New Method For Cryopreservation Research Articles

Awaking sleeping islets for a cure of diabetes

Successful transplantation of pancreatic islets can cure diabetes, but limitations are accessibility, quality, and insufficient quantities of islets. Zhan et al.1 developed a new method for cryopreservation of islets with high recovery, viability, functionality and scalability that could provide an on-demand supply for clinical islet transplantation.

Hydrogel Microfiber Encapsulation Enhances Cryopreservation of Human Red Blood Cells with Low Concentrations of Glycerol.

Cryopreservation of red blood cells (RBCs) has been studied as a typical example of cryobiology methodology. To date, a mature and long-term cryopreservation process for RBCs has been developed, which has the weakness of complicated procedures due to high concentrations of glycerol (Gly). Therefore, it is still a research focus to find a new method for cryopreservation of RBCs to reduce the concentrations of cryoprotectants (CPAs). In this study, alginate hydrogels, which have been widely used in preservation research, were selected because of their advantages such as lower cost, cytocompatibility, and crosslinking occurring under mild conditions. With a variety of CPA solutions, the RBC recovery with the cryopreservation of RBCs and RBC-laden hydrogel microfibers was compared in different situations. It was found that hydrogel microfiber encapsulation enhances cryopreservation of human RBCs with low concentrations of CPAs. And Gly can be removed by washing directly with a 0.9% sodium chloride solution. This study validated the feasibility of cryopreservation of RBC-laden hydrogel microfibers. It may provide a new and evolving direction for reducing the concentrations of CPAs used in the preservation of RBCs.

An Improved Encapsulation Method for Cryopreserving Hepatocytes for Functional Transplantation Using a Thermo-reversible Gelation Polymer.

Thermo-reversible gelation polymer (TGP) can be converted into a gel state upon warming and liquid upon cooling. The present study aimed to demonstrate a new method for cryopreservation and encapsulation of rat hepatocytes using a TGP and their successful transplantation. The isolated rat hepatocytes were microencapsulated using TGP, and stored in liquid nitrogen. After cryopreservation, hepatocytes were cultured. Moreover, hepatocytes were transplanted into the spleen without a TGP capsule. The viability of hepatocytes that were cryopreserved in TGP was 71.2±2.3%. The hepatocytes demonstrated adequate survival, maintained their hepatic function in culture, and expressed albumin after transplantation to the rat spleen. We demonstrated a cryopreservation method of rat hepatocyte encapsulation using a TGP gel in the hydrogel state which subsequently allowed successful transplantation of unencapsulated hepatocytes in a sol state TGP gel at low temperature.

Freezing under pressure: A new method for cryopreservation

The ability to preserve cells, tissues, and organs with minimal damage for an extended period of time is essential for advancements in medicine and research. Current methods for cryopreservation are based on using high concentrations (up to 60% v/v) of cryoprotective agents (CPAs) which are toxic to living cells. The effect of pressure and a low concentration of dimethyl sulfoxide (Me2SO) or glycerol, on hemolysis of human red blood cells (RBCs) after freezing and thawing were investigated. Pressure was applied during cooling and freezing the RBCs and a minimum in hemolysis was reached at approximately 120MPa. Either 5% v/v Me2SO or 8% v/v glycerol concentration in combination with 120MPa pressure was sufficient to obtain 8% or less hemolysis of RBCs after cooling at a 35°C/min or a 160°C/min rate. The preliminary results suggest that the method may help to solve the CPAs toxicity problem.

137 Controlling freezing using infrared radiation

Controlling ice growth is a significant tool in many fields such as preventing frost damage to plants, improving cryo-preservation of cells, tissues and organs, the frozen food industry, and cryopreservation procedures. We suggest a control of ice growth in super-cooled solutions by a combination of infrared (IR) radiation and antifreeze materials, such as antifreeze proteins (AFPs), for the purpose of cryobiology and food science. AFPs, which are part of the ice-binding proteins family, inhibit the growth of an ice crystal. As a result, the freezing temperature is reduced below the melting point. The hypothesis is that ice crystals can be stabilized in super-cooled solution by the use of the difference in the IR absorption of ice and water. Additional super-cooling can be achieved by AFPs. We use a homemade temperature-controlled system that allows fine control of the temperature of small amounts of liquid enclosed in a microdevice (Celik, Drori et al., 2013). The system includes an IR radiation to selectively heat and melt ice, due to the ability of IR radiation to heat ice more than water when a particular wavelength is chosen (Ullman, 2001). To measure the temperature within the device, we use a near-IR (NIR) camera and a band filter. This method is based on the temperature dependence of the NIR absorption of water (Kakuta, Fukuhara et al., 2011). Initial results show the ability to melt ice within microfluidic devices. We also were able to measure temperature field within the water using the IR camera. Simulation guided experiments are developed to show the control of ice growth using IR radiation in coexistence of ice crystals in super-cooled water. The combination we propose to control ice within miniature samples with an IR radiation and AFPs, opens the possibility of a new method for cryopreservation of cells and tissues. Source of funding: The Israel Science Foundation (ISF). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

