Dose–injury relationships for cryoprotective agent injury to human chondrocytes

Vitrification of articular cartilage (AC) could enhance tissue availability but requires high concentrations of cyroprotective agents (CPAs). This study investigated relative injuries caused by commonly used CPAs. We hypothesized that the in situ chondrocyte dose–injury relationships of five commonly used CPAs are nonlinear and that relative injuries could be determined by comparing cell death after exposure at increasing concentrations. Human AC samples were used from four patients undergoing total knee arthroplasty surgery. Seventy μm slices were exposed in a stepwise protocol to increasing concentrations of 5 CPAs (max = 8 M); dimethyl sulfoxide (Me₂SO), glycerol (Gly), propylene glycol (PG), ethylene glycol (EG), and formamide (FM). Chondrocyte viability was determined by membrane integrity stains. Statistical analysis included t-tests and nonlinear least squares estimation methods. The dose–injury to chondrocytes relationships for all CPAs were found to be nonlinear (sigmoidal best fit). For the particular loading protocol in this study, the data identified the following CPA concentrations at which chondrocyte recoveries statistically deviated significantly from the control recovery; 1 M for Gly, 4 M for FM and PG, 6 M for Me₂SO, and 7 M for EG. Comparison of individual means demonstrated that Gly exposure resulted in the lowest recovery, followed by PG, and then Me₂SO, FM and EG in no specific order. The information from this study provides an order of damage to human chondrocytes in situ of commonly used CPAs for vitrification of AC and identifies threshold CPA concentrations for a stepwise loading protocol at which chondrocyte recovery is significantly decreased. In general, Gly and PG were the most damaging while DMSO and EG were among the least damaging.     

Survival of tissue balls from the coral Pocillopora damicornis L. exposed to cryoprotectant solutions

In this study, the tolerance of tissue balls (TBs, 100–300 μm in diameter) from the coral Pocillopora damicornis produced using mechanical excision to exposure to cryoprotectant (CPA) solutions was tested. TBs were treated for 20 min at room temperature with solutions of ethylene glycol (EG), methanol (Met), glycerol (Gly) or dimethyl sulfoxide (Me₂SO) at concentrations between 1.0 and 4.5 M. Two parameters were used to evaluate the survival of TBs following CPA treatment. The Undamaged Duration of Tissue Balls (expressed in h) corresponded to the time period during which the membrane surface of TBs remained smooth and their motility was preserved. Tissue Ball Regression (expressed in μm/h) corresponded to the size reduction of TBs over time. TBs tolerated exposure to all CPAs tested at the three lower concentrations employed (1.0 M, 1.5 M and 2.0 M). No survival was achieved following exposure to a 4.5 M CPA solution. At concentrations of 3.0 and 4.0 M, higher Undamaged Duration of Tissue Balls and lower Tissue Ball Regression were obtained following treatment with EG compared to the other three CPAs. Our experiments show that TBs constitute a good experimental material to evaluate CPA toxicity on corals using large numbers of samples. Performing preliminary experiments with TBs may allow reducing the number of tests carried out with less easily available coral forms such as planulae, thereby preserving larval stocks.     

Alginate beads as a tool to handle,cryopreserve and culture isolated human primordial/primary follicles

Background

One major concern of grafting cryopreserved ovarian tissue to restore fertility in cancer patients is the possibility of reintroducing tumor cells. Cryopreservation of isolated primordial/primary follicles (PFs) may circumvent this problem. The aim of our work was to compare dimethyl sulfoxide (ME₂SO) and ethylene glycol (EG) as cryoprotectants (CPAs) for slow-freezing of isolated human PFs in alginate.

Methods

Ovarian biopsies from four women were processed for follicle isolation. PFs were embedded in alginate (5–15 per group). Follicles were frozen-thawed using 1.4 M ME₂SO or 1.5 M EG as CPAs. Fresh and cryopreserved isolated follicles were in vitro cultured (IVC) for 7 days. At different time periods (after isolation, cryopreservation and IVC), follicles were evaluated with live/dead assay (using fluorescent probes) and diameter measurement. Follicle viability was calculated according to the percentage of dead follicular cells and the presence of a live/dead oocyte.

