Li Kaishu, Du Siyuan, Li Haichao, Li Zhaohui, Zhu Qihui, Peng Qian, Liao Baojian, Qi Ling
Department of Neurosurgery, the Affiliated Qingyuan Hospital (Qingyuan People's Hospital), Guangzhou Medical University, Qingyuan, Guangdong 511518, PR China.
Institute of Digestive Disease, the Affiliated Qingyuan Hospital (Qingyuan People's Hospital), Guangzhou Medical University, Qingyuan, Guangdong 511518, PR China.
Biochim Biophys Acta Gen Subj. 2025 Mar;1869(3):130752. doi: 10.1016/j.bbagen.2024.130752. Epub 2025 Jan 8.
Three-dimensional(3D) cell culture systems provide a larger space for cell proliferation, which is crucial for simulating cellular behavior and drug responses in the tumor microenvironment. In this study, we developed a novel 3D co-culture system for cell interactions, utilizing a commercialized bioreactor-microcarrier system. Mesenchymal stem cells (MSCs) were extracted via enzymatic digestion, and markers CD105 and CD31 were identified. Cell growth was observed using AO and immunofluorescence staining. No significant differences in Ki67 and GFAP expression were found between 2D and 3D cultures, though the 3D system offered more space for proliferation and reduced contact inhibition. Therefore, this 3D culture system may represent the tumor microenvironment more accurately than 2D cultures and will facilitate the investigation of the characteristics and functions of exosomes derived from this system. Exosomes are nanoscale vesicles that mediate intercellular communication by transferring molecules such as miRNAs between cells. Exosomes from 3D cultures were collected via ultra-high-speed centrifugation and characterized using nano-flow cytometry, transmission electron microscopy, and western blotting for markers CD9, Alix, and TSG101. PKH26 staining revealed peak exosome uptake by tumor cells at 24 h and complete metabolism by 72 h. Exosomes from 3D cultures inhibited GBM cell proliferation, migration, and invasion. Lastly, miRNA sequencing of exosomes was performed. This study emphasizes the importance of creating 3D co-culture systems to advance cancer research and offers a helpful tool for studying the complex cell interaction environment of GBM and other malignancies.
三维（3D）细胞培养系统为细胞增殖提供了更大的空间，这对于模拟肿瘤微环境中的细胞行为和药物反应至关重要。在本研究中，我们利用商业化的生物反应器 - 微载体系统开发了一种用于细胞相互作用的新型3D共培养系统。通过酶消化提取间充质干细胞（MSC），并鉴定标志物CD105和CD31。使用AO和免疫荧光染色观察细胞生长。尽管3D系统提供了更多的增殖空间并减少了接触抑制，但在2D和3D培养之间未发现Ki67和GFAP表达的显著差异。因此，这种3D培养系统可能比2D培养更准确地代表肿瘤微环境，并将有助于研究源自该系统的外泌体的特征和功能。外泌体是纳米级囊泡，通过在细胞之间转移诸如miRNA等分子来介导细胞间通讯。通过超速离心收集3D培养物中的外泌体，并使用纳米流式细胞术、透射电子显微镜和针对标志物CD9、Alix和TSG101的蛋白质印迹进行表征。PKH26染色显示肿瘤细胞在24小时时对外泌体的摄取达到峰值，并在72小时时完全代谢。3D培养物中的外泌体抑制了胶质母细胞瘤细胞的增殖、迁移和侵袭。最后，对外泌体进行了miRNA测序。本研究强调了创建3D共培养系统以推进癌症研究的重要性，并为研究胶质母细胞瘤和其他恶性肿瘤的复杂细胞相互作用环境提供了一个有用的工具。
