Askari Esfandyar, Barough Mahdieh Shokrollahi, Seyfoori Amir, Razzaghi Mahmood, Ninan Joel Alexander, Murray Aidan B P, Salkhordeh Mahmoud, Tan Yuan, Mei Shirley H J, Akbari Mohsen
Laboratory for Innovations in Micro Engineering (LiME), Department of Mechanical Engineering, Victoria, BC, V8P 5C2, Canada.
Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada.
Adv Healthc Mater. 2025 Sep;14(23):e2404538. doi: 10.1002/adhm.202404538. Epub 2025 Jul 14.
Scaling up cell therapy requires efficient expansion of high-quality cells. Microcarrier(MC)-based systems offer high surface-to-volume ratios and reduce culture media usage. In this study, we developed BrushGel, a temperature-responsive MC composed of gelatin methacryloyl (GelMA) hydrogel particles coated with poly(N-isopropyl acrylamide) (PNIPAM) polymer brushes via covalent grafting. BrushGel was fabricated using a flow-focusing droplet microfluidic device and functionalized using carbodiimide chemistry ( 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide-N-Hydroxysuccinimide, EDC-NHS). The degree of PNIPAM coating was tuned by varying the degree of methacrylation (DOM) of GelMA and the concentration of PNIPAM. Human dermal fibroblast (HNDF) cells cultured on the BrushGel under dynamic conditions showed a 4.9 fold increase in cell density, 12-fold upregulation in COL1A1 gene expression and elevated procollagen protein secretion compared to static culture. Low temperature detachment (4 °C) yeilded up to 65% detachment efficiency with >95% post-detachment viability. Clinical grade human bone marrow-derived mesenchymal stromal/stem cells (MSCs) expnaded 5.3 fold over five days on BrushGel with 69% detachment efficiency and 80% post-harvest viability using 10-fold less enzyme. BrushGel supported over 10 days of culture in spinner flasks, enabling enzyme-minimized, scalable cell expansion. These findings position BrushGel as a promising platfrom for dyanmic cell culture systems in regenerative medicine.
扩大细胞治疗规模需要高效扩增高质量细胞。基于微载体（MC）的系统具有高表面积与体积比，并减少了培养基的用量。在本研究中，我们开发了BrushGel，这是一种温度响应性微载体，由通过共价接枝涂覆有聚（N-异丙基丙烯酰胺）（PNIPAM）聚合物刷的甲基丙烯酰化明胶（GelMA）水凝胶颗粒组成。BrushGel使用流动聚焦液滴微流控装置制造，并使用碳二亚胺化学（1-乙基-3-（3-二甲基氨基丙基）碳二亚胺-N-羟基琥珀酰亚胺，EDC-NHS）进行功能化。通过改变GelMA的甲基丙烯酰化程度（DOM）和PNIPAM的浓度来调节PNIPAM涂层的程度。与静态培养相比，在动态条件下于BrushGel上培养的人皮肤成纤维细胞（HNDF）细胞密度增加了4.9倍，COL1A1基因表达上调了12倍，前胶原蛋白分泌增加。低温脱离（4°C）产生了高达65%的脱离效率，脱离后活力>95%。临床级人骨髓间充质基质/干细胞（MSCs）在BrushGel上五天内扩增了5.3倍，使用的酶减少了10倍，脱离效率为69%，收获后活力为80%。BrushGel在转瓶中支持超过10天的培养，实现了酶用量最小化、可扩展的细胞扩增。这些发现使BrushGel成为再生医学中动态细胞培养系统的一个有前景的平台。
