Protocol of mesenchymal stem cell inoculation to nonwoven fabric scaffold

Abstract

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\xa0mm; depth, 0.1\xa0mm). A cell suspension inoculum had a volume of 10\xa0µL, which was close to the void volume of the disc, and resulted in a high initial (24\xa0h) cell adhesion efficiency. Use of green fluorescent protein expressing rat MSCs and fluorescence microscopy revealed that adding an additional 10\xa0μL of medium at 0–2\xa0h after the cell inoculation made the initial horizontal distribution of cells in the NWF disc more uniform. Addition of 10\xa0μL of the medium after 1 and 2\xa0h of hMSC inoculation (0.15\u2009×\u2009103 cells/cm2 NWF-fiber) markedly increased the final cell density (21\xa0days) from 2.48 to 7.45\u2009×\u2009103 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.