CN114958753A - 一种舌癌类器官的培养基、培养方法及鉴定方法 - Google Patents
一种舌癌类器官的培养基、培养方法及鉴定方法 Download PDFInfo
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Abstract
本发明公开了一种舌癌类器官的培养基、培养方法及鉴定方法。所述的培养基根据舌癌细胞生长特点,采用含不同成分的类器官培养基进行培养,筛选出针对舌癌类器官生长所必需的关键细胞因子、信号通路调控因子、小分子抑制剂及其使用浓度;同时还优化了培养方法,使舌癌类器官的构建成功率从66.7%提高至100%,再对舌癌类器官进行组织学、遗传学、致瘤性等方面鉴定,验证了本发明提供的舌癌类器官培养基及优化后的培养方法可以成功稳定培养出与来源组织在组织形态、组织病理学特征、肿瘤异质性等各方面高度一致的舌癌类器官模型,且可稳定传代、冻存,能为舌癌基础研究、抗癌药物研发、精准医疗等提供理想的体外研究模型。
Description
技术领域
本发明涉及生物医药技术领域,具体是一种舌癌类器官的培养基、培养方法及鉴定方法。
背景技术
口腔癌是头颈部最常见的恶性肿瘤之一,约占全球肿瘤病例的52%,其中舌癌的发病率占口腔癌首位。已有大量研究证实口腔癌的发病率在全球范围内呈上升趋势,且患病年龄趋于年轻化,其恶性程度高,发病隐匿,易复发转移,为治疗带来了极大困难。另外,由于舌是重要的发音、咀嚼等功能器官,如无法控制病情将会给患者带来巨大的痛苦。近些年,随着治疗技术的不断进步,舌癌治疗除了手术、放化疗之外,靶向治疗和免疫治疗开始兴起,但患者之前存在个体差异,对同种药物的敏感性不同,而很多药物本身会对患者造成巨大的毒副作用,如何筛选最适宜的药物,为患者“量身打造”出最佳的治疗方案是现在舌癌治疗的最大难题。
体外细胞模型是肿瘤研究的基础,目前最常见的是二维单层肿瘤细胞系或者人源性异种移植瘤模型。随着细胞培养技术的发展,人源肿瘤类器官模型应运而生,其区别于传统细胞模型,是指将患者来源的肿瘤组织消化为单个细胞,利用3D细胞培养技术诱导构建出类器官样结构。类器官直接来源于临床肿瘤患者样本,具有多种细胞类型,且3D的生长模式更符合肿瘤在体内的实际生长情况,已有大量文献表明肿瘤类器官能高度模拟体内细胞生长增殖方式,与来源肿瘤组织在组织学、遗传学以及生理功能上保持高度一致性,可在体外稳定传代,并能长期保持肿瘤异质性,是目前肿瘤研究的最佳细胞模型。类器官培养周期短、经济成本低、可高度还原来源组织的真实情况,可作为肿瘤患者的“替身”,在肿瘤发生发展分子机制、耐药机制等基础研究,抗肿瘤药物研发,临床精准医疗等方面具有巨大潜力。
现有的关于舌癌组织的培养仍主要局限于传统的二维原代舌癌细胞培养,其在培养过程中会丢失肿瘤异质性,且在形态学、组织学、遗传学上均会发生较大改变,因而造成使用该细胞取得的研究成果往往与临床试验存在较大差距。此外,运用常规的鉴定方法也难以证明原代舌癌细胞与来源组织的一致性。而舌癌类器官很好的解决了上述问题,但如果无稳定成熟的构建方案或缺乏适宜的培养基,也将会为舌癌类器官培养造成巨大困难。目前,国内外关于舌癌类器官构建的相关报道十分罕见,且构建成功率低、培养基成分复杂经济成本高,针对以上问题优化舌癌类器官培养的具体操作步骤、培养基配方及各成分使用浓度和鉴定方法尤为关键。
发明内容
针对上述问题,本发明提供了一种舌癌类器官的培养基、培养方法及鉴定方法。本发明根据舌癌细胞生长特点,优化了培养基的成分,筛选出针对舌癌类器官生长所必需的关键细胞因子、信号通路调控因子、小分子抑制剂及其使用浓度;同时还优化培养方法,能够成功稳定培养出与来源组织在组织形态、组织病理学特征、肿瘤异质性等各方面高度一致的舌癌类器官模型,且可稳定传代、冻存,为舌癌基础研究、抗癌药物研发、精准医疗等提供了理想的体外研究模型。
为了实现以上目的,本发明采用的技术方案如下:
一种舌癌类器官的培养基,所述培养基包括如下成分:Advanced DMEM/F12、B27、GluMax、HEPES、EGF、Nicotinamide、N-Acetylcysteine、Y27632、A8301、Noggin、Heregulin1和R-Spondin-1;各组分的含量为:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A83015 μM,Noggin 100ng/ml,Heregulin 1 100ng/ml,R-Spondin-1 100ng/ml。所述培养基的原料试剂均为市售产品。
本发明的另一目的在于提供:一种舌癌类器官的培养方法,包括如下步骤:
(1)将新鲜舌癌肿瘤组织分别用含有青链霉素和氟康唑的PBS冲洗干净,取上皮组织剪切为0.5-2mm3的碎块,将剪切好的组织碎块移入含有酶解液的离心管并封口,最后将离心管置于摇床中消化至酶解液浑浊、组织碎块基本消失为止;
