组织工程皮肤的冷冻干燥保存方法研究

发布时间：2018-06-25 08:52

  本文选题：海藻糖 + 水凝胶　； 参考：《西北大学》2012年硕士论文

【摘要】：组织工程皮肤具有良好的皮肤缺损修复效果,然而产品的生产周期较长,更重要的是由于含有活细胞,其有效保存时间较短,严重限制了其临床应用和产业化。因此迫切需要寻找新的有效保存方法。 深低温冷冻保存已成功应用于活细胞的长期保存,细胞在使用时快速复温,可以有效维持其存活状态。然而对于组织工程皮肤,深低温冷冻保存并未取得突破性进展,困难在于复温过程中的重结晶对细胞产生致命的冰晶损伤,因为生物组织具有较低的热传导率和较高的比热,其内部的热传导性不均一,复温速度很难达到避免冰晶损伤所需的速度。海藻糖是一种非还原性二糖,越来越多的研究发现海藻糖对干燥状态下的生物体具有保护作用。 我们设想先将种子细胞负载海藻糖和DMSO再构建组织工程皮肤,海藻糖可以保护细胞在干燥条件下存活,DMSO保护细胞在程序降温和冷冻过程中存活,组织工程皮肤低温冻结后,在真空条件下使其水分升华而成为干燥状态,为组织工程皮肤的长期保存提供了一种新的方法,这种方法既可以使TES能够稳定储存,也在复水恢复活性的过程中避免冰晶损伤,目前未见文献报道。 在本研究中,我们首先优化了海藻糖负载种子细胞成纤维细胞的条件并评价了负载海藻糖的成纤维细胞干燥保存的效果。进一步,我们用负载海藻糖的成纤维细胞与鼠尾胶原凝胶构建了组织工程皮肤,将其冷冻干燥后,进行了一系列显微、超微结构观察,荧光染色,活力测定以及皮肤缺损修复实验,评价冷冻干燥保存方法对组织工程皮肤的保存效果,初步建立组织工程皮肤保存的新方法。 1.我们从保存液中海藻糖的浓度,孵育时间和孵育温度三个方面优化了成纤维细胞负载海藻糖的条件,获得最佳导入条件为海藻糖200mM,在37℃条件下孵育8小时。在此条件下负载海藻糖的细胞,经过冷冻干燥过程后仍可保存30%的活性,经过人工风干过程可保留40%的活性,荧光染色及电镜观察显明细胞膜基本保存完整。 2.我们用负载海藻糖的细胞成功构建了组织工程皮肤。经过冷冻干燥保存后,组织工程皮肤仍保留有26%的活性；SEM显示组织工程皮肤的结构基本保持完整；在C57BL/6J小鼠背部全层皮肤缺损修复中,负载海藻糖的组织工程皮肤伤口愈合速率较没有负载海藻糖的组别提前约30%,而与正常组织工程皮肤无明显差别。利用海藻糖的干燥保护作用而引入的冷冻干燥保存,能有效保存组织工程皮肤的活性并利于其缺损修复效果的发挥,为组织工程皮肤的保存提供了新的方法。
[Abstract]:Tissue engineered skin has good skin defect repair effect, but the production cycle of the product is longer, more importantly, because of the existence of living cells, its effective preservation time is shorter, which seriously limits its clinical application and industrialization. Therefore, there is an urgent need to find new effective conservation methods. Cryopreservation has been successfully applied to the long-term preservation of living cells. However, for tissue engineered skin, cryopreservation has not made a breakthrough. The difficulty is that recrystallization in the process of rewarming causes fatal ice crystal damage to cells, because biological tissues have lower thermal conductivity and higher specific heat. The internal heat conductivity is uneven and the rewarming velocity is difficult to avoid ice crystal damage. Trehalose is a kind of non-reducing disaccharide. More and more researches have found that trehalose has protective effect on dry organisms. We imagine that seed cells are first loaded with trehalose and DMSO to construct tissue engineered skin. Trehalose can protect cells from surviving under dry conditions. DMSO protects cells from surviving during programmed cooling and freezing. Tissue engineered skin is frozen at low temperature. In vacuum condition, the moisture is sublimated to a dry state, which provides a new method for the long-term preservation of tissue engineering skin. This method can not only make tes stored stably, but also avoid ice crystal damage in the process of rehydration. At present, there is no literature report. In this study, we first optimized the conditions of trehalose loaded seed cell fibroblasts and evaluated the effect of trehalose loaded fibroblast dry preservation. Furthermore, we constructed tissue engineered skin from fibroblasts loaded with trehalose and collagen gel from rat tail. After freeze-drying, we made a series of microscopic, ultrastructural observation and fluorescent staining. To evaluate the effect of freeze-drying preservation on tissue engineered skin and to establish a new method for tissue engineering skin preservation. We optimized the conditions of the fibroblasts loading trehalose from three aspects: the concentration of trehalose, the incubation time and the incubation temperature. The optimum conditions were obtained as follows: trehalose 200 mm, incubated at 37 鈩，

本文编号：2065367