使用3D生物印刷创建心脏组织展示球体的机械整合
Summary
该协议描述了不使用生物材料的心脏组织的3D生物印迹。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。
Abstract
该协议描述了仅使用细胞而不使用生物材料的心脏组织的3D生物印迹。首先分离心肌细胞，内皮细胞和成纤维细胞，计数并以所需细胞比例混合。它们在超低附件96孔板中的各个孔中共培养。在3天内，殴打球体。然后使用真空抽吸由喷嘴拾取这些球体，并使用3D生物打印机在针阵列上组装。然后允许球体在针阵列上熔断。三维生物印迹三天后，球状体作为完整的贴片被去除，已经自发地发生跳动。 3D生物印迹的心脏贴片显示组件球体的机械整合，并且在心脏组织再生和心脏病的3D模型中是非常有希望的。
Introduction
3D生物印迹有很多不同的方法， 1，2，3 。 3D生物打印通常通过印刷技术1进行分类，例如喷墨生物印刷，微压缩生物印刷，激光辅助生物印刷，方法的组合或更新的方法。 3D生物印迹也可以分为无支架或支架依赖的方法4 。 3D生物印迹的大多数方法是支架依赖的，其中需要生物材料， 例如生物蛋白5或支架6 。然而，支架依赖的3D生物印迹面临许多问题和局限性，如脚手架材料的免疫原性，专有生物蛋白的成本，降解产物的速度和毒性等。
SCAF已经尝试使用球体的无折叠心脏组织工程8 ，有可能克服支架依赖组织工程的这些缺点。然而，正如作者在该论文中所承认的那样，在生物制造过程中难以强力地处理和定位固定位置的球体。伴随使用3D生物印迹和基于球形的组织工程有可能克服这些困难。在本协议中，我们描述了没有其他生物材料的心脏组织的3D生物印迹，仅使用球形形式的细胞。
基于无支架的基于球体的3D生物打印机9具有使用真空抽吸拾取单个球体的能力，并将其定位在针阵列上。 3D生物印刷中针阵列上的定位球体的概念是从古代日本使用针阵（被称为“ kenzan ”）的灵感来的nese艺术的花卉安排， ikebana。该系统允许球体精确地定位在任何配置中，并导致单个球体在短时间内熔合在一起以产生3D生物印刷组织。因此，该方法允许容易地操纵球体，对于无支架器官生物制造的未来具有潜在的影响。
Protocol
Representative Results
Discussion
It is important to use beating, functional spheroids for 3D bioprinting. If spheroids are not beating, continuing to use them will invariably result in a non-functional 3D bioprinted patch.
One benefit of this approach is the ability to manipulate the cell content of the patch by varying the total number of cells and the percentage of cardiomyocytes, endothelial cells, and fibroblasts in the spheroids. This allows for many different types of cardiac patches to be printed, with varying histolog…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
作者承认以下资金来源：神经科学心血管研究基金和马里兰州干细胞研究基金（2016-MSCRFI-2735）。
Materials
| Geltrex | Invitrogen | A1413202 | |
| Trypsin/EDTA 0.05% | Thermo Fisher | 15400054 | |
| Defined Trypsin inhibitor 0.0125% | Thermo Fisher | R007100 | |
| RPMI Cell Media | Invitrogen | 11875-093 | RPMI supplemented with B27 constitutes HIPSC-CM culture media |
| B-27 Supplement | Thermo Fisher | 17504044 | RPMI supplemented with B27 constitutes HIPSC-CM culture media |
| Countess Automated Cell Counter | Invitrogen | C10227 | |
| Human cardiac fibroblasts (adult ventricular type) | Sciencell | 6310 | |
| Human umbilical vein endothelial cells | Lonza | CC-2935 | |
| PrimeSurface ultra-low attachment 96-well U-bottom plates | Akita Sumitomo Bakelite Co. | MS-9096UZ | |
| Regenova Bio 3D Printer | Cyfuse Biomedical K.K. | N/A | www.cyfusebio.com/en/ |
| Trypan Blue Solution, 0.4% | Thermo Fisher | 15250061 | |
| Troponin T Antibody | Thermo Fisher | 701620 | |
| Connexin 43 (Cx43) Antibody | Chemicon | MAB3068 | |
| ProLong Gold Antifade Mountant with DAPI | Thermo Fisher | P36935 |
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