CN105848793A - 材料选择性的递送至细胞 - Google Patents
材料选择性的递送至细胞 Download PDFInfo
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- CN105848793A CN105848793A CN201480056295.2A CN201480056295A CN105848793A CN 105848793 A CN105848793 A CN 105848793A CN 201480056295 A CN201480056295 A CN 201480056295A CN 105848793 A CN105848793 A CN 105848793A
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Abstract
根据所述细胞的物理性质分离或鉴别一种细胞能够包括提供一种细胞悬浮液;使悬浮液通过一种含有收缩部分的微流体通道;使细胞悬浮液通过收缩部分;并且,使所述细胞悬浮溶液与一种化合物进行接触。所述收缩部分被调节尺寸从而与相对较小的细胞相比优先使一种相对较大的细胞变形。
Description
相关申请的交叉引用
本申请要求享有2013年8月16日提交的美国临时专利申请号为61/866,972的权益,该申请的全部内容已经通过引用并入本文。
政府支持
本发明获得由美国国立卫生研究院授予的R01GM101420-01A1,美国国立卫生研究院授予的RC1EB011187-02,国立癌症研究所授予的P30-CA-14051,以及国家科学基金会授予的GRFP主要基金号为#1122374的政府支持。该政府部门在本发明中享有某些权利。
技术领域
本发明领域涉及材料的尺寸选择性递送至细胞。
发明背景
材料的细胞内递送是一种挑战。依赖于纳米颗粒、电场、成孔化学等的现有技术能够递送一些材料到某些细胞类型中,但是通常针对目标细胞的物理特性是以完全无差别方式进行。通过发展依赖于目标细胞的生物特性的选择性递送方法,人们能够在研究、诊断或者治疗应用的递送活性中运用多种更为有效的控制。举例来说,循环肿瘤细胞(CTC)是在血液中发现的认为是介导转移、或者癌症的扩散的肿瘤细胞,到身体中的较远位点。大约90%的由于癌症导致人类死亡是由于转移。CTC的识别与特征是用于理解、治疗或者预防转移癌症的关键。此外,本领域众所周知相对于周围血细胞而言这些细胞具有不同的物理特性。
发明内容
本发明主题提供了基于物理特性用于选择性为一种或多种细胞递送材料的装置、系统和方法,所述物理特性例如是尺寸、体积、直径、胞液粘度或膜硬度。举例来说,材料可以以细胞尺寸依赖的方式进行递送。含有不同尺寸细胞的细胞悬浮液可穿过有目标递送材料(例如,染料,蛋白质,核苷酸等等)存在的装置,并且这些材料能够选择性地递送到在种群中的较大细胞。通过较大细胞的细胞膜的选择性破坏的数据中的递送机制是因为其是在通道收缩部分中变形,而较小的细胞不会变形从而足以引起膜破坏。
在一些示例性应用中,能够得到相对于非肿瘤细胞而言标记的细胞。细胞通过一种装置用于使用荧光染料或者其他检测的标记物进行尺寸选择性标记。所述细胞任选用抗体进行染色,例如肿瘤细胞选择性抗体比方说抗CD45抗体,从而提供在癌症细胞和血细胞(大部分血细胞是CD45+)之间的进一步对比。样品通过一种细胞分选器,例如一种标准荧光活化细胞分选器(FACS)。
