Background/Objectives:Cancer stem cells (CSCs) represent a minor yet critical subpopulation within tumors, endowed with self-renewal and differentiation capacities, and are implicated in tumor initiation, progression, metastasis, therapeutic resistance, and recurrence. Reliable in vitro functional assays to characterize CSCs are pivotal for the development of personalized oncology strategies. This study sought to establish and validate a microfluidic device (MD) platform for the enrichment, functional assessment, and therapeutic evaluation of CSC populations derived from experimental models and primary tumor samples.Methods:Murine (LM38LP) and human (BPR6) breast cancer cell lines were cultured within MDs to promote sphere formation. CSC enrichment was confirmed through the expression analysis of pluripotency-associated genes (Oct4, Sox2, Nanog, and CD44) by quantitative PCR (qPCR) and immunofluorescence. Sphere number, size, and gene expression profiles were quantitatively assessed before (control) and after chemotherapeutic exposure. To validate the MD platform against conventional scale, parallel experiments were performed in 12 well plates. To extend translational relevance, three primary canine tumor samples (solid thyroid carcinoma, simple tubular carcinoma, and reactive lymph node) were mechanically disaggregated and processed within MDs for CSC characterization.Results:The MD platform enabled the consistent enrichment of CSC populations, showing significant modulation of sphere growth parameters and stemness marker expression following chemotherapeutic treatment. Beyond its comparability with conventional culture, the MD also supported immunofluorescence staining and allowed real-time monitoring of individual cell growth. Sphere formation efficiency (SFE) and CSC marker expression were similarly demonstrated in primary veterinary tumor cultures, highlighting the device’s cross-species applicability.Conclusions:Microfluidic-based sphere assays represent a robust, reproducible, and scalable platform for the functional interrogation of CSC dynamics and therapeutic responses. This methodology holds great promise for advancing CSC-targeted therapies and supporting personalized oncology in both human and veterinary settings.
**背景/目的：** 肿瘤干细胞（CSCs）是肿瘤中占比微小但至关重要的亚群，具有自我更新和分化能力，参与肿瘤的发生、进展、转移、治疗抵抗及复发。建立可靠的体外功能实验以表征CSCs对于制定个体化肿瘤治疗策略至关重要。本研究旨在建立并验证一种微流控装置（MD）平台，用于富集、功能评估及治疗评价来自实验模型和原代肿瘤样本的CSC群体。 **方法：** 将小鼠（LM38LP）和人源（BPR6）乳腺癌细胞系在MD中培养以促进球体形成。通过定量PCR（qPCR）和免疫荧光分析多能性相关基因（Oct4、Sox2、Nanog和CD44）的表达，确认CSC富集情况。在化疗暴露前（对照组）和暴露后，定量评估球体数量、大小及基因表达谱。为验证MD平台相对于常规规模培养的可靠性，在12孔板中进行了平行实验。为拓展转化应用价值，对三例原代犬肿瘤样本（实体性甲状腺癌、单纯性管状癌和反应性淋巴结）进行机械解离，并在MD中处理以进行CSC表征。 **结果：** MD平台能够稳定富集CSC群体，并在化疗处理后显示出球体生长参数和干细胞标志物表达的显著调控。除与传统培养方法具有可比性外，MD还支持免疫荧光染色，并可实时监测单个细胞的生长。在原代兽医肿瘤培养物中同样证实了球体形成效率（SFE）和CSC标志物表达，凸显了该装置的跨物种适用性。 **结论：** 基于微流控的球体实验为CSC动态及治疗响应的功能研究提供了一个稳健、可重复且可扩展的平台。该方法在推动CSC靶向治疗及支持人类与兽医领域的个体化肿瘤学方面具有广阔前景。
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