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.
Keywords:hydrogel microsphere
;
pancreatic cancer
;
tumor microenvironment
;
in vitro 3D culture model
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. Journal of Shanghai Jiao Tong University (Medical Science)[J], 2023, 43(1): 79-87 doi:10.3969/j.issn.1674-8115.2023.01.010
胰腺癌主要起源于胰腺导管上皮细胞及腺泡细胞,恶性程度极高,起病隐匿,早期诊断困难,进展迅速,生存时间短,是预后最差的恶性肿瘤之一,被称为“癌中之王”。胰腺癌患者90%以上是胰腺导管腺癌(pancreatic ductal adenocarcinoma,PDAC)。PDAC的总生存率仅为8.5%,并且可选择的治疗方案很有限[1]。吉西他滨联合白蛋白紫杉醇和FOLFIRINOX(5-氟尿嘧啶、亚叶酸、伊立替康和奥沙利铂)是中晚期PDAC患者的2个一线治疗方案。然而,这些治疗措施为患者带来的生存获益仍十分有限,患者的总生存率仅为6~11个月[2-3]。近年来,特异靶向关键分子途径的药物有望改善肿瘤的治疗效果[4-5]。目前,多聚ADP核糖聚合酶(poly ADP ribose polymerase,PARP)抑制剂是美国食品药品监督管理局(Food and Drug Administration,FDA)批准的针对胰腺癌的唯一靶向药[6]。因此,为了进一步提高胰腺癌患者的精准化治疗水平,迫切需要开发新的胰腺肿瘤靶向药物并建立高效的体外模型进行后续药物筛选。
水凝胶微球来自上海交通大学医学院附属瑞金医院/上海市伤骨科研究所崔文国教授实验室制作[18]。新鲜胰腺癌组织来源于上海交通大学医学院附属瑞金医院胰腺中心行胰腺切除术的胰腺癌患者。肾上皮细胞293T、胰腺癌细胞8988、正常胰腺上皮细胞HPNE均购自中国科学院细胞库,且经过短串联重复序列(short tandem repeat,STR)鉴定,支原体检测呈阴性,保存在添加了10%胎牛血清和抗生素的DMEM培养基中。
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
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
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
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
The study was designed by JIANG Lingxi and MA Fangfang. The experiments were performed by QIN Jiejie and REN Lingjie. The manuscript was drafted and revised by TANG Xiaomei, LIU Jia and SHI Minmin. All the authors have read the last version of paper and consented for submission.
利益冲突声明
所有作者声明不存在利益冲突。
All authors disclose no relevant conflict of interests.
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... 水凝胶微球来自上海交通大学医学院附属瑞金医院/上海市伤骨科研究所崔文国教授实验室制作[18].新鲜胰腺癌组织来源于上海交通大学医学院附属瑞金医院胰腺中心行胰腺切除术的胰腺癌患者.肾上皮细胞293T、胰腺癌细胞8988、正常胰腺上皮细胞HPNE均购自中国科学院细胞库,且经过短串联重复序列(short tandem repeat,STR)鉴定,支原体检测呈阴性,保存在添加了10%胎牛血清和抗生素的DMEM培养基中. ...