CN112626025A - 一种三维肿瘤细胞耐药模型及其制备方法 - Google Patents
一种三维肿瘤细胞耐药模型及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种三维肿瘤细胞耐药模型及其制备方法,该三维模型由肝癌细胞悬液滴于多孔水凝胶阵列内进行三维培养形成。本发明通过三维培养制备的肿瘤细胞球体具有获得性耐药,表现在对药物的敏感性降低、药物半数抑制浓度增加及迁移能力显著增加。本发明成功的制备出三维肿瘤细胞耐药模型,且制备方法简单易行、条件要求低、培养时间短,具有较好的推广应用价值。
Description
技术领域
本发明涉及三维细胞培养领域,特别涉及一种三维肿瘤细胞耐药模型及其制备方法。
背景技术
耐药一直是影响各种疾病尤其是肿瘤治疗的主要障碍之一。因此,现有技术中建立了大量的体外模型用于耐药性的研究。
然而,传统的二维(2D)培养不能很好地模拟实体肿瘤细胞与微环境的相互作用,同时由于成本高、供应不足和伦理问题,动物模型的应用也受到很大限制。目前,肿瘤细胞球体被认为是一种很有前途的体外三维(3D)模型,可以广泛应用于耐药性评估。受益于细胞聚集的结构特征,这些肿瘤细胞球体相比2D模型通常对肿瘤治疗(化疗和放射治疗)具有更高的抵抗力。鉴于肿瘤细胞球体与体内实体瘤的相似性,利用肿瘤细胞球体探讨肿瘤获得性耐药产生的机制,并且开发克服耐药机制的方法,对临床抗肿瘤治疗意义重大。
因此,在本发明中,我们开发了一种基于工程化机理的基于水凝胶多孔基底的三维肿瘤细胞耐药模型及其制备方法。
发明内容
本发明所要解决的技术问题是针对上述现有技术的不足,提供一种三维肿瘤细胞耐药模型及其制备方法。
为实现上述技术目的,本发明采取的技术方案为:
本发明提供了一种三维肿瘤细胞耐药模型的制备方法,包括如下步骤:
(1)平板培养肝癌肿瘤细胞系:在培养瓶中培养肝癌Huh7细胞和耐药Huh7R细胞,用胰酶消化,消化后离心收集细胞,重悬;
(2)计数:调整细胞密度,混匀后将其注入到多孔水凝胶阵列内,待细胞自然沉降3-5分钟,加入培养基放到细胞培养箱中培养;
(3)培养肿瘤细胞球体:培养细胞至形成肿瘤细胞球体,在显微镜下观察是否形成紧密的肿瘤细胞球体;
(4)肿瘤细胞球体的分离收集:在多孔水凝胶阵列中加入胰酶,在细胞培养箱中消化后,在显微镜下观察细胞球体是否脱离多孔阵列,待大部分细胞球体脱落后用终止液终止,离心收集细胞球体。
步骤(1)中,消化时,肝癌Huh7细胞和耐药Huh7R细胞的细胞密度达底部面积的80%-90%。
步骤(1)中,离心收集细胞的条件为1000rpm离心5min;重悬为采用1毫升含血清的培养基重悬。
步骤(2)中,计数时将细胞密度调整为2×105cells/ml。
步骤(2)中,所述的多孔水凝胶为聚乙二醇二丙烯酸酯PEGDA,分子量为700KDa,其具有抗黏附性和良好的生物相容性;所述的培养基与二维培养相同,为10%FBS的DMEM培养基;所述的细胞培养箱为含5%二氧化碳的37℃细胞培养箱。
步骤(3)中,所述形成肿瘤细胞球体的培养时间为5-7天,期间第3天更换培养液,单层细胞在多孔凝胶阵列中形成紧密的肿瘤细胞球体。
步骤(4)中,具体包括以下步骤:(1)弃去培养上清;(2)PBS清洗;(3)胰酶消化10分钟;(4)离心弃上清;(5)PBS再次清洗离心后收集肿瘤细胞球体。
本发明还保护采用所述制备方法制备得到的三维肿瘤细胞耐药模型。
本发明还提供了该三维肿瘤细胞耐药模型的应用,用于研究肝癌细胞对药物的敏感性,用于研究肝癌细胞获得性耐药的机制。
与现有技术相比,本发明的有益效果在于:
1)本发明在较短的时间内就可培养出来具有紧密三维结构的细胞球聚体,并且这种方法获得的细胞球聚体在表型、药物敏感度上都与二维细胞有显著差异,相对于传统的二维耐药模型能更真实的模拟实体瘤内部环境,相较于动物模型方法更简单;
2)本发明借助的PEGDA水凝胶具有良好的生物相容性、抗黏附性以及高吸水性,不会对细胞产生不良影响,且通过这种水凝胶支架获得的细胞球聚体易分离收集,方便下一步实验。此外,多孔阵列方法简单,可以高通量的生成大小均一的细胞球聚体,可用于大规模生产。因此,本发明利用三维培养技术构建的细胞球聚体在耐药研究、高通量药物筛选等方面有良好的应用前景。
