Long-term maintenance of Pinus nigra embryogenic cultures through cryopreservation

Embryogenic tissues of Pinus nigra have been cryopreserved using a two step slow-freezing method. In the first experiment, 20 cell lines were included and the effect of the duration of cryostorage (1 h vs. 1 year) on regrowth was compared. After a short-term storage (1 h in liquid nitrogen, LN) out of 20 cell lines tested 15 showed regrowth (75%) with individual frequencies 10–100%. Long term storage (1 year in LN) resulted in regrowth of 14 cell lines (70%) while the individual frequencies reached 10–100%. One year storage had no negative influence on the fresh mass accumulation evaluated 2–3 months after thawing. Another 20 cell lines were included in the second experiment with the aim to study the correlation between cryotolerance and maturation capacity of cell lines. Between maturation capacity and cryotolerance expressed as regrowth frequencies of individual cell lines, no correlation has been found.     

Enhanced Formation of Roots and Subsequent Promotion of Growth of Shoots on Cryopreserved Nodal Segments of Asparagus officinalis L

This study was designed to investigate the effects of cryopreservation on the survival, organogenesis, and growth of plants regenerated from nodal segments of asparagus (Asparagus officinalis L.) that had been cut from cultures in vitro. The addition of dimethylsulfoxide (Me2SO) to the freezing solution at 8-16% (v/v) with or without a sugar (glucose, sorbitol, or sucrose) was effective for successful cryopreservation by a slow prefreezing method. Frequencies of root formation (average, 59.3%) from cryopreserved and surviving nodal segments were significantly higher (P < 0.005) than those (average, 13.9%) from nodal segments that had only been treated with freezing solution supplemented with 12% (v/v) Me2SO and various sugars without freeze-thawing. The increased frequency of root formation from cryopreserved nodal segments appears to have been induced by the freeze-thaw step of the cryopreservation procedure. Numbers and lengths of shoots derived from cryopreserved nodal segments were initially lower but were subsequently higher, after 60-90 days of culture, than those of shoots derived from nodal segments without freeze-thawing. The promotion of growth of shoots from cryopreserved nodal segments seemed to have been due to the increased percentage of root formation. Histological observations revealed that only dome-shaped meristematic tissue and a few cells of cladophyll primordia survived in cryopreserved nodal segments that had been cultured for 5 days after thawing. Many mitochondria and well developed rER were observed in these cells. Disorganization and/or physiological changes might have occurred in the surviving tissues and/or cells of the cryopreserved nodal segments that were responsible for the subsequent increased formation of roots. Copyright 1998 Academic Press.     

Improved cryopreservability of stallion sperm using a sorbitol-based freezing extender

Cryopreservation of stallion semen is often associated with poor post-thaw sperm quality. Sugars are among the important components of a freezing extender and act as non-permeating cryoprotectants. This study aimed to compare the quality of stallion sperm frozen with glucose, fructose or sorbitol-containing freezing extenders. Semen was collected from six stallions of proven fertility and cryopreserved using a freezing extender containing different types of monosaccharide sugars (glucose, fructose or sorbitol). After thawing, the semen was examined for sperm motility, viability, acrosome integrity, plasma membrane functionality and sperm longevity. The fertility of semen frozen in the presence of sorbitol was also tested by artificial insemination. Sperm quality was significantly decreased following freezing and thawing (P < 0.05). Fructose was inferior for protecting sperm during cryopreservation when compared to sorbitol and glucose (P < 0.05). Although the viability, motility and acrosome integrity of sperm cryopreserved with a glucose-containing extender did not significantly differ from sperm frozen in the sorbitol-based extender when examined at 2 and 4 h post-thaw, all of these parameters plus plasma membrane functionality were improved for sperm frozen in the sorbitol extender than in the glucose extender when examined 10 min post-thaw. Two of four mares (50%) inseminated with semen frozen with a sorbitol-containing freezing extender became pregnant. It is concluded that different sugars have different abilities to protect against cryoinjury during freezing and thawing of stallion sperm. This study demonstrated that an extender containing sorbitol as primary sugar can be used to successfully cryopreserve equine sperm; moreover, the quality of frozen-thawed sperm appeared to be better than when glucose or fructose was the principle sugar in the freezing extender.     

