Preparation Of Cell Microcarriers By Electrostatic Drip Method

Cell microcarriers usually refer to microbeads that can be applied to adherent-dependent cell growth.The materials used to prepare cell microcarriers can be natural polymer materials or synthetic polymer materials.Because of the large specific surface area,cell microcarriers provide a larger growth area for cells,and can achieve a large number of cell growth and expansion in a smaller volume.In foreign countries,microcarriers have been successfully used in practical production.However,the development of microcarriers in China is relatively late and there are few microcarriers to be commercialized.The microcarriers used in related industries can only rely on imports and the prices are quite expensive.Some biopharmaceuticals,vaccines,and antibodies all need to produce large amounts of recombinant proteins from cells in vitro.However,the growth of cells requires the growth interface provided by microcarriers,which inevitably leads to high prices of recombinant protein-based products.Cell microcarriers not only increase the production rate of cells,but also greatly increase the cost of products.Therefore,it is urgent to develop a preparation technology of cell microcarriers to reduce the cost of products.Chitosan is a kind of natural polymer with good properties,low price,wide source and good biocompatibility.It has been widely used in food industry,biomedicine and other fields.In this paper,a kind of monodisperse chitosan cell microcarrier was prepared by electrostatic drop injection using chitosan as raw material.By overcoming the viscosity and surface tension of chitosan solution,the chitosan cell microspheres with uniform particle size and good sphericity were obtained.On this basis,the growth factors which can bind to chitosan cell microcarriers specifically are introduced to further enhance the biological activity of microcarriers,so that they can better meet the growth needs of adherent-dependent cells.In order to obtain chitosan cell microcarriers with excellent conditions,the preparation parameters of chitosan cell microcarriers were optimized in this paper.The parameters were optimized,such as the concentration of chitosan solution,the inner diameter of the needle,the voltage,the heating temperature,and the stability in different environments.A smooth,translucent chitosan cell microcarrier with a particle size distribution of 290~320?m was obtained.Under the condition of conventional high temperature and high pressure sterilization at 121 ?for 20 min,there was no deformation at high temperature and high pressure for five consecutive times,and it could be stored in distilled water for a long time,and the properties were stable.The above results show that the chitosan cell microcarriers prepared by electrostatic drop injection have uniform particle size distribution,good sphericity,and can be sterilized by conventional methods,showing a yellowish translucent shape.After adhesion and proliferation of cells,the growth can be observed directly by optical microscope.After the physical and chemical properties of the chitosan cell microcarriers were tested,the recombinant growth factor bFGF and the recombinant growth factor ChtBD-bFGF with specific binding ability were prepared by genetic engineering,and the recombinant growth factors were used in the chitosan cell microcarrier.To make the growth factor have the ability to bind specifically to the material,a chitin binding domain(ChtBD)was introduced at the end of the growth factor,which can bind to the material specifically.After purification,the concentration of bFGF was 400?g/mL,and the concentration of ChtBD-bFGF was 570?g/mL.Finally,chitosan cell microcarriers were soaked with 40?g/mL growth factors.Mouse osteoblast cell line(MC3T3-E1)and mouse embryonic fibroblast cell line(NIH3T3)were cultured in 48-well plate with 200?L/well microcarrier and 20000/well cell number.The results showed that both MC3T3-E1 cells and NIH3T3 cells could grow well on the microcarriers,and ChtBD-bFGF could promote the adhesion and proliferation of cells better.The self-made chitosan cell microcarriers can be used in the culture of these two kinds of adherent-dependent cells.