Combined automated culture system for tubular structure assembly and maturation for vascular tissue engineering

Abstract

Development of the robust yet practical tissue culture systems for vascular grafting is one of the major challenges for biomedical engineering. The following questions should be solved in priority: (1) flexibility of the tissue reservoir allowing dynamic stretching, (2) seeding of cells and extraction of tissues should be easy and safe, (3) system must allow morphological observation in real time, (4) maintenance of metabolic activity of tissues should be performed automatically. In our study, we attempted to solve these problems designing in vivo-like culture chamber made of PDMS, and developed an integrated system with a perfusion bioreactor and a small digital microscope. We developed disposable cell chamber with following qualities: transparent, autoclave-sterilizable, non cell-adhesive, and having low autofluorescence. The polyacetal mold made it possible to prepare a chamber hosting the tissue of the desired shape and size. In our case, a porous tube made from PTFE was fixed inside the chamber and tubular cell culture space was prepared for loading of preformed cell spheroids. Perfusion of the media within the porous tube continuously supplied the nutrients and oxygen to the spheroids. Growth and fusion of the cell spheroids inside the chamber can be observed real time by the small digital microscope and analyzed retrospectively by time-laps movies. We loaded batches up to 900 goat fibroblast spheroids into the system; growth, development and morphological fusion of the spheroids were followed out for 10 days. Removal of the tissue without disturbing its structural integrity was possible, and histological analysis revealed reasonable fusion degree between spheroids, and 72.5 % cell survival rate as estimated by TUNEL staining. In conclusion, our system has the basic performance necessary for culturing cell spheroids for tissue-engineered vascular graft.