(601c) Designing Cryoprotectant Addition and Removal Protocols for Vitrification of Tissue-Engineered Constructs Using a Mathematical Model

In the field of Tissue Engineering, many different technologies are being investigated with the goal of bringing replacement tissues from the benchtop to the clinic. One of the most critical technologies, cryopreservation, is often overlooked. Cryopreservation will be necessary for the storage of any natural or artificial tissue. Any tissue headed for clinical use will require sterility and quality control testing as well as transport. It will be necessary to maintain both the cells and extracellular matrix during this time. The most common type of cryopreservation, conventional freezing, allows for ice formation, which may be damaging to the extracellular matrix. More recently, vitrification has been used as a cryopreservation method that completely avoids ice formation. Vitrification utilizes high concentrations of cryoprotectants (CPAs) and rapid cooling to achieve a vitreous, or glassy, state. Because of the high concentrations necessary to achieve reliable vitrification, the design of addition and removal protocols is critical for the success of this process. Cytotoxicity and cellular osmotic excursions must be minimized so as to minimize cell death, but it is also important to ensure that a vitrifiable concentration of CPAs is present throughout the construct and within the cells in the construct. Currently, most protocols are based primarily on the prior experience of the researcher who makes a ?best guess? at a protocol that will maintain cellular osmotic excursions within a tolerable limit. This trial-and-error approach may allow coarse adjustment of protocols but does not allow for finer tuning that may result in better performing addition and removal procedures. A mathematical model would allow for fine tuning, as well as eliminate the cumbersome and time-consuming trial-and-error experiments. A mathematical model also allows much more freedom to investigate than experiment. More complex situations such as ramp or step changes in temperature or other conditions may be difficult to accomplish experimentally but may also prove to have significant benefit. Therefore, using a model should give insight into how beneficial complex conditions may be and whether they should be pursued experimentally. Previously, a model based on mass transport and cell permeability with experimentally determined parameters has been used to design addition and removal protocols (Mukherjee 2008). Here, we present an expanded version of this model, which additionally incorporates heat transfer and CPA cytotoxicity?both critical processes?in order to give a more complete picture of the effects of temperature and time on the addition and removal protocol. The hypothesis is that a model including non-isothermal conditions and CPA-mediated cytotoxicity will improve the design of addition and removal procedures by minimizing cell death. The basic structure of a model comprised of mass transport through the matrix and cell membrane, and heat transport through the tissue, can be used for a variety of natural and artificial tissues with the appropriate parameter values in each case. In this work, the model was applied to two tissue systems, which represent extreme situations in the CPA effective diffusivity through the matrix. The first system considered was an engineered tissue composed of murine insulinomas microencapsulated in hydrogel beads. In this, the effective diffusivity through the microcapsules was high, 80% of the bulk solution diffusivity, and the cells were suspended in the matrix. Several model parameters were experimentally determined and incorporated into the model. Cell permeability parameters were determined by measuring cell volume as a function of time after the abrupt addition of dimethyl sulfoxide (DMSO) or propanediol (PD) in a cell suspension. Effective diffusivities through the matrix were determined by adding a CPA into a suspension of capsules and measuring the decline in bulk concentration over time. Cytotoxicity studies were done with DMSO, PD and vitrification cocktails in order to determine the relationship between cell viability and concentration, temperature and time. The second system considered was natural articular cartilage which consists of chondrocytes in a dense extracellular matrix with an effective diffusivity only 30% of the bulk solution. Parameter values for this system were obtained from the literature. Currently, mathematical simulations have focused on situations that are easy to realize experimentally, as well as more complex non-isothermal conditions. Ramp changes are of particular interest as temperature affects both mass transfer and cytotoxicity and may change the overall performance of the addition and removal protocol. Simulating different situations also allows for determination of the sensitivity of the protocol. For example, the effects of over-exposure of CPAs differ based on the temperature and the CPA itself. Protocols using the less cytotoxic CPAs should be less sensitive to temperature changes. Unfortunately, the less cytotoxic CPAs also generally require higher concentrations to achieve a glassy state. Therefore, a balance must be found that minimizes the cytotoxicity while still ensuring vitrification. Use of the model to investigate these allows determination of the most critical factors for each system. Vitrification itself depends upon sufficient mass transfer and heating and cooling rates. The mathematical model describing the addition and removal of CPAs allows for design of protocols that achieve a sufficient concentration of CPAs throughout the construct and cells. This makes simulation of the addition and removal protocols critical to the successful vitrification of a tissue-engineered construct. The mathematical model was used to describe the performance of different addition and removal protocols for two vastly different constructs at isothermal and non-isothermal conditions. The results from these simulations allow insight into the future design and implementation of different vitrification solutions for use in the preservation of natural and engineered tissues.