干细胞微流控芯片的设计、制备、检测与应用研究
摘要
干细胞在细胞治疗、再生医学和癌症研究方面的临床应用前景和意义已被全世界所公知，但其在伦理道德、分化潜能、免疫排斥和安全性等方面尚存亟待解决的问题，而干细胞机理研究作为实现可控增殖与分化、降低免疫排斥和避免成瘤的基础研究，已引起越来越高的重视。微流控芯片以其所具备的独特优势，能够在几微米至几百微米尺度下实现对细胞的操纵控制，从而实现干细胞在可控微环境/小生境下的可控增殖与分化，是干细胞机理研究不可替代的手段之一。目前使用微流控芯片研究干细胞的工作刚刚起步，领域内的参考文献尚少，虽已取得了一些研究成果，但还未能形成统一、高效、稳定地在微流控芯片上操纵和培养干细胞的方法。本论文试图通过自行设计并制备的一种微流控芯片，来探索在微流控芯片上操纵控制干细胞的方法，并探索干细胞在微流控芯片上的培养方法。
本论文从设计、制备和应用等方面对干细胞微流控芯片进行了研究，并针对在微流控芯片中干细胞需要进行呼吸作用、剪切力会对干细胞产生影响、制备过程中芯片材料的选择、制备效率的提高和成本的降低、微流控芯片易用性的提高、干细胞注入微流控芯片、干细胞在微流控芯片中的培养等问题进行了探索。
在设计方面，本论文详细分析了干细胞在非芯片条件下培养的7个基本条件，并探讨了这7个基本条件在微流控芯片培养条件下实现的方法；考虑到微流控芯片培养条件对干细胞的影响以及在微流控芯片上操纵干细胞和培养干细胞对芯片结构的要求等因素，对微流控芯片的芯片结构进行了总体设计，并利用有限元分析软件对气室区和培养区进行了优化设计。结果表明：当气室结构中入口和出口的距离越长时，气室中流速较小部分所占比例越小，流速相对较大部分所占比例越大，越有利于迅速换气并减少气体残留，并且这一规律与入口、出口和气室三者的位置关系无关；通过理论计算可以得出，在常用电动微量注射装置驱动的微流控芯片中，当培养室直径在1mm量级上时，干细胞所受剪切力在10-2dynes/cm2量级上，并且这一量级上的剪切力远远小于已有报道的会对干细胞产生影响的平均剪切力值。
在制备方面，本论文简单介绍了制备微流控芯片的材料、方法和流程，并介绍了常见的加工、键合、封装和检测技术；对微流控芯片在制备过程中，在芯片材料的选择、制备效率和成本、易用性等方面存在的问题进行了分析，并针对这些问题进行了研究。在微结构的加工过程中，采用光盘压印的方法进行加工；在基板和盖片的键合过程中，采用热压键合的方法进行；在微流控芯片的封装过程中，采用微针连接微流控芯片和外部设备；并分别对加工效率、键合效率和封装效率进行了估算。结果表明：PC材料最适合作为干细胞微流控芯片的制备材料；通过再键合，可以提高键合效率，从而提高了微流控芯片的制备效率并降低了制备成本；通过微针连接微流控芯片和外部设备，可以实现微流控芯片中微观结构与外部设备中宏观结构的连接，解决微流控芯片微观结构与外部宏观接口不匹配、难于封装的问题，并提高微流控芯片的易用性。
在应用方面，本论文选用ICR小鼠骨髓间充质干细胞作为实验细胞；并且选用低温湿法灭菌作为微流控芯片的灭菌处理方法；为方便实验操作，设计了一套手动微量注射装置。利用微量注射装置，将单细胞和多个细胞注入到微流控芯片中，并与非芯片条件下刚刚完成接种还未贴壁的细胞进行了对比。同时，为了验证微流控芯片气室区和培养区的优化结果与实际情况是否相符，选用单细胞作为微流控芯片中液体流速的标记物，对单细胞移动速度进行了测量，并将所得测量值与同等条件下液体流速的计算值进行了对比。此外，在将单细胞和多个细胞注入到微流控芯片中以后，还对单细胞和多个细胞进行了培养，并与非芯片条件下培养的细胞进行了对比。结果表明：注入后，微流控芯片中和非芯片条件下的细胞形态一致；单细胞移动速度的测量值与液体流速的计算值基本一致，验证了对微流控芯片气室区和培养区进行优化所得出的结果；培养中，微流控芯片中和非芯片条件下的细胞形态一致；微流控芯片中有限的培养面积可能抑制了ICR小鼠骨髓间充质干细胞的增殖，并且这种对细胞增殖的抑制或促进作用可能与细胞种类有关。
Stem cells are essential for cell therapy, regenerative medicine and cancerresearch, which are well known. However, there are also problems to be solved inethics and morals, differentiation potency, immunological rejection and safety. Themechanism study of stem cells, which are important for controlling proliferation anddifferentiation, reducing immune rejection and avoiding tumorigenesis, has drawnmore and more attention. Microfluidic chips with its unique advantages could beemployed to control proliferation and differentiation of stem cells in amicroenvironment, which makes microfluidic chips one of the irreplaceable tools inmechanism studies of stem cells. The research of stem cells in microfluidic chips hasjust started currently. The references in this field are still less. Moreover, there are notstandard, efficient and stable methods to control and culture stem cells in microfluidicchips. This paper attempts to explore the methods of controlling and culturing stemcells in homemade microfluidic chips.
