CN114774359A - A kind of cervical squamous cell carcinoma organoid culture medium and construction method thereof - Google Patents
A kind of cervical squamous cell carcinoma organoid culture medium and construction method thereof Download PDFInfo
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- CN114774359A CN114774359A CN202210298185.3A CN202210298185A CN114774359A CN 114774359 A CN114774359 A CN 114774359A CN 202210298185 A CN202210298185 A CN 202210298185A CN 114774359 A CN114774359 A CN 114774359A
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Abstract
本发明涉及类器官培养技术领域，具体公开了一种宫颈鳞癌类器官培养基及其构建方法。宫颈鳞癌类器官培养基，包括以下组分：基础培养基、HEPES缓冲对、青霉素链霉素溶液、营养添加剂和特异性因子；所述特异性因子包括烟酰胺、N‑乙酰半胱氨酸、Y‑27632、Noggin因子、A83‑01、EGF、FGF10、Forskolin和氢化可的松。本发明针对宫颈鳞癌开发出宫颈鳞癌类器官培养基，提高了宫颈癌类器官的培养成功率及长期维持率，所培养出宫颈鳞癌类器官可维持原肿瘤组织的组织学形态及遗传学特征；开发的宫颈鳞癌类器官培养基有利于为宫颈癌研究提供新的思路。
The invention relates to the technical field of organoid culture, and specifically discloses a cervical squamous cell carcinoma organoid culture medium and a construction method thereof. Cervical squamous cell carcinoma organoid culture medium, comprising the following components: basal medium, HEPES buffer pair, penicillin-streptomycin solution, nutritional supplements and specific factors; the specific factors include nicotinamide, N-acetylcysteine , Y‑27632, Noggin Factor, A83‑01, EGF, FGF10, Forskolin and Hydrocortisone. The invention develops a cervical squamous cell carcinoma organoid culture medium for cervical squamous cell carcinoma, improves the culture success rate and long-term maintenance rate of the cervical carcinoma organoid, and the cultured cervical squamous cell carcinoma organoid can maintain the histological morphology and genetics of the original tumor tissue. The developed cervical squamous cell carcinoma organoid medium is beneficial to provide new ideas for cervical cancer research.
Description
技术领域technical field
本发明涉及类器官培养技术领域，尤其是涉及一种宫颈鳞癌类器官培养基及其构建方法。The invention relates to the technical field of organoid culture, in particular to a cervical squamous cell carcinoma organoid culture medium and a construction method thereof.
背景技术Background technique
宫颈癌是最常见的妇科肿瘤之一。2021年全球肿瘤统计分析提示，在所有的妇科肿瘤中，宫颈癌的发生率及病死率均占第四位。宫颈癌最常见的病理类型是鳞状细胞癌和腺癌，分别占所有病例的70％和25％。鳞癌起源于宫颈鳞状上皮，腺癌起源于柱状上皮。迄今为止，宫颈癌研究依赖于数量有限的细胞系、异种移植和转基因的小鼠模型。广泛使用的细胞系如hela，siha经过长期的体外培养选择，已经部分失去对临床病人特征的概括，对临床前的研究价值有限。而异种移植及转基因模型，因价格高昂及培养周期较长等原因无法在研究中实现普遍应用。因此，我们建立一种经济简便有病人代表性的模型来进行宫颈癌的研究。Cervical cancer is one of the most common gynecological tumors. The 2021 global tumor statistical analysis suggests that among all gynecological tumors, cervical cancer ranks fourth in both incidence and mortality. The most common pathological types of cervical cancer are squamous cell carcinoma and adenocarcinoma, which account for 70% and 25% of all cases, respectively. Squamous cell carcinoma originates from cervical squamous epithelium, while adenocarcinoma originates from columnar epithelium. To date, cervical cancer research has relied on a limited number of cell lines, xenografts, and transgenic mouse models. Widely used cell lines such as hela and siha have partially lost their generalization of clinical patient characteristics after long-term in vitro culture selection, and have limited value for preclinical research. However, xenotransplantation and transgenic models cannot be widely used in research due to high prices and long culture periods. Therefore, we established an economical and simple patient-representative model for cervical cancer research.
