Conventional Vitrification Research Articles

Multi‐Scale Ice Inhibition Platform Enables Full‐Cycle Cryogenic Protection for Mouse Oocyte

AbstractCryopreservation of metaphase II (MII) oocytes is crucial to preserve female fertility. However, the existing vitrification technology just provides partial cryogenic protection for oocytes and must use high concentrations of toxic penetrating cryoprotective agents (CPAs, up to 4.8 M). Here, a multi‐scale ice inhibition platform is proposed, which effectively suppresses ice crystal morphology, ice recrystallization (IR), temperature gradient, and devitrification. This platform achieves mouse oocyte survival rate of 98.6% using the low concentration of CPA (3.1 M) while also possessing batch oocyte cryopreservation ability. In contrast, recovered oocytes show a much more modest variation of genes compared with the conventional method (85 vs. 1396 genes), retaining normal fertilization, embryonic development, and birth to healthy offspring. Diverging from the conventional vitrification method with partial cryogenic protection capability, this platform introduces full‐cycle ice inhibition, offering promising prospects for achieving high‐quality and scalable fertility preservation.

Cryopreservation and assessment of genetic fidelity Acorus calamus Linn., an endangered medicinal plant

BACKGROUND: Acorus calamus Linn. is a medicinally valuable monocot plant belonging to the family Acoraceae. Over-exploitation and unscientific approach towards harvesting to fulfill an everincreasing demand have placed it in the endangered list of species. OBJECTIVE: To develop vitrification-based cryopreservation protocols for A. calamus shoot tips, using conventional vitrification and V cryo-plate. MATERIALS AND METHODS: Shoot tips (2 mm in size) were cryopreserved with the above techniques by optimizing various parameters such as preculture duration, sucrose concentration in the preculture medium, and PVS2 dehydration time. Regenerated plantlets obtained post-cryopreservation were evaluated by random amplified polymorphic DNA (RAPD) to test their genetic fidelity. RESULTS: The highest regrowth of 88.3% after PVS2 exposure of 60 min was achieved with V cryo-plate as compared to 75% after 90 min of PVS2 exposure using conventional vitrification. After cryopreservation, shoot tips developed into complete plantlets in 28 days on regrowth medium (0.5 mg L-1 BAP, 0.3 mg L-1 GA3, and 0.3 mg L-1 ascorbic acid). RAPD analysis revealed 100% monomorphism in all cryo-storage derived regenerants and in vitro donor (120-days-old) plants. CONCLUSION: Shoot tips of A. calamus that were cryopreserved had 88.3% regrowth using V cryo-plate technique and the regerants retained genetic fidelity.

Ultra-Fast Vitrification: Minimizing the Toxicity of Cryoprotective Agents and Osmotic Stress in Mouse Oocyte Cryopreservation.

Globally, women have been adopting oocyte cryopreservation (OC) for fertility preservation for various reasons, such as inevitable gonadotoxic treatment for specific pathologic states and social preferences. While conventional vitrification (C-VIT) has improved the success rate of OC, challenges of possible toxicities of high-concentration cryoprotective agents and osmotic stress persist. To overcome these challenges, we evaluated the ultra-fast vitrification (UF-VIT) method, which reduces the equilibration solution stage exposure time compared to C-VIT by observing mouse oocyte intracellular organelles and embryonic development. Consequently, compared to fresh mouse oocytes, UF-VIT presented significant differences only in endoplasmic reticulum (ER) intensity and mitochondrial (MT) distribution. Meanwhile, C-VIT showed substantial differences in the survival rate, key ER and MT parameters, and embryonic development rate. UF-VIT exhibited considerably fewer negative effects on key MT parameters and resulted in a notably higher blastocyst formation rate than C-VIT. Meiotic spindle (spindle and chromosomes) morphology showed no significant changes between the groups during vitrification/warming (VW), suggesting that VW did not negatively affect the meiotic spindle of the oocytes. In conclusion, UF-VIT seems more effective in OC owing to efficient cytoplasmic water molecule extraction, osmotic stress reduction, and minimization of cell contraction and expansion amplitude, thus compensating for the drawbacks of C-VIT.

