The relevance of ice crystal formation for the cryopreservation of tissues and organs.

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.

Cryopreservation of articular cartilage. Part 3: The liquidus-tracking method.

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant.

Principles of cryopreservation.

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase.

The relevance of ice crystal formation for the cryopreservation of tissues and organs.

This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.

Reduction of cryoprotectant toxicity in cells in suspension by use of a sodium-free vehicle solution.

Propane-1,2-diol (PD) possesses physico-chemical properties that make it a useful biological vitrifying agent but, although of relatively low toxicity, it still has substantial damaging effects on cells. This study aimed to identify possible toxic mechanisms using primary cell cultures from vascular tissue: these were exposed to the cryoprotectant at room temperature to avoid any possibility of hypothermic injury. Toxicity was evaluated by measuring the ability of the cells to divide in culture after exposure to the cryoprotectant.

Vitrification of rabbit tissues with propylene glycol and trehalose.

A previous study had suggested the use of a mixture of propanediol and trehalose for the preservation of tissues by vitrification. In this paper, we describe experiments in which stepwise procedures were developed for adding these cryoprotectants to high final concentrations in two rabbit tissues-carotid artery and cornea. The tissue concentration of the additives was measured at the end of each step so that the temperature of the next step could be chosen to reduce toxicity but avoid freezing.

Principles of cryopreservation.

Cryopreservation is the use of very low temperatures to preserve structurally intact living cells and tissues. Unprotected freezing is normally lethal and this chapter seeks to analyze some of the mechanisms involved and to show how cooling can be used to produce stable conditions that preserve life. The biological effects of cooling are dominated by the freezing of water, which results in the concentration of the solutes that are dissolved in the remaining liquid phase.

Further work on the cryopreservation of articular cartilage with particular reference to the liquidus tracking (LT) method.

The cryopreservation of articular cartilage with survival of living cells has been a difficult problem. We have provided evidence that this is due to the formation of ice crystals in the chondrons. We have developed a method in which the concentration of the cryoprotectant dimethyl sulphoxide (Me(2)SO) is increased progressively, in steps, as cooling proceeds so that ice is never allowed to form, but the very high concentrations of Me(2)SO required at low temperatures are reached only at those low temperatures.

Analysis of the permeation of cryoprotectants in cartilage.

Some tissues, such as cartilage and cornea, carry an internal fixed negative charge, leading to a swelling pressure that is balanced by tensile stress in the tissue matrix. During the addition and removal of cryoprotectants the changes in osmotic pressure will cause the tissue to deform. Because of the fixed charge and osmotic deformation, the permeation process in such tissues differs from ordinary diffusion processes.

The preservation of tissues for transplantation.

This paper is a written version of a lecture given during the celebration of Professor Rudolf Klen's 90th birthday. Dr. Klen played by far the major part in the introduction and the development of Tissue Banking in Europe.

Optimising cryopreservation protocols for haematopoietic progenitor cells: a methodological approach for umbilical cord blood.

Current cryopreservation protocols for haematopoietic cells have developed largely empirically and there is no consensus on an optimal method of preservation. These protocols, though providing sufficient cells to permit engraftment, can lead to cell loss of the order of 50 percent. In the context of umbilical cord blood such losses are unacceptable.

Cryopreservation of articular cartilage. Part 3: the liquidus-tracking method.

Although it is relatively straightforward to cryopreserve living isolated chondrocytes, at the present time there is no satisfactory method to preserve surgical grafts between the time of procurement or manufacture and actual use. In earlier papers we have established that the cryoprotectants dimethyl sulphoxide or propylene glycol do penetrate into this tissue very rapidly. Chondrocytes are not unusually susceptible to osmotic stress; in fact they appear to be particularly resistant.

Cryopreservation of articular cartilage. Part 2: mechanisms of cryoinjury.

Although isolated chondrocytes can be cryopreserved by standard methods, at the present time there is no satisfactory method that will preserve living chondrocytes in situ in surgical grafts, between the time of procurement or manufacture and actual use; survival of living chondrocytes in situ is inadequate at best and is also very variable. The first step in identifying the cause of this discrepancy was to establish that the cryoprotectants we had chosen to use, dimethyl sulphoxide and propylene glycol, do actually penetrate into the tissue rapidly. They do.

Cryopreservation of articular cartilage. Part 1: conventional cryopreservation methods.

There is increasing interest in the possibility of treating diseased or damaged areas of synovial joint surfaces by grafts of healthy allogeneic cartilage. Such grafts could be obtained from cadaver tissue donors or in the future they might be manufactured by 'tissue engineering' methods. Cartilage is an avascular tissue and hence is immunologically privileged but to take advantage of this is the graft must contain living cells.

The role of vitrification techniques of cryopreservation in reproductive medicine.