在一些示例性应用中,能够得到依据它们的细胞周期的细胞的标记,因为在的种群中接近分裂的细胞比那些刚刚经历过分裂的细胞更大。向在种群中的较大细胞的染料的递送能够用于鉴定在细胞周期较后面的阶段过程中的单个细胞。
在一些示例性应用中,用于血癌(例如淋巴瘤)的治疗可以实现,因为淋巴瘤细胞通常比周围血液细胞大,因此一种细胞内毒素能够递送到淋巴瘤细胞中而不是健康的周围血细胞中。从而,这可以诱导患病细胞的选择性地死亡。
标记的细胞可以通过荧光或者磁性纯化技术进行分离。使用机器人操纵的流式细胞仪或者微阵列能够被用于根据荧光性选择细胞,同时能够使用磁铁柱、微流分离系统、或磁力清扫器来分离磁标记的颗粒。
细胞可以基于相对尺寸或者直径从而进行识别。因此,相对较大的细胞选择性地或者优先地选取标记,这是由于在较大的细胞中细胞膜的破坏程度相对较大,即,相对于较小的细胞,较大的细胞以一种较大程度进行变形。由于较大细胞的较大程度的细胞膜破坏,相对于较小细胞而言,至少10%、25%、50%、2-倍、5-倍、10-倍、100-倍或者更多的有效负载分子获取进入到较大细胞的内部(细胞质)。作为根据上述的方式的可检测的标记的摄取的结果以及按照标记的摄取进行的后续分类,与在外周血中的纯度水平相比,多种肿瘤细胞可被增强100倍、1,000倍、或者高达10,000倍或者更多。纯度的评估是通过靶向/连接到已知标记上的抗体进行的,其中所述标记通过肿瘤细胞被表达/过表达。可选择地,针对标记的抗体,没有被肿瘤细胞所表达,但是会被血细胞所表达/过表达(CD45是一个示例)。两种方式都有助于提供增加的对比度从而分类所感兴趣的细胞。
具有高尺寸-标记荧光和低CD45荧光的样品被捕获作为候选/潜在的CTC。FACS输出在本质上是相对的。一种“高”信号在基线控制信号至少的荧光强度的最小的十进位(十倍较高的水平),并且一种“低”信号是在阳性控制种群之下的十进位。
本发明所述的装置和方法提供了一种解决方案,用于解决用于怎样从来源于患者的血液样本中的1-10百万的白血球中识别和/或分离大约1或者更多(2、5、10、100、1,000或者更多)的CTC的这一长期存在的问题。例如,每毫升血液1CTC是与癌症患者临床相关的。综上所述,用于分离或者识别一种循环肿瘤细胞的方法包括提供一种细胞悬浮液的步骤;使溶液通过微流体通道,所述微粒体通道包括收缩部分,所述收缩部分调节尺寸用于优选地使循环的肿瘤细胞变形,而不是白细胞变形;使细胞悬浮液通过收缩部分;并且是细胞悬浮溶液与可检测标记进行接触。所述溶 液可通过一种微流体通道,所述微流体通道包括一种收缩部分,所述收缩部分被调节尺寸从而优选地将一种化合物递送到具有与其他细胞组相比相对不同的物理特性的细胞组中。所述物理特性可以包括细胞尺寸、直径、胞液浓度和/或细胞膜硬度(例如通过传送时间测定法进行测定,较硬的细胞通过给定的微通道与较为不硬的细胞相比更慢,如在Sharei et al.,2012,Anal.Chem.84(15):6438-6443;Cross et al.,2007,Nature Nanotechnology2:780-783中所描述的那样)。所述接触可以发生在变形治疗之后。或者所述材料可以在变形治疗之前与细胞进行预混合。CTC和白血球都会变形;但是,较大的细胞变形至一种较大程度并且因此,分子可选择性地递送到这些细胞中,从而治疗或者标记它们。