Development of a new vitrification solution, VSL, and its application to the cryopreservation of gentian axillary buds

Vitrification methods are convenient for cryopreserving plant specimens, as the specimens are plunged directly into liquid nitrogen (LN) from ambient temperatures. However, tissues and species with poor survival are still not uncommon. The development of vitrification solutions with high survival that cover a range of materials is important. We attempted to develop new vitrification solutions using bromegrass cells and found that VSL, comprising 20% (w/v) glycerol, 30% (w/v) ethylene glycol, 5% (w/v) sucrose, 10% (w/v) DMSO and 10 mM CaCl₂, gave the highest survival following cryopreservation, as determined by fluorescein diacetate staining. However, the cryopreserved cells showed little regrowth, for unknown reasons. To check its applicability, VSL was used to cryopreserve gentian axillary buds and the performance was compared with those of conventional vitrification solutions. Excised gentian stem segments with axillary buds (shoot apices) were two-step precultured with sucrose to induce osmotic tolerance prior to cryopreservation. Gentian axillary buds cryopreserved using VSL following the appropriate preculturing approach exhibited 78% survival (determined by the regrowth capacity), which was comparable to PVS2 and PVS1 and far better than PVS3. VSL had a wider optimal incubation time (20–45 min) than PVS2 and was more suitable for cryopreserving gentian buds. The optimal duration of the first step of the preculture was 7–11 days, and preculturing with sucrose and glucose gave a much higher survival than fructose and maltose. VSL was able to vitrify during cooling to LN temperatures, as glass transition and devitrification points were detected in the warming profiles from differential scanning calorimetry. VSL and its derivative, VSL+, seem to have the potential to be good alternatives to PVS2 for the cryopreservation of some materials, as exemplified by gentian buds. Mitsuteru Suzuki, Pramod Tandon and Masaya Ishikawa contributed equally to the work.     

Freeze–thaw induced genotoxicity in buffalo (<Emphasis Type="Italic">Bubalus bubalis</Emphasis>) spermatozoa in relation to total antioxidant status

Use of cryopreserved semen has become an important tool in assisted reproduction but freezing and thawing cause sub-lethal damage to spermatozoa. This is detrimental to sperm because of the membrane damage including permeability and integrity. An excess generation of reactive oxygen species (ROS) creates oxidative stress due to reduced antioxidant status of the cryopreserved spermatozoa. In the present study fresh buffalo semen was collected and divided into two aliquots. One aliquot was used for fresh semen analysis and the other was cryopreserved in Tris-egg yolk-citrate extender. The semen samples were used to study different sperm quality parameters like motility, viability, membrane integrity and total antioxidant status. The DNA integrity in fresh and cryopreserved spermatozoa was also studied using comet assay. The sperm quality parameters like post-thaw sperm motility, viability, membrane integrity and total antioxidant status of cryopreserved spermatozoa were significantly lowered (P < 0.05) compared to fresh spermatozoa. The DNA fragmentation in cryopreserved spermatozoa was significantly higher (P < 0.01) as compared to fresh spermatozoa. The results show that the irreversible DNA damage occurs in spermatozoa during cryopreservation.     

