Tissue engineering in cardiovascular surgery: MTT, a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshes1

Objective: Currently used valve substitutes for valve replacement have certain disadvantages that limit their long-term benefits such as poor durability, risks of infection, thromboebolism or rejection. A tissue engineered autologous valve composed of living tissue is expected to overcome these shortcomings with natural existing biological mechanisms for growth, repair, remodeling and development. The aim of the study was to improve cell seeding methods for developing tissue-engineered valve tissue. Methods: Human aortic myofibroblasts were seeded on polyglycolic acid (PGA) meshes. Cell attachment and growth of myofibroblasts on the PGA scaffolds with different seeding intervals were compared to determine an optimal seeding interval. In addition, scanning electron microscopy study of the seeded meshes was also performed to document tissue development. Results: There was a direct correlation between cell numbers assessed by direct counting and MTT(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltertra-zolium bromide) assay. Both attach rate and cell growth seeded on meshes with long intervals (24 and 36 h) were significantly higher than those seeded with short intervals (2 and 12 h) (P≪0.01), there was no significant difference between 24- and 36-h seeding interval. Scanning electron microscopy also documented more cell attachment with long seeding intervals resulting in a more solid tissue like structure. Conclusion: It is feasible to use human aortic myofibroblasts to develop a new functional tissue in vitro. Twenty-four hours is an optimal seeding interval for seeding human aortic myofibroblasts on PGA scaffolds and MTT test is a rapid and reliable quantitative method to assess the optimal human cell seeding on polymeric meshe