CN116179492A - Serum-free special medium for pancreatic cancer organoids - Google Patents
Serum-free special medium for pancreatic cancer organoids Download PDFInfo
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Abstract
本发明提供一种胰腺癌类器官无血清专用培养基，涉及器官培养皿技术领域。该胰腺癌类器官无血清专用培养基，包括基础培养基和培养添加物，所述基础培养基为含有1％B—内酰胺类抗生素、1％HEPES缓冲液、1％GlutaMax溶液的DMEM/F12培养基，所述培养添加物包括TGF‑β、TNF‑α、EGF、PDGF、VEGF、FGF、IL‑1、IL‑6，含壳聚糖纳米粒子，CD3抗体和TLR7/8激动剂，所述培养添加物的终浓度为TGF‑β：0.01‑0.08μg/mL，TNF‑α：0.02‑0.09μg/mL，EGF：0.04‑0.1μg/mL，PDGF：0.05‑0.08μg/mL，VEGF；0.06‑0.1μg/mL，FGF：0.02‑0.08μg/mL，IL‑1：0.01‑0.05μg/mL，IL‑6：0.03‑0.07μg/mL。通过在基础培养基中加入一些含壳聚糖纳米粒子、CD3抗体和TLR7/8激动剂，能够使生物活性增强，提高促进类器官细胞增殖、细胞存活率以及细胞的生长和增殖，提高该培养基的实用性。The invention provides a serum-free special culture medium for pancreatic cancer organoids, and relates to the technical field of organ culture dishes. The pancreatic cancer organoid serum-free special medium includes basal medium and culture supplements, the basal medium is DMEM/F12 containing 1% B-lactam antibiotics, 1% HEPES buffer, and 1% GlutaMax solution Culture medium, described culture supplement comprises TGF-beta, TNF-alpha, EGF, PDGF, VEGF, FGF, IL-1, IL-6, containing chitosan nanoparticle, CD3 antibody and TLR7/8 agonist, all The final concentration of the culture supplements is TGF-β: 0.01-0.08 μg/mL, TNF-α: 0.02-0.09 μg/mL, EGF: 0.04-0.1 μg/mL, PDGF: 0.05-0.08 μg/mL, VEGF; 0.06‑0.1 μg/mL, FGF: 0.02‑0.08 μg/mL, IL‑1: 0.01‑0.05 μg/mL, IL‑6: 0.03‑0.07 μg/mL. By adding some chitosan-containing nanoparticles, CD3 antibodies and TLR7/8 agonists to the basal medium, the biological activity can be enhanced, the proliferation of organoid cells, the survival rate of cells and the growth and proliferation of cells can be improved, and the culture can be improved. base practicality.
Description
技术领域technical field
本发明涉及器官培养皿技术领域，具体为一种胰腺癌类器官无血清专用培养基。The invention relates to the technical field of organ culture dishes, in particular to a special serum-free culture medium for pancreatic cancer organoids.
背景技术Background technique
胰腺癌的预后极差，五年总生存率仅为10％左右。目前的治疗措施如吉西他滨、紫杉醇或奥沙利铂应答率很低，复发极为常见。此外，胰腺癌的隐匿进展和转移也是预后不良的主要原因。因此，迫切需要工具和模型来为个别患者确定更有效的治疗方案。随着分子生物学技术的进展，已经知道胰腺癌是一种高度异质性疾病，包括不同的亚型，这些亚型在遗传表型、病理特征和临床表现均不相同，因此构建基于患者来源的肿瘤体外模型，能够准确模拟体内肿瘤的生物学特征和对药物治疗的反应，对于肿瘤研究、药物测试及筛选有着巨大的价值，利用胰腺癌患者来源的肿瘤组织建立个体化的类器官肿瘤模型，为患者筛选敏感的化疗药物或靶向药物，是胰腺癌个体化治疗的一个重要途，类器官是细胞的三维组装体，包含一种以上的细胞类型，能够至少表现出细胞所述器官的生理特性。由于其保持了亲本肿瘤的关键特征，可以利用类器官进行药物筛选、预测患者放化疗反应等。The prognosis of pancreatic cancer is extremely poor, with a five-year overall survival rate of only about 10%. Response rates to current treatments such as gemcitabine, paclitaxel, or oxaliplatin are low, and relapse is extremely common. In addition, the occult progression and metastasis of pancreatic cancer are also the main reasons for the poor prognosis. Therefore, tools and models are urgently needed to identify more effective treatment options for individual patients. With the advancement of molecular biology techniques, it has been known that pancreatic cancer is a highly heterogeneous disease, including different subtypes, which are different in genetic phenotype, pathological characteristics and clinical manifestations, so the construction is based on the source of patients The tumor in vitro model can accurately simulate the biological characteristics of tumors in vivo and the response to drug treatment, which is of great value for tumor research, drug testing and screening. The tumor tissue derived from pancreatic cancer patients is used to establish individualized organoid tumor models Screening sensitive chemotherapeutic drugs or targeted drugs for patients is an important way for individualized treatment of pancreatic cancer. Organoids are three-dimensional assemblies of cells, including more than one cell type, which can at least express the characteristics of the organ described by the cells. physiological characteristics. Because it maintains the key features of the parental tumor, organoids can be used for drug screening, prediction of patient radiotherapy and chemotherapy responses, etc.
