Krüger-Genge A, Fuhrmann R, Jung F, Franke R P
Institute of Biomaterial Science and Berlin-Brandenburg Center for Regenerative Therapies, Helmholtz-Zentrum Geesthacht, Teltow, Germany.
Abteilung Biomaterialien, Zentralinstitut für Biomedizinische Technik, Universität Ulm, Albert-Einstein-Allee, Ulm, Germany.
Clin Hemorheol Microcirc. 2015;61(2):151-6. doi: 10.3233/CH-151992.
The evaluation of the interaction of human, venous endothelial cells (HUVEC) with body foreign materials on the cellular level cannot be performed in vivo, but is investigated in vitro under standard culture conditions. To maintain the vitality, proliferation and morphology of HUVEC seeded on body foreign substrates over days, the cell culture medium is usually exchanged every second day. It is well known, that alterations in the microenvironment of cells bear the risk of influencing cell morphology and function. In the current study the influence of cell culture medium exchange on HUVEC cytoskeletal microfilament structure and function was investigated.
HUVEC in the third passage were seeded on extracellular matrix (ECM) - which was secreted from bovine corneal endothelial cells on glass- until functional confluence was reached. The experiment started 11 days after HUVEC seeding with an exchange of the cell culture medium followed by a staining of the actin microfilaments with phalloidin-rhodamin 1.5 and 5 minutes after medium exchange. The microfilaments were documented by use of an Olympus microscope (IMT-2) equipped with a UV lamp and online connected to a TV chain (Sony XC 50 ST/monochrome) implying an OPTIMAS - Image analysis system. Prostacyclin was analysed in the cell culture supernatant.
1.5 min after culture medium exchange in the functionally confluent cultures a slight disturbance of the actin microfilament structure with a broadening of the marginal filament band, a partial disconnection of cell-cell contacts and the appearance of intercellular fenestrations were observed. 5 minutes after medium exchange a redevelopment of the slightly disturbed microfilament structure with a condensation and narrowing of the marginal filament band was seen. 12 h later a further consolidation of the microfilament structure occurred. In addition, a perturbation of the cultured HUVEC occurred after cell culture medium exchange. The prostacyclin concentration in the supernatant increased significantly after 1.5 min to 466 ± 543 pg·mL-1 (p < 0.001) and after 5 min to 408 ± 458 pg·mL-1 (p < 0.001), while in control cells the prostacyclin concentration did not change remaining in the range of 50 ± 48.9 pg·mL-1.
This study revealed that the exchange of the cell culture medium led to a rapid disturbance of the HUVEC with stress fiber formation, disconnection of cell-cell contacts and an altered prostacyclin secretion, which had regressed nearly completely after 12 hours. Therefore, the evaluation of HUVEC on body foreign materials should be performed not earlier than 12 hours after cell culture medium exchange to avoid a misinterpretation of the endothelial cell morphological state. This procedure minimizes the risk of a misinterpretation of the endothelial cell morphology - caused by the culture medium exchange and not by the interaction between biomaterials and HUVEC.
在细胞水平上评估人体静脉内皮细胞（HUVEC）与体内异物的相互作用无法在体内进行，而是在标准培养条件下于体外进行研究。为了在数天内维持接种在体内异物底物上的HUVEC的活力、增殖和形态，通常每隔一天更换一次细胞培养基。众所周知，细胞微环境的改变有影响细胞形态和功能的风险。在本研究中，研究了更换细胞培养基对HUVEC细胞骨架微丝结构和功能的影响。
将第三代HUVEC接种于细胞外基质（ECM）上，该细胞外基质由牛角膜内皮细胞分泌于玻璃上，直至达到功能汇合。在接种HUVEC 11天后开始实验，更换细胞培养基，然后在更换培养基后1.5分钟和5分钟用鬼笔环肽-罗丹明对肌动蛋白微丝进行染色。使用配备紫外灯并在线连接到电视链（索尼XC 50 ST/单色）的奥林巴斯显微镜（IMT-2）记录微丝，这意味着使用了OPTIMAS图像分析系统。分析细胞培养上清液中的前列环素。
在功能汇合的培养物中更换培养基1.5分钟后，观察到肌动蛋白微丝结构略有紊乱，边缘丝带变宽，细胞间接触部分断开，出现细胞间窗孔。更换培养基5分钟后，可见微丝结构略有紊乱后重新发展，边缘丝带凝聚变窄。12小时后微丝结构进一步巩固。此外，更换细胞培养基后培养的HUVEC出现扰动。更换培养基1.5分钟后，上清液中前列环素浓度显著增加至466±543 pg·mL-1（p<0.001），5分钟后增加至408±458 pg·mL-1（p<0.001），而对照细胞中前列环素浓度未发生变化，保持在50±48.9 pg·mL-1范围内。
本研究表明，更换细胞培养基会导致HUVEC迅速受到干扰，形成应力纤维，细胞间接触断开，前列环素分泌改变，12小时后几乎完全恢复。因此，对体内异物上的HUVEC进行评估应在更换细胞培养基12小时后进行，以避免对内皮细胞形态状态的错误解读。该程序可将因更换培养基而非生物材料与HUVEC之间的相互作用导致的内皮细胞形态错误解读的风险降至最低。