癌症的研究依赖于稳定的细胞供应-可在实验室中培养的正常细胞和癌性细胞。肿瘤类器官是近年来发展的体外3D培养模型，该模型是在体外利用基质胶或水凝胶等材料模拟细胞外基质，给细胞提供三维支架，外源性添加小分子、生长因子等物质形成干细胞生态环境，诱导来源于组织或干细胞自我更新分化形成三维细胞结构，类器官模型与来源组织密切相关，再现源组织的细胞结构和遗传特质。构建患者来源的肿瘤类器官可很好地在体外复现患者肿瘤的遗传异质性和生物学特性，结合下一代测序在类器官上进行高通量药物筛选可指导患者个体化治疗方案。在肿瘤机制研究中，类器官模型可作为细胞实验和体内实验之间的补充，患者来源的类器官也可成为动物模型和人体临床试验间的桥梁。Cancer research relies on a steady supply of cells—normal and cancerous cells that can be grown in the lab. Tumor organoids are an in vitro 3D culture model developed in recent years. This model uses materials such as matrigel or hydrogel to simulate extracellular matrix in vitro, provides a three-dimensional scaffold for cells, and exogenously adds small molecules, growth factors and other substances to form. The stem cell ecological environment induces the self-renewal and differentiation of the derived tissue or stem cells to form a three-dimensional cell structure. The organoid model is closely related to the source tissue and reproduces the cell structure and genetic characteristics of the source tissue. The construction of patient-derived tumor organoids can well reproduce the genetic heterogeneity and biological characteristics of patient tumors in vitro, and high-throughput drug screening on organoids combined with next-generation sequencing can guide individualized treatment plans for patients. In the study of tumor mechanisms, organoid models can be used as a complement between cell experiments and in vivo experiments, and patient-derived organoids can also serve as a bridge between animal models and human clinical trials.
然而现有技术中现存培养宫颈鳞癌类器官的培养成功率较低，仅有50％的成功率，因此，有必要提出一种培养成功率较高的宫颈鳞癌类器官培养基。However, in the prior art, the success rate of culturing cervical squamous cell carcinoma organoids is low, only 50%. Therefore, it is necessary to propose a cervical squamous cell carcinoma organoid culture medium with a high culture success rate.
发明内容SUMMARY OF THE INVENTION
本发明的目的在于克服上述现有技术的不足之处而提供一种宫颈鳞癌类器官培养基及其构建方法。针对宫颈鳞癌开发出本发明的宫颈鳞癌类器官培养基可以提高培养宫颈癌类器官的成功率及长期维持率，有利于为宫颈癌研究提供新的思路。The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a cervical squamous cell carcinoma organoid culture medium and a construction method thereof. The development of the cervical squamous cell carcinoma organoid culture medium of the present invention for cervical squamous cell carcinoma can improve the success rate and long-term maintenance rate of cervical carcinoma organoid culture, and is beneficial to provide new ideas for cervical cancer research.
为实现上述目的，本发明采取的技术方案为：To achieve the above object, the technical scheme adopted in the present invention is:
本发明提供了一种宫颈鳞癌类器官培养基，包括以下组分：The invention provides a cervical squamous cell carcinoma organoid culture medium, comprising the following components:
基础培养基、HEPES缓冲对、青霉素链霉素溶液、营养添加剂和特异性因子；Basal medium, HEPES buffer pair, penicillin-streptomycin solution, nutritional supplements and specificity factors;
所述特异性因子为烟酰胺、N-乙酰半胱氨酸、Y-27632、Noggin因子、A83-01、EGF、FGF10、Forskolin和氢化可的松。The specific factors were nicotinamide, N-acetylcysteine, Y-27632, Noggin factor, A83-01, EGF, FGF10, Forskolin and hydrocortisone.
本发明发明人经过大量研究及试验发现，开发了针对宫颈鳞癌类器官的培养基，提高了宫颈鳞癌类器官的培养成功率及长期维持率，使得宫颈鳞癌类器官培养成功率从50％提高至73％，且较好的维持了宫颈鳞癌类器官的生物活性，其类器官的生长活性和形态不受影响，所培养出宫颈鳞癌类器官可维持原肿瘤组织的组织学形态及遗传学特征。After extensive research and experiments, the inventors of the present invention found that they developed a culture medium for cervical squamous cell carcinoma organoids, which improved the success rate and long-term maintenance rate of cervical squamous cell carcinoma organoids, and made the success rate of cervical squamous cell carcinoma organoids cultured from 50% to 50%. % increased to 73%, and the biological activity of cervical squamous cell carcinoma organoids was well maintained, the growth activity and morphology of the organoids were not affected, and the cultured cervical squamous cell carcinoma organoids could maintain the histological morphology of the original tumor tissue and genetic characteristics.
其中，培养基添加HEPES缓冲对可以促进类器官的生物活性，并且还能防止培养基pH波动对细胞生长不利的影响。加入上述特异性因子可以协同提高宫颈鳞癌类器官的培养成功率。Among them, the addition of HEPES buffer to the medium can promote the biological activity of organoids, and can also prevent the adverse effects of medium pH fluctuations on cell growth. Adding the above specific factors can synergistically improve the success rate of cervical squamous cell carcinoma organoid culture.
