Large-scale column-free purification of bovine F-ATP synthase.

Mammalian F-ATP synthase is central to mitochondrial bioenergetics and is present in the inner mitochondrial membrane in a dynamic oligomeric state of higher oligomers, tetramers, dimers, and monomers. In vitro investigations of mammalian F-ATP synthase are often limited by the ability to purify the oligomeric forms present in vivo at a quantity, stability, and purity that meets the demand of the planned experiment. We developed a purification approach for the isolation of bovine F-ATP synthase from heart muscle mitochondria that uses a combination of buffer conditions favoring inhibitor factor 1 binding and sucrose density gradient ultracentrifugation to yield stable complexes at high purity in the milligram range. By tuning the glyco-diosgenin to lauryl maltose neopentyl glycol ratio in a final gradient, fractions that are either enriched in tetrameric or monomeric F-ATP synthase can be obtained. It is expected that this large-scale column-free purification strategy broadens the spectrum of in vitro investigation on mammalian F-ATP synthase.

Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer's disease plasma biomarkers.

The reliability of plasma biomarkers of Alzheimer's disease (AD) can be compromised by protease-induced degradation. This can limit the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). In this study, we conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions. We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 h. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0 h or 24 h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA and P100 tubes, followed by storage at RT for 0 h or 24 h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays. Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improves the stability of Aß42 and Aß40 across all approaches. However, the Aß42/Aß40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches. Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aß42 and Aß40, and the Aß42/40 ratio for the IP-MS assay. These findings have crucial implications for preanalytical procedures, particularly in resource-limited settings.

Centrifugation-Mediated Crystal Growth of Attractive Colloids for Band Edge Lasing.

Attractive depletion interactions are utilized to organize colloidal particles into crystalline arrays with high crystallinity through spontaneous phase separation. However, uncontrolled nucleation frequently leads to the formation of crystalline grains with varied crystal orientations, which hampers the optical performance of photonic crystals. Here, colloidal crystals have been engineered with uniform orientation and high surface coverage by applying centrifugal force during the depletion-induced assembly of polystyrene particles. The centrifugal force encourages the particles to move toward the bottom surface, which fosters heterogeneous nucleation and supports rapid crystal growth, yielding densely-packed and uniformly-arranged crystal grains with high reflectivity. This study has observed that the nucleation and crystal growth behavior is significantly influenced by the salt concentration. Based on the pair potentials, the transition boundary has been quantitatively analyzed between fluid and crystal phases and identified the threshold for homogeneous nucleation. Utilizing the high-reflectivity colloidal crystals, band-edge lasing is achieved by dissolving the water-soluble dye into the aqueous suspensions. Upon optical excitation, a lasing emission characterized is observed by a narrow spectral width at the short-wavelength band edge. Notably, the laser wavelength can be adjusted by altering the salt concentration or particle diameter, offering a versatile approach to tuning the optical properties.

Monodispersed Renewable Particles by Cascade and Density Gradient Size Fractionation to Advance Lignin Nanotechnologies.

Control over particle size and shape heterogeneity is highly relevant to the design of photonic coatings and supracolloidal assemblies. Most developments in the area have relied on mineral and petroleum-derived polymers that achieve well-defined chemical and dimensional characteristics. Unfortunately, it is challenging to attain such control when considering renewable nanoparticles. Herein, a pathway toward selectable biobased particle size and physicochemical profiles is proposed. Specifically, lignin is fractionated, a widely available heterogeneous polymer that can be dissolved in aqueous solution, to obtain a variety of monodispersed particle fractions. A two-stage cascade and density gradient centrifugation that relieves the need for solvent pre-extraction or other pretreatments but achieves particle bins of uniform size (~60 to 860 nm and polydispersity, PDI<0.06, dynamic light scattering) along with characteristic surface chemical features is introduced. It is found that the properties and associated colloidal behavior of the particles are suitably classified in distinctive size populations, namely, i) nanoscale (50-100 nm), ii) photonic (100-300 nm) and iii) near-micron (300-1000 nm). The strong correlation that exists between size and physicochemical characteristics (molar mass, surface charge, bonding and functional groups, among others) is introduced as a powerful pathway to identify nanotechnological uses that benefit from the functionality and cost-effectiveness of biogenic particles.

High-speed centrifugation rather than Lipoclear reagent can be used for removing the interference of lipemia on serological tests of infectious diseases: AIDS, hepatitis B, hepatitis C, and syphilis by chemiluminescent microparticle immunoassay.

