Challenges and coping strategies for the clinical translation of stem cell-derived exosomes

doi:10.3877/cma.j.issn.2095-1221.2025.05.007

Abstract

Abstract:

Stem cell-derived exosomes, as a cell-free therapeutic approach, circumvent the risks associated with live cell transplantation and offer enhanced biological safety. Through engineering modifications, they can augment tissue targeting and therapeutic efficacy, making them a promising new treatment strategy that could potentially replace stem cells. In response to the clinical translation challenges, such as improving the yield, batch stability, therapeutic efficacy, and activity maintenance of stem cell-derived exosomes, this review proposes coping strategies including the use of 3D culture combined with immortalized cell lines, the development of engineered exosome empowerment systems, and innovative exosome preservation techniques aiming to further advance the clinical translation process of stem cell-derived exosomes in standardized production, therapeutic applications, and long-term storage.

Key words: Stem cells, Exosomes, Clinical translation, Engineering, Preservation

Lin Ding, Minli Liang, Jiaqi Zhong, Furong Li. Challenges and coping strategies for the clinical translation of stem cell-derived exosomes[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2025, 15(05): 301-311.

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References 69

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生物反应器类型	核心结构/原理	关键优势	主要局限
搅拌式生物反应器	机械搅拌桨驱动培养液与微载体混合	混合效率高，均质性好；易联用微载体，兼容性强；支持高细胞密度（达1×10⁷个细胞/mL）	剪切力可能造成细胞损伤，需优化桨叶设计（如螺旋桨式）与转速控制策略
固定床式生物反应器	细胞贴附于多孔载体（如海绵状基质）内部生长	提供低剪切力环境，适合脆弱细胞（如神经干细胞）；载体稳定性高，利于长期培养	载体内部易形成营养/氧气梯度，导致深层细胞坏死风险升高；载体清洗与细胞回收困难
气升式生物反应器	底部通入气体驱动培养液循环（无机械搅拌）	剪切力极低，细胞存活率高；结构简单，不易污染	大规模培养时氧传输效率不足（气泡对氧溶解能力有限）；循环控制精度要求高
膜式生物反应器（中空纤维）	模拟毛细血管结构，利用选择性渗透膜实现物质交换	维持稳态微环境，保护细胞活性；高效回收高质量外泌体；工艺重复性好，易放大生产	膜污染可能导致通量下降，需定期维护；初始设备投资成本高

生物反应器类型	核心结构/原理	关键优势	主要局限
搅拌式生物反应器	机械搅拌桨驱动培养液与微载体混合	混合效率高，均质性好；易联用微载体，兼容性强；支持高细胞密度（达1×10⁷个细胞/mL）	剪切力可能造成细胞损伤，需优化桨叶设计（如螺旋桨式）与转速控制策略
固定床式生物反应器	细胞贴附于多孔载体（如海绵状基质）内部生长	提供低剪切力环境，适合脆弱细胞（如神经干细胞）；载体稳定性高，利于长期培养	载体内部易形成营养/氧气梯度，导致深层细胞坏死风险升高；载体清洗与细胞回收困难
气升式生物反应器	底部通入气体驱动培养液循环（无机械搅拌）	剪切力极低，细胞存活率高；结构简单，不易污染	大规模培养时氧传输效率不足（气泡对氧溶解能力有限）；循环控制精度要求高
膜式生物反应器（中空纤维）	模拟毛细血管结构，利用选择性渗透膜实现物质交换	维持稳态微环境，保护细胞活性；高效回收高质量外泌体；工艺重复性好，易放大生产	膜污染可能导致通量下降，需定期维护；初始设备投资成本高

