CryoLetters Volume 43 - Issue 1

Contents

CryoLetters 43 (1), 1-9 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110112

PERSPECTIVE: Temperature-dependent density and thermal expansion of cryoprotective agent

Prem K Solanki1 and Yoed Rabin1*

  1. Biothermal Technology Laboratory, Department of Mechanical Engineering, Carnegie Mellon University, Pittsburgh, PA 15213, USA

Abstract

Density is a key thermophysical property, affecting the response of materials to temperature changes in different ways, consistent with the phase of state. In fluids, temperature variation across the domain leads to colder areas being heavier than warmer areas, where buoyancy effects drive fluid flow and thereby increase heat transfer. This phenomenon is known as natural heat convection, which in general is a more efficient heat transfer mechanism than heat conduction in the absence of flow. In solids, where the material is locked in place, colder areas tend to contract while warmer areas tend to expand, leading the material to deform. When this deformation is constrained by the geometry of the domain and/or its container, mechanical stresses develop. This phenomenon is known as thermomechanical stress (or thermal stress), which can lead to structural damage such as fractures. The picture becomes even more complex during vitrification (or glass formation), where the material gradually changes from liquid to an amorphous solid over a significant temperature range. There, due to temperature variation across the domain, fluid mechanics and solid mechanics effects may coexist. It follows that characterization of the density as a function of temperature is crucial for the analyses of thermal, fluid, and mechanical effects during cryopreservation, with the goals of protocol planning, optimization, and preserving structural integrity. For this purpose, the current study focuses on the density of the material and its companion property of thermal expansion. Specifically, this paper reviews literature data on thermal expansion of cryoprotective agents (CPAs), discusses the mathematical relationship between thermal expansion and density, and presents new calculated density data. This study focuses on the CPA cocktails DP6, VS55, M22, and their key ingredients at various concentrations, including DMSO, propylene glycol, and formamide. Data for DP6 combined with a selection of synthetic ice modulators (SIMs) are further presented.

Keywords: density; synthetic ice modulators; thermal expansion; thermo-fluids; thermo-mechanics; vitrification

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Contents

CryoLetters 43 (1), 10-14 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110312

Effect of retinol in the vitrification medium on viability of vitrified ovine preantral follicles and expression of key developmental and apoptosis related genes

Kalpana Kaushik1, PSP. Gupta1*, P. Johnson1, Kavya Krishna1, S. Nandi1, S. Mondal1, J. Nikhil Kumar Tej1, Somoskoi Bence2 and Sandor Cseh2

  1. ICAR-National Institute of Animal Nutrition and Physiology, Bengaluru, India
  2. Department and Clinic of Obstetrics & Reproduction, University of Veterinary Science, Budapest, Hungary

Abstract

Background

Vitrification increases the production of reactive oxygen species (ROS) and the antioxidants in the vitrification solution may be beneficial by reducing excessive ROS production.

Objective

To evaluate the effect of retinol supplementation in vitrification solution on viability, apoptosis and development-related gene expression in vitrified sheep preantral follicles.

Materials and methods

Preantral follicles were isolated and randomly assigned into one of five groups: Group1, control fresh preantral follicles; Group 2, vitrification treatment; Group 3, vitrification + 2 μM retinol; Group 4, vitrification + 5 μM retinol; Group 5, vitrification + 10 μM retinol. Preantral follicles were placed in vitrification solutions and then plunged into liquid nitrogen (-196°C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue exclusion method and for gene expression.

Results

Vitrification with 5 μM retinol positively affected viability in comparison with vitrification without retinol (P < 0.05). There was no significant difference in viability among the Group 1, Group 2, Group 3 and Group 5. Expression of apoptotic genes BAX and Casp 3 were higher in the vitrified group, and vitrification with 5 μM retinol (Group 4) is comparable to the control fresh. Expressions of other apoptosis-related genes (i.e., BCL2L1, BAD and BAK) showed significant difference between the control fresh group and the vitrification group with 5 μM retinol. Expression of Annexin5 was also significantly different among various groups. The expression of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in the Group vitrified with 5 μM retinol.

Conclusion

The supplementation of 5 μM retinol in vitrification solution was beneficial for the vitrification of ovine preantral follicles.

