当前位置: X-MOL 学术Nat. Protoc. › 论文详情
Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)
Generation and biobanking of patient-derived glioblastoma organoids and their application in CAR T cell testing
Nature Protocols ( IF 14.8 ) Pub Date : 2020-11-09 , DOI: 10.1038/s41596-020-0402-9
Fadi Jacob 1, 2, 3 , Guo-Li Ming 1, 4, 5, 6 , Hongjun Song 1, 4, 5, 7, 8
Affiliation  

Glioblastoma tumors exhibit extensive inter- and intratumoral heterogeneity, which has contributed to the poor outcomes of numerous clinical trials and continues to complicate the development of effective therapeutic strategies. Most in vitro models do not preserve the cellular and mutational diversity of parent tumors and often require a lengthy generation time with variable efficiency. Here, we describe detailed procedures for generating glioblastoma organoids (GBOs) from surgically resected patient tumor tissue using a chemically defined medium without cell dissociation. By preserving cell-cell interactions and minimizing clonal selection, GBOs maintain the cellular heterogeneity of parent tumors. We include details of how to passage and cryopreserve GBOs for continued use, biobanking and long-term recovery. In addition, we describe procedures for investigating patient-specific responses to immunotherapies by co-culturing GBOs with chimeric antigen receptor (CAR) T cells. It takes ~2–4 weeks to generate GBOs and 5–7 d to perform CAR T cell co-culture using this protocol. Competence with human cell culture, tissue processing, immunohistology and microscopy is required for optimal results.



中文翻译:

源自患者的胶质母细胞瘤类器官的生成和生物库及其在 CAR T 细胞测试中的应用

胶质母细胞瘤肿瘤表现出广泛的肿瘤间和肿瘤内异质性,这导致了许多临床试验的不良结果,并继续使有效治疗策略的开发复杂化。大多数体外模型不保留亲本肿瘤的细胞和突变多样性,并且通常需要较长的生成时间和可变的效率。在这里,我们描述了使用化学定义的培养基从手术切除的患者肿瘤组织中生成胶质母细胞瘤类器官 (GBO) 的详细程序,而无需细胞解离。通过保留细胞间相互作用和最小化克隆选择,GBOs 保持了母体肿瘤的细胞异质性。我们包括如何通过和冷冻保存 GBO 以供继续使用、生物库和长期恢复的详细信息。此外,我们描述了通过将 GBO 与嵌合抗原受体 (CAR) T 细胞共培养来研究患者对免疫疗法的特异性反应的程序。使用该协议生成 GBO 需要约 2-4 周,使用该协议执行 CAR T 细胞共培养需要 5-7 天。获得最佳结果需要具备人类细胞培养、组织处理、免疫组织学和显微镜检查的能力。

更新日期:2020-11-09
down
wechat
bug