Moghaddam Sevil Vaghefi, Abedi Fatemeh, Lotfi Hajie, Salehi Roya, Barzegar Abolfazl, Eslaminejad Mohamadreza Baghaban, Khalili Mostafa, Alizadeh Effat
Clinical Research Development, Unit of Tabriz Valiasr Hospital, Tabriz University of Medical Sciences, Tabriz, Iran.
Cellular and Molecular Research Center, Research Institute for Prevention of Non-Communicable Diseases, Qazvin University of Medical Sciences, Qazvin, Iran.
J Biol Eng. 2023 Apr 6;17(1):27. doi: 10.1186/s13036-023-00346-8.
Utilizing both medium enrichment and a thermos-responsive substrate to maintain the cell-to-cell junctions and extracellular matrix (ECM) intact, cell sheet technology has emerged as a ground-breaking approach. Investigating the possibility of using sodium selenite (as medium supplementation) and PCL-PEG-PCL (as vessel coating substrate) in the formation of the sheets from rat bone marrow-derived mesenchymal stem cells (rBMSCs) was the main goal of the present study. To this end, first, Polycaprolactone-co-Poly (ethylene glycol)-co-Polycaprolactone triblock copolymer (PCEC) was prepared by ring-opening copolymerization method and characterized by FTIR, H NMR, and GPC. The sol-gel-sol phase transition temperature of the PCEC aqueous solutions with various concentrations was either measured. Next, rBMSCs were cultured on the PCEC, and let be expanded in five different media containing vitamin C (50 µg/ml), sodium selenite (0.1 µM), vitamin C and sodium selenite (50 µg/ml + 0.1 µM), Trolox, and routine medium. The proliferation of the cells exposed to each material was evaluated. Produced cell sheets were harvested from the polymer surface by temperature reduction and phenotypically analyzed via an inverted microscope, hematoxylin and eosin (H&E) staining, and field emission scanning electron microscopy (FESEM). Through the molecular level, the expression of the stemness-related genes (Sox2, Oct-4, Nanog), selenium-dependent enzymes (TRX, GPX-1), and aging regulator gene (Sirt1) were measured by q RT-PCR. Senescence in cell sheets was checked by beta-galactosidase assay. The results declared the improved ability of the rBMSCs for osteogenesis and adipogenesis in the presence of antioxidants vitamin C, sodium selenite, and Trolox in growth media. The data indicated that in the presence of vitamin C and sodium selenite, the quality of the cell sheet was risen by reducing the number of senescent cells and high transcription of the stemness genes. Monolayers produced by sodium selenite was in higher-quality than the ones produced by vitamin C.
利用中等浓度富集和热响应性底物来保持细胞间连接和细胞外基质（ECM）完整，细胞片技术已成为一种开创性的方法。本研究的主要目标是研究使用亚硒酸钠（作为培养基补充剂）和聚己内酯-聚乙二醇-聚己内酯（PCL-PEG-PCL，作为血管包被底物）从大鼠骨髓间充质干细胞（rBMSCs）形成细胞片的可能性。为此，首先通过开环共聚法制备了聚己内酯-聚（乙二醇）-聚己内酯三嵌段共聚物（PCEC），并通过傅里叶变换红外光谱（FTIR）、核磁共振氢谱（¹H NMR）和凝胶渗透色谱（GPC）对其进行了表征。测定了不同浓度的PCEC水溶液的溶胶-凝胶-溶胶相变温度。接下来，将rBMSCs接种在PCEC上，并在含有维生素C（50 μg/ml）、亚硒酸钠（0.1 μM）、维生素C和亚硒酸钠（50 μg/ml + 0.1 μM）、Trolox以及常规培养基的五种不同培养基中进行扩增。评估了暴露于每种材料的细胞的增殖情况。通过降低温度从聚合物表面收获产生的细胞片，并通过倒置显微镜、苏木精和伊红（H&E）染色以及场发射扫描电子显微镜（FESEM）进行表型分析。在分子水平上，通过实时定量聚合酶链反应（q RT-PCR）测定干性相关基因（Sox2、Oct-4、Nanog）、硒依赖性酶（TRX、GPX-1）和衰老调节基因（Sirt1）的表达。通过β-半乳糖苷酶测定检查细胞片中的衰老情况。结果表明，在生长培养基中存在抗氧化剂维生素C、亚硒酸钠和Trolox的情况下，rBMSCs的成骨和成脂能力得到了提高。数据表明，在维生素C和亚硒酸钠存在的情况下，通过减少衰老细胞数量和干性基因的高转录，细胞片的质量得到了提高。亚硒酸钠产生的单层细胞质量高于维生素C产生的单层细胞。
