CN116948974A - 一种病人源性头颈部鳞癌类器官培养方法及其应用 - Google Patents
一种病人源性头颈部鳞癌类器官培养方法及其应用 Download PDFInfo
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Abstract
本发明提供一种病人源性头颈部鳞癌类器官培养方法及其应用,方法包括以下步骤:收集头颈部鳞癌患者手术或活检的新鲜肿瘤组织样本;将组织样本放入组织储存液后转移到实验室处理;将肿瘤组织进一步剪碎成碎片移入组织解离仪器配套C管内解离后离心过滤获得单细胞肿瘤样本沉淀;用类器官培养基重悬单细胞肿瘤组织沉淀后放置于碎冰中,待单细胞悬液冷却后与基质胶混合,将混合好的单细胞悬液在冰上操作加入预冷后的AggreWellTM400 24孔板中离心,将离心后的AggreWellTM400 24孔板移入37℃ CO2培养箱中并加入预热培养基孵育,实现类器官生长。本申请将基质胶肿瘤细胞混合液移入AggreWellTM400 24孔板离心后培养,培养时间短,所需组织样本量少,存活率高,培养成功率大幅度提升。
Description
技术领域
本发明涉及生物医学技术领域,具体涉及一种病人源性头颈部鳞癌类器官培养方法及其应用。
背景技术
头颈部鳞癌(Head and neck squamous cell carcinoma,HNSCC)是颌面部最常见的恶性肿瘤,发病后常造成其功能和外形的破坏,器官保存在头颈部鳞癌的治疗中有重要意义。约60%的HNSCC在诊断时即为局部晚期,目前标准的治疗是放化疗或手术后进行放疗,伴或不伴化疗。尽管采用标准的多学科治疗,但肿瘤耐药、高失败率和疾病复发仍然是不良生存结局的原因,治疗后前两年复发率达50%。在多学科治疗中,每种治疗方式的贡献不能得知,且疗效评估需等待治疗后的检查。因此,肿瘤医生一直在探究治疗方式上的改进,如果治疗效果能够在治疗前进行预判,无疑能够减少一些不必要的治疗,从而达到个体化治疗的目的。但受限于患者作为研究对象的不可操控性,而实验动物与人差异巨大,目前从基础到临床的转化效率极低。
肿瘤类器官的兴起为转化医学提供了全新的技术平台。类器官(Organoid)是由干细胞或者病人源性的肿瘤组织在特定的3D体外微环境下培养而来的、高度模拟体内真实器官特征的小型化的体外器官模型。由于其可以模拟原位器官在体内的各种特征且高度保留患者的个体特异性,目前在再生医学、基因编辑、精准医疗、器官发育、疾病建模等方面表现出了良好的应用潜力。相比于2D疾病模型,类器官在阐明疾病的发展、稳态和发病机制方面更具优势。2013年,类器官被《科学》杂志评为年度十大技术。对于类器官的认知,国内外也已经传出许多认同的声音:Cell、Nature、Science系列杂志及美国科学院院刊PNAS多次报道肿瘤类器官与原位肿瘤一致,并含有相同基因突变谱,并通过基因测序和PDX小鼠肿瘤模型验证类器官能够在体外预测药物敏感性。
目前,已报道的肿瘤类器官有胰腺癌、卵巢癌、乳腺癌和结直肠癌等多种肿瘤。亦有少量研究尝试头颈部鳞癌类器官构建,例如公布号为CN116004539A、公布日为2023.04.25的发明专利申请,公开了一种头颈部鳞癌相关成纤维细胞与类器官共培养系统及其构建方法与应用;Tanaka等人[Tanaka N,Osman AA,Takahashi Y,et al.Head andneck cancer organoids established by modification of the CTOS method can beused to predict in vivo drug sensitivity[J].Oral Oncol.2018Dec;87:49-57.]在2018年报道从头颈部鳞癌患者的手术标本中建立肿瘤类器官模型。而本申请的发明人经过研究发现,这些技术方案均是将肿瘤解离成单细胞,然后与基质胶混匀后直接体外培养,均匀分散在基质胶内的单细胞成长为够实验使用类器官所需时间长,对样本的要求高,培养成功率较低,成本大,且目前还未见运用患者来源头颈部鳞癌类器官做放疗敏感性检测的报道。
