Cryopreservation of sperm of an indigenous endangered fish species Nandus nandus (Hamilton, 1822) for ex-situ conservation

This study dealt with the development of cryopreservation protocol for Nandus nandus, which entailed a number of experiments. Sperm was collected by sacrificing males. The collected sperm was suspended in extenders. Activation of sperm motility was evaluated in different osmolalities of NaCl. Motility of sperm decreased as the osmolality of the extender increased and was completely inhibited at almost 319 mOsmol/kg. To evaluate the toxicity of cryoprotectant, sperm was incubated with DMSO, methanol and ethanol at 5%, 10% and 15% concentrations, respectively, for 5–35 min. Five and ten percent of cryoprotectants produced better motility during 5 and 10 min incubation. Sperm incubated with 15% cryoprotectant seemed to be toxic and this concentration was excluded in the subsequent trials. Three extenders, namely, Alsever’s solution, egg-yolk citrate and urea egg-yolk and three cryoprotectants, DMSO, methanol and ethanol were employed to preserve the sperm. Alsever’s solution with 10% DMSO showed best performance producing 90.0 ± 1.8% and 75.0 ± 2.5% equilibration and post-thaw motility followed by that of 82.5 ± 4.2% and 62.5 ± 5.5% with Alsever’s solution plus methanol, respectively. Between two diluents, sperm preserved with Alsever’s solution plus DMSO produced highest fertilization (76.7 ± 3.3%) and hatching (43.8 ± 7.9%) while fresh sperm yielded 83.3 ± 6.7% and 64.0 ± 10.4% fertilization and hatching, respectively. The protocol developed through the study can be applied for long-term conservation of genetic materials of the endangered fish N. nandus and the cryopreserved sperm can be used in artificial breeding for generating new individuals.     

Cryopreservation of kangaroo spermatozoa using alternative approaches that reduce cytotoxic exposure to glycerol

Alternative techniques for the cryopreservation of kangaroo spermatozoa that reduced or eliminated the need for glycerol were investigated including; (1) freezing spermatozoa with 20% glycerol in pre-packaged 0.25 mL Cassou straws to enable rapid dilution of the glycerol post-thaw, (2) investigating the efficacy of 20% (v/v) dimethyl sulphoxide (DMSO) and dimethylacetamide (DMA—10%, 15% and 20% v/v) as cryoprotectants and (3) vitrification of spermatozoa with or without cryoprotectant (20% v/v glycerol, 20% v/v DMSO and 20% v/v DMA). Immediate in-straw post-thaw dilution of 20% glycerol and cryopreservation of spermatozoa in 20% DMSO produced no significant improvement in post-thaw viability of kangaroo spermatozoa. Spermatozoa frozen in 20% DMA showed post-thaw motility and plasma membrane integrity of 12.7 ± 1.9% and 22.7 ± 5.4%, respectively, while kangaroo spermatozoa frozen by ultra-rapid freezing techniques showed no evidence of post-thaw viability. The use of 10–20% DMA represents a modest but significant improvement in the development of a sperm cryopreservation procedure for kangaroos.     

Minimally manipulated whole human umbilical cord is a rich source of clinical-grade human mesenchymal stromal cells expanded in human platelet lysate

Background aimsMesenchymal stromal cells (MSC) have recently been identified as a therapeutic option in several clinical conditions. Whereas bone marrow (BM) is considered the main source of MSC (BM-MSC), the invasive technique required for collection and the decline in allogeneic donations call for alternative sources. Human umbilical cord (UC) represents an easily available source of MSC (UC-MSC).MethodsSections of full-term UC were transferred to cell culture flasks and cultured in 5% human platelet lysate (PL)-enriched medium. Neither enzymatic digestion nor blood vessel removal was performed. After 2 weeks, the adherent cells were harvested (P1), replated at low density and expanded for two consecutive rounds (P2 and P3).ResultsWe isolated and expanded MSC from 9/9 UC. UC-MSC expanded with a mean fold increase (FI) of 42 735 ± 16 195 from P1 to P3 in a mean of 29 ± 2 days. By processing the entire cord unit, we theoretically could have reached a median of 9.5 × 10¹⁰ cells (ranging from 1.0 × 10¹⁰ to 29.0 × 10¹⁰). UC-MSC expressed standard surface markers; they contained more colony-forming unit (CFU)-fibroblast (F) and seemed less committed towards osteogenic, chondrogenic and adipogenic lineages than BM-MSC. They showed immunosuppressive properties both in vitro and in an in vivo chronic Graft versus Host disease (cGvHD) mouse model. Both array-Comparative Genomic Hybridization (CGH) analysis and karyotyping revealed no chromosome alterations at the end of the expansion. Animal studies revealed no tumorigenicity in vivo.ConclusionsUC constitute a convenient and very rich source of MSC for the production of third-party ‘clinical doses’ of cells under good manufacturing practice (GMP) conditions.     