作为本发明所述宫颈鳞癌类器官培养基的优选实施方式，宫颈鳞癌类器官培养基包括以下组分：As a preferred embodiment of the cervical squamous cell carcinoma organoid medium of the present invention, the cervical squamous cell carcinoma organoid medium comprises the following components:
基础培养基、HEPES缓冲对10～12mM、青霉素链霉素溶液1％～2％、营养添加剂1％～3％、烟酰胺10～12mmol/L、N-乙酰半胱氨酸1～1.5mmol/L、Y-27632 2～10μM、Noggin因子80～120ng/ml、A83-01 450～550nmol/L、EGF5～10ng/ml、FGF10 80～120ng/ml、Forskolin 8～12μM和氢化可的松400～600ng/ml。Basic medium,
作为本发明所述宫颈鳞癌类器官培养基的优选实施方式，宫颈鳞癌类器官培养基包括以下组分：As a preferred embodiment of the cervical squamous cell carcinoma organoid medium of the present invention, the cervical squamous cell carcinoma organoid medium comprises the following components:
基础培养基、HEPES缓冲对10mM、青霉素链霉素溶液1％、营养添加剂3％、烟酰胺10mmol/L、N-乙酰半胱氨酸1.25mmol/L、Y-27632 10μM、Noggin因子100ng/ml、A83-01500nmol/L、EGF 5ng/ml、FGF10 100ng/ml、Forskolin10μM和氢化可的松500ng/ml。Basal medium, HEPES buffer to 10mM, penicillin-
作为本发明所述宫颈鳞癌类器官培养基的优选实施方式，所述基础培养基为advanced DMEM/F12；营养添加剂为Glutamax、B27和N2中的至少一种。更优选地，营养添加剂为Glutamax、B27和N2的复配。营养剂的添加有助于宫颈鳞癌类器官的培养，使得宫颈鳞癌类器官生长形态更佳。As a preferred embodiment of the cervical squamous cell carcinoma organoid medium of the present invention, the basal medium is advanced DMEM/F12; the nutrient additive is at least one of Glutamax, B27 and N2. More preferably, the nutritional additive is a combination of Glutamax, B27 and N2. The addition of nutrients is helpful for the culture of cervical squamous cell carcinoma organoids, which makes the growth morphology of cervical squamous cell carcinoma organoids better.
作为本发明所述宫颈鳞癌类器官培养基的优选实施方式，所述营养添加剂和特异性因子的体积比为10：(0.5～1.5)。更优选地，营养添加剂和特异性因子的体积比为10：0.8。As a preferred embodiment of the cervical squamous cell carcinoma organoid culture medium of the present invention, the volume ratio of the nutritional additive and the specific factor is 10:(0.5-1.5). More preferably, the volume ratio of nutritional additive and specificity factor is 10:0.8.
本发明的营养添加剂和特异性因子以特定体积比复配时，可以提高宫颈鳞癌类器官的培养效果。When the nutritional additive and the specific factor of the present invention are compounded in a specific volume ratio, the culture effect of the cervical squamous cell carcinoma organoid can be improved.
第二目的，本发明提供了一种宫颈鳞癌类器官模型的构建方法，包括以下步骤：The second object, the present invention provides a method for constructing a cervical squamous cell carcinoma organoid model, comprising the following steps:
1)获得宫颈癌组织，机械及酶解消化宫颈癌组织，过滤离心，保留细胞沉淀，去除红细胞，得细胞团块；1) Obtain cervical cancer tissue, mechanically and enzymatically digest cervical cancer tissue, filter and centrifuge, retain cell sediment, remove red blood cells, and obtain cell mass;
2)将细胞团块重悬于基质胶中，接种，固定；然后置于上述宫颈鳞癌类器官培养基中培养，每2-3天观察换液，每7-21天进行传代。2) The cell aggregates were resuspended in Matrigel, inoculated and fixed; then placed in the above-mentioned cervical squamous cell carcinoma organoid culture medium, and the medium was observed every 2-3 days, and passaged every 7-21 days.
作为本发明所述宫颈鳞癌类器官模型的构建方法的优选实施方式，步骤1)中，采用浓度为0.5mg/ml的胶原酶II消化宫颈癌组织。As a preferred embodiment of the method for constructing the cervical squamous cell carcinoma organoid model of the present invention, in step 1), the cervical cancer tissue is digested with collagenase II at a concentration of 0.5 mg/ml.
