CN115491357A - 一种子宫内膜癌类器官无血清专用培养基 - Google Patents
一种子宫内膜癌类器官无血清专用培养基 Download PDFInfo
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Abstract
本发明涉及肿瘤类器官培养技术领域。具体涉及一种子宫内膜癌类器官无血清专用培养基和培养方法。所述培养基为一种无需添加R‑Spondin、Noggin重组蛋白或WNT3A条件培养基的无血清专用培养基,由基础培养基和添加物组成,基础培养基为含有1%青链霉素、1%HEPES、1%GlutaMa的Advanced DMEM/F12培养基。添加物包括FGF2、FGF10,胰岛素、氢化可的松、雌激素,Y‑27632、SB202190,BPE、B27supplement。将患者子宫内膜癌样本用胶原酶消化,重悬于CultrexTM growth factor reduced BME type2中,加入培养板,37度20分钟固化,加入所述培养基,放入培养箱,每4天更换培养基,7天即可获得所需子宫内膜癌类器官。本发明所述培养基可支持子宫内膜癌细胞体外生长,呈现典型球状或不规则团块状肿瘤类器官形态,为后续进一步研究提供较好研究样本。
Description
技术领域
本发明涉及一种肿瘤类器官培养技术领域,尤其涉及一种子宫内膜癌类器官的无血清专用培养基和培养方法。
背景技术
子宫内膜癌是发生于子宫内膜的一种恶性肿瘤,在妇科恶性肿瘤中占20%~30%的比例,根据国家癌症中心发布的报告,在我国每年新发子宫内膜癌7.1万人,死亡约1.7万人,位列女性癌症新发病例数第八位。特别是随着生活水平的提高,子宫内膜癌的发病率呈上升趋势,2000年~2016年期间上升率达到3.5%,位列女性癌症第三位。子宫内膜癌是一种高度异质性的恶性肿瘤,病因目前还不是十分清楚。手术和化疗是主要治疗手段,但有约20~30%的患者缺乏有效和持续的治疗方案,也缺少适当的临床前和临床研究模型。随着类器官技术的发展,构建基于患者来源的肿瘤类器官(Patient- Derived Organoid,PDO)体外模型,能够准确模拟体内肿瘤的生物学特征和对药物治疗的反应,对于肿瘤研究、药物测试及新药筛选有着巨大的价值。传统上的肿瘤细胞系模型建立成功率低、二维培养方法导致肿瘤细胞丧失三维结构、缺少异质性,从而无法准确复制在体肿瘤生物学特征。异种移植动物模型(PDX)需要花费较长时间和资金、耗费大量实验资源、不适合进行大规模培养及动物特异性肿瘤进化等缺点,也限制了更广泛的使用。因此建立基于肿瘤类器官技术的三维肿瘤体外模型,可以满足临床个体化治疗和肿瘤研究的需求。
Boretto,M.与同事于2019年发表的文献公开了一种子宫内膜癌类器官的培养基和培养方法(Patient-derived organoids from endometrial disease captureclinical heterogeneity and are amenable to drug screening.Nat Cell Biol,21(8),1041-1051),并验证了子宫内膜癌类器官是临床研究和药物敏感检测的有效工具。所公开的培养方法使用含有Wnt激动剂R-spondin 1的条件培养基和BMP抑制剂 Noggin重组蛋白及多种生长因子作为添加物的培养基,成功建立了子宫内膜癌类器官。虽然传统上认为WNT3A、R-Spondin和Noggin 对培养的类器官增殖和自我更新是必需的调节蛋白,但是文献报道的方法在培养基种添加Wnt激动剂R-spondin 1条件培养基制备过程复杂、无法准确控制所需蛋白含量,BMP抑制剂Noggin调控蛋白价格昂贵、且主要生产企业均位于国外,这导致子宫内膜癌类器官专用培养基存在生产成本较高、主要添加成分需要进口、采购周期长且供应周期易受外部环境影响等问题。这些问题极大的阻碍了子宫内膜癌类器官的大规模培养和更广范围的应用研究。因此,迫切希望开发更低成本、更稳定易用的子宫内膜癌类器官专用培养基。
发明内容
为了解决上述问题,本发明提供了一种全新的子宫内膜癌类器官无血清专用培养基的组成和培养方法,其目的在于,提供一种无需制备WNT3A等条件培养基、无需添加Wnt激动剂R-Spondin及 BMP抑制剂Noggin等昂贵重组蛋白的子宫内膜癌类器官无血清专用培养基。
为实现上述目的,本发明提供的方案是这样实现的:一方面,本发明所述子宫内膜癌类器官无血清专用培养基由基础培养基和培养添加物两部分组成。基础培养基为含有1%青-链霉素、1%HEPES 缓冲液、1%GlutaMax溶液的Advanced DMEM/F12培养基。
