Comparison Between Bioprinted Tumor Disks and Pre‐cast Hydrogels or Decellularized Natural ECM as 3D Models to Study Artificial Melanoma In‐vitro
Authors: Lyden Timothy , Hultgren Heather , Beethe Nadina , Jaskens Randy , LeBeau Michelle , Thrun Lori 
Published: May 2022

During the Spring and Summer of 2021, the UWRF Tissue and Cellular Innovation Center engaged in a series of new collaborative research projects using biofabrication methods to model and study in‐vitro 3D artificial tumor and stromal tissues. In this presentation, we report on results obtained from efforts to bioprint cancer tumor disks and larger cup‐like structures using mouse melanoma cell lines B16F1 and B16F10, both of which are considered broadly applicable cell models for this type of tumor. Our previous studies in 3D tumor modeling, using other methods, have established a robust library of experience and data using these cells. In these new studies, bioprinting methods were applied to establish tumor structures which contained specific cell numbers as well as initial mono‐cellular distribution patterns. Both of these characteristics have been very difficult to precisely control in earlier studies with precast or molded hydrogels made of chitosan and hyaluronic acid or decellularized natural marine sponge ECM. In these new studies, three distinct bioinks were tested and applied using a Cellink “Inkcredible” model printer. This unit was loaned to the TCIC by Century College for the duration of this research project. Two shapes of tumor tissues were designed and tested in this work. The first were disks with a diameter of 5 mm and thickness of 2.5 mm and the second was a larger “cup‐like” structure which had a diameter of 10 mm and height of 3 mm with a “cup” or “bowl” in the center that was 8 mm in diameter and had a depth of 2 mm. Several commercial bioinks, produced by the Cellink company were tested in the course of this work, including Cellink (alginate/cellulose), GelMa‐C (gelatin‐methacrylate/cellulose) and GelMa (gelatin‐methacrylate). These successfully generated tumor disks which were maintained in culture for at least 2‐3 weeks. In some cases, printed tissues were maintained for up to 8 weeks in culture. In all cases, these structures produced artificial tumor tissues which displayed significantly similar overall morphological features to those seen in earlier studies using non‐printed constructs. In addition, during the course of culture, significant tumor tissue progression was evident and lead to substantial development of further 3D features including branching pigmented and non‐pigmented structures and sphere‐like projections. Following 1‐2 weeks in culture, these tended to be released into the media and generally settled to the chamber floor where they essentially “explanted” and generated new post‐scaffold monolayers. The GelMa disks also offered a unique observational opportunity in that these were essentially clear and could be viewed throughout the full depth until tissues developed to such a degree as to obscure the lower tissues levels. This provided a unique insight into the internal organization of the developing tumors which we have not previously been able to visualize in real‐time. Future studies will focus on this capacity to study tumor development, progression and the onset of metastasis.