Dynamic seeding of perfusing human umbilical vein endothelial cells (HUVECs) onto dual-function cell adhesion ligands: Arg-Gly-Asp (RGD)-streptavidin and biotinylated fibronectin.
Dynamic seeding of perfusing human umbilical vein endothelial cells (HUVECs) onto dual-function cell adhesion ligands: Arg-Gly-Asp (RGD)-streptavidin and biotinylated fibronectin.
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DOI：
10.1021/la803963r
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发表时间：
2009-05-19
期刊：
影响因子：
--
通讯作者：
Reichert WM
中科院分区：
文献类型：
--
作者：
Anamelechi CC;Clermont EC;Novak MT;Reichert WM
用高亲和力配体修饰的表面可用于促进内皮细胞的快速附着;然而，标准的平衡细胞分离研究不太适合评估这些初始粘附事件。在这里，动态接种和细胞保留方法被用来检查灌注的人脐静脉内皮细胞（HUVECs）的初始附着裸露的聚四氟乙烯-AF基板，基板预吸附与纤连蛋白单独，或基板共预吸附两个双功能细胞粘附配体：生物素化纤连蛋白（bFN）和RGD-链霉亲和素突变体（RGD-SA）。细胞附着作为细胞胰蛋白酶消化（整合素消化），表面蛋白制剂，和细胞灌注率的函数进行评价。表面共预吸附bFN和RGD-SA显示最高密度的附着细胞灌注后8分钟和最高百分比保留时，受到剪切流在60达因/平方厘米2 2分钟。表面没有配体处理显示最低的细胞附着和保留在所有情况下的流动。用温和的0.025%胰蛋白酶/乙二胺四乙酸（EDTA）胰蛋白酶处理的HUVEC在灌注后显示出更大的细胞粘附，在剪切流后显示出更高的保留百分比。用表面等离子体共振（SPR）技术研究了这两个双功能配体对α5β1和αvβ3整合素的亲和力。动态细胞接种研究证实，双功能配体系统促进HUVEC的粘附和保留在短时间点时，使用灌注测定测试。SPR研究表明，这两种配体对α5β1和αvβ3整联蛋白表现出相同的亲和力，但组合的配体比单独测试的两种配体结合更多的总整联蛋白。
Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately.
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DOI：
10.1002/jbm.820250209
发表时间：
1991-02-01
期刊：
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH
影响因子：
--
作者：
MASSIA, SP;HUBBELL, JA
通讯作者：
HUBBELL, JA
影响因子：
56.9
作者：
MIYAMOTO, S;AKIYAMA, SK;YAMADA, KM
通讯作者：
YAMADA, KM
影响因子：
3.9
作者：
Anamelechi, Charles C.;Clermont, Edward E.;Reichert, William M.
通讯作者：
Reichert, William M.
影响因子：
3
作者：
Hynd, Matthew R.;Frampton, John P.;Shain, William
通讯作者：
Shain, William
影响因子：
7.5
作者：
Howe, A;Aplin, AE;Juliano, RL
通讯作者：
Juliano, RL