Transwell-Hypoxia Method Facilitates the Outgrowth of 3D-Printed Collagen Scaffolds Loaded with Cryopreserved Patient-Derived Melanoma Explants

[Image: see text] A previous study from our laboratory demonstrated the effects of in vitro three-dimensional (3D)-printed collagen scaffolds on the maintenance of cryopreserved patient-derived melanoma explants (PDMEs). However, it remains unknown whether 3D-printed collagen scaffolds (3D-PCSs) can be harmonized with any external culture conditions to increase the growth of cryopreserved PDMEs. In this study, 3D-PCSs were manufactured with a 3DX bioprinter. The 3D-printed collagen scaffold-on-frame construction was loaded with fragments of cryopreserved PDMEs (approximately 1–2 mm). 3D-PCSs loaded with patient-derived melanoma explants (3D-PCS-PDMEs) were incubated using two types of methods: (1) in transwells in the presence of a low concentration of oxygen (transwell-hypoxia method) and (2) using a traditional adherent attached to the bottom flat surface of a standard culture dish (traditional flat condition). In addition, we used six different types of media (DMEM high glucose, MEM α, DMEM/F12, RPMI1640, fibroblast basal medium (FBM), and SBM (stem cell basal medium)) for 7 days. The results reveal that the culture conditions of MEM α, DMEM/F12, and FBM using the transwell-hypoxia method show greater synergic effects on the outgrowth of the 3D-PCS-PDME compared to the traditional flat condition. In addition, the transwell-hypoxia method shows a higher expression of the MMP14 gene and the multidrug-resistant gene product 1 (MDR1) than in the typical culture method. Taken together, our findings suggest that the transwell-hypoxia method could serve as an improved, 3D alternative to animal-free testing that better mimics the skin’s microenvironment using in vitro PDMEs.