Natural Cryoprotective and Cytoprotective Agents in Cryopreservation: A Focus on Melatonin

Abstract

Cryoprotective and cytoprotective agents (Cytoprotective Agents) are fundamental components of the cryopreservation process. This review presents the essentials of the cryopreservation process by examining its drawbacks and the role of cytoprotective agents in protecting cell physiology. Natural cryoprotective and cytoprotective agents, such as antifreeze proteins, sugars and natural deep eutectic systems, have been compared with synthetic ones, addressing their mechanisms of action and efficacy of protection. The final part of this article focuses melatonin, a hormonal substance with antioxidant properties, and its emerging role as a cytoprotective agent for somatic cells and gametes, including ovarian tissue, spermatozoa and spermatogonial stem cells.

Keywords: DMSO; antioxidant; cryopreservation; cytoprotection; gametes; melatonin; stem cell.

Figure 1

Membrane alterations caused by cryopreservation. The cryopreservation process can cause alterations of the cellular membrane and specific changes in phospholipid organization and bilayer integrity, as well as modifications of the membrane ultrastructure. The oxidative damage of membrane lipids caused by reactive oxygen species (ROS) overproduction is a key underlying event in cellular damage consequent to cryopreservation. An excess of free radicals can derive, for example, from the Fenton reaction or other cellular oxidative stress processes that ultimately sustain lipid peroxidation and membrane impairment. These include an excess of Ca2⁺ influx into the cytoplasm from the extracellular environment and from the endoplasmic reticulum. Moreover, oxidative stress induces a rapid depolarization of the inner mitochondrial membrane potential and subsequent impairment of oxidative phosphorylation, as well as the release of cytochrome c, which is a main trigger of the intrinsic pathway of apoptotic cell death. Membrane lipid peroxidation may lead to the formation of eicosanoids associated with ferroptotic signaling. Ferroptosis is an iron-dependent cell death program that is prevented by the activity of glutathione peroxidase 4 (GPx4) [16].

Figure 2

Cryopreservation and CPA−induced cell injury. When cryopreservation is not carried out under optimal conditions, the cell can undergo a series of alterations. These can be the result of osmotic, mechanical or ROS-induced injuries. The osmotic injury is caused by cell dehydration, while the mechanical injury is due to the formation of ice crystals within the cell that cause its disruption. Finally, ROS overproduction triggers the suppression of antioxidant mechanisms and the oxidative damage of cell components, such as membrane lipids and nucleic acids. The utilization of CPA prior to the freezing process, can prevent these injuries, but at the same time, it can interfere with membrane proteins and phospholipid stability and function.

Figure 3

Melatonin cytoprotection function. Melatonin protects the cell from oxidative stress by inducing the expression of antioxidant genes (up arrow) such as glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT) and by increasing the total antioxidant capacity (T-AOC) in the presence of a stimulation of respiratory complexes activity. These effects are mediated by the stimulation of transcriptional proteins, such as nuclear factor erythroid 2 (Nrf2), the activity of which is particularly important for the CPA and stress adaptation effects of melatonin. Melatonin exerts antiapoptotic effects by decreasing (down arrow) Caspase 3 and Bax activity and by increasing (up arrow) the expression of B-cell lymphoma protein 2, e.g., Bcl-2. Other effects include the modulation of autophagy-related proteins. Moreover, melatonin acts directly on mitochondria, acting as an ROS scavenger, inhibiting the activity of MPTPs proteins, and activating UCP proteins. Upward and downward arrows correspond to “increased” and “decreased” expression or function of the reported parameters, respectively.