Through enabling an efficient supply of cells and tissues in the health sector on demand, cryopreservation is increasingly becoming one of the mainstream technologies in rapid translation and commercialization of regenerative medicine research. Cryopreservation of tissue-engineered constructs (TECs) is an emerging trend that requires the development of practically competitive biobanking technologies. In our previous studies, we demonstrated that conventional slow-freezing using dimethyl sulfoxide (Me2SO) does not provide sufficient protection of mesenchymal stromal cells (MSCs) frozen in 3D collagen-hydroxyapatite scaffolds. After simple modifications to cryopreservation protocol, we report on significantly improved cryopreservation of TECs. Porous 3D scaffolds were fabricated using freeze-drying of mineralized a collagen suspension and following chemical crosslinking. Amnion-derived MSCs from common marmoset monkey Callithrix jacchus were seeded onto scaffolds in static conditions. Cell-seeded scaffolds were subjected to 24 h pre-treatment with 100 mM sucrose and slow freezing in 10% Me2SO/20%FBS alone or supplemented with 300 mM sucrose. Scaffolds were frozen 'in air' and thawed using a two-step procedure. Diverse analytical methods were used for the interpretation of cryopreservation outcome for both cell-seeded and cell-free scaffolds. In both groups, cells exhibited their typical shape and well-preserved cell-cell and cell-matrix contacts after thawing. Moreover, viability test 24 h post-thaw demonstrated that application of sucrose in the cryoprotective solution preserves a significantly greater portion of sucrose-pretreated cells (more than 80%) in comparison to Me2SO alone (60%). No differences in overall protein structure and porosity of frozen scaffolds were revealed whereas their compressive stress was lower than in the control group. In conclusion, this approach holds promise for the cryopreservation of 'ready-to-use' TECs.

中文翻译：

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“空气中”冷冻对 Callithrix jacchus 间充质基质细胞解冻后恢复的影响和 3D 胶原-羟基磷灰石-支架的特性

通过在卫生部门按需高效供应细胞和组织，冷冻保存正日益成为再生医学研究快速转化和商业化的主流技术之一。组织工程构建体 (TEC) 的冷冻保存是一种新兴趋势，需要开发具有实际竞争力的生物样本库技术。在我们之前的研究中，我们证明使用二甲基亚砜 (Me2SO) 的常规慢速冷冻不能为冷冻在 3D 胶原-羟基磷灰石支架中的间充质基质细胞 (MSC) 提供足够的保护。在对冷冻保存方案进行简单修改后，我们报告了显着改善的 TEC 冷冻保存。多孔 3D 支架是使用矿化胶原悬浮液的冷冻干燥和随后的化学交联制造的。在静态条件下，将来自普通狨猴 Callithrix jacchus 的羊膜来源的 MSC 接种到支架上。细胞接种的支架用 100 mM 蔗糖进行 24 小时预处理，并在单独的 10% Me2SO/20% FBS 或补充有 300 mM 蔗糖中缓慢冷冻。支架在“空气中”冷冻并使用两步程序解冻。使用不同的分析方法来解释细胞接种和无细胞支架的冷冻保存结果。在两组中，细胞在解冻后都表现出其典型的形状和保存完好的细胞-细胞和细胞-基质接触。而且，解冻后 24 小时的生存力测试表明，与单独使用 Me2SO (60%) 相比，在冷冻保护溶液中应用蔗糖可保留显着更多的蔗糖预处理细胞 (超过 80%)。冷冻支架的整体蛋白质结构和孔隙率没有差异，而它们的压缩应力低于对照组。总之，这种方法有望用于“即用型”TEC 的低温保存。