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Molecular Oncology Mar 2016Circulating tumor cells, a component of the "liquid biopsy", hold great potential to transform the current landscape of cancer therapy. A key challenge to unlocking the... (Review)
Review
Circulating tumor cells, a component of the "liquid biopsy", hold great potential to transform the current landscape of cancer therapy. A key challenge to unlocking the clinical utility of CTCs lies in the ability to detect and isolate these rare cells using methods amenable to downstream characterization and other applications. In this review, we will provide an overview of current technologies used to detect and capture CTCs with brief insights into the workings of individual technologies. We focus on the strategies employed by different platforms and discuss the advantages of each. As our understanding of CTC biology matures, CTC technologies will need to evolve, and we discuss some of the present challenges facing the field in light of recent data encompassing epithelial-to-mesenchymal transition, tumor-initiating cells, and CTC clusters.
Topics: Animals; Cell Separation; Centrifugation; Electrophoresis; Filtration; Humans; Lab-On-A-Chip Devices; Neoplasms; Neoplastic Cells, Circulating
PubMed: 26897752
DOI: 10.1016/j.molonc.2016.01.007 -
American Journal of Physiology.... Jun 2022Despite decades of experience from high-gravitoinertial (G) exposures in aircraft and centrifuges, information is scarce regarding primary cardiovascular adaptations to...
Despite decades of experience from high-gravitoinertial (G) exposures in aircraft and centrifuges, information is scarce regarding primary cardiovascular adaptations to +Gz loads in relaxed humans. Thus, effects of G-training are typically evaluated after regimens that are confounded by concomitant use of anti-G straining maneuvers, anti-G suits, and pressure breathing. Accordingly, the aim was to evaluate cardiovascular adaptations to repeated +Gz exposures in the relaxed state. Eleven men underwent 5 wk of centrifuge G training, consisting of 15 × 40 min +Gz exposures at G levels close to their individual relaxed G-level tolerance. Before and after the training regimen, relaxed G-level tolerance was investigated during rapid onset-rate (ROR) and gradual onset-rate (GOR) G exposures, and cardiovascular responses were investigated during orthostatic provocation and vascular pressure-distension tests. The G training resulted in: ) a 13% increase in relaxed ROR G tolerance ( < 0.001), but no change in GOR G tolerance, ) increased pressure resistance in the arteries and arterioles of the legs ( < 0.001), but not the arms, and ) a reduced initial drop in arterial pressure upon ROR high G, but no change in arterial pressure under basal resting conditions or during GOR G loading, or orthostatic provocation. The results suggest +Gz adaptation via enhanced pressure resistance in dependent arteries/arterioles. Presumably, this reflects local adaptations to high transmural pressures, resulting from the +Gz-induced exaggeration of the intravascular hydrostatic pressure gradients.
Topics: Acceleration; Adaptation, Physiological; Aerospace Medicine; Centrifugation; Humans; Hypergravity; Male
PubMed: 35470711
DOI: 10.1152/ajpregu.00043.2022 -
Cells Sep 2022A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and...
A focal advantage of cell sheet technology has been as a scaffold-free three-dimensional (3D) cell delivery platform capable of sustained cell engraftment, survival, and reparative function. Recent evidence demonstrates that the intrinsic cell sheet 3D tissue-like microenvironment stimulates mesenchymal stem cell (MSC) paracrine factor production. In this capacity, cell sheets not only function as 3D cell delivery platforms, but also prime MSC therapeutic paracrine capacity. This study introduces a "cell sheet multilayering by centrifugation" strategy to non-invasively augment MSC paracrine factor production. Cell sheets fabricated by temperature-mediated harvest were first centrifuged as single layers using optimized conditions of rotational speed and time. Centrifugation enhanced cell physical and biochemical interactions related to intercellular communication and matrix interactions within the single cell sheet, upregulating MSC gene expression of connexin 43, integrin β1, and laminin α5. Single cell sheet centrifugation triggered MSC functional enhancement, secreting higher concentrations of pro-regenerative cytokines vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and interleukin-10 (IL-10). Subsequent cell sheet stacking, and centrifugation generated cohesive, bilayer MSC sheets within 2 h, which could not be accomplished within 24 h by conventional layering methods. Conventional layering led to H1F-1α upregulation and increased cell death, indicating a hypoxic thickness limitation to this approach. Comparing centrifuged single and bilayer cell sheets revealed that layering increased VEGF production 10-fold, attributed to intercellular interactions at the layered sheet interface. The "MSC sheet multilayering by centrifugation" strategy described herein generates a 3D MSC-delivery platform with boosted therapeutic factor production capacity.
