Kappler Benjamin, Anic Petra, Becker Matthias, Bader Andreas, Klose Kristin, Klein Oliver, Oberwallner Barbara, Choi Yeong-Hoon, Falk Volkmar, Stamm Christof
Berlin-Brandenburg Center for Regenerative Therapies (BCRT), Charité - Universitätsmedizin Berlin, Berlin, Germany.
DZHK (German Centre for Cardiovascular Research), Partner Site Berlin, Berlin, Germany.
J Mater Sci Mater Med. 2016 Jul;27(7):120. doi: 10.1007/s10856-016-5730-5. Epub 2016 Jun 7.
Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66 μm (15-157 μm) by laser diffraction, and 57 μm (20-182 μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39 ± 12 %, P < 0.05) and proliferation (16 ± 3 %, P < 0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240 ± 32 %; BrdU uptake, 45 ± 14 %; LDH release, -72 ± 7 %; P < 0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25 °C, 1 mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.
新鲜分离的人心脏细胞外基质片层（cECM）已被证明可支持干细胞增殖和组织特异性谱系定向分化。我们现在开发了一种方案，用于标准化生产耐用的、具有生物功能的人cECM微粒和相应的水凝胶，并测试了其对处于缺血样条件下的收缩细胞的细胞保护作用。通过三步方案对人心室心肌进行去细胞处理，包括Tris/EDTA、SDS和血清孵育（cECM）。经过速冻和冻干后，制备微粒并通过激光衍射、动态图像分析（DIA）和质谱进行表征。此外，通过胃蛋白酶消化制备cECM水凝胶。使用小鼠HL-1心肌细胞测定基线细胞支持特性，并在缺氧和葡萄糖/血清剥夺条件下测试ECM产品的细胞保护作用。在cECM中，糖蛋白（血小板反应蛋白1、纤连蛋白、胶原蛋白和巢蛋白-1）和蛋白聚糖（皮肤桥蛋白、光蛋白和 mimecan）得以保留，但也检测到残留的细胞内和血源性蛋白质。通过激光衍射测定，微粒的中位费雷特直径为66μm（15 - 157μm），通过DIA和结晶紫染色测定为57μm（20 - 182μm）。在常氧条件下，HL-1细胞在cECM微粒上生长时，代谢活性增强（39±12%，P<0.05），增殖增加（16±3%，P<0.05）。在模拟缺血期间，cECM微粒发挥了明显的细胞保护作用（MTS转化，240±32%；BrdU摄取，45±14%；LDH释放，-72±7%；各P<0.01）。当cECM微粒溶解形成水凝胶时，细胞保护作用最初消失。然而，修改制备过程（在pH 2和25°C下用胃蛋白酶消化，最终cECM浓度为1mg/ml）恢复了cECM的细胞保护活性。人心肌的细胞外基质可进行处理，以产生标准化的耐用微粒，对心肌样细胞发挥特定的细胞保护作用。使用经过处理的cECM可能有助于优化未来的临床级心肌组织工程方法。
