CN113248748A - 用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法 - Google Patents
用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法 Download PDFInfo
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Abstract
本发明提供一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法。该方法包括：混合多个纳米粒子及有机高分子单体，以形成混合液；将混合液置于基材上，以形成液层；以及使液层中的有机高分子单体进行聚合反应，以形成复合材料薄膜固定于基材上。本发明制备的复合材料薄膜利于体外扩增循环肿瘤细胞，且该复合材料薄膜具有良好的耐用性。
Description
技术领域
本发明涉及用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法及体外扩增循环肿瘤细胞的应用。
背景技术
当癌细胞由原位肿瘤细胞脱离进入循环系统(如血液)中，这些血液中的癌细胞称为循环肿瘤细胞(circulating tumor cells，CTC)。CTC计数是一种新兴的癌症生物标记方式，许多研究证实此种方法可预测癌症的预后(prognosis)，并以监控细胞数量作为患者对于化学治疗与标靶治疗反应是否有效的依据。目前相关的临床运用大多以CTC数量来判断病程进展。然而，少数研究论文学理上虽然被证实CTC可以即时且直接反映病患对于药物的治疗反应，但此方式得到的CTC数量非常受限，仍致无法广泛应用。主要原因为受限于缺乏适合的技术可将CTC数量扩增，少量CTC无法进行足够样本数的精确基因检测与药物敏感性测试。而且，体外活体培养CTC的成功率相当低(小于20％)且耗时长达六个月以上，使其临床应用受限。突破CTC数量上的瓶颈已被视为肿瘤转移研究和临床应用最为迫切的研究项目。
发明内容
本发明要解决的技术问题在于，针对现有技术存在的上述不足，提供一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法，以利于体外扩增循环肿瘤细胞。
本发明解决其技术问题所采用的技术方案是提供一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法，包括：混合多个纳米粒子及有机高分子单体，以形成混合液，其中纳米粒子的粒径小于400纳米，有机高分子单体选自由苯乙烯、羧酸化苯乙烯、苯乙烯磺酸、甲基丙烯酸甲酯、己内酯、乳酸－羟基乙酸共聚物及其组合所构成的群组；将混合液置于基材上，以形成液层；以及，使液层中的有机高分子单体进行聚合反应，以形成复合材料薄膜固定于基材上。
在一实施例中，纳米粒子为无机纳米粒子。
在一实施例中，纳米粒子的材料为金属、金属氧化物或硅氧化物。
在一实施例中，纳米粒子选自由金粒子、银粒子、钛粒子、二氧化钛粒子、硅胶粒子(Silica particles)、聚二甲基硅氧烷粒子及其组合构成的群组。
在一实施例中，混合液还包含溶剂。
在一实施例中，使液层中的有机高分子单体进行聚合反应的步骤是对于液层进行加热处理，以使有机高分子单体进行聚合反应，加热处理的温度介于50℃与99℃之间。
在一实施例中，将混合液置于基材上的步骤包括：于基材上涂布混合液，以形成液层。
在一实施例中，该方法还包括：在形成液层之后及使液层中的有机高分子单体进行聚合反应之前，对液层进行静置处理。
在一实施例中，静置处理的时间为至少一小时。
本发明制备方法，用于制备用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜，以利于体外扩增循环肿瘤细胞，且该复合材料薄膜具有较强的抗冲刷能力，因而具有良好的耐用性。
附图说明
图1为依据本发明的一实施例的一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法的流程图。
图2为依据本发明的一实施例的一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法的步骤示意图。
图3为依据本发明的一实施例的一种体外扩增循环肿瘤细胞的方法的流程图。
图4为依据本发明的一实施例的一种体外扩增循环肿瘤细胞的方法的步骤示意图。
图5A及图5B分别为本发明的比较例1及实验例1的培养容器在经过冲刷测试后的示意图。
图6A及图6B为本发明的实验例1的培养容器在经过冲刷测试后的薄膜SEM图。
图7为非小型细胞肺癌患者的循环肿瘤细胞在本发明的实验例1的培养容器中培养第四周后的生长情形。
