Conventional Freezing Protocols Research Articles

Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation

The development of novel cell-based therapies has increased the necessity to improve the long-term storage of cells. The current method of cryopreservation is far from optimal, causing ice-associated mechanical and osmotic damage to sensitive cells. Cell encapsulation is emerging as a new strategy to overcome those current limitations; however, few data are applicable to slow freezing, with conflicting results and multiple experimental conditions. The objective of this research work was to evaluate the impact of capsule size and encapsulation method on cell survival and functionality after a conventional freezing protocol. To this end, cells were encapsulated in alginate beads of different sizes, spanning the range of 200–2000 µm thanks to multiple extrusion techniques and conditions, and further cryopreserved using a slow cooling rate (−1°C/min) and 10 % DMSO as cryoprotectant. Our data show that there is a strong correlation between bead size and cell survival after a slow cooling cryopreservation process, with cell viabilities ranging from 7 to 70 % depending on the capsule size, with the smallest capsules (230 µm) achieving the highest level of survival. The obtained results indicate that the beads’ diameter, rather than their morphology or the technique used, plays a significant role in the post-thawing cell survival and functionality. These results show that a fine control of cell encapsulation in alginate hydrogels is required when it comes to overcoming the current limitations of long-term preservation techniques by slow cooling.

Comparative Study between Slow Conventional Freezing and Cryoprotectant-Free Vitrification of Human Spermatozoa in Large Volume

Background: Cryopreservation is the collection, freezing, and long term storage of sperm, and is a highly effective method of protecting male fertility. Cryopreservation of semen has been widely used as a vital method for fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. Objective: The aim of this study was to compare the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume, to conventional freezing protocol as regards post thawing motility, vitality and sperm DNA fragmentation. Patients and methods: This study included a total of 20 male patients seeking seminal fluid analysis, attending at the andrology laboratory of a specialized IVF center (ADAM International Hospital for Fertility and Sterility, Giza, Egypt. Patients were presented with the diagnoses of normozoospermia, oligozoospermia (either isolated or combined with asthenozoospermia or teratozoospermia). Results: Motility of (in a large volume (300 µl) in the absence of permeable cryoprotectants displayed significant statistically lower levels as compared to conventional Sperm Freezing. It was shown in different groups at different times (post thawing and 1-hour and 24-hour) of assessment that motility of vitrified spermatozoa decreases in comparison with slow conventional freezing as we go from the baseline. DNA fragmentation of vitrified spermatozoa showed higher levels as compared to conventional slow freezing but there is no significant statistical difference between vitrification and conventional slow freezing in DNA fragmentation. Conclusion: It could be concluded that vitrification technique was quite far away from comparison with slow conventional freezing protocol, and still need for further modifications and wide scale of study to achieve the good results.

Comparative Study between Slow Conventional Freezing and Cryoprotectant-Free Vitrification of Human Spermatozoa in Large Volume

Background: Cryopreservation is the collection, freezing, and long term storage of sperm, and is a highly effective method of protecting male fertility. Cryopreservation of semen has been widely used as a vital method for fertility preservation of male patients before undergoing chemotherapy, radiotherapy, and/or surgery that may lead to testicular failure or ejaculatory dysfunction. Objective: The aim of this study was to compare the aseptic technology of cryoprotectant-free vitrification of human spermatozoa in large volume, to conventional freezing protocol as regards post thawing motility, vitality and sperm DNA fragmentation. Patients and methods: This study included a total of 20 male patients seeking seminal fluid analysis, attending at the andrology laboratory of a specialized IVF center (ADAM International Hospital for Fertility and Sterility, Giza, Egypt. Patients were presented with the diagnoses of normozoospermia, oligozoospermia (either isolated or combined with asthenozoospermia or teratozoospermia). Results: Motility of (in a large volume (300 µl) in the absence of permeable cryoprotectants displayed significant statistically lower levels as compared to conventional Sperm Freezing. It was shown in different groups at different times (post thawing and 1-hour and 24-hour) of assessment that motility of vitrified spermatozoa decreases in comparison with slow conventional freezing as we go from the baseline. DNA fragmentation of vitrified spermatozoa showed higher levels as compared to conventional slow freezing but there is no significant statistical difference between vitrification and conventional slow freezing in DNA fragmentation. Conclusion: It could be concluded that vitrification technique was quite far away from comparison with slow conventional freezing protocol, and still need for further modifications and wide scale of study to achieve the good results.