Results

A total of 841 PFs were isolated, embedded in alginate and cryopreserved with ME₂SO (n = 424) or EG (n = 259), or used as controls (n = 158). After 7 days of IVC, a significant increase in follicle size was observed in the fresh and ME₂SO groups, but not in the EG group. The percentage of totally viable PFs was not significantly different before or after seven days of culture in fresh (100% and 82%) or ME₂SO (93.2% and 85.1%) tissue. The EG group showed significantly lower viability before (63.9%) and after IVC (66.2%) than the fresh and ME₂SO groups.

Conclusions

Our results show that 1.4 M ME₂SO yields better preservation of isolated PF viability after thawing and 7 days of IVC than 1.5 M EG. Alginate constitutes an easy, safe hydrogel matrix to handle and cryopreserve isolated human follicles using ME₂SO as a CPA.     

Preliminary studies on cryopreservation of snakehead (Channa striata) embryos

This paper reports the findings of the ongoing studies on cryopreservation of the snakehead, Channa striata embryos. The specific objective of this study was to collect data on the sensitivity of C. striata embryo hatching rate to low temperatures at two different developmental stages in the presence of four different cryoprotectants. Embryos at morula and heartbeat stages were selected and incubated in 1 M dimethyl sulfoxide (Me₂SO), 1 M ethylene glycol (EG), 1 M methanol (MeOH) and 0.1 M sucrose solutions at different temperatures for a period of time. Embryos were kept at 24 °C (control), 15 °C, 4 °C and −2 °C for 5 min, 1 h and 3 h. Following these treatments, the embryos were then transferred into a 24 °C water bath until hatch to evaluate the hatching rate. The results showed that there was a significant decrease of hatching rate in both developmental stages following exposure to 4 °C and −2 °C at 1 h and 3 h exposure in each treatment. Heartbeat stage was more tolerant against chilling at −2 °C for 3 h exposure in Me₂SO followed by MeOH, sucrose and EG. Further studies will be conducted to find the best method to preserve embryos for long term storage.     

Permeability of the equine embryonic capsule to ethylene glycol and glycerol in vitro

Poor survival of cryopreservation by equine expanded blastocysts may involve low penetration of the embryonic capsule by cryoprotective agents (CPAs). This study characterized the permeation and accumulation rates of the CPAs ethylene glycol (EG) and glycerol (GLY) across isolated capsule in vitro, using a dual-chambered Valia-Chien permeation apparatus. Pieces of Days 14 to 18 ±1 capsules separated media in the “donor” chamber containing either 1.5 M EG (n = 6), 0.74 M EG (n = 5), 0.87 M GLY (n = 7), or 0.15 M NaCl (saline, SAL) (n = 6), from the “recipient” chamber. Concentrations of CPA, determined by gas chromatography, allowed calculation of the capsule's apparent permeability (P_app) to those CPAs. Permeation of capsule by 1.5 M EG was significantly more rapid than by 0.87 M GLY, or 0.74 M EG; permeation by both CPAs was significantly slower than by SAL. Accumulation of CPA in the recipient chamber depended more on initial donor chamber concentration, rather than type, of CPA. Accumulation rates for CPAs and SAL were linear only when capsule was present, demonstrating that their permeation through capsule was more complex than simple diffusion. Successful cryopreservation of equine expanded blastocysts has been previously linked to lengths of step-wise exposures to CPAs. Based on the present results, we inferred that alternative CPAs, more capable of permeating the capsule, or alternative methods of ensuring CPA entry into the cells, may also be required.     

