A 3D printed hydrogel to promote human keratinocytes' spheroid-based growth

Tissue engineering uses cells and biomaterials to develop bioartificial tissue substitutes for different purposes. For example, although several skin models have been developed for pharmaceutical and cosmetic research and skin wound healing, there are few studies on 3D cultures of keratinocytes in 3D printed scaffolds. So, this work aimed to develop a 3D-printed hydrogel scaffold to promote human keratinocyte growth. Mesh 3D scaffolds were printed using an extrusion-based method with a 20% gelatin/5% alginate hydrogel, where HaCaT cells were cultured for 7 days. Scaffolds kept their structure for over 1 week, and their stiffness only decreased after 7 days, showing good mechanical and structural characteristics and biodegradability (27% weight lost). Viable keratinocytes (MTT assay) are aggregated into spheroids, a 3D model capable of mimicking in vivo cell properties and phenotypes. Spheroids were formed on 47% of scaffolds pores and grew over time, showing promising cell proliferation. F-actin staining showed cells' irregular and interconnected shapes and organization over time. This method offers an easy and inexpensive solution for keratinocyte spheroid formation, which may be helpful in tissue engineering as a cell delivery system, for pharmacological or basic research, or wound healing medical applications.