CN114480287A - 一种复发肺癌穿刺样本类器官培养基及其应用 - Google Patents
一种复发肺癌穿刺样本类器官培养基及其应用 Download PDFInfo
- Publication number
- CN114480287A CN114480287A CN202210207476.7A CN202210207476A CN114480287A CN 114480287 A CN114480287 A CN 114480287A CN 202210207476 A CN202210207476 A CN 202210207476A CN 114480287 A CN114480287 A CN 114480287A
- Authority
- CN
- China
- Prior art keywords
- lung cancer
- culture medium
- culture
- concentration
- organoid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0693—Tumour cells; Cancer cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0688—Cells from the lungs or the respiratory tract
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/05—Inorganic components
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/32—Amino acids
- C12N2500/33—Amino acids other than alpha-amino carboxylic acids, e.g. beta-amino acids, taurine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/38—Vitamins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/30—Organic components
- C12N2500/46—Amines, e.g. putrescine
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2500/00—Specific components of cell culture medium
- C12N2500/60—Buffer, e.g. pH regulation, osmotic pressure
- C12N2500/62—DMSO
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/15—Transforming growth factor beta (TGF-β)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/18—Liver cell growth factor (LCGF, Gly-His-Lys)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/30—Hormones
- C12N2501/345—Gastrin; Cholecystokinins [CCK]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/40—Regulators of development
- C12N2501/415—Wnt; Frizzeled
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/70—Enzymes
- C12N2501/72—Transferases (EC 2.)
- C12N2501/727—Kinases (EC 2.7.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/90—Polysaccharides
- C12N2501/91—Heparin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/998—Proteins not provided for elsewhere
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2503/00—Use of cells in diagnostics
- C12N2503/02—Drug screening
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
- G01N2500/10—Screening for compounds of potential therapeutic value involving cells
Abstract
本发明公开了一种复发性肺癌穿刺样本类器官的培养基及其培养方法。通过调整培养基各组分的含量,能够实现对复发性肺癌穿刺样本的类器官培养,大大提高了肺癌类器官培养成功率,缩短培养周期,为揭示复发性肺癌发病机理和筛选抗癌药物奠定了基础。
