Roosens Annelies, Puype Inès, Cornelissen Ria
Department of Basic Medical Sciences, Tissue Engineering Group, Ghent University, Ghent, Belgium.
J Mol Cell Cardiol. 2017 May;106:45-54. doi: 10.1016/j.yjmcc.2017.03.004. Epub 2017 Mar 18.
The major challenge of working with valvular interstitial cells in vitro is the preservation or recovery of their native quiescent state. In this study, a biomimetic approach is used which aims to engineer small volume, high quality valve microtissues, having a potential in regenerative medicine and as a relevant 3D in vitro model to provide insights into valve (patho)biology.
To form micro-aggregates, porcine valvular interstitial cells were seeded in agarose micro-wells and cultured in medium supplemented with 250μM Ascorbic Acid 2-phosphate for 22days. Histology showed viable aggregates with normal nuclei and without any signs of calcification. Aggregates stained strongly for GAG and collagen I and reticular fibers were present. ECM formation was quantified and showed a significant increase of GAG, elastin and Col I during aggregate culture. Cultivation of VIC in aggregates also promoted mRNA expression of Col I/III/V, elastin, hyaluronan, biglycan, decorin, versican MMP-1/2/3/9 and TIMP-2 compared to monolayer cultured VIC. Phenotype analysis of aggregates showed a significant decrease in α-SMA expression, and an increase in FSP-1 expression at any time point. Furthermore, VIC aggregates did not show a significant difference in OCN, Egr-1, Sox-9 or Runx2 expression.
In this study high quality valvular interstitial cell aggregates were generated that are able to produce their own ECM, resembling the native valve composition. The applied and completely cell driven 3D approach overcomes the problems of VIC activation in 2D, by downregulating α-SMA expression and stimulating a homeostatic quiescent VIC state.
体外培养瓣膜间质细胞的主要挑战在于维持或恢复其天然静止状态。在本研究中，采用了一种仿生方法，旨在构建小体积、高质量的瓣膜微组织，其在再生医学中具有潜力，并可作为相关的三维体外模型，以深入了解瓣膜（病理）生物学。
为形成微聚集体，将猪瓣膜间质细胞接种于琼脂糖微孔中，并在补充有250μM抗坏血酸2 - 磷酸的培养基中培养22天。组织学检查显示聚集体存活，细胞核正常，无任何钙化迹象。聚集体对糖胺聚糖和I型胶原染色强烈，且存在网状纤维。对细胞外基质形成进行了定量分析，结果显示在聚集体培养过程中糖胺聚糖、弹性蛋白和I型胶原显著增加。与单层培养的瓣膜间质细胞相比，聚集体中瓣膜间质细胞的培养还促进了I/III/V型胶原、弹性蛋白、透明质酸、双糖链蛋白聚糖、核心蛋白聚糖、多功能蛋白聚糖、基质金属蛋白酶-1/2/3/9和金属蛋白酶组织抑制因子-2的mRNA表达。聚集体的表型分析显示，在任何时间点，α - 平滑肌肌动蛋白表达显著降低，而FSP - 1表达增加。此外，瓣膜间质细胞聚集体在骨钙素、早期生长反应蛋白-1、Sox - 9或Runx2表达上无显著差异。
在本研究中，生成了高质量的瓣膜间质细胞聚集体，其能够产生自身的细胞外基质，类似于天然瓣膜组成。所应用的完全由细胞驱动的三维方法通过下调α - 平滑肌肌动蛋白表达并刺激稳态静止的瓣膜间质细胞状态，克服了二维培养中瓣膜间质细胞激活的问题。