Incorporation of DMSO and dextran-40 into a gelatin/alginate hydrogel for controlled assembled cell cryopreservation

A new cell cryopreservation strategy for cell-assembling constructs was proposed. With this strategy, different concentrations of dimethysulfoxide (DMSO) and dextran-40 were directly incorporated into the cell/gelatin/alginate systems, prototyped according to a predesigned structure, cryopreserved at −80 °C for 10 days and followed a thawing process at 17 °C. The rheological properties, bonding water contents and melting points of the gelatin/alginate hydrogel systems were changed with the addition of different amounts of DMSO. The microscopy analysis, (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrasodium bromide (MTT) and hematoxylin and eosin (HE) staining indicated that the cell numbers were progressively in a selected DMSO concentration range. With DMSO 5% (v/v) alone, the metabolic rate in the construct attained (81.3 ± 5.7)%. A synergistic effect was achieved with the combination of the DMSO/gelatin/alginate and dextran-40/gelatin/alginate hydrogel systems. These results indicated that the inclusion of DMSO and dextran-40 in the hydrogel could effectively enhance the cell preservation effects. This cryopreservation strategy holds the ability to be widely used in organ manufacturing techniques.     

Role of high-fat diet in regulation of gene expression of drug metabolizing enzymes and transporters

AimsLate phase ischemic preconditioning (LPC) protects the heart against ischemia–reperfusion (I/R) injury. However, its effect on myocardial tissue oxygenation and related mechanism(s) is unknown. The aim of the current study is to determine whether LPC attenuates post-ischemic myocardial tissue hyperoxygenation through preserving mitochondrial oxygen metabolism.Main methodsC57BL/6 mice were subjected to 30 min coronary ligation followed by 60 min or 24 h reperfusion with or without LPC (3 cycles of 5 min I/5 min R): Sham, LPC, I/R, and LPC + I/R group. Myocardial tissue Po₂ and redox status were measured with electron paramagnetic resonance (EPR) spectroscopy.Key findingsUpon reperfusion, tissue Po₂ rose significantly above the pre-ischemic level in the I/R mice (23.1 ± 2.2 vs. 12.6 ± 1.3 mm Hg, p < 0.01). This hyperoxygenation was attenuated by LPC in the LPC + I/R mice (11.9 ± 2.0 mm Hg, p < 0.01). Activities of NADH dehydrogenase (NADH-DH), succinate-cytochrome c reductase (SCR) and cytochrome c oxidase (CcO) were preserved or increased in the LPC group, significantly reduced in the I/R group, and conserved in the LPC + I/R group. Manganese superoxide dismutase (Mn-SOD) protein expression was increased by LPC in the LPC and LPC + I/R mice compared to that in the Sham control (1.24 ± 0.01 and 1.23 ± 0.01, p < 0.05). Tissue redox status was shifted to the oxidizing state with I/R (0.0268 ± 0.0016/min) and was corrected by LPC in the LPC + I/R mice (0.0379 ± 0.0023/min). Finally, LPC reduced the infarct size in the LPC + I/R mice (10.5 ± 0.4% vs. 33.3 ± 0.6%, p < 0.05).SignificanceThus, LPC preserved mitochondrial oxygen metabolism, attenuated post-ischemic myocardial tissue hyperoxygenation, and reduced I/R injury.     

Cryopreservation of sperm from seven-band grouper,Epinephelus septemfasciatus

In the present study, we examined methods for the cryopreservation of Epinephelus septemfasciatus spermatozoa. The percent motility, average path velocity, and linearity of movement (LIN) of fresh and corresponding post-thaw sperm were evaluated. Sperm motility was investigated using computer-assisted sperm analysis. Five percent dimethyl sulphoxide (Me₂SO) with 95% fetal bovine serum (FBS) was the most successful cryoprotectant diluent with a comparative post-thaw motility of 77.6 ± 8.5%; 5% dimethyl formamide was also effective. Fetal bovine serum was significantly better as an extender when compared with artificial seminal plasma, glucose, and trehalose solution. Sperm tolerated a wide range of cooling rates (from 27.1 to 94.3 °C min^?1); however, the post-thaw motility of sperm cooled to ?30 °C was significantly lower than that of other cooled temperatures (?40 to ?70 °C). The velocity of post-thaw sperm was significantly lower than that of fresh sperm, although LIN remained the same. For effective cryopreservation of seven-band grouper sperm, samples should be diluted in 5% Me₂SO with 95% FBS and cooled to at least ?40 °C before immersion in liquid nitrogen.     

