制备纤维蛋白三维支架干细胞培养应用
Summary
这项工作的具体编制三维纤维支架plutipotent干细胞培养和分化。这种支架可用于筛选各种生物化合物对干细胞的行为的影响，以及修改为包含药物输送系统。
Abstract
干细胞被发现在三维微环境中自然产生的体内 ，它通常被称为干细胞小生境1。三维生物材料支架内培养干细胞提供了一种方法，准确地模仿这些微环境，提供文化，用聚苯乙烯以及更换工程的组织2的方法比传统的2D方法的优势。虽然已为大多数细胞培养实验中使用的二维组织培养聚苯乙烯，三维生物材料支架可以更密切地复制在体内环境中，使更准确的细胞极性建立和拥有生化和机械性能类似于软组织的微环境。 3一个多种自然派生和合成生物材料支架已被用于支持干细胞生长的3D环境调查。虽然合成支架材料可以合成有一个更大的ŕ安格机械和化学性能，往往有更大的重复性，通常由天然生物材料在细胞外基质含有细胞粘附的结合位点，并随时支持细胞培养的结果发现蛋白质和多糖。纤维蛋白支架，通过聚合获得的血浆蛋白纤维蛋白原生产，已被广泛研究了各种组织工程的应用， 在体外和体内 4。这种支架可以修改使用多种方法纳入控释系统提供治疗的因素5。以前的工作表明，这种支架可以用来成功地培养胚胎干细胞，并基于此脚手架的文化系统，可用于筛选内6,7种子的干细胞分化的各种生长因子的影响。
该协议的细节过程polymerizinĞ纤维支架纤维蛋白原使用凝血酶酶活性的解决方案。这个过程需要2天才能完成，包括纤维蛋白原的解决方案，以消除，抑制聚合的柠檬酸隔夜透析的步骤。这些详细的方法依赖于胚胎和诱导多能干细胞培养确定为最佳的纤维蛋白原浓度。其他团体作进一步调查的细胞类型和应用范围广泛的纤维蛋白支架-证明了这种方法的8-12的多功能性。
Protocol
Discussion
上面详细介绍该协议产生的多能干细胞培养小鼠胚胎和诱导多能干细胞的三维纤维支架，专门提供了一个方法。这三维生物材料为基础的文化系统，更准确地模仿体内发现的干细胞小生境，作为一个结果，它可以用来筛选生物的线索，以确定对干细胞分化6的影响。我们的观察表明，干胚体细胞在体外培养2周保持完全降解之前，播种时，这些支架。为了进一步提高这些支架的稳定，…
Divulgations
The authors have nothing to disclose.
Acknowledgements
笔者想承认“诱导多能干细胞的行为控制的组织工程支架材料”NSERC发现格兰特402462。
Materials
Equipment needed:
Analytical balance
pH meter
Tissue culture incubator (37°C, 5% CO2)
Stir plate
Spectrophotometer
Sterile tissue culture hood
Tris buffered saline (pH 7.4) (Need 4 L plus enough for dissolving fibrinogen)
50 mM calcium chloride solution
Sterile conical tubes (15 or 50 mL)
35 mm Petri dishes
Dialysis tubing (7,000 MW cutoff)
Dialysis clips
5.0 μm syringe filters
Individually wrapped sterile 0.22 μm syringe filters
Syringe
24 well tissue culture plates
| Name of the reagent | Company | Catalogue number | Comments |
| Fibrinogen (human) | Calbiochem | 341578 | |
| Thrombin (human) | Sigma | T7009 |
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