Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.
Long-term culture, genetic manipulation and xenotransplantation of human normal and breast cancer organoids.
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DOI：
10.1038/s41596-020-00474-1
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发表时间：
2021-04
期刊：
影响因子：
14.8
通讯作者：
Rios AC
中科院分区：
文献类型：
--
作者：
Dekkers JF;van Vliet EJ;Sachs N;Rosenbluth JM;Kopper O;Rebel HG;Wehrens EJ;Piani C;Visvader JE;Verissimo CS;Boj SF;Brugge JS;Clevers H;Rios AC
类器官技术彻底改变了对人体器官发育、疾病和针对个体的治疗反应的研究。虽然详细的方案可用于从各种器官产生和长期繁殖人类类器官，但对于乳腺组织缺乏这种方法。在这里，我们提供了一种优化的、高度通用的方案，用于长期培养来自正常人乳腺组织或乳腺癌（BC）组织的类器官，以及一组45个生物库样本的培养条件，包括涵盖所有主要疾病亚型（三阴性、雌激素受体阳性/孕激素受体阳性和人表皮生长受体2阳性）的BC类器官。此外，我们还提供了通过Lipofectamine 2000、电穿孔或慢病毒进行遗传操作的方法，以及随后的类器官选择和克隆培养。最后，我们介绍了一种优化的小鼠原位类器官移植方法，其中包括注射类器官和雌激素颗粒，无需手术。从组织碎片衍生类器官直到第一次分裂需要7-21天;遗传操作的克隆类器官培养物的产生需要14-21天;并且用于异种移植的类器官扩增需要>4周。
Organoid technology has revolutionized the study of human organ development, disease and therapy response tailored to the individual. Although detailed protocols are available for the generation and long-term propagation of human organoids from various organs, such methods are lacking for breast tissue. Here we provide an optimized, highly versatile protocol for long-term culture of organoids derived from either normal human breast tissues or breast cancer (BC) tissues, as well as culturing conditions for a panel of 45 biobanked samples, including BC organoids covering all major disease subtypes (triple-negative, estrogen receptor-positive/progesterone receptor-positive and human epidermal growth receptor 2-positive). Additionally, we provide methods for genetic manipulation by Lipofectamine 2000, electroporation or lentivirus and subsequent organoid selection and clonal culture. Finally, we introduce an optimized method for orthotopic organoid transplantation in mice, which includes injection of organoids and estrogen pellets without the need for surgery. Organoid derivation from tissue fragments until the first split takes 7–21 d; generation of genetically manipulated clonal organoid cultures takes 14–21 d; and organoid expansion for xenotransplantation takes >4 weeks.
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影响因子：
14.8
作者：
Cattaneo CM;Dijkstra KK;Fanchi LF;Kelderman S;Kaing S;van Rooij N;van den Brink S;Schumacher TN;Voest EE
通讯作者：
Voest EE
影响因子：
64.8
作者：
Nik-Zainal S;Davies H;Staaf J;Ramakrishna M;Glodzik D;Zou X;Martincorena I;Alexandrov LB;Martin S;Wedge DC;Van Loo P;Ju YS;Smid M;Brinkman AB;Morganella S;Aure MR;Lingjærde OC;Langerød A;Ringnér M;Ahn SM;Boyault S;Brock JE;Broeks A;Butler A;Desmedt C;Dirix L;Dronov S;Fatima A;Foekens JA;Gerstung M;Hooijer GK;Jang SJ;Jones DR;Kim HY;King TA;Krishnamurthy S;Lee HJ;Lee JY;Li Y;McLaren S;Menzies A;Mustonen V;O'Meara S;Pauporté I;Pivot X;Purdie CA;Raine K;Ramakrishnan K;Rodríguez-González FG;Romieu G;Sieuwerts AM;Simpson PT;Shepherd R;Stebbings L;Stefansson OA;Teague J;Tommasi S;Treilleux I;Van den Eynden GG;Vermeulen P;Vincent-Salomon A;Yates L;Caldas C;van't Veer L;Tutt A;Knappskog S;Tan BK;Jonkers J;Borg Å;Ueno NT;Sotiriou C;Viari A;Futreal PA;Campbell PJ;Span PN;Van Laere S;Lakhani SR;Eyfjord JE;Thompson AM;Birney E;Stunnenberg HG;van de Vijver MJ;Martens JW;Børresen-Dale AL;Richardson AL;Kong G;Thomas G;Stratton MR
通讯作者：
Stratton MR
影响因子：
64.5
作者：
Sachs, Norman;de Ligt, Joep;Clevers, Hans
通讯作者：
Clevers, Hans
影响因子：
82.9
作者：
Kopper, Oded;de Witte, Chris J.;Clevers, Hans
通讯作者：
Clevers, Hans
影响因子：
50.3
作者：
Neve, Richard M.;Chin, Koei;Gray, Joe W.
通讯作者：
Gray, Joe W.