Prevotella bivia as a source of lipopolysaccharide in the vagina

ObjectivesTo compare vaginal lipopolysaccharides (LPS) concentrations between patients with and without bacterial vaginosis (BV), to evaluate the correlation between Prevotella bivia colonization density and LPS concentration, and to determine the impact of LPS on loss of dopamine neurons (DA).MethodsVaginal washes obtained from patients with (n = 43) and without (n = 59) BV were tested for quantity of P. bivia cells using quantitative PCR and for concentrations of LPS using the Limulus Amebocyte Lysate gel clot method. Prevotella bivia, Gardnerella vaginalis and Escherichia coli sonicated cell extracts were also tested for LPS production. DA neuron cells obtained from embryonic day (E) 14.5 pregnant rats were exposed to fluid from eight vaginal washes; tyrosine hydrolase immunoreactive staining was applied for visualization and cell counts.ResultsThe median LPS concentrations were dramatically higher among patients who had symptoms of BV compared to those who did not have symptoms (3235.0 vs 46.4 EU/ml, respectively, P < 0.001); patients who had BV also had much higher colonization densities of P. bivia (0.06 ± 0.36 vs 5.4 ± 2.2 log₁₀ CFU/ml, respectively, P < 0.001).Prevotella bivia cell lysates resulted in a higher LPS concentration (10,713.0 ± 306.6 EU/ml) than either E. coli (4679.0 ± 585.3 EU/ml) or G. vaginalis (0.07 ± 0.01 EU/ml of LPS).The loss of DA neuron was 20–27% in cultures treated with vaginal washes from BV-negative patients and 58–97% in cultures treated with vaginal washes from patients with BV.ConclusionP. bivia produces high LPS concentration, which may create a toxic vaginal environment that damages DA neurons.     

Different roles of zinc plus arachidonic acid on insulin sensitivity between high fructose- and high fat-fed rats

AimsThis study was to determine the effects of zinc plus arachidonic acid (ZA) treatment on the insulin action in the specific ZA target organs using hyperinsulinemic euglycemic clamp method.Main methods18 Sprague–Dawley rats weighing ~ 130 g were divided into 3 groups of 6 rats and treated them with 1) normal rat chow, 2) high fructose (60.0%) diet only, or 3) the same fructose diet plus drinking water containing 10 mg zinc plus 50 mg arachidonic acid (AA)/L. In a separate study, male Wistar rats weighing ~ 250 g were fed normal rat chow (n = 4) or high fat (66.5%) diet with drinking water containing zero (n = 9) or 10 mg AA plus 20 mg zinc /L (n = 9). After 4 week treatment, insulin action was assessed using the hyperinsulinemic eguglycemic clamp technique.Key findingsHigh fructose feeding impaired suppression of hepatic glucose output by insulin compared to controls during the clamp procedure (4.39 vs. 2.35 mg/kg/min; p < 0.05). However, ZA treatment in high fructose-fed rats showed a significant improvement of hepatic insulin sensitivity compared to non-treatment controls (4.39 vs. 2.18 mg/kg/min; p < 0.05). Glucose infusion rates in Wistar rats maintained on a high fat diet (HFD) were significantly lower compared to control rats (22.8 ± 1.3 vs. 31.9 ± 1.4 mg/kg/min; p < 0.05). ZA treatment significantly improved (~ 43%) peripheral tissue insulin sensitivity in HFD fed animals (26.7 ± 1.3 [n = 9] vs. 22.8 ± 1.3 mg/kg/min; p < 0.05).SignificanceThese data demonstrate that ZA treatment is effective in improving glucose utilization in hyperglycemic rats receiving either a high-fructose or a high-fat diet.     

