上海交通大学学报(医学版)

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基于水凝胶微球建立胰腺癌原代细胞的3D培养模型

马芳芳(), 秦洁洁, 任灵杰, 唐笑梅, 刘佳, 施敏敏, 蒋玲曦()   

  1. 上海交通大学医学院附属瑞金医院胰腺疾病研究所,上海 200025
  • 收稿日期:2022-05-18 接受日期:2022-08-21 出版日期:2022-12-19 发布日期:2022-12-19
  • 通讯作者: 蒋玲曦 E-mail:mafang704@163.com;jlx12120@rjh.com.cn
  • 作者简介:马芳芳(1993—),女,硕士生;电子信箱:mafang704@163.com
  • 基金资助:
    上海市教育委员会高峰高原学科建设项目(20200037)

Establishment of a 3D culture model in vitro of pancreatic cancer primary cells using hydrogel microspheres

MA Fangfang(), QIN Jiejie, REN Lingjie, TANG Xiaomei, LIU Jia, SHI Minmin, JIANG Lingxi()   

  1. Institute of Pancreatic Diseases, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Received:2022-05-18 Accepted:2022-08-21 Online:2022-12-19 Published:2022-12-19
  • Contact: JIANG Lingxi E-mail:mafang704@163.com;jlx12120@rjh.com.cn
  • Supported by:
    Shanghai Municipal Education Commission—Gaofeng Clinical Medicine Grant Support(20200037)

摘要:

目的·利用水凝胶微球和新鲜胰腺癌组织原代细胞构建模拟肿瘤微环境的体外培养新模型。方法·记录水凝胶微球的形态分布情况,倒置荧光显微镜下观察并拍照,通过Image J软件计算微球的直径,统计得到粒径分布图。肾上皮细胞(293T)、胰腺癌细胞(8988)、正常胰腺上皮细胞HPNE均在DMEM完全培养基中生长,当细胞长满到80%~90%时传代。DMEM培养基和微球浸提液培养293T细胞,通过CCK-8法检测2种培养基培养的293T细胞的增殖曲线,探究水凝胶微球的生物相容性。在超净工作台中剪碎新鲜胰腺肿瘤组织,用透明质酸酶和胶原蛋白酶Ⅰ裂解胰腺癌肿瘤组织,在37 ℃水浴锅中间隔振荡消化为单细胞。水凝胶微球与胰腺细胞在DMEM完全培养基中共培养3 d,半数细胞用4%多聚甲醛固定,鬼笔环肽和DAPI染色,普通荧光显微镜下观察微球的形态和细胞分布;剩余一半细胞用于悬浮细胞和黏附细胞计数。水凝胶微球与原代细胞在培养基中共培养7 d,用免疫荧光法观察基于水凝胶微球建立的胰腺癌原代细胞体外培养模型中的细胞组成。用石蜡包埋胰腺癌组织,随后进行石蜡组织切片,苏木精-伊红染色组织切片,用显微镜观察胰腺癌组织结构。结果·水凝胶微球大小均一,微球粒径约200 μm,293T细胞的增殖曲线表明水凝胶微球具有良好的生物相容性。水凝胶微球与胰腺细胞系共培养结果表明水凝胶微球表面具有较强的细胞亲和力,能够为胰腺细胞提供支撑点,使其黏附在微球表面正常生长。水凝胶微球与消化后的胰腺癌新鲜组织单细胞共培养成功建立胰腺癌体外3D培养模型。该模型具有与胰腺肿瘤组织相似的细胞组成,包含胰腺导管上皮细胞、相似比例的肿瘤干细胞、内皮细胞、成纤维细胞等。结论·基于水凝胶微球建立的胰腺癌原代细胞3D培养模型具有胰腺癌肿瘤微环境的重要特征。

网络首发地址:https://kns.cnki.net/kcms/detail//31.2045.R.20221216.1451.002.html

网络首发时间:2022-12-19 14:54:40

关键词: 水凝胶微球, 胰腺癌, 肿瘤微环境, 3D体外培养模型

Abstract:

Objective ·To establish an in vitro culture model mimicking tumor microenvironment using hydrogel microspheres and fresh primary pancreatic cancer cells. Methods ·The morphological distribution of the hydrogel microspheres was recorded, observed and photographed under an inverted fluorescence microscope. The diameter of the microspheres was calculated by Image J, and the particle size distribution map was obtained by statistics. Renal epithelial cells (293T), pancreatic cancer cells (8988), and normal pancreatic epithelial cells (HPNE) were grown in DMEM complete medium, and passaged when the cells were 80%?90% confluent. 293T cells were cultured in DMEM medium and microsphere extract, and the proliferation curve of 293T cells cultured in the two mediums was detected by CCK-8 method to explore the biocompatibility of hydrogel microspheres. Fresh pancreatic tumor tissue was cut in the ultra-clean workbench, and pancreatic cancer tumor tissue was lysed by hyaluronidase and collagenase Ⅰ, and digested into single cell with interval shaking in a 37 ℃ water bath. The hydrogel microspheres and pancreatic cells were co-cultured in DMEM complete medium for 3 d, 1/2 of the cells were fixed with 4% paraformaldehyde, and stained with phalloidin and DAPI, and the morphology and cell distribution of the microspheres were observed under a common fluorescence microscope. The remaining 1/2 cells were used for suspension and adherent cell counting. The hydrogel microspheres and primary cells were co-cultured in the medium for 7 d, and the cell composition in the established culture model of in vitro pancreatic cancer primary cells based on the hydrogel microspheres was observed by immunofluorescence method. The pancreatic cancer tissue was embedded in paraffin, followed by paraffin tissue sectioning, hematoxylin and eosin staining of the tissue section, and observation of the pancreatic cancer tissue structure with a microscope. Results ·The size of the hydrogel microspheres was uniform, and the diameter of the microspheres was about 200 μm. The proliferation curve of 293T cells showed that the hydrogel microspheres had good biocompatibility. The co-culture experiments of hydrogel microspheres and pancreatic cell lines showed that the surface of hydrogel microspheres had strong cell affinity, which could provide support points for pancreatic cells to adhere to the surface of the microspheres and to grow normally. Through co-culture of hydrogel microspheres with fresh pancreatic cancer cells, a 3D culture model of pancreatic cancer cells in vitro was successfully established. Composition of cell types in this model was similar to that in the corresponding primary tumor tissue, which included pancreatic ductal epithelial cells, tumor stem cells, endothelial cells, fibroblasts and other cells. Conclusion ·The in vitro 3D culture model of primary pancreatic cancer cells based on hydrogel microspheres has important characteristics of pancreatic cancer tumor microenvironment.

Key words: hydrogel microsphere, pancreatic cancer, tumor microenvironment, in vitro 3D culture model

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