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.     

Cryopreservation procedure does not modify human carotid homografts mechanical properties: an isobaric and dynamic analysis

The viscoelastic and inertial properties of the arterial wall are responsible for the arterial functional role in the cardiovascular system. Cryopreservation is widely used to preserve blood vessels for vascular reconstruction but it is controversially suspected to affect the dynamic behaviour of these allografts. The aim of this work was to assess the cryopreservation's effects on human arteries mechanical properties. Common carotid artery (CCA) segments harvested from donors were divided into two groups: Fresh (n = 18), tested for 24–48 h after harvesting, and Cryopreserved (n = 18) for an average time of 30 days in gas-nitrogen phase, and finally defrosted. Each segment was tested in a circulation mock, and its pressure and diameter were registered at similar pump frequency, pulse and mean pressure levels, including those of normotensive and hipertensive conditions. A compliance transfer function (diameter/pressure) derived from a mathematical adaptive modelling was designed for the on line assessment of the arterial wall dynamics and its frequency response. Assessment of arterial wall dynamics was made by measuring its viscous (η), inertial (M) and elastic (E) properties, and creep and stress relaxation time constant (τ_C and τ_SR, respectively). The frequency response characterization allowed to evaluate the arterial wall filter or buffer function. Results showed that non-significant differences exist between wall dynamics and buffer function of fresh and cryopreserved segments of human CCA. In conclusion, our cryopreservation method maintains arterial wall functional properties, close to their fresh values.     

Activation of Caspases in Human Spermatozoa during Cryopreservation – An Immunoblot Study

Cellular stress to ejaculated spermatozoa such as cryopreservation is known to induce caspase-derived, apoptotic signaling. Therefore, the proenzymes and active forms of caspases 1, 3, 8 and 9 were examined by western blot technique in unfrozen and frozen human spermatozoa of infertility patients and of healthy donors. Twenty-two semen samples derived from healthy donors and 26 semen samples of unselected infertility patients were divided into 3 parts, two of them were cryostored at −196 °C with 7% or 14% (v/v, final concentration) of glycerol. The caspases were detected by immunoblots with polyclonal rabbit-anti-caspases-antibodies after 15% sodium dodecyl sulfate-polyacrylgel electrophoresis (SDS-PAGE) under reducing conditions. For evaluation of the differences between amounts of caspase protein the luminol/H₂O₂ method was applied. A significant increase of activated caspase-1 in donors, of caspase-8 in patients and caspase-9 in patients and donors after cryopreservation were found, whereas, the application of 14% glycerol resulted in higher amounts of activated caspase than did 7% glycerol. Possibly, glycerol may also contribute to activation of caspases via direct toxic effects to mitochondria during cryopreservation of spermatozoa. This finding strongly supports an hypothesis of a higher mitochondria-derived apoptosis-sensitivity of spermatozoa in patients than in healthy donors during cryopreservation. Inactive caspase-3 was reduced subsequent to cryopreservation in patients (p<0.05) and non-significant in donors (p<0.05). Active caspase-3 was detectable in all samples but without significant differences between the three assays. It is concluded that mechanisms associated with apoptotic processes deserve attention in cryopreservation of spermatozoa in order to conserve vital sperm functions after thawing.     

Cancellous bone homograft storage with aluminium-polyethylene bags

In order to transport and cryopreserve human tissues, it is essential to have an easy-to-use recipient where tissues can be kept in sterile conditions. Here we show the results obtained by using Macopharma’s tissue freezing bags, an aluminium-polyethylene multilayer bag, in our tissue bank of the Centro Comunitario de Sangre y Tejidos de Asturias. Five hundred and twenty-seven cancellous bone homografts were obtained from hospitals located 120 km around our Bank. The homografts were submitted to bacteriological controls and sent to our bank in these bags. They were stored at −70 °C and sent in dry ice to about 50 hospitals, where the tissue was bacteriologically controlled and grafted. Furthermore, the behaviour of these bags at −140 °C (vapour nitrogen) or −196 °C (liquid nitrogen) was tested. Our results indicate that Macopharma aluminium-polyethylene bags are suitable for the transporting and cryopreserving of cancellous bone homografts. These bags could also be used for keeping tissues in nitrogen containers.     