The aim of this study was to investigate the interference of lipemia on measurement of HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, anti-HCV, HIV Ag/Ab, and anti-TP in serum by chemiluminescent microparticle immunoassay (CMIA) and compare lipemia removing performance between high-speed centrifugation and Lipoclear reagent. Mixed native serum samples (NSs) and hyperlipemia serum samples (HLS) were prepared for the investigated parameters. The levels of these parameters in NS and HLS were determined by CMIA on an Abbott ARCHITECT i2000SR immunoassay analyzer. HBsAg, anti-HBs, and anti-TP were affected with relative bias >12.5% (acceptable limit) when the level of triacylglycerol (TG) was higher than 27.12 mmol/L in HLS. Clinically unacceptable bias were observed for HBeAg and anti-HBe in HLS with TG higher than 40.52 mmol/L. However, anti-HCV and HIV Ag/Ab were not interfered in severe lipemia with TG < 52.03 mmol/L. In addition, the Lipoclear reagent did not reduce the interference of lipemia with relative bias from -62.50% to -18.02%. The high-speed centrifugation under the optimized condition of 12 000g for 10 min successfully removed the interference of lipemia with relative bias from -5.93% to 0% for HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and anti-TP. To conclude, high-speed centrifugation can be used for removing the interference of lipemia to measure HBsAg, anti-HBs, HBeAg, anti-HBe, anti-HBc, and anti-TP. Accordingly, a standardized sample preanalytical preparation of the patients and other screening participants as well as a specimen examination procedure for removing lipemia interference on the serological tests was recommended.

An experimentally informed computational model of neurovestibular adaptation to altered gravity.

Transitions to altered gravity environments result in acute sensorimotor impairment for astronauts, leading to serious mission and safety risks in the crucial first moments in a new setting. Our understanding of the time course and severity of impairment in the early stages of adaptation remains limited and confounded by unmonitored head movements, which are likely to impact the rate of adaptation. Here, we aimed to address this gap by using a human centrifuge to simulate the first hour of hypergravity (1.5g) exposure and the subsequent 1g readaptation period, with precisely controlled head tilt activity. We quantified head tilt overestimation via subjective visual vertical and found ∼30% tilt overestimation that did not decrease over the course of 1 h of exposure to the simulated gravity environment. These findings extended the floor of the vestibular adaptation window (with controlled vestibular cueing) to 1 h of exposure to altered gravity. We then used the empirical data to inform a computational model of neurovestibular adaptation to changes in the magnitude of gravity, which can offer insight into the adaptation process and, with further tuning, can be used to predict the temporal dynamics of vestibular-mediated misperceptions in altered gravity.

Bayesian cell therapy process optimization.

Optimizing complex bioprocesses poses a significant challenge in several fields, particularly in cell therapy manufacturing. The development of customized, closed, and automated processes is crucial for their industrial translation and for addressing large patient populations at a sustainable price. Limited understanding of the underlying biological mechanisms, coupled with highly resource-intensive experimentation, are two contributing factors that make the development of these next-generation processes challenging. Bayesian optimization (BO) is an iterative experimental design methodology that addresses these challenges, but has not been extensively tested in situations that require parallel experimentation with significant experimental variability. In this study, we present an evaluation of noisy, parallel BO for increasing noise levels and parallel batch sizes on two in silico bioprocesses, and compare it to the industry state-of-the-art. As an in vitro showcase, we apply the method to the optimization of a monocyte purification unit operation. The in silico results show that BO significantly outperforms the state-of-the-art, requiring approximately 50% fewer experiments on average. This study highlights the potential of noisy, parallel BO as valuable tool for cell therapy process development and optimization.

Understanding exosomes: Part 2-Emerging leaders in regenerative medicine.