Fabrication of Super‐Sized Metal Inorganic‐Organic Hybrid Glass with Supramolecular Network via Crystallization‐Suppressing Approach

AbstractMetal coordination compound (MCC) glasses [e.g., metal‐organic framework (MOF) glass, coordination polymer glass, and metal inorganic‐organic complex (MIOC) glass] are emerging members of the hybrid glass family. So far, a limited number of crystalline MCCs can be converted into glasses by melt‐quenching. Here, we report a universal wet‐chemistry method, by which the super‐sized supramolecular MIOC glasses can be synthesized from non‐meltable MOFs. Alcohol and acid were used as agents to inhibit crystallization. The MIOC glasses demonstrate unique features including high transparency, shaping capability, and anisotropic network. Directional photoluminescence with a large polarization ratio (≈47 %) was observed from samples doped with organic dyes. This crystallization‐suppressing approach enables fabrication of super‐sized MCC glasses, which cannot be achieved by conventional vitrification methods, and thus allows for exploring new MCC glasses possessing photonic functionalities.

Fabrication of Super-Sized Metal Inorganic-Organic Hybrid Glass with Supramolecular Network via Crystallization-Suppressing Approach.

Metal coordination compound (MCC) glasses [e.g., metal-organic framework (MOF) glass, coordination polymer glass, and metal inorganic-organic complex (MIOC) glass] are emerging members of the hybrid glass family. So far, a limited number of crystalline MCCs can be converted into glasses by melt-quenching. Here, we report a universal wet-chemistry method, by which the super-sized supramolecular MIOC glasses can be synthesized from non-meltable MOFs. Alcohol and acid were used as agents to inhibit crystallization. The MIOC glasses demonstrate unique features including high transparency, shaping capability, and anisotropic network. Directional photoluminescence with a large polarization ratio (≈47 %) was observed from samples doped with organic dyes. This crystallization-suppressing approach enables fabrication of super-sized MCC glasses, which cannot be achieved by conventional vitrification methods, and thus allows for exploring new MCC glasses possessing photonic functionalities.

P-075 Comparison of sperm retrieval efficiency by three cryopreservation techniques using two commercial freezing media in patients with oligozoospermia

Abstract Study question Which cryopreservation method and commercial freezing media offer a higher efficiency of sperm retrieval in patients with oligozoospermia? Summary answer In patients with oligozoospermia, higher sperm retrieval was observed when using the slow freezing technique regardless of the commercial freezing medium used. What is known already Cryopreservation is a process that involves the use of low temperatures to preserve cell structure and function, reducing metabolic activity [1]. Until now, the cryopreservation of sperm has not provided complete protection, since motility decreases by half and even more in infertile patients [2]. Previous publications have reported a deleterious effect when using the slow freezing method, having a higher impact in samples with severe oligoasthenoteratozoospermia or alterations in quantity, motility, and morphology than in normozoospermic samples [3]. Additionally, vitrification has been compared with slow protocols, finding conflicting results while others have reported that these techniques are comparable [4]. Study design, size, duration We conducted a pilot, prospective and comparative study on 20 patients with oligospermia that attended the Centro Universitario de Medicina Reproductiva of the Hospital Universitario “Dr. José Eleuterio González” from July to October of 2021. Participants/materials, setting, methods We included 20 men with oligozoospermia. We compared slow freezing, conventional, and droplet vitrification with two commercial freezing mediums. For which, 1 mL of the semen sample was divided into 2 aliquots. Each of the two freezing media was added at 1:1 ratio into a semen aliquot and later, 300 ul were used to cryopreserve with each of the techniques and subsequently were thawed and assessed for sperm recovery efficiency. Main results and the role of chance A total of 20 samples were evaluated. The patients had the diagnosis of oligozoospermia at the moment of the evaluation. When using the Freezing medium TYB (Irvine Scientific), the slow freezing method had a statistically significant higher sperm recovery than the vitrification techniques. Accordingly, there was no difference between standard and droplet vitrification. When using the Sperm Freezing Medium (InVitro Care, Inc) no statistical difference in sperm recovery was observed for any of the three assessed freezing methods. Interestingly, with the slow freezing technique, no difference in recovery efficiency was observed regardless of the commercial freezing medium used. Our preliminary findings, highlight the use of the slow freezing technique. Limitations, reasons for caution We conducted a pilot study that included 20 patients with oligozoospermia. Therefore, special caution must be taken due to the limited number of patients. Nevertheless, our results showed positive results for the use of the slow freezing method, which has been discontinued possibly by a detrimental effect. Wider implications of the findings The slow freezing method for sperm preservation contrasts with what has been used lately and offers patients with poor semen parameters for instance oncologic patients a chance to maintain their reproductive potential Trial registration number not applicable

