An in vitro study of cryopreserved and fresh human arteries: a comparison with ePTFE prostheses and human arteries studied non-invasively in vivo

The surgical options in arterial reconstruction are: the use of autologous arteries; autologous veins; or expanded polytetrafluoroethylene (ePTFE) grafts. However, the development of intimal hyperplasia when using veins or ePTFE grafts has been associated with graft failure. Since autologous arteries are not always available, the use of cryopreserved arteries has to be considered. The aims of this study were: (a) to compare the viscoelastic properties of stored cryopreserved arteries and fresh arteries by in vitro analysis; and (b) to compare the viscoelastic properties of arteries measured non-invasively in normotensive patients, with fresh arteries, cryopreserved arteries, and ePTFE segments. The viscoelastic studies were performed in normotensive patients using stress-strain analysis with non-invasive measurement of pressure and diameter in the common carotid artery, and in vitro measurements of pressure and diameter in arteries and prostheses. The in vitro studies showed that the elastic modulus (E), viscous modulus (eta), Stiffness Index (SI), Peterson modulus (Ep), and the pulse wave velocity (PWV) values for human cryopreserved carotid arteries were similar to the values obtained non-invasively in normotensive subjects (P>0.05) and to human fresh vessels (P>0.05). In vitro, the SI, Ep, PWV, and E values of ePTFE were significantly higher than the observed values in subjects and with fresh and cryopreserved arteries (P<0.05); on the other hand the ePTFE eta values were the lowest (P<0.05). We concluded that cryopreserved arteries have similar viscoelastic properties to those obtained in vivo in the arteries of normotensive subjects and in vitro in fresh arteries. Consequently, we conclude that the cryopreservation procedure does not modify the mechanical properties of the arterial wall.     

Functional properties of fresh and cryopreserved carotid and femoral arteries,and of venous and synthetic grafts: comparison with arteries from normotensive and hypertensive patients

The ideal arterial graft must share identical functional properties with the host artery. Surgical reconstruction of the common carotid artery (CA) is performed in several clinical situations, using expanded polytetrafluoroethylene prosthesis (ePTFE) or saphenous vein (SV) grafts. At date there is interest in obtaining an arterial graft that improves the results of that nowadays available. The use of a fresh or cryopreserved/defrosted artery appears as an interesting alternative. However, if the fresh and cryopreserved/defrosted arteries allow an adequate viscoelastic and functional matching with the host arteries needs to be established. The aims were to compare the viscoelastic and functional performance of: (1) conduits used in CA reconstruction (SV and ePTFE) with those of the fresh and cryopreserved/defrosted CA and femoral arteries (FA), and (2) normotensive and hypertensive patients’ arteries with those of the arterial substitutes in vitro analyzed. Pressure, diameter and wall thickness of the CA were recorded in 15 normotensive and 15 hypertensive patients (in vivo studies), and in SV, fresh and cryopreserved/defrosted CA and FA (obtained from 15 donors), and ePTFE segments (in vitro studies). From stress–strain relationship we calculated elastic and viscous modulus, and the characteristic impedance. The local buffer and conduit functions were quantified as the viscous/elastic quotient and the inverse of the characteristic impedance. Fresh and cryopreserved/defrosted CA and FA were more alike, both in viscoelastic and functional levels, respect to normotensive and hypertensive patients’ arteries, than the ePTFE and SV grafts. CA and FA cryografts could be considered an important alternative for carotid reconstruction.     

Cryopreservation of human ovarian tissue: Comparison of novel direct cover vitrification and conventional vitrification

Objective

To compare the effect of novel direct cover vitrification (DCV) and conventional vitrification (CV) for human ovarian tissue.

Study design

Ovarian biopsy specimens obtained from 12 patients were randomly allocated into five groups: Fresh, DCV1, DCV2, DCV3 and CV. Three concentrations of cryoprotectants were used in DCV group. The equilibration solution of DCV1, DCV2, DCV3 was 5% EG + 5% DMSO + DPBS, 7.5% EG + 7.5%DMSO + DPBS, 10% EG + 10% DMSO + DPBS, respectively. And the vitrification solution of DCV1, DCV2, DCV3 was 10% EG + 10% DMSO + DPBS, 15%EG+15% DMSO + DPBS, 20% EG + 20% DMSO + DPBS, respectively. The equilibration solution and the vitrification solution of CV group was same as DCV3 group. The effects of cryopreserved procedure on human ovarian tissue were studied by histology, TUNEL assay, transmission electron microscopy (TEM) and heterotopic allograft.

