Figure 5: hMSC proliferation rate during long-term expansion in di?erent animal-component-free culture systems
hMSC expansion on the FN1 motifs surface ensured a signifcantly faster
proliferation rate with a short doubling time and high population
doubling number in animal-component-free conditions. Results are
expressed as cumulative cell population doubling numbers over 10
successive passages on the FN1 motifs surface and two competitor
surfaces in an ACF culture system. Cells expanded on a TC treated
surface in a traditional serum-containing culture system served as a
reference. Results represent a mean of three independent cell counts
(n=3). For each experimental condition, the mean cell population
doubling time and the mean population doubling number per passage are
indicated.
Even after long-term expansion on the FN1 motifs surface, hMSC-BM maintained their specifc immunophenotype and continued to express high levels of mesenchymal markers such as CD105, CD73, CD90 and CD44, while lacking the expression of hematopoietic lineage markers (< 1% CD11b, CD34, CD45, CD79 α, and HLA-DR positive hMSCs in the entire cell population) according to the standard criteria for MSC identifcation recommended by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (Figure 6) [5]. The hMSC marker expression profle obtained on the FN1 motifs surface is comparable to other animal-component-free culture systems as well as a traditional serum-containing culture system (Table 1).
Table 1: Comparison of hMSC-specifc cell surface antigen expression profle after long-term expansion in di?erent culture systems.
Figure 6: Flow cytometry analysis
of hMSC markers after long-term expansion on Eppendorf CCCadvanced FN1
motifs surface in an animal-component-free environment
After long-term expansion across 10 successive passages on the FN1
motifs surface in animal-component-free medium, hMSCs showed a typical
expression profle of characteristic surface markers as evaluated by ?ow
cytometry analysis: more than 95% of cells of the total cell population
expressed the mesenchymal markers CD90, CD73, CD105 and CD44, while
lacking the expression of hematopoietic surface markers CD45, CD34,
CD11b, CD19 and HLA-DR. Unstained cells and isotype controls were
prepared in order to validate staining specifcity. Isotype controls (in
black) were used to determine the percentage of positive cells for each
of the markers of interest.
The functional multipotency of hMSCs after long-term ex pansion on the FN1 motifs surface in an ACF culture system was ultimately confrmed by their in vitro di?erentiation into cells of three mesoderm lineages, the osteogenic, adipogenic and chondrogenic lineages (Figure 7).
Figure 7: Multi-lineage
di?erentiation potential of hMSC-BM after long-term expansion on the
Eppendorf CCCadvanced FN1 motifs surface in an animal-component-free
environment
After 5 successive passages on the FN1 motifs surface under
animal-component-free conditions, hMSCs maintained their multi-lineage
di?erentiation potential as examined by specifc ?uorescent staining of
three specifc mesoderm lineages, respectively: hMSCs successfully
di?erentiated into osteogenic (Alizarin Red staining – 21 days
post-induction), adipogenic (Oil Red O staining – 21 days
post-induction) and chondrogenic (Alcian blue staining – 14 days
post-induction) lineages. Scale bar indicates 100 μm.
Conclusion
The ready-to-use Eppendorf CCCadvanced FN1 motifs sur face efciently
supports long-term hMSC-BM expansion in a completely defned,
animal-component-free culture system. During the expansion process
across 10 successive passages hMSCs maintain a stable and robust
proliferation rate with their characteristic morphology without signs of
replicative senescence. The undi?erentiated hMSCs retain their typical
marker expression profle as well as their multi-lineage mesodermal
di?erentiation potential. The suitability of the FN1 motifs surface to
support efcient hMSC-BM proliferation in di?erent commercial xeno-free
culture media facilitates the establishment of an animal-component-free
environment for hMSC cultivation.
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