CN113278588A - 一种口腔鳞癌类器官培养基以及培养方法 - Google Patents
一种口腔鳞癌类器官培养基以及培养方法 Download PDFInfo
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Abstract
本发明公开了一种口腔鳞癌类器官的培养方法,包括以下步骤:S1、解离酶配置;S2、配置培养基,培养基由基础培养基DMEM/F12、R‑spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成;S3、临床操作获取样品,通过取材或手术切除获得肿瘤标本,切取组织,用无菌生理盐水冲洗3次,将组织放入DMEM培养基中浸泡1小时,继续解离;S4、组织预处理:将步骤S3中获得的样品置于无菌6孔板中,用一次性无菌手术刀片将样品组织块切碎;S5、组织解离消化:加入步骤S1中配好的解离酶,重复离心‑去除上清这一过程3次以充分去除解离酶,最后一次离心前将细胞与样品组织块混合;S6、类器官培养。
Description
技术领域
本发明涉及生物医药技术领域,更具体地说,涉及一种口腔鳞癌类器官培养基以及培养方法。
背景技术
口腔鳞状细胞癌(Oral Squamous Cell Carcinoma,OSCC)是口腔颌面部最常见的恶性肿瘤,2020年全球OSCC年新发病例约37.77万,死亡17.78万,其5年生存率仅60%左右。对于中晚期尤其是复发/转移性的OSCC患者,化疗辅助下的综合治疗是标准化的治疗方案,例如顺铂和紫杉醇是一线化疗用药,但治疗效果并不尽如人意,而靶向治疗药物对OSCC的治疗效果也有待进一步验证,因此,目前急需开发出更为有效的个体化精准治疗方案。
同时,肿瘤类器官(tumor organoid)模型能够很好地预测原位肿瘤的药物敏感性;类器官(organoid)是一种能够自发组织、3D培养的体外细胞模型;类器官能够模拟体内器官的微观结构和功能,类器官由多能干细胞或成体干细胞在多种复杂的细胞因子诱导下发育而来;肿瘤类器官,是利用体外培养类器官的方法来培养原代肿瘤细胞;肿瘤类器官能够维持肿瘤在体内生长的形态,其基因表达谱与体内肿瘤相似性约90%;肿瘤类器官不仅能够保留原发肿瘤的特点,保持不同病人肿瘤之间的差异性,而且能够保持同一个病人原发肿瘤内肿瘤细胞的异质性。此外,肿瘤类器官还能准确地预测原发肿瘤的药物敏感性,为未来个体化治疗提供参考。更重要的是,肿瘤类器官的建立只需一周时间,药物敏感性实验也仅需10天,具有极强的临床时效性,是未来进行肿瘤临床药物筛选的最佳选择。
然而,口腔鳞癌与其他肿瘤相比培养难度在于,一方面口腔鳞癌处于带菌环境,类器官出现细菌、支原体乃至真菌污染的风险较高;另一方面口腔鳞癌病理类型以中高分化为主,高分化肿瘤细胞角化程度较好,肿瘤干性较低,构建类器官对比其他低分化肿瘤存在更高难度,口腔鳞癌类器官生长缓慢且培养成功率较低。
发明内容
本发明旨在提供一种口腔鳞癌类器官培养基以及培养方法,旨在解决现有技术中口腔鳞癌类器官培养中污染比例较高、培养成功率低及生长缓慢的问题。
为实现此目的,本发明提供了一种口腔鳞癌类器官培养基,由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,8-12mmol/L;Glutamax 0.8-1.2×;A83-01,400-600nmol/L;EGF,35-60ng/mL;Noggin,80-120ng/mL;FGF10,8-12ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1-1.5mmol/L;nicotinamide,8-12mmol/L;PGE2,0.8-1.2μmol/L;Butylated hydroxyanisole,4-10ng/ml;青霉素-链霉素-两性霉素B混合溶液0.8-1.2X;B27 supplement;Prostaglandin E2,0.8-1.2umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%
优选的,由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,10mmol/L;Glutamax 1×;A83-01,500nmol/L;EGF,50ng/mL;Noggin,100ng/mL;FGF10,10ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1.25mmol/L;nicotinamide,10mmol/L;PGE2,1μmol/L;Butylated hydroxyanisole,5ng/ml;青霉素-链霉素-两性霉素B混合溶液1X;B27supplement;Prostaglandin E2,1umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%