作为本发明所述宫颈鳞癌类器官模型的构建方法的优选实施方式，步骤2)中，将细胞团块以1万细胞/10μl的密度重悬于基质胶中。As a preferred embodiment of the method for constructing the cervical squamous cell carcinoma organoid model of the present invention, in step 2), the cell mass is resuspended in Matrigel at a density of 10,000 cells/10 μl.
第三目的，本发明提供了上述培养基在培养宫颈鳞癌类器官中的用途。The third object, the present invention provides the use of the above-mentioned culture medium in culturing cervical squamous cell carcinoma organoids.
与现有技术相比，本发明包括以下有益效果：Compared with the prior art, the present invention includes the following beneficial effects:
本发明针对宫颈鳞癌开发出宫颈鳞癌类器官培养基，提高了宫颈癌类器官的培养成功率及传代后维持率，所培养出宫颈鳞癌类器官可维持原肿瘤组织的组织学形态及遗传学特征；开发的宫颈鳞癌类器官培养基有利于为宫颈癌研究提供新的思路。The invention develops a cervical squamous cell carcinoma organoid culture medium for cervical squamous cell carcinoma, improves the culture success rate and the post-passage maintenance rate of the cervical carcinoma organoid, and the cultured cervical squamous cell carcinoma organoid can maintain the histological morphology and the original tumor tissue. Genetic characteristics; the developed cervical squamous cell carcinoma organoid medium is beneficial to provide new ideas for cervical cancer research.
附图说明Description of drawings
图1为经过实施例1的宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官的光学显微镜图；Fig. 1 is the optical microscope picture of the cervical squamous cell carcinoma organoid obtained after the cervical squamous cell carcinoma organoid culture medium of
图2为经过实施例2的宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官的光学显微镜图；Fig. 2 is the optical microscope picture of the cervical squamous cell carcinoma organoid obtained after the cervical squamous cell carcinoma organoid culture medium of
图3为实施例2与对比例1-12的宫颈鳞癌类器官培养基对宫颈鳞癌类器官尺寸影响的对比统计图；Fig. 3 is a comparative statistical diagram of the effect of the cervical squamous cell carcinoma organoid culture medium of Example 2 and Comparative Examples 1-12 on the size of cervical squamous cell carcinoma organoid;
图4为经过对比例13的宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官的光学显微镜图。4 is an optical microscope image of cervical squamous cell carcinoma organoids obtained after cultured in the cervical squamous cell carcinoma organoid medium of Comparative Example 13.
具体实施方式Detailed ways
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.
在以下实施例和对比例中，所使用的实验方法如无特殊说明，均为常规方法，所用的材料、试剂等，如无特殊说明，均可从商业途径得到。In the following examples and comparative examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources.
以下实施例和对比例中，营养添加剂包括Glutamax、B27和N2；特异性因子为烟酰胺、N-乙酰半胱氨酸、Y-27632、Noggin因子、A83-01、EGF、FGF10、Forskolin和氢化可的松。In the following examples and comparative examples, nutritional supplements include Glutamax, B27 and N2; specific factors are nicotinamide, N-acetylcysteine, Y-27632, Noggin factor, A83-01, EGF, FGF10, Forskolin and hydrogenated Cortisone.
在以下实施例和对比例中，青霉素链霉素溶液(100×青霉素链霉素混合液)、Hepes缓冲对、Glutamax、B27、N2均购自英潍捷基(上海)贸易有限公司；烟酰胺(nicotinamide)、N-乙酰半胱氨酸(N-acetylcysteine)、Forskolin、A83-01购自Sigma-Aldrich公司；Noggin因子、EGF、FGF10、R-spondin 1、CHIR99021购自PeproTech公司；Y-27632、氢化可的松(Hydrocortisone)购自stem cell公司。In the following examples and comparative examples, penicillin-streptomycin solution (100× penicillin-streptomycin mixture), Hepes buffer pair, Glutamax, B27, N2 were purchased from Yingweijieji (Shanghai) Trading Co., Ltd.; nicotinamide (nicotinamide), N-acetylcysteine (N-acetylcysteine), Forskolin, A83-01 were purchased from Sigma-Aldrich; Noggin factor, EGF, FGF10, R-
实施例1、宫颈鳞癌类器官培养基
本实施例提供了一种宫颈鳞癌类器官培养基，包括基础培养基Advanced DMEM/F12、12mM Hepes缓冲对、2％青霉素链霉素溶液、1％Glutamax、1％B27、1％N2、12mmol/L烟酰胺(nicotinamide)、1.25mmol/L N-乙酰半胱氨酸(N-acetylcysteine)、2μM Y-27632、100ng/ml Noggin因子、500nmol/L A83-01、10ng/ml EGF、100ng/ml FGF10、10μMForskolin和500ng/ml氢化可的松。特异性因子各组分的浓度以其在宫颈鳞癌类器官培养基中的浓度为准。This example provides a cervical squamous cell carcinoma organoid culture medium, including basal medium Advanced DMEM/F12, 12mM Hepes buffer pair, 2% penicillin-streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 12 mmol /L nicotinamide, 1.25mmol/L N-acetylcysteine, 2μM Y-27632, 100ng/ml Noggin factor, 500nmol/L A83-01, 10ng/ml EGF, 100ng/ ml FGF10, 10 μM Forskolin and 500 ng/ml hydrocortisone. The concentration of each component of specific factor is based on its concentration in cervical squamous cell carcinoma organoid culture medium.
