To obtain a large number of human mesenchymal stem cell (hMSCs) for allograft, nonwoven fabrics (NWF) were used as a cell culture scaffold. NWF are three-dimensional fiber aggregates formed by heat bonding and have a high surface area for cell adhesion and elongation. Inoculation hMSC was done to a center of NWF disc (diameter, 15.1 mm; depth, 0.1 mm). A cell suspension inoculum had a volume of 10 µL, which was close to the void volume of the disc, and resulted in a high initial (24 h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10 μL of medium at 0–2 h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10 μL of the medium after 1 and 2 h of hMSC inoculation (0.15 × 103 cells/cm2 NWF-fiber) markedly increased the final cell density (21 days) from 2.48 to 7.45 × 103 cells/cm2 NWF-fiber and fold increase in cell density by 16–48-fold. In conclusion, the addition of an additional medium after inoculation made the initial cells distribution in NWF more uniform, which might result in higher final cell density.