Description
技术领域
本发明涉及细胞培养领域,具体的涉及一种复发性肺癌类器官的培养基及其培养方法。
背景技术
肺癌居全球范围内癌症发病和死因之首,每年全球有180万新发肺癌病例,160万例死亡病例,因分期和地区不同,肺癌患者5年生存率在4%-17%。在中国的癌症死亡率中,肺癌仍然排名第一,2020年有72万国人死于肺癌,约占全球死亡人数一半。肺癌有临床症状发现时往往是晚期,因此患者已经丧失了手术根治的机会,一般选择靶向治疗,化疗或者放疗。肺癌是目前拥有最多靶向药的一个癌种,但众多的靶向药也给临床选药带来了纠结,面对同一个突变时,究竟哪种靶向药是最合适的,二代基因测序并不能给临床一个满意的回答。另外针对化疗药物的选择,目前只能根据指南和临床医生的经验选择,缺乏明确的个性化治疗的指导依据。
同时肺癌病人绝大多数在治疗过程中会出现靶向药物耐药,临床往往采取再次基因测序证实是否有新的突变继而采用新的靶向药物或化疗药物,在反复的耐药过程中,病人往往出现无药可用的局面,临床医生也束手无策,只能让病人直接试药,有没有一种可以代替病人试药的体外模型,这是临床肺癌治疗迫切需要解决的难题。
类器官是将具有干性潜能的细胞进行3D培养,从而形成相应器官的类似组织,类器官能最大程度地模拟体内组织结构及功能并能长期传代培养。目前已成功建立了肺、胃、小肠、大肠、肺脏、胰腺、前列腺等肿瘤类器官模型。肿瘤类器官的遗传背景与其来源肿瘤组织遗传背景高度一致:肿瘤类器官中的遗传突变与对应肿瘤活组织标本中的突变高度匹配,并与以往大规模的肿瘤突变分析结果相一致。肿瘤类器官也可以在基因表达谱、蛋白质谱、病理形态、肿瘤异质性等方面很好地模拟患者原位肿瘤。另外多项研究结果表明,类器官对药物的反应可以用来预测患者的临床疗效。例如一项关于囊性纤维化的研究表明,其类器官的药物反应情况与已发表的临床试验数据结果一致。2018年Science杂志一项最新研究表明,肿瘤类器官对药物的反应与转移性胃肠道肿瘤患者临床疗效高度一致,提示88%阳性预测率,100%的阴性预测率,为肿瘤类器官作为精准医疗的筛药工具提供了有力的支撑。
虽然原发肺癌类器官培养国内外已经有很多报道,但是复发肺癌穿刺样本因肿瘤细胞较少,原有培养体系不适合而培养成功率较低,因此复发肺癌穿刺样本类器官的成功培养和稳定药物敏感性测试可为复发肺癌的精准医疗提供可靠保证。
发明内容
本发明的目的在于,提供一种适宜复发性肺癌穿刺样本的类器官培养的培养基,进而获得复发肺癌类器官,以便能够研究复发肺癌发病机制和病程以及筛选相关抗癌药物。
为解决上述技术问题,本发明第一方面提供一种复发肺癌的类器官培养基,所述培养基成分包括如下成分:
DMEM/F12K/1640培养基(1:1:1-2),抗坏血酸(1-100mg/L),L-谷氨酸(1-100mg/L),亚硒酸钠(0.002-0.5mg/L),乙醇胺盐酸盐(2-200mg/L),偏钒酸铵(1×10-4-20×10- 4mg/L),四水合氯化锰(5×10-5-50×10-5mg/L)。
R-spondin1(50-1000ng/ml),Noggin(10-200ng/ml),N2:B27(0.1-2),谷氨酰胺补剂(1×),HEPES(1×),烟酰胺(1-100mM),NormocinTM(1×),N-乙酰半胱氨酸(0.1-10mM),Wnt3a(10-500ng/ml)。
优选的,所述培养基中还包括肝素,进一步优选的,所述肝素浓度为10-200IU/ml。
优选的,所述培养基中还包括A83-01,进一步优选的,所述A83-01浓度为0.1-50μM。
优选的,所述培养基中还包括EGF,进一步优选的,所述EGF浓度为1-200ng/ml。
优选的,所述培养基中还包括FGF2,进一步优选的,所述FGF2浓度为1-100ng/ml。
优选的,所述培养基中还包括Gastrin I,进一步优选的,所述Gastrin I浓度为25nM。
优选的,所述培养基中还包括HGF,进一步优选的,所述HGF浓度为5-500ng/ml。
优选的,所述培养基中还包括CHIR99021,进一步优选的,所述CHIR99021浓度为5-20μM。
优选的,所述培养基中还包括DMSO,进一步优选的,所述DMSO浓度为培养基体积比的0.01-2%。
本发明另一方面,提供了一种培养复发肺癌穿刺样本的培养方法,包括使用上述培养基以及优选的组分选择性的添加,能够对多种的复发肺癌穿刺样本实现类器官培养。
具体的,所述培养方法如下:
1.取复发肺癌穿刺样本,4℃保存于专用保存液中,24小时内低温转移至操作室;
2.样本低温破碎,酶解消化;
3.低温离心去除上清,冰PBS重悬;
4. 70g低温离心5-10min,去除上清,冰PBS重悬,此过程重复3-5次;
5. 200g低温离心10min,去除上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
6. 300g低温离心10min,去除上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入肺癌培养液,每3天更换一次相应培养液;
7.肿瘤类器官培养6-12天后,每孔添加1ml冰PBS,反复吹打,移入15ml离心管;
8. 300g低温离心10min,去上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除
9.离心结束后,冰Matrigel重悬,1:3接种扩增;