其中，营养添加剂和特异性因子的体积比为10：0.8。Among them, the volume ratio of nutritional additives and specificity factors is 10:0.8.
实施例2、宫颈鳞癌类器官培养基
本实施例提供了一种宫颈鳞癌类器官培养基，包括基础培养基Advanced DMEM/F12、10mM Hepes缓冲对、1％青霉素链霉素溶液、1％Glutamax、1％B27、1％N2、10mmol/L烟酰胺(nicotinamide)、1.25mmol/L N-乙酰半胱氨酸(N-acetylcysteine)、10μM Y-27632、100ng/ml Noggin因子、500nmol/L A83-01、5ng/ml EGF、100ng/ml FGF10、10μM Forskolin和500ng/ml氢化可的松。特异性因子各组分的浓度以其在宫颈鳞癌类器官培养基中的浓度为准。This example provides a cervical squamous cell carcinoma organoid medium, including basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin-streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 10 mmol /L nicotinamide, 1.25mmol/L N-acetylcysteine, 10μM Y-27632, 100ng/ml Noggin factor, 500nmol/L A83-01, 5ng/ml EGF, 100ng/ ml FGF10, 10 μM Forskolin and 500 ng/ml hydrocortisone. The concentration of each component of specific factor is based on its concentration in cervical squamous cell carcinoma organoid culture medium.
其中，营养添加剂和特异性因子的体积比为10：0.5。Among them, the volume ratio of nutritional additives and specificity factors is 10:0.5.
实施例3、宫颈鳞癌类器官培养基
本实施例提供了一种宫颈鳞癌类器官培养基，包括基础培养基Advanced DMEM/F12、10mM Hepes缓冲对、1％青霉素链霉素溶液、0.3％Glutamax、0.3％B27、0.4％N2、10mmol/L烟酰胺(nicotinamide)、1.0mmol/L N-乙酰半胱氨酸(N-acetylcysteine)、5μMY-27632、80ng/ml Noggin因子、450nmol/L A83-01、5ng/ml EGF、80ng/ml FGF10、8μMForskolin和400ng/ml氢化可的松。特异性因子各组分的浓度以其在宫颈鳞癌类器官培养基中的浓度为准。This example provides a cervical squamous cell carcinoma organoid culture medium, including basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin-streptomycin solution, 0.3% Glutamax, 0.3% B27, 0.4% N2, 10 mmol /L nicotinamide, 1.0mmol/L N-acetylcysteine, 5μMY-27632, 80ng/ml Noggin factor, 450nmol/L A83-01, 5ng/ml EGF, 80ng/ml FGF10, 8 μM Forskolin and 400 ng/ml hydrocortisone. The concentration of each component of specific factor is based on the concentration in cervical squamous cell carcinoma organoid culture medium.
其中，营养添加剂和特异性因子的体积比为10：1.5。Among them, the volume ratio of nutritional additives and specificity factors is 10:1.5.
实施例4、宫颈鳞癌类器官培养基
本实施例提供了一种宫颈鳞癌类器官培养基，包括基础培养基Advanced DMEM/F12、10mM Hepes缓冲对、1％青霉素链霉素溶液、1％Glutamax、1％B27、1％N2、10mmol/L烟酰胺(nicotinamide)、1.5mmol/L N-乙酰半胱氨酸(N-acetylcysteine)、10μM Y-27632、120ng/ml Noggin因子、550nmol/L A83-01、10ng/ml EGF、120ng/ml FGF10、12μMForskolin和600ng/ml氢化可的松。特异性因子各组分的浓度以其在宫颈鳞癌类器官培养基中的浓度为准。This example provides a cervical squamous cell carcinoma organoid medium, including basal medium Advanced DMEM/F12, 10mM Hepes buffer pair, 1% penicillin-streptomycin solution, 1% Glutamax, 1% B27, 1% N2, 10 mmol /L nicotinamide, 1.5mmol/L N-acetylcysteine, 10μM Y-27632, 120ng/ml Noggin factor, 550nmol/L A83-01, 10ng/ml EGF, 120ng/ ml FGF10, 12 μM Forskolin and 600 ng/ml hydrocortisone. The concentration of each component of specific factor is based on its concentration in cervical squamous cell carcinoma organoid culture medium.
