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At least 523 records · Page 29Linked to original sources
PubMed2004

A novel use of centrifugal force for cell seeding into porous scaffolds.

A novel rotor was constructed to allow for the seeding of porous scaffolds via centrifugal force. Using cell seeding times of 10 min, this method placed significantly (roughly 3-fold) more cells into poly(glycolic acid) scaffolds than 24 h of spinner flask seeding or static seeding. There were no significant differences in the mitochondrial activity per cell between the 3 seeding methods. Cell distribution was noted to be homogeneous throughout the scaffold thickness for the centrifugation method, as opposed to surface seeding for the spinner flask method. Centrifugation was especially efficient at low cell concentrations (1.33 x 10(5) cells/ml). This system is useful for the seeding of biomaterials having cylindrical or planar geometries, and may be used under conditions that require low cell numbers and/or short seeding time periods.

Bioreactors↗
PubMed2006

Evaluation of single and double centrifugation tube methods for concentrating equine platelets.

The aim of this study was to evaluate single and double centrifugation tube methods for concentrating equine platelets. Whole blood samples were collected from clinically normal horses and processed by use of single and double centrifugation tube methods to obtain four platelet concentrates (PCs): PC-A, PC-B, PC-C, and PC-D, which were analyzed using a flow cytometry hematology system for hemogram and additional platelet parameters (mean platelet volume, platelet distribution width, mean platelet component concentration, mean platelet component distribution width). Concentrations of transforming growth factor beta 1 (TGF-beta(1)) were determined in all the samples. Platelet concentrations for PC-A, PC-B, PC-C, and PC-D were 45%, 44%, 71%, and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF-beta(1) concentrations for PC-A, PC-B, PC-C, and PC-D were 38%, 44%, 44%, and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and for obtaining potentially therapeutic TGF-beta(1) levels.

Animals↗
PubMed2006

Increases in fecal coliform bacteria resulting from centrifugal dewatering of digested biosolids.

In many countries, the classification of biosolids for disposal purposes can be based, in part, on fecal coliform levels, with alternative criteria also available based on the stabilization process used, such as anaerobic digestion. The assumption that these alternative criteria provide equivalent protection may be flawed. This paper demonstrates that fecal coliform levels determined after digestion do not always indicate the bacterial levels after the same biosolids have been dewatered by centrifugation. In samples from mesophilic digestion, half had significant increases in coliform numbers (P<0.05) with up to one order of magnitude increase during centrifugation, suggesting coliform regrowth. Thermophilically digested samples had significant increases of several orders of magnitude during dewatering, more likely from reactivation of viable but non-culturable coliforms than from regrowth. In other cases, centrifugation induced coliform regrowth or reactivation upon incubation and storage of dewatered samples, but not digested samples. These 2-3 order of magnitude increases occurred with both 25 and 37 degrees C incubations. Coliform increases continued for up to 5 days, then gradually declined. However, by day 20 coliform numbers were still 2 orders of magnitude greater than when originally sampled. The magnitude of the increases could be due either to regrowth or reactivation, but the nature of the longer-term increases--also seen in biosolids/soil mixtures--suggests regrowth. Differences in numbers between digested and dewatered samples could not be duplicated with high shear processing in lab-scale devices, with nitrogen purging to remove volatile or gaseous constituents, or with redilution using centrate. They could not be attributed to enumeration methods, to interference of Bacillus spp. on apparent coliform counts, or to temperature changes. The increases have practical implications in the use of fecal coliform or alternative criteria to define pathogen content in biosolids.

Centrifugation↗
PubMed1997

Centrifugation does not alter spatial distribution of 'BEP4' mRNA in paracentrotus lividus EGG.

Paracentrotus lividus unfertilized eggs were centrifuged in a sucrose gradient, so to split each into two parts: a nucleated light fragment and an anucleated heavy fragment. Northern blot analyses utilizing a bep4 probe as animal marker and H2A histone gene and 12S-mit RNA as controls indicate that the eggs are elongated along the animal-vegetal axis during centrifugation and thereafter split into an animal and a vegetal half. Treatment of the eggs with colchicine before centrifugation abolishes the animal localization of bep4 mRNA.

Animals↗
PubMed1990

Enhanced penetration of zona-free hamster ova by sperm prepared by Nycodenz and Percoll gradient centrifugation.