Cryopreservation of sperm from farmed Pacific abalone,Haliotis discus hannai

This study aimed to improve a sperm cryopreservation protocol for farmed Pacific abalone, Haliotis discus hannai. Dimethyl sulfoxide (Me₂SO), glycerol, ethylene glycol (EG), propylene glycol (PG), and methanol were chosen as cryoprotectants (CPAs). Four different equilibration time (5, 10, 30, and 60 min), and two types of equilibration temperature (4 °C and 20 °C) were selected at the present experiment. Most equilibration temperatures with each CPA showed significant differences among different equilibration time. Post-thaw sperm motility of five CPAs showed no significant difference at two equilibration temperature. Based on these results, 8% Me₂SO, 8% EG, 6% PG, 2% glycerol, and 2% methanol were chosen to determine optimal conditions for sperm cryopreservation of H. discus hannai. The highest post-thaw sperm motility (8% Me₂SO: 50.6%, 8% EG: 45.6%, 2% glycerol: 44.5%, 6% PG: 28.7%, 2% methanol: 25.4%) was achieved after exposing sperm to liquid nitrogen (LN₂) vapor for 10 min at 5 cm above the LN₂ surface and then submerging them in LN₂ for at least 2 h followed by thawing at 60 °C with seawater and recovering them at 20 °C with seawater. In this study, 8% Me₂SO and 2% glycerol were chosen to check post-thaw sperm quality to estimate percentages of plasma membrane integrity (PMI), mitochondrial potential analysis (MP), and acrosome integrity (AI) using fluorescent techniques. No significant difference in PMI, MP, and AI was found between sperm cryopreserved with 8% Me₂SO and those cryopreserved with 2% glycerol. The current study has demonstrated that 8% Me₂SO was optimal for sperm cryopreservation for H. discus hannai with 5 min of equilibration time, 5 cm of rack height and 60 °C of thawing temperature. The present research provides more effective cryopreservation methods for H. discus hannai sperm than previous studies.     

Improved cryopreservation yield of pancreatic islets using combination of lower dose permeable cryoprotective agents

Islet transplantation has been shown to restore normoglycemia in animal models and for type 1 diabetic patients in clinical trials. One method of storing islets intended for transplantation is via cryobanking at very low temperatures (−196 °C). Cryobanking islets without the use of cryoprotecting agents (CPAs) contributes to cellular shear stress and cell death. Although current CPA protocols vary, high concentrations of these agents are toxic to islets cells. This study tested the effects of the permeating CPA dimethyl sulfoxide (Me₂SO) with the addition of ethylene glycol (EG), both at reduced concentrations, on rat and human islet cell yield, viability, and glucose stimulated insulin release (GSIR). To test this, islets were treated using three combinations of CPAs (2M ME₂SO, 1M ME₂SO + 1M EG, and 1M ME₂SO + 0.5M EG). Next, fresh islets, 2M ME₂SO islets, and 1M ME₂SO + 0.5M EG isolated rat islets were transplanted into streptozotocin-induced (STZ) diabetic mice. Our data showed that cryopreservation with a reduced concentration of ME₂SO (1M ME₂SO + multimolar EG) achieved a higher percent yield and viability when compared to the current standard 2M ME₂SO treatment for both rat and human islets. Furthermore, STZ-induced diabetic mice achieved normoglycemia after transplantation with 1000 islet equivalents (IE), an average 12 days sooner, with islets cryopreserved with reduced-concentration (ME₂SO + 0.5M EG), compared to islets preserved with 2M ME₂SO. In conclusion, reduced concentration of penetrating CPAs during islet cryopreservation increases islet yield and viability in vitro and reduces delay before normoglycemia in diabetic mice.     

Cytotoxicity effects of cryoprotectants as single-component and cocktail vitrification solutions

