Merivaara Arto, Koivunotko Elle, Manninen Kalle, Kaseva Tuomas, Monola Julia, Salli Eero, Koivuniemi Raili, Savolainen Sauli, Valkonen Sami, Yliperttula Marjo
Drug Research Program, Division of Pharmaceutical Biosciences, Faculty of Pharmacy, University of Helsinki, 00014 Helsinki, Finland.
HUS Medical Imaging Center, Radiology, University of Helsinki and Helsinki University Hospital, 00290 Helsinki, Finland.
Polymers (Basel). 2022 Dec 17;14(24):5530. doi: 10.3390/polym14245530.
Nanofibrillated cellulose (NFC) hydrogel is a versatile biomaterial suitable, for example, for three-dimensional (3D) cell spheroid culturing, drug delivery, and wound treatment. By freeze-drying NFC hydrogel, highly porous NFC structures can be manufactured. We freeze-dried NFC hydrogel and subsequently reconstituted the samples into a variety of concentrations of NFC fibers, which resulted in different stiffness of the material, i.e., different mechanical cues. After the successful freeze-drying and reconstitution, we showed that freeze-dried NFC hydrogel can be used for one-step 3D cell spheroid culturing of primary mesenchymal stem/stromal cells, prostate cancer cells (PC3), and hepatocellular carcinoma cells (HepG2). No difference was observed in the viability or morphology between the 3D cell spheroids cultured in the freeze-dried and reconstituted NFC hydrogel and fresh NFC hydrogel. Furthermore, the 3D cultured spheroids showed stable metabolic activity and nearly 100% viability. Finally, we applied a convolutional neural network (CNN)-based automatic nuclei segmentation approach to automatically segment individual cells of 3D cultured PC3 and HepG2 spheroids. These results provide an application to culture 3D cell spheroids more readily with the NFC hydrogel and a step towards automatization of 3D cell culturing and analysis.
纳米原纤化纤维素（NFC）水凝胶是一种多功能生物材料，适用于例如三维（3D）细胞球体培养、药物递送和伤口治疗。通过冷冻干燥NFC水凝胶，可以制造出高度多孔的NFC结构。我们对NFC水凝胶进行冷冻干燥，随后将样品重新配制成各种浓度的NFC纤维，这导致了材料的不同硬度，即不同的机械信号。在成功进行冷冻干燥和重构后，我们表明冷冻干燥的NFC水凝胶可用于原代间充质干/基质细胞、前列腺癌细胞（PC3）和肝癌细胞（HepG2）的一步法3D细胞球体培养。在冷冻干燥和重构的NFC水凝胶以及新鲜NFC水凝胶中培养的3D细胞球体之间，在活力或形态方面未观察到差异。此外，3D培养的球体显示出稳定的代谢活性和近100%的活力。最后，我们应用基于卷积神经网络（CNN）的自动细胞核分割方法来自动分割3D培养的PC3和HepG2球体中的单个细胞。这些结果为使用NFC水凝胶更轻松地培养3D细胞球体提供了一种应用，并朝着3D细胞培养和分析的自动化迈出了一步。