Exosomes are the smallest subset of extracellular signaling vesicles secreted by most cells with the ability to communicate with other tissues and cell types over long distances. Their use in regenerative medicine has gained tremendous momentum recently due to their ability to be utilized as therapeutic options for a wide array of diseases/conditions. Over 5000 publications are currently being published yearly on this topic, and this number is only expected to dramatically increase as novel therapeutic strategies continue to be developed. Today exosomes have been applied in numerous contexts including neurodegenerative disorders (Alzheimer's disease, central nervous system, depression, multiple sclerosis, Parkinson's disease, post-traumatic stress disorders, traumatic brain injury, peripheral nerve injury), damaged organs (heart, kidney, liver, stroke, myocardial infarctions, myocardial infarctions, ovaries), degenerative processes (atherosclerosis, diabetes, hematology disorders, musculoskeletal degeneration, osteoradionecrosis, respiratory disease), infectious diseases (COVID-19, hepatitis), regenerative procedures (antiaging, bone regeneration, cartilage/joint regeneration, osteoarthritis, cutaneous wounds, dental regeneration, dermatology/skin regeneration, erectile dysfunction, hair regrowth, intervertebral disc repair, spinal cord injury, vascular regeneration), and cancer therapy (breast, colorectal, gastric cancer and osteosarcomas), immune function (allergy, autoimmune disorders, immune regulation, inflammatory diseases, lupus, rheumatoid arthritis). This scoping review is a first of its kind aimed at summarizing the extensive regenerative potential of exosomes over a broad range of diseases and disorders.

Essential principles for blood centrifugation.

Currently, autologous platelet concentrates (APCs) are frequently used for soft- and hard-tissue regeneration, not only within the oral cavity, but also extra-orally including chronic wounds, burns, joints, dermatological conditions, among others. The benefits of APCs are largely influenced by the treatment strategy but also their preparation. This paper therefore discusses in detail: the physical properties of blood cells, the basic principles of blood centrifugation, the impact of the centrifugation protocol (rotations/revolutions per minute, g-force, variation between centrifuges), the importance of timing during the preparation of APCs, the impact of the inner surface of the blood tubes, the use/nonuse of anticoagulants within APC tubes, the impact of the patient's hematocrit, age, and gender, as well as the important requirements for an optimal centrifugation protocol. All these variables indeed have a significant impact on the clinical outcome of APCs.

The stability of 65 biochemistry analytes in plasma, serum, and whole blood.

OBJECTIVES: The pre-analytical stability of various biochemical analytes requires careful consideration, as it can lead to the release of erroneous laboratory results. There is currently significant variability in the literature regarding the pre-analytical stability of various analytes. The aim of this study was to determine the pre-analytical stability of 65 analytes in whole blood, serum and plasma using a standardized approach. METHODS: Blood samples were collected from 30 healthy volunteers (10 volunteers per analyte) into five vacutainers; either SST, Li-heparin, K2-EDTA, or Na-fluoride/K-oxalate. Several conditions were tested, including delayed centrifugation with storage of whole blood at room temperature (RT) for 8 h, delayed centrifugation with storage of whole blood at RT or 4 °C for 24 h, and immediate centrifugation with storage of plasma or serum at RT for 24 h. Percent deviation (% PD) from baseline was calculated for each analyte and compared to the maximum permissible instability (MPI) derived from intra- and inter-individual biological variation. RESULTS: The majority of the analytes evaluated remained stable across all vacutainer types, temperatures, and timepoints tested. Glucose, potassium, and aspartate aminotransferase, among others, were significantly impacted by delayed centrifugation, having been found to be unstable in whole blood specimens stored at room temperature for 8 h. CONCLUSIONS: The data presented provides insight into the pre-analytical variables that impact the stability of routine biochemical analytes. This study may help to reduce the frequency of erroneous laboratory results released due to exceeded stability and reduce unnecessary repeat phlebotomy for analytes that remain stable despite delayed processing.

Platelet rich fibrin as a bioactive matrix with proosteogenic and proangiogenic properties on human healthy primary cells in vitro.

Blood concentrates like platelet rich fibrin (PRF) have been established as a potential autologous source of cells and growth factors with regenerative properties in the field of dentistry and regenerative medicine. To further analyze the effect of PRF on bone tissue regeneration, this study investigated the influence of liquid PRF matrices on human healthy primary osteoblasts (pOB) and co-cultures composed of pOB and human dermal vascular endothelial cells (HDMEC) as in vitro model for bone tissue regeneration. Special attention was paid to the PRF mediated influence on osteoblastic differentiation and angiogenesis. Based on the low-speed centrifugation concept, cells were treated indirectly with PRF prepared with a low (44 g) and high relative centrifugal force (710 g) before the PRF mediated effect on osteoblast proliferation and differentiation was assessed via gene and protein expression analyses and immunofluorescence. The results revealed a PRF-mediated positive effect on osteogenic proliferation and differentiation accompanied by increased concentration of osteogenic growth factors and upregulated expression of osteogenic differentiation factors. Furthermore, it could be shown that PRF treatment resulted in an increased formation of angiogenic structures in a bone tissue mimic co-culture of endothelial cells and osteoblasts induced by the PRF mediated increased release of proangiogenic growth factors. The effects on osteogenic proliferation, differentiation and vascularization were more evident when low RCF PRF was applied to the cells. In conclusion, PRF possess proosteogenic, potentially osteoconductive as well as proangiogenic properties, making it a beneficial tool for bone tissue regeneration.

