Effective cryopreservation of neural stem or progenitor cells without serum or proteins by vitrification.

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 microl sterile straw placed in 500 microl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Effective Cryopreservation of Neural Stem or Progenitor Cells without Serum or Proteins by Vitrification.

Development of effective cryopreservation protocols will be essential to realizing the potential for clinical application of neural stem and progenitor cells. Current cryopreservation protocols have been largely employed in research, which does not require as stringent consideration of viability and sterility. Therefore, these protocols involve the use of serum and protein additives, which can potentially introduce contaminants, and slow cooling with DMSO/glycerol-based cryopreservation solutions, which impairs cell survival. We investigated whether serum- and protein-free vitrification is effective for functional cryopreservation of neurosphere cultures of neural stem or progenitor cells. To protect the samples from introduction of other contaminants during handling and cryostorage, an original "straw-in-straw" method (250 µl sterile straw placed in 500 µl straw) for direct immersion into liquid nitrogen and storing the samples was also introduced. The protocol employed brief step-wise exposure to vitrification solution composed of ethylene glycol (EG) and sucrose (40% v/v EG, 0.6 M sucrose) and removal of vitrification solution at room temperature. Evaluation of the effects of vitrification revealed that there were no differences between control and vitrified neural stem or progenitor cells in expression of the neural stem or progenitor cell markers, proliferation, or multipotent differentiation. This sterile method for the xeno-free cryopreservation of murine neurospheres without animal or human proteins may have the potential to serve as a starting point for the development of cryopreservation protocols for human neural stem and progenitor cells for clinical use.

Vitrification successfully preserves hepatocyte spheroids.

This is the first report on low-temperature preservation of self-assembled cell aggregates by vitrification, which is both a time- and cost-effective technology. We developed an effective protocol for vitrification (ice-free cryopreservation) of hepatocyte spheroids that employs rapid stepwise exposure to cryoprotectants (10.5 min) at room temperature and direct immersion into liquid nitrogen (-196 degrees C). For this, three vitrification solutions (VS) were formulated and their effects on vitrified-warmed spheroids were examined. Cryopreservation using ethylene glycol (EG)-sucrose VS showed excellent preservation capability whereby highly preserved cell viability and integrity of vitrified spheroids were observed, through confocal and scanning electron microscopy imaging, when compared to untreated control. The metabolic functions of EG-sucrose VS-cryopreserved spheroids, as assessed by urea production and albumin secretion, were not significantly different from those of control within the same day of observation. In both the vitrification and control groups, albumin secretion was consistently high, ranging from 47.57 +/- 14.39 to 70.38 +/- 11.29 microg/10(6) cells and from 56.84 +/- 14.48 to 71.79 +/- 16.65 microg/10(6) cells, respectively, and urea production gradually increased through the culture period. The efficacy of vitrification procedure in preserving the functional ability of hepatocyte spheroids was not improved by introduction of a second penetrating cryoprotectant, 1,2-propanediol (PD). Spheroids cryopreserved with EG-PD-sucrose VS showed maintained cell viability; however, in continuous culture, levels of both metabolic functions were lower than those cryopreserved with EG-sucrose VS. EG-PD VS, in which nonpenetrating cryoprotectant (sucrose) was excluded, provided poor protection to spheroids during cryopreservation. This study demonstrated that sucrose plays an important role in the effective vitrification of self-assembled cell aggregates. In a broad view, the excellent results obtained suggest that the developed vitrification strategy, which is an alternative to freezing, may be effectively used as a platform technology in the field of cell transplantation.

Vitrification as a prospect for cryopreservation of tissue-engineered constructs.

Cryopreservation plays a significant function in tissue banking and will presume yet larger value when more and more tissue-engineered products will routinely enter the clinical arena. The most common concept underlying tissue engineering is to combine a scaffold (cellular solids) or matrix (hydrogels) with living cells to form a tissue-engineered construct (TEC) to promote the repair and regeneration of tissues. The scaffold and matrix are expected to support cell colonization, migration, growth and differentiation, and to guide the development of the required tissue. The promises of tissue engineering, however, depend on the ability to physically distribute the products to patients in need. For this reason, the ability to cryogenically preserve not only cells, but also TECs, and one day even whole laboratory-produced organs, may be indispensable. Cryopreservation can be achieved by conventional freezing and vitrification (ice-free cryopreservation). In this publication we try to define the needs versus the desires of vitrifying TECs, with particular emphasis on the cryoprotectant properties, suitable materials and morphology. It is concluded that the formation of ice, through both direct and indirect effects, is probably fundamental to these difficulties, and this is why vitrification seems to be the most promising modality of cryopreservation.

Vitrification of encapsulated hepatocytes with reduced cooling and warming rates.