Effect of storage of domestic cat (Felis catus) epididymides at 5 °C on sperm quality and cryopreservation

Postmortem sperm recovery from the epididymides may constitute a powerful tool for the conservation of valuable genetic material. The domestic cat (Felis catus) is a good model for wild felids and, using this model, we have explored the effect of epididymides storage time on sperm motility and percentage of intact acrosomes upon sperm recovery and after cryopreservation. We also examined the effect of time of sperm equilibration with glycerol before freezing on sperm motility and the percentage of intact acrosomes. Motility varied between sperm recovered from epididymides that were stored for different times. Significant differences were seen in the sperm motility index (SMI) before freezing (55.91 ± 2.02, 48.21 ± 1.47, and 43.03 ± 1.32) and after thawing (51.81 ± 3.02, 41.90 ± 2.14, and 42.35 ± 1.95) of sperm recovered from epididymides stored for 0, 48, or 72 h, respectively. The percentage of intact acrosomes did not vary significantly with storage time (average 60.33 ± 1.38% before and 52.50 ± 1.91% after freezing, respectively). The percentage of normal sperm after different storage times did not differ (average 19.22 ± 1.25% normal sperm after recovery). When epididymides were stored for 72 h, time of sperm equilibration with glycerol (30 vs. 120 min) resulted in significant differences in both motility (SMI = 39.17 ± 2.76 and 45.00 ± 2.65, respectively) and the percentage of intact acrosomes (45.76 ± 4.91% and 60.67 ± 3.64%, respectively) after thawing. In conclusion, best results are achieved when sperm are recovered from epididymides within 24 h of cool storage and when they are equilibrated with glycerol during 120 min before freezing. The current results should be useful in the further development of techniques for the rescue and cryostorage of epididymal spermatozoa of endangered felids.     

Contaminated liquid nitrogen vapour as a risk factor in pathogen transfer

Liquid nitrogen in storage containers will gather particulate matter from the atmosphere, or the surfaces of containers placed into it, with time. Some of these accumulating particles may be pathogenic organisms and it can be demonstrated that their viability may be conserved by immersion in the liquid nitrogen. This contamination constitutes a risk to the status of stored samples that can, largely, be avoided by the use of appropriate techniques for sealing sample containers and sterilizing their outer surfaces. The present study uses fungal spores and organic crystals to demonstrate that such particles contained in liquid nitrogen are released back into the environment when nitrogen vapour cools programmable freezers or dry shippers. This demonstrates that storage in the vapour phase above liquid nitrogen still carries a real risk of sample, or facility, contamination. Regardless of the safety of the stored sample, this is a potential source of cross-contamination between repositories or experimental sites, both locally and internationally, that could have serious consequences in clinical and agricultural situations. This study provides evidence to suggest that this possibility, as yet unquantified, should be included in risk analysis of storage protocols for reproductive materials. The risk becomes diverse when, for example, semen and embryos are frozen at an agricultural site and the dry shipper can co-transport spores of contaminating crop plant pathogens to the destination site.     

Improvement of liquid and frozen-thawed semen quality of Nili-Ravi buffalo bulls (Bubalus bubalis) through supplementation of fat

The aim of the study was to investigate the effects of dietary fat on quality of liquid and frozen-thawed semen of Nili-Ravi buffalo bulls. Adult bulls (n = 21) were fed a balanced ration (Con; n = 7) or the same ration either containing sunflower oil (SF-O; n = 7) or whole sunflower seeds (SF-S; n = 7) for 63 days. Body weight and body condition score of each bull was recorded on days 0, 30 and 60 of the experiment. Semen was collected on days 39, 46, 53 and 60, frozen by a fast method and stored at −196 °C for 24 h. Sperm motility was assessed using a bright field microscope. Plasma membrane integrity of fresh and frozen-thawed spermatozoa was assessed using a hypo-osmotic swelling (HOS) assay. The concentration of spermatozoa and volume of semen was not different among groups on various days of collection. Sunflower-enriched diets did not affect the motility and number of HOS-positive spermatozoa in the fresh semen. Motility and HOS of post-thawed spermatozoa were higher (p < 0.05) in bulls fed the sunflower-enriched diets. Similarly, diets did not affect the body condition score and body weight of bulls. In conclusion, feeding of sunflower oil or sunflower seed as fat sources can improve the quality of buffalo bull spermatozoa.     

