Large Scale Expansion And Differentiation Of Human Mesenchymal Stromal Cells

Thermo Scientific™ Nunclon™ Delta treated Thermo Scientific™ Nunc™ Cell Factory™ systems is an effective format for the easy and rapid expansion of hMSCs. The Nunclon Delta surface is a fully synthetic chemical surface available with many options of sizes, shapes and surface area. hMSCs were removed from liquid nitrogen and plated directly onto the surfaces to be evaluated. T-25 cell culture flasks were used to determine the optimal seeding density prior to expansion of hMSC on larger formats. For expansion, 1-layer Nunc Cell Factory system, also with the Nunclon Delta surface, was seeded from a fresh cryovial. These cells were in turn used to seed a 4-layer Cell Factory systems. To ensure that the cells maintained multipotency after expansion, the expanded cells were seeded in Thermo Scientific Nunc Multidish 48-well plates and differentiated into osteoblasts or adipocytes. The seeding density chosen for expansion was 350 cells/ cm² because it was the lowest seeding concentration displaying good exponential growth. The reported expansion protocol was able to increase the cell number by 136-fold, however, since Cell Factory systems are available with up to 40 layers, the expansion potential is virtually limitless. Expansion of hMSCs on the Nunclon Delta surface using mesenchymal stem cell basal medium significantly enhances the osteogenic and adipogenic potential of hMSCs and number of differentiated cells relative to α-MEM medium. Additionally, the use of for cell passage gave a slightly higher yield of adipocytes compared to cells passaged using trypsin. The methodology presented here can be applied to the large scale expansion of other cell types for use in various applications using Cell Factory systems.