The Keck Center for Tissue Engineering, Department of Bioengineering, College of Engineering, University of Utah, 20 S 2030 E Building, 570 BPRB, Room 108D, Salt Lake City, UT 84112, USA.
Biomaterials. 2010 Dec;31(36):9595-603. doi: 10.1016/j.biomaterials.2010.08.072. Epub 2010 Oct 14.
Extracellular matrix derived from human and animal tissues is being used to repair and reconstruct a variety of tissues clinically. The utility of such constructs is limited by the geometry, composition and constitutive properties of the tissue or organ from which the ECM is harvested. To address this limitation, we have developed an approach to isolate extracellular matrix in bulk from populations of living cells grown in culture on three-dimensional substrates. Human biopsy derived fibroblasts were seeded within open-cell foams and cultured in-vitro for periods up to three weeks, after which the synthetic component was removed by incubation in a water miscible solvent. After several wash steps and lyophilization, a white, lacy, multi-molecular construct was isolated. Tandem mass spectroscopy showed that it contained 22 extracellular matrix constituents, including such proteins and proteoglycans as collagen type I and type III, fibronectin, transforming growth factor beta, decorin and biglycan among others. On average 47 mg of construct was isolated for each gram of synthetic substrate initially seeded with cells. The biomaterial harvested from human tracheal fibroblasts had an elastic modulus (250 kPa) and a composition similar to that of human vocal fold tissue, and supported reseeding with human tracheal derived fibroblasts. An important finding was that the approach was useful in isolating ECM from a variety of cell lineages and developmental stages including skin fibroblasts, brain derived astrocytes and mesenchymal stem cells. The results, together with the archival literature, suggest that the approach can be used to produce a range of cell derived constructs with unique physical and chemical attributes for a variety of research and medical applications.
从人和动物组织中提取的细胞外基质，正被用于临床修复和重建各种组织。然而，这些构建物的应用受到组织或器官的几何形状、组成和本构特性的限制。为了解决这一限制，我们开发了一种从三维基质上培养的活细胞群体中批量分离细胞外基质的方法。将人活检来源的成纤维细胞接种在开孔泡沫中，并在体外培养长达三周，之后通过在可与水混溶的溶剂中孵育来去除合成物。经过几次洗涤和冻干步骤后，分离出一种白色、多分子的花边状结构。串联质谱分析显示，它包含 22 种细胞外基质成分，包括胶原蛋白 I 型和 III 型、纤连蛋白、转化生长因子β、核心蛋白聚糖和 biglycan 等蛋白质和蛋白聚糖。平均而言，每克最初接种细胞的合成底物可分离出 47 毫克的构建物。从人气管成纤维细胞中收获的生物材料具有弹性模量（250kPa）和组成，类似于人声带组织，并支持人气管衍生的成纤维细胞重新接种。一个重要的发现是，该方法可用于从多种细胞谱系和发育阶段（包括皮肤成纤维细胞、脑源性星形胶质细胞和间充质干细胞）中分离细胞外基质。这些结果，结合存档文献，表明该方法可用于生产一系列具有独特物理和化学特性的细胞衍生构建物，适用于各种研究和医疗应用。
