Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels
Fine-tuning of a three-dimensional microcarrier-based angiogenesis assay for the analysis of endothelial-mesenchymal cell co-cultures in fibrin and collagen gels
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DOI：
10.1007/s10456-006-9037-x
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发表时间：
2006-01-01
期刊：
影响因子：
9.8
通讯作者：
Lelkes, Peter I.
中科院分区：
文献类型：
--
作者：
Dietrich, Franziska;Lelkes, Peter I.
成功的组织工程的先决条件是功能性微血管网络的存在。我们假设，这种网络可以创建和量化在体外设置共培养内皮细胞（EC）与组织特异性的“旁观者细胞”在3-D凝胶矩阵。为了检验这一假设，我们采用了先前描述的基于微载体的体外血管生成测定（V. Nehls和D. Drenckhahn，1995，Microvasc Res 50：311-322）。在优化该测定时，我们注意到初始EC微载体覆盖率取决于EC类型和用于用EC包被微载体珠的接种技术。只有当牛主动脉内皮细胞（BAECs）在轨道摇床上轻轻搅拌下混合到微载体表面的汇合EC单层。在将BAEC覆盖的微载体珠嵌入到胶原蛋白或纤维蛋白凝胶的类胶原蛋白排列中后，我们评估了在不同血清浓度下的细胞生长的迁移距离和芽形成。胶原基质中1%胎牛血清（FBS）和纤维蛋白基质中0.1% FBS时可定量的芽形成最高。在较高的血清浓度下，过量的细胞迁移和簇的形成阻止了发芽的定量分析。在对血管生成试验进行微调后，我们将BAEC与脂肪组织来源的成纤维细胞（FB）和血管平滑肌细胞（SMC）共培养。虽然FB能够增加BAEC在两种基质中的平均迁移距离，但SMC增强了BAEC在纤维蛋白中的迁移，而不是在胶原凝胶中。相比之下，两种细胞类型的纤维蛋白凝胶中新形成的芽的数量增加。我们的结论是，在这个模型中，旁观者细胞增强EC网络的形成在一个矩阵依赖的方式。此外，这些结果强调了仔细选择给定体外血管生成模型的实验参数的重要性。
A prerequisite for successful tissue engineering is the existence of a functional microvascular network. We hypothesized that such networks can be created and quantified in an in vitro setting by co-culturing endothelial cells (ECs) with tissue-specific 'by-stander cells' in 3-D gel matrices. To test this hypothesis we adapted a previously described in vitro microcarrier-based angiogenesis assay (V. Nehls and D. Drenckhahn, 1995, Microvasc Res 50: 311-322). On optimizing this assay, we noted that the initial EC-microcarrier coverage depended on EC type and seeding technique employed to coat the microcarrier beads with the ECs. A confluent EC monolayer on the microcarrier surfaces formed only when bovine aortic endothelial cells (BAECs) were admixed to the beads under gentle agitation on an orbital shaker. After embedding BAEC-covered microcarrier beads into a sandwich-like arrangement of collagen or fibrin gels, we assessed cellular outgrowth at different serum concentrations in terms of migration distance and sprout formation. Quantifiable sprout formation was highest at 1% fetal bovine serum (FBS) in collagen matrices and at 0.1% FBS in fibrin matrices. At higher serum concentration, excess cell migration and formation of clusters prevented quantitative analysis of sprouting. Following the fine-tuning of this angiogenesis assay, we co-cultured BAECs with adipose tissue-derived fibroblasts (FBs) and vascular smooth muscle cells (SMCs). While FBs were able to increase the average migration distance of BAECs in both matrices, SMCs enhanced BAEC migration in fibrin, but not in collagen gels. By contrast, the number of newly formed sprouts in fibrin gels was increased by both cell types. We conclude that in this model bystander cells enhance EC network formation in a matrix-dependent manner. Additionally, these results stress the importance of carefully selecting the experimental parameters of a given in vitro angiogenesis model.