应用搅拌式生物反应器体外扩增人胎盘间充质干细胞的实验研究

发布时间：2018-04-02 06:07

本文选题：生物反应器　切入点：胎盘间充质干细胞　出处：《中国医科大学》2008年硕士论文

【摘要】： 目的近年来研究发现,间充质干细胞(mesenchymal stem cells,MSCs)不仅支持造血系统,还可以向中胚层和外胚层来源的组织分化,在特定的培养条件下可向骨、成骨、脂肪细胞、神经细胞及肌细胞分化,且MSCs的免疫原性较低,在异基因移植中,用不相关供者的MSCs做滋养层支持造血干细胞(haemopoietic stemcell,HSC)体外扩增并不引起异体淋巴细胞反应。因而,MSCs在临床上具有广泛的应用前景。目前,在实验室里,MSCs的体外扩增主要采用小规模的静态培养方式,静态培养有其固有的局限性:培养环境不均一、培养参数不能及时监测和调控以及不利于大规模的培养等。搅拌式生物反应器培养技术可以为细胞的体外扩增提供均衡的营养环境,也可以在培养过程中对细胞代谢和培养液进行监控。人胎盘来源的间充质干细胞(human placenta-derived mesenchymal stem cells,hPDMSCs)不仅来源广泛,且对其研究不会涉及伦理道德和法律问题,因此成为人类MSCs新的来源。本实验应用搅拌式生物反应器体外扩增hPDMSCs,通过检测细胞体外扩增速率、细胞表面标记物、细胞周期、细胞代谢及培养液的PH和渗透压的变化,探讨体外大量扩增干细胞的方法。方法 1、分离培养hPDMSCs:取足月剖宫产胎儿胎盘子体面组织,PBS冲洗去除血迹,采用组织微块贴壁培养法分离培养hPDMSCs。 2、Cytodex-3型微载体及搅拌式生物反应器的预处理:以10mg/ml称取适量的cytodex-3微载体,PBS浸泡过夜、清洗、高压灭菌、0.2%明胶包被后备用。无水乙醇浸泡搅拌式生物反应器过夜,三蒸水反复冲洗,高压灭菌后备用。 3、搅拌式生物反应器体外扩增hPDMSCs:分别将1×1O~7 cells接种于微载体上制备成细胞悬液,在培养瓶内预培养24h后,将该悬液转移至搅拌式生物反应器内继续培养,逐渐调整搅拌式生物反应器至适当的转数,同时设置静态培养的培养瓶为对照组。每2d半量换液一次。 4、hPDMSCs的生长与代谢变化的检测:每日分别从培养瓶中和生物反应器内取样,检测细胞的扩增速率和培养液的PH、乳酸含量、葡萄糖含量、渗透压。 5、流式细胞仪检测细胞表面标记物与细胞周期:取应用搅拌式生物反应器培养前、后的hPDMSCs,流式细胞仪检测CD13、CD14、CD29、CD31、CD44、CD45、CD73、CD90、CD105、CD166、HLA-DR、HLA-ABC的表达情况及细胞周期的变化并进行比较。结果 1、胎盘中提取的MSC形态:呈梭形或成纤维状,传至20代时仍可稳定生长。 2、人胎盘组织来源的细胞细胞表面标记特征:表达CD13、CD29、CD44、CD73、CD90、CD105、CD166、HLA-ABC,不表达CD14、CD31、CD45、HLA-DR。 3、hPDMCs的生长与代谢的变化 (1)hPDMCs在cytodex-3型微载体上的贴附情况:对照组hPDMCs较为明显的黏附在微载体的周围,但仅看到少数微载体间的搭桥现象,且参与的微载体的个数较少;而在搅拌式生物反应器中,微载体大都通过细胞基质黏附在一起,呈现明显的搭桥现象。 (2)hPDMCs的生长曲线:对照组中的hPDMCs开始以较为平缓的趋势扩增,而且细胞密度一直呈现升高的趋势并在第4d达峰值,随后,细胞的密度就出现较为明显的下降趋势;搅拌式生物反应器中的hPDMCs在刚接入的48h内,其扩增趋势较对照组略低,随后细胞出现明显扩增的迹象。 (3)hPDMCs的扩增倍数:hPDMCs在搅拌式生物反应器中每代可以扩增10.55±1.62倍,明显高于对照组的6.10±0.11倍的扩增值(P＜0.05)。 (4)营养物质的消耗与代谢:搅拌式生物反应器内的葡萄糖消耗较对照组的高,但乳酸的生成却较对照组的低。 (5)培养液的PH值的变化:反应器内的PH变化幅度较对照组的小。 (6)培养液的渗透压的变化:反应器和对照组内培养液的渗透压均维持在(280-320)mmol/kg之间。 4、hPDMCs表面标记特征及细胞周期在生物反应器培养前、后的变化:流式细胞仪检测应用搅拌式生物反应器培养前、后的细胞表面标记特征及细胞周期无明显改变(P＞0.05)。结论 1、胎盘中存在着丰富的MSCs,不仅来源广泛,且对其研究不会涉及伦理道德和法律问题,为人类提供了新的干细胞种子来源。 2、搅拌式生物反应器为hPDMSCs提供了一种理想的体外扩增的三维培养体系,与静态的二维培养体系相比较,在这个三维培养体系中,细胞营养物质相对均一,使细胞表现出良好的扩增能力,同时扩增后的细胞干细胞特性无明显变化。因而,搅拌式生物反应器培养技术为体外获得高数量的hPDMSCs提供了一种安全、有效的培养方法,同时为生物反应器的研发提供了生物学依据。
[Abstract]:Purpose

