Feasibility Study Of Polysaccharide-based Hydrogels For Breast Cancer Organoid Carriers

Background:Breast cancer organoids are an emerging breast cancer model in recent years,which play an important role in the mechanism research,drug sensitivity detection and new drug development of breast cancer.At present,there is no unified regulation for the selection of 3D culture vectors for breast cancer organoids in vitro,which largely affects the success rate of breast cancer organoid culture.Matrigel is an extracellular matrix extract derived from poorly differentiated chondrosarcoma in mice.Because it can promote the growth and differentiation of tumor cells,it is used by most scholars as a carrier for three-dimensional culture of breast cancer organoids in vitro.However,recent studies have shown that Matrigel is not suitable for the in vitro culture of breast cancer organoids due to its batch variability,murine tumor origin,complex protein composition,and difficult-to-control mechanical properties.Natural polysaccharide-based hydrogels are macromolecular materials that can swell into a three-dimensional network-like structure.Because of their transparent components,controllable physicochemical properties,and large water content,they make up for the shortcomings of Matrigel.A new vector for in vitro three-dimensional culture of cancer organoids.Currently,there is no specific natural polysaccharide-based hydrogel for in vitro 3D culture of breast cancer organoids.Objective:1.Developed a polysaccharide-based hydrogel carrier and characterized its performance;2.Assess whether the polysaccharide-based hydrogel meets the biological safety standards;3.Characterize the breast cancer cell mass cultured in the natural polysaccharide-based hydrogel carrier,and compare it with the breast cancer cell mass cultured in Matrigel,and evaluate the natural polysaccharide-based hydrogel carrier as a substitute for matrigel feasibility of the product for in vitro three-dimensional culture of breast cancer organoids.Methods:1.Modification of natural materials chitosan and sodium alginate to prepare N-carboxyethyl chitosan(N-Carboxyethyl Chitosan,CEC)and oxidized sodium alginate(Oxidized Sodium Alginate,OSA),through the Schiff formation between the amino group on CEC and the aldehyde group on OSA base reaction to form polysaccharide-based hydrogel;2.By adjusting the concentration of CEC in natural polysaccharide-based hydrogels,and comprehensively considering the gelation time,in vitro degradation time,water content,microscopic morphology under scanning electron microscope,pore morphology,porosity and elastic modulus,we found The natural polysaccharide-based hydrogel ratio that best matches the survival of breast cancer cells;3.Biosafety testing of polysaccharide-based hydrogels through in vivo and in vitro experiments;4.Breast cancer cells were seeded in natural polysaccharide-based hydrogel and Matrigel,respectively.The breast cancer cell clusters in natural polysaccharide-based hydrogel and Matrigel were compared from the aspects of cell morphology detection,dead and alive detection,proliferation detection,and functional expression detection;5.SPSS 26.0 software and Origin Pro 9.0 software were used to process data,measurement data were expressed as mean ± standard deviation,and independent samples t test was used for measurement between groups.All experiments were repeated more than 3 times,with statistical significance at P < 0.05(***: P < 0.001;**: P < 0.01;*: P < 0.05).Results:1.The developed natural polysaccharide-based hydrogels conform to the three-dimensional culture of breast cancer cells in vitro in terms of gel formation time,in vitro degradation time,water content,pore shape,porosity and elastic modulus;2.The natural polysaccharide-based hydrogel developed meets the biological safety standards;3.Breast cancer cell clusters cultured in natural polysaccharide-based hydrogels were compared with breast cancer cell clusters cultured in Matrigel in terms of cell morphology,survival rate,proliferation and functional expression.In terms of cell morphology,the diameter of MCF-7 cell clusters in polysaccharide-based hydrogels was significantly smaller than that of MCF-7 cell clusters in Matrigel,but the smaller diameter of cell clusters ensured that in the later stage of culture(> 7 D)The survival of cells in the center of the cell mass is beneficial to the long-term culture of the cell mass.There was no significant difference between the two in terms of dead and alive、proliferation and functional expression of MCF-7 cell clusters.Conclusion:1.The polysaccharide-based hydrogel composed of CEC and OSA has the advantages of controllable physical and chemical properties and good biocompatibility;2.The natural polysaccharide-based hydrogel can be used as a carrier for in vitro three-dimensional culture of breast cancer organoids.