其中，营养添加剂和特异性因子的体积比为10：0.8。Among them, the volume ratio of nutritional additives and specificity factors is 10:0.8.
实施例5、一种宫颈鳞癌类器官模型的构建方法
一种宫颈鳞癌类器官模型的构建方法，包括以下步骤：A method for constructing a cervical squamous cell carcinoma organoid model, comprising the following steps:
1)样本预处理：将刚切除/活检得到的宫颈癌组织标本用生理盐水冲洗3-5次除去表面杂质，装进含浓度为2％双抗及两性霉素B的Advanced DMEM/F12培养基中保存，冰上运输；1) Sample pretreatment: The freshly excised/biopsy cervical cancer tissue samples were rinsed with normal saline for 3-5 times to remove surface impurities, and loaded into Advanced DMEM/F12 medium containing 2% double antibody and amphotericin B stored in medium and transported on ice;
2)样本消化：在生物安全柜中，将样本转移至10cm无菌培养皿中加入1ml0.5mg/ml胶原酶II(消化液，基底：含2％双抗及两性霉素B的Advanced DMEM/F12培养基)，用灭菌眼科剪将组织剪碎至1-5mm3大小，将组织块及消化液转移至50ml离心管中，按组织体积适量补充消化液，37度，120rpm震荡消化120mins；2) Sample digestion: In a biological safety cabinet, transfer the sample to a 10cm sterile petri dish and add 1ml of 0.5mg/ml collagenase II (digestion solution, base: Advanced DMEM/ with 2% double antibody and amphotericin B). F12 medium), use sterilized ophthalmic scissors to shred the tissue to a size of 1-5mm 3 , transfer the tissue block and digestion solution to a 50ml centrifuge tube, and supplement appropriate amount of digestion solution according to the tissue volume, 37 degrees, 120rpm, shaking and digesting for 120mins;
3)样本解离：用同体积的PBS终止消化，将消化后的组织悬液用100μm的细胞筛过滤，收集细胞筛上未过滤组织碎片于50ml离心管中，加入3ml tryple(依碎片多少加减)，37度，120rpm震荡消化10mins；3) Sample dissociation: stop the digestion with the same volume of PBS, filter the digested tissue suspension with a 100 μm cell sieve, collect the unfiltered tissue fragments on the cell sieve in a 50 ml centrifuge tube, add 3 ml tryple (according to the number of fragments) Minus), 37 degrees, 120rpm shaking digestion 10mins;
4)细胞过滤：用同体积的PBS终止消化，将消化后的组织悬液再次用100μm的细胞筛过滤，收集两次过滤的滤液，4度600g离心5min，保留细胞沉淀去除上清；4) Cell filtration: stop the digestion with the same volume of PBS, filter the digested tissue suspension with a 100 μm cell sieve again, collect the filtrate filtered twice, centrifuge at 600g at 4 degrees for 5 min, retain the cell pellet and remove the supernatant;
5)裂解红细胞(可选)：肉眼可见细胞沉淀内混有红细胞，加入1-2ml红细胞裂解液，重悬细胞，室温裂解2min；5) Lysate erythrocytes (optional): the cell pellet can be seen mixed with erythrocytes, add 1-2ml of erythrocyte lysis solution, resuspend the cells, and lyse at room temperature for 2 minutes;
6)细胞清洗：用10mlPBS终止裂解，4度600g离心5min，保留细胞沉淀去除上清，重复两次；6) Cell washing: stop the lysis with 10ml PBS, centrifuge at 600g at 4°C for 5min, retain the cell pellet and remove the supernatant, repeat twice;
7)接种：取类器官培养基及基质胶(体积比例为1:2)以1万细胞/10μl的密度重悬细胞沉淀，以30μl/孔接种至48孔板中，小心将孔板倒置，于37度培养箱中固定30mins，待胶凝固后加入200ul培养基覆盖胶滴；7) Seeding: Take organoid culture medium and Matrigel (volume ratio of 1:2) to resuspend the cell pellet at a density of 10,000 cells/10 μl, and inoculate 30 μl/well into a 48-well plate. Carefully invert the well plate, Fix in a 37°C incubator for 30mins, add 200ul medium to cover the glue drop after the glue solidifies;
8)培养：于上述实施例1-4中宫颈鳞癌类器官培养基下培养，培养条件为37℃、5％CO2浓度，每2-3天观察换液，每7-21天进行传代。8) Cultivation: cultured in the cervical squamous cell carcinoma organoid medium in the above embodiment 1-4, the culture conditions are 37 ° C, 5% CO concentration, the medium is observed every 2-3 days, and the passage is carried out every 7-21 days. .