Keywords: gene expression; preantral follicle; retinol; sheep; vitrification

Contents

CryoLetters 43 (1), 18-24 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110512

Cryopreservation of Pyramimonas mucifera

Karabo Mokoena*, Stuart Sym and David Mycock

  • School of Animal, Plant and Environmental Sciences, University of the Witwatersrand, Johannesburg, Private Bag 3, Wits, 2050, South Africa

Abstract

Background

It is important to appreciate microalgal diversity, better understand their ecosystem functioning and therefore implement conservation measures. The National Biodiversity Act of South Africa has a marine and coastal component which promotes such investigations.

Objective

To develop a cryostorage method for the marine unicellular algal species Pyramimonas mucifera.

Materials and methods

Cell viability, measured by propidium iodide, was used to determine both optimal exposure time to 10 % DMSO and survival following thawing of cryopreserved cells. Cryopreservation was achieved by a two-step cooling method.

Results and discussion

A 30-min DMSO exposure was selected for P. mucifera, as cells following such treatment retained cell shape and integrity. Although density was significantly reduced after cryopreservation, the surviving cells were capable of returning to viability levels equal to those of the untreated control (>90%).

Conclusion

Cultures of P. mucifera can be successfully cryopreserved and propidium iodide provides a useful indication of culture vitality.

Keywords: dimethyl sulphoxide; propidium iodide; Pyramimonas mucifera; two-step cooling method

Contents

CryoLetters 43 (1), 25-31 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110712

The pattern of cell survival in the pig liver following one freeze-thaw cryosurgery cycle

Jianfei Ye1, Franco Lugnani2*, Ling Yuan3, John GJ Zhao4, Diana Zhang4 and Boris Rubinsky5

  1. Department of Surgery, Tianjin Haibin People’s Hospital, Tianjin, P.R. China
  2. Hippocrates D.O.O, Divaca, Slovenia
  3. Tianjin Institute of Medical Science, Tianjin, P.R. China
  4. Medinux (Tianjin) Technologies Co., Ltd., Tianjin, P.R. China
  5. Department of Mechanical Engineering, University of California Berkeley, Berkeley, CA, USA

Abstract

Background

It is well established that in cryosurgery some cells can survive one freeze thaw cycle and that surviving cells are found at the margin of the frozen lesion. Numerous techniques are being developed to ensure the survival of frozen cells to the margin of the frozen region.

Objective

We thought that it would be of fundamental interest to observe the pattern of cell survival in a liver treated with one freeze-thaw cycle.

Materials and methods

We performed six ultrasound-guided single freeze-thaw cryosurgery procedures on the liver of four Landrace pigs, using two cryosurgery probes separated by 25 mm inserted in parallel. Treated organs were removed 24 hours after the cryosurgery and processed for histology with hematoxylin and eosin. The tissues were analyzed with a digital slice scanner.

Results

We found an unexpected pattern of cell survival; sheets of live cells, about 200 μm in width, that follow the network of interlobular connective tissue septae to a distance of several millimeter from the outer edge of the one freeze-thaw cycle cryosurgery treated lesion. The sheets of live cells surround lobule cores that have undergone complete coagulative necrosis. In addition, larger blood vessels, as far as 5 mm from the outer rim of the treated lesion, have a major and complex effect on cell survival with large areas of completely necrotic and completely alive cells intermixed.

Conclusion

This study may have value as a baseline for developing new cryosurgery protocols designed to ablate cells to the margin of the frozen lesion.

Keywords: cryosurgery; interlobular connective tissue septae; large blood vessels; tissue ablation

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Contents

CryoLetters 43 (1), 32-41 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110212

Cryoprotectant with a mitochondrial derived peptide, humanin, improves post-thaw quality of buffalo spermatozoa

Rahul Katiyar1*, Subrata Kumar Ghosh1*, Abhishek Kumar1, Megha Pande1, Amare Eshetu Gemeda1, Rupali Rautela1, Sanjeev Kumar Bhure2, S.K. Dhara3, Karikalan Mathesh4, Neeraj Srivastava1 and M.K. Patra1

  1. Division of Animal Reproduction,
  2. Division of Biochemistry,
  3. Division of Veterinary Biotechnology,
  4. Centre for Wildlife,
    ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, Uttar Pradesh, India

Abstract

Background

Semen cryopreservation results in deleterious effects on spermatozoa, including lipid peroxidation and a reduction in the total antioxidant components of seminal plasma. The ultimate outcome of these changes is a reduction in post-thaw semen quality. A mitochondrial derived peptide, humanin, a potent cytoprotective and antioxidant agent was used in the present study.