发明内容
针对现有病人源性头颈部鳞癌类器官构建技术方案中培养时间长,对样本的要求高,培养成功率低,成本大的技术问题,本发明提供一种病人源性头颈部鳞癌类器官培养方法。
为了解决上述技术问题,本发明采用了如下的技术方案:
一种病人源性头颈部鳞癌类器官培养方法,包括以下步骤:
样本收集:收集包括口腔、喉、舌、鼻咽在内的头颈部鳞癌患者的手术或活检的新鲜肿瘤组织样本;
样本处理:将组织样本放入组织储存液后转移到实验室,在生物安全柜内用PBS清洗收集的组织样本至少三次后转移到培养皿中,用无菌剪刀修剪掉多余的脂肪、肌肉和坏死的组织;随后将组织剪碎,随机取一部分组织速冻于-80℃用于DNA分离,再选取一部分组织用福尔马林固定用于组织病理分析和免疫组化分析,剩余的组织用于类器官构建;
单细胞样本制备:将肿瘤组织进一步剪碎成1~3mm的碎片,移入组织解离仪器配套的C管内,按照仪器说明书要求加入相应的酶混合均匀;将C管拧紧倒置于组织解离仪器上,启动预设组织解离程序;待组织解离程序结束后,取出C管,重悬样本;将样本通过70um滤网,并用20ml DMEM清洗滤网,离心滤液300g×7分钟,获得单细胞肿瘤样本沉淀;
类器官构建:AggreWellTM400 24孔板用抗黏附剂处理后放置冰上备用,用类器官培养基重悬单细胞肿瘤组织沉淀,浓度控制在1.5×106个细胞每1毫升,随后放置于碎冰中,待单细胞悬液冷却后与基质胶按1:1混合;将混合好的单细胞悬液在冰上操作加入预冷后的AggreWellTM400 24孔板中,然后4℃离心100g×3分钟,将离心后的AggreWellTM400 24孔板移入37℃CO2培养箱中孵育30分钟,待混合液凝固后加入37℃预热的培养基,再放入37℃CO2培养箱中孵育,3~5天能传代,每2~3天更换一次培养基,直到类器官长至100~150um。
进一步,所述步骤类器官构建中还包括:每2~3天更换一次培养基的同时,前2周的培养中添加10μmol/L Y-27632以帮助类器官中干细胞生长。
进一步,所述类器官构建步骤之后还包括类器官传代:吸掉上层的培养基,吸取类器官胶层,转移至1.5ml的EP管中,再用细胞回收液清洗后转移到EP管中,放置冰上1小时或待基质胶完全溶解,4℃离心300g×5分钟回收类器官球沉淀;用TrypLE Express重悬类器官球并在37℃孵育,每5分钟吹打20次帮助类器官球解离,待解离成单细胞的时候用100um滤网过滤,加入20ml Advanced DMEM+/+/+停止TrypLE消耗,300g×5分钟回收单细胞沉淀;沉淀用新鲜的类器官培养基重悬,之后步骤同类器官构建。
进一步,所述类器官传代步骤之后还包括类器官冻存:在类器官传代2~3天后,用细胞回收液去除基质胶,回收类器官颗粒,用无血清冻存液重悬至10000个颗粒每1毫升,然后放入液氮冻存。
进一步,所述方法还包括类器官鉴定步骤:
组织病理和免疫组化:待类器官长至100~150um时,去除基质胶,预包埋PDO,制成石蜡切片,进行HE染色及免疫组化,并与对应肿瘤组织切片进行对比;
DNA分离:运用DNA分离试剂盒提取DNA并测量浓度;
DNA测序:选取患者做类器官和对应肿瘤组织共600种癌症高突变基因panel测序,鉴定类器官与来源肿瘤保真性。
本发明还提供一种根据前述的病人源性头颈部鳞癌类器官培养方法培养出的头颈部鳞癌类器官在化疗药物和放射治疗敏感性检测中的应用。
与现有技术相比,本发明提供的一种病人源性头颈部鳞癌类器官培养方法及其应用具有以下优点:
1、与现有的直接体外培养相比,本发明方法将基质胶(Matrigel)肿瘤细胞混合液移入AggreWellTM400 24孔板离心后培养,由于该孔板包含400μm大小漏斗状微孔,少量细胞悬浮液经离心后可聚集在微孔漏斗底部,从而避免干细胞数量太少,无法存活的缺点,因而所需要的组织样本量少,能够满足通过活检样本培养人源类器官的需求;
