Simple and Cost-Effective Generation of 3D Cell Sheets and Spheroids Using Curvature-Controlled Paraffin Wax Substrates

Abstract

Abstract The challenges associated with animal testing in pharmaceutical development have driven the search for alternative in vitro models that mimic human tissues more accurately. In this study, we present a simple and cost-effective method for generating 3D cell sheets and spheroids using curvature-controlled paraffin wax films, which are easily accessible laboratory materials that eliminate the need for extracellular matrix (ECM) components or thermo-responsive polymers. By adjusting the curvature of the paraffin wax film, we successfully generated human periodontal ligament fibroblast (HPdLF) cell sheets and bone marrow-derived mesenchymal stem cell (hBMSC) spheroids. Key parameters, such as cell density, substrate curvature, and incubation time, were identified as critical factors for optimizing the formation of these 3D structures. In addition, the use of quantum dots (QDs) for cell tracking enabled long-term visualization and distinction between different cell types within complex tissue-like structures. We further demonstrated that wrapping the hBMSC spheroids with HPdLF cell sheets partially replicated the connective tissue structure of the periodontal ligament surrounding the tooth root. This highlights the potential of this platform for the construction of more sophisticated tissue-mimicking assemblies. In conclusion, curvature-controlled paraffin wax films provide a versatile and practical approach for 3D cell cultures. This simplifies the generation of both cell sheets and spheroids, offering a promising tool for tissue engineering and regenerative medicine applications, where precise cell-to-cell interactions are essential. Graphical abstract

Keywords

• Paraffin wax
• Cell sheet
• Spheroids
• Periodontal ligament cell
• Mesenchymal stem cell
• Quantum dots