Results

The percentages of morphologically normal and viable follicles of DCV2 were significantly higher than DCV1, DCV3 and CV groups (P < 0.05). TUNEL assay demonstrated that the incidence of apoptotic cell in vitrification ovarian tissue was significantly higher than fresh tissue (P < 0.05), but there were no difference in various groups with cryopreservation. TEM showed that less damage was detected in DCV2 group. After grafting, the follicle density of DCV2 was greater than DCV1, DCV3 and CV groups (P < 0.05).

Conclusions

The novel cover vitrification with optimal concentration of cryoprotectants is superior to conventional vitrification. It is suitable for human ovarian tissue fragments with high efficiency and facility.     

Cryopreservation of an artificial human oral mucosa stroma. A viability and rheological study

The aim of this study was to evaluate the viability and biomechanical properties of artificial human oral mucosa stroma (HOMS) subjected to cryopreservation with different cryoprotectant solutions.     

Cryopreservation of isolated human hepatocytes for transplantation: State of the art

Hepatocytes isolated from unused donor livers are being used for transplantation in patients with acute liver failure and liver-based metabolic defects. As large numbers of hepatocytes can be prepared from a single liver and hepatocytes need to be available for emergency and repeated treatment of patients it is essential to be able to cryopreserve and store cells with good thawed cell function. This review considers the current status of cryopreservation of human hepatocytes discussing the different stages involved in the process. These include pre-treatment of cells, freezing solution, cryoprotectants and freezing and thawing protocols. There are detrimental effects of cryopreservation on hepatocyte structure and metabolic function, including cell attachment, which is important to the engraftment of transplanted cells in the liver. Cryopreserved human hepatocytes have been successfully used in clinical transplantation, with evidence of replacement of missing function. Further optimisation of hepatocyte cryopreservation protocols is important for their use in hepatocyte transplantation.     

Morphological and biochemical analysis of human cardiac valve allografts after an increment of the cryostorage temperature

Cryopreserved human cardiac valve allografts could suffer lethal damages if the temperature is elevated during cryostorage. This work describes the functional and morphological alterations suffered by human cardiac valve allografts after a gradual increment of the cryostorage temperature from −147 °C to −47 °C due to a technical failure. Three experimental groups of human pulmonary and aortic allografts were compared: fresh, cryopreserved (−147 °C) and cryopreserved with temperature changes from −147 °C up to −47 °C and back to −147 °C. Fibroblast functionality was studied to asses the degree of valvular damages. Collagen network was also analyzed with bright light field and polarized microscopy; an immunohistochemistry for procollagen I was performed and the MTT colorimetric assay was used to evaluate fibroblast mitochondrial enzymatic activity. Porcine heart grafts valves were used to set the MTT colorimetric assay.With bright light field microscopy, disorganized collagen network was seen together with interstitial edema in cryopreserved groups. Polarized microscopy showed that fresh allografts had abundant collagen type I and III, cryopreserved group had less amount of collagen type I and in allografts that suffered cryopreservation temperature elevation collagen type I synthesis could not been demonstrated. Procollagen I was present in fibroblast cytoplasm of fresh group, but it was diminished in cryopreserved group and was absent in the group that suffered temperature elevation.Temperature changes during the cryopreservation period of human cardiac valve allografts induced fibroblast activity reduction. When the cryopreservation temperature is elevated during cryostorage, fibroblasts lost their functionality and the allografts may be not suitable for transplant.     

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22–25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 °C for 36 h. Small pieces of ovarian tissue (1 × 1–1.5 × 0.7–1 mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-β and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-β concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.     

Freezing without surrounding cryomedium preserves the endothelium and its function in human internal mammary arteries

