Turner William S, Sandhu Nabjot, McCloskey Kara E
School of Engineering, University of California, Merced;
School of Engineering, University of California, Merced.
J Vis Exp. 2014 Oct 3(92):e51044. doi: 10.3791/51044.
Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.(2,3) Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.(4) As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.(2,5,6) A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the "tissue". Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.
许多组织，如成体人类心脏，受损后无法充分再生。（2,3）组织工程学策略提出创新方法来协助身体恢复和修复。例如，组织工程方法或许能够减轻心肌梗死后的心脏重塑，并有可能将心脏整体功能提高到接近心肌梗死前的正常水平。（4）与任何功能性组织一样，心脏组织的成功再生涉及多种细胞类型的恰当递送以及有利于植入细胞/组织移植物整合和存活的环境线索。工程组织应考虑多个参数，包括：可溶性信号、细胞间相互作用以及作为递送载体评估的基质材料、它们对细胞存活的影响、材料强度以及细胞与组织组织化的促进作用。仅采用直接注射移植细胞的研究忽略了这些基本要素。（2,5,6）结合这些要素的组织设计尚未开发出来。在此，我们展示一种整合设计的实例，即使用带有两种不同类型生物衍生材料的图案化细胞片层叠，这两种材料包含目标器官细胞类型和内皮细胞，以增强“组织”中的新血管形成。尽管这些研究聚焦于生成类心脏组织，但这种组织设计只需进行最少的设计和材料更改就能应用于心脏以外的许多器官，并且旨在成为用于再生治疗的现成产品。该方案包含五个详细步骤。使用温度敏感型聚（N - 异丙基丙烯酰胺）（pNIPAAM）包被组织培养皿。然后，在包被的平板/微图案表面培养组织特异性细胞，以形成具有强侧向粘附力的细胞片。第三步，通过将多孔基质与促新生血管形成的水凝胶和内皮细胞结合，为组织创建基础基质。最后，将细胞片从pNIPAAM包被的培养皿中提起并转移到基础元件上，制成完整构建体。