Topics: Centrifugation; Connexin 43; Gene Expression; Hepatocyte Growth Factor; Integrin beta1; Interleukin-10; Mesenchymal Stem Cells; Vascular Endothelial Growth Factor A
PubMed: 36139414
DOI: 10.3390/cells11182840 -
Cytotherapy Jun 2022Cell viability is an important release criterion in the manufacturing of cell therapy products. Low cell viability can have significant impact on product quality and...
BACKGROUND
Cell viability is an important release criterion in the manufacturing of cell therapy products. Low cell viability can have significant impact on product quality and manufacturing efficiency. Counterflow centrifugation technology has been applied to the manufacturing of cell therapy products, to enable cell separation based on size and density. This study evaluated the utility of counterflow centrifugation technology for dead cell removal to improve cell viability of the final product.
METHODS
Jurkat cell cultures with low and high dead cell burden were subjected to counterflow centrifugal elutriation to determine the correlation between process parameters (e.g., flow rate, centrifugal force) and processing outcomes (i.e., cell recovery and viability). Subsequently, the optimized parameters were applied to dead cell elutriation using expanded T cells and freshly isolated human amniotic epithelial cells (hAECs). The efficiency of dead cell removal, cell function and post-thaw viability were compared.
RESULTS
Elutriation using a low flow rate allowed better control of viable cell recovery from both low and high dead cell burden cultures of Jurkat cells. The viability of T cells and hAECs was improved by counterflow centrifugal processing, from 80.67% ± 2.33 to 94.73% ± 1.19 and 79.19% ± 5.35 to 90.34% ± 3.59, respectively. Processing increased the proliferation rate of T cells, while the metabolic activity of hAECs was unchanged.
CONCLUSION
Counterflow centrifugal elutriation can be added as an integrated step to the automated wash-and-concentrate protocol for cell manufacturing to remove dead cells and improve cell viability of the final product.
Topics: Cell Separation; Cell Survival; Cell- and Tissue-Based Therapy; Centrifugation; Humans
PubMed: 35248475
DOI: 10.1016/j.jcyt.2022.01.008 -
Journal of Applied Physiology... Nov 2019Short-radius centrifugation combined with exercise has been suggested as a potential countermeasure against spaceflight deconditioning. Both the long-term and acute...
Short-radius centrifugation combined with exercise has been suggested as a potential countermeasure against spaceflight deconditioning. Both the long-term and acute physiological responses to such a combination are incompletely understood. We developed and validated a computational model to study the acute cardiovascular response to centrifugation combined with lower body ergometer exercise. The model consisted of 21 compartments, including the upper body, renal, splanchnic, and leg circulation, as well as a four-chamber heart and pulmonary circulation. It also included the effects of gravity gradient and ergometer exercise. Centrifugation and exercise profiles were simulated and compared with experimental data gathered on 12 subjects exposed to a range of gravitational levels (1 and 1.4G measured at the feet) and workload intensities (25-100 W). The model was capable of reproducing cardiovascular changes (within ± 1 SD from the group-averaged behavior) due to both centrifugation and exercise, including dynamic responses during transitions between the different phases of the protocol. The model was then used to simulate the hemodynamic response of hypovolemic subjects (blood volume reduced by 5-15%) subjected to similar gravitational stress and exercise profiles, providing insights into the physiological responses of experimental conditions not tested before. Hypovolemic results are in agreement with the limited available data and the expected responses based on physiological principles, although additional experimental data are warranted to further validate our predictions, especially during the exercise phases. The model captures the cardiovascular response for a range of centrifugation and exercise profiles, and it shows promise in simulating additional conditions where data collection is difficult, expensive, or infeasible. Artificial gravity combined with exercise is a potential countermeasure for spaceflight deconditioning, but the long-term and acute cardiovascular response to such gravitational stress is still largely unknown. We provide a novel mathematical model of the cardiovascular system that incorporates gravitational stress generated by centrifugation and lower body cycling exercise, and we validate it with experimental measurements from human subjects. Simulations of experimental conditions not used for model development corroborate the model's predictive capabilities.
Topics: Blood Pressure; Centrifugation; Ergometry; Exercise; Heart Rate; Humans; Hypovolemia; Models, Cardiovascular; Patient-Specific Modeling
PubMed: 31343946
DOI: 10.1152/japplphysiol.00314.2019 -
Journal of Applied Microbiology Aug 2022To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures.
AIMS
To provide a reliable, reproducible and centrifuge-free filtration protocol for clarification of large volumes of bacterial cultures.
