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We report a protocol for the layer-by-layer fabrication of adherent and non-adherent 3-dimensional (3-D) cell cultures. Adherent Madin Darby canine kidney (MDCK) epithelial cell cultures were patterned onto Petri dishes, and non-adherent cell cultures were physically encapsulated in alginate hydrogel layer above the adherent cells. The layer-by-layer, CAD/CAM fabrication process results in soft transfer and does not harm adherent cells. We have developed a technique to fabricate a multi-layer cell culture system to study the interactions between adherent and non-adherent cell types.