Three-dimensional Culture Of MCF-7 Cells In Matrigel/Gel/?-PGA/HA Hydrogels

The three-dimensional cell culture method establishes the connection between cells and extracellular matrix by using biomaterials to construct a micromilieu similar to the in vivo tissue scaffold(or matrix)capable of simulating the growth of cells in vivo.It is not only providing the matrix secretion and cell function under the cell proliferation micromilieu,activities and other material structural basis,but also to achieve the cell culture of intuitive and controllable conditions,which effectively complement the cells in the two-dimensional culture of the deficiencies.The 3D cell culture methods are more convenient for exploring cells in the physiological and pathological conditions of growth and proliferation,signal transduction and other molecular mechanisms.Meanwhile,hydrogels have become a favored biomaterial for their good water retention properties,stable mechanical properties,and biocompatibility in many established 3D cell culture systems.At present,hydrogel stents have been widely used in the culture of cartilage tissue,bone tissue,myocardial tissue,and liver tissue and so on.Here,a new type of composite hydrogel was constructed.The characteristics of the hydrogel were tested and the complex hydrogel was optimized for culture tumor cells which observe the growth characteristics,invasion and metastasis of tumor cells in the material.Other researchers in the laboratory has found that the interaction between March5 protein and estrogen receptor ? in 2D culture.So this subject used breast cancer cell MCF-7 as a research object to compare and analyze the effect of March5 on the expression of estrogen receptor ? both in 3D culture and 2D culture in order to understand the molecular mechanisms of the breast carcinogenesis.The experimental process is as follows:Firstly,a 3D cultured gelatin/hyaluronic acid/?-polyglutamic acid(Gel/?-PGA/HA)hydrogel scaffold was constructed.Then we found that the prepared hydrogel scaffold can perform 3D culture of breast tumor cells MCF-7,but the proliferation efficiency is slow,and the colloid degradation rate and proliferation rate can not match.Secondly,Gel/?-PGA/HA hydrogel scaffold was optimized by adding Matrigel.The study found that the optimized hydrogel scaffold can be used to breast cancer cell MCF-7,and the optimized MTT assay was verified.The hydrogel material can better perform the cultivation of breast cancer cell MCF-7.Finally,the effect of March5 on the expression of ER? gene and protein levels was explored in a 3D system.The effect of March5 on the expression of ER?,a key factor in breast cancers,were detected by real-time quantitative PCR and Western blot.The above results indicate that the optimized Marigel/Gel/?-PGA/HA can form a 3D structure,and its structure is more uniform than that of Gel/?-PGA/HA hydrogel,MCF-7 cells can grow well in the prepared Marigel/Gel/?-PGA/HA hydrogels.The status of MCF-7 cells is different from that of 2D cultures.Spherical agglomerates can form in the colloid,and as the number of culture days increases,the agglomeration of the cells gradually becomes larger.In the 3D culture of MCF-7 cell lines,the effects of March5 on endogenous and exogenous ER? protein levels and mRNA levels were investigated through cell death experiments,immunoblotting and co-immunoprecipitation experiments.The results showed that March5 could down-regulate the protein level of ER? and March5 could upregulate the transcription level of ER?,but its mechanism was not clear and needed further study.