| Part 1:Facile preparation of a novel mulberry silk fibroin scaffold for three-dimensional tumor cell cultureObjective: Three-dimensional(3D) culture methods, building a bridge between the traditional two-dimensional(2D) monolayer cell culture and the in-vivo conditions, launch a new era in cell culture. A reliable and stable method for 3D cell culture is required for further cytology research or chemosensitivity tests. In this study, we aim to use a novel material, the mulberry silk fibroin(SF), to build a scaffold for 3D gastric cancer cell culture model, without adding any toxic agents.Methods: A biocompatible scaffold based on mulberry silk fibroin was fabricated by a simple freeze-drying method, under mild conditions. Scanning electron microscopy and confocal laser scanning microscope were used to study the microstructure of the SF scaffold. After MKN45 cell lines were seeded on the scaffold, the fluorescent staining and histological method were used to investigate the cell morphology and distribution during the cell culture. We also investigated the stability of the scaffold in the medium after a long time culture.Results: We successfully build a 3D gastric cancer cell culture model based on the silk fibroin scaffold. The porous microstructure of the scaffold was observed under laser scanning confocal microscope and scanning electron microscope. The pores inside ranged from 31.8 to 108.5 μm. To validate the growth status of the MKN45 cells in the opaque scaffold, we used the fluorescent dye imaging method during the cell culture. There are only sporadic single cells on day 1, while on day 21, the MKN45 cells formed large irregular spheroids and multi-cellular aggregates, under the confocal laser microscope. The HE staining photographs were in agreement with the observations under confocal laser microscope, on day 21.Conclusions: The mulberry silk fibroin is a good biomaterial to build a stable scaffold for 3D gastric cell culture.Part 2:A novel rat tail collagen gel droplet method for three-dimensional cell culture and chemosensitivity test in gastric cancerObjective: We aim to establish a novel rat tail collagen gel droplet model for gastric cancer cell 3D culture, and further investigate the possibility of the chemosensitivity test based on that model.Methods: We choose rat tail collagen to build a cell-embedded gel droplet and we investigated the growth pattern of BGC823 gastric cancer cells inside under the optical microscope. On day 8, after being stained and fixed, the plate containing the droplets was soaked and air dried. The high-definition images of the droplets were taken under the microscope(Zeiss Observer A1). We further calculated the integrated optical density(IOD) of stained area in droplet image, and compared the surviving rate of two group treated by CDDP with different concentrations.Results: The BGC823 cells formed 3D colonies in the rat tail droplet after 8 days and the 3D cell culture model was successfully established. The IOD of stained area in each droplet image was calculated and a strong correlation between IOD and living cell number was observed. Further more, there was significant difference in the density of shade of the dyed droplets after treated with cisplatin of different concentrations, revealing that the further chemosensitivity test based on this model was possible. Then we finally used this method to calculate the inhibition ratio of 0.2ug/ml cisplatin group and 2ug/ml cisplatin group.Conclusions: We successfully established a rat tail collagen gel droplet model for gastric cancer cell 3D culture, and it is possible to proceed a novel chemosensitivity test based on that model, which has potential value for clinical application. |
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