Cryoprotectant (CPA) cytotoxicity constitutes a challenge in developing cryopreservation protocols, specifically in vitrification where high CPA concentrations are necessary to achieve the ice-free, vitreous state. Few cytotoxicity studies have investigated vitrification-relevant concentrations of CPAs, and the benefits and disadvantages of cocktail solutions and of incorporating non-permeating solutes have not been fully evaluated. In this study, we address these issues by determining the cytotoxicity kinetics for dimethylsulfoxide (Me₂SO) and 1,2-propanediol (PD) on alginate-encapsulated βTC-tet mouse insulinomas for a range of concentrations and temperatures. Cytotoxicity kinetics were also determined for two cocktails, DPS (3 M Me₂SO + 3 M PD + 0.5 M sucrose) and PEG400 (1 M Me₂SO + 5 M PD + 0.34 M poly(ethylene)glycol with M.W. of 400). PD was found to be more cytotoxic than Me₂SO at higher concentrations and temperatures. This was reflected in PEG400 being more cytotoxic at room temperature than PEG400 at 4 °C or DPS at either temperature. Addition of non-permeating solutes increased the cytotoxicity of cocktails. Furthermore, results indicate that CPA cytotoxicity may not be additive and that combining CPAs may increase cytotoxicity synergistically. Finally, when comparing cytotoxic effects towards encapsulated HepG2 and βTC-tet cells, and towards βTC-tet cells in capsules and in monolayers, CPAs appear more cytotoxic towards cells with higher metabolic activity. The incorporation of these results in the rational design of CPA addition/removal processes in vitrification is discussed.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Recombinant Dendroides canadensis antifreeze proteins as potential ingredients in cryopreservation solutions

Expanding cryopreservation methods to include a wider range of cell types, such as those sensitive to freezing, is needed for maintaining the viability of cell-based regenerative medicine products. Conventional cryopreservation protocols, which include use of cryoprotectants such as dimethylsulfoxide (Me₂SO), have not prevented ice-induced damage to cell and tissue matrices during freezing. A family of antifreeze proteins (AFPs) produced in the larvae of the beetle, Dendroides canadensis allow this insect to survive subzero temperatures as low as −26 °C. This study is an assessment of the effect of the four hemolymph D. canadensis AFPs (DAFPs) on the supercooling (nucleating) temperature, ice structure patterns and viability of the A10 cell line derived from the thoracic aorta of embryonic rat. Cryoprotectant solution cocktails containing combinations of DAFPs in concentrations ranging from 0 to 3 mg/mL in Unisol base mixed with 1 M Me₂SO were first evaluated by cryomicroscopy. Combining multiple DAFPs demonstrated significant supercooling point depressing activity (∼9 °C) when compared to single DAFPs and/or conventional 1 M Me₂SO control solutions. Concentrations of DAFPs as low as 1 μg/mL were sufficient to trigger this effect. In addition, significantly improved A10 smooth muscle cell viability was observed in cryopreservation experiments with low DAFP-6 and DAFP-2 concentrations in combination with Me₂SO. No significant improvement in viability was observed with either DAFP-1 or DAFP-4. Low and effective DAFP concentrations are advantageous because they minimize concerns regarding cell cytotoxicity and manufacturing cost. These findings support the potential of incorporating DAFPs in solutions used to cryopreserve cells and tissues.     

Statistical prediction of the vitrifiability and glass stability of multi-component cryoprotective agent solutions

Long-term biologic storage of articular cartilage has proven elusive due to cellular degradation over time or acute damage during attempts at cryopreservation. Vitrification is one option that may result in successful cryopreservation but difficulty with cryoprotective agent (CPA) toxicity at high concentrations of a single cryoprotectant has hindered development of successful protocols. This study was designed to determine the vitrifiability and glass stability of solutions containing combinations of commonly used CPAs and to document CPA interactions that occur. One hundred and sixty-four multi-CPA combination solutions of 6-9 M were evaluated for vitrifiability and glass stability using direct visualization after immersion in liquid nitrogen for 30 min and upon warming. Binary and ordinal logistic regression analysis was used to statistically analyze each CPA for its ability to vitrify and its effect on glass stability in multi-component CPA solutions. Propylene glycol had the greatest incremental contribution to vitrification while formamide had the least contribution. A threshold was established whereby the ability of a solution to vitrify could be determined by calculation. Glass stability was not as clearly defined due to variability in the results; however, contributions of interactions between CPAs to the glass stability of solutions were determined. This study provided values that predict if a solution will vitrify. Furthermore, the glass stability of solutions containing multiple CPAs do not behave as linear additions of binary solutions and interactions between CPAs have a significant effect on the glass stability of these solutions. These variables should be considered when designing vitrification solutions.     