PURPOSE: Cryopreserved human blood vessels may become important tools in bypass surgery. Optimal cryopreservation of an arterial graft should, therefore, preserve both histological and physiological characteristics of smooth muscle and endothelium comparable to the unfrozen artery. METHODS: Rings from human internal mammary arteries (IMA) were investigated in vitro either unfrozen or after immersion into a cryomedium (RPMI 1640 containing 1.8M Me2SO and 0.1M sucrose) and cryostorage with and without surrounding medium. RESULTS: In unfrozen IMA, neither contractile responses to noradrenaline (NA) nor endothelium-dependent relaxant responses to acetylcholine (ACH) was modified after exposure of the IMA to cryomedium or during activation of protein kinase C by phorbol-12,13-dibutyrate (PDBu). Exposure to cryomedium with gradually increasing Me2SO content before starting the cooling process did not improve the post-thaw functional activity of the artery. Optimal post-thaw recovery of contractile responses to NA and PGF(2alpha) was observed after freezing at a speed of -1.2 and -3 degrees C/min in arteries stored with and without surrounding cryomedium. Compared to unfrozen controls, the ACH-induced endothelium-dependent relaxation during active tone induced by 3 microM PGF(2alpha) reached 16 and 56% after freezing with and without surrounding medium. All functional data were reflected by electron microscopy images showing considerably better preservation of the endothelial layer after freezing without medium. CONCLUSION: Freezing of human arteries at a mean cooling rate of -3 degrees C/min and storage without surrounding medium offers the prospect of optimal preservation of both smooth muscle and endothelial function in cryopreserved human IMA.     

Cryopreservation of turkey semen by the pellet method: effects of variables such as the extender, cryoprotectant concentration, cooling time and warming temperature on sperm quality determined through principal components analysis

This study was designed to identify the best pellet cryopreservation procedure for the cryosurvival of turkey semen among 192 different treatments established by variations and permutations of seven conditions used in the freezing/thawing process. These conditions were: diluent (IGGKPh, SPh or Tselutin); dilution rate (1:3 vs. 1:4); cooling time (45 vs. 60 min); dimethylacetamide (DMA) concentration as cryoprotectant (6 vs. 8%); equilibration time in DMA (1 vs. 5 min); semen drop volume (50 vs. 80 μL) and thawing temperature (60 vs. 75 °C). Through principal components analysis (PCA), post-thaw sperm quality data (mobility, viability and membrane functional integrity) were reduced to a single output variable (Sperm Quality) indicating overall post-thaw semen quality. All treatments induced a significant reduction in semen quality after warming (P < 0.01), though one set of seven conditions, or treatment, was identified by PCA to generate the highest Sperm Quality score and a further five treatments yielded a score not significantly different (P > 0.05) from this best score. Although still not fulfilling the requirements for commercial application, our findings serve to identify the critical steps in turkey sperm cryopreservation that need to be assessed in future studies.     

The human genome project: Towards an analysis of the empirical,ethical, and conceptual issues involved

In this paper I claim that the goal of mapping and sequencing the human genome is not wholly new, but rather is an extension of an older project to map genes, a central aim of genetics since its birth. Thus, the discussion about the value of the HGP should not be posed in global terms of acceptance or rejection, but in terms of how it should be developed. The first section of this paper presents a brief history of the project. The second section distinguishes among four kinds of issues relevant to an evaluation of the HGP: those economic and organizational issues related to the feasibility of the project; the ethical questions arising in the development of the project and the application of the data gathered; the empirical issues relevant to the scientific value of the project; and conceptual issues like reductionism and determinism relevant to understand the nature and scope of the project. In a third section, I analyze in detail whether the HGP and, more generally, molecular biology is reductionistic.     

Interrelationships among human aldo-keto reductase: immunochemical, kinetic and structural properties

We have propsed earlier a three gene loci model to explain the expression of the aldo-keto reductases in human tissues. According to this model, aldose reductase is a monomer of α subunits, aldehyde reductase I is a dimer of α, β subunits, and aldehyde reductase II is a monomer of δ subunits. Using immunoaffinity methods, we have isolated the subunits of aldehyde reductase I (α and β) and characterized them by immunocompetition studies. It is observed that the two subunits of aldehyde reductase I are weakly held together in the holoenzyme and can be dissociated under high ionic conditions. Aldose reductase (α subunits) was generated from human placenta and liver aldehyde reductase I by ammonium sulfate (80% saturation). The kinetic, structural and immunological properties of the generated aldose reductase are similar to the aldose reductase obtained from the human erythrocytes and bovine lens. The main characteristic of the generated enzyme is the requirement of Li₂SO₄(0.4 M) for the expression of maximum enzyme activity, and its K_m for glucose is less than 50 mM, whereas the parent enzyme, aldehyde reductase I, is completely inhibited by 0.4 M Li₂SO₄ and its K_m for glucose is more than 200 mM. The β subunits of aldehyde reductase I did not have enzyme activity but cross-reacted with anti-aldehyde reductase I antiserum. The β subunits hybridized with the α subunits of placenta aldehyde I, and aldose reductase purified from human brain and bovine lens. The hybridized enzyme had the characteristics properties of placenta aldehyde reductase I.     