Human placenta - derived mesenchymal stem cells ( hPDMSCs ) can be used to amplify hPDMSCs in vitro .

method

( 1 ) separating and culturing hPDMSCs : taking the fetal placenta sub - body surface tissues in full - term cesarean section , washing and removing blood stains by PBS , and separating and culturing hPDMSCs by using tissue micro - block adherent culture method .

2 . Pre - treatment of Cytodex - 3 microcarriers and stirred bioreactor : Take a proper amount of cytodex - 3 microcarriers in 10 mg / ml , soak overnight in PBS overnight , clean , sterilize under high pressure , 0.2 % gelatin capsule was used for backup .

3 . In vitro amplification of hPDMSCs by a stirred bioreactor : 1 脳 1O - 7 cells were seeded onto the microcarriers to prepare the cell suspension . After the culture was pre - cultured for 24 h , the suspension was transferred to the stirred bioreactor to continue the culture , and the stirred bioreactor was gradually adjusted to the appropriate number of revolutions , while the culture flask with static culture was set as the control group .

4 . Detection of growth and metabolic changes of hPDMSCs : sampling from the culture flask and bioreactor , respectively , detecting the amplification rate of the cells and PH , lactic acid content , glucose content and osmotic pressure of the culture solution .

5 . The expression of CD13 , CD14 , CD29 , CD31 , CD44 , CD166 , CD73 , CD90 , CD105 , CD166 , HLA - DR , HLA - ABC and the changes of cell cycle were detected by flow cytometry .

Results

1 . MSC morphology extracted from placenta : fusiform or fibrous , and can be stably grown in 20 passages .

2 . Human placental tissue - derived cell surface labeling features : CD13 , CD29 , CD44 , CD73 , CD90 , CD105 , CD166 , HLA - ABC , CD14 , CD31 , CD45 , HLA - DR were not expressed .

3 . Changes of growth and metabolism of hPDGFs

( 1 ) The attachment of hPDGFs on the cytodex - 3 microcarriers : the control group of the microcarriers was obviously adhered to the periphery of the microcarriers , but only a small number of microcarriers were observed , and the number of the participating microcarriers was less ; and in the stirred bioreactor , the microcarriers were adhered together by the cell matrix , showing obvious bridging phenomenon .

( 2 ) The growth curve of hPDGFs showed that the growth curve of hPDGFs in the control group began to be expanded with a relatively gentle trend , and the density of cells had always increased and peaked at the 4th day , then the density of the cells appeared to decrease obviously . The amplification tendency of hPDLBs in the stirred bioreactor was slightly lower than that of the control group , and then there were some signs of obvious amplification of the cells .

( 3 ) The amplification factor of hPDMD1was 10.55 卤 1.62times in the stirred bioreactor , which was significantly higher than that of the control group ( 6.10 卤 0.11 ) ( P < 0.05 ) .

( 4 ) Consumption and metabolism of nutrient substances : glucose consumption in stirred bioreactor was higher than that of the control group , but the production of lactic acid was lower than that of the control group .

( 5 ) The PH value of the culture fluid varied : the pH change in the reactor was smaller than that of the control group .

( 6 ) The osmotic pressure of the culture fluid varied : the osmotic pressure of the culture solution in the reactor and the control group was maintained between ( 280 - 320 ) mmol / kg .

4 . The marked characteristics of the surface markers and the cell cycle before and after the bioreactor were cultured : flow cytometry showed that the cell surface markers and cell cycle did not change significantly ( P > 0.05 ) before and after the application of stirred bioreactor .

Conclusion

1 . There are abundant MSCs in the placenta , which is not only a wide source , but also does not involve ethics and legal issues . It provides a new source of stem cells for human beings .

2 . The stirred bioreactor provides an ideal three - dimensional culture system for hPDMSCs , and compared with the static two - dimensional culture system , the cell nutrient substances are relatively uniform in the three - dimensional culture system , so that the cells exhibit good amplification capability , and the characteristics of the expanded cell stem cells are not obviously changed .

【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329.2

【参考文献】