Bandarra-Tavares Hélder, Franchi-Mendes Teresa, Ulpiano Cristiana, Morini Sara, Kaur Navjot, Harris-Becker Abigail, Vemuri Mohan C, Cabral Joaquim M S, Fernandes-Platzgummer Ana, da Silva Cláudia L
Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal; Associate Laboratory i4HB - Institute for Health and Bioeconomy at Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal.
Cell and Gene Therapy, Thermo Fisher Scientific, Cell Biology, Frederick, Maryland, USA.
Cytotherapy. 2024 Jul;26(7):749-756. doi: 10.1016/j.jcyt.2024.03.001. Epub 2024 Mar 11.
BACKGROUND & AIMS: Cell therapies based on mesenchymal stromal cells (MSCs) have gained an increasing therapeutic interest in the context of multiple disorders. Nonetheless, this field still faces important challenges, particularly concerning suitable manufacturing platforms. Here, we aimed at establishing a scalable culture system to expand umbilical cord-derived Wharton's jelly MSC (MSC(WJ)) and their derived extracellular vesicles (EVs) by using dissolvable microcarriers combined with xeno(geneic)-free culture medium.
MSC(WJ) isolated from three donors were cultured at a starting density of 1 × 10 cells per spinner flask, i.e., 2.8 × 10 cells per cm of dissolvable microcarrier surface area. After a 6-day expansion period of MSC(WJ), extracellular vesicles (EVs) were produced for 24 h.
Taking advantage of an intermittent agitation regimen, we observed high adhesion rates to the microcarriers (over 90% at 24 h) and achieved 15.8 ± 0.7-fold expansion after 6 days of culture. Notably, dissolution of the microcarriers was achieved through a pectinase-based solution to recover the cell product, reducing the hurdles of downstream processing. MSC identity was validated by detecting the characteristic MSC immunophenotype and by multilineage differentiation assays. Considering the growing interest in MSC-derived EVs, which are known to be mediators of the therapeutic features of MSC, this platform also was evaluated for EV production. Upon a 24-h period of conditioning, secreted EVs were isolated by ultrafiltration followed by anion-exchange chromatography and exhibited the typical cup-shaped morphology, small size distribution (162.6 ± 30.2 nm) and expressed EV markers (CD63, CD9 and syntenin-1).
Taken together, we established a time-effective and robust scalable platform that complies with clinical-grade standards for the dual production of MSC(WJ) and their derived EV.
基于间充质基质细胞（MSC）的细胞疗法在多种疾病的治疗中越来越受到关注。然而，该领域仍面临重大挑战，特别是在合适的生产平台方面。在此，我们旨在建立一种可扩展的培养系统，通过使用可溶解的微载体与无（异）基因培养基相结合，来扩增脐带华通氏胶间充质基质细胞（MSC(WJ)）及其衍生的细胞外囊泡（EVs）。
从三名供体分离的MSC(WJ)以每个转瓶1×10个细胞的起始密度进行培养，即每平方厘米可溶解微载体表面积2.8×10个细胞。在MSC(WJ)扩增6天后，生产细胞外囊泡（EVs）24小时。
利用间歇性搅拌方案，我们观察到对微载体的高粘附率（24小时时超过90%），并在培养6天后实现了15.8±0.7倍的扩增。值得注意的是，通过基于果胶酶的溶液实现了微载体的溶解，以回收细胞产物，减少了下游处理的障碍。通过检测特征性的MSC免疫表型和多谱系分化试验验证了MSC的身份。鉴于对MSC衍生的EVs的兴趣日益增加，已知其为MSC治疗特性的介质，该平台也用于评估EV的生产。在24小时的条件培养期后，通过超滤随后进行阴离子交换色谱法分离分泌的EVs，其呈现典型的杯状形态、小尺寸分布（162.6±30.2nm）并表达EV标志物（CD63、CD9和syntenin-1）。
综上所述，我们建立了一个符合临床级标准的高效且强大的可扩展平台，用于双重生产MSC(WJ)及其衍生的EV。
