The effect of amorphous calcium carbonate as a culture media supplement on embryonic development of murine sibling embryos.

PURPOSE: The aim of this study was to compare the addition in culture media of stabilized amorphous calcium carbonate (ACC) versus calcium chloride (CaCl2) or calcium carbonate in crystalline form (CCC) on growth rates among sibling mouse embryos. METHODS: We evaluated the effect of different ACC concentrations on the rates of embryo compaction at 60 h, blastocyst rate at 84 h and percentage of fully hatched at 108 h following hCG injection. As ACC is stabilized by tripolyphosphate (TPP), we also evaluated the addition of TPP alone to the culture media. Finally, we compared supplemented ACC culture media to one-step SAGE and Irvine cleavage media. RESULTS: The results revealed that ACC accelerates the compaction and blastocyst rates, as well as the percentage of fully hatched embryos in a dose-dependent manner, with an increased positive effect at 2.5 mM. The magnitude of the effect for ACC-supplemented media on the embryo developmental rate was between 30 to 40% (p < 0.01) faster for each stage, compared to both SAGE and Irvine one-step standard media. Embryos cultured with SAGE or Irvine media with or without supplementation of CaCl2 or CCC, did not produce the same improvements as observed with ACC. CONCLUSION: In conclusion, the ACC demonstrates a rapid modulation effect for restoring media optimal pH. ACC can inhibit cathepsin B activity during in vitro culture of fibroblast cells. The beneficial impact of ACC on cleavage mouse embryos is likely due to an improved buffering effect causing slower pH media variations, which may enhance quality and implantation potential of embryos following in vitro culture.

Gene expression analysis of ovine prepubertal testicular tissue vitrified with a novel cryodevice (E.Vit).

PURPOSE: Testicular tissue cryopreservation prior to gonadotoxic therapies is a method to preserve fertility in children. However, the technique still requires development, especially when the tissue is immature and rather susceptible to stress derived from in vitro manipulation. This study aimed to investigate the effects of vitrification with a new cryodevice (E.Vit) on cell membrane integrity and gene expression of prepubertal testicular tissue in the ovine model. METHODS: Pieces of immature testicular tissue (1 mm3) were inserted into "E.Vit" devices and vitrified with a two-step protocol. After warming, tissues were cultured in vitro and cell membrane integrity was assessed after 0, 2, and 24 h by trypan blue exclusion test. Controls consisted of non-vitrified tissue analyzed after 0, 2, and 24 h in vitro culture (IVC). Expression of genes involved in transcriptional stress response (BAX, SOD1, CIRBP, HSP90AB1), cell proliferation (KIF11), and germ- (ZBDB16, TERT, POU5F1, KIT) and somatic- (AR, FSHR, STAR) cell specific markers was evaluated 2 and 24 h after warming. RESULTS: Post-warming trypan blue staining showed the survival of most cells, although membrane integrity immediately after warming (66.00% ± 4.73) or after 2 h IVC (59.67% ± 4.18) was significantly lower than controls (C0h 89.67% ± 1.45). Extended post-warming IVC (24 h) caused an additional decrease to 31% ± 3.46 (P < 0.05). Germ- and somatic-cell specific markers showed the survival of both cell types after cryopreservation and IVC. All genes were affected by cryopreservation and/or IVC, and moderate stress conditions were indicated by transcriptional stress response. CONCLUSIONS: Vitrification with the cryodevice E.Vit is a promising strategy to cryopreserve prepubertal testicular tissue.

The Near Future of Vitrification of Oocytes and Embryos: Looking into Past Experience and Planning into the Future.

Currently vitrification is the method of choice for low-temperature preservation of oocytes and embryos. However, that was not the case until about 10 years ago when freezing methods were used relatively successfully for embryos and investigated (unsuccessfully) for oocyte preservation. In this paper we will review the history of oocyte and embryo cryopreservation and look into ways and methods for overcoming and improving the vitrification method since it suffers from inherent disadvantages since it is a cumbersome, time-consuming and costly procedure.

High post-thaw survival of ram sperm after partial freeze-drying.

