Mathematical modeling of cryoprotectant addition and removal for the cryopreservation of engineered or natural tissues

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 ensure CPA equilibration throughout the sample while still minimizing CPA exposure and maximizing cell survival.

Benefits and Constraints of Vitrification Technologies for Cryopreservation of Bovine in vitro Fertilized Embryos

“…Cryopreservation strategies focus on the use of cryprotectants (CPAs) as supplements prior to cooling and the rates of cooling/ warming of the temperature of the sample [11]. Vitrification can eliminate intracellular ice crystals [12], but high concentrations of CPAs are required to achieve the vitreous state [13,14]. CPAs function in a dual capacity by decreasing freezing temperature and increasing viscosity so that instead of crystallizing the syrupy solution becomes an amorphous ice; it 'vitrifies' .…”

Advances in the slow freezing cryopreservation of microencapsulated cells

“…Although this method works for the thawing of microencapsulated cells, more complex investigations of CPA dilution after thawing should be investigated to improve cell function and diminish the CIOCD phenomena. In this regard, slow freezing could take advantage of vitrification, in which the thawing protocols have been deeply investigated to upgrade the thawing process [54]. Moving forward, cooling has been described extensively in the reviewed studies,…”

Fatigue damage to pig erythrocytes during repeated swelling and shrinkage

“…However, because of its time-consuming, complex operation; lower cell recovery rate; and mechanical stress damage [21,22], scientists are exploring the washing routines of the variety of washing machines [15]. In addition, various alternative approaches have been also proposed, mainly including the dialysis-based method [23][24][25] dilution-filtration method [26][27][28][29], and theoretical work has been performed [30]. In the literature, most of the work focuses on the effects of the diluent addition time, the environmental temperature, the CPA type, the CPA concentration and the cell polydispersity on the volume excursion, the osmotic tolerance limits, and the cell membrane integrity [6,18,[31][32][33][34][35].…”

“…The volume of erythrocytes was then recorded at5,10,15,20,25,30, and 45 min using an inverted microscope. The size of the erythrocytes was analyzed using Image-Pro Plus 6.…”