INSERM UMR-MD-1197 « Interactions cellules souches-niches: physiologie, tumeurs et réparation tissulaire » Institut André Lwoff/Université Paris-Saclay, Hôpital Paul Brousse, 14, Avenue Paul-Vaillant Couturier, 94807 Villejuif, France.
INSERM UMR-MD-1197 « Interactions cellules souches-niches: physiologie, tumeurs et réparation tissulaire » Institut André Lwoff/Université Paris-Saclay, Hôpital Paul Brousse, 14, Avenue Paul-Vaillant Couturier, 94807 Villejuif, France; Institut de Recherche Biomédicale des Armées, Centre de Transfusion Sanguine des Armées, 1 rue Lt Raoul Batany, 92140 Clamart, France.
J Control Release. 2023 Mar;355:501-514. doi: 10.1016/j.jconrel.2023.01.073. Epub 2023 Feb 14.
A new paradigm has emerged recently, which consists in shifting from cell therapy to a more flexible acellular "extracellular vesicle (EV) therapy" approach, thereby opening a new and promising field in nanomedicine. Important technical limitations have still to be addressed for the large-scale production of clinical-grade EV. Cells are cultured in media supplemented with human platelet lysate (hPL) (xenogenic-free) or GMP-grade fetal calf serum (FCS). However, these additives contain high amounts of EV that cannot be separated from cell-secreted -EV. Therefore, cells are generally maintained in additive-free medium during the EV secretion phase, however this can substantially limit their survival. In the present work, we developed a method to prepare vesicle-free hPL (EV-free hPL) or vesicle-free FCS (EV-free FCS) using tangential flow filtration (TFF). We show a very efficient EV depletion (>98%) for both pure hPL and FCS, with a highly conserved protein content. Culture medium containing our EV-free additives supported the survival of human bone marrow MSC (BM-MSC). MSC could survive at least 216 h, their conditioned medium being collected and changed every 72 h. Both the cell survival and the cumulative EV production were substantially higher than in the starving conditions classically used for EV production. In EV-free hPL containing medium, we show that purified EV kept their morphologic and molecular characteristics throughout the production. Finally, we tested our additives with 3 other cell types, human primary Endothelial Colony Forming Cells (ECFC) and two non-adherent human cell lines, Jurkat and THP-1. We confirmed that both EV-free hPL and FCS were able to maintain cell survival and EV production for at least 216 h. Our method provides therefore a new option to help producing large amounts of EV from virtually any mammalian cells, particularly those that do not tolerate starvation. This method can apply to any animal serum for research and development purpose. Moreover, EV-free hPL is clinical-grade compatible and allows preparing xenobiotic-free media for massive therapeutic EV production in both 2D (cell plates) and 3D (bioreactor) setting.
最近出现了一种新的范式，即从细胞治疗转向更灵活的无细胞“细胞外囊泡 (EV) 治疗”方法，从而开辟了纳米医学的一个新的有前途的领域。为了大规模生产临床级 EV，仍然需要解决重要的技术限制。细胞在补充有人血小板裂解物 (hPL)（无异种）或 GMP 级胎牛血清 (FCS) 的培养基中培养。然而，这些添加剂含有大量无法与细胞分泌的 -EV 分离的 EV。因此，在 EV 分泌阶段，细胞通常在无添加剂的培养基中维持，但这可能会大大限制它们的存活。在本工作中，我们开发了一种使用切向流过滤 (TFF) 制备无囊泡 hPL（无 EV hPL）或无囊泡 FCS（无 EV FCS）的方法。我们证明了对于纯 hPL 和 FCS，EV 的去除效率非常高 (>98%)，且蛋白质含量高度保守。含有我们无 EV 添加剂的培养基支持人骨髓间充质干细胞 (BM-MSC) 的存活。MSC 至少能存活 216 小时，每隔 72 小时收集和更换其条件培养基。细胞存活和累积 EV 产量都大大高于经典的 EV 生产饥饿条件。在含有无 EV hPL 的培养基中，我们表明，在整个生产过程中，纯化的 EV 保持了其形态和分子特征。最后，我们用 3 种其他细胞类型（人原代内皮集落形成细胞 (ECFC) 和 2 种非贴壁人细胞系 Jurkat 和 THP-1）测试了我们的添加剂。我们证实，无 EV hPL 和 FCS 都能够至少维持 216 小时的细胞存活和 EV 生产。因此，我们的方法为从几乎任何哺乳动物细胞大量生产 EV 提供了一种新的选择，特别是那些不能耐受饥饿的细胞。该方法可适用于任何动物血清，用于研究和开发目的。此外，无 EV hPL 与临床级兼容，允许在 2D（细胞板）和 3D（生物反应器）设置中制备无异源生物的大量治疗性 EV 生产用培养基。
