人心脏组织对细胞外基质自组装水凝胶的处理体外和体内应用
1Charité – Universitätsmedizin Berlin,corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, 2Berlin-Brandenburg Center for Regenerative Therapies (BCRT), 3German Center for Cardiovascular Research (DZHK), 4Deutsches Herzzentrum Berlin (DHZB), 5Department of Cardiovascular Surgery, Charité – Universitätsmedizin Berlin,corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health
Summary
该协议描述了人类心肌的完整细胞, 同时保留了其胞外基质成分。细胞外基质的进一步处理导致微粒和细胞自组装水凝胶的生产。
Abstract
脱细胞细胞外基质制剂对研究细胞基质相互作用和促进再生细胞治疗的应用非常有用。一些商业胞外基质产品可作为水凝胶或膜, 但这些不具有组织特异性的生物活性。由于灌注细胞通常是不可能与人类心脏组织, 我们开发了3步浸泡细胞过程。在手术过程中获得的人心肌切片首先采用无洗涤剂高裂解缓冲液, 然后用离子洗涤剂、十二烷基硫酸钠进行孵化, 并利用其内在 dnasei 活性来完成。胎儿牛血清。这项技术的结果是在心脏细胞外基质的无细胞板, 主要保存纤维组织结构和生物成分, 这表明, 提供了具体的环境线索, 以心电池群体和多能干细胞细胞.心脏胞外基质片可进一步加工成微粒粉, 无需进一步化学修饰, 或者, 通过短期胃蛋白酶消化, 保存成自组装的心脏细胞外基质水凝胶。活性.
Introduction
细胞外基质 (ECM) 不仅提供结构支持, 而且对生物细胞和组织功能1也很重要。在心脏, ECM 参与调节病理反应, 如纤维化, 炎症, 血管生成, 心肌收缩功能和生存能力, 和居民祖细胞的命运。除了其主要成分-纤维糖蛋白, 多糖, 和蛋白-它包含了一系列分泌生长因子, 细胞因子, 和膜囊泡含有核酸和蛋白质2,3。
最近已经清楚的是, 脱细胞 ECM 制剂不仅对研究细胞基质相互作用, 而且对潜在的治疗细胞基础的应用是非常宝贵的。为治疗性细胞产品或工程组织提供适当环境的重要性现已得到广泛承认。试图结合细胞悬浮液或活性化合物与定义的 biopolymeric 凝胶4,5,6或与小鼠肉瘤细胞分泌的蛋白鸡尾酒 (即,基质, Geltrex)7. 然而, 前者的生物活性有限, 后者在 GMP 级别的过程中存在问题, 且缺乏心脏 ECM (cECM)8、9、10、的组织特异性生物活性.11,12,13。
心肌的细胞先前是通过冠状动脉血管灌注的整个心脏,14,15。虽然这是可能的在动物的心脏, 完整的人的心脏是很少可利用的。因此, 一个浸泡过程, 允许处理在手术室获得的组织样本受到青睐。我们的 “3 步” 协议包含3单独的孵化步骤, 即溶解, 增溶, 和 DNA 去除。它产生人心肌 ECM 与主要保存蛋白质和多糖构成16,17。这些 cECM 切片允许体外研究细胞-基质相互作用, 但不适合潜在的人类规模的治疗应用。然后, 制造过程被扩展到生产冻干 cECM 微粒或 cECM 水凝胶18。
该协议允许从外科标本中获得的人心肌细胞, 保留心肌细胞外基质 (ECM) 的主要成分及其生物活性。在细胞基质相互作用的实验研究中, 或在细胞基心肌再生方法需要合适的环境时, 推荐使用具有组织特异性生物活性的人心脏 ECM。原则上, 也可以将此协议与 GMP 级别的条件相适应, 以便在将来的治疗应用中使用经过处理的 cECM 是可行的。
Protocol
《研究议定书》符合《赫尔辛基宣言》所述的道德原则, 并得到了 charit 医科大学机构审查委员会和道德问题小组的批准。所有患者都提供了书面的、知情的同意, 以供实验研究使用心脏组织。 1. 人心肌切片的制备 获得左心室心肌 (大小取决于手术类型) 直接从手术室和运输在冷 PBS 在一个无菌容器。 在无菌条件下, 用无菌手术刀将所有脂肪组织取出10刀片。 …
Representative Results
3步的人心肌细胞的协议, 导致了近完全去除细胞材料, 同时保留了关键的 ecm 成分和纤维结构的 ecm。细胞后, 通过颜色的变化 (图 1A), 明显去除组织中的细胞。组织学分析与 H & E 和马尾松三污渍显示完全没有剩余完整的细胞 (图 1B)。单个 ECM 成分的定量分析显示, 单独的 DNA 清除和更好地保存总胶原蛋白、弹力蛋白和多糖, 与 SDS (<…
Discussion
在制备人体心肌 ECM 时, 其目的是实现以下目标: 去除相关的免疫细胞材料, 保护 ecm 的完整性和生物活性, 不育, 最终产品的非毒性, GMP-过程相容性, 以及产品在处理方面的适用性。将我们的3步细胞协议与进一步加工成微粒或自组装水凝胶结合, 获得了具有特定生物活性的人体心脏 ECM 材料, 易于处理, 并具有多个应用程序在体外和在体内, 类似于从几个动物种类中显示的 ECM 产品<sup class=…
Disclosures
The authors have nothing to disclose.
