Effects Of 3D Culture On The Biological Clock Of U2OS Cells

Three-dimensional(3D)cell culture technology is a kind of culture method that can make cells grow in three-dimensional space under external conditions,which can better simulate the growth condition and environment of cells in vivo.The cells cultured in 3D were different from those cultured in 2D in cell adhesion,apoptosis and gene expression.3D cell culture not only preserves the physical structure of the natural cell microenvironment,but also provides cellcell and cell-matrix interactions,providing a safer and more reliable method for cell-level research.Hydrogels can provide nutrients to cells and regulate cell growth and differentiation.With advantages of high plasticity and convenient clinical application,hydrogels are widely used as scaffolds for 3D cell culture.3D cell culture technology plays an important role in drug discovery and pharmacology applications,cancer biology applications and cell viability studies.Studies at the cellular level are crucial in the field of biological clocks,but 2D cultures are often used and 3D studies are lacking.In this paper,U2 OS cells were cultured in 3D using three different hydrogels(Gelatin,Gel MA,and HAMA-Gel MA)and their hardness(soft,medium,stiff).The effects of 3D culture on core Clock genes(bmal1,clock,cry1,cry2,per1,per2,per3)of U2 OS cells were studied from two aspects of hydrogel type and hydrogel hardness.According to the time and frequency scanning,the energy storage modulus(G’)is greater than the loss modulus(G"),and the energy storage modulus(G’)is in a stable state with the change of angular frequency,indicating that the nine hydrogels are successfully crosslinked and have good stability.According to the cell viability test results,cells in 2D culture were in a stretched state,while cells in 3D culture were closer to the body in a spherical shape.Compared with 2D culture,3D culture decreased the expression of clock genes,except the bmal1 gene in the medium hydrogel and the stiff hydrogel of the Gelatin,the per1 gene and the per2 gene in the soft hydrogel of the Gel MA,and the per2 gene in the medium hydrogel.The expression of bmal1,per1 and per2 genes in stiffhydrogel was not significantly different from that in 2D culture.According to the rhythmic expression of clock genes,there was no significant difference in the period and phase of clock genes in 2D and 3D culture,except that the amplitude of per1 gene had no significant difference compared with 2D culture,and the amplitude of other clock genes decreased compared with 2D culture.Real-time monitoring of bmal1 gene expression using Lumicycle showed that the expression of Bmal1 gene was lower than that of 2D culture when U2 OS cells were cultured in3 D with three kinds of hardness of Gel MA hydrogel,regardless of the hardness,which was consistent with the above results.These results indicate that culture mode has significant effect on the expression of clock genes,while hydrogel type and hydrogel hardness have little effect on the expression of clock genes.