附图说明
图1为二维培养和三维培养得到的肝癌细胞在不同浓度仑伐替尼药物处理48小时后细胞生存率及药物半数浓度(IC50),其中,图(a)是肝癌细胞系Huh7二维培养和三维培养的生存率对比,图(b)是耐药株Huh7R二维培养和三维培养的生存率对比,图(c)是肝癌细胞系Huh7和耐药株Huh7R二维培养的IC50值,图(d)是耐药株Huh7R二维培养和三维培养的IC50值。
图2为二维培养和三维培养得到的肝癌细胞在相同浓度仑伐替尼药物处理不同时间后的荧光染色图,其中,图(a)是肝癌细胞系Huh7和耐药株Huh7R二维培养的荧光染色图,图(b)是肝癌细胞系Huh7和耐药株Huh7R三维培养的荧光染色图。
图3为二维培养和三维培养得到的肝癌细胞在不同浓度仑伐替尼药物处理48小时后的荧光染色图,其中,图(a)是肝癌细胞系Huh7和耐药株Huh7R二维培养的荧光染色图,图(b)是肝癌细胞系Huh7和耐药株Huh7R三维培养的荧光染色图。
图4为二维培养和三维培养得到的肝癌细胞Transwell迁移光镜图,其中,图(a)是二维培养的肝癌细胞系Huh7和耐药株Huh7R在200倍光镜下的迁移图片,图(b)是三维培养的肝癌细胞系Huh7和耐药株Huh7R在200倍光镜下的迁移图片。
具体实施方式
为了使本领域技术领域人员更好地理解本发明的技术方案,下面结合附图对本发明的实施例作进一步详细描述。
下述实施例中所使用的实验方法,如无特殊说明,均为常规方法,所用的试剂、方法和设备,如无特殊说明,均为本技术领域常规试剂、方法和设备。
本发明提供了一种三维肿瘤细胞耐药模型及其制备方法,包括如下步骤:
(1)平板培养肝癌肿瘤细胞系:在培养瓶中待肝癌Huh7细胞和耐药Huh7R细胞密度达到底部80%-90%时,用1毫升胰酶消化,1000rpm离心5min收集细胞,并计数。
(2)计数:将细胞密度调整为2x105cells/ml,混匀后用移液枪注入到多孔水凝胶阵列内,带细胞自然沉降3-5分钟,加入与二维培养相同的培养基放到含5%二氧化碳的37℃细胞培养箱中。
(3)肿瘤细胞球体培养:待培养到第三天进行培养基换液,培养5-7天后在显微镜下观察是否形成紧密的肿瘤细胞球体。
(4)肿瘤细胞球体收集:每个多孔阵列加入2毫升的胰酶,在37℃细胞培养箱中消化10分钟后,在显微镜下观察细胞球体是否脱离多孔阵列,待大部分细胞球体脱落后用终止液终止,离心收集细胞球体,并用移液枪吹打,得到单细胞悬液。
以下为具体实施例:
实施例1
二维培养和三维培养得到的肝癌细胞在不同浓度仑伐替尼药物处理48小时后细胞生存率的变化对比实验:
二维培养和三维培养的细胞经消化后,按每孔1500个细胞、100μl的培养基种到96孔板中。并设置空白组、对照组、和处理组,每组设置5个重复孔,空白组只加培养基、对照组加细胞和培养基、处理组加细胞和含药的培养基,待细胞种下去24小时后弃去培养上清,在空白组和对照组加培养基,处理组加不同浓度的含药培养基。孵育48小时后弃掉上清,在每孔加入90μl的培养基和10μl的CCK-8试剂混合液,孵育3h。在酶标仪下检测450nm波长下的吸光度。计算细胞生存率=(处理组OD值-空白组OD值)/(对照组OD值-空白组OD值),结果如图1a、b所示。结果显示随着药物浓度增加,细胞生存率都下降,但是与二维培养的相比,三维培养的细胞在相同浓度下生存率更高,说明三维培养的模型更耐药。此外如图1c、d所示,通过计算对仑伐替尼药物的半数抑制浓度(IC50)显示三维培养的细胞球聚体的IC50是二维培养的15.11(Huh7-3D/Huh7-2D=29.52μM/1.953μM)和2.38倍(Huh7R-3D/Huh7R-2D=47.3μM/19.87μM)。结果显示三维培养的细胞具有更好的耐药性。
实施例2
二维培养和三维培养得到的肝癌细胞在相同浓度仑伐替尼药物处理不同时间后及在不同浓度药物处理相同时间后的荧光染色实验:
