There is an ongoing search for alternative tissue culture sera to engineer autologous tissues, since use of fetal bovine serum (FBS) is limited under Good Tissue Practice guidelines. We compared FBS with human platelet-lysate (PL) in media for in vitro cell culture. A threefold increase in duplication rate was found when human, saphenous vein-derived myofibroblasts were cultured in PL, whereas expression of marker proteins (alpha-smooth muscle actin, vimentin, desmin, and nonmuscle myosin heavy chain) was similar. Heat shock protein 47 mRNA expression was increased in PL cells, and type III collagen fibers were seen on PL-cell monolayers but not on cells cultured in FBS. These results imply a more efficient collagen fiber production. We also found higher levels of proteins involved in tissue repair and collagen remodeling, which could explain increased production of proteases and protease inhibitors by PL cells. Our findings indicate that PL is beneficial due to the increased duplication rate, in addition to the increased matrix production and remodeling. This could lead to production of strong tissue with properly organized collagen fibers, which is important for heart valve tissue engineering.