Survival ofEscherichia coli after storage in various frozen menstrua

The effect of storage at –9 C onEscherichia coli was examined. In buffer or water, survival after three days was less than 40%. Dimethylsulfoxide (DMSO) (10%) and glycerol (10%) were very protective with over 90% survivors. Variability of replicate samples was greater with frozen than with non-frozen suspensions.With a slide culture technique, it was found that the time required for the thawed cells to complete their first division was increased up to a time equivalent to over two divisions, dependent upon the protective storage menstrua.Injury as shown by inability to grow on a minimal medium after thawing was negligible when the cells were frozen in DMSO or glycerol. Cells stored in frozen buffer were sensitive to a 20 min treatment with actinomycin D following thawing but cells frozen in glycerol or DMSO showed little death or injury. The results suggest that an alteration of the cell envelope is initially responsible for death by freezing.This work was supported in part by U.S. Public Health Service Research Grant EF-428 from the Division of Environmental Engineering and Food Protection.     

Optimal conditions for the preservation of mouse lymph node cells in liquid nitrogen using cooling rate techniques

The factors that affect the survival of mouse lymphocytes throughout a procedure for storage at ?196 °C have been studied both for the improvement of recovery and the possible extension to the mouse system of cell selection by freezing. After thawing, the survival of cells cooled at different rates in dimethyl sulphoxide (DMSO, 5 or 10%, $\text{v}\text{v}$) was assessed from the [³H]thymidine incorporation in response to phytohaemagglutinin and concanavalin A. Before freezing the protection against freezing damage increased with time (up to 20 min) in DMSO (5%, $\text{v}\text{v}$) at 0 °C. Superimposed upon this effect was toxicity due to the DMSO. During freezing and thawing the cooling rate giving optimal survival was 8 to 15 °C/min for cells in DMSO (5%) and 1 to 3 °C/min for DMSO (10%). Omission of foetal calf serum was detrimental. Rapid thawing (>2.5 °C/min) was superior to slow thawing. After thawing dilution at 25 or 37 °C greatly improved cell survival compared with 0 °C; at 25 °C survival was optimal (75%) at a moderate dilution rate of 2.5 min for a 10-fold dilution in FCS (10%, $\text{v}\text{v}$) followed by gentle centrifugation (50g).Dilution damage during both thawing and post-thaw dilution may be due to osmotic swelling as DMSO and normally excluded solutes leave the cell. The susceptibility of the cell membrane to dilution damage may also be increased during freezing. The need to thaw rapidly and dilute at 25 °C after thawing is probably due to a decrease in dilution stress at higher temperatures. Optimisation of dilution procedures both maximised recovery and also widened the range of cooling rates over which the cells were recovered. These conditions increase the possibility of obtaining good recovery of a mixed cell population using a single cooling procedure. Alternatively, if cell types have different optimal cooling rates, stressful dilution may allow their selection from mixed cell populations.     

Freezing of rat lymphocytes. I. The effects of dimethyl sulfoxide and freezing on the phytohemagglutinin- and pokeweed mitogen-responding lymphocyte subpopulations.

Rat spleen and lymph node lymphocytes have been frozen with dimethyl sulfoxide (DMSO) at 1 °C/min and stored at ?196 °C for 10 min. The functional recovery of the cell populations was monitored by the mitogenic response (uptake of [³H]thymidine) to phytohemagglutinin (PHA) or pokeweed mitogen (PWM) in culture after thawing. With 5 to 10% DMSO in the freezing medium, frozen-thawed lymph node cells were found to retain about 40% of their response to PHA. In contrast, frozen-thawed spleen cells responded better to PHA than fresh cells. The response to PWM was markedly decreased in both spleen and lymph node cell cultures.A similar effect was observed when DMSO was added to the culture medium of fresh spleen cells, i.e., an augmentation of the response to PHA and a suppression of the response to PWM. However, the concentrations of DMSO needed to induce this effect was more than 10-fold higher than that present in the culture medium after freezing and thawing.Since removal of adherent cells from the spleen cell population also produced an augmentation of the response to PHA, it is suggested that the freezing procedure and DMSO may have an inhibitory effect on suppressor cell functions present in spleen cell populations.     