Objective

To evaluate the efficacy of a mitochondrial-derived peptide, humanin to improve the post-thaw quality of buffalo spermatozoa.

Materials and methods

A total of 18 ejaculates from three Murrah buffalo bulls (n=6 each) were collected. Each ejaculate was divided into four aliquots. The first aliquot was diluted with standard EYTG dilutor (Group I, control), whereas the other three aliquots were diluted with EYTG supplemented with 2 μM (Group II), 5 μM (Group III) and 10 μM humanin (Group IV), respectively. Semen was evaluated for physico-morphological and functional attributes such as progressive motility, viability, abnormality, acrosome integrity, plasmamembrane integrity of fresh samples, pre-freeze and post-thaw stages. Oxidative stress parameters [lipid peroxidation (LPO) and total antioxidant capacity (TAC)] were also measured at the pre-freeze and post-thaw stages.

Results

Humanin supplementation resulted in significantly higher (p≤0.05) post-thaw motility in all treatment groups and, higher (p≤0.05) viability in Groups III and IV in comparison to the control at the post-thaw stage. Spermatozoa with intact acrosome and plasma membrane were higher (p≤0.05) in Groups III and IV as compared to Groups I and II. The LPO levels at the post-thaw stage were found to be lower (p≤0.05) in all treatment groups versus the control group, whereas, higher (p≤0.05) TAC values were recorded in Groups III and IV in comparison to the control and Group II.

Conclusion

Humanin supplementation in the extender improved the freezabilty of buffalo spermatozoa.

Keywords: buffalo; cryopreservation; freezability; humanin; lipid peroxidation; semen

Contents

CryoLetters 43 (1), 42-49 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110412

Comparative molecular dynamics study on interaction of acetamide and glycerol with phospholipid bilayer

Youqing Kong, Bin Ye, Lei Yang, Xiangnong Liu and Cai Gao*

  • Department of Refrigeration and Cryogenics Engineering, Hefei University of Technology, Hefei, China

Abstract

Background

The exact mechanisms that acetamide and glycerol interact with cell membrane remains a matter of debate.

Objective

To investigate the microscopic interactions of acetamide and glycerol with phospholipid bilayers at various temperatures.

Materials and methods

Molecular dynamics simulations of a hydrated dipalmitoyl-phosphatidylcholine (DPPC) bilayer in the presence of glycerol and acetamide were performed. The system contains 128 lipids and about 700 cryoprotectant molecules, and simulations extended to 15 ns.

Results

When compared to glycerol, acetamide shows a stronger affinity with water rather than the lipid bilayer.

Conclusion

The knowledge of the mixing dynamics of present system helps to develop better cryoprotective formulas and to propose more optimal cooling/warming protocols.

Keywords: acetamide; cryoprotectant; glycerol; lipid bilayer; molecular dynamics

Contents

CryoLetters 43 (1), 50-57 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110612

Drug release, stability and efficiency of vitamin E loaded in liposomes for bovine sperm protection in cryopreservation medium

Lamia Taouzinet*12, Sofiane Fatmi123, Allaeddine Khellouf2, Malika Lahiani-Skiba3, Mohamed Skiba3 and Mokrane Iguer-Ouada2

  1. Technology Pharmaceutical Laboratory, Department of Processes Engineering, Faculty of Technology, Université de Bejaia, 06000 Bejaia, Algeria
  2. Associated Laboratory in Marine Ecosystems and Aquaculture, Faculty of Nature and Life Sciences, Université de Bejaia, 06000 Bejaia, Algeria
  3. Technology Pharmaceutical and Bio pharmaceutics Laboratory, UFR Medicine and Pharmacy, Rouen University, 22 Blvd. Gambetta, 76183, Rouen, France

Abstract

Background

It is known that a considerable number of drugs in clinical use or under development are water-insoluble drugs with poor bioavailability. The liposomal delivery system has drawn attention as one of the noteworthy approaches to increase both dissolution and absorption because of its biocompatibility and ability to encapsulate hydrophobic molecules in the lipid domain. However, several drawbacks have been reported, the most common is liposome structural instability.