The effect of sperm penetration capacity after selection procedures using Percoll (Pharmacia AB, Uppsala, Sweden) and Nycodenz (Nycomed Diagnostics, Oslo, Norway) gradient centrifugation was compared with double-washed and swim-up in 47 subfertile men. The results of sperm motility, velocity, and amplitude lateral head displacement showed no significant improvement with the centrifugation procedures. The sperm penetration assay results obtained with double-washed and swim-up technique were poor (2.7% +/- 1.7%), however, a significant enhancement was obtained by Percoll (16.3% +/- 3.7%) and Nycodenz (15.8% +/- 3.3%) processing. Nycodenz centrifugation allowed sperm penetration of zona-free hamster ova at comparable rates to Percoll separation.

Adult↗
PubMed1989

Human sperm selection by glass wool filtration and two-layer, discontinuous Percoll gradient centrifugation.

Glass wool filtration and two-layer, discontinuous Percoll (Pharmacia, Uppsala, Sweden) density gradient centrifugation resulted in an average recovery of 50% to 70% of the progressively motile and about 50% of the hypoosmotic swelling (HOS)-positive spermatozoa. Glass wool filtration tended to be more successful than Percoll centrifugation when the ejaculates were asthenozoospermic or produced a suspect/abnormal HOS test. After selection, the acrosin activity increased approximately two- to threefold, but no significant improvement in the percentage of normal sperm forms occurred. Experiments with mixtures of untreated and frozen-thawed ejaculates confirmed that glass wool filtration is more effective in removing nonmotile and HOS-negative spermatozoa than the two-layer Percoll centrifugation technique when the percentage of these types of spermatozoa in the ejaculate is high. The simplicity of these techniques and the good recovery of apparently viable spermatozoa makes these methods more desirable than other, more complicated techniques or procedures that yield a lower recovery of motile spermatozoa.

Cell Separation↗
PubMed2003

Centrifugation facilitates transduction of green fluorescent protein in human monocytes and macrophages by adenovirus at low multiplicity of infection.

Due to their phagocytic and poorly proliferative nature, it has been difficult to transfect human monocytes and macrophages. Adenoviral vectors have recently allowed transduction of a high percentage of human macrophages, but only after CSF upregulation of the integrins, alphavbeta3 or alphavbeta5, during culture for 48 h, a time allowing significant monocyte to macrophage differentiation. In our hands, after 24-h incubation with M-CSF (20 ng/ml) and a further 24-h incubation with an adenoviral vector encoding green fluorescent protein (AdV-GFP) [multiplicity of infection (MOI)=50:1], only 35% of CD14-positive cells express GFP. We report that centrifugation of these cells with AdV-GFP at 2000 x g for 1 h at 37 degrees C significantly enhanced the number of cells expressing GFP (to 65%) and the level of GFP expression per transduced cell (fivefold). The viability of the cells was not compromised (<5 % CD14-positive cells were 7-aminoactinomycin D (7AAD)-positive after 24 h AdV-GFP exposure at MOI=50:1). Centrifugation allowed efficient transduction of monocytes and macrophages with an MOI at least tenfold lower than otherwise required and did not activate the transduced cells or affect their ability to produce TNFalpha or IL-1beta in response to lipopolysaccharide (LPS). This methodology was also suitable for transducing large numbers of in vitro monocyte-derived macrophages (MDMac) and macrophages isolated from synovial fluids with up to 75-80% of CD14-positive cells transduced after 24-h exposure to AdV-GFP (50:1) and centrifugation (2000 x g). This methodology should provide significant expression of transgenes in human monocytes and macrophages.

Adenoviridae↗
PubMed2002

Piglets born from centrifuged and vitrified early and peri-hatching blastocysts.

