组织工程：建设多细胞三维支架的层状细胞片交货
Summary
For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.
Abstract
许多组织中，如成人心，都无法破坏后，以充分再生。2,3策略在组织工程中提出的创新，以协助身体恢复和修复。例如，TE方法可能能够减轻心肌梗死（MI）后心脏重塑和可能增加总的心脏功能，以接近正常的预MI水平。4与任何功能性组织，心脏组织的再生成功涉及适当的递送多种细胞类型与环境因素有利于整合植入的细胞/组织移植和生存。工程化组织应处理的多个参数包括：评价为运载工具，其对细胞存活的影响，材料强度和细胞 – 组织机构的便利可溶信号，细胞 – 细胞相互作用，并且基质材料。研究采用直接注射移植的细胞不仅无视这些基本要素。2,5,6一种组织设计结合这些成分还有待开发。这里，我们提出的使用图案化的细胞片层的分层与两种不同类型的含有靶器官的细胞类型和内皮细胞用于增强新血管形成中的“组织”生物衍生材料集成设计的例子。虽然这些研究集中在心脏样组织的产生，此组织设计可以应用于许多器官比心脏以最小的设计和材料的变化等，并意指是断开的，现成的产品用于再生疗法。该协议包含五个详细步骤。一种温度敏感聚（N -isopropylacrylamide）（PNIPAAM）用于涂层的组织培养皿。然后，特定组织细胞的涂装板/微图案的表面的表面上培养，以形成细胞片层具有很强的横向粘连。第三，基础矩阵的组织由多孔基体结合新生血管permissi创建已经凝胶和内皮细胞。最后，将细胞片从PNIPAAm的涂覆菜肴解除，并转移到基座元件，使得整个结构。
Introduction
Injection of cells and/or single materials alone has shown variable success in other organ systems and limited success in cardiac regeneration.5,7-12 Currently, stem cell-derived cells are delivered to damaged tissue using a variety of delivery methods including: direct cell injection into tissue and perfusion into the blood supply.13-17 Others have implanted cells alone, materials alone and/or in combination with material carriers to help regenerate damaged organs.18-21 This design combines multiple strategies that provide material strength, patterning in multiple materials and multiple cell types.
Specifically, the base acellularized fibrous matrix provides the foundational physical strength to the construct, making it suitable for suturing in into the patient, if necessary. The void spaces in the base matrix are filled with endothelial cells in a neovascular permissive hydrogel22 for rapidly establishing vascularization of the implanted construct. This composite is then integrated with pre-patterned cell sheets that allow enhanced cell-to-cell communication, more closely mimic the native tissue.1,23-25 The overall production process for the layered cellular patch is outlined by the flowchart in Figure 1.
Protocol
Representative Results
Discussion
在该协议中的关键步骤包括：涂布板面与温敏聚合物和冷却板后操纵的细胞片层。因为不同的细胞表现出不同的物理性质，如粘合性，升降时间应为每个不同的细胞类型进行了优化。本协议的第二个，也是最显著挑战性成分，集中在电池板，为组织组装方法的一个重要方面的操作。在电池板的单细胞层是相当脆弱的，并且可以很容易地撕开，如果操作用钳子。此外，当细胞片层中的地方不被保持?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was funded by a New Faculty Award II from the California Institute of Regenerative Medicine (CIRM; RN2-00921-1), NIH-funded National Research Award (F32-HL104924), and CIRM Training Grant (TG21163). Materials were provided by: Glycosan Biosystems Inc / BioTime and Dr. Stephen Badylak (University of Pittsburgh)
Materials
| Table of Reagents: | |||
| Reagent | Company | Catalogue number | Comments |
| Calcein-AM | Invitrogen | C3099 | Cell tracker / live dye |
| Lysotracker Red | Invitrogen | L7528 | Cell tracker |
| Neutral Red | Sigma | N7005 | Visible Cell dye |
| pNIPAAM | Sigma Aldrich | 412780250 | Poly(N-isopropylacrylamide) |
| Toluene | Sigma Aldrich | 244511-1L | |
| Hexane | Sigma Aldrich | 296090-1L | |
| RAOSMC | Lonza | R-ASM-580 | Rat Aortic Smooth Muscle Cells |
| SmGM2 | Lonza | CC-4149 | Smooth Muscle Media |
| HUVEC | Invitrogen | C-003-5C | Human Venous Endothelial Cells |
| HyStem | Glycosan/Biotime | ———— | |
| Isopropyl alcohol | VWR International | BDH1133-4LP | |
| Trypsin | Corning Cellgro | 25-053-C1 | |
| PBS | Gibco | 14287-072 | |
| FBS | Gibco | 16140-071 | |
| Table of Specific Equipment: | |||
| Equipment | Company | Catalogue number | Comments (optional) |
| Filter paper | Ahlstrom | 6310-0900 | |
| Buchner Funnel | Sigma Aldrich | Z247308 | |
| UpCell Plates | Nunc | 2014-11 | |
| UV light. | Jelight Company | UVO Cleaner Model No.42 |
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