Abstract

Stacked cell culture has emerged as a successful 3D technique in tissue engineering, demonstrated by prototypes developed with various scaffold materials. This study aimed to enhance cell culture by integrating an alginate membrane with a 3D-printed stacking platform composed of polylactic acid material. The platform featured an optimized three-layer structure using L929 cells and was evaluated against MG-63 cells. Cells were seeded on alginate membrane layers comprising a mixture of alginate, agarose, gelatin, and glycerol (1.5:0.25:1:1%w/v). Increased alginate content resulted in greater weight loss without significantly affecting the degradation rate. In contrast, reducing agarose content accelerated membrane degradation and halved tensile strength (from ~6 to ~3 MPa). Cell viability, Live/Dead staining, and SEM imaging assessed the performance of the stacked culture across layers. The alginate membrane exhibited biocompatibility with L929 cells over 28 days, as verified by Live/Dead staining and SEM imaging on Days 7 and 14. The stacked culture maintained over 70% cell viability per layer for both L929 and MG-63 cells compared to single-layer culture. MG-63 cells, representing osteoblastic differentiation, showed enhanced outcomes, with bone-related genes (RUNX2, OCN, BMP2, COL1A2) exhibiting 1–2-fold increased expression, particularly in the upper layers, compared to general medium. These findings highlight the potential of stacked cell culture to support cell growth and differentiation across layers. This prototype offers promise for tissue engineering applications, with future studies aiming to utilize it for stem cell culture and large-scale 3D tissue development.