Protective effects of iodixanol during bovine sperm cryopreservation

The aim of cryopreservation is to maintain cellular integrity, thereby enabling resumption of proper biological functioning after thawing. Here we propose OptiPrep™ (60% iodixanol in water) as a protectant during sperm cryopreservation using pooled bull semen as the model. We evaluated OptiPrep concentration effect and its relation to cryopreservation by comparing frozen-thawed and chilled samples. Semen, extended in Andromed^® with 0 (control), 1.25%, 2.5%, and 5% OptiPrep™, was compared after either chilling or freezing in large volume by directional freezing. Sample evaluation included sperm motility upon thawing and after 3 h incubation at 37 °C for frozen-thawed samples and after 3 h and 6 h of chilling for chilled samples; viability, acrosomal integrity, and hypoosmotic swelling were also tested for frozen-thawed and chilled samples. Chilled samples with 5% OptiPrep™ showed inferior viability (P = 0.047) and 3 h motility (P = 0.017) relative to that for chilled samples with 2.5% OptiPrep and inferior viability (P = 0.042), acrosomal integrity (P = 0.045), and 0 h motility (P = 0.024) relative to that for chilled samples with 1.25% OptiPrep. The 1.25%, 2.5%, and control samples did not differ. In frozen-thawed samples, 2.5% OptiPrep was superior to all other concentrations for 3 h motility (control, P = 0.007; 5% OptiPrep, P = 0.005; 1.25% OptiPrep, P = 0.004) and to 1.25% OptiPrep for acrosomal integrity (P = 0.001). In a search for a protection mechanism, we measured glass transition temperature (T_g) of Andromed^® and of Andromed^® with 1.25%, 2.5%, and 5% OptiPrep™. Andromed^® (-58.78 °C) and 1.25% OptiPrep™ (-58.75 °C) groups had lower mean T_g than that of the 2.5% (-57.67 °C) and the 5% (-57.10 °C) groups. Directional cryomicroscopy revealed that the presence of iodixanol alters ice crystal formation into an intricate net of dendrites. Thus, iodixanol appears to possess cryoprotective properties by helping spermatozoa maintain motility and membrane integrity, possibly through altering ice crystals formation into a more hospitable environment and increasing the glass transition temperature.     

Double freezing of bovine semen

Fertility of bull spermatozoa cryopreserved in large volume by directional freezing technique, thawed, repackaged in straws and refrozen over liquid nitrogen vapor (double freezing, DF) was compared to conventional single freezing in straws (CF). Semen was collected from 6 bulls, 4 of which were selected for the field trial. Each semen collection was split into two parts, one frozen by CF and the other by DF. In vitro semen evaluations included motility (fresh, upon thawing and after 3 h incubation at 37 °C), viability and acrosome integrity. A total of 3610 cows and heifers were randomly inseminated by either CF or DF at about equal numbers. In vitro sperm analysis indicated no difference between CF and directional freezing in large volume and both were superior to DF (P < 0.001). Between-bull variations in fresh semen and in their reaction to CF or DF were apparent. Logistic regression analysis revealed that freezing method, bull, parity and inseminating technician, all had significant effect on pregnancy outcome (P ≤ 0.001 for all). Conception rate (CR) was 32.98% for CF and 28.05% for DF. Only in one bull conception rate by CF was significantly superior to DF (P < 0.05). When divided into heifers, primi- and pluriparous cows, only the difference in CR between the pluriparous cows was significant (P = 0.005). In conclusion, acceptable CR can be achieved by DF technique. These can be improved by selecting suitable bulls. The DF technique can be utilized in storage, sperm sexing and genome resource banking.     

Vitality and genetic fidelity of white-rot fungi mycelia following different methods of preservation

Basidiomycetes present specific problems with regard to their preservation, because most of them do not form resistant propagules in culture but exist only as mycelium. Usually these fungi can only be preserved by serial transfer on agar (labour-intensive procedures that can increase the danger of variation or loss of physiological or morphological features), or cryopreserved in liquid nitrogen (expensive). Cryopreservation at −80 °C and lyophilisation could be good alternatives.In this work we set up and tested six protocols of cryopreservation at −80 °C, and 12 protocols of lyophilisation on 15 isolates of white-rot fungi (WRF) belonging to 10 species. The tested protocols were mainly characterized by the use of different growth media, protectants, time and number of perfusion with protectants and finally by the typology and origin of the samples to be cryopreserved (mycelium/agar plug, whole colony) or to lyophilise (mycelium/agar plug, mycelium fragment, whole colony). Cryopreservation and lyophilisation outcomes were checked, at morphological (macro- and microscopic features), physiological (growth rate and laccase, Mn-independent and Mn-dependent peroxidases activities) and genetic level (Amplified Fragment Length Polymorphisms analysis - AFLP). Vitality of all fungi was successfully preserved by all cryopreservation protocols at −80 °C, and by two lyophilisation methods. Our results showed that cryopreservation at −80 °C did not produce morphological changes in any isolate, while two isolates were affected by lyophilisation. None of the physiological features were lost, even though growth rate and enzyme activities were somehow influenced by all preservation methods. AFLP analysis showed that only the two isolates that varied in their morphology after lyophilisation produced a different DNA fingerprint pattern in comparison with that obtained before lyophilisation. These findings provide evidence that cryopreservation at −80 °C and lyophilisation are suitable alternatives to liquid nitrogen cryopreservation for preservation of some WRF strains.