CN117286027A - 基于液滴微流控的高效率定量线粒体转移的系统与方法 - Google Patents
基于液滴微流控的高效率定量线粒体转移的系统与方法 Download PDFInfo
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Abstract
一种基于液滴微流控以定量控制线粒体转移的系统,其包括用于生成含有游离线粒体和单细胞的液滴的生成模块;用于在显微镜下观察生成液滴的观察模块;以及用于收集生成的液滴的收集模块。转移到受体细胞中的所需线粒体数量是精确医学中的一个重要问题,本发明可以精确地在单细胞水平上控制转移线粒体的数量,帮助在进行线粒体基因相关疾病的细胞治疗之前,确定使受体细胞功能明显改善所需的外来线粒体数量。
Description
技术领域
本发明涉及基于液滴微流控系统进行线粒体转移,更具体地说,涉及基于液滴微流控系统的高效率定量线粒体转移。
背景技术
突变型与野生型线粒体DNA(mtDNA)的比率称为异质性系数,此系数决定了线粒体相关病症的严重程度。在肌肉组织中,当异质性系数超过一定水平或线粒体功能失调时,会产生较少的ATP和过量的活性氧(ROS),进而引发肌肉萎缩、无力和耐力丧失。先前的临床和临床前动物研究显示,线粒体损伤的增加与不良的骨骼肌健康状况之间存在联系。自二十世纪90年代以来,细胞疗法(特别是成肌细胞移植)已被提出用于改善受伤骨骼肌的再生,然而,成肌细胞移植的早期临床试验结果大多显示失败,主要是由于宿主免疫细胞(如CD8+T淋巴细胞)的存在,而导致大量细胞死亡,免疫细胞的积累不但会造成肌膜损伤和激活肌纤维中的胱天蛋白酶3,还会进一步诱导肌纤维的凋亡。因此,需要开发新的治疗骨骼肌病症的方法,例如调节巨噬细胞和化学诱导干细胞,而如何恢复或改善线粒体功能以促进肌肉再生是具有吸引力的方法。
除了为细胞产生能量外,线粒体也与细胞增殖、衰老、凋亡、先天免疫、钙稳态、甚至干细胞分化潜力相关。mtDNA的突变会损害细胞和组织的功能,在自然界中,自发性的线粒体转移可以通过不同的机制在健康细胞和受损细胞之间发生,以保护受损细胞并恢复其细胞功能。线粒体转移是一种改变细胞中mtDNA的技术,自从Clark和Shay首次公开后,引起了越来越多的关注。线粒体转移目前已被用于治疗mtDNA相关疾病的细胞疗法,相较于其他修改线粒体基因组的技术,如mitoZFNs和mitoTALENs,线粒体转移更容易执行并且实际上更有效,透过将外源线粒体转移到受体细胞中可以降低突变型与野生型mtDNA的比率,并恢复或改善细胞和组织的功能。先前的研究显示,可以通过共培养或显微注射的方式将外源的游离线粒体递送至细胞中,在与游离线粒体共培养的方法中,受体细胞通过内吞作用吞噬游离的线粒体,内吞作用是一种从周围环境中摄取纳米到数微米的物体的细胞活动,游离的线粒体在受体细胞周围随机移动,当接触到细胞时有机会被细胞吞噬,因此这种现象是一种随机和偶发的过程,共培养方法的转移效率受细胞外游离的线粒体数量的影响,尽管在先前的研究中,最高可以达到28%,但即使在等量的游离线粒体的情况下,转移进细胞的线粒体数量也是相当不平均的(有的受体细胞有1个而有的受体细胞有60个),尽管共培养方法是一个相当简单的过程,但其是否成功取决于许多不可控的因素,这也可能是先前试验中受体细胞的细胞代谢恢复率不理想(约0.2%)的潜在原因之一。为了减少不可控因素的影响,基于自动化光镊的操纵系统(基于OT的操纵系统)被用于进行线粒体质量和数量可控的线粒体转移,基于OT的操纵系统可以精确地获取健康的线粒体,并将它们运送到目标受体细胞。然而,这种方法存在通量低的限制,这使得基于OT的操纵系统难以用于临床应用。
