To show that the MSCs were still viable after five days of growth on collagen-coated microcarriers in the Mobius CellReady
3L bioreactor, the growth capability of MSCs harvested from the suspension culture was investigated. MSCs were withdrawn from
the bioreactor and trypsinized for 5 minutes at 37°C to remove the cells from the microcarriers. The suspension was passed
through a 100 μm sieve, and the cells were seeded on gelatin-coated tissue cultureware for propagation. The cell number versus
time and bright field images of these cells are depicted in Figure 6. Cells retained their ability to propagate for multiple
passages and there was no observable lag in growth after the cells were transitioned from the collagen microcarriers back
to 2D gelatin-coated flasks. The MSCs show the morphology that is classically indicative of MSCs and this can be observed
during various points of this culture in the bright field images. Another signal of healthy stem cells is the capacity of
sustained growth after microcarrier culture; this was observed, as expected. Our next studies aimed to use additional genotypic
and phenotypic analyses to confirm that these cells were definitively MSCs.
Figure 6: (A) Cells removed from the microcarriers retained the ability to propagate when seeded onto gelatin coated flasks;
(B) Cells removed from the microcarriers had consistent morphology.
In addition to growth and morphology, MSCs typically express several markers at high levels, while not expressing others.
To determine whether the MSCs remain MSCs after the 3-L bioreactor culture, mRNA from the cells was isolated and the expression
levels of several genes were probed using RT-PCR. The MSC characterization genes that were investigated were positive markers
CD44, CD105, and CD90, and negative marker CD19. The relative expression of these genes was compared with samples of cells
derived from the 3-L single-use bioreactor, spinner flasks, static cells grown on 3D microcarriers, and gelatin-coated tissue
culture flasks (2D gel). Cells expanded in the 3-L bioreactor showed similar levels of gene expression of the MSC characterization
genes when compared with cells grown on gelatin-coated tissue culture flasks.
High RNA levels of the positive markers were observed for all culture conditions, while expression of markers chosen as negative
controls was not observed. The housekeeping gene GAPDH was present in similar levels between the samples. Differentiation
of the MSCs in the 3-L bioreactor would have caused the gene expression of the characteristic markers to change; that these
genes were expressing similar amounts of messenger RNA across the samples indicated that the cells remained undifferentiated.
A second method to characterize MSCs is to examine protein expression levels of MSC characterization surface proteins. Cells
taken from the 3-L bioreactor and dissociated from the beads were seeded onto gelatin-coated glass slides, along with control
MSCs; these cells were then exposed to antibodies specific for these surface proteins. Labeled secondary antibodies (red)
were incubated with these samples and then counterstained with nuclei marker DAPI (blue) so that immunofluorescent images
could be collected. CD44 and CD90 protein levels of cells taken from the bioreactor were comparable to the levels observed
in cells that were grown on gelatin in tissue culture flasks. The expression of negative control markers CD14 and CD19 was
not observed. Results from both MSC characterization experiments are shown in Figure 7. In subsequent studies, a broader array
of positive and negative surface markers and quantification of positive cells using flow cytometry can be used.
Figure 7: Mesenchymal stem cell characterization. (A) Similar RNA levels were found from all culture configurations; (B) Similar
levels of protein were also observed in mesenchymal stem cells grown in the 3-L single-use bioreactor compared with gelatin-coated
To show that the MSCs retained their ability to differentiate, a study was performed to coax MSCs towards adipocytes. MSCs
removed from microcarriers from the 3-L bioreactor culture and 2D control cultures were compared to evaluate their ability
to differentiate. The adipocyte differentiation was conducted over three weeks; cells were fixed and the differentiation was
assessed via oil red staining of lipid vacuoles present in adult adipose cells (see Figure 8). Cells from the Mobius CellReady
3L and gelatin control cultures both contained the characteristic vacuoles indicative of adipocytes, indicating that the cells
had some of the same differentiation capabilities as the control MSCs. Future experiments will include additional differentiation
protocols to study the differentiation of the cells to additional lineages.
Figure 8: : Lipid vacuoles of apidocytes were stained red after differentiating cells removed from the 3-L bioreactor and
cells grown on gelatin following a two-week differentiation.
Results indicate that MSCs are capable of being expanded in the 3-L bioreactor on collagen-coated microcarriers. Cells grown
in the bioreactor showed similar growth rates to control cells that are grown on tissue cultureware. The MSCs produced from
these cultures also express similar levels of characteristic stem cell genes as observed in the RNA and protein expression.
They also retain the ability to grow for multiple passages and to be differentiated to adipocytes at similar degrees as control
MSCs grown in 2D gelatin cultures.