Rotational and translational motions of human spermatozoa: angle dependence of dynamic laser light scattering

We have studied how the dynamic components of laser light scattered from human spermatozoa depend on the scattering angle. This was done by investigating the halfwidth of the intensity autocorrelation function. A model of the spermatozoa as freely rotating and translating linear objects was adequate to describe the scattered light. Rotational motions determined the halfwidth of the intensity autocorrelation function at very small scattering angles and contribution from translational motions was dominant at scattering angles larger than 20 degrees. The contribution from translational motions increased with increasing scattering angle. We found a nearly linear relationship between the translation speed and the rotation frequency. However, the ratio between the two properties varied more than expected from the methodological error. Therefore we introduced a propelling efficacy as a concept to describe the swimming efficiency. This property might contain important information about the swim characteristics.Abbreviations ACF Autocorrelation function - _1/2 halfwidth - RGD Rayleigh-Gans-Debye - SD Standard deviation Correspondence to: P. Thyberg     

Vitrification of carnation in vitro: Changes in cell wall mechanical properties,cellulose and lignin content

Vitrification of internodes of carnation was brought about by culturing in liquid medium. Cell wall extensibility of these internodes was kinetically followed in comparison to that of normal plants using the constant stress method. Liquid culture induced increased immediate and total deformation capacities of the walls from the second day. Measurements indicated that these deformation capacities involved plastic properties rather than elastic ones. These changes were paralleled by decreased relative levels of cellulose and lignin.     

Expansion of human hematopoietic stem cells for transplantation: trends and perspectives

Umbilical cord blood transplantation is clinically limited by its low progenitor cell content. Ex vivo expansion has become an alternative to increase the cell dose available for transplants. Expansion has been evaluated in several ways such as static cultures combining growth factors or mimicking the natural microenvironment using co-culture systems. However, static cultures have a small volume capacity and therefore large-scale expansion has been addressed using bioreactors. These and other biotechnological approaches for the expansion of hematopoietic progenitors and their utility to study several aspects of hematopoietic stem cell biology are discussed here.     

Proteomic analysis of recurrent spontaneous abortion: Identification of an inadequately expressed set of proteins in human follicular fluid

Recurrent spontaneous abortion (RSA), defined as the loss of three or more consecutive pregnancies prior to the 20th week of gestation, affects up to 5% of the child-bearing population. To investigate the proteins associated with RSA, the protein expression in human follicular fluid was analyzed using 2-DE. Follicular fluid contains a variety of biologically important proteins for oocyte fertilization and follicle maturation in the mammalian reproductive process. Therefore, it can be used as a provisional source for identifying proteins involved in RSA. In this study, we identified five aberrantly expressed proteins (complement component C3c chain E, fibrinogen gamma, antithrombin, angiotensinogen, and hemopexin precursor) in follicular fluid from RSA patients with MALDI-TOF-MS and nano-LC MS/MS. Western blot analysis confirmed that the protein expression level of fibrinogen gamma and antithrombin was less in follicular fluid from RSA patients than those from normal controls. Semiquantitative RT-PCR and real-time PCR analyses revealed that mRNA level of these coagulation factors was also decreased significantly in chorionic villi of RSA patients compared with normal samples. Taken all together, it is likely that coagulation factors (fibrinogen gamma and antithrombin) play an important role in maintaining the normal pregnancy.     

Conformational flexibility in calcitonin: The dynamic properties of human and salmon calcitonin in solution

We have studied the dynamic properties of human (h) and salmon (s) calcitonin (CT) in solution. For both hormones, distance geometry in torsion-angle space has been used to generate three-dimensional structures consistent with NMR data obtained in sodium dodecyl sulfate micelles. For sCT and hCT we used, respectively, 356 and 275 interproton distances together with hydrogen-bonds as restraints. To better characterize their flexibility and dynamic properties two fully unrestrained 1100-ps molecular dynamics (MD) simulations in methanol were performed on the lowest-energy structures of both hormones. Statistical analyses of average geometric parameters and of their fluctuations performed in the last 1000 ps of the MD run show typical helical values for residues 9–19 of sCT during the whole trajectory. For hCT a shorter helix was observed involving residues 13–21, with a constant helical region in the range 13–19. Angular order parameters S() and S() indicate that hCT exhibits a higher flexibility, distributed along the whole chain, including the helix, while the only flexible amino acid residues in sCT connect three well-defined domains. Finally, our study shows that simulated annealing in torsion-angle space can efficiently be extended to NMR-based three-dimensional structure calculations of helical polypeptides. Furthermore, provided that a sufficient number of NMR restraints describes the system, the method allows the detection of equilibria in solution. This identification occurs through the generation of 'spurious' high-energy structures, which, for right-handed -helices, are likely to be represented by left-handed -helices.     