BACKGROUND: Recrystallization damages occur when a frozen sample is held at high subzero temperatures and when the warming process is too slow. METHODS: In this work, ram semen diluted in two different concentrations of sugar solutions (Lyo A consisted of 0.4 M sorbitol and 0.25 M trehalose, and the second, Lyo B composed of 0.26 M sorbitol and 0.165 M trehalose) in egg yolk and Tris medium were compared after freezing 10 µL samples to: (1) - 10, - 25, and - 35 °C and thawing. (2) Freezing to - 10 and - 25 °C, holding for 1 h and then thawing, and (3) freezing to - 10 and - 25 °C and drying for 1 h at these temperatures at a vacuum of 80 mTorr, prior thawing. For drying, we used a new freeze-drying apparatus (Darya, FertileSafe, Israel) having a condensation temperature below - 110 °C and a vacuum pressure of 10-100 mTorr that is reached in less than 10s. RESULTS: Results showed that samples in Lyo B solution frozen at - 25 °C had significantly higher sperm motility in partially freeze-dried samples than frozen samples (46.6 ± 2.8% vs 1.2 ± 2.5%, P < 0.001). Moreover, partially dried samples in Lyo B showed higher motility than Lyo A at - 25 °C (46.6 ± 2.8% vs 35 ± 4%). Cryomicroscopy and low-temperature/low-pressure environmental scanning electronic microscope demonstrated that the amount of the ice crystals present in partially dried samples was lower than in the frozen samples. CONCLUSION: Holding the sperm at high subzero temperatures is necessary for the primary drying of cells during the freeze-drying process. Rapid freeze-drying can be achieved using this new device, which enables to reduce recrystallization damages.

A new, simple, automatic vitrification device: preliminary results with murine and bovine oocytes and embryos.

PURPOSE: This paper reports the use of a novel automatic vitrification device (Sarah, Fertilesafe, Israel) for cryopreservation of oocytes and embryos. METHODS: Mice oocytes (n = 40) and embryos (8 cells, n = 35 and blastocysts, n = 165), bovine embryos (2PN, n = 35), and MII oocytes (n = 84) were vitrified using this automated device. A total of 42 (2 cells) mice embryos, 20 (2PN) bovine embryos, and 150 MII bovine oocytes were used as fresh controls and grown to blastocysts. Upon rewarming, all were assessed for viability, cleavage, blastocyst, and hatching rates. RESULTS: Ninety-five % (38/40) of the mice MII oocytes regained isotonic volumes and all (100%) the surviving were viable. Rewarmed 8-cell mice embryos had 95% (33/35) blastulation rate and 80% (28/35) hatched. Rewarmed mice blastocysts had 97% survival rate (160/165) and 81% (135/165) hatched. Fresh control mice embryos had 100% (42/42) blastulation and 73% (21/42) hatching rates. Bovine embryos' survival was 100% with 54% (19/35) cleavage and 9% (3/35) blastulation rate. Fresh control bovine embryos had 65% (13/20) cleavage and 20% (4/20) blastulation rate. Vitrified bovine oocytes had 100% survival (84/84), 73% (61/84) cleavage, and 7% (6/84) blastocysts' rates; fresh control had 83% (125/150) cleavage and 11% (17/150) blastocysts' rates. CONCLUSION: This novel automatic vitrification device is capable to produce high survival rates of oocytes and embryos. We anticipate that as the demand for vitrification of gametes, embryos, and reproductive tissues increases worldwide, the availability of an automated vitrification device will become indispensable for standardization, simplification, and reproducibility of the entire process.

New methods for cooling and storing oocytes and embryos in a clean environment of -196°C.

It is well documented that oocyte vitrification using open systems provides better results than closed systems. However, its use is limited owing to risks of contamination posed by direct exposure to liquid nitrogen and cross-contamination when stored in liquid nitrogen tanks. A device that produces clean liquid air (CLAir) having similar a temperature as liquid nitrogen and a sterile storage canister device (Esther) that keeps samples sealed in their own compartment while in regular liquid nitrogen tanks were developed. The following experiments were performed: temperature measurements, bioburden tests, vitrification and storage experiments with mice embryos and human oocytes. Results showed similar cooling rates for liquid nitrogen and liquid air. Bioburden tests of CLAir and Esther showed no contamination, while massive contamination was found in "commercial" liquid nitrogen and storage canisters. Mice blastocysts had a survival rate of over 90%, with 80% hatching rate after vitirification in CLAir and 1 week storage in Esther, similar to the fresh (control) results. Human oocytes vitrified in CLAir and in liquid nitrogen for three consecutive vitrification/warming cycles showed 100% survival, seen as re-expansion in both groups. These new systems represent a breakthrough for safe vitrification using open systems and a safe storage process generally.