2、本发明方法将基质胶(Matrigel)肿瘤细胞混合液移入AggreWellTM40024孔板离心后培养,由于该孔板包含1200个400μm大小漏斗状微孔,细胞悬浮液经离心后聚集在微孔漏斗底部,因而肿瘤细胞更容易成球生长,存活率高,类器官培养成功率大幅度提升,同时也能满足类器官培养高通量的需求;
3、本发明方法中肿瘤细胞成球生长,增殖能力更快,3~5天就能传代,满足短期内实验所需;
4、本发明方法培养出的头颈部鳞癌类器官能够保留原代肿瘤的遗传特征,能用于药物和放射治疗敏感性检测。
附图说明
图1是本发明提供的病人源性头颈部鳞癌类器官培养方法流程示意图。
图2是本发明提供的类器官生长时间形态示意图。
图3是本发明提供的类器官形态、HE染色、免疫组化、DNA测序结果及与对应肿瘤组织的对照示意图。
图4是本发明提供的不同剂量放射治疗照射后类器官的存活状态示意图。
图5是本发明提供的6-MV-X射线、2Gy/次、1次/天、连续照射3天后不同时间类器官的形态变化示意图。
具体实施方式
为了使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解,下面结合具体图示,进一步阐述本发明。
请参考图1所示,本发明提供一种病人源性头颈部鳞癌类器官培养方法,包括以下步骤:
样本收集:收集包括口腔、喉、舌、鼻咽在内的头颈部鳞癌患者的手术或活检的新鲜肿瘤组织样本(收集的组织样本获得按照国家和地方研究伦理委员会的指导方针,经机构研究伦理委员会批准);
样本处理:将组织样本放入组织储存液后转移到实验室,在生物安全柜内用PBS清洗收集的组织样本至少三次后转移到6cm的培养皿中,用无菌剪刀修剪掉多余的脂肪、肌肉和坏死的组织;随后将组织剪碎,随机取一部分组织例如5mm3速冻于-80℃用于DNA分离,再选取一部分组织例如5mm3用福尔马林固定用于组织病理分析和免疫组化分析,剩余的组织用于类器官构建;
单细胞样本制备:将肿瘤组织进一步剪碎成1~3mm的碎片,移入美天旎组织解离仪器配套的C管内(gentleMACS C Tube),按照仪器说明书(MACS Tumor Dissociation Kithuman,Order no.130-095-929)要求加入相应的酶混合均匀;将C管拧紧倒置于组织解离仪器(gentleMACSTMOcto Dissociator with Heaters)上,启动预设组织解离程序(Tough,37C_h_TDK_3);待组织解离程序结束后,取出C管,重悬样本;将样本通过70um滤网(MACSSmart Strainer,70um),并用20ml DMEM(培养基)清洗滤网,离心滤液300g×7分钟,获得单细胞肿瘤样本沉淀;
类器官构建:AggreWellTM400 24孔板用抗黏附剂处理后放置冰上备用,用类器官培养基重悬单细胞肿瘤组织沉淀,浓度控制在1.5×106个细胞每1毫升,随后放置于碎冰中,待单细胞悬液冷却后与基质胶(Matrigel)按1:1混合;将混合好的单细胞悬液在冰上操作加入预冷后的AggreWellTM400 24孔板中,然后4℃离心100g×3分钟,将离心后的AggreWellTM400 24孔板移入37℃CO2培养箱中孵育30分钟,待混合液凝固后加入37℃预热的培养基,再放入37℃CO2培养箱中孵育,3~5天能传代,每2~3天更换一次培养基,直到类器官长至100~150um,具体类器官生长时间形态示意图请参考图2。
作为具体实施例,所述步骤类器官构建中还包括:每2~3天更换一次培养基的同时,前2周的培养中添加10μmol/L Y-27632以帮助类器官中干细胞生长。
作为具体实施例,所述类器官构建步骤之后还包括类器官传代:吸掉上层的培养基,吸取类器官胶层,转移至1.5ml的EP管中,再用细胞回收液清洗后转移到EP管中,放置冰上1小时或待基质胶完全溶解,4℃离心300g×5分钟回收类器官球沉淀;用TrypLE Express重悬类器官球并在37℃孵育,每5分钟吹打20次帮助类器官球解离,待解离成单细胞的时候用100um滤网过滤,加入20ml Advanced DMEM+/+/+停止TrypLE消耗,300g×5分钟回收单细胞沉淀;沉淀用新鲜的类器官培养基重悬,之后步骤同类器官构建,由此可把类器官一代一代地传下去。
