"Introduction: The extracellular matrix (ECM) is a highly organize structure that represents the major structural component of the tumor microenvironment. The ECM is a non-cellular network of proteins that provide to the tumor cells the capability to invade, proliferate and migrate. Indeed, the tumor microenvironment represents a dynamic scaffold, and the study of the ECM structure represents the first step to be overcome to understand cellular, molecular, and immunologic mechanisms of tumor response and resistance. A lot of well-known biomaterials were proposed to mimic the 3D tumor structure, however the cell adhesion to this structure did not represent the real tumor microenvironment due to the lack of the complex structure and interaction typical of the tumor.
To overcome these limitations, in this work was proposed to recreate a 3D tumor structure using the decellularized matrix of a native tumor as a scaffold that is colonized by different cell types allowing the study of the interactions between the tumor cells and immune system or the screening of the drugs for targeted therapeutic models.
Methodology: Murine B16f10 melanoma cells were expanded and injected subcutaneously to C57Black/6 mice to create a tumor. After two weeks we harvested the tumor and started the decellularization process (United States Patent 6743574B1.2004) using a cryotube placed in a tilling system (static system) and a dynamic system using a bioreactor (U-CUP). At the decellularization process follows the recolonization of tumor matrix by cells (GFP-MDA and NHI3T3). After 7 days the recellularized tumor were recovered and histological analysis was performed.
Results: The decellularization process removed all the cells from the tissue demonstrated by the absence of DAPI positivity in the slides and was not detrimental to the extracellular fibers, as confirmed by trichrome staining and collagen type VI. The decellularization process developed with both the methods was well.
To test the efficacy of the decellularized tissue as scaffolds for 3D culture, we executed different recellularization experiments. After 7 days of culture, in static system the cell attachment was limited just to the border of the tissue. In dynamic condition the cells appear more integrated into the decellularized scaffold. These differences were due to the collapse of the structure in static condition demonstrated by structural analysis of the decellularized tissue compared to the fresh one that showed an increase of ECM. This hypothesis was confirmed with SEM images, that showed a collapse of the matrix caused by fibres twisted on themselves, generating a complex net, impenetrable from cells.
Conclusions: Our result demonstrate that the murine melanoma decellularized tissue is suitable for 3D in vitro tumor microenvironment reproducibility when is treated in dynamic system that recreates conditions favorable to cell penetration into the decellularized matrix. So, this method could provide a new method to obtain a patient-personalized tumor, adequate for drug screening that will eventually drive to the correct drug therapy for each patient."