本发明还提供了一种口腔鳞癌类器官的培养方法,包括以下步骤:
S1、解离酶配置:5mg/ml的胶原酶I和10ug/ml的DNA酶I以无血清DMEM培养基为溶剂配置;
S2、配置上述培养基;
S3、临床操作获取样品:
通过取材或手术切除获得肿瘤标本,切取0.5cm×0.5cm×0.5cm组织,用无菌生理盐水冲洗3次,将组织放入含有20%双抗的预冷的DMEM培养基中浸泡1小时,继续解离,全程冰上保存运输;
S4、组织预处理:
将步骤S3中获得的样品置于无菌6孔板中,用一次性无菌手术刀片将样品组织块切碎;
S5、组织解离消化:
加入3ml步骤S1中配好的解离酶,将样品组织块移至15ml离心管中,在37度的温度下,以转速140rpm于恒温摇床中震荡消化10分钟;将已消化的上清转移到新的15ml离心管中,冰上保存;在剩余样品组织块沉淀内再加入3ml解离酶,继续在37度温度下以转速140rpm消化10分钟;重复上一操作,共计分3次消化,每次加入3ml解离酶;将消化好的组织和消化液的混合溶液分为2管,并在4度温度下以1400rpm离心5分钟,离心后去掉上清,并加入5ml DMEM培养基,重复离心-去除上清这一过程3次以充分去除解离酶,最后一次离心前将细胞与样品组织块混合;
S6、类器官培养:
在细胞沉淀中先加入50ul步骤S2中获得的培养基,将细胞吹匀,后加入基质胶600ul,用截断枪头轻柔吹打沉淀细胞团使其重悬;充分混匀后,将混有基质胶的细胞团分别均匀点入6孔板中,每孔约4-5个液滴,彼此分散间隔;放入细胞培养箱中10分钟待基质胶充分凝固,基质胶凝固后,加入步骤S2中的培养基2ml,使得液面没过基质胶,每3天换一次新鲜培养基,培养3天后,获得口腔鳞癌类器官。
优选的,所述基质胶为Matrigel。
优选的,S1中所述的解离酶在4度的温度下保存。
相比于现有技术,本发明的优点在于:
采用本发明的口腔鳞癌类器官培养基,使得口腔鳞癌类器官培养中受到污染的比例大大降低,提高了工作效率且适用范围广;采用本发明的口腔鳞癌类器官培养方法,加强了类器官培养前的组织预处理,使得口腔鳞癌类器官生长迅速且培养成功率大大提高。
附图说明
图1为本发明的颊鳞癌类器官在显微镜下的细胞形态学观察图;
图2为本发明的舌鳞癌类器官在显微镜下的细胞形态学观察图。
具体实施方式
下面将结合本发明实施例中的附图,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。
本发明实施例采用的EGF、FGF10、Gastrin I、N-acetylcysteine、R-spondin1conditioned media、Wnt3A conditioned media购自Peprotech公司;
本发明实施例采用的Noggin、Gastrin I、N-acetylcysteine、nicotinamide、PGE2、B27 supplement、Prostaglandin E2、Butylated hydroxyanisole购自Sigma公司;
本发明实施例采用的A83-01、N-acetylcysteine购自MCE公司;
本发明实施例采用的Penicillin-Streptomycin-Amphotericin B Solution(青霉素-链霉素-两性霉素B混合溶液)购自Beyotime公司。
实施例1
一种口腔鳞癌类器官培养基,由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,8-12mmol/L;Glutamax 0.8-1.2×;A83-01,400-600nmol/L;EGF,35-60ng/mL;Noggin,80-120ng/mL;FGF10,8-12ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1-1.5mmol/L;nicotinamide,8-12mmol/L;PGE2,0.8-1.2μmol/L;Butylatedhydroxyanisole,4-10ng/ml;青霉素-链霉素-两性霉素B混合溶液0.8-1.2X;B27supplement;Prostaglandin E2,0.8-1.2umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%。
