本文選題：低溫保護(hù)劑 + 微流控技術(shù)　； 參考：《中國(guó)科學(xué)技術(shù)大學(xué)》2017年碩士論文

【摘要】：低溫保存的細(xì)胞已經(jīng)被廣泛應(yīng)用于細(xì)胞治療、組織再生和生殖醫(yī)學(xué)等領(lǐng)域。在細(xì)胞的低溫保存過(guò)程中,低溫保護(hù)劑可以延長(zhǎng)細(xì)胞的保存時(shí)間以及防止細(xì)胞的低溫?fù)p傷,但是低溫保護(hù)劑對(duì)細(xì)胞有毒性并且對(duì)人體有副作用,因此在細(xì)胞使用之前去除低溫保護(hù)劑十分必要。當(dāng)前,從低溫保存的細(xì)胞中去除低溫保護(hù)劑的方法主要有離心法、透析法和超濾法。離心法操作簡(jiǎn)單,但離心力易導(dǎo)致細(xì)胞損傷和結(jié)塊,從而造成細(xì)胞損失;透析法可以減少細(xì)胞滲透壓損傷,但低溫保護(hù)劑的洗滌效率較低;超濾法可快速清除低溫保護(hù)劑,但細(xì)胞回收率較低。這些方法通常適合洗滌大體積的細(xì)胞懸浮液(30～500mL),但不適合處理微量的細(xì)胞樣品。近年來(lái),隨著微流控技術(shù)的迅速發(fā)展,已有文獻(xiàn)開(kāi)始研發(fā)低溫保護(hù)劑洗滌芯片以處理微量的冷凍復(fù)溫細(xì)胞,但這些芯片存在諸多不足(比如低溫保護(hù)劑洗滌效率不高)。因此,本文提出了一種基于稀釋-過(guò)濾法去除細(xì)胞低溫保護(hù)劑的微系統(tǒng)(該微系統(tǒng)也稱為多步稀釋-過(guò)濾微裝置)。系統(tǒng)設(shè)計(jì)的核心理念是:基于微流控技術(shù),含有低溫保護(hù)劑的細(xì)胞懸浮液用含有不同氯化鈉濃度的稀釋液逐級(jí)稀釋,細(xì)胞內(nèi)的低溫保護(hù)劑跨膜輸運(yùn)至細(xì)胞外溶液,細(xì)胞外含有低溫保護(hù)劑的溶液通過(guò)過(guò)濾逐級(jí)去除。本文的主要內(nèi)容如下:(1)設(shè)計(jì)并加工了多步稀釋-過(guò)濾微裝置。本文微裝置設(shè)計(jì)用于從豬紅細(xì)胞中清除甘油。在設(shè)計(jì)中,利用梯度網(wǎng)絡(luò)產(chǎn)生稀釋液,利用特斯拉混合器實(shí)現(xiàn)溶液混合,利用基爾霍夫電壓-電流定律確定設(shè)計(jì)參數(shù)。本文的微裝置用有機(jī)玻璃加工而成。(2)理論分析了本文設(shè)計(jì)的微裝置的性能。首先,基于低溫保護(hù)劑跨細(xì)胞膜輸運(yùn)模型,模擬了微裝置低溫保護(hù)劑洗滌時(shí)細(xì)胞體積的變化和甘油濃度的變化。其次,利用稀物質(zhì)輸運(yùn)模型,研究了稀釋液產(chǎn)生區(qū)和稀釋-過(guò)濾執(zhí)行區(qū)中混合器的性能。(3)實(shí)驗(yàn)研究了本文設(shè)計(jì)的微裝置的性能。通過(guò)細(xì)胞實(shí)驗(yàn),確定了微裝置在設(shè)計(jì)條件下的甘油清除率、細(xì)胞存活率和細(xì)胞回收率,探索了甘油濃度、細(xì)胞壓積比和過(guò)濾膜孔徑對(duì)微裝置性能的影響。通過(guò)無(wú)細(xì)胞熒光實(shí)驗(yàn),確認(rèn)了稀釋液產(chǎn)生區(qū)和稀釋-過(guò)濾執(zhí)行區(qū)的功能。本文研究結(jié)果表明,在微裝置中,細(xì)胞的體積一直保持在細(xì)胞能承受的安全范圍內(nèi)(0.5～1.51倍的初始體積),細(xì)胞內(nèi)的甘油可被有效清除,混合器能夠?qū)崿F(xiàn)兩股溶液的充分快速混合。在設(shè)計(jì)條件下(細(xì)胞懸浮液流量為0.2 mL/min,甘油初始濃度為20%w/v,細(xì)胞壓積比為24%v/v),甘油清除率達(dá)到約80%,細(xì)胞存活率達(dá)到約90%,但細(xì)胞回收率相對(duì)較低,約40%。當(dāng)甘油濃度較低時(shí),三個(gè)性能指標(biāo)均較高;當(dāng)細(xì)胞密度較高或過(guò)濾膜的孔徑較大時(shí),甘油清除率較高,但細(xì)胞存活率和回收率較低。本文的工作有助于研發(fā)生物芯片以去除低溫保存細(xì)胞中的低溫保護(hù)劑。
[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.
【學(xué)位授予單位】：中國(guó)科學(xué)技術(shù)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R318.52

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本文編號(hào)：1770158