利用PDMS微孔阵列研究三维空间因素对脂肪干细胞体外培养增殖及形态的影响
发布时间：2019-06-18 15:36
【摘要】：微孔阵列(Microwell arrays)是利用微尺度技术(Microscale technologies)在不同材料(玻璃、硅、PDMS、PLGA等)表面进行微米级加工并生成大量微孔的技术,本实验拟应用聚二甲基硅氧烷(polydimethylsiloxane, PDMS)为微孔支架材料制备微阵列细胞培养试验平台,对组织工程种子细胞——大鼠脂肪干细胞(Rat Adipose Stem Cells, RADSCs)在不同孔径微孔中细胞增殖情况及形态学改变进行研究,以期验证微孔阵列这一新兴实验平台在干细胞组织工程研究中的可行性。同时,初步明确大鼠脂肪干细胞在微孔阵列三维培养平台上的增殖及形态学特点,为此平台在干细胞及组织工程领域的应用打下基础。本实验采用聚二甲基硅氧烷(poly-dimethylsiloxane, PDMS)为微孔支架材料,在其表面加工生成40μm、60μm和80μm等不同孔径微阵列,并将RADSCs单细胞接种其上。利用光学显微镜、扫描电镜及激光扫描共聚焦显微镜等手段,分别对各组中细胞的贴附能力、增殖情况、形态改变进行观察及检测。 结果：1、硅晶圆模板经酸性双氧水清洗后,再倒模后形成的PDMS微阵列支架基本符合设计要求。2、ADSCs直接种植在未经预处理的支架上时细胞贴附能力明显下降,而经10%胎牛血清(Fetal Bovine Serum, FB S)培养基完全浸泡预处理后,细胞在PDMS表面的帖附及存活能力提高。3、体外培养120h时,其他条件保持恒定,不同孔径(40μm、60μm、80μm)的微孔内细胞生长总数具有显著性差异。4、随培养时间的延长在40μm微孔内生长的ADSCs更倾向于铺满孔底,60μm和80μm微孔内生长的ADSCs更倾向于沿孔壁-孔底拐角延伸生长。5、ADSCs在平面及微孔内生长,伸展形成“伪足”样及“桥”样结构,其内部丝状肌动蛋白(F-actin)排列方向与此结构长轴平行。 结论：利用微加工技术可以制作具有精细、统一表面结构的微孔阵列。将大鼠脂肪干细胞(ADSCs)悬液以一定浓度种植于PDMS微孔阵列内,细胞可在微孔内呈单个生长。细胞于PDMS表面及微孔内爬附生长表现“伪足”样及“桥”样结构,其内部F-actin纤维排列方向与此结构长轴平行。不同孔径微孔对ADSCs增殖及形态均存在影响,其中40μm微孔较60μm和80μm微孔更适宜ADSCs生长。
[Abstract]:Microwell arrayals are a technique of microscale processing on the surface of different materials (glass, silicon, PDMS, PLGA, etc.) and produce a large number of micro-pores. Polydimethylsiloxanes (polydimethylsiloxanes) are to be used in the experiment. The cell proliferation and morphological changes of rat adipose-derived stem cells (RADSCs) in different pore sizes were studied. In order to verify the feasibility of this new experimental platform in the research of stem cell tissue engineering. At the same time, the proliferation and the morphological characteristics of the adipose-derived stem cells of the rat on the three-dimensional culture platform of the micro-well array are clearly defined, and the foundation is laid for the application of the platform in the field of stem cell and tissue engineering. Polydimethylsiloxane (PDMS) was used as the material of the micro-porous scaffold, and different pore size microarrays such as 40. m u.m,60. m u.m and 80. m The adhesion, proliferation and morphological changes of the cells in each group were observed and tested by means of an optical microscope, a scanning electron microscope and a laser scanning confocal microscope. Results:1. After the silicon wafer template was cleaned by acid hydrogen peroxide, the PDMS micro-array stent formed after remolding was basically in accordance with the design requirements.2. The cell adhesion ability was significantly reduced when the ADSCs were directly planted on the unpretreated scaffold and 10% fetal bovine serum (Fetal Boven Ser.) The number of cells in the microwells with different pore size (40. mu.m,60. mu.m,80. mu.m) was significantly different from the total number of cells in the microwells with different pore sizes (40. mu.m,60. mu.m,80. mu.m). .4. The ADSCs grown in the 40 & mu; m micro-pores with the extension of the culture time tended to extend along the cell wall-bottom corner of the cell wall. The ADSCs tend to grow along the hole wall-hole bottom corner.5. The ADSCs grow in the plane and in the micro-pores to form the "pseudo-foot"-like sample and the "bridge"-like sample. The structure, the orientation of the filamentous actin (F-actin) in the structure and the long axis of the structure Parallel. Conclusion: Micro-machining technology can be used to make fine and uniform surface structure. The rat adipose-derived stem cell (ADSCs) suspension is planted in the PDMS microporous array at a certain concentration, and the cells can be in the micro-pores. The growth of the cells in the surface of the PDMS and the inside of the micro-pores shows the "pseudo-foot"-like structure and the "bridge"-like structure, and the inner F-actin fiber is arranged in the direction of the arrangement of the inner F-actin fibers. The micro-pores of 40. mu. m are more suitable for AD than 60. m u.m and 80. m