Cryopreservation of zona-intact porcine embryos has been relatively unsuccessful to date, although some success has been obtained with lipid reduced morulae and early blastocysts. This study adapted some vitrification protocols used successfully with late blastocysts for use with early zona-intact blastocysts, using actin depolymerization, centrifugation, and open-pulled (OPS) straws. Initially, Day 6 peri-hatching blastocysts were collected, cultured for 40 min in 7.5 microg/ml cytochalasin B and vitrified in 6.5 M glycerol and 6% BSA (VS1) in either heat-sealed (HS) or open straws (OS). The post-thaw survival of those stored in HS was 15.4% after 24 and 48 h in vitro; storage in OS significantly improved survival (58.8% for both 24 and 48 h). When similar stage blastocysts were cultured in cytochalasin B and vitrified with 8 M ethylene glycol and 7% polyvinylpyrrolidone (PVP; VS2) in OS, survival was 44.4 and 33.3% for 24 and 48 h, respectively. Day 5 late morulae and early blastocysts were collected, cultured with cytochalasin B, and centrifuged or left intact (control), then vitrified with VS1 in HS or OS, or vitrified in VS2 in OS only. None of the intact control embryos survived thawing and 48 h culture in vitro. Centrifuged early blastocysts vitrified with VS1 showed good post-thaw survival in culture when stored in HS (62.8 and 60.5% for 24 and 48 h, respectively), or OS (75 and 63.6%). When vitrified with VS2 in OS, survival improved (80 and 76.7%). Peri-hatching blastocysts were vitrified in VS1, and early blastocysts were vitrified with VS1 and VS2. All blastocysts were stored in OS. The embryos were recovered and transferred to Day 4 and 5 pseudopregnant recipients (for Day 5 and 6 blastocysts, respectively). Of the five recipients receiving peri-hatching blastocysts, two became pregnant and delivered a total of eight piglets. All three recipients of early blastocysts vitrified in VS1 had a delayed return to estrus; while of the four receiving embryos vitrified with VS2, two were delayed in returning to estrus, and one was confirmed pregnant after 45 days. A litter of five piglets, one male and four female, was produced at 116 days of gestation. To our knowledge, this is the first litter of piglets produced from early blastocysts vitrified without micromanipulation to remove polarized lipid droplets.

Animals↗
PubMed2002

Influence of centrifugation or low extension rates prefreezing on the fertility of ram semen after cervical insemination.

We compared the fertility of thawed ram semen, frozen according to different prefreezing semen handling protocols and previously well-defined in vitro, after cervical artificial insemination (AI) during natural estrus in Corriedale sheep. Following primary extension 1 + 1, we adjusted the final sperm concentration before packaging (200 x 10(6)/straw) either by centrifugation, in order to reconcentrate the extended semen (Protocol 1: P1), or without centrifugation, by adjusting the final sperm number by stepwise extension (Protocol 2: P2). We evaluated sperm motility (assessed both subjectively and with a computer-assisted sperm analysis instrument [CASA]), membrane integrity (SYBR-14/PI), and capacitation status (chlortetracycline [CTC]) in vitro in three pooled straws of frozen-thawed semen. Three hundred Corriedale ewes, having shown spontaneous estrus during the breeding season (i.e., April, in the southern hemisphere) under extensive management conditions in Uruguay, were cervically inseminated with thawed semen from the same freezing operations as studied in vitro. The semen evaluation in vitro yielded higher percentages (P < 0.05) of damaged spermatozoa in the samples where sperm numbers were adjusted by extension before freezing (P2), compared with when adjustment was done by centrifugation (P1). However, due to the higher sperm concentration finally achieved by P2, the calculated total number of viable spermatozoa was almost equal in the two AI doses. We observed no differences in fertility between P1 and P2 for either nonreturn rates (NRRs) 21 (30.8 vs. 29.7%) and 36 (28.5 vs. 27.8%) days after AI or lambing rate (21.9 vs. 21.4%), respectively. Fertility did not differ significantly between the two different procedures of adjusting sperm numbers prior to freezing. This may indicate that the simplified protocol with adjusted extension of the semen, resulting in higher numbers of viable spermatozoa, should be the procedure of choice when freezing ram semen under field conditions. Further studies aimed at improving the modified protocol need to be performed.

Animals↗
PubMed2004

Separation of canine epididymal spermatozoa by Percoll gradient centrifugation.

