Establishment of the culture condition to maintain the primary Human Colorectal Cancer microenvironment by using a 3D perfusion bioreactor

The screening and assessment of new cancer drugs has for many years carried out on conventional 2D monolayer cell culture that cannot represent the complexity of a tissue. On the other hand, the generation of patient derived xenograft (PDX) determines the loss of human-cancer associated stroma and the interaction with murine environment. In this context, the use of a 3D in-vitro systems based on human specimens that maintain the complexity of the in-vivo tissue is needed. Bioreactors that use a pump system to perfuse media directly through a scaffold are known as perfusion bioreactor. The possibility of direct perfusion allows active delivery of nutrient in complex systems. By continuously removing spent media and replacing it with new media, nutrient levels are maintained for optimal growing conditions and cell waste product is removed to avoid toxicity. Moreover perfusion-based bioreactor reduces mass transfer limitations, particularly in the central part of the scaffold. Thus, active perfusion would benefit cellular proliferation and viability. Using colorectal cancer tissue (CRC), we hypothesize that a 3D perfused scaffold-based bioreactor culture system would preserve both the transformed epithelial (EpCAM+) and the non-transformed stromal (Vimentin+ and CD45+) components and, also, keep cells proliferating (Ki67+). We demonstrated the possibility of using a perfusion-based bioreactor system to maintain alive and proliferating freshly surgical excised CRC tissue fragments in porous 3D collagen type-1 scaffolds. After 10 days of culture the tissue partially maintained its original architecture with typical neoplastic disorganization. Phenotypic analysis confirmed that expanded tissues included epithelial, stromal and hematopoietic cells. Tumor cell proliferation, as provide by Ki67 staining, was assessed. Taken together, our results indicate that culture of primary tumor fragments within perfused bioreactors can be successfully achieved over a short-time period allowing the preservation of the diverse cellular components of the CRC specimens. These ex-vivo generated tissues might mirror features of the original tumor more effectively than 2D or 3D static cultures, and of PDX, thus possibly representing useful tools for drug testing and for the evaluation of sensitivity to chemotherapies for personalized medicine