Viability and engraftment of hematopoietic progenitor cells after long-term cryopreservation: effect of diagnosis and percentage dimethyl sulfoxide concentration

Background aimsWe carried out a retrospective analysis of viability by diagnosis and dimethyl sulfoxide (DMSO) concentration in patients who had undergone autologous transplants using hematopoietic progenitor cells (HPC) after long-term storage (up to 17.8 years).MethodsViability was tested using flow cytometry for HPC that were harvested and preserved using a controlled rate freezer and 5% or 10% DMSO with human serum albumin, then stored in liquid nitrogen. Data from 262 samples were analyzed (249 myeloma patients and 13 other diagnoses): 100 consecutively thawed samples with a storage time of <1 year (all 10% DMSO), 50 consecutive samples stored for 1–4.9 years (10% DMSO), 50 samples stored for 5–9 years (5% DMSO) and all samples stored and used for transplant after >9 years (60 samples, 5% DMSO; two samples, 10% DMSO).ResultsNo statistically significant difference in viability between the 5% DMSO and 10% DMSO groups was observed (P = 0.08), so the 1–4.9 years and 5–9 years were combined and the three groups (<1 year, 1–9 years and >9 years) were compared using an anova test. There was no difference in viability based on cryostorage period (P = 0.23) or between myeloma and other diagnoses (P = 0.45). No difference was seen in time to White blood cell (WBC) engraftment (P = 0.10) or to platelet engraftment between groups (P = 0.52).ConclusionsThese data suggest that long-term storage in 5% DMSO and human serum albumin is safe.     

La Cryoconservation de la laitance de la carpe, Cyprinus carpio

Cryo-preservation of carp, Cyprinus carpio, sperm Deep-freezing trials of carp sperm were carried out by varying several factors such as the basic saline solution, the cryoprotectors added (glycerol, propanediol, ethylene glycol and DMSO), the media (Menezo-INRA B², egg yolk, urea) and the deep-freezing and dilution rates. The success of deepfreezing was judged by the percentage of motile spermatozoa, intensity of motility, fertilizing ability and morphological integrity of the spermatozoa studied under the scanning electron microscope. DMSO was the best cryoprotector and the mineral composition of the dilution medium the least important factor, but there was noticeable improvement after organic compounds were added. The following mixture has been proposed: NaCl 100 mM + KC1100 mM, Tris 20 mM, pH 8: 37%, Menezo medium B² INRA: 15%, urea 5%, DMSO: 10%, fresh sperm: 33%. Optimal deep-freezing rate was: 5°C/min from 2 to-7°C and 25°C/min from-7 to-70°C. In these conditions, about 70 to 80% of the spermatozoa were motile after thawing compared to fresh control sperm, but fertilizing ability was not more than 30 to 40% of that with fresh sperm. The percentage of spermatozoa considered intact was 66% after thawing as against 83% for fresh control sperm. The motility and fertilizing ability of deep-frozen sperm were significantly improved when the dilution rate at insemination was reduced from ¹/₁₀₀ to 1/2.     

Cryopreservation of a water-bloom forming cyanobacterium, Microcystis aeruginosa f. aeruginosa

A method for the Cryopreservation of Microcystis aeruginosa f. aeruginosa is described. For the five strains tested, dimethyl sulfoxide (DMSO) (3% v/v) was the only effective cryoprotectant for freezing to, and thawing from -196°C and allowed the successful recovery (>50%) of all the strains. The viability of frozen material was independent of the period of storage in liquid nitrogen. The strain NIES-44 (National Institute for Environmental Studies) had a recovery level of greater than 90% at 3–10% (v/v) DMSO in both two step and rapid cooling methods. The other three strains, NIES-87, 88 and 89 had greater than 60% of viability after freeze/thawing in presence of both 3% and 5% DMSO concentrations. On the other hand, the strain NIES-90 showed approximately 50% of viability in only 3% DMSO solution after two step cooling to and thawing from -196°C. This strain was damaged by greater than 4% DMSO and by rapid cooling to -196°C. It was found that cold shock injury and the cytotoxicity of DMSO were different at a strain level.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Tropical tannin-rich fodder intake modifies saliva-binding capacity in growing sheep

