CryoLetters

CryoLetters is a bimonthly, international journal for low temperature science and technology

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CryoLetters 42 (4), 188-201 (2020)
© CryoLetters, editor@cryoletters.org

PERSPECTIVE:
FREEZING PROTOCOLS FOR THE CRYOPRESERVATION OF IMMATURE TESTICULAR TISSUE – A SYSTEMATIC REVIEW

Bríd Ní Dhonnabháin¹ and Natalie Getreu¹*

¹ EGA Institute for Women’s Health, University College London, London, United Kingdom.
* Corresponding author's E-mail: natalie.getreu@ucl.ac.uk

Abstract

Increasing numbers of childhood cancer survivors reach adulthood making therapy induced infertility a growing concern. Sperm cryopreservation is not possible prior to puberty. Testicular tissue cryopreservation has been proposed as an alternative fertility preservation method for prepubertal males but no standardised cryopreservation procedure for immature tissue has been agreed to date.  Here we review the current literature of cryopreservation protocols to determine which method best preserves the morphology and function of immature testicular tissue; and to examine which tissue intervention, grafting or tissue culture, is mostly likely to restore fertility. Embase, Medline, and Web of Science were systematically searched using relevant MeSH headings and search terms for testis, cryopreservation, and fertility preservation. This systematic search returned 4748 unique entries which were screened for relevance. Eleven studies were found to be eligible and were included in the systematic review. We found that cryopreservation protocols differ in freezing rate and cryoprotectant media, the optimum combination of which for ITT has yet to be determined. Further investigations must be carried out to decipher which method best preserves tissue integrity and function and which application method is most likely to induce spermatogenesis.

Keywords: cryopreservation; fertility preservation; oncofertility; testis.

Download the paper: FREEZING PROTOCOLS FOR THE CRYOPRESERVATION OF IMMATURE TESTICULAR TISSUE – A SYSTEMATIC REVIEW (PDF)

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CryoLetters 42 (4), 202-209 (2020)
© CryoLetters, editor@cryoletters.org

EVALUATION OF DROPLET-VITRIFICATION, VACUUM INFILTRATION VITRIFICATION AND ENCAPSULATION- DEHYDRATION FOR THE CRYOPRESERVATION OF Syzygium maire ZYGOTIC EMBRYOS

Karin van der Walt^1,2* Peter Kemp², Svetla Sofkova-Bobcheva²,
David Burritt³ and Jayanthi Nadarajan^2,4

¹ Otari Native Botanic Garden and Wilton’s Bush Reserve, Wellington City Council, 160 Wilton Road, Wellington 6012.
² Massey University of New Zealand, Private Bag 11222, Palmerston North, New Zealand.
³ University of Otago, Department of Botany, P.O. Box 56, Dunedin, New Zealand.
⁴ The New Zealand Institute for Plant and Food Research Limited, Private Bag 11600, Palmerston North, New Zealand.
*Corresponding author’s E-mail: Karin.vanderwalt@wcc.govt.nz

Abstract

BACKGROUND: Syzygium maire is a threatened tree species with limited information on long-term storage options for its recalcitrant seed. OBJECTIVE: To evaluate the cryopreservation of S. maire zygotic embryo axes (EA) using dehydration, encapsulation-dehydration as well as PVS2 vitrification using droplet vitrification (DV) and the novel droplet vacuum infiltration vitrification (DVIV) methods. MATERIALS AND METHODS: Excised naked and sodium alginate encapsulated EA were desiccated to various moisture contents (MC) using a laminar flow cabinet. Moisture content, embryo survival and plantlet formation, before and after cryopreservation, were assessed at 1 h intervals during the desiccation period (0–6 h). The influence of PVS2, using DV and DVIV, was assessed for various desiccation times and temperatures. RESULTS: Encapsulated EA desiccated to 31% and 37% MC survived but no plantlets formed following cryopreservation. Exposure to PVS2 using the DV method had a negative impact on embryo survival and plantlet formation, while DVIV resulted in improved results for non-cryopreserved EA. However, neither PVS2 vitrification method resulted in embryo survival following cryopreservation. CONCLUSION: S. maire embryos are sensitive to desiccation and likely require physical, chemical or a combination of protection methods to increase embryo survival and plantlet formation following cryopreservation

Keywords: Myrtaceae; New Zealand native species; PVS2; recalcitrant seeds; vitrification.