按照实施例5的构建方法，通过在上述实施例1-2宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官在倒置的光学显微镜下观察如图1-2所示。经过实施例3-4宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官在倒置的光学显微镜下的形态与图1-2类似，故不再阐述。According to the construction method of Example 5, the cervical squamous cell carcinoma organoids obtained by culturing in the cervical squamous cell carcinoma organoid medium of the above-mentioned Example 1-2 were observed under an inverted optical microscope as shown in Figures 1-2. The morphology of cervical squamous cell carcinoma organoids obtained after culturing in the culture medium of cervical squamous cell carcinoma organoids in Example 3-4 is similar to that in Fig. 1-2 under an inverted optical microscope, so it will not be described again.
对比例1-12Comparative Examples 1-12
在实施例2的基础上，对比例1-12为分别减去或添加如表1的特异性因子构成的宫颈鳞癌类器官培养基，然后再按照实施例5宫颈鳞癌类器官模型的构建方法培养宫颈鳞癌类器官7天后，在倒置显微镜下随机挑选5个视野进行类器官的直径测定。On the basis of Example 2, Comparative Examples 1-12 are respectively subtracted or added the cervical squamous cell carcinoma organoid culture medium composed of specific factors as shown in Table 1, and then constructed the cervical squamous cell carcinoma organoid model according to Example 5 Methods After culturing cervical squamous cell carcinoma organoids for 7 days, 5 fields of view were randomly selected under an inverted microscope to measure the diameter of the organoids.
表1Table 1
对比例1-12的宫颈鳞癌类器官培养基对宫颈鳞癌类器官形成的影响如图3所示，其中图3中“-”代表较实施例2培养基减去对应因子，“+”代表较实施例2培养基添加对应因子。The effect of the cervical squamous cell carcinoma organoid culture medium of Comparative Examples 1-12 on the formation of cervical squamous cell carcinoma organoids is shown in Figure 3, wherein "-" in Figure 3 represents the medium of Example 2 minus the corresponding factor, "+" It represents the addition of corresponding factors to the medium of Example 2.
结果显示，经过对比例1-12宫颈鳞癌类器官培养基培养后的宫颈鳞癌类器官直径不及实施例2宫颈鳞癌类器官培养基培养效果，其中对比例1-3、5-10分别去掉Noggin、A83-01、EGF、FGF7、FGF10、N-乙酰半胱氨酸、烟酰胺、氢化可的松、Forskolin、Y-27632中的一种，宫颈鳞癌类器官培养效果不及实施例2，说明Noggin、A83-01、EGF、FGF7、FGF10、N-乙酰半胱氨酸、烟酰胺、氢化可的松、Forskolin和Y-27632相互协同促进宫颈鳞癌类器官培养效果，提高培养宫颈癌类器官的成功率。对比例4的培养基添加FGF7因子，对比例11的培养基添加R-spondin 1，对比例12的培养基添加R-spondin 1和CHIR99021，这三种培养基培养后的宫颈鳞癌类器官大小不及实施例2，说明不是任意特异性因子的添加就能获得本发明的技术效果。The results show that the diameter of cervical squamous cell carcinoma organoids cultured in the medium of comparative examples 1-12 is not as good as that of the culture medium of cervical squamous cell carcinoma organoids of Example 2, wherein comparative examples 1-3 and 5-10 are respectively Remove one of Noggin, A83-01, EGF, FGF7, FGF10, N-acetylcysteine, nicotinamide, hydrocortisone, Forskolin, Y-27632, the effect of cervical squamous cell carcinoma organoid culture is not as good as Example 2 , indicating that Noggin, A83-01, EGF, FGF7, FGF10, N-acetylcysteine, nicotinamide, hydrocortisone, Forskolin and Y-27632 synergistically promote the effect of cervical squamous cell carcinoma organoid culture, improve the culture of cervical cancer Organoid success rates. The medium of Comparative Example 4 was supplemented with FGF7 factor, the medium of Comparative Example 11 was supplemented with R-
同时观察各例培养基中类器官的传代后维持率(将类器官重新消化成单细胞悬液种于基质胶中，单细胞若再长成类器官则定义为传代成功，长期维持定义为：类器官培养超过10代)，发现除实施例1-4及对比例12外，其余所有对比例均无法传过3代。At the same time, observe the post-passage maintenance rate of the organoids in the medium of each case (the organoids were re-digested into single-cell suspensions and seeded in Matrigel, if the single cells regrown into organoids, it was defined as successful passage, and long-term maintenance was defined as: Organoids were cultured for more than 10 generations), and it was found that except for Examples 1-4 and Comparative Example 12, all the other Comparative Examples could not be passed on for 3 generations.