Vitrification by Transient Vacuum Flashing Spray Cooling of Liquid Nitrogen

BACKGROUND: The transient vacuum flashing spray cooling of liquid nitrogen (LN2 ) on a microstructured surface can provide ultra-fast cooling rate and may improve cell survival rates. OBJECTIVE: To utilize flashing spray cooling of LN2 instead of film boiling to improve further cell vitrification. METHOD: This study analyzed the effects of the three key parameters (flow rate of liquid nitrogen, ambient pressure, and spray distance) on the cooling process by experimentation. RESULTS: The experimental results showed that the vacuum flashing spray cooling of LN2 can gain higher cooling rates than that achieved by film boiling in conventional vitrification methods. The three parameters all affected the vacuum flash evaporation spray cooling of LN2, and their effect trends were not monotonous but followed a parabolic trend that increased and then decreased. That is, the three parameters all have optimum values to the cooling process. CONCLUSION: Vacuum flash evaporation spray cooling can develop the ultra-fast cooling rates needed to enhance cell vitrification.

Cryopreservation of Testicular Tissue from Adult Red-Rumped Agoutis (Dasyprocta leporina Linnaeus, 1758).

Simple SummaryTesticular tissues are composed of many types of germ cells, including early stages which can be grown up to fully formed spermatozoa following grafting or in vitro culture. The systematic banking of testicular tissues at freezing temperatures is useful for future use in assisted reproduction and to improve the reproductive management of rare mammalian species. The present study explored testicular tissue cryopreservation in the red-rumped agouti by slow freezing or vitrification methods, using different combinations of cryoprotectants. Solid-surface vitrification using the combination of dimethyl sulfoxide and ethylene glycol was the most effective protocol to preserve testicular cell morphology and proliferative potential.This study measured the effects of different freezing techniques and permeating cryoprotectants on the preservation of testicular tissues from adult red-rumped agoutis. Tissue biopsies (3.0 mm3) from five individuals were allocated to different experimental groups: control (non-cryopreserved); slow freezing (SF), solid-surface vitrification (SSV), and conventional vitrification (CV). Each method used dimethyl sulfoxide (DMSO), ethylene glycol (EG), or a DMSO + EG combination. Morphology, viability, mitochondrial activity, and proliferative potential were assessed in fresh and frozen tissue samples. Testicular morphology was better using SSV with a combination of DMSO and EG. Across the different cryopreservation approaches, as well as cryoprotectant combinations, cell viability was comparable. Regarding mitochondrial activity, DMSO + EG/SSV or CV, and DMSO + EG/CV were similar to the EG/SF group, which was the best group that provided values similar to fresh control groups. Adequate preservation of the proliferative potential of spermatogonia, Leydig cells, and Sertoli cells was obtained using SSV with DMSO + EG. Overall, the use of SSV with DMSO + EG was the best protocol for the preservation of testicular tissues from adult red-rumped agoutis.