举例来说,所述标记包括一种可检测标记的,例如,荧光性或者磁性示踪的材料,例如一种染料或者颗粒。所述染料或者颗粒不需要是肿瘤特异性的。可选择地,它们差异地连接到肿瘤细胞上(例如,与非肿瘤细胞相比,至少20%、50%、2倍、5倍或者更多)。但是,所述方法的特异性是依据一种发现,其中肿瘤细胞比白血球稍大,并且所述装置具有高度尺寸选择性。这种尺寸差异依赖于肿瘤类型。例如,肿瘤细胞通常比白血球大50%-400%。因此,所递送的材料优选地进入到细胞中,所述细胞是足够大的从而能够通过细胞的尺寸特异性变形而被标记。然后所递送的标记再依次被检测从而识别CTC。
在一个实施例中,所述悬浮液包括全血。作为选择地,所述细胞悬浮液是一种细胞的混合物,其在生理盐水溶液中而不是全血中。典型地,所述细胞悬浮液包括具有患有肿瘤细胞风险或者具备被诊断为具有肿瘤的受试者的全血。例如,患者怀疑具有、被诊断具有、或者怀疑或诊断患有黑色素瘤、结肠癌、前列腺癌、乳腺癌、肝癌、肺癌、胰腺癌、脑或血液的转移性疾病。因此,CTC的早期检测在临床上是重要的,因为这种检测表示一种可能向转移性疾病反向发展的患者的早期识别。
可选择地,在这些细胞通过所述装置之前,完成红细胞溶解作为一种预处理步骤。
所述装置的特征在于使肿瘤细胞区别于非肿瘤细胞(例如,正常的红细胞或者白细胞)的物理参数。举例来说,收缩部分包括从4μm-10μm的宽度,1μm-100μm的长度以及串联的1-10个收缩部分。细胞的估算速度可以是在10mm/s至10m/s的范围内。为了推进或者推动所述细胞悬浮液通过装置,所述方法进一步包括将一种压力施加至细胞。压力被用于趋势细胞悬浮液通过装置,并且通过收缩点的传输是使细胞变形并且导致细胞膜破坏,并且进而递送。
所述方法包括将一种可检测的化合物诱导至肿瘤细胞中。因此,所述细胞悬浮液包括一种有效负载,或者所述方法进一步包括,在其通过收缩部分之后,在一种含有有效负载的溶液中培养所述细胞悬浮液一段预设的时间。举例来说,所述有效负载包括磁性颗粒例如纳米颗粒、荧光颗粒例如一种量子点或者碳纳米管、或者荧光染料或者蛋白质、或者针对荧光蛋白编码的基因材料(DNA或者RNA)或者其他能够检测的化合物(例如荧光素酶)。可替代的,可以递送上述材料的组合,从而完成检测以及细胞的同时操作。举例来说,能够递送一种荧光颗粒从而完成排序和共递送DNA、RNA或者蛋白质,从而方便随后的肿瘤细胞的生存并且刺激它们的生长和增殖分选后,从而使得能够进一步的对培养的转移细胞的研究。
在本发明中,还涉及以一种用于从非肿瘤细胞区分肿瘤细胞的系统,包括定义了内腔的微流体通道,并且被配置从而使悬浮在缓冲液中的肿瘤细胞能够经此通过,并且与非肿瘤细胞相比对肿瘤细胞进行收缩。非肿瘤细胞可以被变形到某些程度;但是,关键在于对肿瘤细胞进行变形从而足以致使细胞膜破裂,反之非肿瘤细胞由于其较小的相对尺寸从而不会进行足以致使细胞膜破裂的变形。较小细胞的细胞膜不会被破坏或者相比于较大的细胞的细胞膜而言破坏较少。例如,在某些情况下,较大和较小细胞都会被破坏但是较小的细胞相对于较大的细胞会吸收较少的材料。微流体 通道包括细胞变形收缩部分,其中所述收缩部分的直径是细胞直径的函数。所述收缩部分进行尺寸化从而相比于非肿瘤细胞会优先地使肿瘤细胞变形。这种优选变形被设计为选择性地促进目标材料递送的肿瘤细胞,与非肿瘤细胞相比。选择性的递送使得一种物质富集在期望的肿瘤种群中,通过分拣/富集方法,例如流式细胞仪(FACS)、显微操纵、磁力分离、细胞培养。
该方法是在生理学温度下完成,例如37℃,室温下,例如20℃,或者任选的在0-4℃温度下。在某些情况中,后者是优选的,因为其能够产生较好的递送性能,由于延迟细胞膜修复并且通过降低细胞的内吞活性从而最大程度减小内吞作用的背景。根据上文所述,细胞悬浮液是全血或者在作为递送缓冲液的生理缓冲溶液(例如磷酸盐缓冲盐水(PBS)或组织培养基)中的任何哺乳动物细胞悬浮液。在某些实施例中,PBS是优选的,因为降低了在组织培养基上的Ca或者Mg的作用。
