【摘要】：干細胞治療有望解決包括退行性細胞或組織疾病,肌肉、骨和軟骨缺損以及癌癥在內的一系列醫(yī)學難題。臍帶間充質干細胞(human umbilical cord mesenchymal stem cells, hUCMSCs)是近年來發(fā)現(xiàn)的一類具有高度自我更新與分化潛能的干細胞。由于hUCMSCs來源廣泛,取材方便,免疫排斥性低,被認為是干細胞治療的理想種子細胞。然而,一次成功的治療需要約109個細胞,這意味著分離出的干細胞必須在短時間內經(jīng)過有效的體外擴增,獲得大量高質量的干細胞,方能達到細胞治療的要求。 通過傳統(tǒng)的二維平面擴增臍帶間充質干細胞,較難在短時間內得到足夠的細胞量,且過多傳代次數(shù)造成干細胞質量下降。三維微載體培養(yǎng)可以提供足夠的比表面積用于細胞擴增,因而得到大量干細胞,同時模擬三維環(huán)境以維持干細胞的未分化特性。本文探討了三維微載體培養(yǎng)法在干細胞體外擴增中的特點以及應用價值。 研究人臍帶間充質干細胞在二維孔板、三維微載體Cytodex-3與Cultispher-S上培養(yǎng)擴增的特點。傳代培養(yǎng)人臍帶間充質干細胞,流式細胞術鑒定免疫表型,將hUCMSCs以4×104/ml的密度分別接種至孔板、含有2g / L Cytodex-3載體的轉瓶以及含有2g / L Cultispher-S載體的轉瓶中,連續(xù)培養(yǎng)7天,期間取樣,描電鏡觀察微載體表面結構及細胞貼附,cck-8法計數(shù)繪制生長曲線,DAPI染色觀察細胞在微載體上的生長分布,代謝動力學檢測,成骨誘導液連續(xù)誘導培養(yǎng)14天,堿性磷酸酶(ALP)檢測,茜素紅染色分析。 實驗結果表明,hUCMSCs均一地高表達間充質細胞表面標記物CD29、CD44及CD105,表達干細胞標志CD90,不表達造血干細胞標志CD14及CD45。生長曲線顯示,經(jīng)過7天擴增培養(yǎng)后,細胞最大收獲量出現(xiàn)在大孔載體Cultispher-S上,密度為(8.0±0.32)×105 /ml,其次為無孔載體Cytodex-3,為(5.8±0.23)×105 /ml,二維孔板內最低,為(4.1±0.1)×105 /ml。培養(yǎng)后期Cultispher-S上的細胞出現(xiàn)結團,載體與載體間已形成細胞橋,且細胞分泌大量細胞外基質(ECM)。代謝動力學表明,Cultispher-S上生長的細胞對于葡萄糖的利用度最為有效。三種培養(yǎng)條件下擴增后的細胞均具有向成骨細胞分化的潛質。此外,Cultispher-S的成分為明膠蛋白,可在人體內降解,因此避免了收獲細胞時使用胰酶將細胞與載體分離的步驟,減少胰酶對干細胞造成的損傷。綜上結果表明,三維微載體擴增干細胞較二維平皿法能夠收獲更多干細胞; Cultispher-S微載體較Cytodex-3更適合臨床上擴增培養(yǎng)間充質干細胞使用。
[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 鹵0.32) 脳 10 ~ 5 / ml, followed by Cytodex-3, without pore vector (5.8 鹵0.23) 脳 10 ~ 5 / ml,. The lowest in 2-D hole plate was (4.1 鹵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.
【學位授予單位】：上海交通大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R329

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