CryoLetters Volume 46 - Issue 5

Contents

CryoLetters 46 (5), 286-300 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110112

PERSPECTIVE: Potential impact of the forthcoming European Union substances of human origin regulation on the programme of allotransplantation of cryopreserved vascular tissue grafts

Pavel Měřička1,11*, Miroslava Jandová1,2, Jiří Gregor1, Pavla Paterová3, Šárka Rudolfová1, Pavel Navrátil Jr.4, Igor Guňka5, Roman Keleman6, Libor Janoušek7,8, Barry Fuller9 and Miroslav Špaček10

  1. Tissue Bank, University Hospital Hradec Králové, Czech Republic.
  2. Department of Histology and Embryology, Faculty of Medicine in Hradec Králové, Charles University, Czech Republic.
  3. Department of Clinical Microbiology, Charles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic.
  4. Department of Urology, Charles University in Prague, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic.
  5. Department of Surgery, Charles University, Faculty of Medicine in Hradec Králové and University Hospital Hradec Králové, Czech Republic.
  6. Department of Organ Retrieval and Transplantation Databases, Institute of Clinical and Experimental Medicine, Prague, Czech Republic.
  7. Department of Transplantation Surgery, Institute of Clinical and Experimental Medicine, Prague, Czech Republic.
  8. First Faculty of Medicine, Charles University, Prague, Czech Republic.
  9. Division of Surgery & Interventional Sciences, UCL Medical School, London, UK.
  10. Second Department of Surgery – Department of Cardiovascular Surgery General University Hospital and First Faculty of Medicine, Charles University, Prague, Czech Republic.
  11. Faculty of Medicine in Hradec Králové, Czech Republic.

Abstract

The Czech vascular tissue transplantation programme has operated under the conditions set by European Union (EU) directives since 2013. The aim of this analysis was to assess the impact of the new EU regulation on it and to analyse potential non-conformance. The impact of the new regulation was assessed based on the texts of the regulation and the recommendations of the European Directorate for Quality of Medicines and Healthcare (EDQM) and the European Centre for Disease Control (ECDC). Areas requiring improvements in the future were identified. The non-compliance analysis was performed on a group of 30 vascular tissue donations. We found that the areas requiring attention are: 1) inclusion of West Nile Fever testing of donors; 2) more frequent particle counting and aeroscopy during processing; 3) determination of shelf life on the basis of a cryostability study; 4) implementation of the ISBT 128 standards for product labelling; 5) use of European Good Tissue Practice II (EURO GTP II) and Microbiologcal Risk Critical Assessment (MiRCA) tools for risk assessment; 6) implementation of the clinical outcome monitoring plan; and 7) staff participation in EDQM e-learning courses. The analysis showed a high level of compliance with the EDQM microbiological criteria for tissue procurement and processing. The temperature during processing complied with the EDQM limits, while relatively high non-compliance with the relative humidity limits occurred. In conclusion, the new regulation, which should be implemented by 2027, represents an important step towards improving vascular tissue transplantation safety. Our plans aim to meet its requirements within this period.

Keywords: allotransplantation; cryopreservation; European Union directive; European Union regulation; substances of human origin; tissue establishment; vascular grafts.

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Contents

CryoLetters 46 (5), 301-310 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110312

Progressive techniques for wild mammal oocyte cryopreservation and recovery

Maria Valéria de Oliveira Santos, Sueli de Oliveira Lima, Antonia Beatriz Mendonça Pereira, Ana Livia Rocha Rodrigues and Alexsandra Fernandes Pereira*

  • Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoró, RN, Brazil.

Abstract

The loss of wild biodiversity has encouraged the development of assisted reproduction techniques, such as in vitro fertilization, intracytoplasmic sperm injection and somatic cell nuclear transfer. The recovery and cryopreservation of oocytes derived from antral follicles are important steps to ensure the efficiency of these techniques. The capability of embryonic development depends on the success of these steps, especially for wild mammals, whose availability is limited, and accessibility is low. In general, it is possible to obtain from a few units to dozens of oocytes depending on the species and collection technique used. The cryopreservation protocols for domestic species are used as a starting point for studies on phylogenetically close wild species; however, the results are modest. Studies show viability after thawing ranging from 37-70% and metaphase II does not exceed 42%. Currently, the main goal is to optimize these results by improving or comparing different cryopreservation and recovery methods. The susceptibility of oocytes to injury during cryopreservation shows inter-specific differences. Optimal methods differ even between species that belong to the same phylogenetic group. Moreover, vitrification has been a promising technique for establishing biobanks. Protocols for evaluating the efficiency of these processes have been studied in several species. Therefore, this review discusses the use and development status of oocyte recovery and cryopreservation techniques in wild mammals and evaluates the success and perspectives of conservation of immature or matured oocytes in different species. The factors that impact successful oocyte cryopreservation must be established for each species.