10.待肿瘤类器官稳定传代后,重复步骤9,一半类器官做药物敏感性实验,一半类器官用细胞冻存液冻存。
优选的,步骤2中,先将肺癌穿刺样本切成3mm×3mm大小,冰PBS反复清洗5次后再进行酶解;进一步优选的,穿刺样本块切成1mm3碎片,
优选的,酶解消化使用的是胶原酶进行消化;进一步优选的,酶解消化条件为:37℃,胶原酶消化30-60min;
优选的,步骤2中,酶解消化后终止酶解,具体操作如下:加入与酶解液等体积冰PBS,移液管反复吹打直至组织碎片消失,镜下观察为均匀的细胞团;进一步优选的,低温低速离心后重悬细胞团,显微镜计数细胞团数量,根据需要接种的孔数转移相应的细胞量至另一15ml离心管;
优选的,步骤2中,酶解消化使用的是TrypLE Express,进一步优选的,酶解消化的操作为:根据类器官数量加1-5ml不等的TrypLE Express,消化10-15min,加入AdvanceDMEM/F12培养液终止,300g离心10min;
优选的,步骤3中,300g离心10min,去除上清;
优选的,步骤6中,Matrigel重悬调整细胞浓度,1000-2000/50μl接种入24孔板,待Matrigel凝固后加入相应的培养基。
第三方面,本发明公开了一种利用上述培养基进行肺癌类器官模型的制备,通过制备得到的肺癌类器官模型,能够更直观的展示肺癌原发病理机制以及癌细胞的侵袭机制。并且利用制备获得的所述类器官模型用于筛选治疗复发性肺癌的药物。
本发明和现有技术相比,具有以下有益效果:
本发明公开的培养基可以进行组分的调整,能够对复发肺癌穿刺样本进行培养获得肺癌类器官,并且能够利用所述培养基制备肺癌类器官模型,大大缩短了培养时间和提高了类器官成功率,并且降低了对国外培养基的依赖,降低了培养基成本,为后续了解肺癌发病机制以及筛选药物奠定了基础。
附图说明
图1为不同培养基培养复发肺癌穿刺样本类器官比较图。
图2为两种改良培养基与CR培养基培养复发肺癌获得的类器官。
图3为本申请肺癌穿刺样本类器官培养基培养获得的类器官药物筛选实验。
具体实施方式
下面将结合示意图对本发明公开的培养基以及培养方法进行更详细的描述,其中表示了本发明的优选实施例,应该理解本领域技术人员可以修改在此描述的本发明,而仍然实现本发明的有利效果。因此,下列描述应当被理解为对于本领域技术人员的广泛知道,而并不作为对本发明的限制。
从市场购买各种培养基组分,规格和来源具体如下:
DMEM培养基,F12K培养基,1640培养基,抗坏血酸(A103539,Aladdin),L-谷氨酸(G6013,Sigma),亚硒酸钠(S5261,Sigma),乙醇胺盐酸盐(E120635,Aladdin),偏钒酸铵(A111829,Aladdin),四水合氯化锰(M112543,Aladdin),R-spondin1(SinoBiological),Noggin(SinoBiological),N2(Gibco),B27(Gibco),谷氨酰胺补剂(Gibco),HEPES(Gibco),烟酰胺(Sigma),NormocinTM(Invivogen),N-乙酰半胱氨酸(Sigma),Wnt3a(RD),A83-01(Tocris),Gastrin I(Tocris),EGF(Prerotech),HGF(Prerotech),FGF2(RD),肝素(Sigma),DMSO(Sigma),CHIR99021(Tocris)。
实施例1、改良培养基1
DMEM/F12K/1640培养基(1:1:1),抗坏血酸(10mg/L),L-谷氨酸(50mg/L),亚硒酸钠(0.05mg/L),乙醇胺盐酸盐(20mg/L),偏钒酸铵(5×10-4mg/L),四水合氯化锰(7×10-5mg/L)。
R-spondin1(500ng/ml),Noggin(20ng/ml),N2:B27(1:1),谷氨酰胺补剂(1×),HEPES(1×),烟酰胺(20mM),NormocinTM(1×),N-乙酰半胱氨酸(2mM),Wnt3a(500ng/ml),A83-01(10μM),Gastrin I(25nM),EGF(100ng/ml),DMSO(1%)。
实施例2、改良培养基2
DMEM/F12K/1640培养基(1:1:2),抗坏血酸(2mg/L),L-谷氨酸(100mg/L),亚硒酸钠(0.1mg/L),乙醇胺盐酸盐(50mg/L),偏钒酸铵(20×10-4mg/L),四水合氯化锰(15×10-5mg/L)。
R-spondin1(250ng/ml),Noggin(100ng/ml),N2:B27(1:2),谷氨酰胺补剂(1×),HEPES(1×),烟酰胺(5mM),NormocinTM(1×),N-乙酰半胱氨酸(2mM),Wnt3a(200ng/ml),FGF2(150ng/ml),肝素(50IU/ml),HGF(100ng/ml),CHIR99021(20μM)。
实施例3、本发明的2种培养基和原发肺癌培养基培养复发肺癌穿刺样本类器官培养比较和药物敏感筛选。
将上述实施例1和2所述的培养基和传统的原发肺癌(Cell Reports)培养基培养不同的复发肺癌穿刺样本,观察类器官成功率以及类器官形态。
具体操作方法如下所示:
1.取13例临床复发肺癌穿刺样本,4℃保存于专用保存液中,24小时内低温转移至操作室;
2.样本冰PBS清洗3-5次,每次5min;
3.样本低温切成1mm碎片入37℃预热胶原酶消化液中,消化时间30-60min.