分离方法	原理	优点	缺点
超速离心法	利用不同离心力分步去除细胞、碎片及大囊泡，最终高速沉淀外泌体	标准方法，适用大体积样本量；操作简单，无需特殊试剂	耗时较长；设备昂贵；外泌体易破损；不适用于微量样本
密度梯度离心法	在超速离心基础上，通过蔗糖/碘克沙醇形成密度梯度，利用外泌体与其他颗粒的密度差异进行分离	纯度高；有效去除蛋白污染	耗时较长；设备昂贵；回收率低；操作复杂
聚合物沉淀法	亲水聚合物与外泌体相结合，形成疏水层，降低其溶解度，低速离心即可沉淀	操作简单，无需超离设备；适用各类体积样本	蛋白质等杂质多；聚合物残留干扰下游分析
尺寸排阻色谱法	多孔凝胶柱按分子粒径尺寸分离，大分子外泌体先洗脱	保留完整结构与活性；纯度高，蛋白污染少	步骤繁琐，需多步离心、过滤；耗时长；柱材成本高、易堵塞；不适合大量样本处理
免疫亲和法	抗体修饰磁珠特异性结合外泌体表面标志物（如CD63/CD81）	特异性高，适合亚群研究；纯度高，降低蛋白等杂质干扰	抗体成本高；不适合大量样本处理；抗体结合可能损伤功能活性
超滤法	超滤膜截留外泌体，滤除小分子	成本低；耗时短；适用各类体积样本	纯度低；膜吸附和堵塞导致外泌体损失；剪切力致结构变形

分离方法	原理	优点	缺点
超速离心法	利用不同离心力分步去除细胞、碎片及大囊泡，最终高速沉淀外泌体	标准方法，适用大体积样本量；操作简单，无需特殊试剂	耗时较长；设备昂贵；外泌体易破损；不适用于微量样本
密度梯度离心法	在超速离心基础上，通过蔗糖/碘克沙醇形成密度梯度，利用外泌体与其他颗粒的密度差异进行分离	纯度高；有效去除蛋白污染	耗时较长；设备昂贵；回收率低；操作复杂
聚合物沉淀法	亲水聚合物与外泌体相结合，形成疏水层，降低其溶解度，低速离心即可沉淀	操作简单，无需超离设备；适用各类体积样本	蛋白质等杂质多；聚合物残留干扰下游分析
尺寸排阻色谱法	多孔凝胶柱按分子粒径尺寸分离，大分子外泌体先洗脱	保留完整结构与活性；纯度高，蛋白污染少	步骤繁琐，需多步离心、过滤；耗时长；柱材成本高、易堵塞；不适合大量样本处理
免疫亲和法	抗体修饰磁珠特异性结合外泌体表面标志物（如CD63/CD81）	特异性高，适合亚群研究；纯度高，降低蛋白等杂质干扰	抗体成本高；不适合大量样本处理；抗体结合可能损伤功能活性
超滤法	超滤膜截留外泌体，滤除小分子	成本低；耗时短；适用各类体积样本	纯度低；膜吸附和堵塞导致外泌体损失；剪切力致结构变形

技术名称	核心原理/技术	关键优势	应用场景/特点
超声纳滤技术（EXODUS系统，汇芯生物）	负压振荡+双耦合超声振荡作用于纳米超滤芯片，快速去除游离核酸/蛋白杂质并截留外泌体	全自动操作；支持微量样本（泪液、脑脊液）至规模化样本提取；杂蛋白去除效率> 99%；回收率> 90%	适用于需要高纯度、高回收率且样本量波动大的研究或临床场景
Cytiva^TM superSEC填料（切向流联用，Cytiva公司）	高分辨率尺寸排阻层析+优化流体动力学设计，结合切向流富集	缩短层析循环时间；提升杂质去除效率；纯度指数高于传统填料（如Sepharose^TM CL-2B）	为外泌体规模化生产与临床应用提供可靠解决方案（如生物制药级外泌体纯化）
微流控技术^[47,48]	分离型：双切向流+两种孔径纳米多孔膜；检测型：化学发光检测+杂交链反应	微量样本处理（如临床少量血液）、高活性外泌体获取；高灵敏度外泌体多靶标miRNA检测	适用于临床即时诊断（如肿瘤标志物外泌体快速检测）、微量样本的高精准分离
细胞内泌体提取^[49,50]	低温"冷萃"裂解DPSCs细胞，直接分离内泌体	提取效率是外泌体的16倍；碱性成纤维细胞生长因子、脑源性神经营养因子、表皮生长因子受体等生长因子含量高于外泌体；低成本、操作简单	为细胞囊泡高效提取提供新思路（如干细胞来源囊泡的低成本制备），适用于再生医学研究

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