(2)消化完成后将含组织细胞的酶解液4℃、1500转/分离心5min,弃上清,收集组织细胞沉淀,加入PBS清洗干净后,在4℃、1500转/分离心5-10min,弃上清液,向细胞沉淀中加入舌癌类器官培养基,充分重悬后转移至培养皿中放在37℃细胞培养箱中悬浮培养24小时;
(3)悬浮培养24小时后用100μm细胞筛过滤,4℃、1500转/分离心5min,弃上清,用Matrigel基质胶重悬细胞沉淀,呈胶滴状接种在孔板中;然后将孔板置于37℃细胞培养箱中5-10min,待胶滴完全凝固后再加入舌癌类器官培养基继续培养;
(4)根据前期已建立的舌癌类器官培养体系完成培养,定期更换培养基,当细胞密度达到70-90%时,或单一类器官球体过大细胞有崩解前兆时,用TryplE消化传代,稳定传代后部分冷冻保存。
作为本发明技术方案的优化:所述酶解液包括以下成分:胶原酶I、透明质酸酶IV、DNaseI和DispaseⅡ。所述酶解液各组分的含量:胶原酶I 2mg/ml,透明质酸酶IV 0.1mg/ml,DNaseI 0.05mg/ml,DispaseⅡ1.5mg/ml。
作为本发明技术方案的优化:舌癌类器官培养基优化:分别用含有不同细胞因子的培养基培养舌癌类器官,筛选出针对舌癌类器官生长所必需的关键细胞因子、信号通路调控因子、小分子抑制剂。1号培养基:为基础培养基,包括Advanced DMEM/F12、B27、GluMax、HEPES、EGF、Nicotinamide、N-Acetylcysteine、Y27632、A8301,2号培养基:为1号+FGF10,3号培养基:为2号+Heregulin 1,4号培养基:为3号+Noggin,5号培养基:为4号+R-Spondin-1。最后筛选出最适宜舌癌类器官生长且最经济的培养基成分:Advanced DMEM/F12、B27、GluMax、HEPES、EGF、Nicotinamide、N-Acetylcysteine、Y27632、A8301、Noggin、Heregulin 1和R-Spondin-1。
作为本发明技术方案的进一步优化:所述舌癌类器官培养基各组分的含量为:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,Noggin100ng/ml,Heregulin 1 100ng/ml,R-Spondin-1 100ng/ml。
作为本发明技术方案的进一步优化:步骤(1)所述PBS中青链霉素和氟康唑的百分含量分别是1%和0.2%。
作为本发明技术方案的进一步优化:步骤(2)所述PBS含有Y-27632和BSA,其中Y-27632和BSA用量分别为10μM和0.1%。
本发明另一目的还提供舌癌类器官鉴定方法:将舌癌器官进行细胞包埋、制作细胞切片,通过HE染色比较来源组织与舌癌类器官病理类型是否一致,通过免疫组化比较两者CK5&6、CKpan、ki-67、p63等指标是否一致。通过单细胞测序检测类器官细胞类型、细胞亚群与临床样本的一致性,验证舌癌类器官与来源组织的遗传学稳定性。通过裸鼠移植瘤实验验证舌癌类器官的成瘤性,并制作裸鼠移植瘤病理切片进行HE染色验证移植瘤病理类型是否与舌癌类器官一致。
与现有技术相比,本发明的优点及有益效果包括:
本发明根据舌癌细胞生长特点,采用含不同成分的类器官培养基进行培养,筛选出针对舌癌类器官生长所必需的关键细胞因子、信号通路调控因子、小分子抑制剂及其使用浓度,极大降低了培养成本。本发明还优化了传统培养方法,通过先悬浮培养24小时,再基质胶培养的方法将舌癌类器官的构建成功率从66.7%提高至100%。再通过通过HE染色、免疫组化、单细胞测序、裸鼠移植瘤等方法对舌癌类器官进行组织学、遗传学、致瘤性等方面鉴定,验证了本发明提供的舌癌类器官培养基及优化后的培养方法可以成功稳定培养出与来源组织在组织形态、组织病理学特征、肿瘤异质性等各方面高度一致的舌癌类器官模型,且可稳定传代、冻存,为舌癌基础研究、抗癌药物研发、精准医疗等提供了理想的体外研究模型。
附图说明
图1舌癌类器官在1-5号培养基中生长情况;
图2 1-5号培养基中舌癌类器官直径对比图;
图3未悬浮和悬浮培养24小时培养舌癌类器官生长情况;
图4未悬浮和悬浮培养24小时培养舌癌类器官直径对比;
图5舌癌组织和舌癌类器官HE染色;
图6舌癌类器官免疫组化;
图7舌癌类器官单细胞测序;
图8舌癌类器官裸鼠移植瘤;
图9舌癌类器官裸鼠移植瘤HE染色。
具体实施方式
下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1
一种舌癌类器官的培养基,所述培养基由如下成分组成:
Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,Noggin100ng/ml,Heregulin 1 100ng/ml,R-Spondin-1 100ng/ml。
