基于水凝胶微球建立胰腺癌原代细胞的3D培养模型

doi:10.3969/j.issn.1674-8115.2023.01.010

摘要/Abstract

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。

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

中图分类号:

R541.6

马芳芳, 秦洁洁, 任灵杰, 唐笑梅, 刘佳, 施敏敏, 蒋玲曦. 基于水凝胶微球建立胰腺癌原代细胞的3D培养模型[J]. 上海交通大学学报（医学版）, 2023, 43(1): 79-87.

MA Fangfang, QIN Jiejie, REN Lingjie, TANG Xiaomei, LIU Jia, SHI Minmin, JIANG Lingxi. Establishment of a 3D culture model in vitro of pancreatic cancer primary cells using hydrogel microspheres[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2023, 43(1): 79-87.

图/表 4

图1 GelMA水凝胶微球的表征Note：A. Bright field imaging of hydrogel microspheres under a fluorescence inverted microscope (×100). The microspheres were highly dispersed and uniform in size. B. Particle size distribution of GelMA hydrogel microspheres, with an average particle size of 200 μm. C. There was no significant difference in cell proliferation between the two groups by CCK-8 assay. The ctl was 293T cells cultured in high glucose medium, and the mic group was 293T cells cultured with GelMA porous hydrogel microsphere extract.

Fig 1 Characterization of GelMA hydrogel microspheres

图2 正常胰腺细胞和胰腺癌细胞系均能黏附在水凝胶微球表面生长Note： A. Bright field images of HPNE cells growing on the surface of hydrogel microspheres (×40). B. The number of HPNE cells adhered to the surface of the microspheres and the number of cells suspended in the culture flask were counted. C. Bright field images of 8988 cells growing on the surface of hydrogel microspheres (×40). D. The number of 8988 cells adhered to the surface of the microspheres and the number of cells suspended in the culture flask were counted. E. Fluorescence images of HPNE cells growing on the surface of hydrogel microspheres [DAPI (blue) staining (×100), phalloidin (green) staining (×100), merge (×100, ×200)]. F. Fluorescence images of HPNE cells growing on the surface of hydrogel microspheres [DAPI (blue) staining (×100), phalloidin (green) staining (×100), merge (×100, ×200)]. ①P=0.000, ②P=0.001.

Fig 2 Both normal pancreatic cells and pancreatic cancer cell lines can grow on the surface of hydrogel microspheres

图3 胰腺癌组织来源细胞能在水凝胶微球表面生长Note： A. Schematic diagram of the 3D model of pancreatic cancer primary cells based on hydrogel microspheres. B. Bright field image of a 3D model of pancreatic cancer primary cells (×40). C. The numbers of cells adhered to the surface of the microspheres and suspended in the culture flask were counted. D. Fluorescence images of cells growing on the surface of hydrogel microspheres [DAPI (blue) staining (×100), phalloidin (green) staining (×100), merge (×100, ×200)]. ①P=0.000.

Fig 3 Pancreatic cancer tissue-derived cells can grow on the surface of hydrogel microspheres

图4 胰腺癌原代细胞3D培养模型包含胰腺癌肿瘤微环境的主要细胞成分Note： A. H-E staining pictures of pancreatic cancer tissue sections (×50). B. The percentage of each cell in the tissue. C. Bright field images of pancreatic cancer primary cell 3D culture model (×100). D. The percentage of each cell in the 3D culture model. E?H. Immunofluorescence staining pictures (×100) of pancreatic cancer tissue sections, stained with CD31, SMA, CD133, EpCAM (red), CK19 (green), and DAPI (blue), respectively. I?L. Immunofluorescence staining pictures (×100) of pancreatic cancer primary cell 3D culture model [confocal microscope x, y axis scan synthetic 2D image stained with CD31, SMA, CD133, EpCAM (red), CK19 (green), and DAPI (blue), respectively]. M?P. Immunofluorescence-stained 3D images (×200) of pancreatic cancer primary cell 3D culture model [confocal microscopy x, y, z axis scans of the synthesized 3D structures, stained with CD31, SMA, CD133, EpCAM (red), CK19 (green), and DAPI (blue), respectively].

Fig 4 Pancreatic cancer primary cell 3D culture model contains major cellular components of the pancreatic cancer tumor microenvironment

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