肿瘤细胞治疗研究
英文原题：The electrical and chemical hypoxia-optimized (ECHO) bioreactor system enables scalable production of functionally preserved NK-exosomes validated in a syngeneic lung cancer model.
电化学缺氧优化（ECHO）生物反应器较传统培养将外泌体产量提高 10.1 0.4 倍（n = 3），且未改变颗粒粒径、zeta 电位或经典外泌体标志物表达。
自然杀伤（NK）细胞来源的外泌体是有前景的无细胞免疫治疗手段，但产量低且缺乏可扩展的制造策略，限制了其临床转化。本研究开发了一种改良型垂直轮式生物反应器，通过可控电刺激和模拟缺氧条件提高NK细胞外泌体产量，同时维持其生物学功能。与常规培养相比，电刺激与化学模拟缺氧优化（ECHO）生物反应器使外泌体产量提高10.1±0.4倍（n=3），且未改变颗粒大小、ζ电位或经典外泌体标志物表达。体外实验中，在1×10^10颗粒/mL条件下，NK-Exo和NK-Exo-CE分别使LLC细胞存活率降至37.8±3.1%和18.6±2.4%（p<0.001）；与紫杉醇联合后，存活率进一步降至9.4±1.8%（p<0.001）。在皮下LLC肿瘤模型中，NK-Exo和NK-Exo-CE处理组肿瘤体积分别降至912±104 mm³和824±96 mm³，对照组为1187±132 mm³（p<0.001）；截至第16天，NK-Exo-CE联合紫杉醇组肿瘤体积为348±57 mm³（较对照组约降低3.4倍；p<0.001）。流式细胞术免疫分析显示，联合治疗肿瘤中CD8 T细胞浸润增加（8.9±1.1%升至35.7±2.6%，p<0.001），调节性T细胞减少（28.7±1.8%降至10.4±1.2%，p<0.001）。Western blot分析证实，NK-Exo-CE联合紫杉醇组肿瘤中裂解型caspase-3、裂解型PARP、颗粒酶B、FasL和磷酸化NF-κB-p65升高，而Ki-67和PD-L1降低。总体而言，电刺激与缺氧模拟条件下的生物反应器可实现NK细胞外泌体的可扩展生产，同时不损害其理化完整性或抗肿瘤效力，有望解决细胞外囊泡疗法转化中的关键障碍。
Natural killer (NK) cell-derived exosomes represent promising cell-free immunotherapeutics, yet clinical translation is limited by low production yield and lack of scalable manufacturing strategies. Here, we developed a modified vertical-wheel bioreactor incorporating controlled electrical and hypoxia-mimetic conditioning to enhance NK-exosome production while preserving biological functionality. The Electrical and Chemical Hypoxia-Optimized (ECHO) Bioreactor increased exosome yield by 10.1 0.4 -fold compared to conventional culture (n = 3), without altering particle size, zeta potential, or canonical exosomal marker expression. In vitro, NK-Exo and NK-Exo-CE reduced LLC cell viability to 37.8 3.1% and 18.6 2.4%, respectively, at 1 10 particles/mL (p < 0.001). Combination therapy with paclitaxel further reduced viability to 9.4 1.8% (p < 0.001). In a subcutaneous LLC tumor model, NK-Exo and NK-Exo-CE reduced tumor volumes to 912 104 mm and 824 96 mm , respectively, compared to 1187 132 mm in controls (p < 0.001), while NK-Exo-CE + PTX achieved 348 57 mm (~ 3.4-fold reduction vs. control; p < 0.001) at the end of day 16. Immune profiling as determined by flow cytometry demonstrated increased CD8 T cell infiltration (8.9 1.1% to 35.7 2.6%, p < 0.001) and reduced regulatory T cells (28.7 1.8% to 10.4 1.2%, p < 0.001) in combination-treated tumors. Western blot analysis confirmed elevated cleaved caspase-3, cleaved PARP, Granzyme B, FasL, Phospho-NF- B-p65 and reduced Ki-67 and PDL1 in NK-Exo-CE + PTX tumors. Collectively, electro-hypoxic bioreactor conditioning enables scalable NK-exosome production without compromising physicochemical integrity or antitumor efficacy, addressing a key translational barrier in extracellular vesicle therapeutics.
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