在一个方面,根据细胞的物理性质分离或者识别一种细胞可以包括提供一种细胞悬浮液;是悬浮液通过包括收缩部分的微流体通道;使细胞悬浮液通过收缩部分;并且使细胞悬浮溶液与化合物接触。收缩部分能够被尺寸化从而优选地使与相对较小的细胞相比相对较大的细胞进行变形。
在另一个方面,用于从非肿瘤细胞中区分肿瘤细胞的微流体系统可以包括一种微流体通道,所述微流体通道定义一种内腔并且被配置从而与非肿瘤细胞相比使悬浮在缓冲液中的肿瘤细胞通过并且被收缩。所述微流体通道可以包括一种细胞变形收缩部分。收缩部分的直径可以是细胞直径的函数。
可以包括下述特征中的一个或多个。例如,物理特性可以是尺寸和直径中的一个或多个。细胞悬浮液能够包括下一种或多种:外周血细胞;以及具有不同物理特性的至少两种不同细胞尺寸。细胞悬浮液可以包括外周血细胞的红细胞耗尽的群。较大的细胞可以包括循环肿瘤细胞,并且较小 的细胞可以包括白血球。化合物可以包括0.5kDa至5Mda的分子量。化合物可以包括3kDa至10kDa的分子量。化合物可以包括一种可检测的标记物(例如,量子点、青色素、荧光素、若丹明、以及它们的衍生物,比方说异硫氰酸荧光素(FITC)或者四甲基罗丹明异硫氰酸酯(TRITC)或者NHS-罗丹明,马来酰亚胺活化的荧光素比如说荧光素-5-马来酰亚胺,以及Alexa荧光剂),活性生物分子、和/或毒素(例如,假单胞菌外毒素、白喉毒素、和蓖麻毒素,caspase蛋白质,干扰基本细胞功能的抗体(例如,抗微管蛋白抗体)),用于选择性地杀死目标细胞。所述化合物能够影响细胞功能(例如,转录因子、siRNA、DNA、mRNA、抗体、小分子药物)和/或能够诱导细胞死亡。所述化合物能够在细胞通过收缩部分之后进入到细胞中。所述悬浮液可以包括全血。所述悬浮液可以包括患有包括肿瘤风险的受试者或者诊断具有肿瘤的受试者的全血。所述肿瘤可以包括黑色素瘤、结肠癌、前列腺癌、肺癌、胰腺癌、乳腺癌、肝癌、脑癌、或血液癌症。所述收缩部分可以包括的宽度为4μm-10μm,长度为1μm-100μm,以及串联的1-10个收缩部分。细胞通过收缩部分的速度可以是在10mm/s至10m/s的范围内。能够将压力应用于细胞悬浮液从而驱使细胞通过微流体通道的收缩部分。
所述细胞悬浮液可以包括一种有效载荷,或者所述细胞悬浮液可以在其通过收缩部分之后在预定的时间内在含有有效载荷的溶液中进行温育。所述有效载荷可以包括磁性颗粒、荧光颗粒、例如量子点或者碳纳米管、或者荧光染料或蛋白质、或者编码荧光蛋白的遗传物质(DNA或者RNA)或者其他能够检测的化合物(例如,荧光素酶)。所述收缩部分可以进行尺寸化从而与非肿瘤细胞相比优先使肿瘤细胞变形。
本发明的这些和其他能力连通本发明本身在重新仔细查看下列附图、详细描述和权利要求书得到全面的理解。
附图说明
附图1指的是用于通过快速机械变形的尺寸选择标记CTC的系统的框图。
附图2是柱状图,其显示了将尺寸选择递送微流体平台与用于CD45染色的抗体进行结合会产生样品富集因子,比独立地其他技术好超过一个量级。
附图3A是一种细胞标记的示意图。使用标准红细胞裂解试剂例如eBioscience RBC裂解缓冲液(Cat.No.00-4333)使红血细胞(RBCs)通过RBC细胞溶解从全血中被消耗。所得到的悬浮液流过收缩通道微流体装置,用荧光染料(以及任选其他化合物)进行温育。然后将所述悬浮液用CD45进行标记,并且在荧光激活细胞分选仪(FACS)机器上进行处理从而收集具有用荧光染料标记的非-CD45+细胞。