采用上述培养基培养舌癌类器官的培养方法,包括如下步骤:
(1)取舌癌患者手术或活检的新鲜离体肿瘤组织,将其浸没于含10μM Rho激酶(ROCK)抑制剂Y-27632的4℃PBS中30min内带回实验室进行预处理。将新鲜舌癌肿瘤组织分别用4℃含有青链霉素和氟康唑(用量分别为1%和0.2%)的PBS冲洗干净,以减小污染的风险。将清洗过的组织置于培养皿中,加入少量PBS保证组织处于湿润状态,用灭菌眼科剪剔除肿瘤组织附着的脂肪及肌肉等成分,剩余的上皮组织移入含少量酶解液的Ep管中剪切为约1mm3的碎块,然后将剪切好的组织碎块移入含适量酶解液的15ml离心管中并封口,最后将离心管水平倾斜15°置于37℃摇床中,100转/分消化约30min左右(具体时间视组织实际消化情况而定)至酶解液浑浊、组织碎块基本消失为止;所述酶解液包括以下成分:胶原酶I2mg/ml,透明质酸酶IV 0.1mg/ml,DNaseI 0.05mg/ml,DispaseⅡ1.5mg/ml。
(2)消化完成后将含组织细胞的酶解液4℃、1500转/分离心5min,弃上清,收集组织细胞沉淀,如残留较多红细胞则加入红细胞裂解液裂解,如无则直接加入足量PBS(含Y-27632和BSA)清洗后4℃、1500转/分离心5min。弃上清,向细胞沉淀中加入舌癌类器官培养基,充分重悬后转移至培养皿中放在37℃细胞培养箱中悬浮培养24小时。
(3)悬浮培养24小时后用100μm细胞筛过滤,4℃、1500转/分离心5min,弃上清,用Matrigel基质胶重悬细胞沉淀,呈胶滴状接种在孔板中。然后将孔板置于37℃细胞培养箱中5-10min,待胶滴凝固后再加入舌癌类器官培养基继续培养。
(4)根据前期已建立的舌癌类器官培养体系完成培养,定期更换培养基,当细胞密度达到70-90%时,或单一类器官球体过大细胞有崩解前兆时,用TryplE消化传代,稳定传代后部分冷冻物样本库液氮中,验证可复苏性。
实施例2
本例与实施例1的区别在于:所用的舌癌器官培养基由如下成分组成:AdvancedDMEM/F12 1×,B2750×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM。
实施例3
本例与实施例1的区别在于:所用的舌癌器官培养基由如下成分组成:AdvancedDMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,FGF10 100ng/ml。
实施例4
本例与实施例1的区别在于:所用的舌癌器官培养基由如下成分组成:AdvancedDMEM/F12 1×,B2750×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,FGF10 100ng/ml,Heregulin 1 100ng/ml。
实施例5
本例与实施例1的区别在于:所用的舌癌器官培养基由如下成分组成:AdvancedDMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,FGF10 100ng/ml,Heregulin 1 100ng/ml,Noggin 100ng/ml。
分别按照实施例1-5的方法培养同一患者来源的舌癌类器官,实施例1-5分别标记为1-5号培养基,观察不同方法培养的舌癌类器官生长情况,统计分析各组间舌癌类器官直径是否有差异,明确舌癌类器官生长所必须的成分,检测结果如图1-2所示:Heregulin 1、Noggin、R-spondin-1为舌癌类器官关键细胞因子。
实施例6
本例与实施例1的区别在于:步骤(2)中经PBS清洗后无悬浮培养24小时步骤,直接按步骤(3)完成后续操作。
以可连续传代≥5代为培养成功标准,按照实施例1的培养方法培养7例舌癌类器官,成功7例,成功率为100%;按照实施例6的培养方法培养9例舌癌类器官,成功6例,成功率为66.7%。同时观察两种方法培养的舌癌类器官生长情况,统计不同方法培养的舌癌类器官直径是否有差异,检测结果如图3-4所示:先悬浮培养24小时后再用基质胶培养的方法可提高培养成功率,且舌癌类器官生长更迅速、稳定。
实施例7
鉴定由实施例1所培养出的舌癌类器官,具体方法如下:预热HistoGel标本处理凝胶,待其从固态融化为液态后将其加入到类器官包埋模具中,把含有舌癌类器官的基质胶从孔板中完整的取出,转移到已加有标本处理凝胶的模具中,并使类器官完全浸没其中。将模具置于4℃冰箱5min,待标本处理凝胶完全凝固后脱模,再进行石蜡包埋、切片等操作制作舌癌类器官病理切片。对舌癌类器官病理切片进行HE染色及CK5&6、CKpan、ki-67、p63等指标的免疫组化,验证舌癌类器官与来源组织在组织学上的一致性,结果如图5-6所示:舌癌类器官具有与来源组织一致的组织学特征。将第2代及第8代舌癌类器官分别进行单细胞测序,检测其是否具备肿瘤异质性等遗传特性,结果如图7所示:舌癌类器官可保持肿瘤异质性并具有良好的遗传稳定性。将舌癌类器官接种到免疫缺陷小鼠腋下观察是否致瘤,结果如图8所示:舌癌类器官具有良好的致瘤性。将裸鼠移植瘤制作组织病理切片,验证移植瘤病理类型是否与舌癌类器官一致,结果如图9所示:两者病理类型一致。
