly greater in DO group than in EO group [(54.67±13.26)% (21.67±8.57)%, < 0.05]. Short tandem repeat (STR) test of the nasal mucosal organoids and the source tissue showed a 100% match between them. On day 21 of culture, scanning and transmission electron microscopy of the nasal mucosal organoids identified ultrastructure of cilia in DO group and short villi structure in most of the organoids in EO group. Immunohistochemical staining showed positivity for P63 (basal cells), β-tubulin (ciliated columnar cells), and MUC5AC (goblet cells) in the organoids. Compared with those in EO group, the organoids in DO group showed significantly greater percentages of ciliated cells [(7.95±1.81)% (27.04±5.91)%, < 0.05] and goblet cells [(14.46±0.93)% (39.85±5.43)%, < 0.05) with a similar percentage of basal cells [(56.91±14.12)% (53.42±15.77)%, > 0.05]. The differentiated nasal mucosal organoids in DO group were positively stained for glycogen.
The staged expansion-differentiation culture method allows more stable and prolonged growth of the cultured cells to produce organoids with controllable differentiation closely resembling the morphological structure and functions (ciliary function and secretory function) of the source tissue.
建立一种具有可控分化的人鼻黏膜类器官培养体系，通过分阶段的扩增 - 分化培养来重现源组织的结构和功能。
收集手术切除的中鼻甲和鼻息肉组织的新鲜样本，通过酶消化和过滤分离鼻黏膜上皮细胞，在气液界面进行连续培养以扩增（EO组）或分阶段培养以扩增和分化（DO组）。采用免疫组织化学染色来表征两组鼻黏膜类器官的结构、细胞组成和纤毛功能。使用PAS染色评估DO组中分化的鼻黏膜类器官的分泌功能。
两种类器官培养体系均产生了空泡状或实心球形的三维类器官，其直径随时间逐渐增加。培养第16天，DO组出现更多空泡状类器官，而EO组可见更多实心球形类器官，且DO组的空泡比例显著高于EO组[（54.67±13.26）% 对（21.67±8.57）%，P < 0.05]。对鼻黏膜类器官和源组织进行短串联重复序列（STR）检测，结果显示二者完全匹配。培养第21天，对鼻黏膜类器官进行扫描电镜和透射电镜观察，发现DO组有纤毛超微结构，EO组大多数类器官有短绒毛结构。免疫组织化学染色显示类器官中P63（基底细胞）、β - 微管蛋白（纤毛柱状细胞）和MUC5AC（杯状细胞）呈阳性。与EO组相比，DO组类器官的纤毛细胞百分比[（7.95±1.81）% 对（27.04±5.91）%，P < 0.05]和杯状细胞百分比[（14.46±0.93）% 对（39.85±5.43）%，P < 0.05]显著更高，而基底细胞百分比相似[（56.91±14.12）% 对（53.42±15.77）%，P > 0.05]。DO组中分化的鼻黏膜类器官糖原染色呈阳性。
分阶段的扩增 - 分化培养方法能使培养的细胞更稳定、更持久地生长，从而产生具有可控分化的类器官，其形态结构和功能（纤毛功能和分泌功能）与源组织极为相似。