Systematic parameter optimization of a Me(2)SO- and serum-free cryopreservation protocol for human mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) have great potential for clinical therapy and regenerative medicine. One major challenge concerning their application is the development of an efficient cryopreservation protocol since current methods result in a poor viability and high differentiation rates. A high survival rate of cryopreserved cells requires an optimal cooling rate and the presence of cryoprotective agents (CPA) in sufficient concentrations. The most widely used CPA, dimethylsulfoxide (Me₂SO), is toxic at high concentrations at temperatures >4 °C and has harmful effects on the biological functionality of stem cell as well as on treated patients.Thus, this study investigates different combinations of non-cytotoxic biocompatible substances, such as ectoin and proline, as potential CPAs in a systematic parametric optimization study in comparison to Me₂SO as control and a commercial freezing medium (Biofreeze®, Biochrom). Using a freezing medium containing a low proline (1%, w/v) and higher ectoin (10%, w/v) amount revealed promising results although the highest survival rate was achieved with the Biofreeze® medium. Cryomicroscopic experiments of hMSCs revealed nucleation temperatures ranging from −16 to −25 °C. The CPAs, beside Me₂SO, did not affect the nucleation temperature. In most cases, cryomicroscopy revealed intracellular ice formation (IIF) during the cryopreservation cycle for all cryoprotocols. The occurence of IIF during thawing increased with the cooling rate. In case of hMSC there was no correlation between the rate of IIF and the post-thaw cell survival. After thawing adipogenic differentiation of the stem cells demonstrated cell functionality.     

Spermatozoa from the maned wolf (Chrysocyon brachyurus) display typical canid hyper-sensitivity to osmotic and freezing-induced injury,but respond favorably to dimethyl sulfoxide

We assessed the influences of medium osmolality, cryoprotectant and cooling and warming rate on maned wolf (Chrysocyon brachyurus) spermatozoa. Ejaculates were exposed to Ham’s F10 medium (isotonic control) or to this medium plus NaCl (350–1000 mOsm), sucrose (369 and 479 mOsm), 1 M glycerol (1086 mOsm) or dimethyl sulfoxide (Me₂SO, 1151 mOsm) for 10 min. Each sample then was diluted back into Ham’s medium and assessed for sperm motility and plasma membrane integrity. Although glycerol and Me₂SO had no influence (P > 0.05), NaCl and sucrose solutions affected sperm motility (P < 0.05), but not membrane integrity. Motility of sperm exposed to <600 mOsm NaCl or sucrose was less (P < 0.05) than fresh ejaculate, but comparable (P > 0.05) to the control. As osmolality of the NaCl solution increased, motility decreased to <5%. In a separate study, ejaculates were diluted in Test Yolk Buffer containing 1 M glycerol or Me₂SO and cooled from 5 °C to −120 °C at −57.8 °C, −124.2 °C or −67.0 °C/min, frozen in LN₂, thawed in a water bath for 30 s at 37 °C or 10 s at 50 °C, and then assessed for motility, plasma- and acrosomal membrane integrity. Cryopreservation markedly (P < 0.05) reduced sperm motility by 70% compared to fresh samples. Higher (P < 0.05) post-thaw motility (20.0 ± 1.9% versus 13.5 ± 2.1%) and membrane integrity (51.2 ± 1.7% versus 41.5 ± 2.2%) were observed in samples cryopreserved in Me₂SO than in glycerol. Cooling rates influenced survival of sperm cryopreserved in glycerol with −57.8 °C/min being advantageous (P < 0.05). The findings demonstrate that although maned wolf spermatozoa are similar to domestic dog sperm in their sensitivity to osmotic-induced motility damage, the plasma membranes tolerate dehydration, and the cells respond favorably to Me₂SO as a cryoprotectant.     

