RESULTS: The hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.
CONCLUSIONS: In summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.
结果:利用hADSCs球体和hADSCs明胶甲基丙烯酸酯(GelMA)微球生产三维培养的电动汽车,分别对应于hADSCs球体-EV和hADSCs微球-EV。与hADSC微球-EV相比,hADSC球体-EV表现出优异的生产和功能分子负载。8种miRNA(即hsa-miR-486-5p,hsa-miR-423-5p,hsa-miR-92a-3p,hsa-miR-122-5p,hsa-miR-223-3p,hsa-miR-320a,hsa-miR-126-3p,和hsa-miR-25-3p)以及hADSC球体-EV中hsa-miR-146b-5p的下调显示出可能通过整合的调节机制改善剩余耳软骨细胞的命运并促进软骨形成的潜力。此外,开发了一种快速和创新的管道,用于从hADSC球体的三维动态培养物中分离软骨细胞归巢肽修饰的EV(CHP-EV)。CHP-EV是通过在外泌体表面蛋白LAMP2B的N末端遗传融合CHP而产生的。用波浪运动培养CHP+LAMP2B转染的hADSCs球体,以促进CHP-EV的分泌。使用收获方法来实现CHP-EV的时间依赖性收集。该管道易于设置,可快速用于CHP-EV的隔离。与未标记的电动汽车相比,CHP-EV穿透生物材料支架并特异性地将治疗性miRNA递送至剩余的耳软骨细胞。功能上,CHP-EV对促进细胞增殖具有重要作用,减少M1巨噬细胞浸润的微环境中剩余的耳软骨细胞的细胞凋亡并增强软骨形成。
结论:总之,开发了一种创新的管道,从hADSC球体的三维动态培养中获得CHP-EV。该管道可以定制以增加电动汽车产量和功能分子负载,满足调节M1巨噬细胞浸润微环境中剩余的耳软骨细胞命运的要求。