Amphipathic polymer-mediated uptake of trehalose for dimethyl sulfoxide-free human cell cryopreservation

For stem cell therapy to become a routine reality, one of the major challenges to overcome is their storage and transportation. Currently this is achieved by cryopreserving cells utilising the cryoprotectant dimethyl sulfoxide (Me₂SO). Me₂SO is toxic to cells, leads to loss of cell functionality, and can produce severe side effects in patients. Potentially, cells could be frozen using the cryoprotectant trehalose if it could be delivered into the cells at a sufficient concentration. The novel amphipathic membrane permeabilising agent PP-50 has previously been shown to enhance trehalose uptake by erythrocytes, resulting in increased cryosurvival. Here, this work was extended to the nucleated human cell line SAOS-2. Using the optimum PP-50 concentration and media osmolarity, cell viability post-thaw was 60 ± 2%. In addition, the number of metabolically active cells 24 h post-thaw, normalised to that before freezing, was found to be between 103 ± 4% and 91 ± 5%. This was found to be comparable to cells frozen using Me₂SO. Although reduced (by 22 ± 2%, p = 0.09), the doubling time was found not to be statistically different to the non-frozen control. This was in contrast to cells frozen using Me₂SO, where the doubling time was significantly reduced (by 41 ± 4%, p = 0.004). PP-50 mediated trehalose delivery into cells could represent an alternative cryopreservation protocol, suitable for research and therapeutic applications.     

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells

Background aimsHuman multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited.MethodsWe systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC).ResultsNine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10²² and 9.7 × 10²³, with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan.ConclusionsF-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.     

Involvement of src-kinase activation in ischemic preconditioning induced protection of mouse brain

AimsTo investigate the role of src-kinase in ischemic preconditioning induced reversal of ischemia and reperfusion induced cerebral injury in mice.Main methodsBilateral carotid artery occlusion of 17 min followed by reperfusion for 24 h was employed to produce ischemia and reperfusion induced cerebral injury in mice. Cerebral infarct size was measured using triphenyltetrazolium chloride staining using both by volume and by weight methods differently. Memory was evaluated using elevated plus maze test. Rota rod test was employed to assess motor incoordination.Key findingsBilateral carotid artery occlusion followed by reperfusion produced cerebral infarction and impaired memory and motor co-ordination. Three preceding episodes of bilateral carotid artery occlusion for 1 min and reperfusion of 1 min (ischemic preconditioning) prevented markedly ischemia–reperfusion-induced cerebral injury measured in terms of infarct size (38.5 ± 1.3% and 38.5 ± 2.9% mean infarct of control animals was reduced to 24.3 ± 1.2% and 23.5 ± 1.8% of the preconditioning groups respectively), loss of memory (72.2 ± 3.6 mean transfer latency time of control animals was reduced to 25.6 ± 5.2 of the preconditioning group respectively) and motor coordination (78.3 ± 17.6 s mean falling down latency time of control animals was increased to a mean value of 180.9 ± 6.5 s of the preconditioning groups respectively). SU6656 (2 mg/kg, ip) and PP1 (0.1 mg/kg, ip), highly selective src-kinase inhibitors, attenuated this neuroprotective effect of ischemic preconditioning.SignificanceTherefore, neuroprotective effect of ischemic preconditioning may be due to src-kinase linked mechanism.     

Cryopreservation of composite tissues and transplantation: Preliminary studies

Despite advances in cryobiology, the reliable cryopreservation of complex tissues has not yet been achieved. This study evaluates the viability of cryopreserved composite flaps and demonstrates the feasibility of their transplantation. Epigastric flaps were harvested from male Lewis rats. 1.5 M dimethyl sulfoxide (Me₂SO) was used as the initial cryoprotectant agent (CPA). Samples were frozen at controlled rate to −140 °C and transferred to liquid nitrogen for at least two weeks. Hematoxylin and eosin (H/E) staining, MTT tetrazolium salt assay, and factor VIII immunostaining were used to evaluate the overall histology, epithelial viability, and vascular endothelial integrity, respectively, of cryopreserved flaps. For the in vivo phase, flaps were isotransplanted to 35 recipient animals, divided into three groups: fresh (n = 10), perfused (n = 8), and cryopreserved (n = 17). Blood vessel patency was assessed via Doppler at 1, 7, and 60 days post-transplantation. For in vitro studies, cryopreserved samples (10/10) retained normal cell architecture and vascular endothelial integrity upon H/E and factor VIII staining. The viability index of cryopreserved composite flap skin (n = 10) was 11.17 ± 2.01, which was not significantly different from fresh controls (n = 10, 12.15 ± 1.32). All transplanted flaps in the fresh and perfusion groups survived with healthy color and hair growth at 60 days after operation. Survival in the cryopreserved group ranged from 2 to 60 days, with a mean of 12 days. These results demonstrate that the long term survival of cryopreserved composite tissue transplants is possible. Further studies are needed to refine protocols for the reliable cryopreservation of composite parts.     