Influence of freezing techniques and glycerol-based cryoprotectant combinations on the survival of testicular tissues from adult collared peccaries

The objective of the study was to evaluate the effects of different cryopreservation techniques including glycerol-based cryoprotectant combinations on the structure and viability of testicular tissues from adult collared peccaries. Tissue biopsies (3.0 mm³) from 5 different individuals were allocated to 10 different groups: fresh control; slow freezing (SF), conventional vitrification (CV), or solid-surface vitrification (SSV); each of them using three different combinations of cryoprotectants [dimethyl sulfoxide (DMSO) + ethylene glycol (EG); DMSO + Glycerol; and EG + Glycerol]. After thawing/warming, samples were evaluated for histomorphology, viability, proliferative capacity potential, and DNA integrity. Most effective preservation of testicular histomorphology was achieved using SF and CV with DMSO + EG. However, the use of glycerol-based cryoprotectant combinations increased the occurrence of tubular cell swelling, tubular cell loss and shrinkage from the basal membrane. Cell viability was comparable among cryopreservation methods and cryoprotectant combinations. Regarding cell proliferative capacity, the use of SF with EG + Glycerol and SSV with DMSO + Glycerol impaired the conservation of spermatogonia proliferative potential compared to other treatments. Moreover, CV with DMSO + EG was better than SF with EG + Glycerol for Sertoli cell proliferation potential. Regarding DNA integrity, less damage occurred when using SF with DMSO + EG while more fragmentations were observed when using CV with EG + Glycerol or DMSO + Glycerol as well as SSV with EG + Glycerol or DMSO + Glycerol. In sum, SF and CV appeared to be the most suitable methods for the cryopreservation of adult peccary testicular tissues. Additionally, the use of glycerol-based cryoprotectant combinations did not improve testicular tissues preservation with DMSO + EG being the most efficient option.

Molecular Design of Polyampholytes for Vitrification-Induced Preservation of Three-Dimensional Cell Constructs without Using Liquid Nitrogen.

Current slow-freezing methods are too inefficient for cryopreservation of three-dimensional (3D) tissue constructs. Additionally, conventional vitrification methods use liquid nitrogen, which is inconvenient and increases the chance of cross-contamination. Herein, we have developed polyampholytes with various degrees of hydrophobicity and showed that they could successfully vitrify cell constructs including spheroids and cell monolayers without using liquid nitrogen. The polyampholytes prevented ice crystallization during both cooling and warming, demonstrating their potential to prevent freezing-induced damage. Monolayers and spheroids vitrified in the presence of polyampholytes yielded high viabilities post-thawing with monolayers vitrified with PLL-DMGA exhibiting more than 90% viability. Moreover, spheroids vitrified in the presence of polyampholytes retained their fusibilities, thus revealing the propensity of these polyampholytes to stabilize 3D cell constructs. This study is expected to open new avenues for the development of off-the-shelf tissue engineering constructs that can be prepared and preserved until needed.

Cryopreservation of shoot tips of Gentiana kurroo Royle – a critically endangered medicinal plant of India

Gentiana kurroo Royle, a critically endangered medicinal plant species of India is conserved as in vitro slow growing cultures in the In Vitro Genebank (IVGB) at ICAR-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, India. Shoot tips (about 1 mm in length) excised from 8-weeks-old stock cultures subjected to cold hardening for four weeks at 8 °C (dark) were precultured on Murashige and Skoog (MS) medium supplemented with 5% Dimethylsulfoxide (DMSO) at 8 °C (dark) for two days. Thereafter, shoot tips were dehydrated with PVS2 solution (30 min) at 0 °C and cryopreserved using conventional vitrification (V) and droplet-vitrification (DV) techniques. Average post-thaw regeneration after 4 weeks was ~ 35% by V technique and ~ 60% by DV in all the three accessions tested. Genetic stability assessment on the basis of 40 Inter Simple Sequence Repeats and 30 Expressed Sequence Tagged-Simple Sequence Repeats markers revealed no variation between in vitro-cryopreserved plants and controls, in any of the genotypes. Thus, standardized DV method has the potential for long-term conservation of germplasm of G. kurroo and the three accessions are now safely cryobanked at ICAR-NBPGR. Gentiana kurroo is a critically endangered medicinal plant endemic to North-western Himalayas, India. A protocol to cryopreserve in vitro shoot-tips was developed using droplet vitrification technique with high recovery rates and genetically stable regnerants. The protocol is being applied for cryobanking of Gentiana germplasm at ICAR-NBPGR.