Cryopreservation of Alkaloid-Producing Cell Cultures of Periwinkle (Catharanthus roseus)

A procedure for cryogenic storage of alkaloid producing cell lines of periwinkle, Catharanthus roseus (L.) G. Don., has been developed. The procedure differs from established cryopreservation protocols in several aspects. Specifically, 4-day-old suspension subcultures of three cell lines were precultured in nutrient media supplemented with 1 molar sorbitol for 6 to 20 hours. The cells were then incubated in nutrient media with 1 molar sorbitol plus 5% DMSO in an ice bath for 1 hour and, thereafter, were frozen in this solution at a cooling rate of 0.5°C per minute to −40°C prior to immersion in liquid nitrogen (LN). After rapid thawing in a 40°C water bath, the regrowth of LN stored cells was achieved by transferring them without washing onto filter paper discs over nutrient media solidified with agar for a period of 4 to 5 hours. The filter paper discs with the cells were then transferred to fresh media of the same composition for regrowth. The viability immediately after thawing as evaluated by the 2,3,5-triphenyl tetrazolium chloride method was about 60% of controls. Suspension cultures established from LN stored cells retained the capability for alkaloid synthesis and accumulation.     

Preservation of cell-based immunotherapies for clinical trials

In the unique supply chain of cellular therapies, preservation is important to keep the cell product viable. Many factors in cryopreservation affect the outcome of a cell therapy: (i) formulation and introduction of a freezing medium, (ii) cooling rate, (iii) storage conditions, (iv) thawing conditions and (v) post-thaw processing. This article surveys clinical trials of cellular immunotherapy that used cryopreserved regulatory, chimeric antigen receptor or gamma delta T cells, dendritic cells or natural killer (NK) cells. Several observations are summarized from the given information. The aforementioned cell types have been similarly frozen in media containing 5–10% dimethyl sulfoxide (DMSO) with plasma, serum or human serum albumin. Two common freezing methods are an insulated freezing container such as Nalgene Mr. Frosty and a controlled-rate freezer at a cooling rate of -1°C/min. Water baths at approximately 37°C have been commonly used for thawing. Post-thaw processing of cryopreserved cells varied greatly: some studies infused the cells immediately upon thawing; some diluted the cells in a carrier solution of varying formulation before infusion; some washed cells to remove cryoprotective agents; and others re-cultured cells to recover cell viability or functionality lost due to cryopreservation. Emerging approaches to preserving cellular immunotherapies are also described. DMSO-free formulations of the freezing media have demonstrated improved preservation of cell viability in T lymphocytes and of cytotoxic function in natural killer cells. Saccharides are a common type of molecule used as an alternative cryoprotective agent to DMSO. Improving methods of preservation will be critical to growth in the clinical use of cellular immunotherapies.     

High-efficiency cryopreservation of Araucaria angustifolia (Bertol.) Kuntze embryogenic cultures: ultrastructural characterization and morpho-physiological features

Here we evaluated and characterized the growth dynamics of A. angustifolia embryogenic cultures (EC) submitted to different cryotreatment incubation times through morphological and time-lapse cell tracking analyzes. The EC submitted to cryopreservation protocol were evaluated by regrowth rates, and ultrastructural characterization by transmission electron microscopy (TEM). The results indicated that A. angustifolia EC support all the cryoprotection times evaluated, without cell proliferation inhibition, but with noticeable genotype-dependent response in all tested cell lines. The use of 1M DMSO showed non-inhibitory effects to EC regrowth independent of cell line or cryotreatment incubation time. However, after cryopreservation, Cr01 cell line regrowth was 100 % for all cryotreatments incubation times evaluated (30, 60, 120, 240 min), while Cr02 cell line only showed 100 % regrowth in 240 min of cryotreatment. The 100 % cell regrowth obtained in both cell lines indicates that the proposed protocol can be successful applied to A. angustifolia EC cryopreservation. Cell tracking analysis showed a survival and initial proliferation of embryogenic cells, with the first cell regrowth signs after 30 days in culture. TEM analysis revealed a conspicuous cell wall thickening in embryogenic cells after cryotreatment and after thawing, which may be related to osmotic stress response caused by the cryopreservation process. An increased heterochromatin presence was also observed in cryotreated or after thawing cells, may possibly be acting as a cell defense mechanism, decreasing the DNA vulnerability to cleavage and preserving the cell integrity.