Directional freezing of spermatozoa and embryos.

Directional freezing is based on a simple thermodynamic principle whereby the sample is moved through a predetermined temperature gradient at a velocity that determines the cooling rate. Directional freezing permits a precise and uniform cooling rate in small- and large-volume samples. It avoids supercooling and reduces mechanical damage caused by crystallisation. Directional solidification was used to date for slow and rapid freezing, as well as for vitrification of oocytes and embryos by means of the minimum drop size technique: small drops are placed on a microscope slide that is moved at high velocity from the hot base to the cold base. Sperm samples from a wide range of domestic and wild animals were successfully cryopreserved using the directional freezing method. The bovine sexed semen industry may benefit from the increased survival of spermatozoa after directional freezing.

Vitrification of oocytes: from basic science to clinical application.

Vitrification is a physical process by which a liquid is transformed into a solid of amorphous glass form. It was only at the end of the nineteenth century (1898) that Gustav Heinrich Johann Apollon Tammann pointed out that a large number of substances can be obtained as glasses and suggested that this property might be universal (Tammann, Zeitschrift for Physikalische Chemie; 25: 441-479, 1898). Basically, vitrification is the supercooling of a liquid to a temperature at which the viscosity is so high that it can be defined as being at a solid state. The understanding of the vitrification process has been deepened over the years and has been applied for cryopreservation and currently is the method of choice for preserving oocytes and embryos.

Amorphous Calcium Carbonate Shows Anti-Cancer Properties That are Attributed to Its Buffering Capacity.

AIM: Amorphous calcium carbonate (ACC) is a non-crystalline form of calcium carbonate, and it is composed of aggregated nano-size primary particles. Here, we evaluated its anti-cancer effect postulated relative to its buffering capabilities in lung cancer. METHODS: Tumors were evaluated in vivo using the Lewis lung carcinoma (LLC) mouse cell line and A549 human lung cancer carcinoma cell line. LLC and A549 cells were injected subcutaneously into the right hind leg of mice. Treatments (ACC, cisplatin, vehicle, and ACC with cisplatin, all given via daily IP injections) started once tumors reached a measurable size. Treatments were carried out for 14 days in the LLC model and for 22 and 24 days in the xenograft model (two experiments). LLC tumors were resected from ACC at the end of the study, and vehicle groups were evaluated for cathepsin B activity. Differential gene expression was carried out on A549 cells following 8 weeks of in vitro culture in the presence or absence of ACC in a culture medium. RESULTS: The ACC treatment decelerated tumor growth rates in both models. When tumor volumes were compared on the last day of each study, the ACC-treated animal tumor volume was reduced by 44.83% compared to vehicle-treated animals in the LLC model. In the xenograft model, the tumor volume was reduced by 51.6% in ACC-treated animals compared to vehicle-treated animals. A more substantial reduction of 74.75% occurred in the combined treatment of ACC and cisplatin compared to the vehicle (carried out only in the LLC model). Cathepsin B activity was significantly reduced in ACC-treated LLC tumors compared to control tumors. Differential gene expression results showed a shift towards anti-tumorigenic pathways in the ACC-treated A549 cells. CONCLUSION: This study supports the ACC anti-malignant buffering hypothesis by demonstrating decelerated tumor growth, reduced cathepsin B activity, and altered gene expressions to produce anti-cancerous effects.

Long-Term Survival and Functional Recovery of Cryopreserved Vascularized Groin Flap and Below-the-Knee Rat Limb Transplants.

Effective cryopreservation of large tissues, limbs, and organs has the potential to revolutionize medical post-trauma reconstruction options and organ preservation and transplantation procedures. To date, vitrification and directional freezing are the only viable methods for long-term organ or tissue preservation, but are of limited clinical relevance. This work aimed to develop a vitrification-based approach that will enable the long-term survival and functional recovery of large tissues and limbs following transplantation. The presented novel two-stage cooling process involves rapid specimen cooling to subzero temperatures, followed by gradual cooling to the vitrification solution (VS) and tissue glass transition temperature. Flap cooling and storage were only feasible at temperatures equal to or slightly lower than the VS Tg (i.e., -135°C). Vascularized rat groin flaps and below-the-knee (BTK) hind limb transplants cryopreserved using this approach exhibited long-term survival (>30 days) following transplantation to rats. BTK-limb recovery included hair regrowth, normal peripheral blood flow, and normal skin, fat, and muscle histology. Above all, BTK limbs were reinnervated, enabling rats to sense pain in the cryopreserved limb. These findings provide a strong foundation for the development of a long-term large-tissue, limb and organ preservation protocol for clinical use.