进一步地,所述培养添加物包括生长因子FGF2、FGF10,胰岛素、氢化可的松、雌激素,小分子抑制剂Y-27632、SB202190,其他添加成分牛垂体提取物(BPE)、B27 supplement。
进一步地,所述培养添加物的终浓度为FGF2:0.005- 0.05μg/mL、FGF10:0.005-0.05μg/mL,胰岛素:10-50μg/mL、氢化可的松:0.1-10μg/mL、雌激素2-200nM,小分子抑制剂Y-27632: 0.1-1.0μM、SB202190:0.1-1.0μM,BPE:0.5-50μg/mL、B27 supplement:1%。
进一步地,所述子宫内膜癌类器官无血清专用培养基在使用时配制,在已配制好的基础培养基中,按照终浓度加入所有培养添加物,混合均匀后即可获得。
在本发明的另一个方面中,提供一种子宫内膜癌类器官体外培养的方法,包括以下步骤:
(1)将子宫内膜癌组织样本切成1~3mm3左右大小,用PBS清洗后加入浓度为1mg/mL的胶原酶中,37度消化30分钟左右;
(2)将消化后的细胞按照2×106个细胞/mL的浓度重悬于冷的 CultrexTM growthfactor reduced BME type2中,加入24孔细胞培养板,每孔加40微升,放入37度细胞培养箱20分钟,使含有细胞的 BME固化;
(3)每孔加入上述子宫内膜癌类器官无血清专用培养基400微升,放入5%CO2浓度、37℃细胞培养箱培养,每3~4天更换一次上述子宫内膜癌类器官无血清专用培养基,7天左右即可获得所需子宫内膜癌类器官。
由于本发明所采用培养方法不需要制备条件培养基,采用的培养添加物中去除了价格昂贵的重组蛋白成分,从而可以得到以下有益效果:
(1)本发明所述培养基对于子宫内膜癌类器官生长有较好的支持和促进作用,7天左右即可获得典型的子宫内膜癌类器官,在体外较好的保留了患者来源肿瘤一致性和保留异质性,为进一步进行研究应用奠定了基础;
(2)本发明所述培养基的成本较其他方法有明显下降,主要原因在于去除了价格较为昂贵的Wnt信号调节重组蛋白R-Spondin和发育调控蛋白Noggin等重组蛋白,而使用其他成分替代,从而为以后进行大规模子宫内膜癌类器官培养用于药物筛选等研究应用提供了可能。
(3)本发明所述培养方法步骤清晰简便,不需要单独制备和生产WNT3A条件培养基及其他条件培养基,能够保持培养基成分的稳定性,提高了培养结果的一致性,为后续大规模培养提供了便利条件。
具体实施方式
实施例:
本发明的优选实施方式是,提供一种无需WNT3A等条件培养基、无需添加Wnt通路调控重组蛋白R-Spondin及BMP抑制剂头蛋白Noggin的子宫内膜癌类器官无血清专用培养基及培养方法,所述无血清专用培养基包括基础培养基和培养添加物。所述基础培养基为含有1%青-链霉素、1%HEPES缓冲液、1%GlutaMax溶液的 Advanced DMEM/F12培养基,所述培养添加物由以下终浓度的成分组成:FGF2:0.05μg/mL、FGF10:0.005μg/mL,胰岛素: 50μg/mL、氢化可的松:10μg/mL、雌激素:2nM,小分子抑制剂 Y-27632:0.1μM、SB202190:1.0μM,BPE:50μg/mL、B27 supplement:1%。在使用时将所有培养添加物按照最终浓度加入所述基础培养基,即可获得所述子宫内膜癌类器官无血清专用培养基。所述子宫内膜癌类器官培养方法为,将患者来源子宫内膜癌组织样本切成1~3mm3左右大小,清洗后加入1%胶原酶消化30分钟左右,将消化后的细胞按照2×106个细胞/mL的浓度重悬于冷的 CultrexTMgrowth factor reduced BME type2中,加入24孔细胞培养板,每孔加40微升,放入37度细胞培养箱20分钟,待含有细胞的BME固化后,每孔加入上述子宫内膜癌类器官无血清专用培养基400微升,放入5%CO2浓度、37℃细胞培养箱培养,每4天更换一次上述子宫内膜癌类器官无血清专用培养基,7天左右即可获得所需子宫内膜癌类器官。为后续进一步研究提供了较好的研究样本。
对比例:
