基于稀释—过滤法去除低温保护剂的微系统研究

发布时间：2018-04-18 21:29

本文选题：低温保护剂 + 微流控技术　；参考：《中国科学技术大学》2017年硕士论文

【摘要】：低温保存的细胞已经被广泛应用于细胞治疗、组织再生和生殖医学等领域。在细胞的低温保存过程中,低温保护剂可以延长细胞的保存时间以及防止细胞的低温损伤,但是低温保护剂对细胞有毒性并且对人体有副作用,因此在细胞使用之前去除低温保护剂十分必要。当前,从低温保存的细胞中去除低温保护剂的方法主要有离心法、透析法和超滤法。离心法操作简单,但离心力易导致细胞损伤和结块,从而造成细胞损失;透析法可以减少细胞渗透压损伤,但低温保护剂的洗涤效率较低;超滤法可快速清除低温保护剂,但细胞回收率较低。这些方法通常适合洗涤大体积的细胞悬浮液(30～500mL),但不适合处理微量的细胞样品。近年来,随着微流控技术的迅速发展,已有文献开始研发低温保护剂洗涤芯片以处理微量的冷冻复温细胞,但这些芯片存在诸多不足(比如低温保护剂洗涤效率不高)。因此,本文提出了一种基于稀释-过滤法去除细胞低温保护剂的微系统(该微系统也称为多步稀释-过滤微装置)。系统设计的核心理念是:基于微流控技术,含有低温保护剂的细胞悬浮液用含有不同氯化钠浓度的稀释液逐级稀释,细胞内的低温保护剂跨膜输运至细胞外溶液,细胞外含有低温保护剂的溶液通过过滤逐级去除。本文的主要内容如下:(1)设计并加工了多步稀释-过滤微装置。本文微装置设计用于从猪红细胞中清除甘油。在设计中,利用梯度网络产生稀释液,利用特斯拉混合器实现溶液混合,利用基尔霍夫电压-电流定律确定设计参数。本文的微装置用有机玻璃加工而成。(2)理论分析了本文设计的微装置的性能。首先,基于低温保护剂跨细胞膜输运模型,模拟了微装置低温保护剂洗涤时细胞体积的变化和甘油浓度的变化。其次,利用稀物质输运模型,研究了稀释液产生区和稀释-过滤执行区中混合器的性能。(3)实验研究了本文设计的微装置的性能。通过细胞实验,确定了微装置在设计条件下的甘油清除率、细胞存活率和细胞回收率,探索了甘油浓度、细胞压积比和过滤膜孔径对微装置性能的影响。通过无细胞荧光实验,确认了稀释液产生区和稀释-过滤执行区的功能。本文研究结果表明,在微装置中,细胞的体积一直保持在细胞能承受的安全范围内(0.5～1.51倍的初始体积),细胞内的甘油可被有效清除,混合器能够实现两股溶液的充分快速混合。在设计条件下(细胞悬浮液流量为0.2 mL/min,甘油初始浓度为20%w/v,细胞压积比为24%v/v),甘油清除率达到约80%,细胞存活率达到约90%,但细胞回收率相对较低,约40%。当甘油浓度较低时,三个性能指标均较高;当细胞密度较高或过滤膜的孔径较大时,甘油清除率较高,但细胞存活率和回收率较低。本文的工作有助于研发生物芯片以去除低温保存细胞中的低温保护剂。
[Abstract]:Cryopreserved cells have been widely used in cell therapy, tissue regeneration and reproductive medicine.During the cryopreservation of cells, cryopreservation agents can prolong the preservation time of cells and prevent the damage of cells at low temperature, but cryopreservation agents are toxic to the cells and have side effects on the human body.Therefore, it is necessary to remove the cryogenic protectant before the cell is used.At present, the methods of removing cryogenic protectants from cryopreserved cells are centrifugation, dialysis and ultrafiltration.Centrifugation is simple, but centrifugal force can easily lead to cell damage and caking, resulting in cell loss; dialysis can reduce cell osmotic pressure damage, but the washing efficiency of cryogenic protectant is low; ultrafiltration method can quickly remove cryogenic protectant.But the cell recovery was low.These methods are usually suitable for washing large volume cell suspensions 3050ml / L, but not for microamounts of cell samples.In recent years, with the rapid development of microfluidic technology, some literatures have started to develop low-temperature protectant scrubbing chips to deal with microamounts of frozen rewarming cells, but these chips have many shortcomings (such as the low temperature protectant washing efficiency is not high.Therefore, a microsystem based on dilution filter method is proposed for the removal of low temperature cell protectants (also known as multistep dilution filtration microdevice).The core idea of the system design is: based on microfluidic technology, cell suspensions containing low temperature protectants are diluted with diluents containing different concentrations of sodium chloride step by step, and the cryogenic protectants in cells are transported to extracellular solutions through membrane.Extracellular solutions containing low temperature protectors are removed step by step through filtration.The main contents of this paper are as follows: 1) A multi-step dilution-filter microdevice is designed and fabricated.This microdevice is designed to remove glycerol from porcine red blood cells.In the design, the gradient network is used to produce the diluent, the Tesla mixer is used to mix the solution, and the Kirchhoff's law of voltage-current is used to determine the design parameters.The performance of the microdevice designed in this paper is analyzed theoretically.Firstly, based on the model of transmembrane transport of cryogenic protectants, the changes of cell volume and glycerol concentration during the washing of cryogenic protectants in microdevices were simulated.Secondly, the performance of the mixer in the dilution generation region and the dilution-filtration executive region is studied by using the dilute material transport model. The performance of the microdevice designed in this paper is studied by experiments.The glycerol clearance rate, cell survival rate and cell recovery were determined by cell experiments. The effects of glycerol concentration, cell volume ratio and filtration membrane pore size on the performance of the microdevice were investigated.The functions of the diluent producing region and the dilution-filtration executive region were confirmed by cell-free fluorescence assay.The results show that in the microdevice, the cell volume has been kept in the safe range of the cell to bear 0.51.51 times of the initial volume, the intracellular glycerol can be effectively removed, the mixer can achieve the full and rapid mixing of two strands of solution.Under the design conditions (the cell suspension flow rate is 0.2 mL / min, the initial concentration of glycerol is 20 weeks / v, the cell product ratio is 24 v / v / m), the clearance rate of glycerol is about 80%, the cell survival rate is about 90%, but the cell recovery is relatively low, about 40%.When the concentration of glycerol was low, the three performance indexes were all higher, and when the cell density was higher or the pore diameter of filtration membrane was larger, the glycerol clearance rate was higher, but the cell survival rate and recovery rate were lower.The work in this paper is helpful for the development of biochip to remove cryogenic protectants from cryopreserved cells.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R318.52

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