During intervertebral disc degeneration (IVDD), due to endplate calcification, diminished oxygen and nutrient concentrations and accumulated lactate are present in the microenvironment of the nucleus pulposus (NP). The disadvantages of 3D layered culture include uneven oxygen and nutrient gradients. In the present study, to mimic the in vivo microenvironment of the NP, a 5-layered 3D culture was constructed using clinical haemostatic gelatine sponges and developed as a NP degeneration (NPD) model. Subsequently, cell distribution as well as expression of NP chondrogenic markers (type II collagen and aggrecan), glycosaminoglycan (GAG) and degeneration markers [e.g. matrix metalloproteinase (MMP) 3] were measured from the top to the bottom layer. However, in a single NP-cell-loaded disc model, the chondrogenic potency in the middle or bottom layer was higher than that in the top layer. To further study the mechanism underlying the degeneration of NP cells in this NPD model, the contribution of secreted metabolites was examined. Lactate identified in the supernatant modulated GAG accumulation and MMP3 expression. Inhibition of lactate influx by the monocarboxylate transporter (MCT)-1 inhibitor, AZD3965, reversed the effect of lactate on GAG accumulation and MMP3 expression and further improved NP cell degeneration in the NPD model. Thanks to the homogenous expression of lactate in the model, it was possible to further identified that the combination of lactate and hypoxia enhanced MMP3 expression. Taken together, multilayered cell-loaded sponges, with oxygen and nutrient gradients as well as lactate accumulation, can represent a 3D multilayered NPD model for exploring potential agents for IVDD.