作为具体实施例,所述类器官传代步骤之后还包括类器官冻存:在类器官传代2~3天后,用细胞回收液去除基质胶,回收类器官颗粒,用无血清冻存液重悬至10000个颗粒每1毫升,然后放入液氮冻存,由此可实现类器官的冷冻存储。
作为具体实施例,所述方法还包括类器官鉴定步骤:
组织病理和免疫组化:待类器官长至100~150um时,去除基质胶,预包埋PDO,制成石蜡切片,进行HE染色及免疫组化,并与对应肿瘤组织切片进行对比;
DNA分离:运用DNA分离试剂盒提取DNA并测量浓度;
DNA测序:选取患者做类器官和对应肿瘤组织共600种癌症高突变基因panel测序,鉴定类器官与来源肿瘤保真性;其具体的类器官形态、HE染色、免疫组化、DNA测序结果及与对应肿瘤组织对照示意图请参考图3。
采用本实施例提供的类器官鉴定,可鉴定出本培养方法培养的头颈部鳞癌类器官能够保留原代肿瘤的遗传特征,即该类器官具有很好的保真性。
请参考图4和图5所示,本发明还提供一种根据前述的病人源性头颈部鳞癌类器官培养方法培养出的头颈部鳞癌类器官在化疗药物和放射治疗敏感性检测中的应用,其具体应用方法如下:
1.在进行化疗药物和放射治疗敏感性检测前2天复苏冻存的类器官。
2.用1:10的稀释的基质胶重悬,然后均匀种植在抗黏附的96孔板中,后置于37℃CO2培养箱中培养。
3.化疗药物敏感性实验:类器官培养2天后,进行加药处理。选择头颈部鳞癌常用的化疗药物顺铂,根据查阅文献,分别设置4个浓度梯度:3μg/mL、6μg/mL、9μg/mL、12μg/mL,作用类器官72小时,后用Calcein-AM/PI活细胞/死细胞双染试剂盒染色类器官,30分钟后用荧光显微镜对结果进行拍摄,用Image J进行图像处理和数据分析。类器官活力计算如下:类器官活力水平=活细胞数/(活细胞数+死细胞数)。
4.放疗敏感性实验:依照辐照剂量,依次分为空白对照、2GY、4GY、8GY、16GY组,分别用不同的96孔板培养,依照分组将类器官置于小动物辐照仪(X-RAD225)下进行相应剂量的射线辐照,在辐照后(24h、48h)用Calcein-AM/PI活细胞/死细胞双染试剂盒染色类器官,30分钟后用荧光显微镜对结果进行拍摄,用Image J进行图像处理和数据分析。类器官活力计算同上。
为了更好理解本发明提供的一种病人源性头颈部鳞癌类器官培养方法,以下将以口腔鳞癌类器官的培养为例进行详细说明。
1、培养流程如图1所示;
2、实验材料准备:组织储存液(MACS,货号:130-100-008),美天旎组织解离仪器配套的C管及解离试剂盒;Advanced DMEM/F12,1%Penicillin-streptomycin,1×B27补充剂购自Life Technologies;10mmol/L HEPES,50ng/mL人EGF,500nmol/L A83-01购自stemcell;1×GlutaMAX(Thermo Fisher Scientific);0.25mmol/L乙酰半胱氨酸,10mmol/L烟酰胺,0.3μmol/L CHIR99021购自MCE;10ng/mL人FGF10,5ng/mL人FGF2购自PeproTech;1μmol/L前列腺素E2(Tocris Bioscience;1μmol/L福斯高林,200ng/ml rhR-spondin,100ng/ml rhNoggin购自R&D Systems;Primocin(Invivogen);抗黏附剂(stemcell);Matrigel(Corning);10μmol/L Y-27632(stemcell);细胞回收液(Corning);TrypLEExpress(Life Technologies);100um滤网(falcon)。配置好的培养基在使用前用0.22μm滤器(Millipore)过滤,该培养基可在4℃冰箱中保存1周。
3、类器官培养步骤:
1)将手术后新鲜口腔鳞癌组织样本放入组织储存液,转移到实验室进行后续处理;
