基于网模的无支架三维心脏组织创建方法
Summary
本协议描述了一种基于净模的方法来创建三维无支架的心脏组织, 结构完整性和同步跳动行为令人满意。
Abstract
该协议描述了一种新的和简单的基于网络模型的方法, 以创建三维 (3 维) 心脏组织没有额外的支架材料。人诱导的多潜能干细胞衍生心肌细胞 (iPSC cms), 人心脏成纤维细胞 (HCFs) 和人脐静脉内皮干细胞 (血管内皮细胞) 被隔离, 并用于产生细胞悬浮与 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞。他们是共同培养的超低附件 “悬挂下降” 系统, 其中包含孔, 以凝聚数以百计的球体一次。在共培养3天后, 细胞聚集并自发形成跳动球体。球体的收获, 种子成一个新的模具腔, 并培养在一个振动筛在孵化器。球体成为成熟的功能组织大约7天后播种。所合成的多层组织包括融合球体, 结构完整性良好, 同步跳动行为。这种新方法具有很好的潜力, 可以作为一种重现性和成本效益高的方法, 为将来的心力衰竭治疗创造出工程组织。
Introduction
目前心脏组织工程的目的是开发一种替代或修复损伤心肌组织的结构和功能的治疗方法1。方法建立3维心脏组织模型, 显示本机心脏组织的重要收缩和电生理特性, 迅速扩大2,3。在研究4、5中探索和使用了各种战略。这些方法包括使用特定的合成和天然生物活性水凝胶, 如明胶, 胶原蛋白, 纤维蛋白和肽6, 生物墨水沉积技术2和生物打印技术7。
研究表明, 无支架的方法可以产生可比的组织作为生物材料的方法, 而不存在的缺点, 纳入外国脚手架材料8。奥伦 Caspi等人证明, 结合各种类型的细胞, 使生成高血管化的人类工程心脏组织9。下巴等人开发了一种3维印刷方法的心脏补丁创建从球体。所产生的斑块由心肌细胞、纤维细胞和内皮干细胞组成, 比例为 70:15:15.10。球体已被证明是有效的 “积木” 的无支架的心脏组织创建, 因为他们耐缺氧, 并拥有足够的机械完整性, 植入11,12。以前的研究已经展示了几种制造球体的方法, 包括使用吊滴法、微调烧瓶13、微流控系统14和非粘附的培养表面, 不涂或涂有琼脂糖微模15。在本协议中, 我们使用悬挂放置装置, 其中包含孔, 以凝聚数以百计的球体一次。
本研究提出了一种新的、高效的无支架的心脏组织创建方法, 包括将球体人工播种到方形模腔中, 并在振动筛上孵化组织以进行成熟。在通常的静态培养条件下, 氧扩散仅限于组织结构的外部, 导致中心坏死。然而, 随着网模, 所有的球体种子进入模具都沉浸在介质中, 不断的射流运动, 使养分和氧气的扩散增加。此外, 这种基于模具的方法允许同时创建不同大小的组织补丁, 用最少的人工努力, 由此产生的组织可以很容易地从模具中删除。这种新的方法允许有效和可重复的创建无支架, 多层心脏补丁。
Protocol
1. 心肌细胞的制备 用基底膜基质和培养人诱导的多能干细胞 (hiPSCs) 6 井板, 如前所述的17。 使用以前描述的方法18将 hiPSCs 区分为 hiPSC CMs。 在 16-18 d 的分化后, 通过冲洗每个井与2毫升的1x 磷酸盐缓冲盐水 (PBS), 不含钙或镁, 然后孵化与1毫升/井的胰蛋白酶或细胞离解试剂 (见表材料) 室温下5分钟。 与胰蛋白酶或细胞离解?…
Representative Results
在我们的实验中, 我们利用细胞悬浮 70% iPSC CMs, 15% HCFs, 15% 血管内皮细胞在 RPMI/B-27 细胞介质中浓度247.5万细胞每毫升。在建立细胞悬浮液后, 我们将4毫升的细胞悬浮液分配给超低附着悬挂系统的每个井, 如协议步骤4.3 所述。在3天的文化在37°c、5% CO2和95% 湿气以后, 使用垂悬的下落系统导致了数以百计的跳动的球体的自发形成。球体很容易被观察到在光显微镜下跳动…
Discussion
该方法的意义在于其重现性和结果多层心肌组织的有效性。在心脏组织工程领域, 目前的目标之一是确定一种方法来构建跳动, 多层和功能3维心脏补丁。我们报告了一个有效的和可重复的方法创建多层心脏组织的直接人工播种的球体, 由心肌细胞, 内皮干细胞, 成纤维形成一个新的网霉菌。本方法所用的网模有各种不同的尺寸, 可用于从 2 x 2 x 1 毫米到 6 x 6 x 1 毫米不等的组织的创建。我们所描述的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者承认以下资金来源: 为心血管研究提供资金的魔力。
Materials
| Human Cardiac fibroblasts (HCF) | Sciencell | 6310 | |
| FM-2 Consists of Basal Medium | Sciencell | 2331 | HCF culture medium |
| Human umbilical vein endothelial cells (HUVEC) | Lonza | CC-2935 | |
| EGM+Bullet Kit | Lonza | CC5035 | HUVEC culture medium |
| E8 media | Invitrogen | A1517001 | HiPSC culture medium |
| Geltrex | Invitrogen | A1413202 | |
| TrypLE Express Enzyme (1X) | Thermo Fisher | 12604013 | Trypsin and Cell dissociation reagent |
| RPMI media | Invitrogen | 11875093 | RPMI media with B-27 supplement is hiPSC-CM culture medium |
| B-27 supplement (50x) | Thermo Fisher | 17504044 | RPMI media with B-27 supplement is hiPSC-CM culture medium |
| Trypan Blue Solution, 0.4% | Thermo Fisher | 15250061 | |
| Novel net mold | TissueByNet Co.,Ltd | NM25-1 | |
| Hanging drop plate | Kuraray Co.,Ltd | MPc350 | |
| 6 well plates | Sigma-Aldrich | CLS-3516 |
References
- Gao, L., et al. Myocardial Tissue Engineering With Cells Derived From Human-Induced Pluripotent Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold. Circulation Research. 120 (8), 1318-1325 (2017).