We evaluated the effect of feeding dietary tannins from Lysiloma latisiliquum fresh forage on the saliva tannin-binding capacity of hair sheep lambs without previous exposure to tannin-rich (TR) fodder. Twenty-four hair sheep lambs (13.6±3.04 kg LW) were fed a tannin-free diet at the beginning of the experimental period (from day 10 to 13). On day 14, lambs were distributed into three groups (n=8): control group (CG), fed with the tannin-free diet (from D10 to D112); tannin short-term group (TST), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D55); tannin long-term group (TLT), fed the basal diet and 650 g of L. latisiliquum forage (from D14 to D112). Saliva samples were collected from the mouth of each lamb in the morning before feeding time on D10 and D14 (baseline period), on D49 and D56 (period 1) and on D97 and D112 (period 2). The tannin binding response of salivary protein (∆% turbidity) was determined with the haze development test (HDT) using either tannic acid or L. latisiliquum forage acetone extract. A turbidity protein index (TPI) was calculated as (∆% turbidity/[salivary protein (mg)]). Differences in HDT and TPI in the different groups were compared by repeated measures ANOVA using Proc Mixed. All groups had similar ∆% turbidity throughout the experiment (P>0.05). At baseline and period 1, the TPI of the different groups was similar (P>0.05). On period 2 the TLT group showed higher TPI compared with CG (P<0.05). Meanwhile, CG and TST showed similar salivary TPI. The saliva of hair sheep lambs consuming TR L. latisiliquum fresh fodder (TLT group) increased their TPI compared with control lambs not exposed to tannins.     

Reusable ω-transaminase sol–gel catalyst for the preparation of amine enantiomers

Heterogeneous ω-transaminase sol–gel catalysts were prepared and characterized in terms of immobilization degree, loading capacity and catalytic behavior in the kinetic resolution of racemic 1-phenylethylamine (a model compound) with sodium pyruvate in phosphate buffer (pH 7.5). The catalyst obtained when ω-transaminase from Arthrobacter sp. was encapsulated from the aqueous solution of the enzyme, isopropyl alcohol and polyvinyl alcohol in the sol–gel matrices, consisting of the 1:5 mixture of tetramethoxysilane and methyltrialkoxysilane, proved to be optimal including the reuse and storage stabilities of the catalyst. The optimized immobilizate was shown to perform well in the kinetic resolution of four structurally different aromatic primary amines in aqueous DMSO (10, v/v-%). The enzyme preparation showed synthetic potential by enabling the catalyst reuse in five consecutive preparative scale kinetic resolutions using 100 mM 1-phenylethylamine in aqueous DMSO (10, v/v-%). It was typical to fresh catalyst preparations that the kinetic resolution tended to exceed 50% before the reaction stopped leaving the (S)-amine unreacted while thereafter in reuse the reactions stopped at 50% conversion as expectable to highly enantioselective reactions.     

Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report.

A comparative study has been made of platelets stored by freeze preservation following treatment with dimethyl sulfoxide (DMSO) or hydroxyethyl starch (HES) with fresh platelets and platelets stored at 4 °C for 48 hr. The indices studied were platelet recovery, pH, light microscope morphology, platelet Factor 3 (PF₃) availability and the hypotonic stress response. The DMSO preserved platelets gave a better response to hypotonic stress and incurred lesser degrees of membrane damage as demonstrated by PF₃ availability. There was however a significantly higher recovery of platelets treated with HES; with DMSO the osmotic damage inflicted during removal caused considerable lysis. Platelets frozen by DMSO or HES gave consistently better in vitro results than platelets stored at 4 °C for 48 hr. A preliminary clinical trial of HES preserved platelets has confirmed haemostatic effectiveness in vivo. HES being relatively nontoxic, platelets can be infused immediately after thawing and with minimal post thaw manipulation, thus maintaining a relatively closed system. It is concluded that cryopreservation with HES is a practical and effective means for long term platelet storage.     

