慢性淋巴细胞性白血病（CLL）细胞高度依赖微环境细胞和信号。淋巴结（LN）是体内CLL增殖和对化疗药物和靶向药物抗性发展的关键场所。我们提出了一个新模型，该模型融入了CLL LN的关键方面，使得能够在保护性生境中研究CLL细胞。我们描述了一个三维（3D）离体培养系统，使用超低附着力板创建从外周血中获得的CLL细胞的球体。从淋巴结样本中观察到的CLL：T细胞比例出发，CLL通过直接刺激和/或间接通过T细胞诱导活化。与二维培养相比，三维培养促进了依赖T细胞的CLL增殖，并能够扩增七周，包括在几周的培养后形成类似滤泡结构。这个模型可以进行高通量药物筛选，我们以Btk抑制剂反应、venetoclax抗性和T细胞介导的细胞毒性为例。总结地说，我们首次呈现了一个仿制LN的体外三维培养模型，用于原发性CLL，可以进行实时药物筛选、动力学生长评估和空间定位等输出。这是第一个在肿瘤微环境的背景下允许测试venetoclax和Bruton酪氨酸激酶抑制剂反应和抗性的体外CLL系统，从而开启了临床实用应用的新可能性。版权©2023作者。由 Wolters Kluwer Health, Inc. 代表欧洲血液学会出版。

Chronic lymphocytic leukemia (CLL) cells are highly dependent on microenvironmental cells and signals. The lymph node (LN) is the critical site of in vivo CLL proliferation and development of resistance to both chemotherapy and targeted agents. We present a new model that incorporates key aspects of the CLL LN, which enables investigation of CLL cells in the context of a protective niche. We describe a three-dimensional (3D) in vitro culture system using ultra-low attachment plates to create spheroids of CLL cells derived from peripheral blood. Starting from CLL:T cell ratios as observed in LN samples, CLL activation was induced by either direct stimulation and/or indirectly via T cells. Compared with two-dimensional cultures, 3D cultures promoted CLL proliferation in a T cell-dependent manner, and enabled expansion for up to 7 weeks, including the formation of follicle-like structures after several weeks of culture. This model enables high-throughput drug screening, of which we describe response to Btk inhibition, venetoclax resistance, and T cell-mediated cytotoxicity as examples. In summary, we present the first LN-mimicking in vitro 3D culture for primary CLL, which enables readouts such as real-time drug screens, kinetic growth assays, and spatial localization. This is the first in vitro CLL system that allows testing of response and resistance to venetoclax and Bruton's tyrosine kinase inhibitors in the context of the tumor microenvironment, thereby opening up new possibilities for clinically useful applications.Copyright © 2023 the Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the European Hematology Association.