Cetinkaya Gaye, Türkoğlu Hilal, Arat Sezen, Odaman Hande, Onur Mehmet A, Gümüşderelioğlu Menemşe, Tümer Aşkin
Department of Biology, Hacettepe University, Beytepe, Ankara, Turkey.
J Biomed Mater Res A. 2007 Jun 15;81(4):911-9. doi: 10.1002/jbm.a.31107.
Embryonic stem (ES) cells have a great interest for tissue engineering because of their pluripotent nature and proliferative capacity. The objective of this study is to constitute a synthetic microenvironment to support the in vitro propagation of murine ES cells in an undifferentiated state. That is why we used a three-dimensional matrix, nonwoven polyester fabric (NWPF), which was formed from poly(ethylene terephthalate) (PET) fibers. NWPF discs were partially hydrolyzed, and then the carboxyl groups were coupled with leukemia inhibitory factor (LIF) in the presence of water-soluble carbodiimide. The effectiveness of immobilization process was checked with ATR-FTIR spectroscopy, fluorimetry, and cell culture studies. ES cell colony morphology, alkaline phosphatase (AP) activity, stage-specific embryonic antigen-1 (SSEA-1) immunoreactivity, and SEM analysis following a 72 - 96-h culture period upon hydrolyzed and LIF-immobilized surfaces were assessed to determine the pluripotent status of ES cells. Results revealed that LIF was active in immobilized form; undifferentiated colonies had not only a significant AP and SSEA-1 immunoreactivity, but also a higher undifferentiated colony ratio on LIF-immobilized surfaces than that of hydrolyzed surfaces. The immobilized LIF protein might be a good model to provide a feeder-free system, but the physical properties of the scaffold is more convenient for differentiation studies.
胚胎干细胞（ES细胞）因其多能性和增殖能力而在组织工程领域备受关注。本研究的目的是构建一个合成微环境，以支持小鼠ES细胞在体外以未分化状态进行增殖。因此，我们使用了一种三维基质——非织造聚酯织物（NWPF），它由聚对苯二甲酸乙二酯（PET）纤维制成。NWPF圆盘经过部分水解，然后在水溶性碳二亚胺存在的情况下，将羧基与白血病抑制因子（LIF）偶联。通过衰减全反射傅里叶变换红外光谱（ATR-FTIR）、荧光测定法和细胞培养研究来检查固定化过程的有效性。在水解并固定了LIF的表面上培养72 - 96小时后，评估ES细胞集落形态、碱性磷酸酶（AP）活性、阶段特异性胚胎抗原-1（SSEA-1）免疫反应性以及扫描电子显微镜（SEM）分析，以确定ES细胞的多能状态。结果表明，固定化形式的LIF具有活性；未分化集落在固定了LIF的表面上不仅具有显著的AP和SSEA-1免疫反应性，而且未分化集落比例高于水解表面。固定化的LIF蛋白可能是提供无饲养层系统的良好模型，但支架的物理性质更便于进行分化研究。