Effect of ovum pick-up medium on blastocyst rate

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

The Effect of Different Vitrification and Staining Protocols on the Visibility of the Nuclear Maturation Stage of Equine Oocytes

In this study, we compared two staining protocols assessing the nuclear chromatin stage of equine oocytes after vitrification using permeable and nonpermeable cryoprotectants. Slaughterhouse-derived oocytes (n = 155) were obtained from a total of 32 mares and in vitro matured in M199 medium for 42 hours at 38.5°C in 5% CO2. In the first experiment, two concentrations of Hoechst 33342 (HO) were tested (10 μg/mL; P1 and 2.5 μg/mL; P2) combined with 50 μg/mL of propidium iodide as staining protocols to evaluate the visibility of matured oocytes (n = 44). In the second experiment, 111 oocytes were evaluated using the staining protocol P2, before (C, control) and after vitrification following a two-step conventional protocol with (15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose; V1) or without (1 M sucrose; V2) using permeable cryoprotectants. Our results showed that P2 provided a higher percentage of oocytes with outstanding visibility of the nuclear chromatin stage (52.17%; P < .05) in comparison with P1 (19.04%). In the second experiment, no cryoprotectant-free vitrified oocytes reached the metaphase II maturation stage. This result was significantly lower (P < .05) than conventional vitrification (15.38%) and both lower in comparison with the nonvitrified control group (42.11%). In conclusion, permeable cryoprotectant-free vitrification of equine oocytes obtained poor results and therefore cannot be considered an alternative to vitrification using permeable cryoprotectants. In addition, a staining protocol with a low concentration of HO is recommended to evaluate the nuclear chromatin stage of equine oocytes after in vitro maturation.

Improved cryopreservation of spermatozoa using vitrification: comparison of cryoprotectants and a novel device for long-term storage

Study questionDoes cryoprotection of spermatozoa using a vitrification protocol with improved cryoprotective agents and a novel device for large storage lead to better outcomes than conventional slow freezing?Summary answerVitrification of human sperm using sucrose and dextran-based cryoprotectant (CPA4) with a new vitrification device resulted in significantly better sperm motility and progressive motility and improved DNA integrity with lower DNA fragmentation compared with conventional slow freezing.What is known alreadyA major limitation to clinical implementation of vitrification is the right balance between the volume of spermatozoa suspension cryopreserved and a standardised use of CPAs for survival of spermatozoa.Study design, size, durationThis was a control versus current clinical practice study using 30 fresh human semen samples to carry out the different cryoprotectant analyses followed by a further 23 semen samples to test the novel vitrification protocol.Participants/materials, setting, methodsAll human specimens fulfilled the following criteria: > 5 million spermatozoa/mL, > 20% total motility, ≥ 1.8 mL in volume, with all participants falling within the age range of 25–45 inclusively. The concentration, progressive motility, non-progressive motility, immotility, and various morphokinetic variables including DAP, DCL, DSL, LIN, and STR were then determined using the IVOS II™ Clinical CASA system (Hamilton Thorne, Beverly, MA, USA) on the basis of the 5th Edition of WHO Laboratory Manual for the Examination and Processing of Human Semen.Main results and the role of chanceAmong the 6 cryopreservation methods in this study, vitrification with the funnel-shaped device using CPA4 best preserves the 13 sperm parameters evaluated by CASA system. Conventional slow freezing and vitrification with the device using seminal plasma also protects sperm quality, but the overall motilities are statistically lower in comparison with the novel vitrification approach with cryoprotective media using the device. DNA fragmentation significantly increased after cryopreservation through the method of conventional slow freezing (p = 0.07). There was no significant difference in DNA fragmentation between fresh control and vitrification (p = 1.000).Limitations, reasons for cautionExtensive training is required to minimise the human error in using the vitrification device to perform cryopreservation. Each operator can only handle one sample at a time with device vitrification, whereas several samples can be processed without the need for special training with conventional slow freezing.Wider implications of the findingsThe presented study shows that a new vitrification method could improve survival sperm rate. Human sperm vitrification using our novel protocol gives higher motility and progression and lower percentage of DNA fragmentation than conventional slow freezing. Our findings indicate that this method could supersede the current clinical practice in particular for patients with oligospermia as it reduces osmotic damage, time, and cost.