The objective was to characterize the separation of canine epididymal spermatozoa on a Percoll gradient. Epididymal spermatozoa were overlaid on a 45 and 90% discontinuous Percoll gradient and centrifuged at 700 x g for 20 min. The Percoll column was separated into six fractions (top to bottom, A-F) after centrifugation. Fractions A-C contained few spermatozoa. Spermatozoa with bent or folded tails and a large amount of granular debris were observed in Fraction B. Fraction D contained many nonmotile spermatozoa, erythrocytes and round epithelial cells. Spermatozoa in Fraction E had significantly lower motility than those in the initial layer. Spermatozoa in Fraction F had motility similar to those before separation. Fraction F contained 40.6% of the motile spermatozoa layered and 67.5% of all motile spermatozoa recovered. There was no significant difference between Fraction F and the initial layer in sperm membrane integrity. In the sperm-oocyte penetration assay, spermatozoa from Fraction F had a significantly higher penetration rate into the immature homologous oocytes than those from Fraction E. Although the recovery rate of the motile spermatozoa was low, the canine epididymal spermatozoa with motility, membrane integrity and penetrating capability could be separated by two-layer discontinuous Percoll gradient centrifugation.

Animals↗
PubMedDate not supplied

Efficacy of periodic centrifugation of primates during 4-week head-down tilt.

Creation of artificial force of gravity (AFG) to counteract the negative consequences of microgravity in manned space missions of extended duration is one of the high-priority problems of space biology and medicine. However, there are a number of especial effects of AFG (namely, structural changes in muscles and bones, and some other system) which need implantation of electrodes and sensors and are possible only with animals. That is why it is of particular interest to make studies with monkeys whose reactions to changed gravity bear much resemblance with human. The purpose of the investigation was development of a protocol of periodic gravity loads as a counter-measure against the hypokinetic syndrome in Macaca mulatta. Two series of experiments were performed. In the series, animals were split into two groups of 6 species each who were motor restrained with the head end tilted downward at 5 degrees (HDT) for 28 days. Monkeys of group-2 were periodically subjected to centrifugation (HDT+G). During the first series of experiments rotation was conducted in the +Gz direction at g-loads from 1.2 to 1.6 units for 30-40 minutes 4-5 times a week. In the second series, g-load was equal to 1.2 units and the animals were rotated 30 min. 2-3 time a week. The criterion of Y-training protocol efficacy was a test +Gz run at 3 units for 30 s. during which functioning of the cardiovascular systems and its controls was evaluated. The test run was performed prior to and after HDT. Following HDT the animals of group HDT+G were more resistant to the test than their counterparts who had not been trained on the centrifuge. Data of the investigation imply that following HDT and HDT+G alike reduced the amount of total bodily fluids (by approximately 5%), the intracellular component (approximately 4%), and plasma volume (by 6-7%). Yet, there are radical differences between the groups in the levels of reduction in extracellular fluids (by 11% and 6.5%, respectively, P<0.05) and the interstitial component (by 11.5% and 6.5, respectively, P<0.05). Prophylactic centrifugation during HDT was also positive to the muscular blood flow in lower extremities.

Adaptation, Physiological↗
PubMed2002

Polyclonal anti-colorectal cancer Fab phage display library selected in one round using density gradient centrifugation to separate antigen-bound and free phage.

A combinatorial Fab phage display library generated from antibody variable (V) region genes of BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837, was used to isolate an anti-colorectal cancer library. In an attempt to preserve as many anti-colorectal cancer specificities as possible, the original Fab phage display library was selected for binding to a suspension of the human colorectal cancer cells using density gradient centrifugation, instead of washes, to separate cell-bound and free phage. The method consists of placing the cell-phage mixture on a layer of fetal bovine serum (FBS) which had been overlaid on a "cushion" of percoll density medium in a soft, polyallomer tube. After centrifugation, free phage remain on top of the serum layer, whereas the colorectal cancer cells with bound phage are recovered from the serum-percoll interface with a syringe. Analysis of the selected phage display library by enzyme linked immunosorbent assay (ELISA) and diagnostic restriction enzyme digests of individual members (fingerprinting), revealed about 90% anti-colorectal cancer diverse clones after only one round of selection. After conversion to a library of full-length antibodies, such an anti-cancer polyclonal library could be useful for therapeutic and/or diagnostic applications. The density gradient centrifugation method presented here holds great promise for the generation of polyclonal antibody libraries (PCALs) to complex antigens. It is also applicable for selection of peptide or other phage display libraries on any insoluble ligand.

Animals↗
PubMed1986

[Standardization of methods for the preparation of blood components: examination of the operational characteristics of various centrifuges].

For a better standardization and comparison of hemocomponents production methods, the A.A. consider the operative characteristics of eight centrifuges used to this purpose in the transfusional centers, according to the method of calculation of the gravities produced during the whole period of centrifugation. The data obtained are used to produce one kind of hemocomponent by several patterns of centrifuges.