Effect of human sperm freezing-thawing process on vitrification and conventional freezing: evaluation of survival, motility, DNA oxidation and mitochondrial activity

Sperm Vitrification is a method for cryopreservation, without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. We developed and evaluated a new method of vitrification, using a combination of 0,25 M trehalose-sucrose. This study aimed to compare the vitrification (V) with conventional freezing protocol (CF) on several sperm parameters: motility, vitality, mitocondryal activity and DNA oxidation.

Effect of egg yolk levels and equilibration periods on freezability of Jamunapari buck semen

Semen ejaculates from adult Jamunapari bucks (2–4 year-old) maintained at the Central Institute for Research on Goats, Makhdoom (UP) were utilized for a study to find out the freezability of buck semen at different levels of egg yolk during different hours of equilibration period by conventional freezing protocol. The ejaculates were extended to maintain sperm concentration approximately 100 million per dose (0.25 ml) in Tris-Citric Acid- Fructose (TCF) diluent having 0, 5, 10, 15 and 20% (v/v) egg yolk and 6% (v/v) glycerol as a cryoprotectant. Filling and sealing of straws were done at 5oC in cold handling cabinet after 2, 3, 4 and 5 h of equilibration period followed by vapour freezing of straws for 10 min at 2 cm above the liquid nitrogen and finally plunged into liquid nitrogen. Post-thaw motility, live sperm count, sperm abnormalities, acrosomal integrity and hypo-osmotic swelling test were conducted to assess freezability. The post-thaw motility, live sperm count, abnormalities, acrosomal integrity and hypo-osmotic swelling (HOS) positive spermatozoa differed significantly (P<0.05) at different levels of egg yolk and equilibration periods. Our results indicated that 10% egg yolk and 4 h of equilibration period is the best combination for semen freezing. The overall results showed that the inclusion of egg yolk significantly improved sperm post-thaw motility, indicating its beneficial effects during the freezing steps of cryopreservation. Furthermore, neither glycerol nor egg yolk alone protected the acrosome and tail membrane; the combination of two significantly (P<0.05) reduced the proportion of acrosome-damaged spermatozoa.

Ovarian Tissue Transplantation: Current State and Research Advancement

During the past decade, babies born from cryopreservation and transplantation of ovarian tissue have emerged as a promising alternative to restore ovarian function and fertility in cancer patients. Despite successful results obtained with conventional freezing protocols to preserve ovarian tissue, attempt for whole ovary transplantation is still under-propelling in large animal and human studies. Patient selection to avoid reimplanting cancer cells in ovarian grafts as a prerequisite safety concern is as important as advanced transplantation technique. Multidisciplinary strategies for the combined isolation immature follicle for culture or in vitro activation of ovarian follicle with the frozen-thawed ovarian tissue raised the diversity for female fertility preservation along with the egg-embryo freezing or in vitro maturation of immature eggs. The purpose of this updated review of fertility preservation techniques is to develop a network among health care professional interested in this challenging field for those who are threatened the reproductive function.