为了更直观的观察二维培养和三维培养的药物敏感性差异,我们通过使用活死染试剂Calcein-AM/PI分别对二维平面培养的细胞和三维培养的细胞球聚体进行染色,按照1/1000细胞培养基体积的Calcein-AM/PI试剂加入到细胞培养基中,37℃孵育1h后,用PBS清洗后进行在荧光显微镜下进行拍照。图2a,b是二维培养和三维培养得到的肝癌细胞和球聚体在相同浓度仑伐替尼药物处理不同时间后的荧光染色图。可以发现在药物处理48h后,细胞都有一定程度的死亡,但相比二维培养的细胞,三维培养的球聚体死亡率大大降低。图3a,b是二维培养和三维培养得到的肝癌细胞在不同浓度药物处理相同时间后的荧光染色图,可以观察到随着药物浓度增加,细胞逐渐死亡且细胞球体变得疏松,但相比二维培养的细胞,三维培养的细胞生存率更高,说明我们构建的三维肿瘤细胞耐药模型比较成功。
实施例3
二维培养和三维培养得到的肝癌细胞Transwell迁移实验:
用胰酶消化对数期的二维培养和三维培养的细胞和球聚体,以1500rpm离心10分钟。弃去上清后加入1ml无血清的培养基混匀。计数后在Transwell上室加入300μl的细胞悬液,密度为2x105个。下室加入800μl的20%FBS的培养基,在37℃培养箱中孵育48小时。取出Transwell上室,将其放入4%甲醛固定液中固定1小时,然后用PBS清洗,再用0.5%结晶紫溶液染色40分钟,然后再用PBS清洗,用棉签擦拭后在200倍镜下观察。选取左上,左下,右上,右下4个视野拍照并计数。
以上所述,仅是本发明的较佳实施例,并非对本发明作任何形式上的限制,任何熟悉本专业的技术人员,在不脱离本发明技术方案范围内,依据本发明的技术实质,对以上实施例所作的任何简单的修改、等同替换与改进等,均仍属于本发明技术方案的保护范围之内。
Claims (10)
1.一种三维肿瘤细胞耐药模型的制备方法,其特征在于,包括如下步骤:
(1)平板培养肝癌肿瘤细胞系:在培养瓶中培养肝癌Huh7细胞和耐药Huh7R细胞,用胰酶消化,消化后离心收集细胞,重悬;
(2)计数:调整细胞密度,混匀后将其注入到多孔水凝胶阵列内,待细胞自然沉降3-5分钟,加入培养基放到细胞培养箱中培养;
(3)培养肿瘤细胞球体:培养细胞至形成肿瘤细胞球体,在显微镜下观察是否形成紧密的肿瘤细胞球体;
(4)肿瘤细胞球体的分离收集:在多孔水凝胶阵列中加入胰酶,在细胞培养箱中消化后,在显微镜下观察细胞球体是否脱离多孔阵列,待大部分细胞球体脱落后用终止液终止,离心收集细胞球体。
2.根据权利要求1所述的三维肿瘤细胞耐药模型的制备方法,其特征在于:步骤(1)中,消化时,肝癌Huh7细胞和耐药Huh7R细胞的细胞密度达底部面积的80%-90%。
3.根据权利要求1所述的三维肿瘤细胞耐药模型的制备方法,其特征在于:步骤(1)中,离心收集细胞的条件为1000rpm离心5min;重悬为采用1毫升含血清的培养基重悬。
4.根据权利要求1所述的制备方法,其特征在于:步骤(2)中,计数时将细胞密度调整为2×105cells/ml。
5.根据权利要求1所述的三维肿瘤细胞耐药模型的制备方法,其特征在于:步骤(2)中,所述的多孔水凝胶为聚乙二醇二丙烯酸酯PEGDA,分子量为700KDa,其具有抗黏附性和良好的生物相容性;所述的培养基与二维培养相同,为10%FBS的DMEM培养基,所述的细胞培养箱为含5%二氧化碳的37℃细胞培养箱。
6.根据权利要求1所述的三维肿瘤细胞耐药模型的制备方法,其特征在于:步骤(3)中,所述形成肿瘤细胞球体的培养时间为5-7天,期间第3天更换培养液,单层细胞在多孔凝胶阵列中形成紧密的肿瘤细胞球体。
7.根据权利要求1所述的三维肿瘤细胞耐药模型的制备方法,其特征在于:步骤(4)中,具体包括以下步骤:(1)弃去培养上清;(2)PBS清洗;(3)胰酶消化10分钟;(4)离心弃上清;(5)PBS再次清洗离心后收集肿瘤细胞球体。
8.一种三维肿瘤细胞耐药模型,其特征在于,采用权利要求1-7任一项所述的制备方法制备得到。
9.权利要求8所述的三维肿瘤细胞耐药模型的应用,其特征在于:用于研究肝癌细胞对药物的敏感性。
10.权利要求8所述的三维肿瘤细胞耐药模型的应用,其特征在于:用于研究肝癌细胞获得性耐药的机制。
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