Cryopreservation by Directional Freezing and Vitrification Focusing on Large Tissues and Organs.

The cryopreservation of cells has been in routine use for decades. However, despite the extensive research in the field, cryopreservation of large tissues and organs is still experimental. The present review highlights the major studies of directional freezing and vitrification of large tissues and whole organs and describes the different parameters that impact the success rate of large tissue and organ cryopreservation. Key factors, such as mass and heat transfer, cryoprotectant toxicity, nucleation, crystal growth, and chilling injury, which all have a significant influence on whole-organ cryopreservation outcomes, are reviewed. In addition, an overview of the principles of directional freezing and vitrification is given and the manners in which cryopreservation impacts large tissues and organs are described in detail.

Current progress in oocyte and embryo cryopreservation by slow freezing and vitrification.

Preservation of female genetics is currently done primarily by means of oocyte and embryo cryopreservation. The field has seen much progress during its four-decade history, progress driven predominantly by research in humans, cows, and mice. Two basic cryopreservation techniques rule the field--controlled-rate freezing, the first to be developed, and vitrification, which, in recent years, has gained a foothold. While much progress has been achieved in human medicine, the cattle industry, and in laboratory animals, this is far from being the case for most other mammals and even less so for other vertebrates. The major strides and obstacles in human and other vertebrate oocyte and embryo cryopreservation will be reviewed here.

Molecular and Histological Evaluation of Sheep Ovarian Tissue Subjected to Lyophilization.

Cryopreservation is routinely used to preserve cells and tissues; however, long time storage brings many inconveniences including the use of liquid nitrogen. Freeze-drying could enable higher shelf-life stability at ambient temperatures and facilitate transport and storage. Currently, the possibility to freeze-dry reproductive tissues maintaining vitality and functions is still under optimization. Here, we lyophilized sheep ovarian tissue with a novel device named Darya and a new vitrification and drying protocol and assessed effects on tissue integrity and gene expression. The evaluation was performed immediately after lyophilization (Lio), after rehydration (LR0h) or after two hours of in vitro culture (IVC; LR2h). The tissue survived lyophilization procedures and maintained its general structure, including intact follicles at different stages of development, however morphological and cytoplasmic modifications were noticed. Lyophilization, rehydration and further IVC increasingly affected RNA integrity and caused progressive morphological alterations. Nevertheless, analysis of a panel of eight genes showed tissue survival and reaction to the different procedures by regulation of specific gene expression. Results show that sheep ovarian tissue can tolerate the applied vitrification and drying protocol and constitute a valid basis for further improvements of the procedures, with the ultimate goal of optimizing tissue viability after rehydration.

Dry biobanking as a conservation tool in the Anthropocene.

Species are going extinct at an alarming rate, termed by some as the sixth mass extinction event in the history of Earth. Many are the causes for this but in the end, all converge to one entity - humans. Since we are the cause, we also hold the key to making the change. Any change, however, will take time, and for some species this could be too long. While working on possible solutions, we also have the responsibility to buy time for those species on the verge of extinction. Genome resource banks, in the form of cryobanks, where samples are maintained under liquid nitrogen, are already in existence but they come with a host of drawbacks. Biomimicry - innovation inspired by Nature, has been a huge source for ideas. Searching methods that Nature utilizes to preserve biological systems for extended periods of time, we realize that drying rather than freezing is the method of choice. We thus argue here in favor of preserving at least part of the samples from critically endangered species in dry biobanks, a much safer, cost-effective, biobanking approach.

Embryo cryopreservation in the presence of low concentration of vitrification solution with sealed pulled straws in liquid nitrogen slush.