图8为非小型细胞肺癌患者的循环肿瘤细胞在本发明的实验例1的培养容器中培养第四周后，取出进行表征鉴定染色情形。在免疫荧光染色照片中，EpCAM为绿色荧光、CD45表现为红色荧光，以及DPAI为蓝色荧光。
具体实施方式
为了使本发明内容的叙述更加详尽与完备，下文针对本发明内容的实施态样与具体实施例提出了说明性的描述，但这并非实施或运用本发明内容具体实施例的唯一形式。以下所揭露的各实施例，在有益的情形下可相互组合或取代，也可在一实施例中附加其他的实施例，而无须进一步的记载或说明。
图1为依据本发明的一实施例的一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法100的流程图。如图1所示，制备此培养容器的复合材料薄膜的方法100包含下列步骤：混合纳米粒子及有机高分子单体，以形成混合液(步骤S102)；将混合液置于基材上，以形成液层(步骤S104)；以及，使液层中的有机高分子单体进行聚合反应，以形成复合材料薄膜固定于基材上(步骤S106)。图2为依据本发明的一实施例的一种用于体外扩增循环肿瘤细胞的培养容器的制备方法的步骤示意图。以下实施例请同时参照图1及图2。
首先，混合纳米粒子22及有机高分子单体24，以形成混合液20(步骤S102)。在步骤S102的一实施例中，纳米粒子22的粒径小于400纳米。在一实施例中，纳米粒子22为无机纳米粒子。在一实施例中，纳米粒子22仅选用一种无机纳米粒子。在一实施例中，纳米粒子22的材料为金属(例如金粒子(Au particles)、银粒子(Ag particles)、钛粒子(Tiparticles)或其他合适的金属粒子)、金属氧化物(例如二氧化钛粒子(TiO2 particles))、硅氧化物(例如硅胶粒子(Silica particles)或聚二甲基硅氧烷粒子(Polydimethylsiloxane particles))或其他合适的材料。
在步骤S102的一实施例中，有机高分子单体24包含苯乙烯(styrene)、羧酸化苯乙烯(carboxylated styrene)、苯乙烯磺酸(styrene sulfonic acid)、甲基丙烯酸甲酯(methylmethacrylate)、己内酯(caprolactone，CL)、乳酸－羟基乙酸共聚物(lactic-co-glycolic acid)、其他合适的有机高分子单体或其组合。
在步骤S102的一实施例中，混合液20更包含溶剂。上述有机高分子单体24可先溶于溶剂中。在一实施例中，溶剂例如但不限于含醇类溶剂(例如甲醇(methanol)或乙醇(Ethanol))、苯环的溶剂(例如甲苯(toluene)、苯(benzene)、二甲苯(xylene)或其他含苯环的溶剂)、非极性溶剂(例如丁酮(Methyl Ethyl Ketone，MEK)、三氯甲烷(Chloroform))、极性溶剂(例如水或其他极性溶剂，如四氢呋喃(Tetrahydrofuran，THF)、二甲基亚砜(Dimethyl sulfoxide，DMSO)、二甲基甲酰胺(Dimethyl formamide，DMF)或丙酮(Acetone))、或其组合。
在形成混合液20(步骤S102)之后，将混合液20置于基材12上，以形成液层201(步骤S104)。在一实施例中，培养容器10包含基材12。在一实施例中，基材12可为培养皿、培养盘、玻璃片或塑胶片，但不限于此。在一实施例中，基材12可例如但不限于具有至少6个孔槽(6-well)的多孔盘或具有至多384个孔槽(384-well)的多孔盘。
在一实施例中，将混合液20置于基材12上的方法包含涂布、喷洒及倒入等方式，但不限于此。在一实施例中，于基材12上涂布混合液20，以形成液层201。
值得注意的是，透过选择上述特定的纳米粒子22的种类、纳米粒子22的粒径、有机高分子单体24的种类及溶剂的种类，液层201中的纳米粒子22会自动规则排列。在一实施例中，在形成该液层201(步骤S104)之后，对液层201进行静置处理，以使液层201中的纳米粒子22有充分的时间进行自组装排列。在一实施例中，在混合液20包含溶剂的情况下，液层201包含溶剂，静置处理有助于液层201中的溶剂挥发，使液层201的浓度提升。在一实施例中，静置处理的时间为至少一小时。
形成液层201(步骤S104)之后，使液层201中的有机高分子单体24进行聚合反应，以于纳米粒子22之间的间隙处形成有机高分子薄膜24’，以形成复合材料薄膜202固定于基材12上(步骤S106)。