Role of proinflammatory markers and NT-proBNP in patients with an implantable cardioverter-defibrillator and an electrical storm

BackgroundSeveral studies have attempted to identify risk factors for the development of an electrical storm (ES), which is defined as ⩾3 separate ventricular tachyarrhythmic (VT/VF) events, but in the majority of studies no triggers have been found. However, little is known about the role of inflammation and NT-proBNP in patients with ES. The aim of this study was therefore to assess the relationship of Interleukin-6 (IL-6), high-sensitivity C-reactive protein (hs-CRP) and NT-proBNP serum concentrations in ICD-patients with or without single spontaneous ventricular tachyarrhythmic events (VT/VF) and in ES.MethodsMarkers were determined in 51 patients without ICD-intervention, in 15 ICD-patients with single VT/VF-episodes during 9-months follow-up and in 20 ICD-patients with ES (blood sampling performed within 60 min after fulfilling ES criteria). VT/VF-episodes were analysed by stored ICD-electrograms.ResultsAll patients had idiopathic dilated cardiomyopathy (n = 23) or coronary artery disease (n = 63). Patients with ES revealed significantly higher mean serum concentrations of all markers (IL-6 15.19 ± 10.34 pg/mL, hs-CRP 20.12 ± 14.4 mg/L, NT-proBNP 4799 ± 4596 pg/mL) compared to baseline values of patients with single VT/VF-events during follow-up (IL-6 8.37 ± 5.8 pg/mL (p = 0.03), hs-CRP 4.7 ± 5.3 mg/dL (p < 0.001), NT-proBNP 1913 ± 2665 pg/mL (p = 0.04)) and compared to baseline values of ICD-patients without device intervention (IL-6 4.62 ± 3.66 pg/mL (p < 0.001), hs-CRP 4.1 ± 3.4 mg/L (p < 0.001), NT-proBNP 1461 ± 2281 pg/mL (p < 0.001)). In 9/20 patients presenting with ES (45%) baseline values were available. All markers were significantly higher during ES compared to event-free determination (IL-6 14.54 ± 10.43 vs. 7.03 ± 2.83 pg/mL (p = 0.04), hs-CRP 19.07 ± 16.07 vs. 6.5 ± 3.9 mg/L (p = 0.02), NT-proBNP 4218 ± 2561 vs. 2099 ± 1279 pg/mL (p = 0.03)).ConclusionsElectrical storm is associated with significantly elevated IL-6, hs-CRP and NT-proBNP serum concentrations in ICD-patients with structural heart disease. Thus, ES may be triggered by proinflammatory activity. Combined intraindividual elevation of determined markers might help to identify patients at risk of impending electrical storm.     

Propofol decreases the axonal excitability in rat primary sensory afferents

AimsThe aim of this present study was to investigate the changes of peripheral sensory nerve excitability produced by propofol.Main methodsIn a recently described in vitro model of rodent saphenous nerve we used the technique of threshold tracking (QTRAC®) to measure changes of axonal nerve excitability of Aβ-fibres caused by propofol. Concentrations of 10 μMol, 100 μMol and 1000 μMol were tested. Latency, peak response, strength-duration time constant (τSD) and recovery cycle of the sensory neuronal action potential (SNAP) were recorded.Key findingsOur results have shown that propofol decreases nerve excitability of rat primary sensory afferents in vitro. Latency increased with increasing concentrations (0 μMol: 0.96 ± 0.07 ms; 1000 μMol 1.10 ± 0.06 ms, P < 0.01). Also, propofol prolonged the relative refractory period (0 μMol: 1.79 ± 1.13 ms; 100 μMol: 2.53 ± 1.38 ms, P < 0.01), and reduced superexcitability (0 μMol: ? 14.0 ± 4.0%; 100 μMol: ? 9.5 ± 5.5%) and subexcitability (0 μMol: 7.5 ± 1.2%; 1000 μMol: 3.6 ± 1.2) significantly during the recovery cycle (P < 0.01).SignificanceOur results have shown that propofol decreases nerve excitability of primary sensory afferents. The technique of threshold tracking revealed that axonal voltage-gated ion channels are significantly affected by propofol and therefore might be at least partially responsible for earlier described analgesic effects.     