METHODS AND RESULTS
Four experiments were designed to compare different techniques enabling clarification of Escherichia coli cultures using as a benchmark the concentration and quality of bacterial outer membrane vesicles (OMVs). The experiments were designed to examine the performance of different extraction methods on large volume (≥1 L) filtrations of bacterial culture media. Performance parameters included filtration flow rates, sterility testing and characterization of the filtrates by: (i) SDS-PAGE, (ii) cryogenic transmission electron microscopy, (iii) nanoparticle tracking analysis and (iv) Qubit protein quantification. The experiments revealed that: (i) addition of the filter aid Diatomaceous Earth to the bacterial cultures improved filtration flow rates significantly and eliminated the need for centrifugation prior to filtration; (ii) sterile filtration was successful as no bacterial passage was identified through the membrane filter; (iii) centrifuge-free filtrates contained an increased amount of OMVs compared to centrifuged filtrates.
CONCLUSIONS
In comparison to conventional centrifuge-based protocols, the clarification method presented has universal applicability for a broad range of microbial extraction procedures, regardless of the volume of culture harvested. Moreover, the decreased amount of OMVs presented in the filtrates following centrifugation step provides an additional argument in favour of a centrifuge-free approach.
SIGNIFICANCE AND IMPACT OF THE STUDY
Sterile filtration is a universal method for the clarification of bacterial cultures. Common challenges related to filtration include filter clogging and long processing times, due to limited centrifugation capacity, which can affect product quality. The proposed protocol is likely to ensure a highly effective filtration process and could be a novel approach in improving the filtrate products without the need of centrifugation.
Topics: Bacteria; Centrifugation; Filtration
PubMed: 35503033
DOI: 10.1111/jam.15608 -
Journal of Chromatography. A Jul 2021Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity...
Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity ligand. A subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty capsids and debris. Empty and full capsid composition of chromatography fractions was evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with respect to capsid separation. Charging with cupric ions maintained resolution between early-eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete elimination of empty capsids. All metal ions bound contaminant subsets that required sodium hydroxide for removal. Columns charged with ferric iron and manganese bound more contaminants than all other metals. Columns charged with calcium, magnesium, barium, and copper bound the least. Contaminant binding on zinc-charged columns was intermediate between the two groups.
Topics: Capsid; Centrifugation, Density Gradient; Cesium; Chlorides; Chromatography, Affinity; Dependovirus
PubMed: 34034104
DOI: 10.1016/j.chroma.2021.462210 -
Journal of Chromatography. B,... Jan 2010Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is... (Review)
Review
Centrifugal precipitation chromatography separates analytes according their solubility in ammonium sulfate (AS) solution and other precipitants. The separation column is made from a pair of long spiral channels partitioned with a semipermeable membrane. In a typical separation, concentrated ammonium sulfate is eluted through one channel while water is eluted through the other channel in the opposite direction. This countercurrent process forms an exponential AS concentration gradient through the water channel. Consequently, protein samples injected into the water channel is subjected to a steadily increasing AS concentration and at the critical AS concentration they are precipitated and deposited in the channel bed by the centrifugal force. Then the chromatographic separation is started by gradually reducing the AS concentration in the AS channel which lowers the AS gradient concentration in the water channel. This results in dissolution of deposited proteins which are again precipitated at an advanced critical point as they move through the channel. Consequently, proteins repeat precipitation and dissolution through a long channel and finally eluted out from the column in the order of their solubility in the AS solution. The present method has been successfully applied to a number of analytes including human serum proteins, recombinant ketosteroid isomerase, carotenoid cleavage enzymes, plasmid DNA, polysaccharide, polymerized pigments, PEG-protein conjugates, etc. The method is capable to single out the target species of proteins by affinity ligand or immunoaffinity separation.
Topics: Ammonium Sulfate; Blood Proteins; Centrifugation; Chemical Precipitation; Chromatography, Affinity; Equipment Design; Humans; Immunosorbent Techniques; Proteins
PubMed: 19541553
DOI: 10.1016/j.jchromb.2009.05.055 -
Theriogenology Feb 2024The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires...