2)在生物安全柜内用PBS清洗收集的组织样本至少三次后转移到6cm的培养皿中,用无菌剪刀修剪掉多余的脂肪、肌肉和坏死的组织;随后将组织剪碎,随机取约5mm3组织速冻于-80℃用于DNA分离,再选取5mm3组织用福尔马林固定用于组织病理分析和免疫组化分析,剩余的组织用于类器官构建;
3)将肿瘤组织进一步剪碎成1~3mm的碎片,移入美天旎组织解离仪器配套的C管内(gentleMACS C Tube),按照仪器说明书(MACS Tumor Dissociation Kit human,Orderno.130-095-929)要求加入相应的酶混合均匀;
4)将C管拧紧倒置于组织解离仪器(gentleMACSTMOcto Dissociator withHeaters)上,启动预设组织解离程序(Tough,37C_h_TDK_3);
5)待组织解离程序结束后,取出C管,重悬样本;
6)将样本通过70um滤网(MACS Smart Strainer,70um),并用20ml DMEM(培养基)清洗滤网,300g离心滤液离心过滤7分钟,获得单细胞肿瘤样本沉淀;
7)按照现有文献资料配置类器官培养基备用,该培养基可在4℃冰箱中保存1周;
8)AggreWellTM400 24孔板用抗黏附剂(stemcell,货号:07010)处理后放置冰上备用;
9)用类器官培养基重悬单细胞肿瘤组织沉淀,浓度控制在1.5×106个细胞每1毫升,随后放置于碎冰中;
10)待单细胞悬液冷却后与基质胶(Matrigel)按1:1混合;
11)将混合好的单细胞悬液加入预冷后的AggreWellTM400 24孔板中(该操作在冰上进行);
12)然后4℃离心100g×3分钟,将离心后的AggreWellTM400 24孔板移入37℃CO2培养箱中孵育30分钟,待混合液凝固后加入37℃预热的培养基,再放入37℃CO2培养箱中孵育;
13)每2~3天更换一次培养基,前2周的培养中添加10μmol/L Y-27632(stemcell,货号:72302)干细胞,以帮助类器官的生长,直到类器官长至100~150um,具体类器官生长时间形态示意图请参考图2。
4、类器官鉴定步骤:
1)组织病理和免疫组化:
a、显微镜下观察类器官的形成和生长状态,待类器官长至100~150um时,去除基质胶,预包埋PDO;
b、待4%多聚甲醛中固定过夜后,常规包埋、脱水,制成石蜡切片,进行HE染色,镜下观察,并与对应肿瘤组织切片进行对比;
c、按免疫组织化学法的试剂盒说明书严格操作,一抗CD133、ki67单克隆抗体工作浓度为1∶1000,每批染色均设立阴性对照(PBS液代替一抗);
d、所有切片采用盲法由2名高年资病理科医师进行独立判定,镜下观察,阳性细胞出现棕褐色染色,在显微镜下随机选择10个高倍视野(×400),每个视野统计100个细胞,计算染色阳性的细胞比例,染色阳性率≤25%为阴性,26%~50%为阳性,≥51%为强阳性;
2)DNA分离:运用DNA分离试剂盒(Promega,货号A2052)提取DNA并测量浓度;
3)DNA测序:选取5例患者做类器官和对应肿瘤组织共600种癌症高突变基因panel测序,鉴定类器官与来源肿瘤保真性;其具体的类器官形态、HE染色、免疫组化、DNA测序结果及与对应肿瘤组织对照示意图请参考图3。
与现有技术相比,本发明提供的一种病人源性头颈部鳞癌类器官培养方法及其应用具有以下优点:
1、与现有的直接体外培养相比,本发明方法将基质胶(Matrigel)肿瘤细胞混合液移入AggreWellTM400 24孔板离心后培养,由于该孔板包含400μm大小漏斗状微孔,少量细胞悬浮液经离心后可聚集在微孔漏斗底部,从而避免干细胞数量太少,无法存活的缺点,因而所需要的组织样本量少,能够满足通过活检样本培养人源类器官的需求;
2、本发明方法将基质胶(Matrigel)肿瘤细胞混合液移入AggreWellTM40024孔板离心后培养,由于该孔板包含1200个400μm大小漏斗状微孔,细胞悬浮液经离心后聚集在微孔漏斗底部,因而肿瘤细胞更容易成球生长,存活率高,类器官培养成功率大幅度提升,同时也能满足类器官培养高通量的需求;