目前在对胰腺癌类器官无血清培养基的培养过程中，大多数都是采用传统的方法，例如在培养基中添加Wnt激动剂R-spond i n、BMP抑制剂Noggi n等调控因子，但是在传统的方法中，培养基中的生物活性较差，导致整个培养基的培养效率较慢，花费的较多，导致其实用性降低。At present, in the culture process of serum-free medium of pancreatic cancer organoids, most of them adopt traditional methods, such as adding regulatory factors such as Wnt agonist R-spond in and BMP inhibitor Noggin in the medium, but in In the traditional method, the biological activity in the culture medium is poor, resulting in slower cultivation efficiency of the whole culture medium and more cost, resulting in reduced practicability.
发明内容Contents of the invention
(一)解决的技术问题(1) Solved technical problems
针对现有技术的不足，本发明提供了一种胰腺癌类器官无血清专用培养基，解决了传统的培养基中的生物活性较差，导致整个培养基的培养效率较慢，花费的较多，导致其实用性降低的问题。Aiming at the deficiencies of the prior art, the present invention provides a special serum-free culture medium for pancreatic cancer organoids, which solves the problem of poor biological activity in the traditional culture medium, which leads to slow culture efficiency of the whole culture medium and high cost , leading to a problem of reduced practicability.
(二)技术方案(2) Technical solution
为实现以上目的，本发明通过以下技术方案予以实现：一种胰腺癌类器官无血清专用培养基，包括基础培养基和培养添加物，所述基础培养基为含有1％B—内酰胺类抗生素、1％HEPES缓冲液、1％G l utaMax溶液的DMEM/F12培养基，所述培养添加物包括TGF-β、TNF-α、EGF、PDGF、VEGF、FGF、I L-1、I L-6，含壳聚糖纳米粒子，CD3抗体和TLR7/8激动剂。In order to achieve the above purpose, the present invention is achieved through the following technical solutions: a special serum-free culture medium for pancreatic cancer organoids, including basal medium and culture supplements, the basal medium is containing 1% B-lactam antibiotics , 1% HEPES buffer, 1% GlutaMax solution of DMEM/F12 medium, the culture supplements include TGF-β, TNF-α, EGF, PDGF, VEGF, FGF, IL-1, IL- 6. Containing chitosan nanoparticles, CD3 antibody and TLR7/8 agonist.
优选的，所述培养添加物的终浓度为TGF-β：0.01-0.08μg/mL，TNF-α：0.02-0.09μg/mL，EGF：0.04-0.1μg/mL，PDGF：0.05-0.08μg/mL，VEGF；0.06-0.1μg/mL，FGF：0.02-0.08μg/mL，I L-1：0.01-0.05μg/mL，I L-6：0.03-0.07μg/mL，含壳聚糖纳米粒子：0.03-0.1μg/mL，CD3抗体：0.02-0.06μg/mL，TLR7/8激动剂：0.01-0.05μg/mL。Preferably, the final concentration of the culture supplement is TGF-β: 0.01-0.08 μg/mL, TNF-α: 0.02-0.09 μg/mL, EGF: 0.04-0.1 μg/mL, PDGF: 0.05-0.08 μg/mL mL, VEGF; 0.06-0.1μg/mL, FGF: 0.02-0.08μg/mL, IL-1: 0.01-0.05μg/mL, IL-6: 0.03-0.07μg/mL, containing chitosan nanoparticles : 0.03-0.1μg/mL, CD3 antibody: 0.02-0.06μg/mL, TLR7/8 agonist: 0.01-0.05μg/mL.