In this paper, the design, fabrication and application of microfluidic chips forstem cells are researched. The problems of stem cells’ breathing, shear force’sinfluence, microfluidic chips’ material selecting, microfluidic chips’ productivity, costand usability, stem cells’ microinjection and culturing are explored.
In the respects of designing microfluidic chips,7basic conditions of culturingstem cells in culture dishes were analyzed. In addition, the methods of realizing7basic conditions in microfluidic chips were analyzed. Based on the analysis,microfluidic chips are designed. Moreover, the air chambers and the culture chambersin microfluidic chips are optimized by finite element method. Results show: when thedistance between the entrance and exit of gas chamber increases, the percentage ofportion with smaller velocity reduces, the percentage of portion with greater velocityincreases, the velocity of changing air increases and the residue of air becomes less,which has nothing to do with the structures of the entrance, the exit and gas chamber;and in microfluidic chips driven by electric micro injection device, when the diameterof culture chamber is1mm more or less, the shear force suffered by stem cells isabout10-2dynes/cm2which has no influence to the stem cells.
In the respects of fabricating microfluidic chips, the material, methods andprocesses of fabricating microfluidic chips are introduced. Some important problems in fabricating microfluidic chips are analyzed: the selection of microfluidic chips’material, the productivity and cost of fabricating microfluidic chips and the usabilityof microfluidic chips. In the processing cover slips, CD/DVD manufacturingtechnology is adopted. In the bonding of cover slips and substrates, the thermalbonding method is adopted. In the packaging of microfluidic chips, the micro needlesare adopted. Meanwhile, the productivity of processing, bonding and packaging isestimated. Results show: the material PC is suited for fabricating microfluidic chips;repeated bonding could increase the productivity and reduce the cost of fabricatingmicrofluidic chips; and using micro needles could make the usability of microfluidicchips easier.
In the respects of applying microfluidic chips, ICR mouse bone marrowmesenchymal stem cells were chosen as the experimental cells and low temperaturesteam sterilization was chosen as the microfluidic chips’ sterilization methods. Inaddition, a manual injection device was designed and made. Using the manualmicroinjection device and an electric microinjection device, a single cell and cellswere microinjected into microfluidic chips. Moreover, the flow velocity inmicrofluidic chips was measured to confirm the optimized results. After themicroinjection and measurement, single cells and cells were cultured in microfluidicchips. Results show: the movement velocity of a single cell is similar to the liquidflow velocity which means the optimized results and the real situation reach a goodagreement; the limited culture area in microfluidic chips may inhibit the proliferationrate of ICR mouse bone marrow mesenchymal stem cells; and the inhibition orpromotion of proliferation rate may relate to the kinds of cells.
本论文从设计、制备和应用等方面对干细胞微流控芯片进行了研究，并针对在微流控芯片中干细胞需要进行呼吸作用、剪切力会对干细胞产生影响、制备过程中芯片材料的选择、制备效率的提高和成本的降低、微流控芯片易用性的提高、干细胞注入微流控芯片、干细胞在微流控芯片中的培养等问题进行了探索。
在设计方面，本论文详细分析了干细胞在非芯片条件下培养的7个基本条件，并探讨了这7个基本条件在微流控芯片培养条件下实现的方法；考虑到微流控芯片培养条件对干细胞的影响以及在微流控芯片上操纵干细胞和培养干细胞对芯片结构的要求等因素，对微流控芯片的芯片结构进行了总体设计，并利用有限元分析软件对气室区和培养区进行了优化设计。结果表明：当气室结构中入口和出口的距离越长时，气室中流速较小部分所占比例越小，流速相对较大部分所占比例越大，越有利于迅速换气并减少气体残留，并且这一规律与入口、出口和气室三者的位置关系无关；通过理论计算可以得出，在常用电动微量注射装置驱动的微流控芯片中，当培养室直径在1mm量级上时，干细胞所受剪切力在10-2dynes/cm2量级上，并且这一量级上的剪切力远远小于已有报道的会对干细胞产生影响的平均剪切力值。
在制备方面，本论文简单介绍了制备微流控芯片的材料、方法和流程，并介绍了常见的加工、键合、封装和检测技术；对微流控芯片在制备过程中，在芯片材料的选择、制备效率和成本、易用性等方面存在的问题进行了分析，并针对这些问题进行了研究。在微结构的加工过程中，采用光盘压印的方法进行加工；在基板和盖片的键合过程中，采用热压键合的方法进行；在微流控芯片的封装过程中，采用微针连接微流控芯片和外部设备；并分别对加工效率、键合效率和封装效率进行了估算。结果表明：PC材料最适合作为干细胞微流控芯片的制备材料；通过再键合，可以提高键合效率，从而提高了微流控芯片的制备效率并降低了制备成本；通过微针连接微流控芯片和外部设备，可以实现微流控芯片中微观结构与外部设备中宏观结构的连接，解决微流控芯片微观结构与外部宏观接口不匹配、难于封装的问题，并提高微流控芯片的易用性。
在应用方面，本论文选用ICR小鼠骨髓间充质干细胞作为实验细胞；并且选用低温湿法灭菌作为微流控芯片的灭菌处理方法；为方便实验操作，设计了一套手动微量注射装置。利用微量注射装置，将单细胞和多个细胞注入到微流控芯片中，并与非芯片条件下刚刚完成接种还未贴壁的细胞进行了对比。同时，为了验证微流控芯片气室区和培养区的优化结果与实际情况是否相符，选用单细胞作为微流控芯片中液体流速的标记物，对单细胞移动速度进行了测量，并将所得测量值与同等条件下液体流速的计算值进行了对比。此外，在将单细胞和多个细胞注入到微流控芯片中以后，还对单细胞和多个细胞进行了培养，并与非芯片条件下培养的细胞进行了对比。结果表明：注入后，微流控芯片中和非芯片条件下的细胞形态一致；单细胞移动速度的测量值与液体流速的计算值基本一致，验证了对微流控芯片气室区和培养区进行优化所得出的结果；培养中，微流控芯片中和非芯片条件下的细胞形态一致；微流控芯片中有限的培养面积可能抑制了ICR小鼠骨髓间充质干细胞的增殖，并且这种对细胞增殖的抑制或促进作用可能与细胞种类有关。
