Clinical demand for MSCs drives the need for development of robust large-scale production. This study demonstrates the utility
of a 3-L single-use bioreactor and collagen-coated microcarriers for the expansion of human bone marrow derived MSCs. This
proof of principle study is a demonstration of the potential for large-scale stem-cell scale-up using stirred bioreactors.
Optimal seeding density and oxygen levels were first established for MSC cultures. A seeding density of 5,000 MSCs per cm2 propagated more effectively than other seeding densities that were tested as did cells grown at low oxygen when compared
with normoxic conditions. Collagen-coated microcarriers led to the highest recovery of viable cells after static culture.
MSCs grown on microcarriers were able to propagate from one microcarrier to another while under stirred agitation in spinner
flasks and were able to retain their proliferation rates after two sequential passages.
MSCs propagated in the 3-L single-use bioreactor for five days, while doubling the working volume, with a 5.2-fold increase
in total cell number from 30 to 150 million cells. MSCs were capable of growing for multiple passages after being removed
from the bioreactor and showed similar levels of gene and protein expression of MSC characterization genes. After differentiating
to adipocytes, both the cells from the 3-L bioreactor and cells grown on gelatin contained lipid vacuoles that stained positively
red, confirming successful differentiation.
The next step to augment the results presented here will be to use multiple MSC lines from different donors to better characterize
the robustness and reproducibility of this process. Additionally, upcoming studies will use a wider and more quantitative
array of stem cell markers at both the RNA and protein levels to more fully characterize the cells produced using this method.
Daniel Kehoe, PhD,* is a research scientist in Process Solutions, Aletta Schnitzler, PhD, is a research scientist in Pharmaceutical Chemical Solutions, Janice Simler, PhD, is a senior research scientist in Process Solutions, Anthony DiLeo, PhD, is director, Corporate Technology Office, and Andrew Ball, PhD, is R&D manager, Systems Pharmacology, all at EMD Millipore, Billerica MA. *To whom correspondance should be addressed, firstname.lastname@example.org
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