优选的，所述基础培养基为无血清培养基。Preferably, the basal medium is a serum-free medium.
优选的，所述在配制好的基础培养基中，按照终浓度加入所有培养添加物，混合均匀后即可获得。Preferably, all culture supplements are added to the prepared basal medium according to the final concentration, and can be obtained after mixing evenly.
一种胰腺癌类器官的培养方法：A method for culturing pancreatic cancer organoids:
将胰腺癌组织样本处理清洗后加入胶原酶消化，然后将消化后的细胞重悬于冷的Cu ltrexTMgrowthfactorreducedBMEtype2中，接种于细胞培养板，待含有细胞的BME固化后，加入1-4所述的一种胰腺癌类器官无血清专用培养基进行培养。After the pancreatic cancer tissue samples were processed and washed, they were digested with collagenase, and then the digested cells were resuspended in cold Cu ltrex TM growthfactorreducedBMEtype2, and seeded on cell culture plates. After the BME containing cells solidified, add the cells described in 1-4 A special serum-free medium for pancreatic cancer organoids.
(三)有益效果(3) Beneficial effects
本发明提供了一种胰腺癌类器官无血清专用培养基。具备以下有益效果：The invention provides a special serum-free culture medium for pancreatic cancer organoids. Has the following beneficial effects:
本发明中，通过在基础培养基中加入一些含壳聚糖纳米粒子、CD3抗体和TLR7/8激动剂，能够使生物活性增强，提高促进类器官细胞增殖、细胞存活率以及细胞的生长和增殖，提高该培养基的实用性。In the present invention, by adding some nano-particles containing chitosan, CD3 antibody and TLR7/8 agonist in the basic medium, the biological activity can be enhanced, and the proliferation of organoid cells, cell survival rate and cell growth and proliferation can be improved. , to improve the practicability of the medium.
具体实施方式Detailed ways
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。The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts belong to the protection scope of the present invention.
实施例一：Embodiment one:
本发明实施例提供一种胰腺癌类器官无血清专用培养基，包括基础培养基和培养添加物，基础培养基为无血清培养基，基础培养基为含有1％B—内酰胺类抗生素、1％HEPES缓冲液、1％G l utaMax溶液的DMEM/F12培养基，培养添加物包括TGF-β、TNF-α、EGF、PDGF、VEGF、FGF、I L-1、I L-6，含壳聚糖纳米粒子，CD3抗体和TLR7/8激动剂，培养添加物的终浓度为TGF-β：0.01μg/mL，TNF-α：0.02μg/mL，EGF：0.04μg/mL，PDGF：0.05μg/mL，VEGF；0.06-μg/mL，FGF：0.02μg/mL，I L-1：0.01μg/mL，I L-6：0.03μg/mL，含壳聚糖纳米粒子：0.03μg/mL，CD3抗体：0.02μg/mL，TLR7/8激动剂：0.01μg/mL，在配制好的基础培养基中，按照终浓度加入所有培养添加物，混合均匀后即可获得。An embodiment of the present invention provides a special serum-free medium for pancreatic cancer organoids, including a basal medium and culture supplements, the basal medium is a serum-free medium, and the basal medium contains 1% B-lactam antibiotics, 1 DMEM/F12 medium with % HEPES buffer, 1% GlutaMax solution, culture supplements including TGF-β, TNF-α, EGF, PDGF, VEGF, FGF, IL-1, IL-6, containing shell Glycan nanoparticles, CD3 antibody and TLR7/8 agonist, culture supplements at final concentrations of TGF-β: 0.01 μg/mL, TNF-α: 0.02 μg/mL, EGF: 0.04 μg/mL, PDGF: 0.05 μg /mL, VEGF; 0.06-μg/mL, FGF: 0.02μg/mL, IL-1: 0.01μg/mL, IL-6: 0.03μg/mL, chitosan-containing nanoparticles: 0.03μg/mL, CD3 antibody: 0.02μg/mL, TLR7/8 agonist: 0.01μg/mL, in the prepared basal medium, add all the culture supplements according to the final concentration, and mix well to obtain.