与共培养方法不同,显微注射是将预装在微针中的游离线粒体直接注射到受体细胞中;因此,由于在递送过程中必需打开细胞膜,可能会对受体细胞造成损害。此外,显微注射技术的通量也很低。
上述方法为研究线粒体转移后细胞功能的恢复或改善机制提供了有用的技术方案,然而,它们仍然不能满足细胞疗法行业对转进了线粒体的细胞的大量需求。共培养技术由于无害而具有相当大的优势,但其低效率和异质性仍是一个主要瓶颈。
液滴微流控是一种将携带化学试剂、细胞或其他生物材料的连续流分散到微米级的离散体积(称为液滴)中的技术,这些液滴是进一步进行化学反应、细胞生命活动、目标检测和材料合成的基本单位。液滴微流控比大体积的分析方法提供了更小且受限制的环境,因此可以更快速地反应和检测分子/颗粒以及与包裹细胞的相互作用。先前的研究已经证明,液滴生成速率可高达每秒数千个液滴,这使得液滴微流控在本质上是属于一种高通量技术。液滴微流控的一个重要应用是单细胞分析,其中单个细胞被包裹在一个液滴中,用于分析细胞生命活动或进行细胞修饰,如抗体分析或基因编辑。
美国专利申请公开号2017/159017A1,名称为:“将外源线粒体引入哺乳动物细胞的方法(Method for introducing exogenous mitochondria into a mammalian cell)”和美国专利美国专利申请公开号2013/149778A1,名称为:“肽介导的线粒体递送系统的方法和应用(Method and Applications of Peptide-Mediated Mitochondrial DeliverySystem)”,应用天然细胞膜吞噬过程(也称为内吞作用)来转移游离的线粒体。
美国专利申请公开号2019/276852A1,名称为:“将外源线粒体递送到细胞中的方法(Method for delivering exogenous mitochondria into cells)”和欧洲专利申请公开号EP3169338A1,名称为:“受体细胞中游离的线粒体的细胞间转移方法(Methods forthe intercellular transfer of isolated mitochondria in recipient cells)”,应用离心法转移线粒体。在这些方法中,游离的线粒体和细胞一起被离心,通过迫使线粒体进入细胞来提高线粒体转移效率。
美国专利号10,760,040B1,名称为:“机械转染装置和方法(Mechanicaltransfection devices and methods)”,应用流体诱导的机械力打开细胞膜并将游离的线粒体递送到细胞中。
传统的显微注射方法会对受体细胞造成物理伤害,共培养技术能够产生细胞疗法所需的细胞数量。然而,共培养技术无法控制转移到每个受体细胞中的线粒体数量,因此没有足够线粒体的细胞可能无法发挥其完整的治疗功能。
发明内容
因此,一种基于液滴微流控的线粒体转移系统提供了高效定量的控制功能。在本发明中,基于液滴微流控的线粒体转移方法不会对受体细胞造成物理伤害(这在显微注射中不可避免),并且显示出高通量的优势,与传统的共培养方法相比,本发明中所提出的方法还可以在单细胞水平上控制转移到受体细胞的线粒体数量,实现高效率和高通量。
本发明提供了一种基于液滴微流控的线粒体转移高效定量控制系统,所述系统包括生成含有游离的线粒体和单细胞的液滴的生成模块;在显微镜下观察生成的液滴的观察模块;以及收集所述生成的液滴的收集模块。
