Cryopreservation of a whole liver

Preservation of vascularized organs such as the liver is limited to 24hrs. before destructive processes disqualify it for transplantation. This narrow time window prevents surgeons from performing optimal pathogen screening and matching tests which often lead to re-transplantation. Numerous problems are associated with viably freezing and thawing a whole liver: complicated geometry, poor heat transfer, release of latent heat and the difficulty of generating a uniform cooling rate. Our past success led us to apply our novel freezing technique to a larger solid organ, the liver. Whole livers were frozen/thawed using a directional solidification apparatuses; viability was tested by means of integrity and functionality in vitro and in auxiliary liver transplantation. Thawed livers were intact with over 80% viability; histology revealed normal architecture, bile production and blood flow following auxillary transplantation where normal. Our results suggest a novel cryopreservation method and may enable better organ donor-recipient matching in the futur

Freeze-Drying of Mononuclear Cells Derived from Umbilical Cord Blood Followed by Colony Formation

BACKGROUND: We recently showed that freeze-dried cells stored for 3 years at room temperature can direct embryonic development following cloning. However, viability, as evaluated by membrane integrity of the cells after freeze-drying, was very low; and it was mainly the DNA integrity that was preserved. In the present study, we improved the cells' viability and functionality after freeze-drying. METHODOLOGY/PRINCIPAL FINDINGS: We optimized the conditions of directional freezing, i.e. interface velocity and cell concentration, and we added the antioxidant EGCG to the freezing solution. The study was performed on mononuclear cells (MNCs) derived from human umbilical cord blood. After freeze-drying, we tested the viability, number of CD34(+)-presenting cells and ability of the rehydrated hematopoietic stem cells to differentiate into different blood cells in culture. The viability of the MNCs after freeze-drying and rehydration with pure water was 88%-91%. The total number of CD34(+)-presenting cells and the number of colonies did not change significantly when evaluated before freezing, after freeze-thawing, and after freeze-drying (5.4 x 10(4)+/-4.7, 3.49 x 10(4)+/-6 and 6.31 x 10(4)+/-12.27 cells, respectively, and 31+/-25.15, 47+/-45.8 and 23.44+/-13.3 colonies, respectively). CONCLUSIONS: This is the first report of nucleated cells which have been dried and then rehydrated with double-distilled water remaining viable, and of hematopoietic stem cells retaining their ability to differentiate into different blood cells

Freeze-Dried Somatic Cells Direct Embryonic Development after Nuclear Transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies

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

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

Automation in Oocyte and Ovarian Tissue Vitrification

Two hundred years have passed since the first description of supercooled water by Gay-Lussac to the recent reports of high survival rates of embryo and oocytes after vitrification. The use of vitrification as a preservation method has increased and improved worldwide in the last decade. However, the vitrification procedure can be further improved, for example, developing an automatic device that will recapitulate the entire manual steps. The ideal automation device should be capable to prepare gametes, embryos, or reproductive tissue slices for vitrification by exposing them to the different vitrification solutions, cooling them in a rapid and safe manner, and later, warming and diluting them from cryoprotectants (CPs). Automation will help to standardize the technique of oocyte, embryos, and tissue vitrification and rewarming and make it reproducible for everyone to use

Successful pregnancies in cows following double freezing of a large volume of semen

The objective of the following paper is to describe a new technology for large volume and double freezing of semen in 12 mL test tubes. Semen from two different bulls was frozen with a new technique using 12 mL test tubes and was refrozen after thawing in mini straws. All freezing was done in a “Multi thermal gradient” (MTG) freezing apparatus, which moves the container at a constant velocity (V) through a thermal gradient (G) producing a controlled cooling rate B = (G) $\times$ (V). Each of the two bulls ejaculated were evaluated for post thaw motility in the lab and then in a field trial which was carried out in a split sample mode. We inseminated 105 cows after a double freezing/thawing cycle, and another 123 cows were inseminated with semen frozen in mini-straws and a conventional method. The results showed a 75 $\pm$ 5% post thaw motility after freezing a 12 mL test tube and 50 $\pm$ 5% after a second freezing/thawing in mini-straws, respectively. Controlled vapour freezing showed a 60 $\pm$ 10% post thaw motility. The results of the field trial showed a pregnancy rate of 44% (47/105) for the double freezing group in comparison to 45.5% (56/123) for the controlled group. These results can be beneficial for large volume freezing, and therefore for bull semen cryobanking in a large volume which will be followed by second freezing in a regular insemination volume

Percentage membrane integrity after freeze-thawing and freeze-drying with different concentrations of EGCG.

<p>This experiment was performed on HUCB units from three different donors, in duplicate (n = 60). Data are presented as mean±SE.</p