Preparation Of Porous Microspheres With Aqueous Two-phase Emulsions As Template For Three-dimensional Cell Culture

With the development of cell culture technology,traditional two-dimensional(2D)cell culture has been unable to meet the needs of biological research,drug screening and clinical applications.Three-dimensional(3D)cell culture is considered to be a better alternative to in vitro culture,due to its ability to simulate the in vivo microenvironment of cell-cell and cell-matrix interactions to maintain normal cell growth,gene expression and cell function.Emulsion template method is a common method for preparing 3D cell culture carrier in recent years.In this paper,porous calcium alginate microspheres with interconnected porous structure were prepared using the aqueous two-phase emulsions stabilized by methoxy polyethylene glycol-albumin(m PEG-BSA)macromolecule protein particles as the template and used as the carrier for 3D cell culture.By comparing the results of the aqueous two-phase emulsions without adding stable particles of m PEG-BSA and adding stable particles of m PEG-BSA over time,it is concluded that the aqueous two-phase emulsions with stable particles of m PEG-BSA can be used as a template for preparing porous calcium alginate microspheres.Moreover,C_Algwt%was optimized by characterizing the morphology of aqueous two-phase-alginic acid(ATPS-Alg)emulsions and porous calcium alginate microspheres to prepare ideal porous calcium alginate microspheres with interconnected porous structures.In addition,through the characterization of the prepared porous calcium alginate microspheres,it is proved that the microspheres have interconnected porous structures.Furthermore,by regulating the emulsifying frequency of the water-in water(W/W)emulsions and the volume ratio of the W/W emulsion to the polyethylene glycol-alginic acid(PEG-Alg)solution,the change rule of the pore size was explored.Finally,fluorescein isothiocyanate-albumin(FITC-BSA),a small molecule substance,was used to simulate the absorption of cellular nutrients and the excretion of metabolic wastes.It was verified that the porous calcium alginate microspheres had excellent semipermeability.It was concluded that the porous calcium alginate microspheres with interconnected porous structure and controllable pore size are expected to be used as a carrier for cell culture in vitro.In order to verify that the microspheres can be used as an excellent carrier for 3D cell culture in vitro,two different cells(Hep G2 and HUVEC)were directly encapsulated into the pores of the porous microspheres.The results showed that both Hep G2 and HUVEC cells encapsulated in porous calcium alginate microspheres had higher cell activity(?90%)and faster proliferation than those encapsulated in general microspheres.In addition,the results of cell distribution and morphology showed that the cells in the porous microspheres were significantly more concentrated than those in the general microspheres.At the same time,the results of investigating of Hep G2 cell functional proteins showed that the concentration of albumin,urea,and cytochrome P450 in the porous calcium alginate microspheres was obviously higher than those in the genenral microspheres,while the concentrations of alanine aminotransferase were significantly lower than those in general microspheres during the culture time.Therefore,it was anticipated that the porous calcium alginate microspheres with a controllable interconnected porous structure could be applied as carries in vitro cell culture,scaffold for tissue engineering.In order to further demonstrate the advantages of porous calcium alginate microspheres in organ-like construction in vitro,we also investigated the cell culture of directly encapsulation of two different cells(Hep G2 and HUVEC)on the outside of the pores of the porous microsphere.The results showed that the activity,proliferation,cell morphology,and measurement results of Hep G2 cell functional protein of the two cells encapsulated outside the pores of the porous microspheres were consistent with the trends in the pores of the porous microspheres,indicating that cell culture could be performed both inside and outside of the pores of the porous calcium alginate microspheres,which provided a possibility for the construction of organ-like cells in vitro in the future.