Type Of Cryoprotectant Research Articles

A rational approach to oocyte cryopreservation

Reports of clinical pregnancies from cryopreserved human oocytes have been steadily increasing in recent years. However, success in terms of births per thawed oocyte remains poor. A wide variety of freezing techniques has been used lately, but modifications to protocols are made on an empirical basis. Methods of cryopreservation are often poorly described or protocols are not strictly adhered to, resulting in variability of outcome. The first stage of a freezing protocol is exposure to cryoprotectant. If performed inappropriately, such exposure can result in damage due to chemical toxicity and/or osmotic stress. Measurement of cell volume change during exposure to cryoprotectants demonstrates the extent of osmotic stress experienced by that cell. Such measurements have been performed during perfusion of murine and human oocytes with cryoprotectant concentrations commonly used for cryopreservation of these cells. It has been demonstrated that changes in the cryoprotectant type, concentration and temperature of exposure can dramatically affect the extent of cell volume change. Even small changes in duration of exposure to cryoprotectant prior to cooling can result in drastic changes in cellular hydration. Such factors will potentially influence the ability of the cell to survive the stresses experienced during the subsequent stages of the cryopreservation protocol.

Cryoinjury in endothelial cell monolayers

Developing successful cryopreservation strategies for corneas have proven to be more difficult than anticipated, because of the resulting loss of viability and detachment of endothelial cells from Descemet’s membrane following cryopreservation of corneas. The objectives of this study are to develop a more detailed understanding of cryoinjury in human corneal endothelial cell (HCEC) monolayers and to examine the effects of storage temperature, cryoprotectant type and concentration, and cooling/warming rates on HCEC monolayers. Monolayers of endothelial cells attached to collagen-coated glass, immersed in an experimental solution (with and without cryoprotectant) were cooled at 1 °C/min to various temperatures (−5 to −40 °C), then thawed directly or cooled rapidly to −196 or to −80 °C before thawing. Cryoprotectants used were dimethyl sulfoxide and propylene glycol in concentrations of 1 and 2 M. Monolayers were assessed for membrane integrity and detachment using SYTO/ethidium bromide fluorescent stain. The presence of cryoprotectants resulted in high recovery of membrane integrity and low monolayer detachment in monolayers thawed directly from temperatures down to −40 °C. In contrast, there was excessive detachment and loss of membrane integrity in monolayers cooled to −196 °C compared to monolayers cooled to −80 °C. Also, increasing cryoprotectant concentrations did not improve recovery of the monolayers. The higher recovery and lower detachment after storage at −80 °C compared to storage at −196 °C suggest that storage temperatures for corneas should be re-evaluated.

Effect of cryoprotectants on the stability and aerosol performance of nebulized aviscumine, a 57-kDa protein

Nebulization of aqueous drug solutions is a suitable delivery system for pulmonary application of proteins because it can easily produce droplets small enough to reach the alveolar region. However, proteins are sensitive to nebulization. Therefore, stabilizers need to be added which on the other hand influence the aerosol performance, such as average droplet size or mass output. This research presents the effect of various cryoprotectants such as Na-polyphosphate, CaCl 2×6H 2O and MgSO 4×7H 2O on the stability and aerosol performance of freeze-dried aviscumine after reconstitution and nebulization using three different nebulizers. Formulations containing Tris-buffer, polysorbate 80, Na 2-EDTA and HES450 were lyophilized and reconstituted with a buffered isotonic solution containing 100 mmol/l Tricine-buffer pH 8, 0.03% (w/v) octanoyl- N-methylglucamide, 150 mmol/l NaCl and a cryoprotectant. The aviscumine activity was determined by a binding assay. The addition of 0.2% Na-polyphosphate to the reconstitution medium led to retention of approx. 73% of the aviscumine activity after 20 min nebulization with the Systam ® ultrasonic nebulizer. It has been observed that 84 and 72% of the activity were retained by the addition of 10 mmol/l CaCl 2×6H 2O using PariBoy ® air-jet and Multisonic ® ultrasonic nebulizer, respectively. In addition, a decrease in the mean droplet size with increasing the cryoprotectant concentration has been observed. A relationship between the average droplet size, surface tension and viscosity depending on the used cryoprotectant type and concentration could be established.

Successful cryopreservation of mouse blastocysts using a new vitrification solution.