Keywords: gamete rescue; in vitro maturation; vitrification; wildlife conservation.

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Contents

CryoLetters 46 (5), 311-320 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110512

Changes in biological activity of placenta extracts depend on its storage temperature

Olena Bobrova1,2*, Stanislav Narozhnyi1, Svetlana Repina1, Kateryna Rozanova1 and Oleg Nardid1

  1. Department of Cryobiophysics, Institute for Problems of Cryobiology and Cryomedicine NAS of Ukraine, Kharkiv, 61016, Ukraine.
  2. Physiology and Cryobiology of Plants, Czech Agrifood Research Center, Praha, 161 00, Czech Republic.

Abstract

Background

Cryopreservation is a critical method for the long-term storage of biological tissues, including human placental extracts (HPEs). Preserving the biological activity of these tissues during storage is essential for medical applications, particularly for their anti-inflammatory and anti-aggregation properties. The impact of storage temperature on the biological efficacy of HPEs remains a crucial area of study.

Objective

To investigate the effect of different cryopreservation temperatures on the preservation of biological activities in human placental extracts and their fractions, with a particular focus on their effects on erythrocyte thermal hemolysis and ADP-induced platelet aggregation.

Materials and methods

Human placentas were collected after normal pregnancies from healthy parturients and cryopreserved at -20°C, -80°C, and -196°C for up to six months. Placental extracts and fractions were analyzed for their impact on erythrocyte thermal stability and platelet aggregation using spectrophotometric methods. Calorimetric studies were conducted to assess the phase state of placenta tissues at low temperatures.

Results

Extracts from placentas stored at -20°C for six months lost their ability to reduce erythrocyte hemolysis and platelet aggregation. Storage at -80°C and -196°C preserved these activities for at least six months. Calorimetric analysis revealed the presence of unfrozen water in placenta tissues at -20°C, contributing to the degradation of biological activity.

Conclusion

Cryopreservation at -80°C or lower effectively preserves the biological activity of placental extracts for extended periods, making these temperatures ideal for long-term storage in medical applications.

Keywords: anti-inflammatory activity; cryopreservation; erythrocyte thermal hemolysis; non-crystallized water; platelet aggregation.

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Contents

CryoLetters 46 (5), 321-330 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110712

Effects of cryolipolysis by vacuum system and by cooling plates: a comparative study

Christiane Rodrigues Tófoli1, Patrícia Froes Meyer2*, Rafaella Rego Maia3, Eneida de Morais Carreiro2, Felipe Pimentel de Almeida4, Cibelle Maria Soares de Sousa5, Priscila da Silveira Palmeira Silva Daumas6 and Ciro Dantas Soares7

  1. Vilha Velha University Center, Vila Velha, Brazil.
  2. International Research Group - IRG, Natal, Brazil.
  3. Federal University of Rio Grande do Norte, Natal, Brazil.
  4. Estácio de Sá University - Fortaleza, Brazil.
  5. Technology College of João Pessoa - FATEC, João Pessoa, Brazil.
  6. Estácio de Sá University - UNESA, Brazil.
  7. Department of Oral Diagnosis, Piracicaba Dental School, University of Campinas, Piracicaba, Brazil.

Abstract

Background

Cryolipolysis reduces the thickness of adipose tissue, but are different ways of applying this technology.

Objective

To compare the histological and immunohistochemical differences between the application of suction cryolipolysis versus plate cryolipolysis in adipose tissue.

Materials and methods

The sample included two participants with localized fat. One participant received a session of cryolipolysis by vacuum system (-11°C, -30 KPA, two cycles of 30 minutes, and three minutes of manual reperfusion); the other participant received cryolipolysis by cooling plates (-5°C, two cycles of 30 minutes, and three minutes of manual reperfusion). After 60 days, participants underwent abdominoplasty surgeries, and tissue fragments were removed and submitted to biopsy.

Results

The treated sides showed a significant reduction of localized fat and activation of the adipocyte apoptosis, evidenced by the presence of CASP3. Moreover, this reduction was associated with an increase in COX-2. The total quantity of collagen was greater on the treated sides, without significant differences between application methods. However, type I collagen was predominant in the vacuum system. The presence of FGF2 indicated activation of pathways of fibroblast proliferation, with a neocollagenesis rate approximately 50% higher in the vacuum system. Analysis showed a greater DRP1 expression in the vacuum system, indicating an increase in metabolic rate due to higher inflammation. The differentiation of adipocytes was significantly present in the side treated with the vacuum system, with a PPAR-γ expression 100% higher.