Quantification of cis and trans influences in [PtX(PPh3)3] complexes. A P NMR study

In [PtX(PPh₃)₃]⁺ complexes (X = F, Cl, Br, I, AcO, NO₃, NO₂, H, Me) the mutual cis and trans influences of the PPh₃ groups can be considered constants in the first place, therefore the one bond Pt-P coupling constants of P_(cis) and P_(trans) reflect the cis and trans influences of X. The compounds [PtBr(PPh₃)₃](BF₄) (2), [PtI(PPh₃)₃](BF₄) (3), [Pt(AcO)(PPh₃)₃](BF₄) (4), [Pt(NO₃)(PPh₃)₃](BF₄) (5), and the two isomers [Pt(NO₂-O)(PPh₃)₃](BF₄) (6a) and [Pt(NO₂-N)(PPh₃)₃](BF₄) (6b) have been newly synthesised and the crystal structures of 2 and 4·CH₂Cl₂·0.25C₃H₆O have been determined. From the ¹J_PtP values of all compounds we have deduced the series: I > Br > Cl > NO₃ > ONO > F > AcO > NO₂ > H > Me (cis influence) and Me > H > NO₂ > AcO > I > ONO > Br > Cl > F > NO₃ (trans influence). These sequences are like those obtained for the (neutral) cis- and trans-[PtClX(PPh₃)₂] derivatives, showing that there is no dependence on the charge of the complexes. On the contrary, the weights of both influences, relative to those of X = Cl, were found to depend on the charge and nature of the complex.     

Cryopreservation of amniotic fluid-derived stem cells using natural cryoprotectants and low concentrations of dimethylsulfoxide

Amniotic fluid-derived stem cells (AFSCs) are a potential cell source for therapeutic applications. They can be easily mass produced, cryopreserved and shipped to clinics for immediate use. However, one major obstacle to the manufacturing of clinical grade stem cells is the need for current good manufacturing practices for cryopreservation, storage, and distribution of these cells. Most current cryopreservation methods used for stem cells include the potentially toxic cryoprotectant (CPA) dimethylsulfoxide (Me₂SO) in the presence of animal serum proteins that prevent direct use of these cells in human therapeutic applications. To avoid any potential cryoprotectant related complications, it will be essential to develop non-toxic CPAs or reduce CPA concentration in the freezing media used. In this study, we assessed the use of disaccharides, antioxidants and caspase inhibitors for cryopreservation of AFSCs in combination with a reduced concentration of Me₂SO. The thawed cells were tested for viability with MTT assays and a growth curve was created to measure population doubling time. In addition, we performed flow cytometry analysis for cell surface antigens, RT-PCR for mRNA expression of stem cell markers, and assays to determine the myogenic differentiation potential of the cells. A statistically significant (p < 0.05) increase in post-thawed cell viability in solutions containing trehalose, catalase and _ZVAD-fmk with 5% Me₂SO was observed. The solutions containing trehalose and catalase with 5% or 2.5% (v/v) Me₂SO produced results similar to those for the control (10% (v/v) Me₂SO and 30% FBS) in terms of culture growth, expression of cell surface antigens and mRNA expression of stem cell markers in AFSCs cryopreserved for a minimum of 3 weeks. Thus, AFSCs can be cryopreserved with 1/4 the standard Me₂SO concentration with the addition of disaccharides, antioxidants and caspase inhibitors. The use of Me₂SO at low concentrations in cell freezing solutions may support the development of clinical trials of AFSCs.     

Determination of the membrane permeability characteristics of Pacific oyster, Crassostrea gigas, oocytes and development of optimized methods to add and remove ethylene glycol

