Pasanen Shiina, Mootha Archana, Hirata Isao, Tanimoto Kotaro, Kato Koichi
Department of Biomaterials, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Department of Orthodontics and Craniofacial Developmental Biology, Graduate School of Biomedical and Health Sciences, Hiroshima University, Hiroshima, Japan.
Biotechnol J. 2025 Jun;20(6):e70057. doi: 10.1002/biot.70057.
Microcarrier-based bioreactors have been studied extensively for the expansion of human mesenchymal stem cells (hMSCs) to be used in regenerative therapy. To maximize the expansion efficiency, we developed microcarriers that tethered basic fibroblast growth factor (bFGF) on their surfaces. Using recombinant DNA technologies, bFGF was fused with a collagen-binding peptide (CBP) and bound to microcarriers that had been coated with type I collagen. In this study, we examined two types of CBP: a 28-mer peptide derived from human osteopontin (CBP1) and an 11-mer peptide derived from human decorin (CBP2). The chimeric proteins, CBP1-bFGF and CBP2-bFGF, were prepared using a bacterial expression system. Their structures were analyzed by circular dichroism spectroscopy and computer-based structure prediction, and their biological functions were assessed in hMSC cultures. Based on the results of these experiments, refolding conditions were optimized to increase the yields of soluble and bioactive CBP1-bFGF and CBP2-bFGF. Microcarriers that were tethered with CBP1-bFGF and CBP2-bFGF promoted hMSC growth without considerably altering their surface marker expression and osteogenic differentiation ability. These findings highlighted the potential of bFGF-tethered microcarriers as essential bioreactor components for hMSC expansion.
基于微载体的生物反应器已被广泛研究用于扩增人类间充质干细胞（hMSC），以用于再生治疗。为了最大化扩增效率，我们开发了在其表面 tether 有碱性成纤维细胞生长因子（bFGF）的微载体。利用重组 DNA 技术，将 bFGF 与胶原结合肽（CBP）融合，并与已用 I 型胶原包被的微载体结合。在本研究中，我们研究了两种类型的 CBP：一种来自人骨桥蛋白的 28 肽（CBP1）和一种来自人核心蛋白聚糖的 11 肽（CBP2）。使用细菌表达系统制备嵌合蛋白 CBP1-bFGF 和 CBP2-bFGF。通过圆二色光谱和基于计算机的结构预测分析它们的结构，并在 hMSC 培养物中评估它们的生物学功能。基于这些实验的结果，优化复性条件以提高可溶性和生物活性 CBP1-bFGF 和 CBP2-bFGF 的产量。与 CBP1-bFGF 和 CBP2-bFGF 相连的微载体促进 hMSC 生长，而不会显著改变其表面标志物表达和成骨分化能力。这些发现突出了 bFGF 连接的微载体作为 hMSC 扩增的重要生物反应器组件的潜力。
