Karkampouna Sofia, Kloen Peter, Obdeijn Miryam C, Riester Scott M, van Wijnen Andre J, Kruithof-de Julio Marianna
Department of Molecular Cell Biology, Cancer Genomics Centre and Centre for Biomedical Genetics, Leiden University Medical Center.
Department of Orthopedic Surgery, Academic Medical Center.
J Vis Exp. 2015 Apr 18(98):52534. doi: 10.3791/52534.
Organ fibrosis or "scarring" is known to account for a high death toll due to the extensive amount of disorders and organs affected (from cirrhosis to cardiovascular diseases). There is no effective treatment and the in vitro tools available do not mimic the in vivo situation rendering the progress of the out of control wound healing process still enigmatic. To date, 2D and 3D cultures of fibroblasts derived from DD patients are the main experimental models available. Primary cell cultures have many limitations; the fibroblasts derived from DD are altered by the culture conditions, lack cellular context and interactions, which are crucial for the development of fibrosis and weakly represent the derived tissue. Real-time PCR analysis of fibroblasts derived from control and DD samples show that little difference is detectable. 3D cultures of fibroblasts include addition of extracellular matrix that alters the native conditions of these cells. As a way to characterize the fibrotic, proliferative properties of these resection specimens we have developed a 3D culture system, using intact human resections of the nodule part of the cord. The system is based on transwell plates with an attached nitrocellulose membrane that allows contact of the tissue with the medium but not with the plastic, thus, preventing the alteration of the tissue. No collagen gel or other extracellular matrix protein substrate is required. The tissue resection specimens maintain their viability and proliferative properties for 7 days. This is the first "organ" culture system that allows human resection specimens from DD patients to be grown ex vivo and functionally tested, recapitulating the in vivo situation.
器官纤维化或“瘢痕形成”因影响大量疾病和器官（从肝硬化到心血管疾病）而导致高死亡率。目前尚无有效的治疗方法，现有的体外工具无法模拟体内情况，使得失控的伤口愈合过程的进展仍然成谜。迄今为止，来自杜普伊特伦挛缩症（DD）患者的成纤维细胞的二维和三维培养是主要可用的实验模型。原代细胞培养有许多局限性；从DD患者获得的成纤维细胞会因培养条件而改变，缺乏细胞背景和相互作用，而这些对于纤维化的发展至关重要，并且不能很好地代表所衍生的组织。对来自对照和DD样本的成纤维细胞进行实时PCR分析表明，几乎检测不到差异。成纤维细胞的三维培养包括添加细胞外基质，这会改变这些细胞的天然状态。作为表征这些切除标本的纤维化、增殖特性的一种方法，我们开发了一种三维培养系统，使用完整的人类精索结节部分切除术标本。该系统基于带有附着硝酸纤维素膜的Transwell板，该膜允许组织与培养基接触，但不与塑料接触，从而防止组织改变。无需胶原蛋白凝胶或其他细胞外基质蛋白底物。组织切除标本在7天内保持其活力和增殖特性。这是第一个“器官”培养系统，它允许对来自DD患者的人类切除标本进行体外培养和功能测试，重现体内情况。