In order for cryopreservation to become a practical tool for aquaculture, optimized protocols must be developed for each species and cell type. Knowledge of a cell’s osmotic tolerance and membrane permeability characteristics can assist in optimized protocol development. In this study, these characteristics were determined for Pacific oyster oocytes and modified methods for loading and unloading ethylene glycol (EG) were tested. Oocytes were found to behave as ideal osmometers and their osmotically inactive fraction (V_b) was calculated to be 0.48. Oocytes exposed to NaCl solutions of 0.6 to 2.3 Osm fertilized at rates equivalent to oocytes left in seawater. This corresponds to volume changes of +27.3 and −38.1 ± 1.2%. The permeability of the oocytes to water (L_p) was determined to be 3.8 ± 0.4 × 10⁻², 5.7 ± 0.8 × 10⁻², and 13.2 ± 1.3 × 10⁻² μm min⁻¹ atm⁻¹, when measured at temperatures of 5, 10 and 20 °C. The respective EG permeability values (P_s) were 9.5 ± 0.1 × 10⁻⁵, 14.6 ± 1.2 × 10⁻⁵, and 41.7 ± 2.4 × 10⁻⁵ cm min⁻¹. The activation energies for L_p and P_s were determined to be 14.5 and 17.5 kcal mol⁻¹, respectively. Different models for EG loading and unloading from oocytes were developed and tested. Post-thaw fertilization did not differ significantly between a published step addition method and single step addition at 20 °C. This represents a considerable reduction in handling. The results of this study demonstrate that the cryobiological characteristics of a given cell type should be taken into account when developing cryopreservation methods.     

Cryopreservation of zebrafish (Danio rerio) primordial germ cells by vitrification of yolk-intact and yolk-depleted embryos using various cryoprotectant solutions

The aim of this study was to examine the effects of partial removal of yolk and cryoprotectant mixtures on the viability of cryopreserved primordial germ cells (PGCs) and elucidated the differentiation ability of cryopreserved PGCs in zebrafish. First, dechorionated yolk-intact and yolk-depleted (partially yolk removed) embryos, PGCs of which were labeled with green fluorescence protein (GFP), were vitrified after serial exposures to pretreatment solution (PS) and vitrification solution (VS) that contained ethylene glycol (EG), dimethyl sulfoxide (Me₂SO) or propylene glycol at 3 and 5 M, respectively. Although partial removal of yolk improved the viability of cryopreserved PGCs, numbers of PGCs with pseudopodial movement were limited (0–2.6 cells/embryo). Next, yolk-depleted embryos were cryopreserved using mixtures of two types of cryoprotectants. The maximum survival rate of PGCs (81%; 9.6 cells/embryo) was obtained from the yolk-depleted embryos vitrified using PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO and 56% (5.3 cells/embryo) of PGCs showed pseudopodial movement. Finally, PGCs recovered from yolk-depleted embryos (wild-type) that were vitrified under the optimum condition were transplanted individually into 236 sterilized recipient blastulae (recessive light-colored). Seven recipients matured and generated progeny with characteristics inherited from the PGC donor. In conclusion, the authors confirmed the beneficial effects of partial removal of yolk on the viability of cryopreserved PGCs and that the viability of the PGCs was improved by using PS and VS that contained two types of cryoprotectants, especially PS containing 2 M EG + 1 M Me₂SO and VS containing 3 M EG + 2 M Me₂SO, and that recovered PGCs retained ability to differentiate into functional gametes.     

Effect of different cryoprotectant agents on spermatogenesis efficiency in cryopreserved and grafted neonatal mouse testicular tissue