Conclusion

The vacuum system seems to be superior to cooling plates in the reduction of localized fat, neocollagenesis, and fibroblast proliferation.

Keywords: adipose tissue; cryolipolysis; esthetics; immunohistochemistry.

Contents

CryoLetters 46 (5), 331-344 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110212

Foliar-spraying with wine pomace extract to alleviate freezing damage in some crops cultivated in northern Japan

Yuna Ozaki, Yutaro Kita, Keita Arakawa, Takashi Suzuki and Yutaka Jitsuyama

  • Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.

Abstract

Background

Wine pomace, a byproduct of wine production, is problematic because of the environmental impact of its disposal. Extracts from wine pomace have been shown to exhibit anti-ice nucleation activity against bacterial ice nucleation caused by Erwinia ananas.

Objective

To investigate whether wine pomace extracts (WPEs) could be used as a frost protection agent for crops.

Materials and methods

Wine pomace from the skins of grapes was extracted and used as WPEs. Adzuki, Chinese yam, and grapevine were selected as target crops to investigate freezing damage. Initially, the leaf discs of the three crops were used to determine droplet freezing temperature. We also investigated whether WPEs inhibited this damage. Second, the survival of whole adzuki seedlings exposed to low temperatures in the presence of E. ananas and WPEs was examined.

Results

In all crops, droplet freezing temperature increased in the presence of E. ananas, and this increase was suppressed following WPE treatment. Adzuki seedlings sprayed with WPE with E. ananas had higher survival rates than those sprayed with ultrapure water.

Conclusion

This study showed that WPEs could prevent lethal damage to crops by reversing the increase in the freezing temperatures of droplets or crops caused by E. ananas.

Keywords: adzuki; anti-ice nucleation activity; E. ananas; freezing damage; wine pomace.

Contents

CryoLetters 46 (5), 345-354 (2025)
© CryoLetters, editor@cryoletters.org

doi.org/10.54680/fr25510110412

Ultrasonic ice seeding as an effective technique for Hep-G2 cell cryopreservation

Zhengxin Peng1,2,3, Weijie Li1,2,3*, Baolin Liu1,2,3 and Jianxin Wang4

  1. Institute of Biothermal Science and Technology, University of Shanghai for Science and Technology, Shanghai, China.
  2. Shanghai Collaborative Innovation Center for Tumor Treatment with Energy, Shanghai, China.
  3. Shanghai Technical Service Platform for Cryopreservation of Biological Resources, Shanghai, China.
  4. Shanghai OriginCell Biological Cryo Equipment Co., Ltd., shanghai, China.

Abstract

Background

Cryopreservation faces challenges from intracellular ice formation (IIF) and solution damage, influenced by cooling rates. Ice seeding mitigates supercooling risks, yet traditional methods like contact nucleation are impractical. Ultrasonic ice seeding emerges as a promising alternative, leveraging cavitation to induce nucleation without disrupting temperature stability.

Objective

To evaluate ultrasonic ice seeding’s efficacy in reducing IIF and enhancing post-thaw survival of Hep-G2 liver cancer cells.

Materials and methods

A cryogenic microscopy platform and ultrasonic device (40 kHz, 500W) were constructed. Hep-G2 cells were cryopreserved using three methods including 10% DMSO as cryoprotectant: cell freezing container (10%-CFC), programmable controlled-rate freezing (10%-PC), and programmable freezing with ultrasonic ice seeding (10%-PC+UIC). Survival rates were assessed via AOPI staining, while intracellular ice formation and cell morphology were analyzed microscopically.

Results

The 10%-PC+UIC group achieved the highest survival rate (93.29 ± 1.20%), surpassing 10%-PC (90.32 ± 1.60%) and 10%-CFC (80.33 ± 3.36%). Ultrasonic ice seeding reduced intracellular ice occurrence to 6.43% versus 20.71% in spontaneous crystallization. Microscopy revealed smaller, uniform ice crystals and controlled cell dehydration/rehydration dynamics, minimizing membrane damage.

Conclusion

Ultrasonic ice seeding significantly improves Hep-G2 cell survival by reducing IIF through controlled nucleation. Its non-contact, contamination-free operation offers practical advantages for large-scale cryopreservation, outperforming traditional methods. This technique holds potential for broader applications in biological and medical storage protocols.

Keywords: cryopreservation; ice-seeding; intracellular ice; ultrasound.