BACKGROUND: Vitrification is becoming the method of choice for embryo cryopreservation. Nevertheless, major problems are still associated with this process such as chemical toxicity and osmotic stress as well as risk of liquid nitrogen (LN) contamination. METHODS: An innovative vitrification method that combines LN slush and sealed pulled straws (SPS) was employed to achieve a high cooling rate, enabling a reduction in cryoprotectant concentration. Open pulled straws were sealed at both ends to prevent direct contact with LN. RESULTS: Ultrarapid cooling of murine embryos at 32 200 degrees C/min in SPS with LN slush yielded a higher blastocyst survival rate (54 +/- 3.5%, 106/196) than cooling at 1700 degrees C/min in 0.25 ml straws (10 +/- 2.1%, 21/197) (P < 0.05). Embryos at the 2-cell stage cryopreserved in 75% vitrification solution (VS) (100% VS contains approximately 5 M ethylene glycol, 0.6 M trehalose and 6% w/v bovine serum albumin) in SPS formed blastocysts at a higher rate (79 +/- 3.6%, 99/126) than cryopreservation in 100% VS (31 +/- 6.5%, 16/51), however, this was not significantly different from the fresh control group (88 +/- 4.6%, 43/49). Early stage embryos at the 2 pronuclei- and 4-8-cell stage formed blastocysts at rates of 68 +/- 4.5 and 60 +/- 3.7%, respectively, after vitrification in 87.5% VS. CONCLUSIONS: This method enables maintenance of high cooling rates as well as reduction of cryoprotectant concentration, despite the use of a sealed container that helps to reduce the potential risk of contamination.

Do physical forces contribute to cryodamage?

To achieve the ultimate goal of both cryosurgery and cryopreservation, a thorough understanding of the processes responsible for cell and tissue damage is desired. The general belief is that cells are damaged primarily due to osmotic effects at slow cooling rates and intracellular ice formation at high cooling rates, together termed the "two factor theory." The present study deals with a third, largely ignored component--mechanical damage. Using pooled bull sperm cells as a model and directional freezing in large volumes, samples were frozen in the presence or absence of glass balls of three different diameters: 70-110, 250-500, and 1,000-1,250 microm, as a means of altering the surface area with which the cells come in contact. Post-thaw evaluation included motility at 0 h and after 3 h at 37 degrees C, viability, acrosome integrity, and hypoosmotic swelling test. Interactions among glass balls, sperm cells, and ice crystals were observed by directional freezing cryomicroscopy. Intra-container pressure in relation to volume was also evaluated. The series of studies presented here indicate that the higher the surface area with which the cells come in contact, the greater the damage, possibly because the cells are squeezed between the ice crystals and the surface. We further demonstrate that with a decrease in volume, and thus increase in surface area-to-volume ratio, the intra-container pressure during freezing increases. It is suggested that large volume freezing, given that heat dissipation is solved, will inflict less cryodamage to the cells than the current practice of small volume freezing.

Techniques of Cryopreservation for Ovarian Tissue and Whole Ovary.

Cryopreservation of ovarian tissue has been considered experimental for many years, but very recently the American Society of Reproductive Medicine is reviewing the process and perhaps soon will remove the label of "experimental" and recognize it as an established method for preserving female fertility when gonadotoxic treatments cannot be delayed or in patients before puberty or when there is desire to cryopreserve more than just few oocytes. This article discusses in detail the 3 methodologies used for cryopreservation: (a) slow freezing, (b) directional freezing, and (c) vitrification.

Cryopreservation and Transplantation of Vascularized Composite Transplants: Unique Challenges and Opportunities.

Vascularized composite allotransplantation is the ultimate reconstructive tool when no other means of reconstruction are available. Despite its immense potential, the applicability of vascularized composite allotransplantation is hampered by high rejection rates and the requirement for high doses of immunosuppressive drugs that are associated with severe adverse effects and death. Because this is a non-life-saving procedure, widespread use of vascularized composite allotransplantation demands methods that will allow the reduction or elimination of immunosuppressive therapy. Efficient methods for the cryopreservation of biological cells and tissues have been sought for decades. The primary challenge in the preservation of viable tissue in a frozen state is the formation of intracellular and extracellular ice crystals during both freezing and thawing, which cause irreversible damage to the tissue. Recent proof-of-concept transplantations of a complete cryopreserved and thawed hindlimb in a rat model have demonstrated the potential of such methods. In the current review, the authors discuss how limb cryopreservation can attenuate or eliminate allograft rejection by either enabling better human leukocyte antigen matching or by adaptation of clinical tolerance protocols such as mixed chimerism induction. Also, the authors discuss the possible advantages of cryopreservation in autologous tissue salvage and cryopreservation following trauma. Clinical-grade cryopreservation may revolutionize the field of reconstruction, organ banking, and complex traumatic limb injury management.