ABSTRACT
Long-term storage of natural tissues or tissue-engineered constructs is critical to allow off-the-shelf availability. Vitrification is a method of cryopreservation that eliminates ice formation, as ice may be detrimental to the function of natural or bioartificial tissues. In order to achieve the vitreous state, high concentrations of CPAs must be added and later removed. The high concentrations may be deleterious to cells as the CPAs are cytotoxic and single-step addition or removal will result in excessive osmotic excursions and cell death. A previously described mathematical model accounting for the mass transfer of CPAs through the sample matrix and cell membrane was expanded to incorporate heat transfer and CPA cytotoxicity. Simulations were performed for two systems, an encapsulated system of insulin-secreting cells and articular cartilage, each with different transport properties, geometry and size. Cytotoxicity and mass transfer are dependent on temperature, with a higher temperature allowing more rapid mass transfer but also causing increased cytotoxicity. The effects of temperature are exacerbated for articular cartilage, which has larger dimensions and slower mass transport through the matrix. Simulations indicate that addition and removal at 4°C is preferable to 25°C, as cell death is higher at 25°C due to increased cytotoxicity in spite of the faster mass transport. Additionally, the model indicates that less cytotoxic CPAs, especially at high temperature, would significantly improve the cryopreservation outcome. Overall, the mathematical model allows the design of addition and removal protocols that insure CPA equilibration throughout the sample while still minimizing CPA exposure and maximizing cell survival.
Subject(s)
Cryopreservation/methods , Cryoprotective Agents , Vitrification , Biological Transport , Cartilage, Articular/metabolism , Cell Membrane Permeability , Cell Survival , Cryoprotective Agents/adverse effects , Cryoprotective Agents/metabolism , Cryoprotective Agents/toxicity , Humans , Ice , Insulin-Secreting Cells/metabolism , Models, Theoretical , Temperature , Tissue EngineeringABSTRACT
Development of optimal cryopreservation protocols requires delivery and removal of cryoprotective agents (CPAs) in such a way that negative osmotic and cytotoxic effects on cells are minimized. This is especially true for vitrification, where high CPA concentrations are employed. In this study, we report on the determination of cell membrane permeability parameters for water (L(p)) and solute (P(s)), and on the design and experimental verification of CPA addition and removal protocols at vitrification-relevant concentrations for a murine insulinoma cell line, betaTC-tet cells. Using membrane permeability values and osmotic tolerance limits, mathematical modeling and computer simulations were used to design CPA addition and removal protocols at high concentrations. The cytotoxic effects of CPAs were also evaluated. Cells were able to tolerate the addition and removal of 2.5M dimethyl sulfoxide (DMSO) and 2.5M 1,2 propanediol (PD) in single steps, but required multi-step addition and removal with 3.0M DMSO, 3.0M PD, and a vitrification-relevant concentration of 3.0M DMSO+3.0M PD. Cytotoxicity studies revealed that betaTC-tet cells were able to tolerate the presence of single component 6.0M DMSO and 6.0M PD and to a lesser extent 3.0M DMSO+3.0M PD. These results determine the time and concentration domain of CPA exposure that cells can tolerate and are essential for designing cryopreservation protocols for free cells as well as cells in engineered tissues.
