基于微流体数字化喷射的细胞微胶囊制备技术研究
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摘要
细胞微胶囊在疾病治疗、组织培养、三维药物筛选、细胞新陈代谢分析等方面有着广阔应用前景。以“节拍化”方式制备细胞微胶囊对单细胞微胶囊的制备以及后续细胞的高效集成化、自动化操作都具有重要意义,现有微流控芯片液滴生成技术存在成本高、通用性差,对细胞活性影响较大等不足。
将高黏度细胞悬浮液分割为微小均匀液滴是细胞微胶囊制备技术的核心。本文以微流体数字化技术为起点,对微流体数字化驱动细胞悬浮液的原理、理论及微流体数字化驱动下细胞悬浮液微流动特性进行了分析,建立了微流体数字化驱动下圆管中细胞悬浮液脉冲运动流量理论模型,定性确定了影响微流体脉冲流动的各种系统参量。
基于微流体数字化技术设计制作了电磁铁为作动器,电磁吸合撞击振动梁产生交替脉冲惯性力的撞击式微流体数字化喷射实验系统,实现了微尺度、低雷诺数微管内高黏度液体以及高黏度固-液两相流体的稳定脉冲喷射。实现了海藻酸钠微胶囊的“节拍化”制备和对固体颗粒“节拍化”的包囊。系统结构简单,具有良好的微结构特性,对液体的极性没有要求。
进行了细胞悬浮液稳定脉冲喷射和“节拍化”包囊细胞研究。以微流体数字化脉冲喷射实现了尺度量级10-103μm细胞的“节拍化”包囊,对不同粒径以及粒径不均匀的细胞都具有良好的适应性。制备的细胞微胶囊平均粒径可小至30μm,相应的粒径标准偏差小于5μm,具有较好单分散性。可通过驱动电压和微喷嘴内径参数控制细胞微胶囊的粒径大小和分布。在细胞的活性影响和粒径的控制等方面优于微流控芯片技术。
进行了微流体数字化喷射制备单细胞微胶囊研究。通过控制管径不大于2倍细胞粒径,将细胞序列整列为单列排列,控制脉冲喷射步长控制喷射的细胞数量,当脉冲喷射步长不大于2倍细胞粒径时,以微流体数字化脉冲喷射可实现单细胞的脉冲喷射和对单细胞“节拍化”包囊。调节驱动电压及细胞与喷嘴内径比,可控制单细胞微胶囊的粒径大小和分布。
设计制作了脉冲喷射用同轴微喷嘴,进行了低雷诺数微流体同轴脉冲喷射特性和稳定喷射实验研究,进行了微流体同轴脉冲喷射“节拍化”包囊细胞研究。确定了稳定同轴脉冲喷射合适内径比和端口相对位置等几何参数,实现了细胞液和海藻酸钠溶液同轴、同步脉冲喷射,以“节拍化”方式实现了细胞共微胶囊的制备。将淋巴细胞与台盼蓝溶液制成共微胶囊,实现了微胶囊中细胞活性的直接检测。
Cell microcapsule is wildly applied in disease treatment, tissue culture, analysis of cell metabolism and3-D drug screening. Preparation of cell microcapsule rhythmically has important significance on integrated and automated manipulating of cell microcapsule subsequently. Droplet formation based on the microfluidic chip is expensive, has bad generality and negative influence on the cell activity.
The key technology in cell microcapsule preparation is to divide the high viscosity cell suspending liquid to unified micro droplets. In this paper, based on the digital of micro-fluids, the pulse inertia driving principle, theory and the flow field characters of cell suspending liquid were analyzed, theoretical model of pulse moving cell suspending liquid in micropipette driven by pulse inertia force was built, the system parameters that had influence on the pulse flow of micro-fluids were determined qualitatively.
Based on the digital of micro-fluids, an experiment system for micro-fluids ejection with pulse inertia force produced by the electromagnet impact on beam was built. Steady ejection of liquid and solid-liquid two-phase flow with high viscosity and low Re in micro-pipette was realized. The sodium alginate microcapsule and microcapsule with solid particles were respectively prepared rhythmically. The system has simple structure and fine micro-structural properties, and is versatile to the polarity of the liquid.
Steady pulse ejection of cell suspending solution and rhythmically cell encapsulating were researched. Based on the digital of micro-fluids pulse ejection,10～103μm scale cells were encapsulated rhythmically and the system was well adapted to different size cells and heterogeneous cells. The average diameter of cell microcapsules prepared could reach as small as30μm, and was smaller than those prepared by other method such as pressure spray, electrostatic spray. The standard deviation could be less than30μm, and the microcapsules prepared were well mono-disperse. The size and size distribution of cell microcapsule could be changed by adjusting driving voltage and inner diameter of the micro-nozzle. This method was superior to microfluidic chip on the cell activity influence and cell microcapsule size control.
Single cell microencapsulating based on the digital of micro-fluids was researched. Cells could be single lined when the nozzle diameter was less than two times of the cell size. The cells number in a capsule could be changed by adjusting the pulse ejection step length. When the step length was less than two times of the cell diameter, a single cell could be dispensed and encapsulated rhythmically. The size and size distribution of cell microcapsule could be changed by adjusting the driving voltage and ratio of the cell size and nozzle diameter.
A kind of coaxial micro-nozzle for pulse ejection was designed and fabricated. The steady pulse ejection state and micro ejection character of low Re coaxial fluid were experimentally studied, and pulse ejection of coaxial fluid and cell microencapsulating rhythmically were researched. The diameter ratio and relative geometric position of the coaxial micro-nozzle in steady ejection state were determined, and the coaxial and synchronous pulse ejection of cell suspending solution and sodium alginate solution was realized. Cell co-microcapsules were prepared through'rhythmically method'. Direct testing of cell activity was realized by co encapsulation of lymphocyte cells and Trepan blue solution.
