Cryobiology最新文献

Osmotic stresses during cryoprotectant loading induce changes in cellular volume, leading to membrane damage or even cell death. Appropriate model-guided mitigation of these osmotic gradients during cryoprotectant loading is currently lacking, but would be highly beneficial in reducing viability loss during the loading process. To address this need, we reformulate the two-parameter formalism described by Jacobs and Stewart for cryoprotectant loading under the constraint of constant cell volume. We then derive simple, concise, analytic solutions to these equations, showing the transient extracellular permeating and nonpermeating cryoprotectant concentrations required to load a cell at constant volume, thus eliminating osmotic stresses during cryoprotectant loading. Additionally, we show analytic approximations of both ramp (linear) as well as step-wise loading and how one can use the hydraulic conductivity L_p, membrane permeability P_s, cell volume V_o, and osmotically inactive fraction to derive cryoprotectant loading protocols that minimize osmotic stress. We also present timescales for water and cryoprotectant transport which can be used to estimate loading times as well as L_p and P_s. We discuss how previous optimized loading strategies are inherently sensitive to parameter uncertainties and biological variability, increasing the likelihood of exceeding critical osmotic limits. By contrast, the proposed protocol provides a larger buffer against deviations, offering a safer and more robust solution to CPA loading. Importantly, we demonstrate that the volume-loss-free CPA loading protocols outlined in this paper occur on the same timescale as conventional and step-loading methods, suggesting that these protocols could be a safer, more efficient alternative for CPA loading.

For infertile patients who are unable to obtain sperm or prepubertal boys who require radiotherapy, testicular tissue freezing can be used for later transplantation and is a potentially effective method of preserving male fertility. Oxidative stress caused by the freezing process is an important cause of tissue damage. Procyanidin B2 (PCB2) is a polyphenolic natural compound widely distributed in plants that is known for its anti-inflammatory, anticancer, and neuroprotective properties, and its antioxidant capabilities are particularly noteworthy. Research has indicated that PCB2 exerts a protective effect on the reproductive system. However, its specific role in mitigating testicular tissue cryoinjury and the underlying mechanisms remain unclear. This study investigated whether adding PCB2 to a vitrified cryoprotective solution of mouse testicular tissue can alleviate the cryoinjury of testicular tissue and its possible mechanism. Our findings revealed that frozen mouse testicular tissue presented decreased cell viability and induced oxidative stress. Conversely, PCB2 effectively mitigated these adverse effects. In addition, PCB2 improved the tubular structural disorganization caused by freezing and increased the expression of proteins related to the junction function of Sertoli cells. Further experiments indicated that PCB2 activated the nuclear respiratory factor 2 (Nrf2)/heme oxygenase 1 (HO-1) antioxidant signaling pathway, increased the activity of downstream antioxidant enzymes, and improved mitochondrial kinetic homeostasis. Additionally, PCB2 ameliorated apoptosis while increasing the expression levels of key enzymes involved in testosterone synthesis. In summary, these results suggest that PCB2 attenuates damage to mouse testicular tissue during freezing by inhibiting oxidative stress and apoptosis.