Objective

To encapsulate alpha tocopherol into liposomes, to determine the new formulation stability and to study the drug-release of alpha tocopherol into the sperm cryopreservation medium.

Materials and methods

The liposomes prepared by an ethanol injection method were characterized for size stability, alpha tocopherol release and sperm motility tests.

Results

The prepared unilamellar vesicles had both narrow size distribution (around 99 nm) and a good physical and chemical stability at 4°C during 12 months. The liposomes did not release the vitamin E immediately, but retained the protectant for 24 hours, probably due to the rigidity of the liposomal fence which was reinforced by adding cholesterol. Then, all vitamin E molecules were released by 48 hours. Release was potentially by Fickian diffusion probably by the creation of mini-ducts due to both agitation and fence hydration. Moreover, semen motility treated with vitamin E liposome preparations was significantly improved compared to all other treatments (including commonly used sperm conservation media).

Conclusion

The stable vitamin E liposomes formulated in this work are a promising alternative for semen cryopreservation protection.

Keywords: alpha tocopherol; drug release; liposome; sperm cryopreservation; stability

Contents

CryoLetters 43 (1), 58-65 (2022)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr22110110812

Exposure of Calophyllum antillanum seeds to liquid nitrogen delays seedling emergence and decreases leaf anthraquinones

Ysmel Entensa1, Abel González-Morales1, Claudia Linares1, José Gerardo Vázquez1, Marcos Edel Martínez-Montero1, Byron E. Zevallos-Bravo2, Elliosha Hajari3, Oscar Vicente4, Ariel Villalobos-Olivera1 and José Carlos Lorenzo1*

  1. Laboratory for Plant Breeding and Conservation of Genetic Resources, Bioplant Centre, University of Ciego de Ávila, Ciego de Ávila, 69450, Cuba.
  2. Universidad Estatal del Sur de Manabí (UNESUM), Ecuador
  3. Plant Improvement; Agricultural Research Council-Tropical and Subtropical Crops; Private Bag X11208, Nelspruit, 1200, South Africa
  4. Universitat Politècnica de València, Institute for the Conservation and Improvement of Valencian Agrodiversity (COMAV), 46022 Valencia, Spain

Abstract

Background

Trees within the Calophyllum genus are multi-use trees that produce valuable wood, phytochemicals with a range of biological activities, and seed oil as a source of biodiesel. As a consequence of climate change, there is a need to develop strategies to preserve valuable plant genetic resources. Cryopreservation represents the most suitable option for the long-term storage of germplasm with minimal space and maintenance requirements.

Objective

To determine appropriate methods to cryopreserve seeds of Calophyllum antillanum and maintain secondary compound production.

Materials and methods

Seeds at a moisture content of 6% were used to evaluate two treatments: seeds immersed in liquid nitrogen and control seeds. Biosynthetic pathway efficiency was assessed post-cryo by determining anthraquinone contents in roots, stems and leaves following 30 and 75 d of seedling growth.

Results

The results indicated that exposure to liquid nitrogen delayed germination and seedling emergence for a period of up to 45 d after seed sowing. By 60 d of cultivation, no significant differences in plant growth were observed for cryostored and control seeds. The levels of anthraquinones, which were also measured in seeds and seedlings, were lower in plants regenerated from cryostored seeds following 30 d of growth, but there were no differences in roots and stems by 75 d of growth. Furthermore, the difference in leaf anthraquinone levels for cryopreserved and control seeds at 75 d was much smaller than at 30 d.

Conclusion

The low initial anthraquinone levels in emerging seedlings correlated with the initial slow growth of cryopreserved seeds.

Keywords: anthraquinones; Calophyllum antillanum; cryopreservation; ex situ conservation; genetic resources