What is the context?The treatment of bone defects is still a challenge in the field of regenerative medicine. In this context, researchers and clinicians are continuously focusing on developing new therapeutic strategies like the use of autologous blood concentrates like Platelet rich fibrin (PRF) to improve regeneration by directly delivering wound healing promoting cells and growth factors to the defect side in order to restore the structure and functional integrity of damaged hard tissue in combination with adequate tissue regeneration.What is new?Focus of the present in vitro study was to further evaluate the potential of PRF paying particular attention to the PRF-mediated effect on osteogenic differentiation and angiogenesis of human primary osteoblasts as well as on a more complex tissue like co-culture consisting of osteoblasts and microvascular endothelial cells. We could demonstrate that PRF is able to support and affect a variety of processes involved in bone tissue regeneration including osteogenic proliferation, osteogenic differentiation as well as angiogenic structure formation.Treatment of PRF resulted in:- increased cell viability*- higher expression of osteogenic differentiation factors*- higher release of osteogenic growth factors*- increased formation of microvessel-like structures*(*compared to untreated control)What is the impact?PRF represents a beneficial autologous tool for regenerative purposes combining proosteogenic and proangiogenic properties. Therefore, PRF might be used for applications in versatile fields of medicine in the context of improving bone tissue regeneration.

Comparative Analysis of T-Cell Spatial Proteomics and the Influence of HIV Expression.

As systems biology approaches to virology have become more tractable, highly studied viruses such as HIV can now be analyzed in new unbiased ways, including spatial proteomics. We employed here a differential centrifugation protocol to fractionate Jurkat T cells for proteomic analysis by mass spectrometry; these cells contain inducible HIV-1 genomes, enabling us to look for changes in the spatial proteome induced by viral gene expression. Using these proteomics data, we evaluated the merits of several reported machine learning pipelines for classification of the spatial proteome and identification of protein translocations. From these analyses, we found that classifier performance in this system was organelle dependent, with Bayesian t-augmented Gaussian mixture modeling outperforming support vector machine learning for mitochondrial and endoplasmic reticulum proteins but underperforming on cytosolic, nuclear, and plasma membrane proteins by QSep analysis. We also observed a generally higher performance for protein translocation identification using a Bayesian model, Bayesian analysis of differential localization experiments, on row-normalized data. Comparative Bayesian analysis of differential localization experiment analysis of cells induced to express the WT viral genome versus cells induced to express a genome unable to express the accessory protein Nef identified known Nef-dependent interactors such as T-cell receptor signaling components and coatomer complex. Finally, we found that support vector machine classification showed higher consistency and was less sensitive to HIV-dependent noise. These findings illustrate important considerations for studies of the spatial proteome following viral infection or viral gene expression and provide a reference for future studies of HIV-gene-dropout viruses.

Heat-stable whey protein isolate made using isoelectric precipitation and clarification.