Mesenchymal stromal cells prolong the lifespan in a rat model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals’ lifespan.MethodsAt the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP⁺ MSC) on the same day, intraspinally (10⁵ cells) and intravenously (2 × 10⁶ cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis.ResultsThe combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP⁺ MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site.ConclusionsIntraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.     

In situ cryopreservation of human embryonic stem cells in gas-permeable membrane culture cassettes for high post-thaw yield and good manufacturing practice

Current strategies for marine pollution monitoring are based on the integration of chemical and biological techniques. The sea urchin embryo-larval bioassays are among the biological methods most widely used worldwide. Cryopreservation of early embryos of sea urchins could provide a useful tool to overcome one of the main limitations of such bioassays, the availability of high quality biological material all year round. The present study aimed to determine the suitability of several permeant (dimethyl sulfoxide, Me₂SO; propylene glycol, PG; and ethylene glycol, EG) and non-permeant (trehalose, TRE; polyvinylpyrrolidone, PVP) cryoprotectant agents (CPAs) and their combination, for the cryopreservation of eggs and embryos of the sea urchin Paracentrotus lividus. On the basis of the CPAs toxicity, PG and EG, in combination with PVP, seem to be most suitable for the cryopreservation of P. lividus eggs and embryos. Several freezing procedures were also assayed. The most successful freezing regime consisted on cooling from 4 to −12 °C at 1 °C/min, holding for 2 min for seeding, cooling to −20 °C at 0.5 °C/min, and then cooling to −35 °C at 1 °C/min. Maximum normal larvae percentages of 41.5% and 68.5%, and maximum larval growth values of 42.9% and 60.5%, were obtained for frozen fertilized eggs and frozen blastulae, respectively.     

Cryopreservation of human fetal liver hematopoietic stem/progenitor cells using sucrose as an additive to the cryoprotective medium

IntroductionHuman fetal liver (HFL) is a valuable source of hematopoietic stem/progenitor cells (HSCs) for the treatment of various hematological disorders. This study describes the effect of sucrose addition to a cryoprotective medium in order to reduce the Me₂SO concentration during cryopreservation of HFL hematopoietic cell preparations.MethodsHuman fetal liver (HFL) cells of 8–12 weeks of gestation were cryopreserved with a cooling rate of 1 °C/min down to −80 °C and stored in liquid nitrogen. The cryoprotectant solutions contained 2% or 5% Me₂SO (v/v) with or without sucrose at a final concentration of 0.05, 0.1, 0.2 or 0.3 M. The metabolic activity of HFL cells was determined using the alamar blue assay. For the determination of the number and survival of hematopoietic progenitors present, cells were stained with CD34 (FITC) and 7-AAD, and analyzed by flow cytometry. The colony-forming activity of HFL hematopoietic stem/progenitor cells after cryopreservation was assessed in semisolid methylcellulose.ResultsThe addition of sucrose to the cryoprotective medium produced a significant reduction in HFL cell loss during cryopreservation. The metabolic activity of HFL cells, cryopreserved with 5% Me₂SO/0.3 M sucrose mixture was comparable to cryopreservation in 5% Me₂SO/10% FCS. Although the inclusion of sucrose did not affect the survival of CD34⁺ cells in HFL after cryopreservation it did improve the functional capacity of hematopoietic stem/progenitor cells.ConclusionThe inclusion of sucrose as an additive to cryoprotective media for HFL cells enables a reduction in the concentration of Me₂SO, replacing serum and increasing the efficiency of cryopreservation.     

Single-platform quality control assay to quantify multipotential stromal cells in bone marrow aspirates prior to bulk manufacture or direct therapeutic use

