3D printing is a rapidly evolving field for biological (bioprinting) and non-biological applications. Due to a high degree of freedom for geometrical parameters in 3D printing, prototype printing of bioreactors is a promising approach in the field of Tissue Engineering. The variety of printers, materials, printing parameters and device settings is difficult to overview both for beginners as well as for most professionals. In order to address this problem, we designed a guidance including test bodies to elucidate the real printing performance for a given printer system. Therefore, performance parameters such as accuracy or mechanical stability of the test bodies are systematically analysed. Moreover, post processing steps such as sterilisation or cleaning are considered in the test procedure. The guidance presented here is also applicable to optimise the printer settings for a given printer device. As proof of concept, we compared fused filament fabrication, stereolithography and selective laser sintering as the three most used printing methods. We determined fused filament fabrication printing as the most economical solution, while stereolithography is most accurate and features the highest surface quality. Finally, we tested the applicability of our guidance by identifying a printer solution to manufacture a complex bioreactor for a perfused tissue construct. Due to its design, the manufacture via subtractive mechanical methods would be 21-fold more expensive than additive manufacturing and therefore, would result in three times the number of parts to be assembled subsequently. Using this bioreactor we showed a successful 14-day-culture of a biofabricated collagen-based tissue construct containing human dermal fibroblasts as the stromal part and a perfusable central channel with human microvascular endothelial cells. Our study indicates how the full potential of biofabrication can be exploited, as most printed tissues exhibit individual shapes and require storage under physiological conditions, after the bioprinting process.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Malignant melanoma is among the tumor entities with the highest increase of incidence worldwide. To elucidate melanoma progression and develop new effective therapies, rodent models are commonly used. While these do not adequately reflect human physiology, two-dimensional cell cultures lack crucial elements of the tumor microenvironment. To address this shortcoming, we have developed a melanoma skin equivalent based on an open-source epidermal model. Melanoma cell lines with different driver mutations were incorporated into these models forming distinguishable tumor aggregates within a stratified epidermis. Although barrier properties of the skin equivalents were not affected by incorporation of melanoma cells, their presence resulted in a higher metabolic activity indicated by an increased glucose consumption. Furthermore, we re-isolated single cells from the models to characterize the proliferation state within the respective model. The applicability of our model for tumor therapeutics was demonstrated by treatment with a commonly used v-raf murine sarcoma viral oncogene homolog B (BRAF) inhibitor vemurafenib. This selective BRAF inhibitor successfully reduced tumor growth in the models harboring BRAF-mutated melanoma cells. Hence, our model is a promising tool to investigate melanoma development and as a preclinical model for drug discovery.

Melanoma is the solid tumor with high mutational burden, which leads the generation of neoantigens and the infiltration of cytotoxic T cells (CTLs) recognizing these antigens. Recent improvement of prognosis of melanoma resulting from anti PD-1 antibody elucidated the importance of the expression of PD-1 on tumor-infiltrating CTLs for melanoma cells to evade the cytotoxic activity of CTLs. Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that responds to a wide range of chemicals and induces the battery of genes associated with detoxification, including CYP1A1 and CYP1B1. Recently Liu and colleagues reported the upregulation of PD-1 on CTLs is induced via AHR activation by binding of AHR ligands released from melanoma cells. Among various solid tumors, melanoma exerts the relatively stronger suppression to anti-tumor immunity. Notably, characteristic biology in melanoma compared to other solid tumors is the ability of melanogenesis, though little is known about the association between melanogenesis and anti-tumor immune response. Therefore, we investigated the correlation among melanogenesis, AHR activation and PD-1 expression in the lesion of melanoma by using published RNA-seq data and immunohistochemistry of biopsy samples. Gene set variation analysis of RNA-seq data from microdissected lesions of melanoma revealed the correlation between the gene set of AHR system and that of melanogenesis in melanoma. Moreover, immunohistochemical analysis revealed CYP1A1 is expressed in Tyrosinase + melanoma cells and their surrounding CTLs, which express PD-1. Furthermore, given the clinical data, the level of CYP1A1 expression in melanoma lesion tends to correlate with the efficacy of immunotherapies including anti PD-1 antibody. These results indicate the melanogenic property of melanoma cells enables them to activate AHR system, which suppresses anti-tumor immunity via the upregulation of PD-1 on CTLs in some way related to melanogenesis.