Vackova Irena, Vavrinova Eliska, Musilkova Jana, Havlas Vojtech, Petrenko Yuriy
Department of Biomaterials and Tissue Engineering, Institute of Physiology of Czech Academy of Science, 14220 Prague, Czech Republic.
Department of Neuroregeneration, Institute of Experimental Medicine of the Czech Academy of Sciences, 14220 Prague, Czech Republic.
Polymers (Basel). 2022 Jun 23;14(13):2553. doi: 10.3390/polym14132553.
The regulatory requirements in cell processing, in the choice of a biomaterial scaffold and in quality control analysis, have to be followed in the clinical application of tissue-engineered grafts. Confirmation of sterility during quality control studies requires prolonged storage of the cell-based construct. After storage, preservation of the functional properties of the cells is an important prerequisite if the cells are to be used for cell-based tissue therapies. The study presented here shows the generation of 3D constructs based on Wharton's jelly multipotent mesenchymal stromal cells (WJ-MSCs) and the clinically-acceptable HyaloFast scaffold, and the effect of two- and six-day hypothermic storage of 3D cell-based constructs on the functional properties of populated cells. To study the viability, growth, gene expression, and paracrine secretion of WJ-MSCs within the scaffolds before and after storage, xeno-free culture conditions, metabolic, qPCR, and multiplex assays were applied. The WJ-MSCs adhered and proliferated within the 3D HyaloFast. Our results show different viability of the cells after the 3D constructs have been stored under mild (25 °C) or strong (4 °C) hypothermia. At 4 °C, the significant decrease of metabolic activity of WJ-MSCs was detected after 2 days of storage, with almost complete cell loss after 6 days. In mild hypothermia (25 °C) the decrease in metabolic activity was less remarkable, confirming the suitability of these conditions for cell preservation in 3D environment. The significant changes were detected in gene expression and in the paracrine secretion profile after 2 and 6 days of storage at 25 °C. The results presented in this study are important for the rapid transfer of tissue engineering approaches into clinical applications.
在组织工程移植物的临床应用中，必须遵循细胞处理、生物材料支架选择以及质量控制分析方面的监管要求。在质量控制研究中确认无菌状态需要对基于细胞的构建体进行长时间储存。储存后，如果要将细胞用于基于细胞的组织治疗，保持细胞的功能特性是一个重要前提。本文介绍的研究展示了基于华通氏胶多能间充质基质细胞（WJ-MSCs）和临床可接受的HyaloFast支架生成三维构建体，以及对基于三维细胞的构建体进行两天和六天低温储存对植入细胞功能特性的影响。为了研究储存前后支架内WJ-MSCs的活力、生长、基因表达和旁分泌分泌情况，采用了无动物源培养条件、代谢分析、定量聚合酶链反应（qPCR）和多重分析方法。WJ-MSCs在三维HyaloFast内黏附并增殖。我们的结果显示，在温和（25°C）或强烈（4°C）低温条件下储存三维构建体后，细胞活力有所不同。在4°C时，储存2天后检测到WJ-MSCs的代谢活性显著下降，6天后几乎完全细胞丢失。在温和低温（25°C）下，代谢活性的下降不太明显，证实了这些条件适用于在三维环境中保存细胞。在25°C储存2天和6天后，基因表达和旁分泌分泌谱发生了显著变化。本研究结果对于将组织工程方法快速转化为临床应用具有重要意义。