一种胰腺癌类器官的培养方法：A method for culturing pancreatic cancer organoids:
将胰腺癌组织样本处理清洗后加入胶原酶消化，将消化后的细胞重悬于冷的CultrexTMgrowthfactorreducedBMEtype2中，接种于细胞培养板，待含有细胞的BME固化后，加入1-4的一种胰腺癌类器官无血清专用培养基进行培养，放入5％CO2浓度、37℃细胞培养箱培养，每4～6天更换一次上述培养基，7天左右即可获得所需胰腺癌类器官。After the pancreatic cancer tissue samples were processed and cleaned, they were digested with collagenase, and the digested cells were resuspended in cold Cultrex TM growthfactor reduced BME type 2, seeded on cell culture plates, and after the BME containing cells solidified, 1-4 of a pancreatic Cancer organoids are cultured in a serum-free special medium, placed in a 5% CO2 concentration, 37°C cell incubator for culture, and the above medium is replaced every 4 to 6 days, and the required pancreatic cancer organoids can be obtained in about 7 days.
实施例二：Embodiment two:
本发明实施例提供一种胰腺癌类器官无血清专用培养基，包括基础培养基和培养添加物，基础培养基为无血清培养基，基础培养基为含有1％B—内酰胺类抗生素、1％HEPES缓冲液、1％G l utaMax溶液的DMEM/F12培养基，培养添加物包括TGF-β、TNF-α、EGF、PDGF、VEGF、FGF、I L-1、I L-6，含壳聚糖纳米粒子，CD3抗体和TLR7/8激动剂，培养添加物的终浓度为TGF-β：0.08μg/mL，TNF-α：0.09μg/mL，EGF：0.1μg/mL，PDGF：0.08μg/mL，VEGF；0.1μg/mL，FGF：0.08μg/mL，I L-1：0.05μg/mL，I L-6：0.07μg/mL，含壳聚糖纳米粒子：0.1μg/mL，CD3抗体：0.06μg/mL，TLR7/8激动剂：0.05μg/mL，在配制好的基础培养基中，按照终浓度加入所有培养添加物，混合均匀后即可获得。An embodiment of the present invention provides a special serum-free medium for pancreatic cancer organoids, including a basal medium and culture supplements, the basal medium is a serum-free medium, and the basal medium contains 1% B-lactam antibiotics, 1 DMEM/F12 medium with % HEPES buffer, 1% GlutaMax solution, culture supplements including TGF-β, TNF-α, EGF, PDGF, VEGF, FGF, IL-1, IL-6, containing shell Glycan nanoparticles, CD3 antibody and TLR7/8 agonist, culture supplements at final concentrations of TGF-β: 0.08 μg/mL, TNF-α: 0.09 μg/mL, EGF: 0.1 μg/mL, PDGF: 0.08 μg /mL, VEGF; 0.1 μg/mL, FGF: 0.08 μg/mL, IL-1: 0.05 μg/mL, IL-6: 0.07 μg/mL, nanoparticles containing chitosan: 0.1 μg/mL, CD3 Antibody: 0.06μg/mL, TLR7/8 agonist: 0.05μg/mL, in the prepared basal medium, add all culture supplements according to the final concentration, and mix well to obtain.
一种胰腺癌类器官的培养方法：A method for culturing pancreatic cancer organoids:
将胰腺癌组织样本处理清洗后加入胶原酶消化，将消化后的细胞重悬于冷的CultrexTMgrowthfactorreducedBMEtype2中，接种于细胞培养板，待含有细胞的BME固化后，加入1-4的一种胰腺癌类器官无血清专用培养基进行培养，放入5％CO2浓度、37℃细胞培养箱培养，每4～6天更换一次上述培养基，7天左右即可获得所需胰腺癌类器官。After the pancreatic cancer tissue samples were processed and cleaned, they were digested with collagenase, and the digested cells were resuspended in cold Cultrex TM growthfactor reduced BME type 2, seeded on cell culture plates, and after the BME containing cells solidified, 1-4 of a pancreatic Cancer organoids are cultured in a serum-free special medium, placed in a 5% CO2 concentration, 37°C cell incubator for culture, and the above medium is replaced every 4 to 6 days, and the required pancreatic cancer organoids can be obtained in about 7 days.
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications and substitutions can be made to these embodiments without departing from the principle and spirit of the present invention. and modifications, the scope of the invention is defined by the appended claims and their equivalents.
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