Residual lipids (RL) in whey protein isolate (WPI) are detrimental to optimal functional applications (like foaming and low turbidity) and contribute to off-flavor development during powder storage. The objective of this research was to prepare an experimental WPI by removing RL without using the traditional microfiltration (MF) process and compare its properties with commercially available WPIs made using MF and some other whey powders. We hypothesize that by adjusting the pH of whey to < 5.0, we would be close to the isoelectric point of any remaining denatured proteins (DP) and phospholipoproteins (PLP), and therefore reduce electrostatic repulsion between these molecules. Further, demineralization of the acidified whey protein solution by ultrafiltration (UF) combined with diafiltration (DF) should reduce ionic hindrance to aggregation and thereby help with the aggregation of these denatured proteins as well as most RL; centrifugation or clarification could be used to remove these materials. Calcium should also be more extensively removed by this approach, which should improve the heat stability of the experimental WPI. Demineralization was achieved on a pilot scale by acidifying liquid (cheese) whey protein concentrate (WPC-34) to pH 4.5 using HCl, and UF the whey protein solution along with extensive DF using acidified (pH∼3.5) reverse osmosis filtered (RO) water. Demineralized whey protein solution was adjusted to various combinations of pH (4.1 to 4.9), conductivities (500 to 2000 µS.cm-1), and protein concentrations (1 to 7%) and then centrifuged at 10,000 × g for 10 min. The effective sedimentation (precipitation) of RL in these treatments was estimated by measuring the turbidity of the supernatants. Maximum precipitation was observed at pH 4.5-4.7. Reducing conductivity via UF/DF increased the precipitation of RL due to reduced ionic hindrance to aggregation Maximum sedimentation of RL was observed at protein concentrations ≤3% because of a higher density difference between the precipitate and serum phase. SDS-PAGE analysis confirmed the sedimentation of PLPs, caseins, and DPs upon isoelectric precipitation at pH ∼4.5, while native whey proteins or undenatured whey proteins remained soluble in the supernatant, unaffected by the pretreatment. To scale up the process, 750 L of fluid WPC34 was acidified and demineralized by UF (volume concentration factor = 1.35) and DF until the permeate solids reached 0.1% (when desired demineralization was achieved), clarified using a pilot-scale desludging clarifier to remove RL, neutralized, ultrafiltered to concentrate the protein, and then spray-dried to produce an experimental WPI (91% protein and 1.8% fat db). In another trial, demineralized UF concentrate was clarified by gravity sedimentation and the supernatant was neutralized, ultrafiltered, and spray-dried to produce a second experimental WPI (91% protein and < 1% fat db). These experimental WPI powders were compared with several commercially available WPI powders to assess functional properties like solubility, heat stability, foamability and foam strength, gelation, and sensory attributes over accelerated storage. Experimental WPI had excellent functional properties, had low turbidity, were highly heat stable and only developed very slight to slight off-flavors upon accelerated storage, their properties were comparable to the WPI manufactured commercially using MF even after accelerated storage.

Delayed centrifugation weakens the in vitro biological properties of platelet-rich fibrin membranes.

OBJECTIVE: To investigate how delayed blood centrifugation affects the composition of the resultant platelet rich fibrin membrane (PRF, a concentrated growth factor preparation) and its biological effects towards gingival fibroblasts. MATERIALS AND METHODS: Blood samples were collected from 18 healthy individuals and centrifuged immediately (T-0), or after a 1-6-minute delay (T-1-6, respectively), to generate PRF. Each PRF membrane was weighed. T-0 and T-6 membranes were incubated for 48 h in cell culture medium at 37 °C to create PRF "releasates" (soluble factors released from the PRF). Human gingival fibroblasts were incubated for 48 h with or without the releasates, followed by RNA isolation and real-time polymerase chain reaction to measure expression of select genes associated with granulation tissue formation, angiogenesis and wound contraction. Additional T-0 and T-6 membranes were used for visualization of leucocyte nuclei and platelets by immunostaining. RESULTS: Immediate centrifugation (T-0) resulted in the largest membranes, T-6 membranes being on average 29% smaller. Leucocytes and platelets were significantly more abundant in T-0 than in T-6 samples. Majority of the fibroblast genes studied were consistently either upregulated or downregulated by the T-0 PRF releasates. However, centrifugation after a 6-minute delay significantly weakened the fibroblast responses. CONCLUSIONS: Delayed centrifugation resulted in smaller PRF membranes with fewer leucocytes and platelets and also significantly reduced on the expression of a set of healing-related gingival fibroblast genes. CLINICAL RELEVANCE: The higher expression of wound healing-related genes in gingival fibroblasts by the immediately-centrifuged PRF membranes may increase their biological properties in clinical use.

Concentrated Deoiled Fat: A Novel Method of Fat Processing to Improve Fat Graft Survival-A Basic Research.

