An Optimized Co-culture Model Of Mouse Blastocysts And Mouse Melanoma Cells

BackgroundIn nineteen eighties, investigators developed an ideal co-culture model to research the development and implantation of embryos. Mouse embryos were co-cultured with human cancer cell lines which were mitosis suppressed by mitomycin C. It showed that cancer cells, as feeder cells, improved the ratio of cleavage and blastocyst formation. But this co-culture model based on deficient malignant cancer cells can't meet requirement of research about the interaction between embryos and malignant cancer cells.Therefore, Hou developed a co-cultured model consisted of mouse embryos and A549 human lung cancer cell line with malignant invasive capacity. Embryos developed and adhered on the cancer cell monolayer, then expanded among cancer cells and finally formed their own growing space which resisted malignant cancer cells invasion. It was a milestone on the way of studying the interaction between embryos and cancer cells. Li and Wang followed this model and broaden its application. They demonstrated that the invasion of embryos in co-culture model was universal and suggested a possible mechanism.The process of tumor cells invasion is similar to the implantation of embryos. They co-express many invasion-related genes as well as follow similar invasion patterns. Several studies demonstrated that malignant cancer cells co-expressed a lot of biomarkers, such as integrins, FAK, MMP-9, CD44, OCT-4 etc. It was the very barrier for further research, to distinguish the embryos from malignant cancer cells by morphology or molecular expression. Thus, it is extremely urgent to find specific biomarkers for distinguishing them in the co-culture model.Enhanced Green florescent protein can be observed under fluorescent microscope expediently. It is stable, non-toxic in many heterogenous organisms, and widely applied in labeling various cells and cancer research. In this subject, we utilized EGFP to mark B16 mouse melanoma cell line via gene transfection and genetic screen. EGFP-B16 cell line was used for co-culture with C57BL/6 mouse blastocysts. This optimized co-culture model of embryos and malignant cancer cells was expected to break some barriers in the research.Objectives1. To transfect the plasmid vector containing EGFP gene into B16 mouse melanoma cells originated from C57BL/6 mouse and screen out stable EGFP-B16 cell line.2. To apply the EGFP-B16 cells to co-culture with embryos obtained from C57BL/6 mouse in order to optimize the co-culture model.Methods 1. The plasmid containing EGFP gene was extracted from E. coli, then transfected into B16 cells by GenEscortⅡtransgenic regent. Positive EGFP-B16 cells was scanned out via G418 combined with monoclonal selection, and amplified positive clones.2. The characteristics of EGFP-B16 cell line were studied after 20 generations. Cells were observed under LSCM. The mRNA and protein of EGFP gene by were detected by RT-PCR and FCM respectively. The proliferative capacity in vitro and in vivo was assessed by MTT and inoculating EGFP-B16 cell into subcutaneous tissue respectively.3. EGFP-B16 cell line were applied to co-culture with mouse embryos obtained from C57BL/6 mouse and co-culture with NIH-3T3 cell line. B16 cells co-culture with mouse embryos, NIH-3T3 co-cultured with mouse embryos were set as control.Results1. 50 percent of B16 cells were observed EGFP-positive 24 hours after transfection. Stable EGFP-B16 cell line was scanned out via G418 combined with monoclonal selection.2. EGFP-B16 cells were cultured for 20 generations. Positive EGFP-B16 cells showed green fluorescence under LSCM, but they look the same as B16 morphologically. RT-PCR displayed the EGFP mRNA in EGFP-B16 cells. FCM detected that there were 99.82 percent EGFP positive cells in EGFP-B16 sample. MTT showed that EGFP-B16 cell line proliferated in a similar rhythm as B16 cell line in vitro. The solid tumor volume of EGFP-B16 in vivo was smaller (p﹤0.05) than that of B16 at the same time.3. LSCM distinguished EGFP-B16 cells from the co-cultured embryo cells clearly and showed a sharp boundary which was vague in control groups. In the co-culture model containing EGFP-B16 and NIH-3T3, with the help of fluorescence microscope we exposed that EGFP-B16 cells invaded NIH-3T3 cells and developed from a seed to a colony. ConclusionEGFP-B16 cell line was established by gene transfection and screened out via G418 combine with monoclonal selection. EGFP-B16 and B16 cell lines were similar in biological characteristics. EGFP-B16 could be used for co-culture with C57BL/6 mouse blastocysts. The optimized co-culture model distinctively displayed the differences between embryos and EGFP-B16 mouse melanoma cells. It was a better model for further research on the interaction between embryos and malignant cancer cells.