CN114457018A - 一种三维乳腺癌类器官模型及其培养方法与用途 - Google Patents
一种三维乳腺癌类器官模型及其培养方法与用途 Download PDFInfo
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Abstract
本发明公开了一种三维乳腺癌类器官模型及其培养方法与用途,利用猪源性肺脏组织作为构建肿瘤模型的支架,利用猪源性组织的生理及组织学特性作为构建块,创建具有脉管系统的多细胞组织,构建得到多孔组织支架,同时利用明胶、海藻酸钠的复合溶胶溶液对支架进行浸泡,再进行冷冻干燥,在多孔组织支架外表面形成保护层,提高支架的机械性能和宏观形状保持能力,以满足肿瘤类器官长期培养的支撑需求;利用水凝胶与扩大培养后的细胞培养上清液混合,提高与支架的生物相容性,同时可通过改变水凝胶成分,针对性的加入促进肿瘤细胞生长的细胞生长因子,有助于体外三维乳腺癌类器官的培养,制备工艺简单,重复性好。
Description
技术领域
本发明涉及肿瘤免疫学技术领域,具体涉及三维乳腺癌类器官模型及其培养方法与用途。
背景技术
传统的肿瘤体外药物敏感测试模型主要有人源肿瘤细胞系和人源肿瘤异种移植模型。人源肿瘤细胞系可快速得到药敏筛选结果,但其培养方式影响原代病人肿瘤特性的保持,药敏筛选的准确率与患者体内反应的一致性相对较低;人源肿瘤异种移植模型可以原代病人肿瘤的特性,可以作为活体肿瘤用于保存和传代,为肿瘤学研究提供非常宝贵的研究标本,但该模型成功率低,筛药测试时间太长、花费太高。近年来兴起的人源肿瘤的类器官模型类器官是将癌症患者手术获取的肿瘤进行体外细胞3D培养,建成肿瘤类器官模型。
该模型维持了肿瘤组织的生理结构和功能特点,同时维持了肿瘤细胞在体内的特征,临床相关性达到95%,且效率高、耗时少,适用于大批量筛选。但是,目前的肿瘤类器官培养技术多局限于基质胶中,该培养方式限制了类器官与外界的气体交换和物质代谢,当类器官形成较大的组织后,循环系统的缺乏和氧气养分交换的局限性严重影响了类器官所需营养物质的吸收以及代谢废物的清除。目前研究人员试图通过改变培养基质成分尝试创造更大的物质交换空间,如摒弃使用的Matrigel换用能够提供更大空腔的水凝胶作为支架。
支架是一种具有生物相容性和化学稳定性的细胞外支持结构,可作为细胞附着、生长和组织形态发生的指导性支持。多孔支架可由脱细胞组织或几种天然ECM蛋白质或生物相容性聚合物(如胶原蛋白、透明质酸、丝蛋白、聚乙二醇(PEG)和聚乳酸)制成。支架通常通过冷冻干燥、静电纺丝、相分离和微尺度大分子自组装来制备。
在支架中培养的肿瘤细胞对化疗的敏感性较低,并产生具有更多侵袭性表型的肿瘤。虽然多孔支架具有用于三维肿瘤细胞培养的ECM的机械和化学特性,但缺点包括在制造的支架中缺乏脉管结构,阻碍长期培养的灌注,以及支架内细胞放置位置的控制不佳。
发明内容
针对上述背景技术中的问题,本发明的一个目的在于提供一种三维乳腺癌类器官模型的培养方法,利用猪源性肺脏组织作为构建肿瘤模型的支架,创建具有脉管系统的多细胞组织支架,同时利用明胶、海藻酸钠的复合溶胶溶液对支架进行浸泡,再进行冷冻干燥,在多孔组织支架外表面形成保护层,提高支架的机械性能和宏观形状保持能力,以满足肿瘤类器官长期培养的支撑需求。
为实现上述目的,本发明的技术方案如下:
一种三维乳腺癌类器官模型的培养方法,具体包括如下步骤:
(1)乳腺癌肿瘤细胞的培养:
A)对脱离人体的患者来源乳腺癌组织的处理:对乳腺癌新鲜样本组织进行处理,并对分离得到的肿瘤细胞进行培养,得到细胞培养上清液并进行过滤,并利用差速贴壁法进行细胞纯化,获得纯化的乳腺癌初代细胞;
B)乳腺癌初代细胞体外扩大培养:将所述乳腺癌初代细胞使用专用培养基继续扩增培养,得到扩大培养后的细胞培养上清液;
(2)多孔组织支架的构建:
A)将整个猪肺组织置于-20~-30℃冷冻固定成型,选取肉眼观察无明显支气管的区域切块,采用纯净水、磷酸盐缓冲液清洗,去除切块中的血丝;
