Abstract

Organ failure is one the biggest problems, doctors face every day. Many patients are not able to get a transplant, but even those who recieved it, may undergo painful process of organ rejection and be on the transplant waiting list again. Organ transplants shortage is severe problem in current medicine that has many ethical and medical issues. To solve this problem, the new direction in regenerative medicine was formed, organ prinitng. The main goal of organ printing is fabrication of organ replacements that would mimic the original ones in terms of complexity and functionality. By direct fabrication and maturation of organs in vitro, the problem of organ shortage can be solved, moreover, based on the advances in cell therapy, these organs can be printed with patients own cells, which will eliminte the problem of transplant rejection. Organ printing is multistep and complex process, composed of three main steps: tissue design, or theoretical modelling of replacement composition; tissue fabrication, or direct cell encapsulation and controlled assembly of building units; at last, tissue maturation to reach desirable functionality of the replacement. In the past decade, there was developed a variety of methods for the second step of organ printing, cell encapsulation, which is practicaly the main procedure for tissue fabrication. However, all these methods of cell encapsulation are complex and they might affect cells viability and functionality, which will result in changed tissue function. Thus, starting from the detailed analysis of the tissue fabrication process (encapsulation and assembly methods) the list of possible cell behavior affectors was composed. Based on this list, we designed a multistep protocol for coherent evaluation of cells behavior parameters, in terms of viability, functionality and activity during the tissue fabrication and its maturation steps. Three main materials were used for this study, two naturally (alginate and modified gelatin) and one synthetically (polyethilene glycol) derived polymers. The encapsulation step was performed with two different methods based on chemical or photo crosslinking of the material. Cell parameteres were evaluated on the molecular level for variety of parameters, including viability, activity, proliferation, stress markers expression, at last ability to adapt artificial environment to the cell functional niche with extracellular matrix markers expression, and proteoglycans. The innovation of the presented study consists in the developing a unique protocol for detailed cell functionality evaluation during the organ printing procedures. In fact, based on the conducted study, it was proved the safety of the encapsulation methods. Moreover, based on the cell parameters post-encapsulation, there was suggested the optimal time for tissue maturation for application of the fabricated structures in organ printing, but also in other fileds, like developmental and pathological biology, or drug screening. Eventully, a novel way of simple blocks assembly into 3D complex structures was developed and proved to be safe for cell parameters. At last, for the future research in organ printing, a detailed study over a cell behavior and functionality has to be performed for every fabrication method, what will improve the organ production process drastically.