Multicellular spheroids have served as a promising preclinical model for drug efficacy testing and disease modeling. Many microfluidic technologies, including those based on water–oil–water double emulsions, have been introduced for the production of spheroids. However, sustained culture and the in situ characterization of the generated spheroids are currently unavailable for the double emulsion-based spheroid model. This study presents a streamlined workflow, termed the double emulsion-pretreated microwell culture (DEPMiC), incorporating the features of (1) effective initiation of uniform-sized multicellular spheroids by the pretreatment of double emulsions produced by microfluidics without the requirement of biomaterial scaffolds; (2) sustained maintenance and culture of the produced spheroids with facile removal of the oil confinement; and (3) in situ characterization of individual spheroids localized in microwells by a built-in analytical station. Characterized by microscopic observations and Raman spectroscopy, the DEPMiC cultivated spheroids accumulated elevated lipid ordering on the apical membrane, similar to that observed in their Matrigel counterparts. Made possible by the proposed technological advancement, this study subsequently examined the drug responses of these in vitro-generated multicellular spheroids. The developed DEPMiC platform is expected to generate health benefits in personalized cancer treatment by offering a pre-animal tool to dissect heterogeneity from individual tumor spheroids.

多细胞球体已成为药物功效测试和疾病建模的有前途的临床前模型。许多微流体技术，包括基于水-油-水双乳液的技术，已被用于生产球体。然而，基于双乳液的球体模型目前无法对生成的球体进行持续培养和原位表征。本研究提出了一种简化的工作流程，称为双乳液预处理微孔培养 (DEPMiC)，结合了以下特点：(1) 通过预处理微流体产生的双乳液有效启动均匀大小的多细胞球体，无需生物材料支架；(2) 产生的球体的持续维护和培养，易于去除油封；(3) 通过内置分析站对定位在微孔中的单个球体进行原位表征。通过显微观察和拉曼光谱表征，DEPMiC 培养的球体在顶端膜上积累了升高的脂质排序，类似于在它们的 Matrigel 对应物中观察到的。由于拟议的技术进步，这项研究随后检查了这些体外生成的多细胞球体的药物反应。开发的 DEPMiC 平台有望通过提供一种动物前工具来剖析单个肿瘤球体的异质性，从而在个性化癌症治疗中产生健康益处。DEPMiC 培养的球体在顶端膜上积累了升高的脂质排序，类似于在它们的 Matrigel 对应物中观察到的。由于拟议的技术进步，这项研究随后检查了这些体外生成的多细胞球体的药物反应。开发的 DEPMiC 平台有望通过提供一种动物前工具来剖析单个肿瘤球体的异质性，从而在个性化癌症治疗中产生健康益处。DEPMiC 培养的球体在顶端膜上积累了升高的脂质排序，类似于在它们的 Matrigel 对应物中观察到的。由于拟议的技术进步，这项研究随后检查了这些体外生成的多细胞球体的药物反应。开发的 DEPMiC 平台有望通过提供一种动物前工具来剖析单个肿瘤球体的异质性，从而在个性化癌症治疗中产生健康益处。