附图3B是一系列共轭3kDa右旋糖酐的cascade蓝的流式细胞素计数点,通过CellSqueeze装置递送至PBMCs(在50psi下的30-6个芯片)、HT-29(在50psi下的30-6个芯片)、SK-MEL-5(在50psi下的10-7个芯片)以及PANC-1(在50psi下的10-7个芯片)。
附图3C是在通过收缩通道之前和之后的一系列Panc-1肿瘤细胞和血细胞的透射光和荧光显微图。预递送的细胞是在染料存在下进行温育,用于矫正背景胞吞作用。所述后递送图像是在递送后24个小时拍照从而证明染料的滞留和细胞在递送后细胞的粘附和生长能力。虽然大的血细胞还可以在处理中得到标记,但是这些数据证明了肿瘤细胞的选择性标记。
附图4是与在PBMC和淋巴瘤混合物中的百分PBMC相比PBMC递送的图,显示了与健康的PBMC相比染料选择性递送至肿瘤细胞。即使当所述悬浮液是以数量级99.9%健康的PBCM时,在某些实施中能够实现高达8倍的递送特异性。在另一些实施中,能够实现更高的特异性。
附图5A是加入到全血(40细胞/ml)并且用CD45复染法(APC)处理的四甲基若丹明右旋糖酐标记的Panc-1-GFP细胞的FACS图。
附图5B是GFP与CD45相比的FACS图,证明根根据GFP荧光PANC-1GFP标记怎样能够单独地被证实。P5的栅极将被作为用于排序候选CTC的基础,P4被用于证实PANC-1GFP细胞的标识。绿色的点是精确命中(P4&P5),红色的点是假阳性(仅有P5),蓝色的点是未命中(仅有P4)。
附图5C是HTB1760的原发肿瘤的组织病理学图像,确定胰腺导管腺癌(PDAC)。
具体实施方案
CTC是肿瘤细胞,其在血液中被发现,并且被认为是癌症转移至远端器官的原因。CTC被认为是对于癌症患者最小侵入性的、“液体活组织检查”,并且是作为用于患者预后和治疗功效的预后指标。这些单一细胞的全面表征提供了转移性转移、治疗性抵抗和肿瘤生物学的更高的理解。
典型的人的红细胞具有大约6.2-8.2μm直径的圆盘,以及在2-2.5μm的最厚点的厚度;并且在0.8-1μm中心的最小厚度,比大多数其他人类细胞小的多。白细胞(白血细胞)包括中心粒细胞(12-14μm直径)、嗜酸粒细胞(12-17μm直径)、嗜碱粒细胞(14-16μm直径)、淋巴细胞(对于静止的直径为平均6-9μm,并且对于激活的直径为10-14μm)、以及单核细胞,最大类型的白血细胞可以在直径上高达20μm。如附图1所示,在CTC和血液细胞之间的尺寸差异通常允许从在循环血液(CTCs~9–20μm;RBC~8μm盘形;白细胞~7–12μm)中的其他细胞区分CTC。参见附图1。随后使用抗体(或者其细胞特异性片段)特异性标记的肿瘤细胞或者其他肿瘤细胞特异性配体提高了方法的选择性。
由于CTC是作为一种在血流中的106-107单核细胞中存在,所以使用了高灵敏度富集技术其依赖于从血细胞中在CTC方面的免疫学或者形态学的差异。免疫学方法通常针对上皮细胞表面标记(例如EpCAM)以及肿瘤特异性蛋白(例如Her2-neu、MUC I/MUC2、癌胚抗原(CEA)、乳腺球蛋白、以及甲胎蛋白)或者用于消除CD45+细胞的目的。微过滤、密度梯度分离和微流体平台是以形态学为基础的方法的示例。CTC的所有这些方法可能下调表面抗原或者表现出不同的形态特征。这些偏见造成该领域的显著挑战,因为所述CTC的子集是对于新陈代谢负责或者是可靠地预后标记仍然很大程度上是未知的。因此,开发能够确保所有候选CTC子类型的高灵敏度方案用于拍摄最为临床相关的候选物是非常重要的。在这里所描述的装置和方法允许感兴趣的CTC子类型的分离和计数。
