PDF(Pubmed)
Abstract：
Creating model systems that replicate in vivo tissues is crucial for understanding complex biological pathways like drug response and disease progression. Three-dimensional (3D) in vitro models, especially multicellular spheroids (MCSs), offer valuable insights into physiological processes. However, generating MCSs at scale with consistent properties and efficiently recovering them pose challenges. We introduce a workflow that automates large-scale spheroid production and enables parallel harvesting into individual wells of a microtiter plate. Our method, based on the hanging-drop technique, utilizes a non-contact dispenser for dispensing nanoliter droplets of a uniformly mixed-cell suspension. The setup allows for extended processing times of up to 45 min without compromising spheroid quality. As a proof of concept, we achieved a 99.3% spheroid generation efficiency and maintained highly consistent spheroid sizes, with a coefficient of variance below 8% for MCF7 spheroids. Our centrifugation-based drop transfer for spheroid harvesting achieved a sample recovery of 100%. We successfully transferred HT29 spheroids from hanging drops to individual wells preloaded with collagen matrices, where they continued to proliferate. This high-throughput workflow opens new possibilities for prolonged spheroid cultivation, advanced downstream assays, and increased hands-off time in complex 3D cell culture protocols.
摘要：
创建在体内组织中复制的模型系统对于理解复杂的生物途径（如药物反应和疾病进展）至关重要。三维(3D)体外模型，尤其是多细胞球体（MCSs），提供对生理过程的宝贵见解。然而,在规模上生成具有一致属性的MCS并有效地恢复它们构成了挑战。我们引入了一种自动化大规模球体生产的工作流程，并允许平行收获到微量滴定板的单个孔中。我们的方法,基于悬垂技术，利用非接触式分配器分配均匀混合的细胞悬浮液的纳升液滴。该设置允许长达45分钟的延长处理时间，而不影响球体质量。作为概念的证明，我们实现了99.3％的球体生成效率，并保持高度一致的球体尺寸，MCF7球体的变异系数低于8%。我们基于离心的液滴转移用于球体收获，样品回收率为100％。我们成功地将HT29球体从悬滴转移到预装胶原蛋白基质的各个孔中，在那里他们继续扩散。这种高通量工作流程为延长球体培养开辟了新的可能性，先进的下游化验，在复杂的3D细胞培养方案中增加了移交时间。