- Borovjagin, A. V., Ogle, B. M., Berry, J. L., Zhang, J. From Microscale Devices to 3D Printing: Advances in Fabrication of 3D Cardiovascular Tissues. Circulation Research. 120 (1), 150-165 (2017).
- Tandon, N., et al. Electrical stimulation systems for cardiac tissue engineering. Nature Protocols. 4 (2), 155-173 (2009).
- Duran, A. G., et al. Regenerative Medicine/Cardiac Cell Therapy: Pluripotent Stem Cells. The Journal of Thoracic and Cardiovascular Surgery. 66 (1), 53-62 (2018).
- Lux, M., et al. In vitro maturation of large-scale cardiac patches based on a perfusable starter matrix by cyclic mechanical stimulation. Acta Biomaterialia. 30, 177-187 (2016).
- Eschenhagen, T., et al. Three-dimensional reconstitution of embryonic cardiomyocytes in a collagen matrix: a new heart muscle model system. The FASEB Journal. 11 (8), 683-694 (1997).
- Moldovan, N. I., Hibino, N., Nakayama, K. Principles of the Kenzan Method for Robotic Cell Spheroid-Based Three-Dimensional Bioprinting. Tissue Engineering Part B: Reviews. 23 (3), 237-244 (2017).
- Sugiura, T., Hibino, N., Breuer, C. K., Shinoka, T. Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model. Journal of Cardiothoracic Surgery. 11 (1), 163 (2016).
- Caspi, O., et al. Tissue engineering of vascularized cardiac muscle from human embryonic stem cells. Circulation Research. 100 (2), 263-272 (2007).
- Ong, C. S., et al. Biomaterial-Free Three-Dimensional Bioprinting of Cardiac Tissue using Human Induced Pluripotent Stem Cell Derived Cardiomyocytes. Scientific Reports. 7 (1), 4566 (2017).
- Kelm, J. M., et al. A novel concept for scaffold-free vessel tissue engineering: self-assembly of microtissue building blocks. Journal of Biotechnology. 148 (1), 46-55 (2010).
- Noguchi, R., et al. Development of a three-dimensional pre-vascularized scaffold-free contractile cardiac patch for treating heart disease. The Journal of Heart and Lung Transplantation. 35 (1), 137-145 (2016).
- Zuppinger, C. 3D culture for cardiac cells. Biochimica et Biophysica Acta. 1863 (7 Pt B), 1873-1881 (2016).
- Langenbach, F., et al. Generation and differentiation of microtissues from multipotent precursor cells for use in tissue engineering. Nature Protocols. 6 (11), 1726-1735 (2011).
- Fennema, E., Rivron, N., Rouwkema, J., van Blitterswijk, C., de Boer, J. Spheroid culture as a tool for creating 3D complex tissues. Trends in Biotechnology. 31 (2), 108-115 (2013).
- Agocha, A., Sigel, A. V., Eghbali-Webb, M. Characterization of adult human heart fibroblasts in culture: a comparative study of growth, proliferation and collagen production in human and rabbit cardiac fibroblasts and their response to transforming growth factor-beta1. Cell Tissue Research. 288 (1), 87-93 (1997).
- Baudin, B., Bruneel, A., Bosselut, N., Vaubourdolle, M. A protocol for isolation and culture of human umbilical vein endothelial cells. Nature Protocols. 2 (3), 481-485 (2007).
- Pimentel, C. R., et al. Three-dimensional fabrication of thick and densely populated soft constructs with complex and actively perfused channel network. Acta Biomaterialia. 65, 174-184 (2018).
- Shimizu, T., et al. Polysurgery of cell sheet grafts overcomes diffusion limits to produce thick, vascularized myocardial tissues. The FASEB Journal. 20 (6), 708-710 (2006).
- Sakaguchi, K., Shimizu, T., Okano, T. Construction of three-dimensional vascularized cardiac tissue with cell sheet engineering. Journal of Controlled Release. 205, 83-88 (2015).
- Ong, C. S., et al. Creation of Cardiac Tissue Exhibiting Mechanical Integration of Spheroids Using 3D Bioprinting. Journal of Visualized Experiments. 125 (e55438), (2017).
- Tan, Y., et al. Cell number per spheroid and electrical conductivity of nanowires influence the function of silicon nanowired human cardiac spheroids. Acta Biomaterialia. 51, 495-504 (2017).
- Karra, R., Poss, K. D. Redirecting cardiac growth mechanisms for therapeutic regeneration. Journal of Clinical Investigation. 127 (2), 427-436 (2017).
- Bartholoma, P., et al. Three-dimensional in vitro reaggregates of embryonic cardiomyocytes: a potential model system for monitoring effects of bioactive agents. Journal of Biomolecular Screening. 10 (8), 814-822 (2005).
Cite This Article
Bai, Y., Yeung, E., Lui, C., Ong, C. S., Pitaktong, I., Huang, C., Inoue, T., Matsushita, H., Ma, C., Hibino, N. A Net Mold-based Method of Scaffold-free Three-Dimensional Cardiac Tissue Creation. J. Vis. Exp. (138), e58252, doi:10.3791/58252 (2018).