一种组合富集的方法结合了免疫学和形态学基础的方法,用于标记和分离具有较少偏差的纯的CTC,并且基于可调的参数。所属方法结合了微流体细胞内递送(附图1)和抗体染色,用于从全血中产生稳定的、高灵敏度纯化的循环肿瘤细胞(附图2),包括一种4μ-10μm宽,1μm-100μm长,以及1-10个串联的收缩部分。细胞的估算速度可以为从10mm/s至10m/s的范围内。所选择的具体的装置参数是有目标肿瘤细胞类型所决定的,例如,不同的装置设计被用来针对黑色素瘤患者或者是结肠癌患者进行CTC的选择。肿瘤细胞尺寸/直径的实施例包括:黑色素瘤~15um,结肠癌~11um,以及胰腺癌~15um。
在这种方法中,一种快速机械变形递送系统利用在多种CTC和周围血细胞之间固有的尺寸差异用于选择性地将荧光、磁性和/或其他的区别材料递送至肿瘤细胞。在进一步的过程中,基于抗体的荧光和/或磁性标记被用于增强候选CTC和周围血细胞之间的对比。通过唯一地将基于尺寸和免疫学的方法与CTC分离结合,这种技术证明了用于分析的从患者样本中无偏置分离候选肿瘤细胞的作用。在某些实施方案中,较大和较小的细胞都能够进行变形,但是较小的细胞膜不会被变形至细胞膜受损 的那一点。例如,为了选择性地递送至在全血细胞中的15μm的肿瘤细胞,其中大部分健康的白细胞是~8μm的尺寸时,可以使用一种6μm宽的收缩部分。这种收缩部分将会同时时两种类型的细胞变形,但是将更优选地使15μm的肿瘤细胞细胞膜扰乱,而不是8μm的血细胞。
临床/转移相关性
目前正在探索将CTC作为用于肿瘤活检的替代指标,用于了解抗性机制并且指导靶向疗法的选择。治疗前和治疗后的CTC的数量和组合物的测量表明治疗效果和预后。所述方法利用了一种稳定的、高通量的、一次性的装置,用于根据细胞尺寸和表面抗原进行CTC的标记。此外,递送各种大分子的能力还能够使其递送分子探针(例如,抗体、量子点、碳纳米管、以及分子信标),其对细胞内环境进行应答,并且因此提供为目标细胞的细胞内特性提供进一步的信息。这种组合方法提供了一种稳定的平台,能够对那些仅仅依靠免疫学或形态学分离的替代方法已经错过的CTC种群进行富集。该技术有利于分离患者的CTC。
实施例
1
全血或者其他的细胞悬浮液同时使用未标记的和/或抗体包覆的磁珠进行处理。然后使用一种作用于罕见的细胞的高保真、磁性富集系统对这些细胞进行分离。也可以使用一种纳米孔技术用于通过从84,672亚纳米级的孔的弹性阵列中对所需要的单个细胞进行成像和自动检索从而实现高纯度的分离。
得到不同表型的单一、活的、纯净、完整的CTC允许表征的宿主从基因组到具有立即临床和转移相关性的功能水平努力。该方法允许一种具有降低偏差的活的、不同的CTC的高度敏感性和特异性富集。
实施例
2
磁性纳米颗粒被递送至肿瘤细胞系&PBMCs。纳米颗粒递送至EpCAM-表达的、上皮癌细胞系,例如HT-29、LNCaP、以及SK-BR-3,是与来源于人类血液的大量外周血单核细胞(PBMC)悬浮液进行比较的。
具有聚乙二醇(PEG)表面涂层的10nm氧化铁纳米颗粒被递送至与全血混合的癌症细胞中,并且所得到的目标细胞混合物通过使用上文所描述的细胞分离装置进行处理。例如,微流体递送系统被用于诱导细胞的快速机械变形从而生成在细胞膜中的瞬态空隙(附图1)。所述方法已经被证明了具有将一系列材料,包括蛋白质、RNA、DNA以及纳米颗粒递送之多种细胞类型中的能力,并且适用于全血、通常会对微流体系统产生问题的介质。