Background aimsThe manufacture of multipotential stromal cell (MSC)-based products is costly; therefore, a rapid evaluation of bone marrow (BM) ‘quality’ with respect to MSC content is desirable. The aim of this study was to develop a rapid single-platform assay to quantify MSC in BM aspirates.MethodsAspirated MSC were enumerated using the CD45^?/low CD271^bright phenotype and AccuCheck counting beads and compared with a classic colony-forming unit–fibroblast (CFU-F) assay. The phenotype of CD45^?/low CD271^bright cells was defined using a range of MSC (CD73, CD105, CD90) and non-MSC (CD31, CD33, CD34, CD19) markers. The effect of aspirated BM volume on MSC yield was also determined.ResultsCD45^?/low CD271^bright cells had a classic MSC phenotype (CD73⁺ CD105⁺ CD90⁺ ). Their numbers correlated positively with CFU-F counted manually (R = 0.81, P < 0.001) or using automatic measurements of surface area occupied by colonies (R = 0.66, P < 0.001). Simultaneous enumeration of CD34 ⁺ cells revealed donor variability ranges compatible with standard International Society of Hematotherapy and Graft Engineering (ISHGE) protocols. Aspirating larger marrow volumes gave a significant several-fold reduction in the frequency of CFU-F and CD45^?/low CD271^bright cells per milliliter. Therefore aspirated MSC yields can be maximized through a standardized, low-volume harvesting technique.ConclusionsAbsolute quantification of CD45^?/low CD271^bright cells was found to be a reliable method of predicting CFU-F yields in BM aspirates. This rapid (< 40 min) procedure could be suitable for intra-operative quality control of BM aspirates prior to volume reduction/direct injection in orthopedics. In the production of culture-expanded MSC, this assay could be used to exclude samples containing low numbers of MSC, resulting in improved consistency and quality of manufactured MSC batches.     

Sperm cryopreservation in guppies and black mollies—A generalized freezing protocol for livebearers in Poeciliidae

In this study, we evaluated various parameters of sperm cryopreservation in two livebearers, guppies (Poecilia reticulata) and black mollies (P. latipinna). Our results suggested a common freezing protocol for the guppies and mollies: suspend sperm in Hanks’ balanced salt solution (HBSS) at 300 mOsm/kg, use 14% glycerol as cryoprotectant, cool at 25 °C/min, and thaw at 40 °C in a water bath for 7 s. Live young were produced from females inseminated with frozen-thawed sperm in both species. In guppies, percent fertilization (F) and the number of embryos (N) produced with cryopreserved sperm (F = 50%, N = 74, from 26 females) were similar to those of fresh controls (F = 54%, N = 61, from 22 females). Interestingly, this same freezing protocol has been used successfully for sperm cryopreservation in green swordtails Xiphophorus helleri, and platyfish of X. couchianus with post-thaw motility as high as 80%. All these species belong to the family of Poeciliidae, and their sperm are similar in morphology exhibiting the absence of acrosome, elongate sperm head, and the long mitochondrial sheaths. Besides their internal fertilization reproduction mode, these fish are also small in size (2–4 cm) and live in a freshwater environment. Sperm cryopreservation in fish has been generally recognized as species specific, and new protocols are required for new species. However, results presented in this study suggested otherwise. Thus, sperm cryopreservation methods optimized for one species may be applicable to others if they are taxonomical closely related species with similar sperm morphology and reproduction mode. Considering the enormous number of fish species on the planet, development of generalized sperm freezing protocols for species in groups could have additional advantages for genetic conservation.     

Low serum zinc levels in patients undergoing coronary angiography correlate with immune activation and inflammation

IntroductionLow serum zinc concentrations are associated with adverse outcomes. To explain this phenomenon we aimed to investigate whether low zinc levels are related to immune activation, renal function and coronary artery disease (CAD).MethodsSerum concentrations of zinc and the immune activation markers neopterin and C-reactive protein (CRP) were measured in 2048 patients derived from the LUdwigshafen RIsk and Cardiovascular Health (LURIC) study, a cohort study among patients referred for coronary angiography.ResultsZinc concentrations did not differ between patients with CAD (mean ± SD: 13.3 ± 2.4 μmol/L) and controls (13.3 ± 2.2 μmol/L; Welch's t test: p = n.s.) but CAD patients had higher neopterin (8.6 ± 7.4 nmol/L) and CRP (9.7 ± 19.6 mg/L) concentrations compared to controls (neopterin: 7.5 ± 4.8 nmol/L, p = 0.0005; CRP: 5.5 ± 10.0 mg/L, p < 0.0001). There was an inverse correlation between serum zinc concentrations and neopterin (Spearman's rank correlation: r_s = ?0.222) and CRP (r_s = ?0.166; both p < 0.0001) concentrations.ConclusionsOur results indicate increased inflammatory processes in patients with low zinc levels. Further studies should clarify whether inflammation related processes such as renal wasting contribute to zinc deficiency and underlie the adverse health consequences of low serum zinc levels.