以上内容是结合具体的/优选的实施方式对本发明所作的进一步详细说明,不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说,在不脱离本发明构思的前提下,其还可以对这些已描述的实施例做出若干替代或变型,而这些替代或变型方式都应视为属于本发明的保护范围。
Claims (9)
1.一种舌癌类器官的培养基,其特征在于:所述培养基包括如下成分:Advanced DMEM/F12、B27、GluMax、HEPES、EGF、Nicotinamide、N-Acetylcysteine、Y27632、A8301、Noggin、Heregulin 1和R-Spondin-1;各组分的含量为:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,Noggin 100ng/ml,Heregulin 1 100ng/ml,R-Spondin-1 100ng/ml。
2.一种舌癌类器官的培养方法,其特征在于:包括如下步骤:
(1)将新鲜舌癌肿瘤组织分别用含有青链霉素和氟康唑的PBS冲洗干净,取上皮组织剪切为0.5-2mm3的碎块,将剪切好的组织碎块移入含有酶解液的离心管并封口,最后将离心管置于摇床中消化至酶解液浑浊、组织碎块基本消失为止;
(2)消化完成后将含组织细胞的酶解液4℃、1500转/分离心5min,弃上清,收集组织细胞沉淀,加入PBS清洗干净后,在4℃、1500转/分离心5-10min,弃上清液,向细胞沉淀中加入舌癌类器官培养基,充分重悬后转移至培养皿中放在37℃细胞培养箱中悬浮培养24小时;
(3)悬浮培养24小时后用100μm细胞筛过滤,4℃、1500转/分离心5min,弃上清,用Matrigel基质胶重悬细胞沉淀,呈胶滴状接种在孔板中;然后将孔板置于37℃细胞培养箱中5-10min,待胶滴完全凝固后再加入舌癌类器官培养基继续培养;
(4)根据前期已建立的舌癌类器官培养体系完成培养,定期更换培养基,当细胞密度达到70-90%时,或单一类器官球体过大细胞有崩解前兆时,用TryplE消化传代,稳定传代后部分冷冻保存。
3.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:所述酶解液包括以下成分:胶原酶I、透明质酸酶IV、DNaseI和DispaseⅡ。
4.根据要求3所述一种舌癌类器官的培养方法,其特征在于:所述酶解液各组分的含量:胶原酶I 2mg/ml,透明质酸酶IV 0.1mg/ml,DNaseI 0.05mg/ml,DispaseⅡ 1.5mg/ml。
5.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:所述舌癌类器官培养基的组成成分如下:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,A8301 5μM,Y27632 10μM,Nicotinamide 10mM,N-Acetylcysteine 1mM。
6.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:所述舌癌类器官培养基包括如下成分:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM。
7.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:所述舌癌类器官培养基由如下成分组成:Advanced DMEM/F12 1×,B27 50×稀释为1×,GluMax 100×稀释为1×,HEPES 1×,EGF 50ng/ml,Nicotinamide 10mM,N-Acetylcysteine 1mM,Y27632 10μM,A8301 5μM,Noggin 100ng/ml,Heregulin 1 100ng/ml,R-Spondin-1 100ng/ml。
8.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:步骤(1)所述PBS中青链霉素和氟康唑的百分含量分别是1%和0.2%。
9.根据权利要求2所述一种舌癌类器官的培养方法,其特征在于:步骤(2)所述PBS含有Y-27632和BSA,其中Y-27632和BSA用量分别为10μM和0.1%。
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