Microcarrier Culture for Efficient Expansion and Osteogenic Differentiation of Human Fetal Mesenchymal Stem Cells

Abstract

Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo.

A Multicompartment Holder for Spinner Flasks Improves Expansion and Osteogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Scaffolds

“…3,7,33,36 Accordingly, in terms of metabolic activity, higher glucose consumption accompanied by higher lactic acid production was observed in dynamic vs. static conditions, although lactic acid values were always below growth-inhibitory concentrations. 3,7,25 Other authors have correlated MSCs growth with glucose and lactic concentrations, concluding that increasing cell numbers indicate result in lower glucose and higher lactic acid concentrations in the medium. 37 One of the most remarkable features of this dynamic culture system is its clear effect on spatial cell distribution within 3D structures.…”

“…This time was essential to prevent cell detachment and death (data not shown), and has been also referred by other authors like Hewitt et al 36 To prevent initial cell loss, other authors have opted to use initial low agitation speed, which was afterward increased. 7 MSCs 3D cultures in the multicompartment holder in spinner flasks were analyzed in what concerns cell viability, metabolic activity, proliferation, and spatial distribution. Although a high number of viable cells was observed, at day 14, the number of cells in the scaffolds' surface appears to be lower in dynamic than in static conditions, which lead us to hypothesize that cells could have migrated toward the center of the scaffolds or removed by the agitation, although this last hypothesis was less probable since the holder is expected to protect samples from stirring.…”

“…The results indicate that the dynamic culture system proposed here clearly promoted an earlier and more homogenous MSCs differentiation along the osteoblastic lineage, in accordance to literature using other dynamic culture systems. 35,39,40 It is known that MSCs differentiation is highly dependent on cell density and the establishment of cell-cell contact, 7,41 which might be favored when scaffold-colonization is more effective. We cannot exclude that MSCs osteogenic differentiation was also promoted by shear-stress related with the use of dynamic conditions.…”

“…2,3 It has been widely explored for the culture of suspension cells, and in some cases for attachment-dependent cells in the form of cell aggregates 4,5 or cell-loaded microcarriers. 3,[6][7][8][9] However, only a few studies described the use of spinner flasks for cell cultures in three-dimensional (3D) scaffolds. In those cases, the 3D constructs have been cultured in different ways: (1) maintained in free floating, 10,11 (2) threaded in stacks onto needles embedded in the stoppers of the spinner flask [12][13][14][15] or (3) directly fixed with clamps to the spinner's wall.…”

Bioactive surfaces from seaweed-derived alginates for the cultivation of human stem cells

“…The diameters of microcarriers vary from 120 to 300 μm (Kong et al 1999;Chen et al 2013;Goh et al 2013). In addition, microcarriers are functionalized with different surface features like charge or protein coatings.…”