Preparation And Detection Of Polyglutamic Acid 3D Cell Culture Hydrogel

In recent years,the development of tissue engineering,an emerging engineering discipline,has received extensive attention from all walks of life.Among them,hydrogel is developing rapidly as an important biological material in tissue engineering.Because of its good water absorption performance and high viscoelasticity,it has been widely used in many industries.Compared with the traditional in vitro twodimensional cell culture(2D culture),in vitro three-dimensional cell culture(3D culture)has attracted much attention because it can better simulate the extracellular matrix(ECM).3D culture hydrogel not only has good biocompatibility,its special structural performance effectively simulates the extracellular matrix to realize 3D cell culture,and the simple responsiveness of the smart gel can change the microenvironment of cell growth and achieve cells are specifically cultured and studied in a three-dimensional space.This experiment intends to use natural protein anion polypeptide ?-polyglutamic acid as raw materials to prepare two different ?-polyglutamic acid composite hydrogels to be used in many biological fields such as biomedicine,tissue engineering and wound repair.The most basic function of this hydrogel is as a cell scaffold,providing mechanical support and growth space for cells,so it should have the characteristics of non-toxicity,high water content and good viscoelasticity.?-Polyglutamic acid(?-PGA)is an amino acid polymer prepared by microbial fermentation.It is mainly derived from Bacillus licheniformis and Bacillus subtilis.It has high water absorption properties and low cost.The raw materials and preparation process are not Will pollute the environment.Polylysine(PLL)has a certain antibacterial and antibacterial effect,and it contains more amino groups and can react easily with ?-polyglutamic acid.Chitosan(CS)is derived from marine crustaceans,which is not only easy to obtain,but also has antibacterial and hydrophilic properties.1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride(EDC)and N-hydroxysulfo-succinimide(NHS),EDC is a commonly used protein coupling agent,NHS is commonly used protein stabilizers,they can make amino and carboxyl groups react to form amide bond.And EDC and NHS are recognized nontoxic crosslinkers.Therefore,in this experiment,the carbodiimide(EDC/NHS)method was used to take the ?-polyglutamic acid obtained by microbial fermentation as the main raw material,cross-linked with polylysine and chitosan separately,and pores were made by freeze-drying technology.Two different ?-polyglutamic acid composite hydrogels have been released: ?-polyglutamic acid/polylysine(?-PGA/PLL)composite hydrogel and ?-polyglutamic acid/chitosan(?-PGA/CS)composite hydrogel,and its physical and chemical properties,pore size and cell compatibility have been systematically studied.The experiment explored the physical and chemical properties of the two ?-polyglutamic acid composite hydrogels through water absorption,fourier transform infrared spectroscopy(FTIR),and rheological properties;through scanning electron microscope(SEM)testing,the pore size of the two ?-polyglutamic acid composite hydrogels varies with the concentration of ?-polyglutamic acid;through the real-time growth of chondrocytes under the Zen CELL owl live cell dynamic imaging and analysis system,the MTT cytotoxicity test and Confocal microscopy observed the 3D growth of 293 T fluorescent cells in two ?-polyglutamic acid composite hydrogels at different concentrations to explore their cytocompatibility.The specific research results are as follows:(1)Preparation of ?-polyglutamic acid composite hydrogel: Taking ?-polyglutamic acid obtained by microbial fermentation as the main raw material,polylysine and chitosan are used as arms respectively,and carbodiimide(EDC/NHS)Method,using freeze-drying technology to successfully prepare ?-polyglutamic acid/polylysine composite hydrogel and ?-polyglutamic acid/chitosan composite hydrogel.Through the test of Fourier transform infrared spectroscopy(FTIR)and rheological properties,it is proved that the two kinds of ?-polyglutamic acid composite hydrogels are successfully prepared and have good viscoelasticity and mechanical stability.(2)The water absorption rate test: The water absorption rate of the ?-PGA/PLL composite hydrogel can be as high as 1590.82 %,and the water absorption rate of the ?-PGA/CS composite hydrogel can be as high as 1560.93 %,which proves that the two kinds of ?-Polyglutamic acid composite hydrogels all have good swelling properties,and the water absorption curves and equations of two gamma-polyglutamic acid composite hydrogels with the change of gamma-polyglutamic acid concentration have been found.(3)Scanning electron microscope(SEM)test: Both ?-polyglutamic acid compound hydrogels have found a hydrogel concentration suitable for cell 3D culture.Among them,the ?-PGA/PLL composite hydrogel according to the curve of its pore size with the concentration of ?-polyglutamic acid proves that when the ?-PGA concentration is between 0.0075-0.0175 g/m L,the pore size is consistent with the 3D culture water of the cell gel requirements.The ?-PGA/CS composite hydrogel has been proved through repeated experiments that when the ?-PGA concentration is about 0.01475 g/m L,its pore size meets the requirements of 3D cell culture hydrogel.(4)The tests of confocal microscope,Zen CELL owl live cell dynamic imaging and analysis system,and MTT cytotoxicity experiment all proved that the two ?-polyglutamic acid composite hydrogels are almost non-toxic to cells,and they are to a certain extent,it can promote cell growth,that is,both ?-polyglutamic acid composite hydrogels have good cell compatibility.In this experiment,two ?-polyglutamic acid composite hydrogels using microbial fermentation products as raw materials were successfully prepared.Both of which have good physical and chemical properties and cell compatibility and both have found their concentrations suitable for cell 3D culture.This experiment can provide a certain theoretical basis for the subsequent biological application of ?-polyglutamic acid composite hydrogel.