B)室温下,将得到的切块前后分别放置于0.1~1%的十二烷基磺酸钠水溶液、0.1~0.5%的TritonX-100溶液中,脱除细胞,并采用磷酸盐缓冲液清洗,得到脱细胞切;
C)将得到的脱细胞切块置于-20~-30℃预冻5~12h,再于-45~-55℃条件下进行第一次冷冻干燥12~24h,得到预制多孔组织支架;
D)按照(6~18):(4~16):100的质量比将明胶、海藻酸钠与水混合均匀,得到复合溶胶溶液,在室温条件下,将得到的预制多孔组织支架浸于复合溶胶溶液中4-10h,取出后室温静置2-6h,得到固化的多孔组织支架;
E)将上述固化的多孔组织支架置于-45~-55℃条件下,对其进行第二次冷冻干燥12~24h,得到稳定的多孔组织支架;
(3)将步骤(1)得到的细胞培养上清液与水凝胶混合,得到含有水凝胶的细胞悬液,然后将所述含有水凝胶的细胞悬液、专用培养基分别加入到步骤(2)构建的支架中进行三维细胞培养,得到三维乳腺癌类器官模型。
进一步地,步骤(3)中所述水凝胶为4℃~120℃下将琼脂或/和海藻酸钠溶解于水中制备成的质量分数在10~50%的水凝胶溶胶。
更进一步地,所述细胞培养上清液与水凝胶按照1:(0.1-1)的比例混合。
更进一步地,步在琼脂或/和海藻酸钠中混入细胞生长因子,所述细胞生长因子重量与琼脂/和海藻酸钠重量的比例是1:(10,000~1,000,000)。
进一步地,在所述步骤(3)前还包括对多孔组织支架的预处理:将所述多孔组织支架进行灭菌处理,再用磷酸盐缓冲液预先润湿。
进一步地,所述专用培养基包括70-90%的基础培养基以及10-30%的胎牛血清。
更进一步地,所述基础培养基选自RPMI-1640培养基、DMEM培养基、F-12培养基或DMEM/F-12培养基中的一种。
本发明的第二个目的在于,提供如上所述的三维乳腺癌类器官模型的培养方法构建的三维乳腺癌类器官模型。
本发明的第三个目的在于,提供如上所述的三维乳腺癌类器官模型在开发乳腺癌肿瘤疫苗、药物中的用途。
与现有技术相比,本发明具有如下优点:
第一,本发明中利用猪源性肺脏组织作为构建肿瘤模型的支架,利用猪源性组织的生理及组织学特性作为构建块,创建具有脉管系统的多细胞组织,构建得到多孔组织支架,同时利用明胶、海藻酸钠的复合溶胶溶液对支架进行浸泡,再进行冷冻干燥,在多孔组织支架外表面形成保护层,提高支架的机械性能和宏观形状保持能力,以满足肿瘤类器官长期培养的支撑需求;
第二,本发明利用水凝胶与扩大培养后的细胞培养上清液混合,提高与支架的生物相容性,同时可通过改变水凝胶成分,针对性的加入促进肿瘤细胞生长的细胞生长因子,有助于体外三维乳腺癌类器官的培养,制备工艺简单,重复性好。
具体实施方式
以下对本发明的具体实施方式进行详细说明。应当理解的是,此处所描述的具体实施方式仅用于说明和解释本发明,并不用于限制本发明。
实施例1
一种三维乳腺癌类器官模型的培养方法,具体包括如下步骤:
(1)乳腺癌肿瘤细胞的培养:
A)对脱离人体的患者来源乳腺癌组织的处理:对乳腺癌新鲜样本组织进行破碎、分离处理,并对分离得到的肿瘤细胞进行培养3天,得到细胞培养上清液并进行过滤,并利用差速贴壁法进行细胞纯化,获得纯化的乳腺癌初代细胞;
B)乳腺癌初代细胞体外扩大培养:将所述乳腺癌初代细胞使用含有10%的胎牛血清的RPMI-1640培养基进行扩增培养,取扩大培养后的细胞培养上清液;
(2)多孔组织支架的构建:
A)将整个猪肺组织置于-25℃冷冻3h,固定成型后,选取肉眼观察无明显支气管的区域切块,采用纯净水、磷酸盐缓冲液清洗,去除切块中的血丝;
B)室温下,将得到的切块前放置于0.5%的十二烷基磺酸钠水溶液中,搅拌12h,初步脱除细胞,再置于0.3%的TritonX-100溶液中,搅拌16h,再次脱除细胞,并采用磷酸盐缓冲液清洗,得到脱细胞切;
C)将得到的脱细胞切块置于-25℃预冻8h,再于-55℃条件下进行第一次冷冻干燥24h,得到预制多孔组织支架;
