Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, Taiwan; Advanced Membrane Materials Center, National Taiwan University of Science and Technology, Taipei, Taiwan; College of Medicine and Health Science, Bahir Dar University, Bahir Dar, Ethiopia.
Department of Materials Science and Engineering, National Taiwan University of Science and Technology, Taipei, Taiwan.
Biomater Adv. 2022 Aug;139:213008. doi: 10.1016/j.bioadv.2022.213008. Epub 2022 Jul 2.
The use of microspheres for culturing adherent cells has been proven as an important method, allowing for obtaining adequate number of cells in limited space and volume of medium for the intended cell-based medical applications. However, the use of proteolytic enzymes for cell harvesting from the microsphere resulted in cell damage and loss of functionality. Therefore, in this study, we developed a novel redox/thermo-responsive dissolvable gelatin-based microsphere for successful cell proliferation and harvesting adequate high-quality cells using non-enzymatic cell detachment methods. Initially, a redox-induced dissolvable gelatin-based microsphere was successfully prepared using disulfide bonds as crosslinking agent, firmly stabilizing gelatin networks and forming a stable microsphere at physiological temperature. The optimized concentration of the crosslinking agent was 1.2 mM, which kept the microsphere stable for >120 h. The microsphere was then coated with PNIPAm-ALA copolymer via physical or chemical means, resulting in a positively charged thermosensitive surface. The positive charge derived from ALA in PNIPAm-ALA copolymer enhanced cell attachment, while the thermosensitive property of the copolymer enabled for temperature induced cell harvesting. When the temperature dropped below the LCST value of PNIPAm-ALA (33.4°C), the copolymer swelled and became more hydrophilic, allowing cells to be readily separated. The addition of reducing agents such as GSH, DTT and L-cysteine resulted in further cleavage of the disulfide bond in the microsphere and dissolution of the microsphere for complete cell detachment. Interestingly, cell attachment and proliferation were enhanced on microspheres coated with PNIPAm-ALA using diselenide as a crosslinking agent, and complete cell detachment was occurred within 15 min after adding 25 mM DTT followed by lowering the temperature (4°C). Therefore, the microsphere fabricated in this study was worthwhile for non-enzymatic cell detachment and has the potential to be used for cell expansion and harvesting adequate live cells of high quality and functionality for tissue engineering or cell therapy.
用于培养贴壁细胞的微球已被证明是一种重要的方法，可在有限的空间和培养基体积内获得足够数量的细胞，用于预期的基于细胞的医学应用。然而，用蛋白酶从微球中收获细胞会导致细胞损伤和功能丧失。因此，在这项研究中，我们开发了一种新型的氧化还原/温敏可溶解明胶基微球，用于成功地进行细胞增殖，并使用非酶细胞分离方法收获足够数量的高质量细胞。最初，使用二硫键作为交联剂成功制备了氧化还原诱导的可溶解明胶基微球，该交联剂可牢固地稳定明胶网络，并在生理温度下形成稳定的微球。交联剂的最佳浓度为 1.2mM，可使微球稳定超过 120 小时。然后通过物理或化学手段将 PNIPAm-ALA 共聚物涂覆在微球上，形成带正电荷的温敏表面。PNIPAm-ALA 共聚物中的 ALA 衍生出的正电荷增强了细胞的附着性，而共聚物的温敏性使细胞能够在温度诱导下收获。当温度降至 PNIPAm-ALA 的 LCST 值（33.4°C）以下时，共聚物溶胀并变得更加亲水，使细胞易于分离。添加还原剂，如 GSH、DTT 和 L-半胱氨酸，会导致微球中二硫键进一步断裂，并使微球完全溶解以实现完全细胞分离。有趣的是，使用二硒键作为交联剂涂覆的微球上细胞附着和增殖增强，并且在添加 25mM DTT 并降低温度（4°C）后 15 分钟内即可完全分离细胞。因此，本研究中制备的微球非常适合非酶细胞分离，并且有可能用于细胞扩增和收获足够数量的高质量和高功能活细胞，用于组织工程或细胞治疗。