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CryoLetters 42 (4), 210-219 (2020)
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EVALUATION OF DIFFERENT CRYOPROTECTANT SOLUTIONS FOR THE CRYOPRESERVATION OF SOMATIC TISSUES OF Dasyprocta leporina (LINNAEUS, 1758)

Luanna Lorenna Vieira Rodrigues¹, Alana Azevedo Borges¹,
Matheus Barbosa do Nascimento¹,
Leonardo Vitorino Costa de Aquino¹,
Maria Diana Cáritas Barros dos Santos¹, Alexandre Rodrigues Silva²,
Moacir Franco de Oliveira³, and Alexsandra Fernandes Pereira¹*

¹ Laboratory of Animal Biotechnology, Federal Rural University of Semi-Arid (UFERSA), Mossoro, RN, Brazil.
² Laboratory of Animal Germplasm Conservation, UFERSA, Mossoro, RN, Brazil
³ Laboratory of Applied Animal Morphophysiology, UFERSA, Mossoro, RN, Brazil
*Corresponding author’s E-mail: alexsandra.pereira@ufersa.edu.br

Abstract

BACKGROUND: Somatic tissue banks represent important tools for the conservation of wild mammals, aiming at the immediate maintenance and safeguarding of biological samples. For agouti, Dasyprocta leporina, studies on the formation of these banks are still scarce, especially regarding protocols of the best cryoprotectant solution employed. OBJECTIVE: To optimize the cryoprotectant solution [ethylene glycol (EG), dimethyl sulfoxide (DMSO), sucrose (SUC)] used for the cryopreservation of agouti somatic tissues. MATERIALS AND METHODS: We treated ear tissues with various cryoprotectant solutions: 3.0 M EG (EG group), 3.0 M EG and 0.25 M SUC (EG-SUC group), 3.0 M DMSO (DMSO group), 3.0 M DMSO and 0.25 M SUC (DMSO-SUC group), 1.5 M EG and 1.5 M DMSO (EG-DMSO group) and 1.5 M EG, 1.5 M DMSO and 0.25 M SUC (EG-DMSO-SUC group). Non-cryopreserved tissues were used as controls. All tissues were analyzed for their ultrastructural and morphometric characteristics by scanning electron microscopy and conventional histology. RESULTS: EG-DMSO-SUC was found to be the optimal cryoprotectant solution in terms of the evaluated parameters, such as thickness of the dermis and skin, number of perinuclear halos, proliferative potential, number of empty lacunas and degenerated chondrocytes. CONCLUSION: Agouti somatic tissue cryopreservation may serve for its conservation and as an experimental model for the development of preservation methods for species of the same genus that are either vulnerable or critically endangered.

Keywords: agouti; biobanks; ear skin and cartilage; tissue vitrification; wild rodents.

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CryoLetters 42 (4), 220-226 (2020)
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COMBINING DIMETHYL SULPHOXIDE (DMSO) WITH DIFFERENT CRYOPROTECTANTS ENSURES BETTER CARTILAGE CELL CRYOPRESERVATION

Emel TÜTEN SEVİM¹ and Sezen ARAT²*

¹Department of Agricultural Biotechnology, Faculty of Agriculture, Akdeniz University, Antalya, Turkey.
²Department of Agricultural Biotechnology, Faculty of Agriculture, Tekirdag Namik Kemal University, Tekirdag, Turkey.
*Corresponding author’s E-mail:sarat@nku.edu.tr

Abstract

BACKGROUND: DMSO is a cryoprotective agent (CPA) that is widely used in the cryopreservation of cells. However, evidence suggests that it is more effective when combined with other CPAs. OBJECTIVE: To investigated the effect of combining serum and balanced solutions with DMSO for cryopreservation. MATERIALS AND METHODS: Cartilage cells cultured with Dulbecco’s Modified Eagle Medium (DMEM) medium were taken from the ears of adult cattle. Then, these cells were frozen by supplementation with different concentrations of fetal bovine serum (FBS) and different balanced solutions with DMSO. RESULTS:  The highest cell viability was obtained using freezing solutions containing 10% DMSO and 40% serum, in dextran 40 or dextrose solution or DMEM. CONCLUSION: Our results indicated that serum concentration is important for cell viability and that supplementing DMSO with dextran or dextrose or DMEM benefits the cryopreservation of bovine cartilage cells.

Keywords: cartilage; cell viability; cryopreservation; DMSO; sugars.

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CryoLetters 42 (4), 227-232 (2020)
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CRYODILUENTS OPTIMIZATION OF Glossogobius giuris (HAMILTON-BUCHANAN) SPERMATOZOA

Leena Grace Beslin

Department of Aquatic Biology and Fisheries, University of Kerala, Kariavattom, Thiruvananthapuram - 695581, India
Author’s E-mail: drblgrace@rediffmail.com
ORCID ID: 000-0002-7987-0572

Abstract

BACKGROUND: Biodiversity conservation by germplasm maintenance by cryobanks is an accepted way of saving species. The edible fish Glossogobius giuris is reported to be threatened in India and needs rehabilitation measures to improve their numbers in natural waters. OBJECTIVE: To investigate the standardization of cryoprotectants for the preservation of male gametes of this species. MATERIALS AND METHODS: Four different cryoprotectants were used: DMSO, glycerol, ethylene glycol and methanol. Different combinations of diluents and milt were processed inside a cold handling unit at -5°C and stored for the short-term. RESULTS: Prior to exposure to cryoprotectants, the motility of fresh sperm was 98.3 ± 2.5%. After 10 min equilibration at room temperature in 7.5 % glycerol, sperm motility was 95.6  1.5%, and 93  3.2% after 180 min at -5°C in this cryoprotectant. In contrast, motility was 653% after equilibration in 12.5 % methanol, and survivability fell to 30.7 0.9% after 180 min storage at -5°C. Analysis by Bonferroni and Holm Multiple Comparison showed highly significant variations between the effect of methanol and the other cryoprotectants. There was a statistically significant fall in motility when using methanol compared to glycerol. CONCLUSION: Glycerol provides greater protection to spermatozoa during cold storage at -5°C, possibly as a result of its membrane stabilizing power.