We have used microencapsulated hepatocytes as model to develop a method of vitreous cryopreservation of large quantities of cell-containing constructs. The method included a pre-equilibration procedure in which the amount of penetrating cryoprotectant was gradually increased by 15% in each step. The optimal vitrification solution consists of 40% ethylene glycol and 0.6M sucrose. The concentration of 1M sucrose used for the first dilution solution with subsequent decrease of sucrose concentration to 0.7 M sucrose and by 0.2-0.15M for each subsequent step. This sucrose dilution procedure had no adverse effect on cell functions. Three cooling rates (400 degrees C/min and above) and three warming rates (650 degrees C/min and above), in combination with the proposed vitrification solution, were equally effective. The optimization of the procedure and solutions allow microencapsulated hepatocytes to be preserved with almost 100% retention of cell functions and no detectable damage to the fragile microcapsules. The de-linking of the cooling/warming rates with the effectiveness of vitrification potentially paves the way for large scale cryopreservation of complex tissue engineered constructs.

Comparison of slow- and rapid-cooling protocols for early-cleavage-stage Macaca fascicularis embryos.

Cryostorage of nonhuman primate embryos by time-consuming slow-cooling methods is often limited to early cleavage stages. Effective rapid-cooling methods have been developed for many species and represent valuable tools for laboratory- and field-based studies of nonhuman primate reproductive biology. However, few rapid-cooling protocols have been applied to nonhuman primate embryos in terms of comparing various developmental stages. Here we compare slow cooling vs. two- and three-step rapid cooling of two-, four-, and eight-cell Macaca fascicularis (Mf) embryos. Rapid cooling was conducted in open pulled straws (OPS) using cooling solutions containing reduced quantities of ethylene glycol (EG) and supplemented with either of two high-molecular-weight polymers, ficoll and dextran. The survival of the slow-cooled embryos, but not the rapid-cooled embryos, was independent of embryonic stage at cryostorage. Slow cooling was associated with greater cell survival (82%) post thaw compared to warming following rapid cooling (18-29%). Slow cooling resulted in a high proportion of embryo survival (18/20; 90%) and cleavage (15/18; 83%) post thaw. Rapid cooling resulted in significantly reduced percentages of embryo survival (26-32%) and embryo cleavage in culture (29-38%) after warming. Conventional slow cooling was more effective than the rapid-cooling protocols employed in this study for cryopreservation of early-cleavage-stage Mf embryos.

Studies on replacing most of the penetrating cryoprotectant by polymers for embryo cryopreservation.

Solutions used for vitrification or rapid cooling of embryos usually contain high concentrations of penetrating cryoprotectants. At these concentrations embryos can tolerate the penetrating cryoprotectants for only short periods of time without damage. This study designed and tested cryoprotectant solutions that combined high polymer concentrations with low penetrating cryoprotectant concentrations. Mouse 2-cell embryo development was not compromised by up to 15-min exposure to 30 wt% solutions of the polymers Ficoll 70,000 MW or dextran 69,000 MW at room temperature. However, our batches of polyvinylpyrrolidone (PVP) 10,000 and PVP 40,000 were embryo-toxic even after extensive dialysis against Milli-Q water. As both Ficoll and dextran contribute to a solution's physical vitrification properties, we formulated vitrifying solutions containing only 11 to 27 wt% ethylene glycol (EG) by including 34 to 49 wt% polymers (27 wt% EG + 34 wt% Ficoll, 27 wt% EG + 34 wt% dextran, 16 wt% EG + 39 wt% Ficoll, or 11 wt% EG + 49 wt% Ficoll, in phosphate-buffered saline (PBS)). Novel solutions were designed for 0.25 ml straw as a viscous matrix for encapsulation of embryos. These yielded high rates of development of 2-cell mouse embryos after rapid cooling and warming (> or = 96% expanded blastocysts in vitro and > or = 62% viable fetuses as assessed on day 15 of gestation in vivo) in all tested solutions. All control 2-cell embryos formed expanded blastocysts in vitro and 78% formed fetuses in vivo. Comparable results were obtained with both 4-cell and 8- to 16-cell mouse embryos. The lower toxicity of Ficoll and dextran may explain why these new solutions gave better results than had previously been reported for solutions containing 7.5% PVP and low concentrations of EG (2 M).

A strategy for rapid cooling of mouse embryos within a double straw to eliminate the risk of contamination during storage in liquid nitrogen.