D)按照12:8:100的质量比将明胶、海藻酸钠与水混合均匀,得到复合溶胶溶液,在室温条件下,将得到的预制多孔组织支架浸于复合溶胶溶液中8h,取出后室温静置4h,得到固化的多孔组织支架;
E)将上述固化的多孔组织支架置于-55℃温度条件下,对其进行第二次冷冻干燥24h,得到稳定的多孔组织支架;
(3)将步骤(1)得到的细胞培养上清液与含10%琼脂的水凝胶溶液混合,得到含有水凝胶的细胞悬液,将所述多孔组织支架进行灭菌处理,再用磷酸盐缓冲液预先润湿;
然后将所述含有水凝胶的细胞悬液、含有10%的胎牛血清的RPMI-1640培养基分别加入到步骤(2)构建的支架中进行三维细胞培养,得到三维乳腺癌类器官模型。
实施例2
一种三维乳腺癌类器官模型的培养方法,具体包括如下步骤:
(1)乳腺癌肿瘤细胞的培养:
A)对脱离人体的患者来源乳腺癌组织的处理:对乳腺癌新鲜样本组织进行破碎、分离处理,并对分离得到的肿瘤细胞进行培养3天,得到细胞培养上清液并进行过滤,并利用差速贴壁法进行细胞纯化,获得纯化的乳腺癌初代细胞;
B)乳腺癌初代细胞体外扩大培养:将所述乳腺癌初代细胞使用含有10%的胎牛血清的DMEM培养基进行扩增培养,取扩大培养后的细胞培养上清液;
(2)多孔组织支架的构建:
A)将整个猪肺组织置于-25℃冷冻3h,固定成型后,选取肉眼观察无明显支气管的区域切块,采用纯净水、磷酸盐缓冲液清洗,去除切块中的血丝;
B)室温下,将得到的切块前放置于0.5%的十二烷基磺酸钠水溶液中,搅拌12h,初步脱除细胞,再置于0.3%的TritonX-100溶液中,搅拌16h,再次脱除细胞,并采用磷酸盐缓冲液清洗,得到脱细胞切;
C)将得到的脱细胞切块置于-25℃预冻8h,再于-55℃条件下进行第一次冷冻干燥24h,得到预制多孔组织支架;
D)按照12:8:100的质量比将明胶、海藻酸钠与水混合均匀,得到复合溶胶溶液,在室温条件下,将得到的预制多孔组织支架浸于复合溶胶溶液中8h,取出后室温静置4h,得到固化的多孔组织支架;
E)将上述固化的多孔组织支架置于-55℃温度条件下,对其进行第二次冷冻干燥24h,得到稳定的多孔组织支架;
(3)将步骤(1)得到的细胞培养上清液与含10%琼脂的水凝胶溶液混合,得到含有水凝胶的细胞悬液,将所述多孔组织支架进行灭菌处理,再用磷酸盐缓冲液预先润湿;
然后将所述含有水凝胶的细胞悬液、含有10%的胎牛血清的DMEM培养基分别加入到步骤(2)构建的支架中进行三维细胞培养,得到三维乳腺癌类器官模型。
实施例3
一种三维乳腺癌类器官模型的培养方法,具体包括如下步骤:
(1)乳腺癌肿瘤细胞的培养:
A)对脱离人体的患者来源乳腺癌组织的处理:对乳腺癌新鲜样本组织进行破碎、分离处理,并对分离得到的肿瘤细胞进行培养3天,得到细胞培养上清液并进行过滤,并利用差速贴壁法进行细胞纯化,获得纯化的乳腺癌初代细胞;
B)乳腺癌初代细胞体外扩大培养:将所述乳腺癌初代细胞使用含有10%的胎牛血清的DMEM/F-12培养基进行扩增培养,取扩大培养后的细胞培养上清液;
(2)多孔组织支架的构建:
A)将整个猪肺组织置于-25℃冷冻3h,固定成型后,选取肉眼观察无明显支气管的区域切块,采用纯净水、磷酸盐缓冲液清洗,去除切块中的血丝;
B)室温下,将得到的切块前放置于0.5%的十二烷基磺酸钠水溶液中,搅拌12h,初步脱除细胞,再置于0.3%的TritonX-100溶液中,搅拌16h,再次脱除细胞,并采用磷酸盐缓冲液清洗,得到脱细胞切;
C)将得到的脱细胞切块置于-25℃预冻8h,再于-55℃条件下进行第一次冷冻干燥24h,得到预制多孔组织支架;
D)按照12:8:100的质量比将明胶、海藻酸钠与水混合均匀,得到复合溶胶溶液,在室温条件下,将得到的预制多孔组织支架浸于复合溶胶溶液中8h,取出后室温静置4h,得到固化的多孔组织支架;
E)将上述固化的多孔组织支架置于-55℃温度条件下,对其进行第二次冷冻干燥24h,得到稳定的多孔组织支架;
(3)将步骤(1)得到的细胞培养上清液与含10%琼脂的水凝胶溶液混合,得到含有水凝胶的细胞悬液,将所述多孔组织支架进行灭菌处理,再用磷酸盐缓冲液预先润湿;