将高黏度细胞悬浮液分割为微小均匀液滴是细胞微胶囊制备技术的核心。本文以微流体数字化技术为起点,对微流体数字化驱动细胞悬浮液的原理、理论及微流体数字化驱动下细胞悬浮液微流动特性进行了分析,建立了微流体数字化驱动下圆管中细胞悬浮液脉冲运动流量理论模型,定性确定了影响微流体脉冲流动的各种系统参量。
基于微流体数字化技术设计制作了电磁铁为作动器,电磁吸合撞击振动梁产生交替脉冲惯性力的撞击式微流体数字化喷射实验系统,实现了微尺度、低雷诺数微管内高黏度液体以及高黏度固-液两相流体的稳定脉冲喷射。实现了海藻酸钠微胶囊的“节拍化”制备和对固体颗粒“节拍化”的包囊。系统结构简单,具有良好的微结构特性,对液体的极性没有要求。
进行了细胞悬浮液稳定脉冲喷射和“节拍化”包囊细胞研究。以微流体数字化脉冲喷射实现了尺度量级10-103μm细胞的“节拍化”包囊,对不同粒径以及粒径不均匀的细胞都具有良好的适应性。制备的细胞微胶囊平均粒径可小至30μm,相应的粒径标准偏差小于5μm,具有较好单分散性。可通过驱动电压和微喷嘴内径参数控制细胞微胶囊的粒径大小和分布。在细胞的活性影响和粒径的控制等方面优于微流控芯片技术。
进行了微流体数字化喷射制备单细胞微胶囊研究。通过控制管径不大于2倍细胞粒径,将细胞序列整列为单列排列,控制脉冲喷射步长控制喷射的细胞数量,当脉冲喷射步长不大于2倍细胞粒径时,以微流体数字化脉冲喷射可实现单细胞的脉冲喷射和对单细胞“节拍化”包囊。调节驱动电压及细胞与喷嘴内径比,可控制单细胞微胶囊的粒径大小和分布。
设计制作了脉冲喷射用同轴微喷嘴,进行了低雷诺数微流体同轴脉冲喷射特性和稳定喷射实验研究,进行了微流体同轴脉冲喷射“节拍化”包囊细胞研究。确定了稳定同轴脉冲喷射合适内径比和端口相对位置等几何参数,实现了细胞液和海藻酸钠溶液同轴、同步脉冲喷射,以“节拍化”方式实现了细胞共微胶囊的制备。将淋巴细胞与台盼蓝溶液制成共微胶囊,实现了微胶囊中细胞活性的直接检测。
Cell microcapsule is wildly applied in disease treatment, tissue culture, analysis of cell metabolism and3-D drug screening. Preparation of cell microcapsule rhythmically has important significance on integrated and automated manipulating of cell microcapsule subsequently. Droplet formation based on the microfluidic chip is expensive, has bad generality and negative influence on the cell activity.
The key technology in cell microcapsule preparation is to divide the high viscosity cell suspending liquid to unified micro droplets. In this paper, based on the digital of micro-fluids, the pulse inertia driving principle, theory and the flow field characters of cell suspending liquid were analyzed, theoretical model of pulse moving cell suspending liquid in micropipette driven by pulse inertia force was built, the system parameters that had influence on the pulse flow of micro-fluids were determined qualitatively.
Based on the digital of micro-fluids, an experiment system for micro-fluids ejection with pulse inertia force produced by the electromagnet impact on beam was built. Steady ejection of liquid and solid-liquid two-phase flow with high viscosity and low Re in micro-pipette was realized. The sodium alginate microcapsule and microcapsule with solid particles were respectively prepared rhythmically. The system has simple structure and fine micro-structural properties, and is versatile to the polarity of the liquid.
Steady pulse ejection of cell suspending solution and rhythmically cell encapsulating were researched. Based on the digital of micro-fluids pulse ejection,10～103μm scale cells were encapsulated rhythmically and the system was well adapted to different size cells and heterogeneous cells. The average diameter of cell microcapsules prepared could reach as small as30μm, and was smaller than those prepared by other method such as pressure spray, electrostatic spray. The standard deviation could be less than30μm, and the microcapsules prepared were well mono-disperse. The size and size distribution of cell microcapsule could be changed by adjusting driving voltage and inner diameter of the micro-nozzle. This method was superior to microfluidic chip on the cell activity influence and cell microcapsule size control.
Single cell microencapsulating based on the digital of micro-fluids was researched. Cells could be single lined when the nozzle diameter was less than two times of the cell size. The cells number in a capsule could be changed by adjusting the pulse ejection step length. When the step length was less than two times of the cell diameter, a single cell could be dispensed and encapsulated rhythmically. The size and size distribution of cell microcapsule could be changed by adjusting the driving voltage and ratio of the cell size and nozzle diameter.
A kind of coaxial micro-nozzle for pulse ejection was designed and fabricated. The steady pulse ejection state and micro ejection character of low Re coaxial fluid were experimentally studied, and pulse ejection of coaxial fluid and cell microencapsulating rhythmically were researched. The diameter ratio and relative geometric position of the coaxial micro-nozzle in steady ejection state were determined, and the coaxial and synchronous pulse ejection of cell suspending solution and sodium alginate solution was realized. Cell co-microcapsules were prepared through'rhythmically method'. Direct testing of cell activity was realized by co encapsulation of lymphocyte cells and Trepan blue solution.
引文
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