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.

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.

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.

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.

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.

High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device.

BACKGROUND: To advance the use of embryo vitrification in veterinary practice, we developed a system in which embryo vitrification, warming and dilution can be performed within a straw. Ovine in vitro produced embryos (IVEP) were vitrified at either early (EBs: n = 74) or fully expanded blastocyst stage (FEBs: n = 195), using a new device named "E.Vit", composed by a 0.25-mL straw with a 50-µm pore polycarbonate grid at one end. Embryos at each stage (EBs and FEBs) were vitrified by either Two-step (TS) or Multi-step (MS; 6 different concentrations of vitrification solutions) protocol. Non-vitrified embryos (n = 102) were maintained in in vitro culture as a control. Warming consisted of placing the straws directly into 1.5 mL tubes containing a TCM-199 solution with three decreasing concentrations of sucrose. Blastocyst re-expansion, embryo survival and hatching rate were evaluated at 2, 24 and 48 h post warming. The number of apoptotic cells was determined by TUNEL assay. RESULTS: Blastocyst re-expansion (2 h) after warming was higher (P < 0.05) in FEBs group, vitrified with the MS and TS methods (77.90% and 71.25%, respectively) compared with the EBs group (MS: 59.38% and TS: 48.50%, respectively). Survival rates of vitrified FEBs after 24 h IVC were higher (P < 0.001) in both methods (MS and TS) than vitrified EBs (MS: 56.25%; TS: 42.42%) and was higher (P < 0.05) in the MS method (94.19%) compared with those in TS (83.75%). After 48 h of culture the hatching rate for FEBs vitrified in MS system (91.86%) was similar to control (91.89%), but higher than FEB TS (77.5%) and EBs vitrified in MS (37.5%) and TS (33.33%). Number of apoptotic cells were higher in EBs, irrespective of the system used, compared to FEBs. The number of apoptotic cells in FEBs vitrified with MS was comparable to the control. CONCLUSIONS: A high survival rate of IVP embryos can be achieved by the new "E.Vit" device with hatching rates in vitro comparable with control fresh embryos. This method has the potential for use in direct embryo transfer in field conditions.

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.

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.

Correction to: High in vitro survival rate of sheep in vitro produced blastocysts vitrified with a new method and device.

[This corrects the article DOI: 10.1186/s40104-019-0390-1.].

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.

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.

Chapter 16 Directional Freezing of Ovarian Tissue and Freeze-Drying of Stem Cells for Fertility Preservation.

Fertility preservation is practiced today for women in their fertile years who wish or need to postpone or prolong their reproductive era. Currently women have two main options to preserve their fertility either by oocyte vitrification or by freezing of ovarian cortical slices. In this chapter we will describe the use of directional freezing for cortical slices and freeze-drying of stem cells. These procedures open the way for the preservation of reproductive cells and tissue for future use in fertility preservation treatments.

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 sperm and associated derived technologies.

Directional freezing has now completed 30 years of development since it was first introduced to cryobiology. In the field of sperm cryopreservation, directional freezing has been shown to be advantageous over slow freezing for numerous domestic and wildlife species. In particular, it was shown that freezing of large volume is possible. Furthermore, double freezing of sperm and freezing of sex-sorted sperm are possible and became the routine in the sex sorted sperm industry. In wild animals, our labs and others showed that sperm from a wide range of terrestrial and aquatic species can be successfully cryopreserved using directional freezing. Finally, we will describe for the first time the successful freeze-drying of human sperm in an aseptic method. Using a device that produces clean liquid air, we froze human sperm in small droplets and then dried them in a bench top lyophilizer that was sterilized prior to use. More than 80% of DNA integrity was found after rehydration.