     

Cryopreservation of apple (Malus×domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification

Axillary shoot tips of apple cv. Golden Delicious isolated from shoot cultures were successfully cryopreserved using the encapsulation-dehydration technique. After encapsulation in alginate gel, embedded shoot tips were dehydrated by exposure to a sterile air flow before being frozen in liquid nitrogen and subsequent slow thawing. A preculture on modified MS medium containing 0.75 M sucrose followed by 6 h of dehydration (21% residual water) led to the highest shoot regrowth of frozen, coated shoot tips (83.7%). Among the sugars tested, sucrose and sorbitol presented the best cryoprotective effect. Four other scion apple varieties and rootstocks were also successfully cryopreserved. Axillary shoot tips of five apple (Malus×domestica Borkh.) scion and rootstock cultivars were cryopreserved using the encapsulation-vitrification technique. Using a one-step freezing method, we successfully cryopreserved axillary shoot tips without the requirement of a cold hardening pretreatment of the shoot cultures. Cryopreserved shoot tips treated with aqueous cryoprotective mixture IV containing 180% (w/v) sucrose and 120% (v/v) ethylene glycol showed the highest shoot regrowth rates, which varied from 64% to 77%, depending on the cultivar. Received: 29 July 1999 / Revision received: 24 September 1999 / Accepted: 26 November 1999     

Effect of different cryoprotectant procedures on the recovery and maturation ability of cryopreserved <Emphasis Type="Italic">Pinus pinea</Emphasis> embryogenic lines of different ages

Strategies for genetic improvement programs of Pinus pinea L, an important tree species of the Mediterranean ecosystem, are focused on increasing pine nut yield. Somatic embryogenesis and cryopreservation of elite genotypes are emerging as key components of advanced forest breeding programs. This study was carried out with embryogenic lines of different ages obtained from selected half-sib families of the species. The effect of three cryoprotectant procedures on the recovery and maturation ability was tested in embryogenic lines that showed different growth rate, two of them at different ages. In general, cryopreservation drastically reduced growth rates of frozen and rewarmed tissues; however, the use of 5% PEG–sucrose–DMSO dramatically increased growth rates of rewarmed embryogenic cultures. Overall, embryogenic lines of stone pine were suitable for cryopreservation. Seven out of eight lines were recovered, although the initial growth rates were variable. Five of six lines including the three oldest ones were recovered using 5% PEG–sucrose–DMSO. No relation was observed between age and growth rate of embryogenic lines and their response to cryopreservation. The line 2F47 showed the most stable response after long-term subculture and recovery after cryopreservation, at different ages. On the contrary, younger embryogenic lines either recovered after cryopreservation or did not, depending on the applied procedure. Maturation of some of the older lines was restored or enhanced after cryopreservation. Somatic embryos were obtained in three out of five tested embryogenic lines recovered from cryopreservation. However, only a few plantlets from cryopreserved lines were regenerated indicating the process must be optimized further before it is a practical adjunct to breeding.     

Cryopreservation of swine colostrum-derived cells

Mesenchymal stromal cells (MSCs) have been demonstrated to possess anti-inflammatory and antimicrobial properties and are of interest in biotechnologies that will require cryopreservation. Recently, MSC-like cells were isolated from colostrum and milk. We used an interrupted slow freezing procedure to examine cryoinjury incurred during slow cooling and rapid cooling of MSC-like cells from swine colostrum. Cells were loaded with either dimethyl sulfoxide (Me₂SO) or glycerol, cooled to a nucleation temperature, ice-nucleated, and further cooled at 1 °C/min. At several temperatures along the cooling path, cells were either thawed directly, or plunged into liquid nitrogen for storage and later thawed. The pattern of direct-thaw and plunge-thaw responses was used to guide optimization of cryopreservation protocol parameters. We found that both 5% Me₂SO (0.65 M, loaded for 15 min on ice) or 5% glycerol (0.55 M, loaded for 1 h at room temperature) yielded cells with high post-thaw membrane integrity when cells were cooled to at least −30 °C before being plunged into, and stored in, liquid nitrogen. Cells cultured post-thaw exhibited osteogenic differentiation similar to fresh unfrozen control. Fresh and cryopreserved MSC-like cells demonstrated antimicrobial activity against S. aureus. Also, the antimicrobial activity of cell-conditioned media was higher when both fresh and cryopreserved MSC-like cells were pre-exposed to S. aureus. Thus, we were able to demonstrate cryopreservation of colostrum-derived MSC-like cells using Me₂SO or glycerol, and show that both cryoprotectants yield highly viable cells with osteogenic potential, but that cells cryopreserved with glycerol retain higher antimicrobial activity post-thaw.     