Restoration of male fertility associated with use of the cryopreserved testicular tissue would be a significant advance in human and animal assisted reproductive technology. The purpose of this study was to test the effects of four different cryoprotectant agents (CPA) on spermatogenesis and steroidogenesis in cryopreserved and allotransplanted neonatal mouse testicular tissue. Hank’s balanced salt solution (HBSS) with 5% fetal bovine serum including either 0.7 M dimethyl sulfoxide (DMSO), 0.7 M propylene glycol (PrOH), 0.7 M ethylene glycol (EG), or glycerol was used as the cryoprotectant solution. Donor testes were collected and dissected from neonatal pups of CD-1 mice (one day old). Freezing and seeding of the testicular whole tissues was performed using an automated controlled-rate freezer. Four fresh (non-frozen) or frozen–thawed pieces of testes were subcutaneously grafted onto the hind flank of each castrated male NCr nude recipient mouse and harvested after 3 months. Fresh neonatal testes grafts recovered from transplant sites had the most advanced rate of spermatogenesis with elongated spermatid and spermatozoa in 46.6% of seminiferous tubules and had higher levels of serum testosterone compared to all other frozen–thawed-graft groups (p < 0.05). Fresh grafts and frozen–thawed grafts in the DMSO group had the highest rate of tissue survival compared to PrOH, EG, and glycerol after harvesting (p > 0.05). The most effective CPA for the freezing and thawing of neonatal mouse testes was DMSO in comparison with EG (p < 0.05) in both pre-grafted and post-grafted tissues based on histopathological evaluation. Likewise, the highest level of serum testosterone was obtained from the DMSO CPA group compared to all other cryoprotectants evaluated (p < 0.05). The typical damage observed in the frozen–thawed grafts included disruption of the interstitial stroma, intercellular connection ruptures, and detachment of spermatogonia from the basement membrane. These findings indicate that neonatal mouse testes were most effectively preserved when frozen with HBSS medium with DMSO and that the type of CPA is a significant factor to obtain the most advanced stages of spermatogenesis and steroidogenesis after cryopreservation, thawing, and transplantation of neonatal mouse testes.     

Permeation of several cryoprotectant agents into porcine articular cartilage

Objective

Osteochondral allografting is an effective method to treat large osteochondral defects but difficulties in tissue preservation have significantly limited the application of this technique. Successful cryopreservation of articular cartilage (AC) could improve the clinical availability of osteochondral tissue and enhance clinical outcomes but cryopreservation of large tissues is hampered by a lack of knowledge of permeation kinetics within these tissues. This study describes the refinement and extension of a recently published technique to measure the permeation kinetics of cryoprotectant agents (CPAs) within porcine AC.

Design

Dowels of porcine AC (10 mm diameter) were immersed in solutions containing 6.5 M concentrations of four commonly used CPAs [dimethyl sulfoxide (DMSO), propylene glycol (PG), ethylene glycol (EG) and glycerol] for different times (1 s, 1, 2, 5, 10, 15, 30, 60, 120, 180 min , 24 h) at three different temperatures (4, 22, and 37 °C). The cartilage was isolated and the amount of CPA within the matrix was determined.

Results

Diffusion coefficients (DMSO = 2.4-6.2 × 10⁻⁶ cm²/s; PG = 0.8-2.7 × 10⁻⁶ cm²/s; EG = 1.7-4.2 × 10⁻⁶ cm²/s; and glycerol = 0.8-2.4 × 10⁻⁶ cm²/s) and activation energies (DMSO = 4.33 kcal/mol, PG = 6.29 kcal/mol, EG = 3.77 kcal/mol, and glycerol = 5.56 kcal/mol) were determined for each CPA.

Conclusion

The results of this experiment provide accurate permeation kinetics of four commonly used CPAs in porcine articular cartilage. This information will be useful for developing effective vitrification protocols for cryopreservation of AC.     

Synthesis, characterization and applications in ethylene polymerization of asymmetric ansa-titanocene complexes. Molecular structure of [Ti{Me2Si(η-C5Me4)(η-C5H3iPr)}Cl2]

The ansa-titanocene complexes, [Ti{Me₂Si(η⁵-C₅Me₄)(η⁵-C₅H₃R)}Cl₂] (R = Me (5), iPr (6), tBu (7), SiMe₃ (8)), were obtained from the reaction of Li₂{Me₂Si(C₅Me₄)(C₅H₃R)} (R = Me (1), iPr (2), tBu (3), SiMe₃ (4)) with [TiCl₄(THF)₂], respectively. Compounds 5-8 have been tested as catalysts in the polymerization of ethylene and compared with the ansa-titanocene complexes [Ti{Me₂Si(η⁵-C₅H₄)₂}Cl₂] and [Ti{Me₂Si(η⁵-C₅Me₄)(η⁵-C₅H₄)}Cl₂]. The resulting polyethylene showed molecular weights of about 200 000 g mol⁻¹ and polydispersity values of approximately 3. In addition, the molecular structure of 6 has been determined by single crystal X-ray diffraction studies.