Protocol Development for Vitrification of Tissue-Engineered Cartilage

The success of tissue-engineered cartilage constructs (TECCs) as treatment options for healing cartilage defects can only be achieved if suitable preservation methods are found that can maintain their viability and function. Simply lowering the temperature of cells and tissues to below their freezing point invariably destroys them due to ice crystals that form in the water-laden cells and tissues. In addition, high salt concentrations that result from removal of water due to ice formation create a toxic imbalance. If the formation of ice crystals can be minimized while still halting metabolic activity of cells at low temperatures, then the viability and functionality of the preserved tissue may be maintained. Several important variables have been studied that affect cryopreservation of cells and tissues to maximize cell survival and tissue functionality: cryoprotectant agents, cryoprotectant concentrations, methods of introduction and elution, cooling and warming rates, and storage temperatures.[1–5] In 1949, Polge et al.[6] discovered a significant breakthrough in the role of glycerol as an intracellular cryoprotectant, which permeates cells to minimize intracellular ice formation. Later on, other intracellular cryoprotectants were discovered such as dimethylsulfoxide (Me2SO)[7] and extracellular cryoprotectants such as starches and sucrose, which protect cell membranes or act as osmotic buffers.[2,8] Formamide, acetamide and other such chemicals reduce the toxicity of cryoprotectants used in high concentration. Amides, in general, are weak cryoprotectants but improve cell viability when combined with cryoprotectants.[9] Combinations of intracellular and extracellular cryoprotectants can have additive or synergistic effects on cell viability upon rewarming of cryopreserved samples.[1] There are two main approaches to cryopreservation for cells, tissues, and organs. The first is the conventional freezing technique where up to 30% of cell water is substituted by a cryoprotective compound, usually Me2SO, permitting the storage of many types of cells in vitrified channels within ice.[1] Conventional freezing relies on slow cooling and warming rates. In contrast, vitrification uses high concentrations of cryoprotectants resulting in greater than 50% replacement of water in the cell or tissue. When combined with fast cooling and rewarming rates, both intracellular and extracellular ice formation is avoided. This method minimizes or prevents formation of ice, creating a glass-like material, while still halting metabolic activity and maintaining an osmotic balance between cells and their environment. The major limitation of vitrification, however, is the potential cytotoxicity of the high cryoprotectant concentrations employed.[1] Vitrification has been shown to provide effective preservation for a number of tissues including oocytes, early embryos, cartilage, skin, blood vessels (natural and engineered), and heart valves.[10–19] Furthermore, vitrified arterial blood vessels and tissue-engineered vascular grafts have been shown to maintain viscoelastic properties similar to fresh vascular tissues while demonstrating superior biomechanical performance when compared with frozen cryopreserved specimens.[15–17] Song et al. achieved significantly better viability by employing vitrification (80%) with a 55% (w/v) vitrification solution (VS), known as VS55, than with conventional freezing (12.8%) employing 0.6 mm thick rabbit articular cartilage.[20] Guan et al. demonstrated that 51% viability occurred when bovine cartilage was vitrified, and only 5% viability when bovine cartilage was preserved without cryoprotectants.[21] Subsequently, we found that VS55 was inadequate for preservation of thicker, large mammal cartilage and that better preservation was obtained using a more concentrated 83% vitrification formulation.[22, 23] Kuleshova et al. demonstrated the need to consider different approaches in the preservation of engineered tissues due to potential interactions between the scaffold material and tissue matrix.[24] There are three main variables affecting cell viability during cryopreservation by vitrification (cooling/warming rates, effective tissue permeation, and cytotoxicity) and protocols that work for one tissue type do not necessarily apply for other tissues or, in the case of cartilage, even the same tissue type from different species. The investigation of long-term storage is an important field in tissue engineering. The main reason for this study was to determine whether vitrification protocols developed for native articular cartilage were effective for tissue-engineered cartilage constructs. TECCs were harvested from a bioreactor, vitrified employing different protocols, and cell viability was assessed after rewarming and cryoprotectant removal. The end goal of this study was to employ a bioreactor, the perfusion concentric cylinder bioreactor, to aid in the cryopreservation of tissue-engineered cartilage.