Krutty John D, Koesser Kevin, Schwartz Stephen, Yun Junsu, Murphy William L, Gopalan Padma
Department of Materials Science and Engineering, University of Wisconsin-Madison, 1500 University Avenue, Madison, Wisconsin 53706, United States.
Department of Biomedical Engineering, University of Wisconsin-Madison, 1500 University Avenue, Madison, Wisconsin 53706, United States.
ACS Biomater Sci Eng. 2021 Feb 8;7(2):617-625. doi: 10.1021/acsbiomaterials.0c00663. Epub 2021 Jan 14.
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--vinyl dimethyl azlactone--glycidyl methacrylate) (P(PEGMEMA--VDM--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.
人间充质基质细胞（hMSC），也称为间充质干细胞，是成体细胞，已在治疗应用中展现出其潜力，其能够向不同谱系分化、调节免疫系统并产生生物制品，这一点尤为突出。由于临床应用需要大量细胞，因此迫切需要用于hMSC的可扩展培养系统。目前大多数扩增hMSC的方法在不使用含血清培养基或苛刻酶的情况下，无法提供临床所需细胞数量的可重复细胞产品。在这项工作中，我们将一种可定制的、薄的合成聚合物涂层——聚（聚乙二醇甲基醚甲基丙烯酸酯-乙烯基二甲基氮杂内酯-甲基丙烯酸缩水甘油酯）（P（PEGMEMA-VDM-GMA），PVG）应用于市售聚苯乙烯（PS）微载体表面，以创建用于大规模细胞扩增的化学成分明确的三维（3D）表面。这些化学成分明确的微载体提供了一个可重复的表面，不依赖于异种血清蛋白的吸附来介导细胞黏附，从而能够在无动物源成分的培养系统中使用。具体而言，这项工作证明了在无动物源成分的培养基中，hMSC对涂覆微载体的黏附性优于PS微载体，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，这些表面能抵抗培养基中及细胞产生的蛋白质的吸附，从而在整个培养期间实现整合素介导的细胞黏附。这一特性使得在不使用裂解酶的情况下，能够使用化学螯合剂（乙二胺四乙酸（EDTA））有效地将细胞从微载体上传代，这是该领域其他微载体产品的一个改进。在这些微载体上对hMSC进行生物反应器培养，能够在一个可扩展的、无动物源成分的环境中，在4天内生产出hMSC。