Enkephalin dipeptidyl carboxypeptidase (enkephalinase) activity: selective radioassay, properties, and regional distribution in human brain

Abstract: The compound [³H-Tyr¹,D-Ala²,Lcu-OH⁵]enkephalin has been synthesised as a potentially selective substrate for enkephalin dipeptidyl carboxypcptidase ( enkephalinase ) activity in brain. lncubations in the presence of homogenates and particulate fractions from rodent and human brain result in the formation of [³H]Tyr-D-Ala-Gly, which can be conveniently isolated by polystyrene bead column chromatography. The enzyme activity responsible for the hydrolysis of the Gly³-Phe⁴ amide bond of this substrate displays close resemblance to that hydrolysing the natural enkephalins at the same level. In addition, enkephalinase activity characterised in postmortem human brain is closely similar to that in rodent brain, with regard to optimal pH and apparent affinities of various substrates and inhibitors, including the potent compound thiorphan. Enkephalinase activity is distributed in a highly heterogeneous fashion among regions of human brain, the highest levels being found in globus pallidus and pars reticulata of the substantia nigra. This distribution is poorly correlated with that of opiate receptor binding sites but displays some resemblance to that of reported Met5-enkephalin levels.     

Sphingolipids in human lens membranes: an update on their composition and possible biological implications

The unique nature of the most abundant phospholipids in human lens membranes remained overlooked until the 1990s when it was possible to discern dihydrosphingomyelins (DHSMs) from the more common sphingomyelins (SMs). Unlike in other mammalian membranes, DHSMs comprise nearly half of the phospholipids in adult human lenses. Compared to SMs with a trans double bond between carbons 4 and 5 of the sphingoid backbone, the absence of this unsaturation site in DHSMs allows the participation of the OH group on C3 in intermolecular H-bonds and leads to stronger interlipid interactions with both neighboring DHSMs and cholesterol. Phospholipid compositional changes with age and lens region observed in mammals with various life spans and lens growth rates, suggest that the highest levels of DHSMs along with the lowest amounts of phosphatidylcholines and SMs are found in lenses with the lowest growth rate, namely human lenses. The participation of phospholipid metabolites in the control of mitosis and elongation of lens cells is plausible and deserves investigation.     

Biodegradable biocomposite films based on whey protein and zein: barrier, mechanical properties and AFM analysis

Whey and zein protein are byproducts of the food industries and have good film making properties. Single and laminated films were produced from zein protein and whey protein and their film properties were studied. Glycerol and olive oil were used as plasticizer for the single and laminated films. The laminated films exhibited higher ultimate tensile strength (UTS) than the single whey protein films (260% and 200% in the whey-zein-glycerol and whey-zein-olive oil films, respectively). The UTS of the whey protein films increased 2-3-fold after lamination. The laminated films showed higher barrier properties than the single whey protein films (180% in the whey protein-zein-glycerol films and 200% in the whey protein-zein-olive oil films in comparison to single whey protein films) and lower than the single zein films.     

Comparative analysis of structural and dynamic properties of the loaded and unloaded hemophore HasA: functional implications

A heme-acquisition system present in several Gram-negative bacteria requires the secretion of hemophores. These extracellular carrier proteins capture heme and deliver it to specific outer membrane receptors. The Serratia marcescens HasA hemophore is a monodomain protein that binds heme with a very high affinity. Its α/β structure, as that of its binding pocket, has no common features with other iron- or heme-binding proteins. Heme is held by two loops L1 and L2 and coordinated to iron by an unusual ligand pair, H32/Y75. Two independent regions of the hemophore β-sheet are involved in HasA-HasR receptor interaction. Here, we report the 3-D NMR structure of apoHasA and the backbone dynamics of both loaded and unloaded hemophore. While the overall structure of HasA is very similar in the apo and holo forms, the hemophore presents a transition from an open to a closed form upon ligand binding, through a large movement, of up to 30 Å, of loop L1 bearing H32. Comparison of loaded and unloaded HasA dynamics on different time scales reveals striking flexibility changes in the binding pocket. We propose a mechanism by which these structural and dynamic features provide the dual function of heme binding and release to the HasR receptor.