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
本文编号：2501598
[Abstract]:Microwell arrayals are a technique of microscale processing on the surface of different materials (glass, silicon, PDMS, PLGA, etc.) and produce a large number of micro-pores. Polydimethylsiloxanes (polydimethylsiloxanes) are to be used in the experiment. The cell proliferation and morphological changes of rat adipose-derived stem cells (RADSCs) in different pore sizes were studied. In order to verify the feasibility of this new experimental platform in the research of stem cell tissue engineering. At the same time, the proliferation and the morphological characteristics of the adipose-derived stem cells of the rat on the three-dimensional culture platform of the micro-well array are clearly defined, and the foundation is laid for the application of the platform in the field of stem cell and tissue engineering. Polydimethylsiloxane (PDMS) was used as the material of the micro-porous scaffold, and different pore size microarrays such as 40. m u.m,60. m u.m and 80. m The adhesion, proliferation and morphological changes of the cells in each group were observed and tested by means of an optical microscope, a scanning electron microscope and a laser scanning confocal microscope. Results:1. After the silicon wafer template was cleaned by acid hydrogen peroxide, the PDMS micro-array stent formed after remolding was basically in accordance with the design requirements.2. The cell adhesion ability was significantly reduced when the ADSCs were directly planted on the unpretreated scaffold and 10% fetal bovine serum (Fetal Boven Ser.) The number of cells in the microwells with different pore size (40. mu.m,60. mu.m,80. mu.m) was significantly different from the total number of cells in the microwells with different pore sizes (40. mu.m,60. mu.m,80. mu.m). .4. The ADSCs grown in the 40 & mu; m micro-pores with the extension of the culture time tended to extend along the cell wall-bottom corner of the cell wall. The ADSCs tend to grow along the hole wall-hole bottom corner.5. The ADSCs grow in the plane and in the micro-pores to form the "pseudo-foot"-like sample and the "bridge"-like sample. The structure, the orientation of the filamentous actin (F-actin) in the structure and the long axis of the structure Parallel. Conclusion: Micro-machining technology can be used to make fine and uniform surface structure. The rat adipose-derived stem cell (ADSCs) suspension is planted in the PDMS microporous array at a certain concentration, and the cells can be in the micro-pores. The growth of the cells in the surface of the PDMS and the inside of the micro-pores shows the "pseudo-foot"-like structure and the "bridge"-like structure, and the inner F-actin fiber is arranged in the direction of the arrangement of the inner F-actin fibers. The micro-pores of 40. mu. m are more suitable for AD than 60. m u.m and 80. m
【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R329
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