Abstract: OBJECTIVE To optimize 3D culturing conditions for the tumor cells based on human liver cancer cells SMMC-7721, find out the proper concentrations of sodium alginate and calcium chloride to form calcium alginate gel. METHODS SMMC-7721 cells were cultured in 96-well plate. The cell survival rate under different concentrations and treatment durations of calcium chloride and sodium citrate solutions were detected by MTT method based on L₉(3⁴) orthogonal design. According to the result of orthogonal optimization, the suitable concentration of sodium alginate was investigated, and optimal conditions of gelatin forming and dissolving were screened. Then cell viability was confirmed under the 3D culture mode. RESULTS The optimal conditions were as follows:1% calcium chloride act on the sodium alginate within 15 min to form gel, 10% sodium citrate act on the calcium alginate gel within 10 min to dissolve gel, which achieved a 94.97% cell survival rate by MTT method. The SMMC-7721 cells cultured in the calcium alginate gel showed a fine state within 72 h, the survival rate was 92.10%, and showed a compact agminated status which was called multicellular spheroid. CONCLUSION The conditions for gel forming and dissolving used for tumor cells 3D culture is compatible for cell culturing, SMMC-7721 cells show a fine growth state, and the agminated status shows some difference compared with the cell cultured in 2D condition. Tumor cells cultured by this method could provide a better way to study the living status for tumor cells and to screen more anti-tumor drugs.