Blood Chemical Analysis↗
PubMed1999

A centrifuge technique for the evaluation of the extent of water movement in wet powder masses.

A centrifuge method has been applied to the assessment of water retention in pharmaceutical powders. Five drug models and microcrystalline cellulose (MCC) were each mixed with different amounts of water and centrifuged at different speeds. The amount of water retained by the wet mass was evaluated by drying the powders to constant weight. Binary mixtures of each of the five model drugs, MCC and water were also processed in the same way. From the amount of water extracted the moisture retention capacity (MRC) was calculated. The MCC retained water more strongly than the different drug models over a wider range of initial water contents. The five drug models, although similar in their chemical structure, were divided into two groups, in terms of their MRC values. 4-HBA and propyl gallate recorded higher MRC values than methyl, propyl and butyl paraben. For the drug models mixed with MCC, the MRC values recorded were similar, though it was still possible to divide the drugs into the two subgroups. A correlation between the MRC value recorded for the different systems and the hydrogen bonding solubility component was found. The application of different centrifuge speeds indicated that within the same material there were different mechanisms of water retention.

Cellulose↗
PubMed1997

Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers.

Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.

Alkaline Phosphatase↗
PubMedDate not supplied

Quantitation of intercellular binding strength by disruptive centrifugation: application to the analysis of adhesive interactions between P815 tumour cells and activated macrophages.

The cell binding assay described in the present paper provides a quantitative measurement of intercellular binding strengths between P815 mastocytoma cells and macrophages activated for cytotoxicity. Conjugates were submitted to a dislodgement force generated by centrifugation tending to remove radiolabelled probe cells from macrophages adhering to a glass support. Non-specific binding occurred between P815 cells and the glass support, but was negligible on siliconed coverslips. The adhesive interactions macrophage/glass were strong enough to resist up to about 1 X 10(-9) N, the maximal dislodgement force tested. Completion of conjugate formation was reached at 37 degrees C after 45 to 60 min and was inhibited at 4 degrees C or in Ca++-free medium. The strengths of intercellular bonds between P815 cells and macrophages varied from 7 X 10(-11) N (the minimal force applied) to more than 9 X 10(-10) N. No clear-cut separation between weak and strong interactions was observed when the centrifugation step was performed at 4 degrees C. In contrast, when centrifugation was carried out at 37 degrees C, a subpopulation of loosely bound tumour targets could be distinguished from more strongly bound P815 cells.

Animals↗
PubMed2001

Evaluation of two in vitro pharmacodynamic simulation models: microfiltration versus centrifugation-filtration.

Pharmacodynamic in vitro models that simulate serum antimicrobial concentrations provide more information about the activity of an antibiotic than MICs or traditional time-kill methods. The aim of this study was to compare two pharmacodynamic simulation models using ATCC strains of five different species and five antibiotics. In the first model (Centriprep-10 system), a filtration-centrifugation process was used to eliminate the antibiotic; in the second model (microfiltration system) no centrifugation was necessary. The antibiotic concentrations tested were similar to those in serum after normal doses of cefuroxime, clarithromycin, ciprofloxacin, gentamicin and cefotaxime. No significant differences were observed in the killing rates between the models except in the case of Haemophilus influenzae and cefotaxime. The new microfiltration model had the following advantages: lack of the carry-over effect, the absence of centrifugation that could damage bacteria and the possibility of increasing the number of incubation periods to give a better fit of the kinetic profile of man.

Anti-Bacterial Agents↗
PubMed1996

Recovery efficiency of Cryptosporidium from water with a crossflow system and continuous flow centrifugation: a comparison study.

A crossflow system and continuous flow centrifugation were tested with regard to the recovery rate of Cryptosporidium oocysts from water. Equal volumes of tap water were contaminated with specific numbers of oocysts. With the crossflow system, an average recovery of 9.8% was obtained when using a concentration of 2 x 10(2) oocysts/L. Recovery rates achieved with the continuous flow centrifuge were more satisfactory: Starting with a concentration of 80 oocysts/L, we obtained an average recovery of 12.9%. The technique of continuous flow centrifugation was found to be more efficient for collection of Cryptosporidium oocysts from water than the crossflow system.

Animals↗