Stem cells are essential for cell therapy, regenerative medicine and cancerresearch, which are well known. However, there are also problems to be solved inethics and morals, differentiation potency, immunological rejection and safety. Themechanism study of stem cells, which are important for controlling proliferation anddifferentiation, reducing immune rejection and avoiding tumorigenesis, has drawnmore and more attention. Microfluidic chips with its unique advantages could beemployed to control proliferation and differentiation of stem cells in amicroenvironment, which makes microfluidic chips one of the irreplaceable tools inmechanism studies of stem cells. The research of stem cells in microfluidic chips hasjust started currently. The references in this field are still less. Moreover, there are notstandard, efficient and stable methods to control and culture stem cells in microfluidicchips. This paper attempts to explore the methods of controlling and culturing stemcells in homemade microfluidic chips.
In this paper, the design, fabrication and application of microfluidic chips forstem cells are researched. The problems of stem cells’ breathing, shear force’sinfluence, microfluidic chips’ material selecting, microfluidic chips’ productivity, costand usability, stem cells’ microinjection and culturing are explored.
In the respects of designing microfluidic chips,7basic conditions of culturingstem cells in culture dishes were analyzed. In addition, the methods of realizing7basic conditions in microfluidic chips were analyzed. Based on the analysis,microfluidic chips are designed. Moreover, the air chambers and the culture chambersin microfluidic chips are optimized by finite element method. Results show: when thedistance between the entrance and exit of gas chamber increases, the percentage ofportion with smaller velocity reduces, the percentage of portion with greater velocityincreases, the velocity of changing air increases and the residue of air becomes less,which has nothing to do with the structures of the entrance, the exit and gas chamber;and in microfluidic chips driven by electric micro injection device, when the diameterof culture chamber is1mm more or less, the shear force suffered by stem cells isabout10-2dynes/cm2which has no influence to the stem cells.
In the respects of fabricating microfluidic chips, the material, methods andprocesses of fabricating microfluidic chips are introduced. Some important problems in fabricating microfluidic chips are analyzed: the selection of microfluidic chips’material, the productivity and cost of fabricating microfluidic chips and the usabilityof microfluidic chips. In the processing cover slips, CD/DVD manufacturingtechnology is adopted. In the bonding of cover slips and substrates, the thermalbonding method is adopted. In the packaging of microfluidic chips, the micro needlesare adopted. Meanwhile, the productivity of processing, bonding and packaging isestimated. Results show: the material PC is suited for fabricating microfluidic chips;repeated bonding could increase the productivity and reduce the cost of fabricatingmicrofluidic chips; and using micro needles could make the usability of microfluidicchips easier.
In the respects of applying microfluidic chips, ICR mouse bone marrowmesenchymal stem cells were chosen as the experimental cells and low temperaturesteam sterilization was chosen as the microfluidic chips’ sterilization methods. Inaddition, a manual injection device was designed and made. Using the manualmicroinjection device and an electric microinjection device, a single cell and cellswere microinjected into microfluidic chips. Moreover, the flow velocity inmicrofluidic chips was measured to confirm the optimized results. After themicroinjection and measurement, single cells and cells were cultured in microfluidicchips. Results show: the movement velocity of a single cell is similar to the liquidflow velocity which means the optimized results and the real situation reach a goodagreement; the limited culture area in microfluidic chips may inhibit the proliferationrate of ICR mouse bone marrow mesenchymal stem cells; and the inhibition orpromotion of proliferation rate may relate to the kinds of cells.
引文
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