示例性的标记分子,例如3kDa和70kDa,荧光标记的、作为模型分子的葡聚糖聚合物,被用于单独依据尺寸在PBMC与两种不同的癌症细胞系之间进行区分。所述结果还表明系统对于磁性颗粒选择性递送至血液中的肿瘤细胞的功效。PEG包覆的氧化铁颗粒被用于磁性标记结肠癌(例如,根据细胞系HT-29所例证的)。进一步的富集是通过将FITC共轭至氧化铁纳米颗粒表面从而直接测量纳米颗粒的摄取来实现的。
PEG包覆的10nm氧化铁纳米颗粒被递送至细胞悬浮液中,所述细胞悬浮液被怀疑含有或者已知含有CTC,例如,一种来源于患者的血液样本,或者细胞系HT-29、LNCaP、和SK-BR-3细胞,其分别于全血进行混合。然后对所得到的标记细胞混合物进行纯化,例如,使用一种高保真磁分离器。所述分离器在具有高氧化铁含量的模型CTC和效率较低标记的PBMC之间进行准确区分。任选地,在治疗、纳米颗粒浓度增加、它们的尺寸改变或者涉及多个处理步骤之前,红血细胞被裂解。
实施例
3
一种基于结合的免疫学和形态学的方法如下所述实施。在基于细胞尺寸通过装置进行处理之后,用一种抗体或其他肿瘤细胞特异性配体例如荧光标记的抗-CD45抗体对细胞进行处理。对三种不同的分离方法的敏感度和特异性进行比较:1)仅装置,2)仅抗-CD45抗体,3)装置+抗-CD45抗体。形态标记(装置)+免疫标记(例如抗-CD45抗体)被发现相对于另外两种单独使用的技术而言可显示出优异的灵敏度(附图2)。例如,能够观察到于单独的抗-CD45抗体相比,其在灵敏度方面的2-5倍的增加和/或在特异性方面的2-5倍的增加。观察到超过一个数量级的富集指数(附图2)。
实施例
4
在一个实施例中,所述装置是由硅和玻璃制成。可替代的,所述装置是使用一种聚合物,例如硅氧烷、PDMS、聚碳酸酯、丙烯酸、聚丙烯、聚苯乙烯制成。任何装置都是被灭菌的(加热或者γ辐射)并且是一次性的。针对多种细胞类型使用材料和参数对设备的性能进行验证。例如,在一定范围的流速下的用PEG包覆的量子点(尺寸在10-50nm范围内)的性能被用于确定是否纳米颗粒和细胞存活率的递送效果。示例的装置在PCT/US2012/060646中进行描述,所述申请已经通过引用全部并入到本文中。
优势
当与现有的方法进行比较时,可以发现该方法具有下述优势。相对于基于抗体的方法时,该方法提供了一种非偏离隔离过程,其适用于大多数癌症类型中并且时独立于任何特定的细胞表面标记物的。所述装置和方法实现了不能够由现有的标记物分离的CTC的识别,并且因此具有显著的诊断和预后意义。
相对于现有的基于尺寸的分离方法,在本发明中所描述的装置和方法提供了高很多的通量,并且是通过改变“W”(附图1)可调的从而捕获特定的CTC尺寸范围。例如,一种6μm宽的收缩部分适用于结肠癌细胞的捕获,而一种7μm和8μm的宽度分别适用于胰腺癌细胞和黑色素瘤细胞的捕获。此外,与现有技术不同,这种系统可以与基于抗体的技术相结合用于增强分离敏感性和/或是CTC的子级多参数分离(例如通过分离一定尺寸+表面标记物的CTC)。
通过使获自一定范围的癌症类型的CTC的有效的、稳定的分离,这种技术将是对抗癌症的一种有价值的平台。这种技术的预后和诊断连理可以使新基因鉴别,所述新基因是癌症发展的关键,并且因此使得新的治疗技术的开发。它也能够提供患者期望寿命和治疗效果的更精确的预测。