实施例2
一种口腔鳞癌类器官培养基,由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,10mmol/L;Glutamax 1×;A83-01,500nmol/L;EGF,50ng/mL;Noggin,100ng/mL;FGF10,10ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1.25mmol/L;nicotinamide,10mmol/L;PGE2,1μmol/L;Butylated hydroxyanisole,5ng/ml;青霉素-链霉素-两性霉素B混合溶液1X;B27 supplement;Prostaglandin E2,1umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%。
实施例3
本实施例提供一种口腔鳞癌类器官的培养方法,包括以下步骤:
S1、解离酶配置:5mg/ml的胶原酶I和10ug/ml的DNA酶I以无血清DMEM培养基为溶剂配置,4度的温度下保存;
S2、配置如实施例1或者实施例2任一实施例的培养基;
S3、临床操作获取样品:
通过取材或手术切除获得肿瘤标本,切取0.5cm×0.5cm×0.5cm组织,用无菌生理盐水冲洗3次,将组织放入含有20%双抗的预冷的DMEM培养基中浸泡1小时,继续解离,全程冰上保存运输;
S4、组织预处理:
将步骤S3中获得的样品置于无菌6孔板中,用一次性无菌手术刀片将样品组织块切碎;
S5、组织解离消化:
加入3ml步骤S1中配好的解离酶,将样品组织块移至15ml离心管中,在37度的温度下,以转速140rpm于恒温摇床中震荡消化10分钟;将已消化的上清转移到新的15ml离心管中,冰上保存;在剩余样品组织块沉淀内再加入3ml解离酶,继续在37度温度下以转速140rpm消化10分钟;重复上一操作,共计分3次消化,每次加入3ml解离酶;将消化好的组织和消化液的混合溶液分为2管,并在4度温度下以1400rpm离心5分钟,离心后去掉上清,并加入5ml DMEM培养基,重复离心-去除上清这一过程3次以充分去除解离酶,最后一次离心前将细胞与样品组织块混合;
S6、类器官培养:
在细胞沉淀中先加入50ul步骤S2中获得的培养基,将细胞吹匀,后加入基质胶600ul,用截断枪头轻柔吹打沉淀细胞团使其重悬;充分混匀后,将混有基质胶(Matrigel)的细胞团分别均匀点入6孔板中,每孔约4-5个液滴,彼此分散间隔;放入细胞培养箱中10分钟待基质胶(Matrigel)充分凝固,基质胶(Matrigel)凝固后,加入步骤S2中的培养基2ml,使得液面没过基质胶(Matrigel),镜下确认观察细胞形态,记录,放回培养箱继续培养。第二天观察肿瘤类器官,部分肿瘤应该已经形成球状结构,每3天换一次新鲜培养基,培养3天后,获得口腔鳞癌类器官,培养2周后,类器官直径约1-2mm,可将其冻存于液氮中。
肿瘤类器官在建立3天后,在显微镜下能观察到肿瘤类器官呈球状,典型口腔鳞癌类器官内为蜂巢状结构,在培养3周后,肿瘤类器官直径长到约1毫米,肉眼可观测到。
常规肿瘤类器官培养中在获得组织后即进入解离步骤,但并不适用于口腔鳞癌,这是由于口腔鳞癌组织位于口腔带菌环境,存在污染风险。本发明提供的方法加强了类器官培养前的组织预处理,因此我们在组织离体后加强了对组织的冲洗,并在含有20%双抗的DMEM培养基中浸泡1小时以降低后续污染风险。不仅如此,真菌污染也是口腔鳞癌有别于内脏肿瘤类器官培养的风险之一,因此我们在培养基中额外加入两性酶霉素B以减少后续真菌污染的风险。
本发明提供的方法改进了以往类器官的培养基配方,以提高口腔鳞癌类器官培养的成功率。口腔鳞癌细胞分化程度较高、干性较低,我们额外增加了抗氧化剂2,6-二叔丁基-4-甲基苯酚,提高表皮生长因子的浓度为50ng/ml促进上皮细胞生长,降低成纤维细胞生长因子的浓度为10ng/ml,增加Prostaglandin E2(前列腺素E2)1umol/L。
肿瘤类器官在建立3天后,在显微镜下能观察到肿瘤类器官呈球状,典型口腔鳞癌类器官内为蜂巢状结构,在培养3周后,肿瘤类器官直径长到约1毫米,肉眼可观测到。图1和图2即为成功培养的口腔鳞癌来源类器官的图像,其呈球状,典型蜂巢状结构,其中图1为本发明的颊鳞癌类器官在显微镜下的细胞形态学观察图,图2为本发明的舌鳞癌类器官在显微镜下的细胞形态学观察图。