Antioxidant and anti-stress compounds improve regrowth of cryopreserved <Emphasis Type="Italic">Rubus</Emphasis> shoot tips

Regrowth of plants after cryopreservation varies, and resulting regrowth ranges from poor to excellent. Oxidative stress is a potential cause of damage in plant tissues. Antioxidants and anti-stress compounds may improve regrowth by preventing or repairing the damage. Lipoic acid (LA), glutathione (GSH), glycine betaine (GB), and polyvinylpyrrolidone (PVP) were tested during cryopreservation of shoot tips using the plant vitrification solution 2 (PVS2) protocol. Two in vitro-grown blackberry cultivars were cold acclimated and then cryopreserved in liquid nitrogen (LN). The antioxidant and anti-stress compounds were added at four critical steps of the protocol: pretreatment, loading, rinsing, and regrowth. Three out of the four compounds significantly improved regrowth of cryopreserved shoot tips. Regrowth ranged from 40% to 50% for controls to >80% for treated shoot tips. LA (4-8 mM) produced high regrowth at pretreatment, loading, and rinsing for ‘Chehalem’ and at all steps for ‘Hull Thornless’. Recovery improved at all steps with GSH (0.16 mM) and GB (10 mM). PVP had a neutral or negative impact on regrowth. Overall addition of LA, GSH, and GB improved regrowth by ∼25% over the shoot tips cryopreserved using the regular PVS2 protocol (control). This study shows that adding non-vitamin antioxidants and anti-stress compounds during the PVS2-vitrification protocol improves regrowth of shoot cultures following cryopreservation. We recommend inclusion of antioxidants as part of standard cryopreservation protocols.     

Reliable and practical methods for cryopreservation of embryogenic cultures and cold storage of somatic embryos of Norway spruce

Somatic embryogenesis (SE) is considered as the most-effective method for vegetative propagation of Norway spruce (Picea abies L. Karst). For mass propagation, a cryopreservation method able to handle large numbers of embryogenic tissues (ETs) reliably and at low costs is needed. The aim of the present study was to compare pretreatments, cryoprotectants and slow-cooling devices for cryopreservation of Norway spruce ETs, with 12 variations of methods and a total of 136 spruce genotypes. Secondly, possible applications for cold storage of mature somatic embryos were studied with the aim of developing a flexible time window for embling production. At best, 100% of the embryogenic lines were recovered following cryopreservation, but the results varied among the sets of lines. Also physiological condition of the tissues, pre-treatment and cryoprotectant applied, as well as the slow-cooling device used were found to affect the recovery. The best option for cryopreservation of Norway spruce is to select fresh growth from young ETs as samples, pretreat them on semi-solid medium with increasing sucrose concentration (0.1 M for 24 h; 0.2 M for another 24 h), apply a mixture of polyethylene glycol 6000, glucose, and dimethylsulfoxide, 10% w/v each, as cryoprotectant and use a programmable freezer with a slow cooling rate (0.17 °C/min). On average, 87% of the genotypes can be recovered, without any effect on their genetic fidelity, as shown by microsatellite markers and embryo production capacity. Mature somatic embryos of Norway spruce can also be safely cold-stored at +4 °C, without adverse effects on their germination ability.     

