Non-small cell lung cancer (NSCLC) is a widespread tumor classified in heterogeneous subtypes according to the cell of origin and molecular features (1). Disease progression is linked to complex microenvironment, which includes several cell types, matrix components (2) and pervasive angiogenesis (3). Traditional 2D culture fails at resuming the complexity of in-vivo model, whereas 3D environment (e.g. scaffolds) sustains cell culture with stimuli alike the physiological ones (4). The aim of the work is the development of an in-vitro device able to mimic the NSCLC niche and using it as more reliable platform for molecular studies and drug screening.
Methacrylate gelatin (GelMA) (5), is used as hydrogel for the establishment of long term cell culture. To mimic NSCLC features, two adenocarcinoma cell lines, A549 and H1299, and normal lung fibroblast MRC-5 are selected. Cells at the density of 1,5x106 cells/ml are dispersed into the formulation and photopolymerized at l=405 nm. Then, biocompatibility of different GelMA hydrogel, are analyzed by MTT assay, calcein AM/PI staining and cytofluorimetric analysis.
Three different GelMA formulations (medium, high and very high), were obtained by modulating its degree of substitution. Methacrylation level and GelMA percentage determine pore size and matrix stiffness, which, in turns, influences cell behavior (6). Consequently, several combinations were tested to identify the preferable culture conditions. Since the aim of the work is the development of scaffolds which resemble tumor microenvironment features, A549 and H1299 cells were grown as mono-culture or co-culture with normal fibroblasts (MRC-5) to determine the importance of the cross-talk between these two cell types. Particularly, two ratios tumor : fibroblast were chosen: 1:1 and 1:2. Then, MTT analysis are performed after 1 – 6 - 14 days of culture. For A549 model, high 10% and very high 10% result the better matrices whereas, for H1299, the percentage must be increased at 12,5%. CalceinAM/PI staining revealed good cell viability at 6 - 14 days, confirming the absence of matrix toxicity. Moreover, the long term ratio of the two cell types was verified by cytofluorimetric analysis.
Our results support the feasibility of using GelMA as matrix for 3D long term cell culture. Further analysis will be done to dissect the crosstalk that interplays between the two cell types and the matrix, which will be compared with the one of in-vivo model. Moreover, since GelMA is a promising ink for 3D-bioprinting, the system will be implemented by the introduction of a 3D printed-microfluidic, which will mimic the physiological vasculature.
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