在另一实施例中,液滴生成模块和液滴观察模块透过导管连接。
在其他实施例中,所述系统被设置在芯片上。
在又一实施例中,所述芯片的长度小于8cm。
在另一实施例中,液滴生成模块包括三个入口。
在另一实施例中,液滴生成模块进一步包括线粒体受体细胞悬浮液、游离的线粒体悬浮液和添加了表面活性剂的氟化油。
在其他实施例中,线粒体受体细胞是受体C2C12细胞。
在另一实施例中,液滴生成模块包括流动聚焦结构,其中所述流动聚焦结构可将线粒体受体细胞悬浮液和所述游离的线粒体悬浮液分离成液滴。
在又一实施例中,液滴生成模块包括波状结构,其中所述流动聚焦结构可从入口将随机分布的细胞排列成一条直线。
在另一实施例中,所述波状结构可提高单细胞包裹率至超过47%。
在另一方实施例中,所述波状结构可抑制多细胞包裹率至小于6%。
在其他实施例中,所述液滴包含游离的线粒体,以及一个单细胞。
在另一实施例中,线粒体的转移效率至少为75%。
在其他实施例中,所述系统在30分钟内可产生至少2×106个包裹在液滴中用于线粒体转移的细胞。
本发明还提供了一种基于液滴微流控的线粒体转移的定量控制方法,其包括以下步骤:制备第一悬浮液、第二悬浮液和油液;将第一悬浮液、第二悬浮液和油液注入基于液滴微流控的线粒体转移的定量控制系统;将第一和第二悬浮液共同包裹在液滴中;收集液滴;以及将第一和第二悬浮液共同培养在液滴中。
在另一实施例中,第一悬浮液是线粒体受体细胞悬浮液。
在又一实施例中,第二悬浮液是游离的线粒体悬浮液。
在其他实施例中,液滴的直径为40μm。
在其他实施例中,所述系统包括波状结构。
在另一实施例中,液滴包含线粒体和线粒体受体细胞。
附图说明
图1A-1G显示系统设置、基于液滴微流控的线粒体转移技术和实验评估流程的示意图。图1A显示基于液滴微流控的线粒体转移技术的系统设置;图1B显示包裹前用于细胞配对的波状结构和包裹后用于混合线粒体和细胞悬浮液的波状结构;图1C显示共同包裹在液滴中的细胞和线粒体;图1D显示波状结构对细胞进行聚焦的演示;图1E显示在液滴中细胞内吞线粒体转移的演示;图1F显示用于液滴生成和观察的芯片;图1G显示完成线粒体转移的受体细胞进行体外成肌分化实验的流程。
图2A-2I显示基于液滴微流控的线粒体转移系统的展示。图2A显示细胞悬浮液和游离的线粒体悬浮液共同流向波状结构;图2B显示用于提高单细胞包裹效率的波状结构;图2C显示用于液滴生成的流动聚焦结构;图2D显示了从供体细胞(C2C12成肌细胞)分离出来的游离线粒体(分离前用MitoTrackerTM Green FM染色);图2E显示由受体C2C12细胞吸收的游离线粒体,以及进一步以共聚焦成像分析一部分液滴,以确认线粒体是否转移成功(受体细胞为红色,转移的线粒体为绿色);图2F显示在不同浓度的细胞悬浮液下使用波状结构的细胞包裹效率,其中1和2分别代表一个和两个或更多个细胞包裹在一个液滴中,L代表0.85×107个细胞/毫升,以及H代表1.7×107个细胞/毫升;图2G显示油/水流速比对液滴尺寸的影响;图2H显示了液滴大小对线粒体转移效率的影响;图2I显示细胞悬浮液流速对细胞活性的影响。图2F-2I的所有数据均以平均值±标准差表示,并通过单因素方差分析和邓恩多重比较检验(Dunn’s multiple comparisons test)进行分析,*p<0.05和**p<0.01,从A到C的红色箭头表示流体在微流控芯片通道中的流动方向。