Mouse blastocysts were exposed to solutions containing four concentrations (10, 20, 30 and 40% v/v) of six permeating cryoprotectants (glycerol, ethylene glycol, propylene glycol, dimethyl sulfoxide, 1,3-butanediol and 2,3-butanediol) in phosphate-buffered saline (PBS) with calf serum (CS) at room temperature (20-22 degrees C). Blastocysts were exposed to these solutions for various periods, diluted into PBS plus CS with or without 1 mol trehalose l-1 solution and their subsequent survival in vitro was examined. Two-way anova showed a significant interaction (P < 0.01) between cryoprotectant type, concentration of cryoprotectant and method of dilution. However, no significant interaction was observed between cryoprotectant type and duration of exposure. Results suggest that cryoprotectant-induced injury to nonfrozen blastocysts is variable and depends on the cryoprotectant used. On the basis of toxicity assays, ethylene glycol was the least harmful and was combined with dimethyl sulfoxide and 1,3-butanediol to produce a new vitrification solution. Mouse blastocysts were successfully cryopreserved using a vitrification solution (designated as VSv) consisting of 20% ethylene glycol, 20% dimethyl sulfoxide and 10% 1,3-butanediol (v/v). Embryos were equilibrated in two steps, first in an equilibration solution (designated as ESv: 10% ethylene glycol, 10% dimethyl sulfoxide and 5% 1,3-butanediol; v/v) and then to VSv or one-step in VSv at different exposure times at room temperature, and then vitrified by direct plunging into liquid nitrogen. High developmental rates were obtained in vitro when the embryos were exposed to ESv and VSv for 3 and 0.5 min, respectively (96.2%) or exposed to VSv for 0.5 min (95.4%). Prolonged exposure time proved detrimental to subsequent embryo development in vitro. When vitrified warmed embryos were transferred immediately to pseudopregnant recipients, the rate of development to normal fetuses did not significantly differ from that of the nonvitrified control (two-step, 54.2 and one-step, 45.0 versus 60.0%, P > 0.05). These results suggest that the simple vitrification solution described in this study is effective for the cryopreservation of mouse blastocysts.

Optimizing conditions for cryopreservation of an insect cell line

A cell line (UM-BGE-2) derived from embryos of the cockroach Blattella germanica was frozen to −196 °C under a variety of conditions and cell viability was assayed after warming. It was found that cell viability was affected by the cooling rate, the warming rate, the controlled cooling endpoint temperature, and the type and concentration of cryoprotectant. The best survival for cells suspended in Grace's tissue culture medium containing 1 M Me 2SO was obtained when cells were cooled at 1 °C/ min to at least −90 °C before being placed in liquid nitrogen and warmed at more than 900 °C/min. Cultures initiated from these frozen cells produce typical growth curves and appear normal after several passages.

Cryopreservation of Human Lymphocytes: A Brief Review and Evaluation of an Automated Liquid Nitrogen Freezer

Successful cryopreservation of human lymphocytes has been previously described. Cryopreserved lymphocytes are useful for a variety of in vitro immunologic studies. This study was performed to determine the applicability and/or advantages of using a programmable freezing system, and compares glycerol versus dimethyl sulfoxide (DMSO) at varying concentrations on post-thaw viability, E-rosetting and immunoglobulin fluorescence. Prefreeze T and B lymphocyte percentages were determined. Cells were then frozen in varying concentrations of glycerol and DMSO. Optimum cryoprotectant type and concentration was determined. Lymphocytes from seven individuals were frozen by the batch method in a mechanical freezer and with the automated liquid nitrogen injection system. Data on post-thaw T and B percentages and viability revealed 10% DMSO and liquid nitrogen control freezing method at 1 C/minute as the best conditions for lymphocyte preservation as reflected by post-thaw in vitro testing.

Variations in myocardial CPK and Na +-K + ATPase following long-term freezing

The activity of the soluble enzyme CPK and the membrane bound enzyme Na +-K + ATPase as a function of storage temperature, time of storage and cryoprotectant type and concentration in canine myocardial tissue was invesigated. Activity of CPK is well preserved at −196 °C and −79 °C and falls off during one month storage at −40 °, −20 °, and 0 °C. Na +-K + ATPase demonstrates a greater liability. After an initial cryoprotectant “activation,” activity drops. In all cases, however, addition of the cryoprotectant preserved activity better than in samples stored only in buffer.

Effects of Storage by Cryopreservation on Types and Duration of Cryopectant Soaking With Different Conditions of Seeds on the Growth of Mangosteen Seeds (Garcinia mangostana L)

Effect Storage In Criopreservasi On Type And Old South Crioprotektan Different Conditions Of Seeds Against Growth of Mangosteen Seeds (Garcinia mangostana L). The aim of the study was to determine the type, duration of soaking and the different conditions of mangosteen seeds and their interaction with the growth of mangosteen seeds (Garcinia mangostana L). This research was carried out at the Laboratory of Applied Sciences in Seed Technology of Labuhanbatu College of Agricultural Sciences (STIPER), starting March to July 2018, using factorial randomized block design with 3 treatment factors namely cryoprotectant type, cryoprotectant immersion and mangosteen fruit flesh. Parameters observed were number of shoots (fruit), shoot height (cm), root length (cm), and sprout dry weight (g). The results showed that the highest application of the parameters was the use of non-fruit flesh with cryoprotectant PVS2 and 180 minutes soaking time. The results of the research were used to develop mangosteen seed storage for the needs of the community, especially mangosteen farmers in Indonesia.