然后将所述含有水凝胶的细胞悬液、DMEM/F-12培养基分别加入到步骤(2)构建的支架中进行三维细胞培养,得到三维乳腺癌类器官模型。
以上显示和描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是本发明的原理,在不脱离本发明精神和范围的前提下本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明的范围内。本发明要求的保护范围由所附的权利要求书及其等同物界定。
Claims (9)
1.一种三维乳腺癌类器官模型的培养方法,其特征在于,具体包括如下步骤:
(1)乳腺癌肿瘤细胞的培养:
A)对脱离人体的患者来源乳腺癌组织的处理:对乳腺癌新鲜样本组织进行处理,并对分离得到的肿瘤细胞进行培养,得到细胞培养上清液并进行过滤,并利用差速贴壁法进行细胞纯化,获得纯化的乳腺癌初代细胞;
B)乳腺癌初代细胞体外扩大培养:将所述乳腺癌初代细胞使用专用培养基继续扩增培养,得到扩大培养后的细胞培养上清液;
(2)多孔组织支架的构建:
A)将整个猪肺组织置于-20~-30℃冷冻固定成型,选取肉眼观察无明显支气管的区域切块,采用纯净水、磷酸盐缓冲液清洗,去除切块中的血丝;
B)室温下,将得到的切块前后分别放置于0.1~1%的十二烷基磺酸钠水溶液、0.1~0.5%的TritonX-100溶液中,脱除细胞,并采用磷酸盐缓冲液清洗,得到脱细胞切;
C)将得到的脱细胞切块置于-20~-30℃预冻5~12h,再于-45~-55℃条件下进行第一次冷冻干燥12~24h,得到预制多孔组织支架;
D)按照(6~18):(4~16):100的质量比将明胶、海藻酸钠与水混合均匀,得到复合溶胶溶液,在室温条件下,将得到的预制多孔组织支架浸于复合溶胶溶液中4-10h,取出后室温静置2-6h,得到固化的多孔组织支架;
E)将上述固化的多孔组织支架置于-45~-55℃条件下,对其进行第二次冷冻干燥12~24h,得到稳定的多孔组织支架;
(3)将步骤(1)得到的细胞培养上清液与水凝胶混合,得到含有水凝胶的细胞悬液,然后将所述含有水凝胶的细胞悬液、专用培养基分别加入到步骤(2)构建的支架中进行三维细胞培养,得到三维乳腺癌类器官模型。
2.根据权利要求1所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,步骤(3)中所述水凝胶为4℃~120℃下将琼脂或/和海藻酸钠溶解于水中制备成的质量分数在10~50%的水凝胶溶胶。
3.根据权利要求2所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,所述细胞培养上清液与水凝胶按照1:(0.1-1)的比例混合。
4.根据权利要求2所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,步在琼脂或/和海藻酸钠中混入细胞生长因子,所述细胞生长因子重量与琼脂/和海藻酸钠重量的比例是1:(10,000~1,000,000)。
5.根据权利要求1所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,在所述步骤(3)前还包括对多孔组织支架的预处理:将所述多孔组织支架进行灭菌处理,再用磷酸盐缓冲液预先润湿。
6.根据权利要求1所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,所述专用培养基包括70-90%的基础培养基以及10-30%的胎牛血清。
7.根据权利要求6所述的一种三维乳腺癌类器官模型的培养方法,其特征在于,所述基础培养基选自RPMI-1640培养基、DMEM培养基、F-12培养基或DMEM/F-12培养基中的一种。
8.采用如权利要求1-7任一所述的三维乳腺癌类器官模型的培养方法构建的三维乳腺癌类器官模型。