Traditional cryopreservation methods allow ice to form and solute concentrations to rise during the preservation process: both ice and high solute concentrations can cause damage. Cryoprotectants are highly soluble, permeating compounds of low toxicity; they reduce the amount of ice that crystallises at any given temperature and thereby limit the solute concentration factor. Vitrification methods use cryoprotectant concentrations that are sufficient to prevent the crystallisation of ice altogether: the material solidifies as an amorphous glass and both ice and solute concentration are avoided.

Banking of non-viable skin allografts using high concentrations of glycerol or propylene glycol.

The aims of this study were to investigate the kinetics of the current glycerol banking method for the preservation of non-viable skin allografts; to improve it with respect to efficiency and microbial safety; and to investigate the possibility of using propylene glycol in place of glycerol to provide a more rapid process. Skin grafts were preserved in 98% v/v glycerol (GLY) according to the method used in the Sheffield Skin Bank. During the addition and removal processes, the amounts of GLY and water in the skin were determined using the Karl Fischer method and HPLC respectively.

Use of peracetic acid to sterilize human donor skin for production of acellular dermal matrices for clinical use.

We previously reported methods for sterilizing human skin for clinical use. In a comparison of gamma-irradiation, glycerol, and ethylene oxide, sterilization with ethylene oxide after treatment with glycerol provided the most satisfactory dermis in terms of structure and its ability to produce reconstructed skin with many of the characteristics of normal skin. However, the use of ethylene oxide is becoming less common in the United Kingdom due to concerns about its possible genotoxicity.

Vitrification of ECV304 cell suspensions using solutions containing propane-1,2-diol and trehalose.

In this paper, we report on the suitability of solutions containing propane-1,2-diol (propylene glycol, PD), sugars, and salts for the vitrification of the human cell line, ECV304. Cooling (at 10 degrees C/min) and rewarming (at 80 degrees C/min) were at rates that are practicable for the tissues to be studied later. Under these conditions, 45% PD in phosphate-buffered saline (PBS) sometimes froze during cooling and always devitrified during rewarming but both events were avoided if the PBS salts were replaced by an osmotically equivalent concentration of sucrose or trehalose.

Cryopreservation of umbilical cord blood: 2. Tolerance of CD34(+) cells to multimolar dimethyl sulphoxide and the effect of cooling rate on recovery after freezing and thawing.

Cryopreservation protocols for umbilical cord blood have been based on methods established for bone marrow (BM) and peripheral blood stem cells (PBSC). The a priori assumption that these methods are optimal for progenitor cells from UCB has not been investigated systematically. Optimal cryopreservation protocols utilising penetrating cryoprotectants require that a number of major factors are controlled: osmotic damage during the addition and removal of the cryoprotectant; chemical toxicity of the cryoprotectant to the target cell and the interrelationship between cryoprotectant concentration and cooling rate.

Cryopreservation of umbilical cord blood: 1. Osmotically inactive volume, hydraulic conductivity and permeability of CD34(+) cells to dimethyl sulphoxide.

Umbilical cord blood (UCB) is an accepted treatment for the reconstitution of bone marrow function following myeloablative treatment predominantly in children and juveniles. Current cryopreservation protocols use methods established for bone marrow and peripheral blood progenitors cells that have largely been developed empirically. Such protocols can result in losses of up to 50% of the nucleated cell population: losses unacceptable for cord blood.

The stability during low-temperature storage of an antifreeze protein isolated from the roots of cold-acclimated carrots.

Natural antifreeze proteins (AFPs) not only inhibit freezing at high subzero temperatures; they have the additional properties of inhibiting the recrystallization of ice during warming and of preventing devitrification. The natural AFP that occurs in the roots of cold-acclimated carrots can be extracted reasonably simply and is non-toxic: it was selected for study as a possible ingredient of the vitrification mixtures that are being developed for use in tissue cryopreservation. For this application, it would be essential for the AFP to remain active during prolonged storage at very low temperatures.

Electromagnetic re-warming of cryopreserved tissues: effect of choice of cryoprotectant and sample shape on uniformity of heating.

A method that has been proposed for the cryopreservation of tissues and organs is to add a cryoprotective agent (CPA) in sufficient concentration to allow vitrification, and to use rapid electromagnetic heating to prevent the formation of ice crystals during the re-warming. We have compared the physical and biological properties of four CPAs, measuring the speed and uniformity of heating in a 36 mm sphere placed in a 434 MHz applicator, and the toxicity to ECV304 endothelial cells. Ethanediol and dimethyl sulfoxide were found to be suitable for rapid, uniform heating but toxic to the endothelial cells at vitrifying concentrations.

Cryopreservation of vascular endothelial cells as isolated cells and as monolayers.

This paper reports the cryopreservation of an immortalized human endothelial cell line (ECV304), either as a single cell suspension or as a confluent layer on microcarrier beads. Cell suspensions were exposed to 10% w/w dimethyl sulfoxide in a high-potassium solution (CPTes) at 0 degrees C. The cells were then cooled to -60 degrees C at controlled rates between 0.