The availability of viable human tissues is critical to support translational research focused on personalized care. Most studies have relied on fresh frozen or formalin-fixed paraffin-embedded tissues for histopathology, genomics, and proteomics. Yet, basic, translational, and clinical research downstream assays such as tumor progression/invasion, patient-derived xenograft, organoids, immunoprofiling, and vaccine development still require viable tissue, which are time-sensitive and rare commodities. We describe the generation of two-dimensional (2D) and three-dimensional (3D) cultures to validate a viable freeze cryopreservation technique as a standard method of highest quality specimen preservation. After surgical resection, specimens were minced, placed in CryoStor^™ media, and frozen using a slow freezing method (−1°C/min in −80°C) for 24 hours and then stored in liquid nitrogen. After 15–18 months, the tissues were thawed, dissociated into single-cell suspensions, and evaluated for cell viability. To generate primary 2D cultures, cells were plated onto Collagen-/Matrigel-coated plates. To develop 3D cultures (organoids), the cells were plated in reduced serum RPMI media on nonadherent plates or in Matrigel matrix. The epithelial nature of the cells was confirmed by using immunohistochemistry for cytokeratins. DNA and RNA isolation was performed using QIAGEN AllPrep kits. We developed primary lines (2D and 3D) of colon, thyroid, lung, renal, and liver cancers that were positive for cytokeratin staining. 3D lines were developed from the same cohort of tumor types in both suspended media and Matrigel matrix. Multiple freeze-thaw cycles did not significantly alter the viability and growth of 2D and 3D lines. DNA/RNA recovery was similar to its fresh frozen cohort. In this study, we validated 2D and 3D tissue cultures as methods to corroborate the feasibility of viable cryopreservation of tumor tissue. This proof-of-principle study, if more widely implemented, should improve accessibility of human viable tumor tissue/cells in a time-independent manner for many basic, preclinical, and translational assays.

中文翻译：

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人体组织的冷冻保存：活组织，细胞系和类器官发育的可再生资源。

可行的人体组织的可用性对于支持专注于个性化护理的转化研究至关重要。大多数研究依靠新鲜的冷冻或福尔马林固定石蜡包埋的组织进行组织病理学，基因组学和蛋白质组学研究。然而，基础的，转化的和临床研究的下游测定，例如肿瘤进展/侵袭，患者来源的异种移植，类器官，免疫分布图和疫苗开发，仍然需要活组织，它们是时间敏感的稀有商品。我们描述了二维（2D）和三维（3D）文化的产生，以验证可行的冷冻冷冻保存技术，作为最高质量标本保存的标准方法。手术切除后，将样品切碎，放入CryoStor ^™培养基，并使用慢速冷冻方法（在-80°C下以-1°C / min的速度）冷冻24小时，然后保存在液氮中。15–18个月后，将组织融化，分离成单细胞悬液，并评估细胞活力。为了产生初级2D培养物，将细胞接种到胶原/基质胶包被的平板上。为了开发3D培养物（类器官），将细胞接种在非粘附平板或Matrigel基质中的降低的血清RPMI培养基中。通过使用细胞角蛋白的免疫组织化学证实了细胞的上皮性质。使用QIAGEN AllPrep试剂盒进行DNA和RNA分离。我们开发了结肠角膜癌，甲状腺癌，肺癌，肾癌和肝癌的主要细胞系（2D和3D），这些细胞系对细胞角蛋白染色呈阳性。在悬浮培养基和基质胶基质中，从相同类型的肿瘤队列中开发了3D细胞系。多个冻融循环并没有显着改变2D和3D线的生存能力和生长。DNA / RNA的回收与其新鲜的冷冻队列相似。在这项研究中，我们验证了2D和3D组织培养作为证实肿瘤组织可行冷冻保存可行性的方法。如果进行更广泛的实施，这项原理验证研究应以与时间无关的方式改善人类生存的肿瘤组织/细胞的可及性，以用于许多基础，临床前和转化试验。