A crucial factor in the tissue engineering of heart valves is an effective cell seeding with uniform cell distribution on biodegradable scaffolds to eventually form functional tissue constructs in vitro. In our laboratory, we developed a new cell-seeding device for optimal cell distribution for tissue-engineered heart valve constructs. In the present study, we developed a new cell-seeding device made of acrylic glass that is completely transparent (University Hospital Benjamin Franklin, Berlin, Germany). The polymeric heart valve scaffold is fixed in a small-volume, cylindrical cell-seeding chamber, and is surrounded by optimal cell suspension. The cell-seeding chamber is placed in a clear acrylic bowl so that it can be rotated in all directions to provide optimal cell distribution to all areas of the heart valve construct. We thus developed a highly isolated cell-seeding device that is driven by an independently developed rotating machine consisting of two independent motors (University Hospital Benjamin Franklin, Berlin, Germany). The whole system provides a high level of sterility and fits into a humidified incubator. Our newly developed cell-seeding device enables sterile conditions and optimal cell distribution for the controlled fabrication of autologous tissue-engineered heart valve constructs.