The in-house derived hMSCs used throughout this study were first characterized through analysis of their cell surface antigen
profiles and their differentiation ability toward adipogenic and osteogenic lineages (see Figure 1). These cells demonstrated the appropriate CD90+ , CD105+ , CD73+ , CD34- , CD45- , CD11b- cell surface antigen profile and the ability to differentiate toward all three lineages.
Figure 2: (a) Human mesenchymal stem cells (hMSCs) can be either expanded first on T-flasks or thawed and directly seeded
into a Mobius CellReady 3-L bioreactor with microcarriers; (b) Morphology of hMSCs on microcarriers after expansion in the
bioreactor (Upper: CellTracker Green; Lower: bright field).
For 3D hMSC expansion, cells can either be expanded first on T-flasks, or thawed and directly seeded in the Mobius CellReady
3-L Bioreactor on microcarriers (see Figure 2). To determine the best parameters for 3D expansion, various cell and microcarrier seeding densities were investigated (see
Figure 3). Optimal cell seeding density was demonstrated at 5 million cells/L and optimal microcarrier seeding density was demonstrated
at a concentration of 15 g/L.
Figure 3: (a) Human mesenchymal stem cell (hMSC) attachment affinity 24 h; (b) Microcarrier concentration of 15 g/L demonstrates
maximum hMSC expansion after 11 d.
To study the effects of aeration on the growth of hMSCs in the bioreactor, 5 million cells/L were seeded per bioreactor, allowed
to grow for 6 d, and then various sparge strategies were executed, including aeration through minisparge and open-tube at
both high and low air-flow rates (see Figure 4). By day 12, cell growth reached a maximum in the control bioreactor, which could either be due to shear force by air bubbles
or a preferred lower oxygen culture.
Figure 4: (a) Sparge strategies; (b) Cell number reached a maximum in the control bioreactor; (C) dO2 level remained fairly constant except in the control bioreactor.
The growth rate of hMSCs was then compared in 2D and 3D cultures (see Figure 5). hMSCs not only proliferated on microcarriers in the bioreactor, but also colonized empty microcarriers. After a one to
three day lag phase, hMSCs expanded quickly in the bioreactor and reached maximum cell number (approximately 600 million cells)
in 12 d, which is roughly 50% greater than expected from growth in 2D culture after 14 d. Flow cytometry data illustrated
no difference in cell surface antigen expression between hMSCs expanded via 2D or 3D culture.
Figure 5: (a) Human mesenchymal stem cells (hMSCs) not only proliferated on initial seeded microcarriers but also colonized
empty microcarriers; (b) hMSCs expanded quickly in the bioreactor and reached maximum cell number (~600 million cells) in
12 d. The grey bar represents the expected cell number from a 10-Stack CellSTACK in 14 d; (C) No difference in cell surface
antigen expression between hMSCs expanded in CellSTACK and a bioreactor with microcarriers (dotted line: isotype control;
dark blue line: bioreactor; light blue line: CellSTACK).
hMSCs were characterized following expansion for two weeks in 2D and 3D cultures (see Figure 6). Expanded cells were exposed to differentiation media toward adipogenic and osteogenic lineages, cytogenetic analysis, and
cell functional analysis. Regardless of method of expansion, expanded hMSCs demonstrated multipotent differentiation abilities,
normal male karyotype, and the ability to secrete important cytokines, including interferon-gamma, interleukin-6 and interleukin-8.
Figure 6: (a) After expansion for two weeks in CellSTACK and Mobius CellReady 3-L bioreactor, human mesenchymal stem cells
(hMSCs) demonstrate multipotent differentiation abilities; (b) Expanded hMSCs retain normal male karyotype; (C) Induced expanded
hMSCs secrete important cytokines, including IFN-γ, IL-6, and IL-8.
Spent media (glutamine, glucose, lactic acid and NH4+) from 2D and 3D culture were analyzed and compared (see Figure 7). Additionally, multiple parameters that potentially affect hMSC expansion in 3D cultures were examined, including the concentration
of lactic acid in the media and feeding strategies (see Figure 7). The capacity to monitor and control the metabolism of cells in a bioreactor facilitates a feeding strategy to maximize
Figure 7: (a) Spent media (glutamine, glucose, lactic acid and NH4+) from bioreactor and CellSTACK were analyzed; (b) Parameters (e.g., concentration of lactic acid in the media, dO2, pH and feeding strategies) that affect hMSCs expansion in bioreactor were studied.
The effect of lactate and glucose concentration and pH on the expansion of hMSC on microcarriers in suspension culture was
tested (data not shown). hMSCs were able to grow under conditions of no glucose by consuming other nutrients in the medium;
however, the growth rate was reduced. hMSCs were able to expand under conditions of high glucose concentration (4.5 g/L) without
significantly reducing growth rate. High concentrations of lactate (> 1/6 g/L) inhibit hMSC growth on microcarriers, which
is consistent with 2D culture results. Growth was significantly reduced at low pH (< 6.8).