Figueroa-Valdés Aliosha I, de la Fuente Catalina, Hidalgo Yessia, Vega-Letter Ana María, Tapia-Limonchi Rafael, Khoury Maroun, Alcayaga-Miranda Francisca
Cells for Cells, Santiago, Chile.
Consorcio Regenero, Chilean Consortium for Regenerative Medicine, Santiago, Chile.
Front Bioeng Biotechnol. 2021 May 26;9:619930. doi: 10.3389/fbioe.2021.619930. eCollection 2021.
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 OxiumEXO, 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, OxiumEXO 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, OxiumEXO-sEV showed a similar expression pattern of the vesicular markers CD63, CD9, and CD81, with respect to sEV derived from the other conditions. The internalization assays in different target cell types and the pharmacokinetic profile of intraperitoneally administered sEV indicated that the higher EV production rate did not affect the uptake kinetics or the systemic biodistribution in healthy mice. In conclusion, the OxiumEXO 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 , supplying the amount and quality of EVs for the development of cell-free therapies.
细胞疗法正显著转向基于旁分泌因子的无细胞治疗，特别是朝着模仿亲代细胞功能效应的小细胞外囊泡（sEV）发展。虽然目前许多基于sEV的应用正处于临床前晚期阶段，但它们能否成功转化取决于克服大规模生产纯化sEV所带来的制造障碍。毫无疑问，与亲代细胞一起使用的培养基在sEV的分泌速率和内容物方面起着关键作用。一个基本要求是使用无血清、无异种蛋白和无血液的培养基，以满足临床级sEV生产的监管要求。在此，我们评估了符合监管要求的培养基OxiumEXO在生产能力、EV特性和功能的保存以及亲代细胞的表型和活力方面的表现。我们与标准DMEM和一种专门为sEV生产开发的市售培养基进行了比较研究。在相似条件下，OxiumEXO使sEV分泌增加了三倍，富集了51至200纳米范围内的颗粒。这些结果是通过直接从条件培养基中定量获得的，以避免分离方法的干扰和变异性，并与所评估的两种培养基进行了比较。获得的更高产量在几个收获时间点（2天、4天和6天）以及不同细胞来源（包括脐带、月经血来源的间充质基质细胞和成纤维细胞）中都是一致的。此外，亲代细胞的干细胞表型和活力保持不变。此外，与源自其他条件的sEV相比，OxiumEXO-sEV显示出类似的囊泡标志物CD63、CD9和CD81表达模式。在不同靶细胞类型中的内化试验以及腹腔注射sEV的药代动力学概况表明，较高的EV产生率并不影响健康小鼠的摄取动力学或全身生物分布。总之，OxiumEXO培养基能够高效、稳健地生产大量sEV，保留了内化到受体靶细胞和生物分布的经典功能特性，为无细胞疗法的发展提供了EV的数量和质量。