在步骤S106的一实施例中，有机高分子单体24进行聚合反应的方法包括自由基聚合法(Free-radical polymerization)、活性阳离子聚合法(active cationicpolymerization)、阴离子聚合法(anionic polymerization)、或缩合聚合法(condensation)等，但不限于此。在一实施例中，使液层201中的有机高分子单体24进行聚合反应(步骤S106)是对于液层201进行加热处理，以使有机高分子单体24进行聚合反应。在一实施例中，加热处理可生成自由基，自由基与有机高分子单体24反应后进行链锁聚合反应，而形成有机高分子薄膜24’。在一实施例中，加热处理的温度介于50℃与99℃之间。在一实施例中，加热时间可为一小时以上。
在一实施例中，复合材料薄膜202包括规则排列的纳米粒子22(即分散相)及位于纳米粒子22之间的间隙处的有机高分子薄膜24’(即连续相)。有机高分子薄膜24’可帮助增加纳米粒子22与纳米粒子22之间和纳米粒子22与基材12表面之间的接着强度，以使纳米粒子22有效固定于基材12的二维平面上。
图3为依据本发明的一实施例的一种体外扩增循环肿瘤细胞的方法的流程图。如图3所示，体外扩增循环肿瘤细胞的方法200包含下列步骤：使细胞液与上述培养容器的复合材料薄膜接触(步骤S202)以及扩增循环肿瘤细胞(步骤S204)。图4为依据本发明的一实施例的一种体外扩增循环肿瘤细胞的方法的步骤示意图。以下实施例请同时参照图3及图4。
首先，使细胞液30与培养容器10的复合材料薄膜202接触(步骤S202)。在步骤S202的一实施例中，细胞液30包含循环肿瘤细胞32及培养液34。在一实施例中，循环肿瘤细胞32由生物体的血液中分离出而得。在一实施例中，对生物体的血液进行分离程序，以取得含有循环肿瘤细胞32的周边血单核细胞(peripheral blood mononuclear cell；PBMC)，然后使用抗体形式的白血球分离试剂去除周边血单核细胞中的多余白血球，再以细胞尺寸大小纯化取得循环肿瘤细胞32，然后再将循环肿瘤细胞32及培养液34放置于培养容器10上，使循环肿瘤细胞32及培养液34与培养容器10的基材12上的复合材料薄膜202接触。上述的生物体的血液来源可为人体，亦可为其他动物，例如猫、犬或其他可豢养的哺乳类动物。循环肿瘤细胞32例如但不限于来自小细胞肺癌、肺癌、乳癌、胰脏癌、肉瘤、黑色素瘤、肝癌、食道癌、大肠直肠癌、鼻咽癌或脑癌的肿瘤细胞。
在一实施例中，培养液34包含干细胞所需培养液。至于培养液34中的其他成分，可依据循环肿瘤细胞32的种类选择合适的成分。在一些实施例中，培养液34包含基底培养液，例如MEM、DMEM或是RPMI1640与其他合适的基底培养液。在一些实施例中，培养液34更包含避免微生物与真菌污染的抗生素。在一实施例中，培养液34更包含一或多种的重组生长因子，例如表皮生长因子与其他已发表文献中所提及支持循环肿瘤细胞生长的补充剂。
细胞液30与培养容器10的复合材料薄膜202接触(步骤S202)之后，循环肿瘤细胞32会附着在纳米粒子22上，并在适当的条件下扩增循环肿瘤细胞32(步骤S204)。
扩增后的扩增循环肿瘤细胞32可应用于评估个人化药物候选物应用。将预测式药物候选物加到扩增后的循环肿瘤细胞32或循环肿瘤细胞团块32’之中，再检测循环肿瘤细胞32或循环肿瘤细胞团块32’的存活率。然后判断此药物候选物是否能降低循环肿瘤细胞32的存活率。若此药物候选物能有效降低循环肿瘤细胞32的存活率，则可作为优选的治疗对应癌症的药物候选或给药选择建议。
此外，在一些实施例中，提供一种扩大培养的循环肿瘤细胞的冷冻保存方法。在一实施例中，将扩大培养的循环肿瘤细胞与含有干细胞生长液的冻存液进行混合后，冷冻保存于-80℃以下的环境或液态氮中。经实验发现，解冻后的循环肿瘤细胞在含有粒子层的细胞培养容器内可恢复其生长活性，且其遗传物质与生化特性在冷冻前后并未受到改变，因此可将其应用于新药开发过程中的毒杀效果测试。
上述由病患血液中所分离出的循环肿瘤细胞无论是在分离后持续培养或是冻存后再进行解冻培养，在含有粒子层的细胞培养容器内进行扩增后到达一定的细胞数目后，其中所包含的肿瘤干细胞(Cancer Stem Cells，CSCs)可应用于药物敏感度的筛选。此外，针对不同癌种的新药开发过程可建立不同个体的肿瘤干细胞毒杀测试平台。
图5A及图5B分别为本发明的比较例1及实验例1的培养容器在经过冲刷测试后的示意图。比较例1是将纳米粒子以自然风干方式形成薄膜，实验例1则是以含有纳米粒子及有机高分子单体的混合液透过图1的制备方法形成薄膜。两种薄膜进行冲刷测试，即以1,000毫升的定量吸管(pipette)重复吸吐三十次，以模拟细胞培养过程中，薄膜受到细胞液中的培养液的冲刷状况。如图5A及图5B所示，在经过冲刷测试后，图5A的比较例1的薄膜破裂并且翘起来脱离基材，但图5B的薄膜仍良好附着于基材上，由此可知聚合而形成的有机高分子薄膜可使薄膜具有较强的抗冲刷能力，使薄膜具有良好的耐用性。
图6A及图6B为本发明的实验例1的培养容器在经过冲刷测试后的薄膜SEM图。如图6A及图6B所示，粒子透过有机高分子薄膜而良好附着于基材上。
