Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford, United Kingdom.
J Biomed Mater Res A. 2011 Nov;99(2):211-20. doi: 10.1002/jbm.a.33177. Epub 2011 Aug 16.
A method to functionalize alginate by introducing monomeric or self-assembling (tetrameric) fibronectin (FN) domains is described, leading to a functional scaffold, which is used for three dimensional (3D) culture of human endometrial stromal cells (EnSCs). EnSCs encapsulated in the functional alginate were cultured under perfusion using the TissueFlex® platform, a multiple parallel microbioreactor system for 3D cell culture. The effect of the novel scaffold and the effect of perfusion were examined. Cell viability, proliferation, and extracellular matrix (ECM) deposition were determined and the results compared with those obtained with cells encapsulated in non-functionalized alginate, and also those without perfusion. Staining for focal adhesions and actin showed maximal cell adhesion only for alginate-tetrameric FN scaffolds under perfusion, associated with a significant increase in cell number over 7 days culture; in contrast to poor cell adhesion and a decrease in cell number for non-functionalized alginate scaffolds (irrespective of perfused/static culture) and 3D static culture (irrespective of the scaffold). Conjugation of alginate to FN was an absolute requirement to attenuate the loss of cell metabolic activity over 7 days culture. ECM deposition for blank alginate and alginate-monomeric FN was similar, but increased around 2-fold and 3-fold for alginate-tetrameric FN under static and perfusion culture, respectively. It is concluded that the requirement for EnSC engagement with multivalent integrin α5β1 ligands and perfused culture are both essential as a first step toward endometrial tissue engineering.
一种通过引入单体或自组装（四聚体）纤维连接蛋白（FN）结构域对海藻酸钠进行功能化的方法被描述，从而得到一种功能性支架，用于三维（3D）培养人子宫内膜基质细胞（EnSCs）。EnSCs 被包裹在功能性海藻酸钠中的细胞在 TissueFlex®平台下进行灌流培养，该平台是一种用于 3D 细胞培养的多平行微反应器系统。研究了新型支架的作用和灌流的作用。测定了细胞活力、增殖和细胞外基质（ECM）沉积，并将结果与包裹在非功能化海藻酸钠中的细胞以及无灌流的细胞进行比较。对焦点粘连和肌动蛋白的染色显示，只有在灌注下，海藻酸钠-四聚体 FN 支架才能实现最大的细胞黏附，同时在 7 天的培养过程中细胞数量显著增加；与非功能化海藻酸钠支架（无论灌流/静态培养）和 3D 静态培养（无论支架如何）相比，细胞黏附不良，细胞数量减少形成鲜明对比。海藻酸钠与 FN 的缀合是减轻 7 天培养过程中细胞代谢活性丧失的绝对要求。空白海藻酸钠和海藻酸钠-单体 FN 的 ECM 沉积相似，但在静态和灌注培养下，海藻酸钠-四聚体 FN 的 ECM 沉积分别增加了约 2 倍和 3 倍。结论是，与多价整合素 α5β1 配体的 EnSC 结合和灌流培养的要求都是子宫内膜组织工程的必要前提。
