Objective: This study optimizes three-dimensional(3D)culture conditions of L02 cells using response surface methodology(RSM) based on the VitroGel system to construct the hepatocytes model in vitro.
Methods: L02 cells were 3D cultured by the VitroGel system. The appropriate level of three key factors(concentration of inoculated cells, culture time and dilution degree of the hydrogel) was determined by single-factor experiment, and the optimal conditions of 3D culture of L02 cells based on the VitroGel system were determined by RSM. During the detection process, the optical density(OD) value of cell viability was used as the detection index, and the cell viability was detected using the cell counting kit-8(CCK-8) assay. The proliferative performance and viability of L02 cells was measured by fluorescent staining assay.
Results: The selected optimal culture conditions by RSM were as follows: concentration of inoculated cells was 1.1 × 10~5/mL, culture time was 9.5 days, and dilution degree of hydrogel was 1∶3.7. The result shows that under optimal conditions, the predicted OD value of cell viability was 2.17 and measured 2.13 with a relative error of 1.84%, indicating that the condition was suitable and reliable. The fluorescent staining and dead and live cells detection results showed the 3D hepatocytes model was successfully constructed.
Conclusion: The optimal conditions for 3D culture of L02 cell based on the VitroGel system were determined by RSM, and a hepatocytes model with high cellular activity was successfully constructed.
Keywords: L02 cell; hydrogel; response surface methodology; the three-dimensional culture.