9.权利要求8所述的三维乳腺癌类器官模型在开发乳腺癌肿瘤疫苗、药物中的用途。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1586636A (zh) * | 2004-07-15 | 2005-03-02 | 浙江大学 | 将水凝胶复合到多孔组织工程支架中的方法 |
CN107988158A (zh) * | 2017-11-27 | 2018-05-04 | 大连理工大学 | 一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用 |
CN110272860A (zh) * | 2019-06-30 | 2019-09-24 | 东南大学苏州医疗器械研究院 | 一种细胞三维培养微环境构建方法及应用 |
CN111001041A (zh) * | 2019-12-10 | 2020-04-14 | 河南亚都实业有限公司 | 一种抗炎抗菌复合皮肤支架材料及制备方法 |
CN113082295A (zh) * | 2021-04-02 | 2021-07-09 | 大连理工大学 | 一种基于皮肤源脱细胞基质衍生支架及其构建方法 |
CN113278587A (zh) * | 2021-04-29 | 2021-08-20 | 潍坊医学院 | 一种三维工程化乳腺癌肺转移模型、构建方法及应用 |
-
2022
- 2022-02-17 CN CN202210144647.6A patent/CN114457018A/zh active Pending
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1586636A (zh) * | 2004-07-15 | 2005-03-02 | 浙江大学 | 将水凝胶复合到多孔组织工程支架中的方法 |
CN107988158A (zh) * | 2017-11-27 | 2018-05-04 | 大连理工大学 | 一种三维肿瘤模型脱细胞多孔支架、构建方法及其应用 |
CN110272860A (zh) * | 2019-06-30 | 2019-09-24 | 东南大学苏州医疗器械研究院 | 一种细胞三维培养微环境构建方法及应用 |
CN111001041A (zh) * | 2019-12-10 | 2020-04-14 | 河南亚都实业有限公司 | 一种抗炎抗菌复合皮肤支架材料及制备方法 |
CN113082295A (zh) * | 2021-04-02 | 2021-07-09 | 大连理工大学 | 一种基于皮肤源脱细胞基质衍生支架及其构建方法 |
CN113278587A (zh) * | 2021-04-29 | 2021-08-20 | 潍坊医学院 | 一种三维工程化乳腺癌肺转移模型、构建方法及应用 |
Non-Patent Citations (4)
Title |
---|
WENFANG LI等: "Detection and Evaluation of Anti-Cancer Efficiency of Astragalus Polysaccharide Via a Tissue Engineered Tumor Model", MACROMOL BIOSCI, vol. 18, no. 11, pages 1 - 12 * |
任荣;张剑飞;司家文;吴稼晟;李伟;史俊;: "海藻酸钠/明胶复合水凝胶用于3D生物打印的初步研究", 中国口腔颌面外科杂志, no. 05, pages 402 - 407 * |
韩小东;王磊;郑军;贺小龙;吉金山;符兆英;: "三维细胞培养生物材料及其在肿瘤组织工程研究中的应用", 中国组织工程研究, no. 42, pages 6371 - 6377 * |
马小军: "海藻酸基海洋生物医用材料", 上海科学技术出版社, pages: 320 - 321 * |
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