(75a) Using Atmospheric Carbon Dioxide Environments to Improve Control of Stem Cell Expansions

Mesenchymal stem cells (MSCs) are found in many tissues in adult mammals, including but not limited to bone marrow. MSCs replenish lost cells of many different lineages throughout the life of an adult organism as well as possess the ability to modulate the immune system. Among other applications, MSCs have already been implemented to prevent graft-versus-host-disease in humans and in mice have improved myocardial compliance after infarction. As the potential applications of MSCs increase, improving expansion practices will be crucial. Already, fed-batch bioreactors allow volumetric scale-up and enable better control of mammalian cell cultures to improve batch-to-batch reproducibility; however, all stem cell medias currently utilize the bicarbonate pH buffering system, which requires elevated carbon dioxide levels that must be supplied from the gas phase along with crucial oxygen. While the ability to dynamically alter culture parameters is beneficial, the challenge of ensuring sufficient mixing of all these components can result in cyclic fluctuations in pH and dissolved oxygen that can temporarily upset culture conditions. The cells can be stressed in the time it takes for the system to rebalance itself, which can be especially detrimental to stem cells, which rely on precise culture conditions to ensure generation and expansion of a desired phenotype. Eliminating carbon dioxide as a requirement in pH control can simplify bioreactor control and minimize these disruptive shocks and improve the consistency with which stem cells can be expanded for both therapeutic and research applications.

This current research developed the first media for MSCs in atmospheric carbon dioxide conditions. A MOPS-HEPES buffered media enabled MSCs to achieve equivalent growth rates to MSCs grown in the standard, bicarbonate-buffered media in 5% carbon dioxide. All cell densities and growth rates between the two conditions were determined to be not significantly different after performing a t-test (p ≤ 0.05). After accomplishing equivalent growth kinetics, MSCs grown in each of the two conditions were differentiated along the osteogenic, adipogenic, and chondrogenic lineages. Imaging helped visualize differentiation and through staining of lineage-specific markers, we confirmed that MSCs grown in each condition successfully differentiated and also that undifferentiated MSCs were negative for differentiated markers. Real time qrt-PCR analysis confirmed gene expression levels for both the undifferentiated and differentiated MSCs were not significantly different between the custom and standard conditions (p ≤ 0.05). This study demonstrated the proof of principle that MSCs can be expanded effectively in atmospheric carbon dioxide. These results are encouraging, but further study is needed to confirm these results for suspension and large-scale stem cell expansions. In addition, serum-free medias that do not rely on the bicarbonate-buffering system will have to be developed before this method can be used to expand MSCs for use in human clinical treatments. By eliminating the requirement of carbon dioxide sparging, bioreactor control can be simplified and allow more consistent culture conditions to be maintained. This will grant the potential to improve yields and batch-to-batch reproducibility of stem cells, which rely on the ability to expand stem cells and ensure emergence of a precise phenotype, as well as other biotechnology-relevant cell lines such as Chinese hamster ovary (CHO) and NS0 cells.