本发明的对比例采用Boretto,M.与同事于2019年发表的文献所描述的方法(Patient-derived organoids from endometrial disease capture clinicalheterogeneity and are amenable to drug screening.Nat Cell Biol,21(8),1041-1051),子宫内膜类器官培养基由DMEM/F12 培养基加左旋谷氨酸及HEPES缓冲液组成,补充1%青霉素-链霉素、1%谷氨酰胺和1% N2 supplement、2% B27 supplement,Chemically defined lipid concentrate、5% R-Spondin-1条件培养基、 100ng/mLNoggin、50ng/mL EGF、20ng/mL HGF、5mmol/L烟酰胺、1.25mmol/L N-乙酰半胱氨酸、40ng/mL IGF、10nM 17-β Estradiol、0.1μmol/L SB202190、0.25μM A83-01和5ng/mL IL-6、 10μmol/L Y-27632二盐酸盐。具体培养方法为,活检组织切成小块,并用不含钙镁的PBS清洗后,用1~2mg/ml胶原酶IV消化1-3 小时,然后在TrypLE中孵育15分钟,离心后,细胞重悬于70%Matrigel/30%DMEM/F12中,每孔20微升于48孔板中,待 Matrigel基质胶固化后,向每个孔中添加250μL生长培养基。本发明采用对比例的方法也能够获得所需子宫内膜癌类器官,所获得的子宫内膜癌类器官呈现典型的肿瘤类器官三维结构,生长良好。
综上,本发明所述子宫内膜癌类器官无血清专用培养基及培养方法能够有效支持子宫内膜癌类器官的体外生长,且生长迅速,所形成的子宫内膜癌类器官呈现典型的三维球状或三维不规则细胞团块,可以生长至直径较大水平依然保持肿瘤类器官形态。与已公开文献所述方法比较,本专利所述培养基去除了价格昂贵的重组蛋白类添加成分,改用其他生长因子和化合物替代,从而极大地降低了专用培养基的成本,不需要制备条件培养基作为肿瘤类器官生长的支持,培养效果与已公开的方法比较相一致。
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。本领域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说明本发明的原理,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,这些变化和改进都属于本发明保护的范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (6)
1.一种子宫内膜癌类器官无血清专用培养基,其特征在于:包括基础培养基和培养添加物,基础培养基为含有1%青-链霉素、1%HEPES缓冲液、1%GlutaMax溶液的AdvancedDMEM/F12培养基,培养添加物包括生长因子FGF2、FGF10,胰岛素、氢化可的松、雌激素,小分子抑制剂Y-27632、SB202190,其他添加物牛垂体提取物BPE、B27 supplement。
2.根据权利要求1所述的一种子宫内膜癌类器官无血清专用培养基,其特征在于,所述培养添加物的终浓度为:FGF2:0.005-0.05μg/mL、FGF10:0.005-0.05μg/mL,胰岛素:10-50μg/mL、氢化可的松:0.1-10μg/mL、雌激素2-200nM,小分子抑制剂Y-27632:0.1-1.0μM、SB202190:0.1-1.0μM,BPE:0.5-50μg/mL、B27 supplement:1%。
3.根据权利要求1所述的一种子宫内膜癌类器官无血清专用培养基,其特征在于,所述基础培养基为无血清培养基。
4.根据权利要求1所述的一种子宫内膜癌类器官无血清专用培养基,其特征在于,所述培养基不需要添加Wnt信号调节重组蛋白R-Spondin或者发育调控蛋白Noggin等重组蛋白,不需要单独制备WNT3A条件培养基。
5.根据权利要求1所述的一种子宫内膜癌类器官无血清专用培养基,其特征在于,在已配制好的基础培养基中,按照终浓度加入所有培养添加物,混合均匀后即可获得。
6.一种子宫内膜癌类器官培养方法,其特征在于,将子宫内膜癌组织样本处理清洗后加入胶原酶消化,将消化后的细胞重悬于冷的CultrexTM growth factor reduced BMEtype2中,接种于细胞培养板,待含有细胞的BME固化后,加入权利要求1~5任意一项所述的培养基,放入5%CO2浓度、37℃细胞培养箱培养,每3~4天更换一次上述培养基,7天左右即可获得所需子宫内膜癌类器官。
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