Xeno-Free Bioreactor Culture of Human Mesenchymal Stromal Cells on Chemically Defined Microcarriers
Xeno-Free Bioreactor Culture of Human Mesenchymal Stromal Cells on Chemically Defined Microcarriers
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化学成分确定的微载体上人间充质基质细胞的无异种生物反应器培养
DOI：
10.1021/acsbiomaterials.0c00663
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发表时间：
2021
期刊：
影响因子：
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通讯作者：
John D. Krutty, Kevin Koesser
中科院分区：
文献类型：
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作者：
John D. Krutty, Kevin Koesser
人间充质干细胞(HMSC)，又称间充质干细胞，是一种成熟的细胞，已显示出其在治疗应用中的潜力，突出表现为它们能够向下分化不同的谱系，调节免疫系统，并产生生物制剂。由于临床应用需要大量的细胞，因此迫切需要可扩展的hMSC培养系统。目前大多数扩增人骨髓间充质干细胞的方法在没有使用含血清的培养液或苛刻的酶的情况下，无法提供符合临床需要的细胞数量的可复制的细胞产品。在这项工作中，我们将一种可定制的薄合成聚合物涂层-聚(聚乙二醇甲基醚甲基丙烯酸酯-RAN-乙烯基二甲基氮杂内酯-RAN-缩水甘油酯甲基丙烯酸酯)(P(PEGMEMA-r-VDM-r-GMA)，PVG)-应用于商用聚苯乙烯(PS)微载体的表面，以创建用于大规模细胞扩张的化学定义的三维(3D)表面。这些化学定义的微载体提供了一个可复制的表面，不依赖于异种血清蛋白的吸附来调节细胞黏附，使它们能够在无异种培养系统中使用。具体地说，这项工作证明了在无异种介质中，hMSC与PS微载体上涂层微载体的粘附性得到了改善，并描述了它们在易于扩展的基于生物反应器的培养系统中的应用。此外，这些表面还能抵抗介质中和细胞产生的蛋白质的吸附，从而在整个培养过程中导致整合素介导的细胞黏附。这一特性允许使用化学螯合剂(乙二胺四乙酸(EDTA))在没有切割酶的情况下有效地从微载体上传代细胞，这比该领域的其他微载体产品有所改进。在这些微载体上进行hMSC的生物反应器培养，可以在可扩展的、无异物的环境中在4天内生产hMSC。
Human mesenchymal stromal cells (hMSC), also called mesenchymal stem cells, are adult cells that have demonstrated their potential in therapeutic applications, highlighted by their ability to differentiate down different lineages, modulate the immune system, and produce biologics. There is a pressing need for scalable culture systems for hMSC due to the large number of cells needed for clinical applications. Most current methods for expanding hMSC fail to provide a reproducible cell product in clinically required cell numbers without the use of serum-containing media or harsh enzymes. In this work, we apply a tailorable, thin, synthetic polymer coating—poly(poly(ethylene glycol) methyl ether methacrylate-ran-vinyl dimethyl azlactone-ran-glycidyl methacrylate) (P(PEGMEMA-r-VDM-r-GMA), PVG)—to the surface of commercially available polystyrene (PS) microcarriers to create chemically defined three-dimensional (3D) surfaces for large-scale cell expansion. These chemically defined microcarriers provide a reproducible surface that does not rely on the adsorption of xenogeneic serum proteins to mediate cell adhesion, enabling their use in xeno-free culture systems. Specifically, this work demonstrates the improved adhesion of hMSC to coated microcarriers over PS microcarriers in xeno-free media and describes their use in a readily scalable, bioreactor-based culture system. Additionally, these surfaces resist the adsorption of media-borne and cell-produced proteins, which result in integrin-mediated cell adhesion throughout the culture period. This feature allows the cells to be efficiently passaged from the microcarrier using a chemical chelating agent (ethylenediaminetetraacetic acid (EDTA)) in the absence of cleavage enzymes, an improvement over other microcarrier products in the field. Bioreactor culture of hMSC on these microcarriers enabled the production of hMSC over 4 days from a scalable, xeno-free environment.