Outcomes of effective cryopreservation. A two-year assessment of survival rates of vitrified and warmed blastocysts. The experience of an IVF clinic in a developing country

Cryopreservation of oocytes and embryos by vitrification is currently the method of choice employed by most facilities that practice assisted reproductive technology (ART) and there are several studies reporting impressive outcomes. This specialized technique, however, is operator-dependent, so results do vary amongst embryologists and clinics. In Nigeria, vitrification is rapidly gaining widespread popularity as many hospitals are gradually incorporating this technique into their ART practice. This study aims to show the success of an IVF clinic in a developing country at implementing conventional vitrification and warming protocols with survival rates of vitrified-warmed blastocysts reassessed. A retrospective study of 171 FET cycles with a total of 485 embryos vitrified and subsequently warmed within a period of two years and three months (October 2015-December 2017). This study was conducted at Medical Art Center (MART), a private fertility clinic in Lagos, Nigeria. Participants included infertile couples going through frozen embryo transfers after prior failed in-vitro fertilization (IVF) cycles and in some cases, patients susceptible to OHSS with previous freeze all cycles. Vitrification and subsequent warming was performed with Medicult vitrification and warming media (Origio/Medicult, Målov, Denmark) using the McGill cryoleaf protocol as described by the manufacturer (Medicult, Denmark). A total of 146 vitrification cycles were carried out for patients who went through frozen embryo transfers within the study period (Oct 2015-Dec 2017). In 171 FET cycles, 485 vitrified blastocysts were warmed with 358 blastocysts surviving the procedure. The freeze-thaw survival rate was 73.8%, and the average survival rate per freeze-thaw method was 80.5%. The total number of embryos transferred was 343 (average of 2.1 embryos per patient). 166 embryo transfers were performed. 2 FET cycles required re-vitrification of embryos without a transfer taking place, and only 3 patients had no surviving embryos. Our study supports existing reports from publications highlighting excellent outcomes with vitrification. We were able to achieve high embryo survival rates; therefore, we have adopted Vitrification as the only method of choice for cryopreserving embryos. It may be recommended to other IVF clinics looking to improve their freeze-thaw survival rates.

Innovative multi-protectoral approach increases survival rate after vitrification of ovarian tissue and isolated follicles with improved results in comparison with conventional method

BackgroundIn recent years, autotransplantation of cryopreserved ovarian tissue became a promising approach to preserve female fertility. The slow freezing is the most effective technique which resulted in greater live birth incidence so far. Despite that, interest to vitrification of the ovarian tissue is swiftly growing, thereby undermining the necessity for further improvements in the technique. In present study, we evaluated possibilities to increase follicle survival rates adopting innovative multi-protectoral vitrification protocols, applied to the slivers of ovarian cortex or isolated early-antral follicles, frozen individually. These experimental protocols have been compared with with validated vitrification and slow freezing ones, clinically used for female fertility preservation.ResultsThe results showed that third tested variation of experimental vitrification protocol, with four cryoprotectants in relatively low concentrations and applied to pieces of ovarian tissue at 0 °C during equilibration, increased survival rate of ovine ovarian tissue and improved results in comparison with conventional vitrification method. This variation of experimental protocol showed significant increase in percentage of follicles with good morphology (69,3%) in comparison with only commercially available vitrification protocol for ovarian tissue (62,1%). Morphology results were confirmed by TUNEL assay. Analysis of estradiol and progesterone production by cultured individual follicles after freezing/thawing revealed that steroids secretion remained significantly higher after multi-protectoral vitrification and slow freezing protocol, when follicles after standard vitrification protocol demonstrated decline in steroidogenic activity.ConclusionsThe multi-protectoral approach represents a workable solution to improve vitrification outcome on ovarian tissue and isolated follicles. The reduction of individual cryoprotectants concentrations, while maintaining their sufficient cumulative level in the final freezing solution, helps to increase efficiency of the procedure. Moreover, equilibration with lower temperatures helped to decrease even further the toxic effects of cryoprotectants and preserve original quality of ovarian tissue. Therefore, multi-protectoral vitrification can be suggested as an improved method for the clinical cryopreservation of ovarian tissue.