A new method for cryopreservation of mouse spermatozoa

A new method for the cryopreservation of mouse spermatozoa was developed using a modified egg-yolk TES-Tris diluent containing 0.1% sodium lauryl sulfate and 1.25% (v/v) glycerol (mouse sperm cryoprotectant, MSC). Epididymal spermatozoa collected from 10-week-old CBA males were frozen at a rate of 5 degrees C min-1 to 4 degrees C and 50 degrees C min-1 to -70 degrees C using a programmable cell freezer. A percentage of the spermatozoa (25%) regained motility after thawing. In vitro fertilization with frozen-thawed spermatozoa resulted in 50% of oocytes developing to the two-cell stage. These two-cell embryos were placed in the oviducts of pseudopregnant recipients (C57BL/CBA) and 16% developed to be viable fetuses, or in the oviducts of pregnant recipients (MF1) and 17% developed to live offspring.

Thawed human hepatocytes in primary culture

In drag metabolism studies, isolated and cultured human hepatocytes provide a useful model for overcoming the difficulty of extrapolating from animal data. In vitro studies with human hepatocytes are scarce because of the lack of livers and suitable methods of storage. After developing a new method for cryopreservation of human hepatocytes, we evaluated the effects of deep freezing storage on their viability, morphology, and functional and toxicological capabilities in classical culture conditions. Freshly isolated human hepatocytes were cryopreserved in medium containing 10% Me 2SO and 20% fetal calf serum, using a Nicool ST20 programmable freezer (−1.9 °C/min for 18 min and −30 °C/min for 4 min). Cells were stored in liquid nitrogen. Viability of thawed human hepatocytes was 50–65% as assessed by erythrosin exclusion test prior to purification on a Percoll density gradient. Morphological criteria showed that thawed human hepatocytes require an adaptation period to the medium after seeding. Functional assessments showed that human hepatocytes which survive freezing and thawing preserve their protein synthesis capabilities and are able to secrete a specific protein, anionic peptidic fraction, which is involved in the hepatic uptake of bile-destined cholesterol. We then studied Midazolam biotransformation to test metabolic functions, and erythromycin toxicity by Neutral Red test (cell viability) and 3-(4,5-dimethylthiazol-2-yl)-diphenyl tetrazolium bromide test (cell metabolism). All of these experiments indicated that thawed human hepatocytes should be used 38 h after seeding for optimum recovery of their functions: membrane integrity, protein synthesis, and stabilization of drug metabolism enzymes.

Freshly isolated or cryopreserved human hepatocytes in primary culture: Influence of drug metabolism on hepatotoxicity

The use of human hepatocytes in drug metabolism studies overcomes the difficulty in extrapolating from animal data. As drug metabolites are often involved in pharmacological activity and toxicity, it is essential to determine these compounds early in the development of a drug. We previously demonstrated that mitoxantrone metabolism exhibits a great interspecies variability: the two main metabolites in humans being dicarboxylic acid (the major metabolite) and monocarboxylic acid. In this paper we analyse their respective cytotoxicities with two endpoint tests: neutral red uptake (cell viability) and MTT reduction (cell metabolism) after a 24- or 48-hr exposure. Results demonstrated less or no toxicity of mono- and dicarboxylic acid derivatives compared with mitoxantrone. In vitro studies using human hepatocytes are scarce because of the limited availability of human livers. After developing a new method for cryopreservation of human hepatocytes, we evaluated them before and after cryopreservation using erythromycin as a test molecule. The cell viability test was more reproducible than the cell metabolism test after 38 hr of cell seeding.

Cryopreservation of porcine corneas with chondroitin sulfate

A new method for cryopreservation of porcine corneoscleral discs has been developed. Instead of the widely used cryoprotectant DMSO, chondroitin sulfate was applied as a cryoprotectant for the first time. Post-thawing evaluation was done after short-term tissue culture. Central cell counts showed a mean endothelial cell density of 2430 cells/mm2 (+/- 383) after freezing with a combination of 2% chondroitin sulfate and 20% fetal calf serum. Corneae which were cryopreserved with DMSO using the method established by Capella and Kaufman showed a mean endothelial cell density of 1672 cells/mm2 (+/- 617). These experiments demonstrated significant cryoprotective properties of chondroitin sulfate. In addition to its application in refrigerated corneal storage at 4 degrees C and organ culture at 32 degrees C, this substance may also be useful in cryopreservation.