Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells
Optimization of fibrin scaffolds for differentiation of murine embryonic stem cells into neural lineage cells
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DOI：
10.1016/j.biomaterials.2006.07.036
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发表时间：
2006-12-01
期刊：
影响因子：
14
通讯作者：
Sakiyama-Elbert, Shelly Elese
中科院分区：
文献类型：
--
作者：
Willerth, Stephanie M.;Arendas, Kelly J.;Sakiyama-Elbert, Shelly Elese
本研究的目的是通过检查细胞接种密度、位置和纤维蛋白原、凝血酶和抑肽酶（蛋白酶抑制剂）的最佳浓度，确定胚胎干细胞（ES）在纤维蛋白支架中增殖和分化的适当细胞培养条件。通过向胚状体（EB）培养物中加入视黄酸4天，诱导小鼠ES细胞成为神经祖细胞。对于解离的EB，最佳的细胞接种密度和位置被确定为250，000个细胞/cm（2），接种在纤维蛋白支架的顶部。对于完整的EB，三维（3D）文化与EB每400 μ L纤维蛋白支架导致更大的细胞增殖和分化比二维（2D）文化。支架聚合的最佳浓度为10 mg/mL纤维蛋白原和2 NIH单位/mL凝血酶。在3D纤维蛋白支架中，最佳抑肽酶浓度对于解离的EB（2D）确定为50 μ g/mL，对于完整的EB确定为5 μ g/mL。此外，在3D培养14天后，EB分化成神经元和星形胶质细胞，如通过荧光化学所指示的。这些条件提供了一个最佳的纤维蛋白支架评估ES细胞分化和增殖的文化，并作为一个平台，用于神经组织工程应用，如治疗脊髓损伤。(c)2006爱思唯尔有限公司保留所有权利。
The objective of this research was to determine the appropriate cell culture conditions for embryonic stem (ES) cell proliferation and differentiation in fibrin scaffolds by examining cell seeding density, location, and the optimal concentrations of fibrinogen, thrombin, and aprotinin (protease inhibitor). Mouse ES cells were induced to become neural progenitors by adding retinoic acid for 4 days to embryoid body (EB) cultures. For dissociated EBs, the optimal cell seeding density and location was determined to be 250,000 cells/cm(2) seeded on top of fibrin scaffolds. For intact EBs, three-dimensional (3D) cultures with one EB per 400 mu L fibrin scaffold resulted in greater cell proliferation and differentiation than two-dimensional (2D) cultures. Optimal concentrations for scaffold polymerization were 10 mg/mL of fibrinogen and 2 NIH units/mL of thrombin. The optimal aprotinin concentration was determined to be 50 mu g/mL for dissociated EBs (2D) and 5 mu g/mL for intact EBs in 3D fibrin scaffolds. Additionally, after 14 days in 3D culture EBs differentiated into neurons and astrocytes as indicated by immunohistochemisty. These conditions provide an optimal fibrin scaffold for evaluating ES cell differentiation and proliferation in culture, and for use as a platform for neural tissue engineering applications, such as the treatment for spinal cord injury. (c) 2006 Elsevier Ltd. All rights reserved.