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Vitrification technique for female germinative tissue cryopreservation and banking.

ABSTRACT: OBJECTIVE:To report on a device designed for the vitrification of germinative tissue, and a systematic vitrification/warming protocol. METHODS:We obtained six fragments of cortical germinative tissue from a human ovary. We randomly chose two fragments and sent them to histological analysis. We vitrified four test samples and stored them for one week in liquid nitrogen (LN), and warmed one week later. We sent the vitrified/warmed fragments to the pathology laboratory, where they analyzed them morphologically under an optical microscope (10-40X). They analyzed the nuclear and cytoplasmic characteristics of the follicular cells, luteal layer, and stroma. The primordial and primary follicles in the fresh and vitrified/warmed fragments were counted and compared with the Mann-Whitney test (p<0.05). RESULTS:There were ovarian follicles in different phases of maturation in both fresh and vitrified/warmed fragments, with a predominance of healthy-looking primordial and primary follicles. In the test fragments, the fusocellular architecture supporting the stromal cells exhibited some foci of edema, and were associated with cells with hydropic degeneration, with cytoplasmic fragmentation and eosinophilia. However, there were no signs of tissue necrosis or autolysis. There was no statistically significant difference between the number of follicles found in the control and test tissue fragments (p>0.05). CONCLUSIONS:There were no significant morphological changes between fresh and vitrified/warmed germinative tissue. The vitrification device and protocol tested were effective in the preservation of human follicles, and should be considered for the banking germinative tissue for the restoration of fertility of women who are submitted to life-saving sterilizing treatments.

SUBMITTER: Almodin CG

PROVIDER: S-EPMC7169907 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Vitrification technique for female germinative tissue cryopreservation and banking.

Almodin Carlos Gilberto CG Radaelli Moacir Rafael MR Almodin Paula Motta PM Mingetti-Câmara Vânia Cibelle VC Silva Carla Graziele da CGD

JBRA assisted reproduction 20200501 2

<h4>Objective</h4>To report on a device designed for the vitrification of germinative tissue, and a systematic vitrification/warming protocol.<h4>Methods</h4>We obtained six fragments of cortical germinative tissue from a human ovary. We randomly chose two fragments and sent them to histological analysis. We vitrified four test samples and stored them for one week in liquid nitrogen (LN), and warmed one week later. We sent the vitrified/warmed fragments to the pathology laboratory, where they an ...[more]

PMID: 31692315

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Project description:BackgroundOrchids are under threat from human activities and climate change, with populations limited to small geographic hotspots. This makes them ideal candidates for ex situ conservation. Orchid seeds are desiccation tolerant, but often have poor longevity in seed banks and cryopreservation of orchid protocorms is complex and expensive. Therefore, simple methods for large-scale storage programs are essential to store orchid seeds of different life forms. Seeds of five species representing epiphytic, lithophytic and terrestrial orchids from the Central Highlands of Madagascar were studied to find a simple and effective system of cryopreservation. The use of a vitrification solution prior to cryopreservation to improve survival was investigated, as well as the use of symbiotic and asymbiotic germination media to maximise germination after cryopreservation. Using the filter paper packet method, dried seeds were stored in vapour phase above liquid nitrogen and recovered after thawing with both symbiotic and asymbiotic media.ResultsThe study revealed that cryoprotection is not essential for the species in this study, which represented a range of lifeforms. Vitrification generally led to a decrease in germination post cryopreservation. The use of a symbiotic germination medium post cryopreservation was found to be successful in the species in which it was tested. However, the use of an asymbiotic medium was successful for all the species in this study.ConclusionsVitrification was not essential for the species in this study as the orchid seeds were already ultralow temperature and desiccation tolerant. However, further studies using more species are required to validate this approach. This may be an ecophysiological or genetic trait of these species. Therefore, this form of dry seed cryopreservation could form part of ex situ orchid seed conservation using a standard method. The methods developed here will store greater genetic diversity compared to what can be achieved with protocorms and are suitable for both asymbiotic and symbiotic recovery after cryopreservation. This will help reduce the time and cost of ex situ conservation, and help develop universal protocols for large genera, compared to custom protocols required for protocorm cryopreservation.

Project description:Sperm cryopreservation by vitrification is a promising approach for small-bodied animals such as zebrafish (Danio rerio). However, most vitrification tools adopted in aquatic research were initially designed for applications other than sperm (such as human embryo freezing) and, thus, pose challenges for adoption to sperm vitrification. Three-dimensional (3D) printing combined with open hardware sharing is an emerging strategy to address challenges in the development of cryopreservation tools. The goal of this study was to develop a 3D printed Vitrification Device for Cryo-Vials (VDCV) that can be integrated with the existing vial storage systems. The VDCV combined the vitrification and handling components to achieve functions of sample handling, vitrification, storage, and identification. The vitrification component featured a base, a stem, and a loop. A total of 36 configurations with various loop lengths (8, 10, and 12 mm); loop widths (2.0, 2.5, 3.0, and 3.5 mm); and support structures (open, transverse, and axial) of the VDCD prototypes were designed, fabricated, and tested. Device handling orientations (horizontal and vertical holding angles prior to and during freezing) were also investigated. Computer simulations estimated that the cooling rate of the samples ranged from 0.6-1.5 × 105 °C/min in all the configurations. Prior to freezing, loops with axial supports produced a minimum of 92% film retention. The overall trends of full vitrification occurrence were observed: horizontal plunging > vertical plunging, and axial support > transverse support and open loop. A loop length of 8 mm had the highest overall vitrification occurrence (86-100%). No significant differences (p = 0.6584) were shown in a volume capacity (5.7-6.0 μL) among the three supporting configurations. A single unit of VDCV can provide loading efficiencies of about 6 × 107 sperm/vial, pooling of samples from 3-6 males/vial, and fertilization for 1800 eggs/vial. The VDCV are low-cost (<$0.5 material cost per unit) and can be customized, standardized, securely labeled, and efficiently stored. The prototypes can be accessed by user communities through open-fabrication file sharing and fabricated with consumer-level 3D printers, thus facilitating community-level standardization.

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Vitrification technique for female germinative tissue cryopreservation and banking.

Publications

Vitrification technique for female germinative tissue cryopreservation and banking.

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