Indra Neil Mukherjee1, Yang Li1, Ying Ching Song2, and Athanassios Sambanis1. (1) Chemical & Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0100, (2) Department of Surgery and Institute of Molecular Medicine and Genetics, Medical College of Georgia, Medical College of Georgia, CA-2125, 1120 15th Street, Augusta, GA 30912
Long-term storage for off-the-shelf availability and quality and sterility control are key components of the manufacturing process of tissue engineered constructs at a clinically relevant scale. Cryopreservation can effectively address this need. The cryopreservation process is generally aided with the use of protective agents called cryoprotectants (CPA). Appropriate delivery and removal of these agents critically contribute to the final outcome of cell survival. CPA-induced cellular damage may arise from osmotic and cytotoxic effects of the CPAs, as these agents need to be used at appropriate concentrations, exposure times and temperature. In order to account for osmotic effects, CPAs are generally added and removed in a stepwise manner. For this, cell membrane permeability parameters and osmotic tolerances are determined and used in computer simulations to model cell volume excursions during stepwise CPA addition and removal to maintain volumes within their osmotic tolerances. In addition, cytotoxicity studies need to be performed to determine acceptable operating CPA concentrations and exposure times at different temperatures. Collectively, these experiments define the domain of conditions within which cryopreservation protocols need to operate for maximal cell viability at the end of the process. For cells in three-dimensional tissue constructs, the transport of mass and heat through the extracellular matrix also need to be accounted for.
We investigated these processes using mouse insulinoma cells by themselves and encapsulated in alginate/ poly-L-lysine/ alginate (APA) beads as a model tissue engineered pancreatic substitute. Experimentally measured cell membrane permeabilities and effective CPA diffusivities were used in a model of the three-dimensional substitute, treated as a two-compartment model, to generate possible cryopreservation protocols. The use of the model for natural and engineered tissues with different cellular properties and, more importantly, different matrix transport characteristics, will be discussed.