Controlled expansion and differentiation of mesenchymal stem cells in a microcarrier based stirred bioreactor
© Sart et al; licensee BioMed Central Ltd. 2011
Published: 22 November 2011
Cell based therapy requires great numbers of cells in a functional state permitting their in vivo implantation for the restoration of tissue homeostasis. Three main parameters are believed to be essential for such a purpose: an appropriate cell population, a suitable scaffold and appropriate physical / biochemical factors enabling proper expansion and in vitro cell differentiation. In recent years, mesenchymal stem cells (MSCs) have been attracting a lot of interest in this field, because of their differentiation potential and their trophic factor secretion abilities. The aim of this work is to perform a rational analysis of key factors involved in the efficient proliferation and differentiation of MSCs, in the context of a stirred microcarrier (MC)-based bioreactor.
Materials and methods
MSCs from external ear (E-MSCs) and bone marrow stroma (BM-MSCs) were extracted from Wistar rats, selected and cultivated on plastic dishes as previously described . The differentiation potential of E-MSCs along the adipogenic, osteogenic and chondrogenic pathways was established and assessed by staining (respectively Oil red O, von Kossa, Alcian Blue) as well as by RT-PCR analysis on marker genes of differentiation (respectively, C/EBPα, osteocalcin, aggrecan) as previously described . MCs (i.e. Cultispher-S, Cytodex-3, Cytopore-2) were prepared and cells were seeded as reported in . Cell counting was performed as follows: (1) after a full digestion of Cultispher-S by trypsin and using trypan blue exclusion counting method, as in ; (2) by crystal violet staining and nuclei counting for Cytodex-3, as in ; or (3) cell counting on Cytopore-2 was performed using MTT according to . The multiplication ratio was calculated as defined elsewhere . Cell cycle was analyzed by FACS, after cell staining with propidium iodide, as in . The actin organization was assed by confocal microscopy after cell staining with phalloidin-rhodamine.
MSC and microcarrier screening
E-MSCs were compared to the “gold standard” BM-MSCs on the basis of their proliferative properties. E-MSCs bear characteristics of progenitor cells: expression of CD73, Sca-1 and Notch-1, and also in vitro differentiation potential into mesodermal cell types such as adipocytes, chondrocytes and osteoblasts (not shown). Thus, these cells are in vitro functionally analogous to BM-MSCs. This cell population was further selected on the basis of its high intrinsic proliferation potential in monolayer culture, a clear advantage in the field of MSC bioprocessing (Table 1).
Multiplication ratios of E-MSCs and BM-MSCs on various culture systems. Multiplication ratios of E-MSCs and percentage of cells in S-phase at day 5 of a 7 day run, under various modes of culture
MSC and MC screening
0.4 ± 0.2
0.16 ± 0.1
-0.1 ± 0.12
-0.5 ± 0.04
2.4 ± 0.1
2 ± 0.3
0.7 ± 0.6
-0.6 ± 0.3
Maximization of MSC proliferation
E-MSCs on Cultispher-S
Mode of culture
% of cells in S-phase at day 5
1.5 ± 0.3
1.4 ± 0.3
2.6 ± 0.2
8 ± 1
3 ± 0.04
15 ± 1
Maximization of MSC proliferation in MC-based stirred bioreactors
According to Table 1, a batch culture mode was not sufficient to promote efficient E-MSC propagation on Cultispher-S. Conversely, cyclic fed-batch increased E-MSC growth span (Table 1). In addition, the use of high levels of growth factors (using a pulsed culture composed of 40% FBS and 1 ng/mL of TGFβ1) increased growth span (Table 1). The beneficial effects of cyclic fed-batch and pulsed culture were linked to a sustainment of the percentage of cells in S-phase of the cell cycle compared to batch culture (Table 1). These results underline that the control of growth factor levels in the medium is the key to maximize E-MSC growth extent.
Sequential proliferation and differentiation in MC-based stirred bioreactors, modulating actin organization
According to these data, it emerges that a correct control of MSC microenvironment in terms of MC composition is necessary to promote these cells’ efficient proliferation via proper actin organization. An efficient MC system must also be combined with adapted biochemical signaling. Indeed, the growth factor content is an essential factor to monitor towards improved MSC growth yield. As we observed that the differentiation step could not be combined with expansion, sequential phases are required for the mass scale production of a given MSC differentiated phenotype. Similarly to the expansion phase, the microenvironment to which MSCs are exposed modulates the efficiency of their differentiation. According to our results, the promotion of an adequate actin organization is one of the essential parameters enabling, in association to biochemical signaling from the differentiation medium, efficient MSC differentiation on MCs.
Taken together, these results open the way toward mass scale production of MSCs suitable for future in vivo applications.
This work was supported by a FSR grant of Université Catholique de Louvain, and an IN.WALLONIA-BRUSSELS INTERNATIONAL (IN.WBI) grant.
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