Fraunhofer Project Group PZ-Syn of the Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (IZI-BB), 14476 Potsdam-Golm, Germany, Institute of Biotechnology, Brandenburg University of Technology Cottbus-Senftenberg, 01968 Brandenburg, Germany.
Institute of Active Polymers and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, 14513 Teltow, Germany.
Int J Mol Sci. 2021 Feb 2;22(3):1493. doi: 10.3390/ijms22031493.
The adherence and shear-resistance of human umbilical venous endothelial cells (HUVEC) on polymers is determined in vitro in order to qualify cardiovascular implant materials. In these tests, variable fractions of HUVEC do not adhere to the material but remain suspended in the culture medium. Nonadherent HUVEC usually stop growing, rapidly lose their viability and can release mediators able to influence the growth and function of the adherent HUVEC. The aim of this study was the investigation of the time dependent behaviour of HUVEC under controlled nonadherent conditions, in order to gain insights into potential influences of these cells on their surrounding environment in particular adherent HUVEC in the context of in vitro biofunctionality assessment of cardiovascular implant materials. Data from adherent or nonadherent HUVEC growing on polystyrene-based cell adhesive tissue culture plates (TCP) or nonadhesive low attachment plates (LAP) allow to calculate the number of mediators released into the culture medium either from adherent or nonadherent cells. Thus, the source of the inflammatory mediators can be identified. For nonadherent HUVEC, a time-dependent aggregation without further proliferation was observed. The rate of apoptotic/dead HUVEC progressively increased over 90% within two days. Concomitant with distinct blebbing and loss of membrane integrity over time, augmented releases of prostacyclin (PGI2, up to 2.91 ± 0.62 fg/cell) and platelet-derived growth factor BB (PDGF-BB, up to 1.46 ± 0.42 fg/cell) were detected. The study revealed that nonadherent, dying HUVEC released mediators, which can influence the surrounding microenvironment and thereby the results of in vitro biofunctionality assessment of cardiovascular implant materials. Neglecting nonadherent HUVEC bears the risk for under- or overestimation of the materials endothelialization potential, which could lead to the loss of relevant candidates or to uncertainty with regard to their suitability for cardiac applications. One approach to minimize the influence from nonadherent endothelial cells could be their removal shortly after observing initial cell adhesion. However, this would require an individual adaptation of the study design, depending on the properties of the biomaterial used.
为了对心血管植入材料进行质量鉴定，我们在体外确定了人脐静脉内皮细胞（HUVEC）在聚合物上的黏附性和抗剪切能力。在这些测试中，部分 HUVEC 没有黏附在材料上，而是悬浮在培养基中。未黏附的 HUVEC 通常停止生长，迅速失去活力，并可释放能够影响黏附 HUVEC 生长和功能的介质。本研究的目的是在受控的非黏附条件下研究 HUVEC 的时间依赖性行为，以便深入了解这些细胞对其周围环境的潜在影响，特别是在心血管植入材料体外生物功能评估中对黏附的 HUVEC 的影响。在基于聚苯乙烯的细胞黏附组织培养板（TCP）或非黏附低附着板（LAP）上生长的黏附或非黏附 HUVEC 产生的数据可计算出培养基中释放的介质数量，这些介质分别来自黏附或非黏附细胞。因此，可以确定炎症介质的来源。对于非黏附的 HUVEC，观察到无进一步增殖的时间依赖性聚集。在两天内，凋亡/死亡 HUVEC 的比例逐渐增加到 90%以上。随着时间的推移，出现明显的起泡和膜完整性丧失，同时检测到前列环素（PGI2，高达 2.91±0.62 fg/细胞）和血小板衍生生长因子 BB（PDGF-BB，高达 1.46±0.42 fg/细胞）的释放增加。该研究表明，非黏附、濒死的 HUVEC 释放介质，可影响周围的微环境，从而影响心血管植入材料体外生物功能评估的结果。忽略非黏附的 HUVEC 有低估或高估材料内皮化潜力的风险，这可能导致相关候选物的丢失，或对其是否适用于心脏应用的不确定性。减少非黏附内皮细胞影响的一种方法是在观察到初始细胞黏附后不久将其去除。然而，这需要根据所使用的生物材料的特性，对研究设计进行单独调整。