采用本发明的口腔鳞癌类器官的培养基,使得口腔鳞癌类器官培养中污染的比例大大降低,提高了工作效率且适用范围广;采用本发明的口腔鳞癌类器官培养方法,加强了类器官培养前的组织预处理,使得口腔鳞癌类器官生长迅速且培养成功率大大提高。
以上所述,仅为本发明较佳的具体实施方式;但本发明的保护范围并不局限于此。任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其改进构思加以等同替换或改变,都应涵盖在本发明的保护范围内。
Claims (5)
1.一种口腔鳞癌类器官培养基,其特征在于:由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,8-12mmol/L;Glutamax 0.8-1.2×;A83-01,400-600nmol/L;EGF,35-60ng/mL;Noggin,80-120ng/mL;FGF10,8-12ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1-1.5mmol/L;nicotinamide,8-12mmol/L;PGE2,0.8-1.2μmol/L;Butylated hydroxyanisole,4-10ng/ml;青霉素-链霉素-两性霉素B混合溶液0.8-1.2X;B27 supplement;Prostaglandin E2,0.8-1.2umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%。
2.根据权利要求1所述的口腔鳞癌类器官培养基,其特征在于:由基础培养基DMEM/F12、R-spondin1条件培养基、Wnt3A条件培养基、无菌水和功能组分组成,所述功能组分在该类器官培养基中的终浓度组成为:HEPES,10mmol/L;Glutamax 1×;A83-01,500nmol/L;EGF,50ng/mL;Noggin,100ng/mL;FGF10,10ng/mL;Gastrin I,0.01μmol/L;N-acetylcysteine,1.25mmol/L;nicotinamide,10mmol/L;PGE2,1μmol/L;Butylatedhydroxyanisole,5ng/ml;青霉素-链霉素-两性霉素B混合溶液1X;B27supplement;Prostaglandin E2,1umol/L;且R-spondin1条件培养基占总体积的10%,Wnt3A条件培养基占总体积的50%。
3.一种口腔鳞癌类器官的培养方法,其特征在于,包括以下步骤:
S1、解离酶配置:5mg/ml的胶原酶I和10ug/ml的DNA酶I以无血清DMEM培养基为溶剂配置;
S2、配置如权利要求1或2任一项所述的培养基;
S3、临床操作获取样品:
通过取材或手术切除获得肿瘤标本,切取0.5cm×0.5cm×0.5cm组织,用无菌生理盐水冲洗3次,将组织放入含有20%双抗的预冷的DMEM培养基中浸泡1小时,继续解离,全程冰上保存运输;
S4、组织预处理:
将步骤S3中获得的样品置于无菌6孔板中,用一次性无菌手术刀片将样品组织块切碎;
S5、组织解离消化:
加入3ml步骤S1中配好的解离酶,将样品组织块移至15ml离心管中,在37度的温度下,以转速140rpm于恒温摇床中震荡消化10分钟;将已消化的上清转移到新的15ml离心管中,冰上保存;在剩余样品组织块沉淀内再加入3ml解离酶,继续在37度温度下以转速140rpm消化10分钟;重复上一操作,共计分3次消化,每次加入3ml解离酶;将消化好的组织和消化液的混合溶液分为2管,并在4度温度下以1400rpm离心5分钟,离心后去掉上清,并加入5mlDMEM培养基,重复离心-去除上清这一过程3次以充分去除解离酶,最后一次离心前将细胞与样品组织块混合;
S6、类器官培养:
在细胞沉淀中先加入50ul步骤S2中获得的培养基,将细胞吹匀,后加入基质胶600ul,用截断枪头轻柔吹打沉淀细胞团使其重悬;充分混匀后,将混有基质胶的细胞团分别均匀点入6孔板中,每孔约4-5个液滴,彼此分散间隔;放入细胞培养箱中10分钟待基质胶充分凝固,基质胶凝固后,加入步骤S2中的培养基2ml,使得液面没过基质胶,每3天换一次新鲜培养基,培养3天后,获得口腔鳞癌类器官。
4.根据权利要求3所述的口腔鳞癌类器官的培养方法,其特征在于:所述基质胶为Matrigel。
5.根据权利要求3所述的口腔鳞癌类器官的培养方法,其特征在于:S1中所述的解离酶在4度的温度下保存。
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