Mouse Embryo Cryopreservation by High-Osmolality Vitrification

In conventional vitrification methods, the embryos are vitrified under considerable supercooling (i.e., under nonequilibrium conditions). This protocol is a refinement of newer equilibrium vitrification methods. The equilibrium vitrification solution contains a high concentration of cryoprotectants that increase its osmolality and allows vitrified embryos to survive not only during storage in liquid nitrogen (LN2) at -196°C but also during temporary holding at -80°C (for at least 5 mo). This high-osmolality vitrification (HOV) method is as simple as conventional vitrification and provides essentially the same high postwarming survival rate. It has several advantages over conventional vitrification: (1) Cryopreserved embryos are less likely to be damaged during handling for warming; (2) samples can be temporarily evacuated to a -80°C freezer and can be successfully recovered after 5 mo at -80°C; (3) samples can be arranged and sorted at -80°C; and (4) vitrified embryos can be transported using dry ice. Also included here is an alternative protocol that describes the use of straws instead of cryotubes.

Cryobanking Pluripotent Stem Cells.

Cryobanking human pluripotent stem cells (hPSCs), be they human embryonic (hESCs) or induced pluripotent stem cells (iPSCs), is essential for their use in research and cell-based therapeutics. Working and master cell banks can be generated with a desired level of quality assurance applied during cell freezing and storage. Conventional vitrification has evolved to more advanced control rate freezing, culminating in a myriad of published protocols with variable proficiencies and clinical efficacies. Notwithstanding, standardized and reliable protocols are necessary for basic science through to applied research and clinical product development. This chapter details several methods for hPSC cryopreservation, suitable for routine application, high-quality research, and adaptable for clinical compliance.

Chapter 17 Sterile Plate-Based Vitrification of Adherent Human Pluripotent Stem Cells and Their Derivatives Using the TWIST Method.

Due to their high biological complexity, e.g., their close cell-to-cell contacts, cryopreservation of human pluripotent stem cells with standard slow-rate protocols often is inefficient and can hardly be standardized. Vitrification that means ultrafast freezing already showed very good viability and recovery rates for this sensitive cell system, but is only applicable for low cell numbers, bears a high risk of contamination, and can hardly be implemented under GxP regulations. In this chapter, a sterile plate-based vitrification method for adherent pluripotent stem cells and their derivatives is presented based on a procedure and device for human embryonic stem cells developed by Beier et al. (Cryobiology 66:8-16, 2013). This protocol overcomes the limitations of conventional vitrification procedures resulting in the highly efficient preservation of ready-to-use adherent pluripotent stem cells with the possibility of vitrifying cells in multi-well formats for direct application in high-throughput screenings.

Preservation of mouse ovarian tissue follicle morphology and ultra-structure after vitrifying in biotechnological protocols.

BackgroundThe aim of the present study was to characterize the morphological and ultrastractural of mouse ovarian tissue with different cryoprotectant solution.ObjectiveAim of this study, is to demonstrae an improved convetional vitrification method on mouse ovarian tissue using different concentrations of ethylene glycol (EG) and/or dimetyl sulfoxide (DMSO) and EG.Materials and methodsMouse ovarian tissue dissected and were randomly assigned to three groups: control, conventional vitrification (CV) and toxicity test. Then ovaries were vitrified by 5%, 10% EG or DMSO CV1-CV4, 5%, 10% EG plus DMSO CV5-CV6 and EG plus DMSO in climbing concentrations CV7. The effect of cryoprotectant solutions on ovarian tissue were evaluated by histological examination hematotoxillin & eosin stain, H&E, viability assessment trypan blue stain and ultrastructural analyses transmission electron microscopy, TEM.ResultsOvarian tissue frozen in CV7 solution showed a higher percentage of morphologically normal follicles or viable follicles than other cryoprotectant solutions P < 0.05. Ultrastructural analysis of ovarian tissue showed that less damage was observed in CV7 and it was very similar to the control group.ConclusionVitrification of ovarian tissue with optimal cryoprotectant solutions such as EG plus DMSO is the most effective for preserving the structural efficiency of ovarian follicles.