本发明人经过大量实验验证，最终获得本发明宫颈鳞癌类器官培养基，即包括以下组分：基础培养基、Hepes缓冲对缓冲对10～12mM、青霉素链霉素溶液1％～2％、营养添加剂1％～3％、烟酰胺10～12mmol/L、N-乙酰半胱氨酸1～1.5mmol/L、Y-27632 0～10μM、Noggin因子80～120ng/ml、A83-01 450～550nmol/L、EGF 5～10ng/ml、FGF10 80～120ng/ml、Forskolin 8～12μM和氢化可的松400～600ng/ml。After a large number of experimental verifications, the inventor finally obtained the cervical squamous cell carcinoma organoid medium of the present invention, which includes the following components: basal medium, Hepes buffer to buffer 10-12 mM, penicillin-
对比例13Comparative Example 13
与实施例2相比，营养添加剂和特异性因子的体积比为10：3，其余组分及浓度与实施例2相同，参考图4。Compared with Example 2, the volume ratio of nutritional additives and specific factors was 10:3, and the remaining components and concentrations were the same as those in Example 2, referring to FIG. 4 .
试验例、宫颈鳞癌类器官培养基对宫颈鳞癌培养成功率的影响Test case, the effect of cervical squamous cell carcinoma organoid medium on the success rate of cervical squamous cell carcinoma culture
本试验例各组别(实施例1-4、对比例11-13)分别纳入60例宫颈鳞癌类器官样本，按照实施例5的构建方法，检测经过上述实施例1-4、对比例11-13宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官的活性，各组培养宫颈癌类器官的成功率如表2所示，其中，培养成功是指在特定的培养条件下消化后的肿瘤细胞或细胞团能在体外形成立体结构即类器官培养成功。Each group (Examples 1-4 and Comparative Examples 11-13) of this test example included 60 cases of cervical squamous cell carcinoma organoid samples respectively. -13 The activity of cervical squamous cell carcinoma organoids obtained after culturing in cervical squamous cell carcinoma organoid medium. After the tumor cells or cell clusters can form a three-dimensional structure in vitro, the organoid culture is successful.
表2Table 2
根据上述结果可知，采用实施例2的宫颈鳞癌类器官培养基培养后获得的宫颈鳞癌类器官有44例可以获得成功，其成功率可以提高至73％(44/60)，实施例1、3-4提高宫颈鳞癌类器官成功培养的效果与实施例2类似。According to the above results, 44 cases of cervical squamous cell carcinoma organoids obtained after culturing with the cervical squamous cell carcinoma organoid medium of Example 2 can be successful, and the success rate can be increased to 73% (44/60). Example 1 The effect of 3-4 on improving the successful culture of cervical squamous cell carcinoma organoids is similar to that of Example 2.
对比例11-12中的培养基新添加了其他特异性因子，其培养宫颈鳞癌类器官的效果不及实施例2，说明特异性因子不是随意可以添加的，有些特异性因子的加入反而会影响类器官培养的效果。The medium in Comparative Examples 11-12 was newly added with other specific factors, and the effect of culturing cervical squamous cell carcinoma organoids was not as good as that in Example 2, indicating that the specific factors were not added at will, and the addition of some specific factors would affect the The effect of organoid culture.
对比例13的培养基改变了营养添加剂和特异性因子的体积比，宫颈鳞癌类器官培养成功率不及实施例2，说明营养添加剂和特异性因子以特定的体积比搭配，可以提高培养宫颈鳞癌类器官的成功率。The medium of Comparative Example 13 changed the volume ratio of nutrient additives and specific factors, and the success rate of cervical squamous cell carcinoma organoid culture was lower than that of Example 2, indicating that the combination of nutrient additives and specific factors in a specific volume ratio can improve the culture of cervical squamous cell carcinoma. Cancer organoid success rates.
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，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实质和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that, The technical solutions of the present invention may be modified or equivalently replaced without departing from the spirit and scope of the technical solutions of the present invention.
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