3、本发明方法中肿瘤细胞成球生长,增殖能力更快,3~5天就能传代,满足短期内实验所需;
4、本发明方法培养出的头颈部鳞癌类器官能够保留原代肿瘤的遗传特征,能用于药物和放射治疗敏感性检测。
最后说明的是,以上实施例仅用以说明本发明的技术方案而非限制,尽管参照较佳实施例对本发明进行了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的宗旨和范围,其均应涵盖在本发明的权利要求范围当中。
Claims (6)
1.一种病人源性头颈部鳞癌类器官培养方法,其特征在于,包括以下步骤:
样本收集:收集包括口腔、喉、舌、鼻咽在内的头颈部鳞癌患者的手术或活检的新鲜肿瘤组织样本;
样本处理:将组织样本放入组织储存液后转移到实验室,在生物安全柜内用PBS清洗收集的组织样本至少三次后转移到培养皿中,用无菌剪刀修剪掉多余的脂肪、肌肉和坏死的组织;随后将组织剪碎,随机取一部分组织速冻于-80℃用于DNA分离,再选取一部分组织用福尔马林固定用于组织病理分析和免疫组化分析,剩余的组织用于类器官构建;
单细胞样本制备:将肿瘤组织进一步剪碎成1~3mm的碎片,移入组织解离仪器配套的C管内,按照仪器说明书要求加入相应的酶混合均匀;将C管拧紧倒置于组织解离仪器上,启动预设组织解离程序;待组织解离程序结束后,取出C管,重悬样本;将样本通过70um滤网,并用20ml DMEM清洗滤网,离心滤液300g×7分钟,获得单细胞肿瘤样本沉淀;
类器官构建:AggreWellTM400 24孔板用抗黏附剂处理后放置冰上备用,用类器官培养基重悬单细胞肿瘤组织沉淀,浓度控制在1.5×106个细胞每1毫升,随后放置于碎冰中,待单细胞悬液冷却后与基质胶按1:1混合;将混合好的单细胞悬液在冰上操作加入预冷后的AggreWellTM400 24孔板中,然后4℃离心100g×3分钟,将离心后的AggreWellTM400 24孔板移入37℃CO2培养箱中孵育30分钟,待混合液凝固后加入37℃预热的培养基,再放入37℃CO2培养箱中孵育,3~5天能传代,每2~3天更换一次培养基,直到类器官长至100~150um。
2.根据权利要求1所述的病人源性头颈部鳞癌类器官培养方法,其特征在于,所述步骤类器官构建中还包括:每2~3天更换一次培养基的同时,前2周的培养中添加10μmol/L Y-27632以帮助类器官中干细胞生长。
3.根据权利要求1所述的病人源性头颈部鳞癌类器官培养方法,其特征在于,所述类器官构建步骤之后还包括类器官传代:吸掉上层的培养基,吸取类器官胶层,转移至1.5ml的EP管中,再用细胞回收液清洗后转移到EP管中,放置冰上1小时或待基质胶完全溶解,4℃离心300g×5分钟回收类器官球沉淀;用TrypLE Express重悬类器官球并在37℃孵育,每5分钟吹打20次帮助类器官球解离,待解离成单细胞的时候用100um滤网过滤,加入20mlAdvanced DMEM+/+/+停止TrypLE消耗,300g×5分钟回收单细胞沉淀;沉淀用新鲜的类器官培养基重悬,之后步骤同类器官构建。
4.根据权利要求3所述的病人源性头颈部鳞癌类器官培养方法,其特征在于,所述类器官传代步骤之后还包括类器官冻存:在类器官传代2~3天后,用细胞回收液去除基质胶,回收类器官颗粒,用无血清冻存液重悬至10000个颗粒每1毫升,然后放入液氮冻存。
5.根据权利要求1所述的病人源性头颈部鳞癌类器官培养方法,其特征在于,所述方法还包括类器官鉴定步骤:
组织病理和免疫组化:待类器官长至100~150um时,去除基质胶,预包埋PDO,制成石蜡切片,进行HE染色及免疫组化,并与对应肿瘤组织切片进行对比;
DNA分离:运用DNA分离试剂盒提取DNA并测量浓度;
DNA测序:选取患者做类器官和对应肿瘤组织共600种癌症高突变基因panel测序,鉴定类器官与来源肿瘤保真性。
6.根据权利要求1所述的病人源性头颈部鳞癌类器官培养方法培养出的头颈部鳞癌类器官在化疗药物和放射治疗敏感性检测中的应用。
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