正交法探索平板离心构建无载体细胞片的可行性

背景:实体器官移植目前在实验室及临床已积极展开,具有良好的治疗效果,但实体组织器官供体来源的匮乏是世界性的难题之一。目的:用平板离心技术构建细胞片以探索一种新的实体组织构建方法。方法:首先用梯度离心力对人皮肤成纤维细胞进行离心力反应和细胞即时贴壁率的探索,设计出不同离心力水平、细胞浓度及离心时间,用正交实验优选最佳离心参数,用优选所得的最佳离心条件进行稳定重复实验。用最佳离心条件进行单层细胞离心实验,自然培养组为对照组,观察离心后单层细胞的活性及增殖活力。用离心法进行多层细胞片的构建,然后进行体外培养及苏木精-伊红染色切片以观察不同层数细胞片的形成情况。结果与结论:最佳构建参数为细胞浓度9×105cm-2,离心转速2200r/min,离心时间4min,细胞种植效率可达(93.75±9.58)%;单层对照显示离心贴壁细胞增殖活性良好;苏木精-伊红染色观察显示可快速形成活性良好的细胞片。提示离心速构法形成了无支架组织,可作为一种方便、快捷、高效的实体组织工程构建方法。

Feasibility of rapidly constructing scaffold-free cell sheets for tissue engineering using orthogonal design method

BACKGROUND:Solid organ transplantation has been actively developed with a good therapeutic effect,but the lack of organ donor source is a worldwide problem.OBJECTIVE:To develop a centrifugal cell seeded method for rapid and efficient tissue engineering construction.METHODS:Different acceleration forces and cell densities and centrifugal times were studied.The orthogonal design method was used to optimize centrifugation parameters for cell seeding.To test stability and reproducibility of the orthogonal design results,experiments were studied using optimization centrifugation parameters.Monolayer cell adherence ratio after centrifugation and harvested cell numbers after cultured for 3 days was counted.Epithelial stratification tissues were constructed in vitro.The cell sheets were constructed on transwell culture insert using centrifugation parameters.Pathological sections were cut and stained with hematoxylin and eosin for 7 consecutive days.RESULTS AND CONCLUSION:The (93.75±9.58)% attached cells were achieved using optimal cell seeding density at 9×105 cells/cm2 with a centrifugation at 2 200 r/min for 4 minutes.Monolayer centrifugal seeded cell sheets showed better cell viability and proliferation compared to naturally seeded cell sheets.The one-layer to four-layer cell sheets were rapidly constructed and showed good viability.Different layers of scaffold-free cell sheets were rapidly constructed using optimal centrifugation procedure.It demonstrates that the centrifugation method is a good way for tissue engineering construction.