Double packaging, in which an inner straw containing the specimen is inserted into an outer, larger straw (here termed 'straw-in-straw') to prevent the inner straw from coming into direct contact with liquid nitrogen provides a simple strategy for reducing or eliminating the potential contamination risk associated with storage in liquid nitrogen. This approach has in the past been used in conjunction with cryopreservation by slow cooling, but has not previously been tested for use throughout an entire rapid cooling and warming procedure. This study determined whether keeping the straw containing the embryos inside a second protecting container throughout the cryopreservation and storage protocol would compromise embryo viability. We established that a cryoprotectant containing a high polymer concentration (35% dextran or Ficoll) together with 25% ethylene glycol (as the penetrating cryoprotectant) was highly effective for day 2 and day 3 mouse embryos in both single and double straws. The survival and development of all cryopreserved embryos, as assessed both in vitro and in vivo, was not statistically different to their untreated controls. This established that a protein/serum-free cryoprotectant solution supplemented with polymers could provide complete protection of mouse embryos. It also shows, for the first time, that embryos can be cooled by direct immersion in liquid nitrogen and warmed by direct immersion into a waterbath within a double straw arrangement to reduce the likelihood of contamination.

Sugars exert a major influence on the vitrification properties of ethylene glycol-based solutions and have low toxicity to embryos and oocytes.

A systematic approach was taken to assess the vitrification properties of ethylene glycol-based solutions supplemented with carbohydrates. Solutions were prepared by weight (gravimetrically) using ethylene glycol as the cryoprotectant, 0.9% NaCl in water, and six different sugars: d-glucose, d(-)-fructose, d-sorbitol, sucrose, d(+)-trehalose, and raffinose. Sugars were added on a molal basis (0. 1, 0.5, and 1 m). Characteristics of the solutions were measured during warming by differential scanning calorimetry using a cooling rate of 100 degrees C/min and a warming rate of 10 degrees C/min. In the absence of carbohydrates a 59 wt% EG-saline solution formed a stable glass. When EG was replaced by an equimolal concentration of glucose, fructose, or sorbitol (monosaccharides) at 0.1, 0.5, or 1.0 m there was no change in the total solute concentration at which vitrification occurred, but the glass transition (Tg) occurred at a higher temperature than in EG-saline alone. When EG was replaced by an equimolal concentration of sucrose or trehalose (disaccharides) both the Tg and the lowest total solute concentration required for vitrification became progressively higher as the molecular weight, or the ratio of sugar to EG in the solutions, increased. At the highest tested disaccharide concentration (1 m) vitrification was achieved at a total solute concentration of 65 wt% (sucrose) and 67 wt% (trehalose). The polysaccharide raffinose significantly modified the vitrification properties of ethylene glycol solutions. When 0.5 or 0.1 m raffinose replaced EG on an equimolal basis the glass transition point was raised more than with either the monosaccharides or the disaccharides. Raffinose allowed vitrification at a total solute concentration of 67 wt% (0.5 m) and 63 wt% (0.1 m). The maturation of immature mouse oocytes, and the development of embryos in media containing 5-7 mM of any sugar was comparable to controls, indicating that they are not toxic. Exposure of freshly collected GV or MII oocytes to sugar concentrations between 0.5 and 1.0 M, for up to 10 min had no significant effect on the proportion which subsequently formed two cells. We conclude that added sugars do contribute to a solutions overall vitrification properties, and their properties should be taken into consideration when vitrification solutions are being designed or modified.

Vitrification properties of solutions of ethylene glycol in saline containing PVP, Ficoll, or dextran.

Vitrification solutions which are used for cells or embryos generally contain cryoprotectant, physiological saline, and one or more macromolecular solutes. The macromolecules modify the vitrification tendencies of these solutions, but there is little detailed information on the vitrification properties of ethylene glycol solutions containing the additives PVP, Ficoll, and dextran. This study therefore added ethylene glycol to 0.9% NaCl in water (saline) and used differential scanning calorimetry to determine the lowest concentration at which the solution would remain vitreous when a warming rate of 10 degrees C/min was used. In the absence of other additives 59 wt% ethylene glycol (EG) in saline formed a stable glass. When ethylene glycol was replaced by the polymers Ficoll and/or dextran on a weight for weight basis, the resulting solution vitrified less readily than an EG-saline solution even though the total solute concentration was kept constant. The total solute concentration required to form a stable vitreous solution increased as the Ficoll 70,000 and 400,000 MW or dextran 78,000 MW content increased (5, 10, and 20 wt%). Ficoll and dextran had little or no effect on the glass transition and melting points of the solutions. In the presence of PVP vitrification occurred at a total solute concentration of 59 wt% (PVP 360,000 MW) or 60 wt% (PVP 40,000 MW) for all three tested PVP concentrations (5, 10, and 20 wt%). Although this indicates that PVP and EG have comparable vitrification properties, the melting and the glass transition temperature of the solutions rose as the PVP content increased. When 1 m sucrose was added to saline and 0, 5, 10, or 20 wt% PVP 40,000 MW vitrification was achieved with 31, 26, 23, and 15% EG, respectively, indicating that the total solute concentration required for vitrification could be estimated with reasonable accuracy from the sum of the individual components. We conclude that the tested polymers differ in how they interact with ethylene glycol-based vitrification solutions.