Landegger Lukas D, Sagers Jessica E, Dilwali Sonam, Fujita Takeshi, Sahin Mehmet I, Stankovic Konstantina M
Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear; Department of Otolaryngology, Harvard Medical School; Department of Otolaryngology, Vienna General Hospital, Medical University of Vienna.
Eaton Peabody Laboratories, Department of Otolaryngology, Massachusetts Eye and Ear; Program in Speech and Hearing Bioscience and Technology, Harvard Medical School.
J Vis Exp. 2017 Jun 20(124):55827. doi: 10.3791/55827.
Vestibular schwannomas are the most common neoplasms of the cerebellopontine angle, making up 6-8% percent of all intracranial growths. Though these tumors cause sensorineural hearing loss in up to 95% of affected individuals, the molecular mechanisms underlying this hearing loss remain elusive. This article outlines the steps established in our laboratory to facilitate the collection and processing of various primary human tissue samples for downstream research applications integral to the study of vestibular schwannomas. Specifically, this work describes a unified methodological framework for the collection, processing, and culture of Schwann and schwannoma cells from surgical samples. This is integrated with parallel processing steps now considered essential for current research: the collection of tumor and nerve secretions, the preservation of RNA and the extraction of protein from collected tissues, the fixation of tissue for the preparation of sections, and the exposure of primary human cells to adeno-associated viruses for application to gene therapy. Additionally, this work highlights the translabyrinthine surgical approach to collect this tumor as a unique opportunity to obtain human sensory epithelium from the inner ear and perilymph. Tips to improve experimental quality are provided and common pitfalls highlighted.
前庭神经鞘瘤是桥小脑角最常见的肿瘤,占所有颅内肿瘤的6 - 8%。尽管这些肿瘤在高达95%的受累个体中会导致感音神经性听力损失,但其导致听力损失的分子机制仍不清楚。本文概述了我们实验室建立的步骤,以促进收集和处理各种原发性人类组织样本,用于前庭神经鞘瘤研究不可或缺的下游研究应用。具体而言,这项工作描述了一个统一的方法框架,用于从手术样本中收集、处理和培养雪旺细胞和神经鞘瘤细胞。这与目前研究认为必不可少的并行处理步骤相结合:收集肿瘤和神经分泌物、保存RNA以及从收集的组织中提取蛋白质、固定组织以制备切片,以及将原代人类细胞暴露于腺相关病毒以应用于基因治疗。此外,这项工作强调了经迷路手术方法收集这种肿瘤,这是一个从内耳和外淋巴中获取人类感觉上皮的独特机会。提供了提高实验质量的提示,并突出了常见的陷阱。