图3A-3B显示在不同浓度的游离线粒体悬浮液的情况下,包裹在每个液滴中的游离线粒体的数量。图3A显示使用三种不同浓度的游离线粒体悬液时,单一液滴中的游离线粒体(以MitoTrackerTM Green标记)的3D重建图像(通过关闭共聚焦显微镜的红色通道,避免显示经Cell MaskDeep Red染色的细胞),其中每一方框都包含一个液滴,左上角标注了游离线粒体的数量;图3B显示在三种不同浓度的游离线粒体悬浮液的情况下,包裹在每个液滴中的游离线粒体的数量。所有数据均以平均值±标准差表示。
图4A-4C显示使用基于液滴的方法对线粒体转移的定量控制。图4A显示分别在0.25、0.5或1.0单位浓度下进行线粒体转移后,受体细胞悬浮液的代表性共聚焦图像,其中一单位浓度的线粒体代表从1×106个细胞中所分离出来并悬浮在10μL线粒体储存试剂中的线粒体的浓度,转移的线粒体在分离前用MitoTrackerTM Green FM标记,受体细胞在包裹至液滴中之前用Cell Mask Deep Red标记,从每单位中选出三个受体细胞,以说明转移的线粒体在细胞内的位置(底部);图4B显示在不同的线粒体浓度下,每个细胞中的平均转移线粒体数量,在共聚焦荧光显微镜下对3D重建图像进行线粒体数量计数;图4C显示转移效率,转移效率被定义为转移到受体细胞中的游离线粒体数量与包裹在液滴中的游离线粒体总数的比率。图4B和图4C中的所有数据均以平均值±标准差表示。
图5A-5D显示线粒体转移对C2C12成肌细胞的成肌分化的体外研究。图5A显示经线粒体转移的C2C12细胞在成肌诱导过程中的代表性图像,C2C12细胞在成肌诱导前进行不同浓度的线粒体转移(每个细胞转移8、14和31个外源分离的游离线粒体分别定义为低线粒体、中线粒体和高线粒体转移组),在诱导开始前和诱导过程的第3天和第7天,对细胞形态和形成的肌管进行成像;图5B-5C显示在第7天,用ImageJ测定肌管的面积和长度,每孔取三个FOV;图5D显示在线粒体转移完成后的第1、2、3和4天,通过MTT分析法测定C2C12细胞的增殖速率。所有值均相对于第0天标准化,数据以平均值±标准差表示,用单因素方差分析随后用邓恩多重比较检验进行分析,N=3。*(或#)表示p<0.05,**(或##)表示p<0.01,***(或###)表示p<0.001,以及****(或####)表示p<0.0001。
具体实施方式
参见附图,图1A示意性地描绘了一种基于液滴微流控系统的高效定量线粒体转移,液滴生成模块10可生成含有游离线粒体和单细胞的液滴40,其中液滴生成模块10还包括波状结构,可将随机分布的细胞从入口处聚焦成一排,以提高单细胞包裹率并抑制多细胞包裹率;元件20是液滴观察模块,其可在显微镜(未示出)下观察生成的液滴;元件30是液滴收集模块,可收集生成的液滴40;液滴生成模块和液滴观察模块可以通过导管50连接。可选地,如图1F所示,系统1被设置在芯片60上。
微流体芯片制造和操作:
透过软光刻法制造设计的芯片。在实验之前,芯片通道涂有表面改性剂,使其具有疏水性,以便稳定地生成和输送油包水液滴。
细胞培养:
C2C12成肌细胞在37℃与5%CO2的培养条件下,培养在具有高葡萄糖(GibcoTM,11965084)且含有10%胎牛血清(GibcoTM,12800058)和1%抗生素-抗霉菌素(GibcoTM,15240096)的达尔伯克改良伊格尔培养基(Dulbecco modified Eagle medium,DMEM)中。
线粒体分离:
使用的线粒体是在每次线粒体转移实验前,按照线粒体分离试剂盒(Beyotime,C3601)的方案从C2C12成肌细胞中新鲜分离出来的。首先,用MitoTrackerTM Green FM(InvitrogenTM,M7514)进行供体C2C12细胞的线粒体的染色,接着用PBS将染色的细胞洗涤三次,再用胰蛋白酶/EDTA溶液(GibcoTM,R001100)将染色的细胞从培养瓶中分离出来,并以500g离心5分钟,去除上清液后,用1ml的细胞裂解试剂(Beyotime,C3601-1)重新悬浮收集的细胞,并在冰浴中放置15分钟,再用玻璃均质机将裂解后的细胞均质化30个循环,接着将均质化的细胞在4℃下以1,000g离心10分钟后,用1ml的细胞裂解试剂(Beyotime,C3601-1)重新悬浮上清液,并再次在4℃下以1,000g离心10分钟,以获得更高的纯度,最后,收集上清液并在4℃下以3,500g离心10分钟,收集的离心块就是分离的游离线粒体。使用线粒体储存试剂(Beyotime,C3601-3)将分离的游离线粒体以所需的浓度悬浮起来,用于进一步的实验。将从1×106个细胞中分离出来并悬浮在10μL线粒体储存试剂中的线粒体设定为一单位的游离线粒体悬浮液。
共聚焦荧光显微镜下细胞和线粒体的3D重建:
将受体细胞和外源分离的游离线粒体在液滴中共培养2小时后,将液滴装入液滴观察模块,如图1A所示;然后,使用共聚焦荧光显微镜(LEICASP8LIA++TRUE共聚焦激光扫描显微镜)拍摄含有单细胞(用Cell Mask Deep Red标记)和游离线粒体(用MitoTrackerTMGreen FM标记)的液滴的影像;之后,使用共聚焦显微镜软件对拍摄的影像进行3D重建;最后,将转移到细胞中的游离线粒体计数为红色细胞区域内的绿色颗粒,而将红色细胞区域外的绿色颗粒计数为未转移的游离线粒体,线粒体转移效率为转移的线粒体与液滴内总线粒体的比率。对从液滴中回收的细胞重复相同的过程,以计数在不同浓度的游离线粒体浓度下转移的线粒体数量。
液滴破碎和细胞收集:
收集的液滴漂浮在氟化油的顶部,在液滴破碎之前,去除试管底部多余的油,然后,将1ml 50%的1H,1H,2H,2H-全氟辛醇(PFO,Thermo ScientificTM,AAB2015609)添加到含有收集的液滴的试管中,并轻轻涡旋1分钟,使液滴合并成本体溶液,之后,将上部本体溶液小心地移至新的试管中并以300g离心3分钟来收集细胞。
C2C12的成肌分化:
为了评估C2C12的分化,将C2C12细胞以5,000个细胞/cm2的浓度接种在六孔板中,并在生长培养基中培养直至达到80%汇合,然后用含有2%马血清(GibcoTM,16050130)的DMEM(GibcoTM,11965084)替换培养基,将细胞保持在分化培养基中直到实验结束,通常在第5天至第7天之间,每两天监测肌管形成,时间点分别为第0天、第3天及第7天。
MTT分析法:
通过MTT分析法测定各C2C12细胞组的细胞增殖率。简而言之,将C2C12细胞以5,000个细胞/cm2的密度接种在96孔板中并培育24小时后,用3-(4,5-二甲基-2-噻唑基)-2,5-二苯基-2H-四唑溴化物(MTT,100μL,0.5mg/mL)在37℃下处理细胞3小时,再以100μLDMSO溶解产生的深蓝色甲臜晶体。用微板读取器测量570nm处的吸光度。
在实验中,采用如图1A中开发的基于液滴的线粒体转移系统。三种溶液,如(1)线粒体受体C2C12细胞悬浮液,(2)游离线粒体悬浮液,和(3)添加了表面活性剂的氟化油,通过三个入口分别将线粒体受体C2C12细胞悬浮液、分离的线粒体悬浮液和添加了表面活性剂的氟化油(Sphere Fluidics,C021)注入液滴生成模块10中,之后,生成的液滴被收集在液滴收集模块30中,其中液滴包括线粒体、线粒体受体细胞。
