García-Aponte Oscar Fabian, Serra Marta, Kahlenberg Simon, Subbiahdoss Guruprakash, Reimhult Erik, Egger Dominik, Kasper Cornelia
Institute of Cell and Tissue Culture Technology, Department of Biotechnology, BOKU University, Muthgasse 18, Vienna, 1190, Austria.
Instituto Superior Técnico - Taguspark Campus, Av. Prof. Doutor Cavaco Silva, Porto Salvo, 2744-016, Portugal.
Adv Healthc Mater. 2025 Aug;14(20):e2405137. doi: 10.1002/adhm.202405137. Epub 2025 Jun 8.
Mesenchymal stem cells (MSCs) hold potential for several applications, but inefficient nonphysiological culturing methods constantly prevent clinical translation. Automated cell encapsulation in small hydrogels (microgels) facilitates physiologically relevant MSC expansion in bioreactors. Unfortunately, encapsulation processes are poorly characterized, biological variability is seldomly considered, and dynamic culturing is marginally explored. Here, a high-throughput millifluidic encapsulation process is introduced and standardized. This platform enables highly viable MSC networks within gelatin methacryloyl microgels. The impact of biological variability and crosslinking variations under strong dynamic culturing conditions is closely monitored through cell proliferation, microgel shrinkage, and metabolic activity. The effect of carboxymethyl cellulose on microgel's architecture is observed with cryogenic scanning electron microscopy. Increased crosslinking controls the formation of an outer layer on the microgels, which improves the microgel's resistance to shrinking, prevents cell proliferation on the material's surface and increases overall MSC expansion. Cell proliferation, microgel shrinkage, glucose uptake, and cell metabolism show interdependencies observable thanks to the high encapsulation output. Cell proliferation and metabolic activity depend strongly on donor-to-donor variability and change during culture. However, metabolic readouts reliably follow cell expansion, which makes this simple and mechanically robust platform promising for large-scale bioreactor applications.
间充质干细胞（MSCs）在多种应用中具有潜力，但低效的非生理性培养方法一直阻碍着其临床转化。在小型水凝胶（微凝胶）中进行自动细胞封装有助于在生物反应器中实现与生理相关的MSCs扩增。不幸的是，封装过程的特征描述不足，很少考虑生物学变异性，对动态培养的探索也很少。在此，引入并标准化了一种高通量微流控封装工艺。该平台能够在甲基丙烯酰化明胶微凝胶中形成高度有活力的MSCs网络。通过细胞增殖、微凝胶收缩和代谢活性，密切监测在强动态培养条件下生物学变异性和交联变化的影响。用低温扫描电子显微镜观察羧甲基纤维素对微凝胶结构的影响。增加交联可控制微凝胶外层的形成，这提高了微凝胶的抗收缩性，防止细胞在材料表面增殖，并增加了整体MSCs扩增。由于高封装产量，细胞增殖、微凝胶收缩、葡萄糖摄取和细胞代谢表现出相互依赖性。细胞增殖和代谢活性强烈依赖于供体间的变异性，并在培养过程中发生变化。然而，代谢读数可靠地跟随细胞扩增，这使得这个简单且机械稳健的平台在大规模生物反应器应用中具有前景。