在这里所描述的CTC分离方法结合了免疫学和以尺寸为基础的分离,从而得到一种高富集因子/回收率以及可调的偏置(标记特异性vs尺寸特异性)。
虽然上文只详细描述了少数的实施例,但是其他的修改也是可能的。上文描述的实施方案可以针对公开的特征的各种组合和子组合,和/或上文描述的几种其他特征的组合或亚组合。此外,附图中的逻辑流程图和/或在此的描述并不要求完全按照所示的特征顺序或步骤次序来实现所需结果。其他的实施方案可落在以下权利要求书的范围内。
Claims (19)
1.一种基于细胞的物理特性用于分离、鉴别、或者操作细胞的方法,所述细胞包括:
提供一种细胞悬浮液;
使所述悬浮液通过一种含有收缩部分的微流体通道,所述收缩通道被调节尺寸从而将化合物递送至具有相对不同的物理特性的细胞组中,而不是其他的细胞组;
使所述细胞悬浮溶液与一种化合物进行接触。
2.根据权利要求1所述的方法,其中所述物理特性是尺寸、直径和膜硬度的其中一种或多种。
3.根据权利要求1所述的方法,其中所述细胞悬浮液包括外周血细胞;以及具有不同物理特性的至少两种不同的细胞种类中的其中一种或多种。
4.根据权利要求1所述的方法,其中所述细胞悬浮液包括一种外周血细胞的红细胞去除的群。
5.根据权利要求1所述的方法,其中所述较大的细胞包括循环的肿瘤细胞并且所述较小的细胞包括白细胞。
6.根据权利要求1所述的方法,其中所述化合物包括0.5kDa至5MDa的分子质量。
7.根据权利要求1所述的方法,其中所述化合物包括3kDa至10MDa的分子质量。
8.根据权利要求1所述的方法,其中所述化合物包括可检测的标记物、活性分子和毒素的其中一种或多种。
9.根据权利要求1所述的方法,其中在所述细胞通过所述收缩部分之后,可检测的标记物进入到所述细胞中。
10.根据权利要求1所述的方法,其中所述悬浮液包括全血。
11.根据权利要求1所述的方法,其中所述悬浮液包括患有肿瘤风险或者被诊断为包括肿瘤的受试者的全血。
12.根据权利要求10所述的方法,其中所述肿瘤包括黑色素瘤、结肠癌、前列腺癌、肺癌、胰腺癌、乳腺癌、肝癌、脑癌、或者血液癌。
13.根据权利要求1所述的方法,其中所述收缩部分包括的宽度为4μm-10μm,长度为1μm-100μm,以及串联的1-10个收缩部分。
14.根据权利要求1所述的方法,其中细胞通过收缩部分的速度是在10mm/s至10m/s的范围内。
15.根据权利要求1所述的方法,进一步包括将压力施用于所述细胞悬浮液用于驱使细胞通过微流体通道的收缩部分。
16.根据权利要求1所述的方法,其中所述细胞悬浮液包括一种有效载荷或者其中所述方法进一步包括在所述细胞悬浮液通过收缩部分之后将其在预定的时间内在含有有效载荷的溶液中进行温育的步骤。
17.根据权利要求16所述的方法,其中所述有效载荷包括磁性颗粒、荧光颗粒、例如量子点或者碳纳米管、荧光染料、荧光蛋白质、编码荧光蛋白质的遗传物质(DNA或者RNA)、其他能够检测的化合物(例如,荧光素酶)、影响细胞功能的化合物以及诱导细胞死亡的化合物的一种或多种。
18.一种用于从非肿瘤细胞中区分肿瘤细胞的微流体通道,包括:
一种定义了内腔的微流体通道,并且被配置从而使悬浮在缓冲液中的肿瘤细胞能够经此通过,并且与非肿瘤细胞相比对肿瘤细胞进行收缩,其中所述微流体通道包括细胞变形的收缩部分,其中所述收缩部分的直径是细胞直径的函数。
19.根据权利要求18所述的系统,其中所述收缩通道被调整尺寸从而与非肿瘤细胞相比优先使肿瘤细胞变形。
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