Keywords: cryoprotectant; glycerol; motility; preservation; spermatozoa.

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CryoLetters 42 (4), 233-244 (2020)
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CRYOPRESERVATION OF POLLEN OF Abelmoschus moschatus Medik. subsp. moschatus AS AN AID TO OVERCOME ASYNCHRONOUS FLOWERING FOR WIDE HYBRIDIZATION WITH CULTIVATED OKRA [A. esculentus (L.) Moench]

Ravi Gowthami¹, Neelam Sharma¹, Krishna Kumar Gangopadhyay¹, Subramani Rajkumar², Pooja Pathania² and Anuradha Agrawal¹*

ICAR-National Bureau of Plant Genetic Resources (NBPGR), Pusa Campus, New Delhi-110012, India:
¹ Division of Germplasm Evaluation;
² Division of Genomic Resources.
*Corresponding author’s E-mail: anuradha.agrawal@icar.gov.in

Abstract

BACKGROUND: Asynchronous flowering is one of the major constraints for hybridization between Abelmoschus moschatus subsp. moschatus, a wild species closely related to cultivated okra [A. esculentus (L.) Moench]. Availability of viable pollen is a prerequisite to facilitate breeding in these species. OBJECTIVES: Pollen cryopreservation was attempted in A. moschatus subsp. moschatus, to overcome the asynchronous flowering barrier during wide hybridization with A. esculentus. MATERIALS AND METHODS: Viability of fresh pollen of A. moschatus subsp. moschatus was assessed using acetocarmine and triphenyl tetrazolium chloride (TTC) test and in vitro germination by sitting drop culture method. Pollen of 10 accessions were stored at four temperatures (25, 4, −20 and −196˚C), in the dark and periodically monitored for viability. The standardized cryopreservation protocol was applied to 24 accessions of A. moschatus subsp. moschatus over three months. In vivo pollen germination of 24 accessions of cryopreserved pollen and its efficacy on fertilizing A. esculentus cv ‘Pusa Sawani’ were recorded and pollen was utilized for hybridization with A. esculentus. RESULTS: Brewbaker and Kwack medium with 15% sucrose was optimal for in vitro pollen germination. Pollen viability assessed by in vitro germination (60-90%) was more reliable compared to acetocarmine (90-99%) and TTC (85-99%) staining tests. Significant negative correlation was found between pollen germination, storage time and temperature (25, 4 and −20°C^{) in all the accessions}. Cryopreserved (-196°C) pollen showed significantly higher viability compared to all the other storage conditions, without viability loss. Successful pollination, fruit and seed set was observed in four out of 24 cross combinations attempted. CONCLUSION: The cryopreservation of pollen of A. moschatus subsp. moschatus and its fertilizing ability offers great potential for a successful wide hybridization programme in okra.

Keywords: in vitro germination; okra; pollen cryopreservation; pollen viability; wide hybridization.

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CryoLetters 42 (4), 245-250 (2020)
© CryoLetters, editor@cryoletters.org

SILVER CLOSED VITRIFICATION SYSTEM VERSUS SLOW FREEZING METHOD FOR THE CRYOPRESERVATION OF HUMAN OVARIAN TISSUE

You Yang^1,#, Yan Wang^1,# and Zhun Xiao¹*

¹ Reproductive Medical Department of West China 2^{nd University Hospital,} Key Laboratory of Birth Defects and Related Diseases of women and Children, Ministry of Education, Sichuan University, Chengdu 610041, China.
^#Contributed equally to this work and are co-first authors
* Corresponding author’s E-mail: xiaozhunok@163.com

Abstract

BACKGROUND: We developed a hand-made silver container as a closed vitrification system (CVS) which avoid bacterial or viral contamination. OBJECTIVE: The aim of this study was to evaluate the preservation outcomes by comparing silver CVS and slow freezing (SF) method. MATERIALS AND METHODS: Donated human ovarian tissues were collected from nine patients. The fragments from each patient were randomly and evenly assigned to three groups: fresh control, silver CVS and SF group. The histology of the thawed ovarian tissue was investigated and the levels of secretion of estradiol (E₂) and progesterone (P₄) in the culture media were used to evaluate the development and function of thawed ovarian tissue. RESULTS: The results showed that the proportion of morphologically normal primordial follicles was higher in silver CVS group than in SF (P<0.005). E2 and P4 concentrations were significantly higher in the silver CVS group than in the SF group at any time point after day 6 (E2: P<0.05; P4: P<0.05). CONCLUSION: This study showed that human ovarian tissue cryopreserved with silver CVS had better morphology and higher E2 and P4 levels during in vitro ovarian tissue growth compared with SF. It implies that silver CVS has a better potential for ovarian tissue development in future clinical use.

Keywords: human ovarian tissue; primordial follicle; slow freezing; steroid hormone; vitrification.

Abstracts

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