Data_Sheet_4_A Chemically Defined, Xeno- and Blood-Free Culture Medium Sustains Increased Production of Small Extracellular Vesicles From Mesenchymal Stem Cells.PDF

Cell therapy is witnessing a notable shift toward cell-free treatments based on paracrine factors, in particular, towards small extracellular vesicles (sEV), that mimic the functional effect of the parental cells. While numerous sEV-based applications are currently in advanced preclinical stages, their promised translation depends on overcoming the manufacturing hurdles posed by the large-scale production of purified sEV. Unquestionably, the culture medium used with the parental cells plays a key role in the sEV’s secretion rate and content. An essential requisite is the use of a serum-, xeno-, and blood-free medium to meet the regulatory entity requirements of clinical-grade sEV’s production. Here, we evaluated OxiumTMEXO, a regulatory complying medium, with respect to production capacity and conservation of the EV’s characteristics and functionality and the parental cell’s phenotype and viability. A comparative study was established with standard DMEM and a commercially available culture medium developed specifically for sEV production. Under similar conditions, OxiumTMEXO displayed a three-fold increase of sEV secretion, with an enrichment of particles ranging between 51 and 200 nm. These results were obtained through direct quantification from the conditioned medium to avoid the isolation method’s interference and variability and were compared to the two culture media under evaluation. The higher yield obtained was consistent with several harvest time points (2, 4, and 6 days) and different cell sources, incluiding umbilical cord-, menstrual blood-derived mesenchymal stromal cells and fibroblasts. Additionally, the stem cell phenotype and viability of the parental cell remained unchanged. Furthermore, OxiumTMEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The in vitro internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV in vivo indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumTMEXO medium sustains an efficient and robust production of large quantities of sEV, conserving the classic functional properties of internalization into acceptor target cells and biodistribution in vivo, supplying the amount and quality of EVs for the development of cell-free therapies.

细胞治疗领域正经历显著转向，逐步转向基于旁分泌因子的无细胞治疗，尤其是可模拟亲代细胞功能效应的小型细胞外囊泡（small extracellular vesicles, sEV）。尽管目前诸多基于sEV的应用已处于高级临床前研究阶段，但这类疗法的预期临床转化仍需克服纯化sEV大规模生产所带来的生产瓶颈。毋庸置疑，亲代细胞所使用的培养基对sEV的分泌速率及其携带的内容物具有关键调控作用。满足监管机构对临床级sEV生产的相关要求，首要前提便是采用无血清、无异种来源成分且无血液的培养基。本研究针对符合监管要求的OxiumTMEXO培养基展开评估，考察其生产能力、对细胞外囊泡特性与功能的维持效果，以及对亲代细胞表型与活力的影响。研究采用标准DMEM培养基与一款专为sEV生产开发的商业化培养基作为对照，开展对比实验。在相同培养条件下，OxiumTMEXO培养基的sEV分泌量提升了三倍，且51~200 nm粒径区间的囊泡占比显著升高。该结果通过直接对条件培养基进行定量获得，以避免分离方法带来的干扰与变异性，并与另外两款受试培养基的结果进行了对比。该高产量结果在多个收获时间点（2、4、6天）以及不同细胞来源中均得到验证，包括脐带来源、月经血来源的间充质基质细胞与成纤维细胞。此外，亲代细胞的干细胞表型与活力未发生改变。进一步研究显示，经OxiumTMEXO培养基培养获得的sEV，其囊泡标志物CD63、CD9与CD81的表达模式与其他培养条件下获得的sEV一致。针对不同靶细胞类型开展的体外内化实验，以及对健康小鼠腹腔注射sEV的体内药代动力学分析均表明，更高的EV生产速率并未影响靶细胞对囊泡的摄取动力学，也未改变小鼠体内的全身生物分布。综上，OxiumTMEXO培养基可实现高效且稳定的大规模sEV生产，同时保留了sEV经典的靶细胞内化功能与体内生物分布特性，可为无细胞治疗的开发提供足量且符合质量要求的细胞外囊泡制剂。