Band 5, Heft 2

Objectives Cold-acclimated organisms accumulate low molecular weight organic solutes such as sugar alcohols and soluble sugars. This study aimed to compare the efficacy of five sugar alcohols and 14 soluble sugars in stabilizing proteins under freezing, freeze-drying, and air-drying stresses. Materials and methods Glucose-6-Phosphate Dehydrogenase (G6PD) was used as the model protein. G6PD solutions with or without sugar alcohols and or sugars were subjected to freezing, freeze-drying, and air-drying stresses. The recovery of G6PD activity was measured to evaluate the protective efficacy of these compounds. Results Without stabilizers, freezing G6PD at -20°C or -80°C reduced enzyme activity by around 24%, while freeze-drying or air-drying reduced activity by 90%-95%. Among the five sugar alcohols tested, pinitol, quebrachitol and sorbitol stabilized G6PD, whereas mannitol and myo-inositol destabilized it. Among 14 soluble sugars, trehalose and raffinose showed slightly lower enzyme recovery after repeated freeze-thaw cycles at -20°C. Most soluble sugars (except arabinose and xylose) protected G6PD during freeze-drying, with di-, tri-, and oligosaccharides generally outperforming monosaccharides. During air-drying, lactose was ineffective, while arabinose, galactose, and xylose were detrimental. Conclusion The study highlights the diverse mechanisms of sugar alcohols and sugars in protein stabilization under stress, offering insights for formulating stable protein- and cell-based drugs.

Cryopreservation is a fundamental technology in biomedical research, regenerative medicine, and tissue engineering, enabling the long-term storage of cells, tissues, and organs. However, its effectiveness is limited by challenges such as intracellular ice formation, cryoprotectant toxicity, and reduced post-thaw viability. This review explores the crucial role of encapsulation in enhancing cryopreservation efficiency, with a focus on recent advances in materials science, bioengineering, and cryobiology. Emerging technologies, such as nanotechnology and stimuli-responsive polymers, are transforming encapsulation strategies. Innovations such as microfluidic systems offer precise control over cooling rates and cryoprotectant distribution, thereby mitigating conventional limitations. The review also addresses current obstacles related to scaling up encapsulation processes and ensuring the long-term biocompatibility and stability of preserved specimens. By synthesizing recent findings, this work provides a comprehensive resource for researchers and clinicians seeking to enhance biopreservation techniques and their applications in contemporary medicine and biotechnology. Finally, the review identifies critical knowledge gaps that must be addressed to improve the efficacy of cryopreservation strategies and advance their clinical translation.

Oocyte cryopreservation is an essential procedure in assisted reproductive technologies, aimed at preserving fertility, particularly for women undergoing IVF treatment or at risk of ovarian damage due to radiation, chemotherapy, or surgery. Despite its growing use, the survival and fertilization rates of cryopreserved oocytes remain suboptimal, largely due to cryo-induced oxidative stress. The generation of Reactive Oxygen Species (ROS) during freezing and thawing causes considerable damage to key cellular components, including proteins, lipids, DNA, and mitochondria. This oxidative stress compromises oocyte quality and reduces developmental potential. To address these challenges, the use of additives - especially antioxidants - has shown significant promise in mitigating oxidative damage. Enzymatic antioxidants such as Superoxide Dismutase (SOD) and Catalase (CAT), along with non-enzymatic antioxidants like glutathione, melatonin, and resveratrol, have demonstrated the ability to neutralize ROS and improve oocyte viability and developmental outcomes. Recent studies highlight the potential of Mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to effectively counteract mitochondrial ROS and enhance cellular defense mechanisms during cryopreservation. This review explores the cellular mechanisms of cryodamage, the role of oxidative stress in oocyte cryopreservation, and the potential of various antioxidant strategies to enhance oocyte survival and function. Developing effective antioxidant supplementation approaches may significantly improve the outcomes of cryopreservation in reproductive medicine.

Cryopreservation of living cells and tissues plays a vital role in biomedical research, clinical applications, biotechnology innovation, the development of new vaccines and drugs, and the conservation of endangered species. While significant technological breakthroughs have been achieved in cooling methods—particularly through vitrification for large tissue and organs—the lack of optimal rewarming technology remains a key obstacle to successful cryopreservation, especially for larger samples such as tissues and organs. The primary challenges during the warming process include non-uniformity heating and insufficient rewarming rates, which can lead to thermal stress-induced structural damage and lethal ice recrystallization, ultimately compromising the integrity and functionality of biological materials. In recent years, various advanced warming techniques have emerged, employing different energy conversion approaches to achieve volumetric heating while minimizing the risk of overheating. These techniques involve thermal, mechanical-thermal, and electromagnetic-thermal energy conversions. However, each method presents its own limitation. This review aims to summarize recent advancements in rewarming technologies for cryopreservation, with a focus on their mechanisms, applications, and the key challenges that must be addressed to enable broader adoption in medical and commercial contexts.

Sugars are widely recognized for their ability to stabilize cell membranes during dehydration. However, the precise mechanisms by which sugars interact with lipid bilayers remain unclear. This mini-review synthesizes four key hypotheses explaining sugar-mediated protection of dehydrated bilayers: the Water Replacement Hypothesis (WRH), Hydration Force Hypothesis (HFH), Headgroup Bridging Hypothesis (HBH), and Vitrification Hypothesis (VH). We argue that these mechanisms are not mutually exclusive but instead operate synergistically under different cellular contexts. We propose that these hypotheses are not mutually exclusive but likely operate under different cellular contexts. Future studies should prioritize the development of biologically realistic membrane models—incorporating diverse lipids, proteins, and asymmetric leaflets—to elucidate the exact roles and mechanisms of sugars in membrane stabilization. Such advancements will enhance our understanding of anhydrobiosis and inform cryopreservation strategies for mammalian cells.

Since the beginning of the 21st century, modern medical technology has advanced rapidly, and the cryomedicine has also seen significant progress. Notable developments include the application of cryomedicine in assisted reproduction and the cryopreservation of sperm, eggs and embryos, as well as the preservation of skin, fingers, and other isolated tissues. However, cryopreservation of large and complex tissues or organs remains highly challenging. In addition to the damage caused by the freezing and rewarming processes and the inherent complexity of tissues and organs, there is an urgent need to address issues related to damage detection and the investigation of injury mechanisms. It provides a retrospective analysis of existing methods for assessing tissue and organ viability. Although current techniques can detect damage to some extent, they tend to be relatively simple, time-consuming, and limited in their ability to provide timely and comprehensive assessments of viability. By summarizing and evaluating these approaches, our study aims to contribute to the improvement of viability detection methods and to promote further development in this critical area.

Organoids are three-dimensional structures derived from stem cells that recapitulate the gene expression profiles and functional characteristics of their tissue of origin, rendering them invaluable tools for disease modeling, drug screening, and precision medicine. Despite their promise, the widespread application of organoids is limited by extended culture durations and technical complexity. Cryopreservation has emerged as a critical strategy to overcome challenges related to the long-term storage and application of organoids, offering a range of preservation approaches tailored to organoid development. Nevertheless, conventional cryopreservation techniques encounter significant limitations when applied to organoids. To address these issues, the development of naturally derived, low-toxicity Cryoprotectants (CPAs), along with the optimization of CPA loading methods and refinement of cooling and warming protocols, is essential to mitigate cryoinjury. Looking forward, the comprehensive enhancement of cryopreservation technologies may facilitate the transformation of organoids into “off-the-shelf” products, enabling scalable production, batch standardization, and centralized distribution. Such advancements will lay the foundation for the establishment of Next-Generation Living Biobanks (NGLB).