图7为非小型细胞肺癌患者的循环肿瘤细胞在本发明的实验例1的培养容器中培养第四周后的生长情形。此非小型细胞肺癌患者的循环肿瘤细胞是以体温(约37℃)与5％至40％的二氧化碳的条件进行循环肿瘤细胞的培养与扩增。此条件使循环肿瘤细胞在小于或等于6周内，细胞数增殖至初始种入的细胞数的2,000倍以上。如图7所示，以光学显微镜观察实验例1的培养容器的复合材料薄膜的表面，可见到循环肿瘤细胞团块或循环肿瘤细胞在薄膜上增殖而形成的聚落(图中箭头和方框标示处)。
图8为非小型细胞肺癌患者的循环肿瘤细胞在本发明的实验例1的培养容器中培养第四周后，取出进行表征鉴定染色情形。在免疫荧光染色照片中，EpCAM为绿色荧光、CD45表现为红色荧光，以及DPAI为蓝色荧光。如图8所示，扩增后的循环肿瘤细胞表现仍有上皮细胞黏着分子(EpCAM)及DAPI的荧光信号，证明扩增后仍然有癌细胞的相关表征，但未表现T细胞和B细胞常见表征(CD45)的荧光信号。
上述实施例仅为例示性说明本发明的原理及其功效，以及阐释本发明的技术特征，而非用于限制本发明的保护范畴。任何本技术领域普通技术人员在不违背本发明的技术原理及精神的情况下，可轻易完成的改变或均等性的安排均属于本发明所主张的范围。
Claims (9)
1.一种用于体外扩增循环肿瘤细胞的培养容器的复合材料薄膜的制备方法，其特征在于，包括：
混合多个纳米粒子及有机高分子单体，以形成一混合液，其中该多个纳米粒子的粒径小于400纳米，该有机高分子单体选自由苯乙烯、羧酸化苯乙烯、苯乙烯磺酸、甲基丙烯酸甲酯、己内酯、乳酸－羟基乙酸共聚物及其组合所构成的群组；
将该混合液置于一基材上，以形成一液层；以及
使该液层中的该有机高分子单体进行聚合反应，以形成该复合材料薄膜固定于该基材上。
2.如权利要求1所述的方法，其特征在于，该多个纳米粒子为无机纳米粒子。
3.如权利要求1所述的方法，其特征在于，该多个纳米粒子的材料为金属、金属氧化物或硅氧化物。
4.如权利要求1所述的方法，其特征在于，该多个纳米粒子选自由金粒子、银粒子、钛粒子、二氧化钛粒子、硅胶粒子、聚二甲基硅氧烷粒子及其组合构成的群组。
5.如权利要求1所述的方法，其特征在于，该混合液还包含一溶剂。
6.如权利要求1所述的方法，其特征在于，该使该液层中的该有机高分子单体进行该聚合反应的步骤是对于该液层进行一加热处理，以使该有机高分子单体进行该聚合反应，该加热处理的温度介于50℃与99℃之间。
7.如权利要求1所述的方法，其特征在于，该将该混合液置于该基材上的步骤包括：于该基材上涂布该混合液，以形成该液层。
8.如权利要求1所述的方法，其特征在于，该方法还包括：在形成该液层之后及使该液层中的该有机高分子单体进行该聚合反应之前，对该液层进行一静置处理。
9.如权利要求8所述的方法，其特征在于，该静置处理的时间为至少一小时。
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| CN110328950A (zh) * | 2019-06-05 | 2019-10-15 | 江苏隆晟医药包装材料有限公司 | 用于细胞培养的容器膜及其制备方法 |
| CN110628721A (zh) * | 2019-05-30 | 2019-12-31 | 佛山市璞狄医疗科技有限公司 | 一种循环肿瘤细胞的分离培养方法及试剂盒 |
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| US20170369838A1 (en) * | 2015-01-26 | 2017-12-28 | Ube Industries, Ltd. | Long-term cell-cultivation using polyimide porous membrane and cell-cryopreservation method using polyimide porous membrane |
| US20190300832A1 (en) * | 2018-03-29 | 2019-10-03 | Taipei Medical University | Method and kit for expanding circulating tumor cells in vitro |
| CN110628721A (zh) * | 2019-05-30 | 2019-12-31 | 佛山市璞狄医疗科技有限公司 | 一种循环肿瘤细胞的分离培养方法及试剂盒 |
| CN110328950A (zh) * | 2019-06-05 | 2019-10-15 | 江苏隆晟医药包装材料有限公司 | 用于细胞培养的容器膜及其制备方法 |
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