微载体培养法在人脐带间充质干细胞体外扩增中的应用

发布时间：2019-01-30 20:12

【摘要】：干细胞治疗有望解决包括退行性细胞或组织疾病,肌肉、骨和软骨缺损以及癌症在内的一系列医学难题。脐带间充质干细胞(human umbilical cord mesenchymal stem cells, hUCMSCs)是近年来发现的一类具有高度自我更新与分化潜能的干细胞。由于hUCMSCs来源广泛,取材方便,免疫排斥性低,被认为是干细胞治疗的理想种子细胞。然而,一次成功的治疗需要约109个细胞,这意味着分离出的干细胞必须在短时间内经过有效的体外扩增,获得大量高质量的干细胞,方能达到细胞治疗的要求。通过传统的二维平面扩增脐带间充质干细胞,较难在短时间内得到足够的细胞量,且过多传代次数造成干细胞质量下降。三维微载体培养可以提供足够的比表面积用于细胞扩增,因而得到大量干细胞,同时模拟三维环境以维持干细胞的未分化特性。本文探讨了三维微载体培养法在干细胞体外扩增中的特点以及应用价值。研究人脐带间充质干细胞在二维孔板、三维微载体Cytodex-3与Cultispher-S上培养扩增的特点。传代培养人脐带间充质干细胞,流式细胞术鉴定免疫表型,将hUCMSCs以4×104/ml的密度分别接种至孔板、含有2g / L Cytodex-3载体的转瓶以及含有2g / L Cultispher-S载体的转瓶中,连续培养7天,期间取样,描电镜观察微载体表面结构及细胞贴附,cck-8法计数绘制生长曲线,DAPI染色观察细胞在微载体上的生长分布,代谢动力学检测,成骨诱导液连续诱导培养14天,碱性磷酸酶(ALP)检测,茜素红染色分析。实验结果表明,hUCMSCs均一地高表达间充质细胞表面标记物CD29、CD44及CD105,表达干细胞标志CD90,不表达造血干细胞标志CD14及CD45。生长曲线显示,经过7天扩增培养后,细胞最大收获量出现在大孔载体Cultispher-S上,密度为(8.0±0.32)×105 /ml,其次为无孔载体Cytodex-3,为(5.8±0.23)×105 /ml,二维孔板内最低,为(4.1±0.1)×105 /ml。培养后期Cultispher-S上的细胞出现结团,载体与载体间已形成细胞桥,且细胞分泌大量细胞外基质(ECM)。代谢动力学表明,Cultispher-S上生长的细胞对于葡萄糖的利用度最为有效。三种培养条件下扩增后的细胞均具有向成骨细胞分化的潜质。此外,Cultispher-S的成分为明胶蛋白,可在人体内降解,因此避免了收获细胞时使用胰酶将细胞与载体分离的步骤,减少胰酶对干细胞造成的损伤。综上结果表明,三维微载体扩增干细胞较二维平皿法能够收获更多干细胞; Cultispher-S微载体较Cytodex-3更适合临床上扩增培养间充质干细胞使用。
[Abstract]:Stem cell therapy is expected to solve a range of medical challenges, including degenerative cell or tissue diseases, muscle, bone and cartilage defects, and cancer. Umbilical cord mesenchymal stem cell (human umbilical cord mesenchymal stem cells, hUCMSCs) is a kind of stem cells with high self-renewal and differentiation potential. HUCMSCs is considered to be an ideal seed cell for stem cell therapy due to its wide range of sources, convenient selection and low immune rejection. However, a successful treatment requires about 109 cells, which means that the isolated stem cells must be effectively expanded in vitro in a short time to obtain a large number of high-quality stem cells in order to meet the requirements of cell therapy. It is difficult to obtain enough cells in a short time by traditional two-dimensional expansion of umbilical cord mesenchymal stem cells, and too many times of passage lead to the decline of stem cell quality. Three-dimensional microcarrier culture can provide sufficient specific surface area for cell expansion, resulting in a large number of stem cells, while simulating three-dimensional environment to maintain the undifferentiated characteristics of stem cells. In this paper, the characteristics and application value of three-dimensional microcarrier culture method in stem cell expansion in vitro were discussed. To study the characteristics of human umbilical cord mesenchymal stem cells (HMSCs) cultured and amplified on two dimensional pore plates, three dimensional microcarriers (Cytodex-3 and Cultispher-S). Human umbilical cord mesenchymal stem cells were cultured and immunophenotype was identified by flow cytometry. HUCMSCs was inoculated with 4 脳 104/ml density into the pore plate, the flask containing 2g / L Cytodex-3 vector and the flask containing 2g / L Cultispher-S vector, respectively. During 7 days of continuous culture, the surface structure and cell adhesion of microcarriers were observed by electron microscope, the growth curve was calculated by cck-8 method, the growth distribution of cells on microcarriers was observed by DAPI staining, and the metabolic kinetics was detected. Osteoblasts were cultured for 14 days. Alkaline phosphatase (ALP) was detected and analyzed by alizarin red staining. The results showed that hUCMSCs highly expressed mesenchymal cell surface markers CD29,CD44 and CD105, expressed stem cell markers CD90, did not express CD14 and CD45. markers of hematopoietic stem cells. The growth curve showed that after 7 days of amplification and culture, the maximum cell yield appeared on the macroporous carrier Cultispher-S, and the density was (8.0 卤0.32) 脳 10 ~ 5 / ml, followed by Cytodex-3, without pore vector (5.8 卤0.23) 脳 10 ~ 5 / ml,. The lowest in 2-D hole plate was (4.1 卤0.1) 脳 10 ~ 5 / ml.. In the later stage of culture, the cells on Cultispher-S appeared knot, the cell bridge was formed between the carrier and the carrier, and the cells secreted a large amount of extracellular matrix (ECM). Metabolic kinetics showed that the cells grown on Cultispher-S were the most effective for glucose utilization. The expanded cells had the potential to differentiate into osteoblasts under the three culture conditions. In addition, the component of Cultispher-S is gelatin protein, which can be degraded in human body, thus avoiding the step of separating the cell from the vector by using trypsin when harvesting cells, and reducing the damage caused by trypsin to stem cells. The results showed that three-dimensional microcarriers could harvest more stem cells than two-dimensional plate method, and Cultispher-S microcarriers were more suitable for clinical amplification of cultured mesenchymal stem cells than Cytodex-3.
【学位授予单位】：上海交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R329

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