Optimization of cryoprotectants for cryopreservation of rat hepatocyte

Rat hepatocytes were cryopreserved in hormonally-defined medium (HDM) containing either fetal bovine serum (FBS), glycerol, dimethyl sulfoxide (DMSO), sucrose or a mixture of these as a cryoprotectant. The best survival was with 10% (v/v) DMSO containing 30% (v/v) FBS using 5 x 10(5) hepatocytes ml(-1) at -70 degrees C for 5 d on type I collagen-coated dishes. After thawing, the cell viability was 81% determined by the MTT-test. The cryopreserved hepatocytes had the capacity of albumin synthesis similar to hepatocytes without cryopreservation. This result shows that cryopreservation of rat hepatocyte can be used for the evaluation of hepatic functions.     

A simple and serum-free protocol for cryopreservation of human umbilical cord as source of Wharton’s jelly mesenchymal stem cells

Mesenchymal stromal cells (MSCs) show promise in cell-based transplantations and regenerative medicine applications. MSCs from Wharton’s jelly (WJ) of umbilical cord can be easily harvested and exhibit greater proliferative activity than bone marrow MSCs. It is important to develop a practical cryopreservation technique to effectively store umbilical cord for potential future applications. Successful cryopreservation would allow access to umbilical cord from the same donor for repeated WJ MSC-based transplantations. For therapeutic applications, one should be able to obtain clinically-relevant quality and quantity of MSCs from cryopreserved tissues. In this study, we optimised a serum-free formulation of 10% dimethyl sulfoxide (DMSO) and 0.2 M sucrose for cryopreservation of umbilical cord tissue. Slow freezing and rapid thawing were adopted. MSCs harvested from WJ of cryopreserved umbilical cord could undergo robust expansion, differentiate to mesodermal lineages and express MSC-characteristic surface antigens. The cumulative cell yield, however, was less compared to corresponding fresh cord tissue.     

Retention of the capacity to produce plants from protoplasts in cryopreserved cell lines of rice (Oryza sativa L.)

A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15–21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.Abbreviations DMSO dimethyl sulfoxide - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphtylacetic acid - FDA fluorescein diacetate     

Cryopreservation of somatic embryos of the herbaceous peony (Paeonia lactiflora Pall.) by air drying

This study was carried out to establish a suitable method for the cryopreservation of somatic embryos of the herbaceous peony. The somatic embryos were obtained from cotyledon and anther cultures on a MS medium supplemented with abscisic acid (ABA) and phenylacetic acid (PAA), respectively. The frequency of somatic embryo formation was the greatest (61%) from the cotyledons cultured on a MS medium supplemented with 1.0 mg l(-1) of ABA. Embryos were also obtained directly from anthers cultured on a MS medium with or without 2.0 mg l(-1) of PAA. For the cryopreservation of peony somatic embryos, the embryos were dried under a stream of sterile air and frozen by immersion in liquid nitrogen. Thawed embryos were germinated into plantlets after placing on a medium containing 0.3 mg l(-1) of gibberellic acid (GA(3)). The frequency of the post-thaw regrowth of cryopreserved somatic embryos was related to their size and desiccation time, the latter ranging from 0 to 2 h. When the somatic embryos were desiccated for 1 h, the frequency of post-thaw regrowth was greater than 66%. The frequency of post-thaw regrowth of the cryopreserved somatic embryos from anthers and cotyledon tissues was generally high when they were 2-3 mm in size. Desiccation may be a suitable method for the cryopreservation of somatic embryos of the herbaceous peony.     