4.样本消化完全后,添加与消化液同体积的冰PBS,反复吹打直至不见碎片组织;
5. 100g低温离心5-10min,去除上清,冰PBS重悬;
6. 70g低温离心5-10min,去除上清,冰PBS重悬,此过程重复3-5次;
7. 200g低温离心10min,去除上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
8. 300g低温离心10min,去除上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入肺癌培养液,每3天更换一次相应培养液;
9.肿瘤类器官培养7-12天后,每孔添加1ml冰PBS,反复吹打,移入15ml离心管;
10. 300g低温离心10min,去上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除
11.离心结束后,冰Matrigel重悬,1:3接种扩增;
12.待肿瘤类器官稳定传代后,重复步骤10,一半类器官做药物敏感性实验,一半类器官用细胞冻存液冻存。
13.复发肺癌类器官扩增后接种于96孔板,50-100个/孔,200μl培养基培养3天;
14.类器官培养3天后更换含化疗药物,靶向药物和联合药物培养基;
15.药物作用6-12天后,CTG方法和拍照展示肺癌类器官被药物抑制情况。
实施例3中所使用的原发肺癌培养基如下所示:
Advance DMEM/F12培养基,10%R-spondin1 conditioned medium,10%Nogginconditioned medium,B27(1×),烟酰胺(10mM),N-乙酰半胱氨酸(1.25mM),A83-01(500nM),SB202190(1μM),FGF7(25ng/ml),FGF10(100ng/ml),Y27632(5μM)。
结果
1、不同培养基培养成功率
13例非小细胞肺癌穿刺样本经上述两种培养基和原发肺癌培养基(CR培养基)培养9-14天后,培养基1和2成功培养9例(P1-3,P5,P7-9,P11,P13)和10例(P1-2,P4-5,P7-9,P11-13)复发肺癌类器官,相同培养时间段,CR培养基成功培养3例(P5,P8,P11)复发肺癌类器官(图1)。
经培养基1和2培养后,穿刺样本培养6-12天即可首次传代,在CR培养基中,穿刺样本需要14-21天才可以进行首次传代。培养基1和2的培养成功率分别为69.2%(9/13)和76.9%(10/13),CR培养基成功率则为23.1%(3/13)。这与已报道的原发肺癌的培养基不太适合复发肺癌类器官的培养类似,进一步证明了本发明培养基针对复发肺癌有较高的类器官培养成功率和相对较短的类器官培养时间(图2)。
2、复发肺癌类器官药物筛选
原发肺癌培养基获得的类器官对多种药物不敏感,而本申请的两种培养基培养获得的类器官对各种药物的敏感性与肺癌组织类似,非常适合进行体外筛药,可以为肺癌复发患者提供用药参考(图3)。
显然,本领域的技术人员可以对本发明进行各种改动和变型而不脱离本发明的精神和范围。这样,倘若本发明的这些修改和变型属于本发明权利要求及其等同技术的范围之内,则本发明也意图包含这些改动和变型在内。
Claims (10)
1.一种复发肺癌细胞类器官培养基,其特征在于,所述培养基包含如下组分:DMEM/F12K/1640培养基1:1:1-2,抗坏血酸1-100mg/L,L-谷氨酸1-100mg/L,亚硒酸钠0.002-0.5mg/L,乙醇胺盐酸盐2-200mg/L,偏钒酸铵1×10-4-20×10-4mg/L,四水合氯化锰5×10-5-50×10-5mg/L,R-spondin1 50-1000ng/ml,Noggin10-200ng/ml,N2:B27(0.1×-2×),谷氨酰胺补剂(1×),HEPES(1×),烟酰胺1-100mM,NormocinTM(1×),N-乙酰半胱氨酸0.1-10mM,Wnt3a10-500ng/ml。
2.根据权利要求1所述的培养基,其特征在于,所述培养基还包括以下物质的一种或多种:A83-01、EGF,Gastrin I。
3.根据权利要求2所述的培养基,其特征在于,所述A83-01浓度为0.1-50μM;所述EGF浓度为10-200ng/ml;所述Gastrin I浓度为5-25nM。
4.根据前述任一权利要求所述的培养基,其特征在于所述培养基中还包括下述物质的一种或多种的组合:肝素,FGF2,HGF,优选的,所述肝素的浓度为1-200IU/ml,所述FGF2浓度为1-100ng/ml;所述HGF浓度为5-500ng/ml。
5.根据前述任一权利要求所述的培养基,其特征在于所述培养基中还包括CHIR99021,优选的,所述CHIR99021浓度为1-20μM,和/或,所述培养基中还包括DMSO,优选的,所述二甲亚砜浓度为培养基体积比的0.01-2%。
6.一种利用上述任一权利要求所述培养基培养复发肺癌细胞类器官的方法,其特征在于,所述方法包括如下步骤:
1).取复发肺癌穿刺样本,4℃保存于专用保存液中,24小时内低温转移至操作室;