如图1B和1C所示,流动聚焦结构用于将两种悬浮液分离成液滴,这两种悬浮液是线粒体受体C2C12细胞悬浮液和游离线粒体悬浮液。如图1D所示,波状结构可从入口将随机分布的细胞排列成一条直线,因此,可以提高单细胞包裹效率;如图1E所示,游离线粒体通过内吞作用被液滴内的受体C2C12细胞所吸收;通过PE管(BD IntramedicTM,BD 427406)连接液滴生成模块和液滴观察模块,整个制造的芯片仅大约8em长,如图1F所示;如图1G所示,线粒体转移完成后,通过液滴破碎处理从液滴中收集受体细胞,并可以立即进行功能实验,以评估不同的线粒体转移数量对C2C12细胞体外成肌分化的影响。
为了在共聚焦荧光显微镜(LEICA SP8LIA++TRUE共聚焦激光扫描显微镜)下进行观察和3D重建,在从供体细胞中分离出线粒体之前,使用浓度为2μM的MitoTrackerTM GreenFM(InvitrogenTM,M7514)进行线粒体染色。
如图2A-2C所示,线粒体分离后,线粒体受体细胞和刚分离的游离线粒体被立即注入微流体芯片,进行包裹和线粒体转移过程。如图2D所示,悬浮液中被标记的游离线粒体为球状结构,直径约为1μm。在生成的液滴中共培养2小时后,图2E显示,受体细胞通过内吞作用吸收游离线粒体。
如上所述,通过使用波状结构,细胞包裹效率的提高超过了泊松分布。图2F显示,在使用细胞浓度为0.85×107个细胞/毫升并且细胞悬浮液流速为300μL/30分钟(游离线粒体悬浮液的流速保持与之一致)的情况下,单细胞包裹效率能够达到约47.8%,而多细胞包裹率被抑制到约5.9%,这意味着单细胞与多细胞的包裹比率提高到8.1,换句话说,是泊松分布的292%(在平均每个液滴包裹的细胞为0.6的条件下)。单细胞包裹效率的提高能够帮助提高通量。本系统能够在30分钟内产生包裹在液滴中可用于线粒体转移的2×106个受体细胞,如图2H所示,线粒体转移效率被定义为转移到细胞中的游离线粒体与包裹在液滴中的总游离线粒体的比率,随着液滴直径从40μm增加到52μm,而从75%略微下降到70%;如图2G所示,通过将油/水相的流速比设置为6,将液滴直径设置为40μm;如图2I所示,用本系统处理后,受体细胞仍能保持相对较高的存活率,如在300μL/30分钟的流速下,存活率为95%。
液滴的封闭微环境限制了游离线粒体的移动距离,增加了游离线粒体与细胞接触的概率,从而使线粒体容易被细胞吸收,提高了线粒体的转移效率。此外,由于游离线粒体比液滴(直径为1-40μm)的尺寸小,因此游离线粒体被均匀地包裹在每个液滴中,包裹在液滴中的游离线粒体的数量可以通过调整游离线粒体悬浮液的浓度来控制。图3A-3B显示,在使用0.25、0.5和1.0单位浓度的游离线粒体悬浮液时,每个液滴中分别包裹了8、22和41个游离线粒体。1.0单位浓度是指从1×106个细胞中分离出来并悬浮在10μL线粒体储存试剂(Beyotime,C3601-3)中的线粒体。
实例
在一个实施例中,使用三种不同浓度的游离线粒体悬浮液(0.25、0.5和1.0单位浓度)来验证本发明的转移效率。图4A和4B显示,在所使用的0.25、0.5和1.0单位浓度下,平均分别有8、14和31个游离线粒体被转移到受体细胞中。图4C显示,在所使用的0.25、0.5和1.0单位浓度下,线粒体的转移效率约为75%。
通过诱导成肌细胞分化成肌管来测试C2C12成肌细胞在线粒体转移后的分化能力。诱导7天后,图5A-5C显示,与对照组、低线粒体转移组和中线粒体转移组(分别对应于每个细胞转移0、8和14个外源分离的游离线粒体)相比,高线粒体转移组(每个细胞转移31个外源分离的游离线粒体)的肌管面积和长度(作为肌生成的指标)都明显增加。此外,MTT分析法表明,在第4天,高线粒体转移组和中线粒体转移组的细胞增殖分别增加了2.5倍和1.5倍,如图5D所示。
工业适用性:
本发明提供的基于液滴微流控系统的线粒体转移是作为一种高效定量的线粒体转移系统提供,因为具有以下优点:
需要转移到受体细胞中的线粒体的数量是精确医学中的一个重要问题。本发明的开发可以在单细胞水平上实现对转移的线粒体数量的精确控制,对线粒体基因有关的疾病进行细胞治疗之前,确定明显改善受体细胞功能所需的外来线粒体数量。
与现有的线粒体转移方法相比,本发明的方法可以产生大量用于细胞疗法目的的,完成定量线粒体转移的细胞。虽然与游离的线粒体共培养的方法也可以产生细胞治疗所需的细胞数量,但它无法控制转移到每个受体细胞中的线粒体数量,因此,一些没有足够线粒体转移的细胞可能无法完全发挥其治疗功能。然而,使用本发明的系统,转移到每个受体细胞中的线粒体是可控的,因此,用于细胞治疗的细胞可以完全发挥其治疗功能。
虽然本公开已参照其具体实施例进行了描述和说明,但这些描述和说明并不是限制性的。本领域的技术人员应理解,在不背离所附权利要求书所定义的本公开的真正精神和范围的情况下,可以做出各种改变并且可以用等效物来替代。图示可能未必按比例绘制。由于制造工艺和公差的原因,本公开中的工艺再现与实际设备之间可能存在差异。本公开可能还有其他未具体说明的实施例。本说明书和附图应被视为说明性的,而不是限制性的。可以进行修改以使特定的情况、材料、物质组成、方法或工艺适应本公开的目标、精神和范围。所有这些修改都旨在落入所附权利要求的范围内。虽然本文公开的方法是参照以特定顺序进行的特定操作来描述的,但应理解,这些操作可以组合、细分或重新排序,以形成等效的方法,而不偏离本公开的教示。因此,除非在此特别指出,否则操作的顺序和分组不是限制。
Claims (10)
1.一种基于液滴微流控的线粒体转移定量控制系统,其特征在于,所述基于液滴微流控的线粒体转移定量控制系统包括:
生成含有游离线粒体和单细胞的液滴的生成模块;
在显微镜下观察生成的液滴的观察模块;以及
收集所述生成的液滴的收集模块。
2.根据权利要求1所述的系统,其特征在于,所述液滴生成模块和液滴观察模块透过导管连接。
3.根据权利要求1所述的系统,其特征在于,所述液滴生成模块包括三个入口。
4.根据权利要求1所述的系统,其特征在于,
所述液滴生成模块进一步包括线粒体受体细胞悬浮液、游离线粒体悬浮液以及添加了表面活性剂的氟化油,其中所述线粒体受体细胞为受体C2C12细胞。
5.根据权利要求1所述的系统,其特征在于,
所述液滴生成模块包括流动聚焦结构,其中所述流动聚焦结构可将所述线粒体受体细胞悬浮液和所述游离线粒体悬浮液分离成液滴;以及
所述液滴生成模块包括波状结构,其中所述波状结构可从入口将随机分布的细胞排列成一条直线。
6.根据权利要求5所述的系统,其特征在于,所述波状结构可提高单细胞包裹率至超过47%。
7.根据权利要求5所述的系统,其特征在于,所述波状结构可抑制多细胞包裹率至小于6%。
8.根据权利要求1所述的系统,其特征在于,所述液滴包含游离线粒体以及一个单细胞。
9.根据权利要求1所述的系统,其特征在于,线粒体转移效率至少为75%。
10.根据权利要求1所述的系统,其特征在于,其中所述系统在30分钟内产生至少2×106个包裹在液滴中用于线粒体转移的细胞。
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