Date of Award


Degree Name

MS in Biomedical Engineering


Biomedical Engineering


College of Engineering


Kristen O'Halloran Cardinal

Advisor Department

Biomedical Engineering

Advisor College

College of Engineering


Cell therapy is the administration of living cells into a patient to prevent or treat a variety of diseases and illnesses. The cell therapy industry is rapidly expanding, and continued research is necessary for manufacturing safe and effective therapies. Although cell therapy manufacturing generally involves cryopreservation processes for storage, there are limited standards for cryopreservation processes and assays required to evaluate cell therapies post-thaw, and limited understanding exists about how a recovery period post-thaw could impact cell health. The overall goal of this thesis was to evaluate the effect of cryopreservation and potential subsequent recovery time on cell viability.

Because of cell therapy research’s novelty within Dr. Kristen Cardinal’s Tissue Engineering Laboratory at Cal Poly, the first aim of this thesis was to establish and implement protocols for cell therapy applicable cell types and evaluation assays. Methods for thawing, culturing, and freezing adipose-derived mesenchymal stem cells (hMSC-ATs) and RAW 264.7 murine macrophages were designed and optimized. Protocols for three viability assays – Trypan Blue, alamarBlue, and MT Cell Viability – were developed for both cell types. The results of the first aim showed successful establishment of new cell types for cell therapy research and development of cell viability assays.

The second aim of this thesis was to establish baseline viabilities for hMSC-ATs and RAW 264.7s post-thaw and assess the impact of protocol timing on cell viability. Documentation of baseline viabilities is important for future hMSC-AT and RAW 264.7 cell therapy-based work. The results of this aim demonstrated cell viabilities of hMSC-ATs in the 90-95% range and of RAW 264.7s in the 36-46% range immediately post-thaw. After a period of culture, cell viabilities of hMSC-ATs were in the 95-99% range and RAW 264.7s were in the 55-78% range. Protocol timing showed

no significant effect on viability of hMSC-ATs and RAW 264.7s up to 100 min. at ambient conditions.

The third aim of this thesis was to assess the impact of recovery time, cell density, and the culture chamber on viability of hMSC-ATs and RAW 264.7s. Trypan Blue viability measurements were taken over a 24 hr. recovery period at different cell densities and culture chambers (petri dish or conical). The results of this aim showed a statistical decrease in hMSC-AT viability after 1 hr. of recovery time and no statistical significance for RAW 264.7s. Densities did not impact either cell types’ viability. Culturing cells in a conical post-thaw showed statistically higher viability for both hMSC-ATs and RAW 264.7s than cells in a petri dish.

Overall, the work of this thesis developed methods for continued cell therapy research and assessment of recovery time in the Tissue Engineering Laboratory. The data provided a foundation for evaluation of freshly thawed cells and recovery time, which will hopefully lead to more cell therapy research at Cal Poly and ultimately improve cell therapy efficaciousness.