Post-thaw viability of bull AI-doses with low-sperm numbers

Use of AI-doses containing low-sperm numbers are increasingly been used to optimise use of elite bulls as well as to accommodate an eventual wider application of sex-sorted semen. Since spermatozoa might, however, suffer from high extension rates, thus compromising fertility, this study evaluated the post-thaw sperm quality of semen from commercial progeny-tested, high-ranked AI-sires whose semen was within acceptable limits of normality, frozen in a split-design to 15 (control, 15M) or 2 × 10 6 total spermatozoa (treatment, 2M) per straw. Assessment post-thaw included computer-evaluated sperm motility (CASA), membrane integrity (SYBR-14/PI), membrane stability (Annexin-V/PI), acrosome integrity (Carboxy-SNARF-1/PI/FITC-PSA), and chromatin integrity (AO of in situ acid-induced DNA denaturation). High extension did not affect the proportions of linearly motile spermatozoa, of membrane integrity or stability nor chromatin integrity, immediately post-thaw. However, high extension clearly affected linear sperm motility following incubation at 38 °C for 30 min, sperm viability when assessed by SNARF and, particularly, acrosome integrity of the otherwise viable spermatozoa. Individual sire variation was evident. Fertility was preliminarily evaluated for one of the less affected bulls in a blind field trial. A total of 109 dairy cows were randomly inseminated with 15M or 2M-straws without differences in pregnancy rate between them (47% versus 43%). This similarity in fertility rates, confirmed the in vitro methods used were appropriate for identifying cryosurvival and further suggested the site of sperm deposition was not crucial for the fertility of low-sperm AI-numbers for this particular sire. However, the inter-bull variation seen calls for caution when cryopreserving low concentrations of bull spermatozoa with conventional freezing protocols.

Cell partitioning during the directional solidification of trehalose solutions

Previous studies have demonstrated that ice/cell interaction influences post thaw viability and specific cryoprotective agents can affect those interactions. Trehalose, a disaccharide, has been shown to have a protective benefit during conventional slow freezing. Existing theories have been put forth to explain the protective benefit of trehalose during desiccation and vitrification, but these theories do not explain the protective benefit observed during conventional freezing protocols. The overall objective of this investigation was to characterize cell/ice interactions in the presence of trehalose using non-planar freezing conditions. To that end, lymphoblasts suspended in phosphate buffered saline solution with various levels of trehalose (0, 10, 100, and 300 mM) were frozen on a directional solidification stage. The partitioning of cells into the interdendritic space or engulfment by an advancing dendrite was determined as a function of velocity and solution composition. For a given temperature gradient, the fraction of cells entrapped into the interdendritic region increased with increasing velocity. With small additions of trehalose (10 mM), the velocity at which cells were entrapped in the interdendritic region increased. At high trehalose concentrations (100, 300 mM), interface morphology was significantly different and cells were engulfed by the advancing interface. Dehydration of cells in the region shortly before and after the interface was significant and depended upon of the type of interaction experienced by the cell (entrapped vs. engulfed). These studies suggest that one potential mechanism for the action of trehalose involves changing the ice/cell interactions during conventional slow freezing.

Do different portions of the boar ejaculate vary in their ability to sustain cryopreservation?

Previous studies have shown sperm quality post-cryopreservation differs depending on the fraction of the seminal plasma boar spermatozoa are fortuitously contained in. As such, spermatozoa contained in the first 10 mL of the sperm-rich fraction (portion I) have better sustained handling procedures (extension, handling and freezing/thawing) than those contained in the ulterior part of a fractionated ejaculate (second portion of the sperm-rich fraction and the post-spermatic fraction, portion II). However, those studies were performed using pooled samples. In the present study, individual ejaculates were used. Split ejaculates (portions I and II) from five boars were frozen and thawed using a conventional freezing protocol, followed by computer-assisted motility and morphology analysis (CASA and ASMA, respectively), as well as an Annexin-V assay for spermatozoa from each boar and ejaculate portion. Significant differences between portions were observed in all ASMA-derived variables, except in one boar. Also significant differences were observed between boars and ejaculate portions in sperm quality post-thaw. We identified, however, boars showing best results of motility and sperm membrane integrity post-thaw in portion I, while in other boar the best results was observed in portion II. It is concluded that the identification of the ejaculate portion more suitable to sustain cryopreservation in each individual boar may be a readily applicable and easy technique to diminish variation in sperm freezability among boars.