2).样本低温破碎,酶解消化;
3).低温离心去除上清,冰PBS重悬,此过程重复3-5次;
4).低温离心去除上清,依据获得细胞情况,1-5ml冰PBS重悬后显微镜下计数细胞;
5).低温离心去除上清,依据细胞计数加入适量冰Matrigel重悬接种于24孔板,加入肺癌培养液,每3天更换一次相应培养液;
6).肿瘤类器官培养6-12天后,每孔添加1ml冰PBS,反复吹打,移入15ml离心管;
7).低温离心去除上清,冰PBS重悬,此步骤重复3-5次,直至Matrigel全部清除;
8).离心结束后,冰Matrigel重悬,1:3接种扩增;
9).待肿瘤类器官稳定传代后,重复步骤8,一半类器官做药物敏感性实验,一半类器官用细胞冻存液冻存。
7.根据权利要求6所述的方法,其特征在于,步骤2)消化使用的是胶原蛋白酶。
8.根据权利要求6-7任一项所述的方法,低温离心去除上清的操作中,温度为4℃,离心转速为70-300g。
9.权利要求1-5任一所述培养基或权利要求6-8任一项所述的方法在制备复发性肺癌类器官模型中的应用。
10.由前述任一权利要求获得的类器官模型在筛选靶向复发性肺癌药物中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210207476.7A CN114480287A (zh) | 2022-03-03 | 2022-03-03 | 一种复发肺癌穿刺样本类器官培养基及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202210207476.7A CN114480287A (zh) | 2022-03-03 | 2022-03-03 | 一种复发肺癌穿刺样本类器官培养基及其应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN114480287A true CN114480287A (zh) | 2022-05-13 |
Family
ID=81485725
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202210207476.7A Pending CN114480287A (zh) | 2022-03-03 | 2022-03-03 | 一种复发肺癌穿刺样本类器官培养基及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN114480287A (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115322948A (zh) * | 2022-07-20 | 2022-11-11 | 创芯国际生物科技(广州)有限公司 | 一种微量原代组织类器官培养前的扩增培养方法 |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106967672A (zh) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | 一种肺及肺癌组织培养方法以及用其构建肺癌小鼠动物模型方法 |
CN112080472A (zh) * | 2020-09-01 | 2020-12-15 | 南通大学 | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 |
CN113122500A (zh) * | 2021-03-18 | 2021-07-16 | 上海诺典生物科技有限公司 | 一种转移性肠癌类器官的培养和应用 |
CN113801838A (zh) * | 2021-08-03 | 2021-12-17 | 上海诺典生物科技有限公司 | 原发性肝细胞癌类器官的持续培养方法和培养基 |
CN113817683A (zh) * | 2021-09-30 | 2021-12-21 | 南京鼓楼医院 | 一种肺癌类器官的培养基及其应用 |
-
2022
- 2022-03-03 CN CN202210207476.7A patent/CN114480287A/zh active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106967672A (zh) * | 2017-03-24 | 2017-07-21 | 四川大学华西医院 | 一种肺及肺癌组织培养方法以及用其构建肺癌小鼠动物模型方法 |
CN112080472A (zh) * | 2020-09-01 | 2020-12-15 | 南通大学 | 一种培养专用于生物医药功能研究的人肺癌类器官3d模型的方法 |
CN113122500A (zh) * | 2021-03-18 | 2021-07-16 | 上海诺典生物科技有限公司 | 一种转移性肠癌类器官的培养和应用 |