Effect of osmotic immobilization on refrigerated storage and cryopreservation of sperm from a viviparous fish, the green swordtail Xiphophorus helleri

In this study, refrigerated storage and cryopreservation of sperm from the green swordtail Xiphophorus helleri were investigated. Previous cryopreservation research in this species utilized motile sperm because unlike in most fish species, Xiphophorus sperm can remain continuously motile after collection for a week with refrigerated storage. However, this species reproduces by internal fertilization, and given the significant requirements for motility within the female reproductive tract and potential limitations on sperm energetic capacities, immobilization of sperm prior to insemination could be used to improve fertilization success. Thus, the goal in this study was to use osmotic pressure to inhibit the motility of sperm after collection from X. helleri, and to test the effect of immobilization on refrigerated storage and cryopreservation. The objectives were to: (1) estimate the motility of sperm at different osmotic pressures, and determine an osmotic pressure suitable for immobilization; (2) cryopreserve the immobilized sperm, and estimate the motility after thawing with or without dilution, and (3) compare motility of non-immobilized and immobilized sperm after thawing, centrifugation, and washing to remove cryoprotectant. Motility was determined when sperm were suspended in 11 different osmotic pressures (24-500 mOsmol/kg) of Hanks' balanced salt solution (HBSS). Motility was observed between 116 and 425 mOsmol/kg. Sperm were not motile when the osmolality was lower than 116 or higher than 425 mOsmol/kg. Motility of the immobilized (non-motile) sperm could be activated by changing the osmotic pressure to 291-316 mOsmol/kg, and motility of immobilized sperm from hypertonic HBSS (425 mOsmol/kg) was significantly higher than that from hypotonic HBSS (145 mOsmol/kg) after 48 h of storage. At an osmolality of 500 mOsmol/kg, HBSS was used as extender to maintain immobilized sperm during cryopreservation with glycerol as the cryoprotectant. High motility (approximately 55%) was obtained in sperm after thawing when cryopreserved with 10-15% glycerol, and dilution of thawed sperm in fresh HBSS (1:4; V:V) was found to decrease the motility significantly. No difference was found in the motility of thawed sperm cryopreserved with 14% glycerol and extended in 310 and 500 mOsmol/kg HBSS. Washing by centrifugation prolonged the motility of thawed sperm from 24 to 72 h in HBSS at 310 and 500 mOsmol/kg. This study showed that sperm from X. helleri could be immobilized by use of specific osmotic pressures, and that the immobilization did not affect sperm motility after thawing. The immobilization of sperm by osmotic pressure could minimize reduction of the energetic capacities necessary for insemination, traversal, and residence within the female reproductive tract, and fertilization.     

Cryopreservation and subsequent viability of human bone marrow in hematologic malignancies: Comparison of two different methods of cell reconstitution

Bone marrow cells collected from patients with hematologic malignancies were cryopreserved using DMSO as a cryoprotective agent. The growth kinetics of hemopoietic stem cells frozen to −196 °C was monitored immediately after thawing by the semisolid agar CFU-C assay and two different methods of cell reconstitution were compared. In the first procedure, thawed cells were plated after the removal of DMSO by washing the cell suspension; in the second, cell suspensions were cultured after a simple 1:1 dilution of DMSO with medium. The numbers of CFU-C per 2 × 10⁵ cells plated was higher by washing out the DMSO in all the groups studied. However, the absolute numbers of CFU-C contained in the whole ampoules after the freezing procedures was approximately the same using both methods. It is concluded that washing the cells only apparently yielded a better cloning efficiency, suggesting that such a procedure led to a higher mature nucleated cell loss with the consequence of a CFU-C concentration. This trend seems particularly evident in cells from the AML and CML patients.     

Cryopreservation and Plant Regeneration from Embryogenic Cultures of Larch (Larixxeurolepis) and Black Spruce (Picea mariana)

A method has been developed for the routine cryopreservationof embryogenic cultures of hybrid larch (Larixxeurolepis) andblack spruce (Picea mariana Mill.). The method involves growingthe cultures in the presence of sorbitol and then briefly exposingthem to DMSO followed by controlled cooling to –40°C.The cultures were then submerged and stored in liquid nitrogen.Growth of the embryogenic cultures was monitored for 14 d afterrapid thawing and plating on to media. The highest relativeincrease in the tissue fresh weight, after storage in liquidnitrogen, was observed when embryogenic cultures of both specieswere pregrown for 24 h in a medium with 0·4 M sorbitoland then treated with 10% DMSO. This pretreatment also ensuredthe shortest lag phase in resuming the growth. The post-thawcultures gave rise to mature somatic embryos which developedinto plants Key words: Larixxeurolepis, Picea mariana, cryopreservation, embryogenic tissue, plant regeneration     

Optimization of cryopreservation of buffalo (Bubalus bubalis) blastocysts produced by in vitro fertilization and somatic cell nuclear transfer

The objective of this study was to optimize cryopreservation conditions for buffalo in vitro produced (IVP) embryos. The in vitro fertilized (IVF) and somatic cell nuclear transferred (SCNT) blastocysts were vitrified with either 40% ethylene glycol (EG), 25% EG + 25% dimethylsulfoxide (DMSO), or 20% EG + 20% DMSO + 0.5 m sucrose, and the IVF blastocysts produced from abattoir-derived ovaries were also slow-frozen with either 10% EG or 0.05 m trehalose dehydrate + 1.8% EG + 0.4% BSA. Cryosurvival rates of blastocysts harvested on various days or at various developmental stages were also examined. In this study: (1) vitrification with 20% EG + 20% DMSO + 0.5 m sucrose had the best cryopreservation efficiency; (2) IVF and SCNT blastocysts had similar cryotolerance (P > 0.05); (3) after thawing, slow-frozen blastocysts reexpanded earlier than the vitrified blastocysts (P < 0.01); (4) cryosurvival rate of expanded blastocysts was higher than that of early blastocysts (P < 0.05); (5) cryosurvival rates of Days 5 to 7 blastocysts (Day 0 = day of IVF or SCNT) were higher than those of Days 8 to 9 blastocysts (P < 0.01); and (6) after embryo transfer, pregnancy rates for fresh and cryopreserved blastocysts were not different (P > 0.05). In conclusion, vitrification of Days 6 to 7 expanded blastocysts with 20% EG + 20% DMSO + 0.5 m sucrose was optimal for cryopreservation of buffalo IVP embryos.     

Toxicity of cryoprotectants to honey bee semen and queens

Given the threats to the intraspecific biodiversity of Apis mellifera and the pressure on bee breeding to come up with disease-tolerant lines, techniques to cryopreserve drone semen are of great interest. Freeze-thawed drone semen of high viability and/or motility has repeatedly been obtained, but fertility of such semen, when it was measured, was always low. The cryoprotective agent (CPA) most frequently used with drone semen is dimethyl sulfoxide (DMSO), although this substance has been suspected of causing genetic damage in sperm. No form of sperm washing is currently performed. Using a membrane permeability assay, we measured the short-term toxicity of four possible replacements for DMSO, 1,3-propane diol, 2,3-butane diol, ethylene glycol, and dimethyl formamide. We also tested whether the practice of inseminating queens with CPA-containing semen affects sperm numbers in the storage organs of queens, or sperm fertility. Finally, we tested whether CPA-toxicity in vivo can be reduced by using mixtures of two CPAs, DMSO, and ethylene glycol. Our results show that, although short-term toxicity of all CPAs tested was low, the presence of single CPAs in insemination mixtures at concentrations required for slow freezing greatly reduced the number of sperm reaching the spermatheca. Contrary to earlier reports, this was also true for DMSO. Ethylene glycol was additionally shown to reduce the viability of spermatozoa reaching the storage organ. Mixtures of DMSO and EthGly performed better than either substance used singly at the same concentration. We conclude that the toxicity of CPAs, including DMSO, on honey bee semen and/or queens has been underestimated in the past. This could partly explain the discrepancy between in vitro and in vivo quality of cryopreserved drone semen, described by others. Combinations of several CPAs and techniques to partly remove CPAs after thawing could help to solve this problem.     