Viability of pulp stromal cells in cryopreserved deciduous teeth

The cryopreservation of exfoliated deciduous teeth and harvesting of stem cells from them as required would reduce the costs and efforts associated with banking stem cells from primary teeth. The aim of this study was determine whether the viability of pulp stromal cells from deciduous teeth was influenced by the cryopreservation process itself or the period of cryopreservation. In total, 126 deciduous teeth were divided into three groups: (1) fresh, (2) cryopreserved for <3 months (cryo<3), and (3) cryopreserved for 3–9 months (cryo3–9). The viability of the pulp tissues was compared among the three groups by evaluating the outgrowth from pulp tissues and cell activity within those pulp tissues. In addition, the terminal deoxynucleotidyl transferase-mediated dUTP–biotin nick end labeling (TUNEL) assay was performed to compare cell apoptosis within fresh pulp tissue and pulp tissue that had been cryopreserved for 4 months. The outgrowth from and cell activity within the pulp tissues did not differ significantly between the fresh and cryo<3 pulp tissues. However, these parameters were significantly reduced in the cryo3–9 pulp tissue. In TUNEL assay, 4-month cryopreserved pulp tissues has more apoptotic cells than fresh group. In conclusion, it is possible to acquire pulp stromal cells from cryopreserved deciduous teeth. However, as the period of cryopreservation becomes longer, it is difficult to get pulp cells due to reduced cell viability.     

Curcumin treatment enhances islet recovery by induction of heat shock response proteins, Hsp70 and heme oxygenase-1, during cryopreservation

Limited recovery of islets post-cryopreservation influences graft survival and transplantation efficiency during diabetes treatment. As curcumin, a potent antioxidant/radical scavenging compound, protects islets against beta cell toxins, we hypothesized that inclusion of curcumin during cryopreservation or during post-thaw culture or both may rescue islets from cryoinjury. To test the effect of curcumin inclusion on islet recovery murine islets were isolated by the collagenase digestion, cultured for 48 h, cryopreserved using dimethylsulphoxide as cryoprotectant -- with or without curcumin (10 microM) -- and then slow cooled to -40 degrees C before immersing them in liquid nitrogen for 7 days. Following rapid thawing with sucrose gradient and 24 h post-thaw culture -- in presence or absence of curcumin (10 microM) -- islet viability and functionality were determined. Islet recovery in curcumin treated groups was significantly higher than in groups where islets were cryopreserved without curcumin. Islets cryopreserved with curcumin also showed more intact islets as well as better morphology as compared to islets cryopreserved without curcumin. Curcumin treated islets also showed significant inhibition of ROS generation as compared to islets cryopreserved without curcumin. Glucose responsiveness and insulin secretion in islets cryopreserved with curcumin was equal to that of the freshly isolated islets as against islets cryopreserved without curcumin. Elevated level of Hsp 70 and HO-1 were observed in islets cryopreserved with curcumin and may contribute to curcumin-induced islet rescue. Hence, we conclude that inclusion of curcumin into cryopreservation medium inhibits ROS generation and corresponding islet damage and dysfunction.     

The proteomic characterization of ram sperm during cryopreservation analyzed by the two-dimensional electrophoresis coupled with mass spectrometry

The aim of this study was to analyze the effects of the cryopreservation process on the protein profile of ram sperm using two-dimensional electrophoresis (2-DE) coupled with mass spectroscopy. Semen was collected from five rams and cryopreserved in a Tris-based extender supplemented with glycerol and egg yolk as the main cryoprotectants. The fresh and post-thaw sperm total proteins were extracted and purified, followed by the 2-DE. The differential proteins in the stained gel were determined by mass spectrometry. The results indicated that there were 39 differential proteins between fresh sperm and frozen-thawed sperm. Among these proteins, the abundance of 28 proteins in fresh sperm was higher than those in post-thaw sperm (P < 0.05). However, 11 proteins in post-thaw sperm were up-regulated instead. The gene ontology (GO) analysis showed that most of differential proteins were implicated in cellular process, metabolism and regulation of the biological process. The networks of protein-protein interaction indicated a strong interaction among these differential proteins, which may be involved in sperm metabolism, acrosomal function, sperm motility, and reducing ROS level. In conclusion, the cryopreservation process modifies the proteome of ram sperm, which may be directly associated with ram sperm cryodamage, consequently influencing their fertility. Additionally, these differential proteins can be used as biomarkers for evaluation of frozen ram semen quality.