CN113801838A (zh) * | 2021-08-03 | 2021-12-17 | 上海诺典生物科技有限公司 | 原发性肝细胞癌类器官的持续培养方法和培养基 |
CN113817683A (zh) * | 2021-09-30 | 2021-12-21 | 南京鼓楼医院 | 一种肺癌类器官的培养基及其应用 |
Non-Patent Citations (1)
Title |
---|
RUOBING ZHANG,等: "Development and Application of Patient-Derived Cancer Organoids in Clinical Management of Gastrointestinal Cancer: A State-of-the-Art Review", FRONTIERS IN ONCOLOGY, vol. 11, pages 1 - 14 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115322948A (zh) * | 2022-07-20 | 2022-11-11 | 创芯国际生物科技(广州)有限公司 | 一种微量原代组织类器官培养前的扩增培养方法 |
CN115322948B (zh) * | 2022-07-20 | 2023-08-29 | 创芯国际生物科技(广州)有限公司 | 一种微量原代组织类器官培养前的扩增培养方法 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Ledur et al. | Culture conditions defining glioblastoma cells behavior: what is the impact for novel discoveries? | |
US11883508B2 (en) | Method for culturing 3-dimensional lung cancer organoid and method for preparing patient-derived xenograft animal model using same | |
CN112094813B (zh) | 培养失分化或未分化甲状腺癌类器官的方法及甲状腺癌培养基 | |
CN112210537B (zh) | 肝癌类器官及其培养方法、培养用培养基和用途 | |
CN110656086A (zh) | 癌类器官的体外培养方法 | |
CN113122500B (zh) | 一种转移性肠癌类器官的培养和应用 | |
CN113278588A (zh) | 一种口腔鳞癌类器官培养基以及培养方法 | |
CN110564686B (zh) | 扩增造血干细胞的组合物、扩增方法、药物组合物和用途 | |
CN111065732A (zh) | 肿瘤类器官模型 | |
CN114480287A (zh) | 一种复发肺癌穿刺样本类器官培养基及其应用 | |
CN112592896A (zh) | 一种肺腺癌类器官的培养液及其培养方法 | |
CN113801838A (zh) | 原发性肝细胞癌类器官的持续培养方法和培养基 | |
CN110564687B (zh) | 用于扩增造血干细胞的组合物、培养基、方法和试剂盒 | |
CN114574443A (zh) | 一种培养复发肾细胞癌类器官的培养基及其应用 | |
Vinel et al. | Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma | |
CN115916240A (zh) | 一种神经类细胞的扩增培养基及培养方法 | |
CN113308437A (zh) | 用于快速培养骨转移癌类器官的培养基、方法以及试剂盒 | |
Regent et al. | Nicotinamide promotes formation of retinal organoids from human pluripotent stem cells via enhanced neural cell fate commitment | |
Wen et al. | Applications of organoid technology to brain tumors | |
CN113943755B (zh) | 构建原位原发食管癌动物模型的方法 | |
CN114561340A (zh) | 一种培养复发乳腺癌类器官的培养基及其应用 | |
CN114958753B (zh) | 一种舌癌类器官的培养基、培养方法及鉴定方法 | |
CN110669732A (zh) | 组合物在将造血祖细胞重编程为造血干细胞中的用途 | |
CN116004722A (zh) | 肝母细胞瘤类器官及其应用 | |
CN114891749A (zh) | 一种胰腺癌类器官的培养基及胰腺癌类器官的培养方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |