Construction And Evaluation Of A Three-dimensional Hepatocarcinoma Cell Culture Model That Simulates The Matrix In The Tumor Microenvironment

Developing three-dimensional(3D)cell culture models to mimic native tumor microenvironments might provide a platform for investigating the mechanism of hepatocellular carcinoma(HCC)metastasis and screening therapeutic drugs.Alginate gel(ALG)beads have been explored as vehicles for 3D in vitro HCC cell culture due to their numerous favorable attributes.In the present study,we modified the traditional gelling bath and incorporated decellularized liver matrix(DLM)with ALG beads to provide a better biomimetic simulation of the matrix in in vivo HCC microenvironment,and investigated their effects on the proliferation,the invasion and metastasis potential of entrapped HCC cells.First,the mass transfer properties of ALG beads were improved by altering the gelling bath.1/1 bath,1/2 bath and 1/3 bath were obtained through diluting the traditional calcium chloride gelling bath(100 mmol/L)by sodium chloride solution(154 mmol/L)at different volume ratios(1:1,1:2,1:3),uniform ALG beads with good sphericity could be stably generated in all three baths.ALG beads had increased size,more homogeneous structure,and better mass transfer properties but reduced mechanical stability with decreased calcium ion concentration and increased sodium ion concentration in gelling baths.Enhanced proliferation of HepG2 cells was detected in beads generated in a 1/2 bath compared to the traditional bath,the relative growth rate of cells increased by 1.6-fold after 15 days,while no significant difference was found in the invasion and metastasis potential of entrapped HCC cells between these two groups.Next,the biological activities were enhanced through the addition of DLM.Porcine DLM were harvested with the removal of cell remnants and the preservation of most ECM components by the combination of physical and chemical treatments.DLM could be digested into a solution form or comminuted into a powder form to mix with alginate solution.In the traditional gelling bath,uniformed composite ALG beads with good sphericity were obtained when the volume ratio of DLM solution and alginate solution was 1:2 or the DLM powder concentration was under 1%(w/v),respectively.The MMP2 and MMP9 activities of HCC cells in DLM solution-ALG(DLMS-ALG)beads were increased when compared with that in traditional ALG beads,which indicated that the addition of DLM solution into ALG beads might improve the invasion and metastasis potential of the entrapped HCC cells.After being cultured in 1%(w/v)DLM powder-ALG(DLMP-ALG)beads,HCCLM3 cells presented gradually enhanced proliferation.The MMP2 and MMP9 activities of HCC cells were significantly improved compared with that in ALG beads,and the promoting effect was much better than that in DLMS-ALG beads.Moreover,the protein expression of urokinase-type plasminogen activator system of cells was significantly increased,and the cell epithelial to mesenchymal transition(EMT)related gene expression was in consistent with the expression of liver cancer tissue.The results achieved in the present study showed that the alteration of ALG bead preparation conditions and the addition of DLM could promote the proliferation,also the invasion and metastasis potential of entrapped HCC cells by improving the simulation of the matrix in native HCC microenvironment,which might provide support for the construction of new 3D in vitro HCC cell culture models.