Effects of Cryopreservation on Cell Viability and Insulin Secretion in a Model Tissue-Engineered Pancreatic Substitute (TEPS)

The use of encapsulated insulin-secreting cells constitutes a promising approach towards the treatment of insulin-dependent diabetes. However, long- term storage for off-the-shelf availability still remains an issue, which can be addressed by cryopreservation. This study investigated cryopreservation of a model tissue-engineered pancreatic substitute by two ice-free cryopreservation (vitrification) solutions (designated VS55 and PEG400) in comparison to a conventional freezing protocol. The model substitute consisted of insulin-secreting mouse insulinoma betaTC3 cells entrapped in calcium alginate/poly-L-lysine/alginate (APA) beads. Cell viability and static insulin secretion from the thawed cryopreserved groups were characterized and compared against fresh controls. Cell viability tests using alamarBlue showed that, compared to the fresh groups, the VS55 had the highest viability (p < 0.05), followed by both the PEG400 (p < 0.001) and the frozen groups (p < 0.001). In response to a square wave of glucose, the static insulin secretion data showed that the VS55 and PEG400 groups had similar induction levels against the fresh group, whereas the frozen group had the poorest secretion rate. Cryosubstitution of capsules showed ice formation in the frozen group but no ice in the vitrified groups. Microscopic observations revealed holes and/or tears within beads subjected to freezing, whereas no such abnormalities were detected in the vitrified samples. Overall, vitrification was found to be a promising preservation procedure for this encapsulated cell system.

9 DOES STRAW CONCENTRATION AFFECT POST-THAW SPERM QUALITY IN AI BULL SIRES?

There is increasing interest in decreasing the number of spermatozoa per AI dose, owing to the discrimination of threshold concentration and fertility, economical revenues and the use of sexed semen. This study evaluated the quality (as sperm viability, acrosome integrity, membrane stability and chromatin stability) post-thaw of semen collected from four elite AI sires and frozen at 15 × 106 (control) or 2 × 106 spermatozoa (spz) per dose to disclose eventual deleterious extension effects. Semen collected via a.v. from 4 élite AI sires was split-processed and frozen in 0.25 mL plastic straws under commercial conditions, at either 2 × 106 or 15 × 106 spz/straw (the latter as control). The semen parameters were within acceptable limits of normality. The post-thaw samples showed acceptable motility (above 50%). Sperm viability and stability were assessed post-thaw using flow cytometry of cells loaded with SYBR-14 and propidium iodide (PI) for sperm viability (membrane integrity), and with carboxy-SNARF-1 (SNARF; Invitrogen AB, Frolunda, Sweden), PI, and FITC-Pisum sativum agglutinin (PSA, triple stain) for acrosome status. Membrane stability status was measured with Annexin-V/PI, while sperm chromatin condensation and stability were assessed following in situ acid-induced DNA denaturation and staining with acridine orange. No significant differences were seen between the two concentrations regarding sperm motility, plasma membrane integrity (SYBR/PI, SNARF), or chromatin stability. The highly extended semen (2 × 106 spz/straw), however, showed a higher (P &lt; 0.05) frequency of spermatozoa with translocated phosphatydil serine (as detected with Annexin-V), indicating their plasma membranes had become unstable. Also, there were more spermatozoa showing acrosomal damage (PSA). In conclusion, low concentrations of spermatozoa do not properly sustain conventional cryopreservation, although damages appear restricted to the sperm membrane. These changes in the stability of the plasma membrane apparently did not affect the viability of the spermatozoa, although they may negatively affect their lifespan. These findings may have an impact on the care that must be taken when cryopreserving low concentrations of spermatozoa with conventional freezing protocols. This work was supported by the Swedish Farmers’ Foundation for Research in Agriculture (SLF) and FORMAS, Stockholm.