Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, UK.
Division of Biomaterials and Tissue Engineering, Eastman Dental Institute, University College London, Royal Free Hospital, London, United Kingdom.
Biotechnol Bioeng. 2022 Sep;119(9):2345-2358. doi: 10.1002/bit.28133. Epub 2022 May 31.
In this study, polycaprolactone (PCL) macrobeads were prepared by an oil-in-water (o/w) emulsion solvent evaporation method with poly(vinyl alcohol) (PVA) as an emulsifier and conjugated to poly(N-isopropylacrylamide) (PNIPAAm) to be used as cell carriers with noninvasive cell detachment properties (thermo-response). Following previous studies with PCL-PNIPAAm carriers, our objectives were to confirm the successful conjugation on homemade macrobeads and to show the advantages of homemade production over commercial beads to control morphological, biological, and fluidization properties. The effects of PCL concentration on the droplet formation and of flow rate and PVA concentration on the size of the beads were demonstrated. The size of the beads, all spherical, ranged from 0.5 to 3.7 mm with four bead categories based on production parameters. The morphology and size of the beads were observed by scanning electron microscopy to show surface roughness enhancing cell attachment and proliferation compared to commercial beads. The functionalization steps with PNIPAAm were then characterized and confirmed by Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersion spectroscopy. PNIPAAm-grafted macrobeads allowed mesenchymal stem cells (MSCs) to spread and grow for up to 21 days. By reducing the temperature to 25°C, the MSCs were successfully detached from the PCL-PNIPAAm beads as observed with fluorescence microscopy. Furthermore, we validated the scalability potential of both macrobeads production and conjugation with PCL, to produce easily kilograms of thermo-responsive macrocarriers in a lab environment. This could help moving such approaches towards clinically and industrially relevant processes were cell expansion is needed at very large scale.
在这项研究中，采用油包水（o/w）乳液溶剂蒸发法制备聚己内酯（PCL）微球，以聚乙烯醇（PVA）作为乳化剂，并与聚（N-异丙基丙烯酰胺）（PNIPAAm）结合，用作具有非侵入性细胞分离特性（温度响应性）的细胞载体。在先前使用 PCL-PNIPAAm 载体的研究之后，我们的目标是确认自制微球上成功的接枝，并展示自制生产相对于商业珠粒的优势，以控制形态、生物学和流化性质。展示了 PCL 浓度对液滴形成的影响以及流速和 PVA 浓度对珠粒尺寸的影响。通过扫描电子显微镜观察到所有球形珠粒的尺寸范围为 0.5 至 3.7mm，并根据生产参数分为四个珠粒类别。珠粒的形态和尺寸通过扫描电子显微镜观察到，表面粗糙度增强了细胞的附着和增殖，与商业珠粒相比。然后通过傅里叶变换红外光谱、扫描电子显微镜和能量色散光谱对 PNIPAAm 的功能化步骤进行了表征和确认。接枝 PNIPAAm 的微球允许间充质干细胞（MSCs）扩散和生长长达 21 天。通过将温度降低到 25°C，可以如荧光显微镜观察到的那样，成功地从 PCL-PNIPAAm 珠粒上分离出 MSCs。此外，我们验证了 PCL 生产和接枝的这两种大珠粒的可扩展性潜力，以在实验室环境中轻松生产公斤级的热响应性大载体。这有助于将这些方法推向临床和工业相关的过程，在这些过程中需要大规模细胞扩增。
