The manual seeding of cells onto a biodegradable scaffold by pipetting is an effective method of cell seeding. However, the widespread use and ultimate clinical utility of this technique is limited by operator variability. This study was conducted to evaluate an operator-independent vacuum-seeding method for use in an upcoming clinical trial. Using bone marrow-derived mononuclear cells, we achieved seeding comparable to manually seeded scaffolds in terms of cellular attachment, distribution, and viability in vacuum-seeded grafts at vacuum pressures of -25 to -50 mmHg. In conclusion, we describe an operator-independent seeding method for use in the clinical setting.