Three-dimensional cell culture for the study of nasal polyps

Abstract Objectives: Three-dimensional (3D) cell cultures have many applications such as stem cell biology research, new drug discovery, cancer, and Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). This disease is characterized by a significant impact on quality of life and productivity. The diversity of factors that act in the progression of CRSwNP point to the creation of a cell culture model that allows the integration of different cell types with extracellular matrix. This work aimed to create a cell culture model in 3 dimensions (spheroids) for the study of Nasal Polyposis. Methods: Nasal polyp tissue from patients diagnosed with CRSwNP was mechanically dissociated using tweezers and a scalpel and the solution containing cells and small aggregates of nasal polyps was transferred to a Petri dish containing 5 mL of culture medium at the concentration of 106 cells/mL. Results: The spheroids were cultivated for 20 days, fixed and analyzed using confocal microscopy. In a 3D culture environment, the spheroids were formed both by clustering cells and from small tissue fragments. In the cultures analyzed, the ciliary beat was present from the dissociation of the cells up to 20 days in culture. Conclusion: Our findings also point to these characteristics showing the environment generated in our study, the cells remained differentiated for a longer time and with ciliary beating. Thus, this work shows that nasal polyp-derived cells can be maintained in a 3D environment, enabling better strategies for understanding CRSwNP in situations similar to those found in vivo. Level of evidence: Laboratory studies.

摘要

### 研究目标
三维（3D）细胞培养具备诸多应用场景，涵盖干细胞生物学研究、新药研发、癌症及伴鼻息肉的慢性鼻鼻窦炎（Chronic Rhinosinusitis with Nasal Polyps, CRSwNP）领域。该疾病会对患者的生活质量与生产力造成显著负面影响，而CRSwNP进展过程中涉及多种复杂调控因素，因此亟需构建可整合不同细胞类型与细胞外基质的细胞培养模型。本研究旨在构建三维（3D）球体（spheroids）细胞培养模型，用于鼻息肉病的相关研究。

### 研究方法
从确诊为CRSwNP的患者体内获取鼻息肉组织，采用镊子与手术刀进行机械解离，将包含细胞与鼻息肉小聚集体的细胞悬液转移至盛有5毫升培养液的培养皿中，调整细胞浓度至1×10⁶个细胞/毫升。

### 研究结果
细胞球体培养周期为20天，经固定处理后采用共聚焦显微镜（confocal microscopy）进行分析。在三维培养环境中，细胞球体既可由细胞集群聚集形成，亦可源自小组织片段。在所分析的培养体系中，从细胞解离阶段直至培养至第20天，均可观察到纤毛摆动现象。

### 研究结论
本研究结果进一步验证了上述特性，表明本研究构建的培养环境可使细胞长时间维持分化状态，并保留纤毛摆动功能。综上，本研究证实鼻息肉来源的细胞可在三维培养环境中稳定存活，为在更贴近体内生理的条件下探究CRSwNP提供了更具优势的研究策略。

### 证据等级
实验室研究（Laboratory studies）