Bladder cancer is the 10th most common cancer worldwide. About 20% of bladder cancers are muscle-invasive and are indication for a radical cystectomy. The use of ileal segment is a standard method for urinary diversion after bladder removal. Use of this method extend surgical procedure and lead to numerous metabolic complications. To overcome these side effects, tissue engineering methods can be utilized to create an artificial urinary conduit. The aim of this study was to develop a method of the tissue-engineered conduit construction for urinary diversion using dynamic bioreactor culture.
Tissue engineered tubular scaffolds were used for construction of the artificial urinary conduits (n=40). Conduits were seeded manually with adipose-derived mesenchymal stromal cells (~10x106/cm2) isolated from porcine fat tissue. Cells were grown on scaffold surface in a bioreactor for 6 days. During the culture pH, pO2 and temperature were continuously monitored. On the basis of culture parameters and glucose values measured each day medium feeding rate was established. ADSCs were seeded after 3rd or 4th passage, before seeding on scaffold mesenchymal stromal cell phenotype was analyzed using flow cytometry (positive markers: CD29, CD44, CD90; negative markers: CD11b, CD31, CD45). After end of experiment analysis of cell growth on scaffold surface was performed using scanning electron and confocal microscopy. Cells were detached from scaffold surface in order to check their number and changes in phenotype after dynamic culture in bioreactor.
ADSCs expressed typical for mesenchymal stem cells markers. Scanning electron microscopy revealed appropriate morphology of cells grew on artificial urinary conduit. Cells were elongated, spindle-shaped with good condition confirmed by production of microvesicles. Analysis using confocal microscopy showed that majority of cells seeded on artificial urinary conduit after the end of 6-days culture were viable. Analysis of culture medium and bioreactor vessel showed that only small number of cells (<10%) detached from tested material during dynamic culture in bioreactor. Cells detached from scaffold surface showed significant changes in cell phenotype in the case of CD29 antigen.
Dynamic culture of tissue-engineered scaffold seeded with ADSCs in bioreactor enables their proper growth. Application of such culture method may allow for construction of artificial organs for clinical application.
The present work was supported by the National Centre for Research and Development (NCBR) in Poland under Agreement no. STRATEGMED1/235368/8/NCBR/2014 (Smart AUCI Project) within the Strategic Programme STRATEGMED “Prevention practices and treatment of civilization diseases.”