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。
Stem cell therapy is promising in solving a series of complicated diseased including degenerative cells or tissue disorders; muscle, bone and cartilage defects; cancer etc. Umbilical cord mesenchymal stem cells (hUCMSCs) are some type of stem cells with high degree of self-renewal and differentiation potential that have recently been discovered. hUCMSCs have variety of sources, easy to isolation, low in immune rejection, and are considered to be the ideal cells for stem cell therapy.
However, 109 cells are required by a successful treatment, which means that isolated stem cells must be effectively expanded in vitro before clinically used. hUCMSCs proliferation using the traditional two-dimensional culture system is hard to meet the need of quantity of cells in a short period of time, and the excessive passage times may cause damage to stem cells. Culture using three-dimensional microcarriers can provide enough growth area, and simulate a 3D environment in order to maintain the characteristics of the undifferentiated stem cells. Our work discussed the features and value of application of three-dimensional microcarrier culture system.
To study the culture features of human umbilical cord mesenchymal stem cells (hUCMSCs) on 2D plate, 3D Cytodex-3 and Cultispher-S. Primary hUCMSCs were passaged for use. Immunophenotypic characteristics of cells were identified by flow cytometry. 4×104/ml hUCMSCs were seeded in plate, spinner flask with Cytodex-3 and spinner flask with Cultispher-S, respectively. In the following 7 days, microcarrier structures and cell morphology were observed by Scanning Electron Microscopic (SEM). Cell number was calculated by cck-8 assay and the growth curve was drawn. Cell distribution on microcarriers was observed by DAPI staining. Metabolic kinetics was measured. Cells under three culture systems were harvested and differentiated in osteogenesis media for 14 days, ALP activity was analyzed and alizarine red staining was conducted. Results showed that hUCMSCs expressed CD29, CD44, CD90 and CD105, but negative for CD14 and CD45. After 7 days proliferation, cells on macroporous Cultispher-S had the highest density which reached(8.0±0.32)×105 /ml, with Cytodex-3 followed of(5.8±0.23)×105 /ml, plate culture was the lowest with (4.1±0.1)×105 /ml. Cells on Cultispher-S at late phase formed aggregates, Cell Bridge could be observed. Large amounts of extracellular matrix (ECM) are secreted by cells. Metabolic kinetics showed that cells on Cultispher-S are most efficient in utilization of glucose. Cells recovered from three culture systems all remain the osteogenic potential. In addition, Cultispher-S is made of collagen which could degrade in human bodies, thus avoid trypsinization procedure used to harvest cells. In summary, 3D culture system can provide larger amount of cells than 2D culture. Cultispher-S was more suitable than Cytodex-3 for clinically expansion of hUCMSCs.
引文
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