Acknowledgements
《研究议定书》符合《赫尔辛基宣言》中概述的道德原则。患者提供了知情同意的使用组织的研究目的, 和组织收集的过程是批准的机构审查委员会和伦理委员会 charit-Universitätsmedizin 柏林 (EA4/028/12)。
Materials
| Balance | DR Precisa, Dietikon, Switzerland | Precisa XR 205SM | |
| Blades Nr.10 Skalpell Nr.3 | InstrumenteNRW, Erftstadt, Germany | SK-10-004 | |
| Cell culture plates (6-well) | Greiner, Frickenhausen, Germany | 657160 | |
| Cryostat CM | Leica, Wetzlar, Germany | 3050S | |
| EDTA | Carl Roth, Karlsruhe, Germany | 8043.3 | |
| Eppendorf reaction tubes (1.5 or 2 ml) | Greiner, Frickenhausen, Germany | 616201, 623201 | |
| Falcon 15ml, 50ml | Greiner, Frickenhausen, Germany | 188271, 227270 | |
| Fetal Bovine Serum (FBS) | Biochrome, Berlin, Germany | S 0115 | |
| Freeze Dry System | Labconco, Kansas City, USA | 7670520 | |
| Freezer (-80°C) | Thermo Scientific, Waltham, MA, USA | Forma 900 Series | |
| HCl | Carl Roth, Karlsruhe, Germany | 281.1 | |
| Microtome Blades Type 819 | Leica, Wetzlar, Germany | 14035838925 | |
| Minilys Homogeniser | PEQLAB Biotechnologie GmbH, Erlangen, Germany | 91-PCSM | |
| NaOH | Carl Roth, Karlsruhe, Germany | K021.1 | |
| Nystatin | PAN Biotech, Aidenbach, Germany | P06-07800 | |
| PBS | Thermo Scientific, Waltham, MA, USA | 14190-094 | |
| Penicillin/streptomycin | Life Technologies, Darmstadt, Germany | 15140122 | |
| Pepsin | Sigma-Aldrich, Taufkirchen, Germany | P6887-1G | |
| Precellys Keramik-Kit 1.4 mm | Peqlab Biotechnolgie, Erlangen, Germany | 91-PCS-CK14 | |
| Rotamax 120 Plate shaker | Heidolph, Schwabach, Germany | 544-41200-00 | |
| SDS | Carl Roth, Karlsruhe, Germany | CN30.3 | |
| Stereo microscope | Leica, Wetzlar, Germany | M125 | |
| Steriflip-GP, 0,22 µm | Merck Millipore, Darmstadt, Germany | SCGP00525 | |
| Stuart analogue rocker & roller mixers | Sigma-Aldrich, Taufkirchen, Germany | Z675113-1EA | |
| Tissue Tek O.C.T compound | Hartenstein, Wurzburg, Germany | TTEK | |
| Transfusion set 200µm | Sarstedt, Nümbrecht, Germany | 798.200.500 | |
| TRIS | Carl Roth, Karlsruhe, Germany | 5429.3 | |
| vedena Skalpellgriff Fig. 3, Standard, 125 mm | Medical Highlights, Rohrdorf, Germany | CV102-003 | |
| Vortex-Genie2 | Scientific Industry, New York, USA | SI-0256 |
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Cite This Article
Becker, M., Maring, J. A., Oberwallner, B., Kappler, B., Klein, O., Falk, V., Stamm, C. Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications. J. Vis. Exp. (130), e56419, doi:10.3791/56419 (2017).