Optimization of the cryopreservation of African clawed frog (Xenopus laevis) sperm

Cryopreservation of testicular sperm in the African clawed frog, Xenopus laevis, was tested using three penetrating cryoprotectants (DMSO, methanol, and glycerol) and three semen diluents (300 mmol/L glucose, 300 mmol/L sucrose, and a motility inhibiting saline [MIS] solution [150 mmol/L NaCl, 3 mmol/L KCL, 1 mmol/L Mg₂SO₄, 1 mmol/L CaCl₂, and 20 mmol/L Tris, pH 8.0]). Three freezing rates and four thawing rates were also tested, and the best freezing/thawing conditions have been determined. The responses of sperm motility, viability, and fertility were assessed. Incubation of the sperm macerates with penetrating cryoprotectants showed that DMSO was the least toxic and methanol the most toxic. Semen in cryodiluents frozen 10 cm above the surface of liquid nitrogen (freezing rate of 20 to 25 °C/min) and thawed at room temperature for 40 sec had significantly higher percentages of motile and viable sperm than that of semen frozen 5 cm or 8 cm above the surface of liquid nitrogen and thawed at 5, 25, or 30 °C for 10, 15, or 60 sec, respectively. Sperm frozen in MIS containing 5% DMSO had a higher hatching rate than that of sperm frozen in sucrose and glucose diluents containing 5% or 10% DMSO and in MIS containing 10% DMSO. Addition of 73 mmol/L sucrose to the sperm extender MIS + 5% DMSO could improve the postthaw sperm motility and fertility. In conclusion, dilution of collected sperm in MIS solution (to have a final concentration of 6.5 × 10⁶ to 8 × 10⁶/mL) containing 5% DMSO and 73 mmol/L sucrose, freezing in a vapor of liquid nitrogen at 10 cm above the surface, and thawing at room temperature for 40 sec was the best cryopreservation protocol. This protocol gave 70% hatching rate, 80% motility rate, and 75% viability rate of fresh hormonally induced sperm.     

Cryopreservation of erythropoietin-responsive cells in murine hematopoietic tissue

The optimal conditions were determined under which maximum survival of murine hematopoietic erythropoietin-responsive cells (ERC) could be ensured during manipulations required for cryopreservation. Cell survival was similar over freezing rates between 2 and 10 °C/min. Optimal cryoprotectants were 10% dimethyl sulfoxide (DMSO) and 20% fetal calf serum; the DMSO was removed by centrifugation after stepwise dilution with 20 vol of medium over a 10-min period. Differing thawing rates for the cell suspensions had minimal effects on survival. “Seeding” the cell suspensions with ice crystals had no effect on ERC recovery. Overall ERC survival varied between 20 and 40%. These results confirm earlier reports that certain ERC populations are more sensitive to damage during cryopreservation than are other hematopoietic progenitor cells.     

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.     

Determination of residual dimethylsulfoxide in cryopreserved cardiovascular allografts

Dimethylsulfoxide (DMSO) is a solvent which protects the structure of allografts during the cryopreservation and thawing process. However, several toxic effects of DMSO in patients after transplantation of cryopreserved allografts have been described. The aim of this study is to determine the residual DMSO in the cardiovascular allografts after thawing and preparation of cryopreserved allografts for clinical application following guidelines of the European Pharmacopoeia for DMSO detection. Four types of EHB allografts (aortic valve-AV, pulmonary valve-PV, descending thoracic aorta-DA, and femoral artery-FA) are cryopreserved using as cryoprotecting solution a 10% of DMSO in medium 199. Sampling is carried out after thawing, after DMSO dilution and after delay of 30 min from final dilution (estimated delay until allograft implantation). After progressive thawing in sterile water bath at 37–42 °C (duration of about 20 min), DMSO dilution is carried out by adding consecutively 33, 66 and 200 mL of saline. Finally, tissues are transferred into 200 mL of a new physiologic solution. Allograft samples are analysed for determination of the residual DSMO concentration using a validated Gas Chromatography analysis. Femoral arteries showed the most important DMSO reduction after the estimated delay: 92.97% of decrease in the cryoprotectant final amount while a final reduction of 72.30, 72.04 and 76.29% in DMSO content for AV, PV and DA, was found, respectively. The residual DMSO in the allografts at the moment of implantation represents a final dose of 1.95, 1.06, 1.74 and 0.26 mg kg^?1 in AV, PV, DA and FA, respectively, for men, and 2.43, 1.33, 2.17 and 0.33 mg kg^?1 for same tissues for women (average weight of 75 kg in men, and 60 kg in women). These results are seriously below the maximum recommended dose of 1 g DMSO kg^?1 (Regan et al. in Transfusion 50:2670–2675, 2010) of weight of the patient guaranteeing the safety and quality of allografts.