The development of endoscopic transcervical catheterization (ETC) in the queen increases the interest in handling fresh and cryopreserved feline semen. The ETC requires a small volume of the insemination dose with a high concentration, not easily reached with the actual frozen technique in this species. Centrifugation is widely used to concentrate spermatozoa for several purposes, but this process is detrimental to spermatozoa. This study verified the effects of conventional and cushioned centrifugation on fresh and cryopreserved feline spermatozoa. To this, semen was collected from 20 toms, grouped in seven pools and diluted. After dilution, the pools were divided into two aliquots, the first used for centrifugation on fresh semen, and the second, after freezing, on cryopreserved semen. Centrifugation regimens were: conventional at 500×g, conventional at 1000×g, and cushioned (iodixanol) at 1000×g. The sperm recovery rate was calculated for the three centrifugation regimens, and sperm kinematics, membrane and acrosome integrity, and plasma membrane stability on viable spermatozoa were assessed as endpoints. The data reported in this study showed that the centrifugation at 500×g resulted in negligible effects on both fresh and cryopreserved spermatozoa, but the lower recovery rate (62.4 ± 3.1 % and 60.2 ± 1.6 %, respectively) underlines the loss of a large proportion of spermatozoa, unfavourable in a species with small total sperm ejaculated. On the other hand, the centrifugation at 1000×g improved the recovery rate (86.9 ± 4.3 % and 89.8 ± 2.4 % in fresh and cryopreserved samples, respectively), but was more deleterious for feline spermatozoa, especially in cryopreserved samples (i.e. total motility of 40.7 ± 5.4 % compared with 57.2 ± 9.8 % in cryopreserved uncentrifuged samples, P < 0.05), resulting in artificial insemination doses of lower quality. The recovery rate in cushioned centrifugation appeared less efficient, likely due to the small volume of feline samples, which makes difficult the separation of sperm pellet and cushioned fluid. Interestingly, in cryopreserved samples centrifuged at 1000×g the number of viable spermatozoa with membrane destabilization (31.3 ± 3.2 %) was greater than uncentrifuged (4.1 ± 0.7 %, P < 0.05) and those centrifuged at 500×g (9.8 ± 1.3 %, P < 0.05), suggesting modifications induced by the cryopreservation amplifies centrifugation sublethal damage on feline spermatozoa. Cushioned centrifugation on cryopreserved samples showed kinematics similar to uncentrifuged samples, but higher viable spermatozoa with membrane destabilization (37.4 ± 3.4 % vs 4.1 ± 0.7 %; P < 0.05). In felines, g-force is crucial for sperm recovery rate during centrifugation, with better results at 1000×g; on the other hand, greater g-forces could have a significant impact on the quality of feline insemination dose, especially in cryopreserved samples.
Topics: Cats; Animals; Male; Semen; Sperm Motility; Semen Preservation; Spermatozoa; Cryopreservation; Centrifugation
PubMed: 38096623
DOI: 10.1016/j.theriogenology.2023.12.009 -
Biochemia Medica Feb 2023In order to deliver high quality results, detection and elimination of possible analytical interferences, such as lipaemia, is crucial. The aim of this study is to...
INTRODUCTION
In order to deliver high quality results, detection and elimination of possible analytical interferences, such as lipaemia, is crucial. The aim of this study is to evaluate the efficacy of high-speed centrifugation in eliminating lipaemic interference and to define own lipaemic index (LI) for the studied biochemical analytes.
MATERIALS AND METHODS
Evaluated analytes were: albumin, alkaline phosphatase, alanine-aminotransferase (ALT), aspartate-aminotransferase (AST), calcium, creatinine, gamma-glutamyltransferase (GGT), glucose, phosphates, total proteins, urea and total bilirubin. Those analytes and LIs have been analysed in duplicate in the Roche Diagnostics-c8000 analyser in samples centrifuged at 3000 rpm/10 minutes in the SL16 (Thermo Scientific, Waltham, USA) centrifuge and according to an own high-speed centrifugation protocol (12,900 rpm/15 minutes) in the MicroCL17R (Thermo Scientific, Waltham, USA) centrifuge. Lipaemia has been measured in each sample. The efficiency of high-speed centrifugation is verified by the Wilcoxon test (P < 0.05). In cases where significant differences are observed, our own LI is calculated. For ALT and AST, it is verified by McNemar test (P < 0.05. For creatinine, both Wilcoxon and McNemar test were applied.
RESULTS
There were statistically significant differences in analyte concentration before and after high-speed centrifugation for: albumin, creatinine, GGT, glucose, phosphates, urea and total bilirrubin. Own LI is calculated. McNemar test shows statistically significant diferences in the proportion of delivered results before and after high-speed centrifugation in ALT, AST and creatinine.
CONCLUSIONS
This study confirms the efficacy of high-speed centrifugation protocol for all the considered analytes, excepting calcium, alkaline phosphatase and total proteins.
Topics: Humans; Calcium; Creatinine; Alkaline Phosphatase; Centrifugation; Hyperlipidemias; Glucose; Alanine Transaminase; Albumins; Phosphates
PubMed: 36627977
DOI: 10.11613/BM.2023.010703