BACKGROUND: Oil compromises graft outcomes via inflammation, which accounts for the unpredictability of volume retention rates as low as 20%. Existing techniques for oil removal are relatively inefficient. In this study, a novel approach was taken to prepare concentrated deoiled fat (CDF) by utilizing flocculation and centrifugation to remove the oil. The hypothesis put forward in this study was that CDF would exhibit improved volume retention and quality by enhancing purification efficiency and reducing inflammation. METHODS: This basic research involved both in vitro and in vivo experiments using samples obtained from women who underwent abdominal liposuction. The CDF was prepared by flocculation and centrifugation. In the vitro experiments, the microstructure of fat was assessed using Calcein acetoxymethyl ester (AM) staining for living cells and propidium iodide (PI) staining for dead nuclei in two groups: Coleman fat group and CDF group. Additionally, the glucose uptake capacity of these two groups was evaluated using the glucose transport test (GTT). In the vivo experiments, the study included three groups: two experimental groups (low-volume concentrated deoiled fat, LCDF; high-volume concentrated deoiled fat, HCDF) and one control group (Coleman fat), with 10 healthy female BALB/c nude mice in each group, 1ml of the graft was injected subcutaneously to each mouse. After 8 weeks, the fat grafts were harvested and subjected to volume evaluation, HE staining and immunostaining for perilipin to assess graft outcomes. RESULTS: In the vitro experiments, the concentration rate of the CDF was found to be 79.6% that of Coleman fat, with 15.1% more oil separated. Cell viability, as assessed by AM/PI staining, did not show a significant difference between the two grafts, but the results of the GTT showed that the tissue viability of the CDF was higher than that of Coleman fat. In the vivo experiments, the CDF had higher volume retention than Coleman fat, as measured by water displacement. Histopathologic scoring indicated that HCDF group and LCDF group had a more intact fat structure with fewer vacuoles, inflammation, and fibrosis compared to Coleman fat. Additionally, the percentages of perilipin-positive area in the LCDF group and HCDF group were higher than in the Coleman group, indicating improved graft quality and outcome with the use of concentrated deoiled fat. CONCLUSIONS: "Concentrated deoiled fat" refers to an autologous fat graft from which oil has been removed by flocculation and centrifugation. This process increases volume retention and viable cells and decreases infiltrated inflammatory cells. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors   www.springer.com/00266 .

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

BACKGROUND: Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. OBJECTIVES: To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. METHODS: BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. RESULTS: The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. CONCLUSIONS: 300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Mechanical Purification of Lipofilling: The Relationship Between Cell Yield, Cell Growth, and Fat Volume Maintenance.

BACKGROUND: The mechanical manipulations of fat tissue represented from centrifugation, filtration, washing, and fragmentation were considered the most effective strategies aiming to obtain purified lipofilling with different impacts both in terms of adipose-derived stem cells amount contained in stromal vascular fraction, and fat volume maintenance. OBJECTIVES: The present work aimed to report results in fat volume maintenance obtained by lipofilling purification based on the combined use of washing and filtration, in a clinical study, and to deeply investigate the adipose-derived stem cells yield and growth capacity of the different stromal vascular fraction extraction techniques with an in vitro approach. METHODS: A preliminary prospective, case-control study was conducted. 20 patients affected by face and breast soft tissue defects were treated with lipofilling and divided into two groups: n = 10 patients (study group) were treated with lipofilling obtained by washing and filtration procedures, while n = 10 (control group) were treated with lipofilling obtained by centrifugation according to the Coleman technique. 6 months after the lipofilling, the volume maintenance percentage was analyzed by clinical picture and magnetic resonance imaging comparisons. Additionally, extracted stromal vascular fraction cells were also in vitro analyzed in terms of adipose-derived stem cell yield and growth capacity. RESULTS: A 69% ± 5.0% maintenance of fat volume after 6 months was observed in the study group, compared with 44% ± 5.5% in the control group. Moreover, the cellular yield of the control group resulted in 267,000 ± 94,107 adipose-derived stem cells/mL, while the study group resulted in 528,895 ± 115,853 adipose-derived stem cells /mL, with a p-value = 0.1805. Interestingly, the study group showed a fold increase in cell growth of 6758 ± 0.7122, while the control group resulted in 3888 ± 0.3078, with a p < 0.05 (p = 0.0122). CONCLUSIONS: The comparison of both groups indicated that washing and filtration were a better efficient system in lipofilling preparation, compared to centrifugation, both in terms of volume maintenance and adipose-derived stem cell growth ability. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

Separation of Lipoproteins for Quantitative Analysis of 14C-Labeled Lipid-Soluble Compounds by Accelerator Mass Spectrometry.

To date, 14C tracer studies using accelerator mass spectrometry (AMS) have not yet resolved lipid-soluble analytes into individual lipoprotein density subclasses. The objective of this work was to develop a reliable method for lipoprotein separation and quantitative recovery for biokinetic modeling purposes. The novel method developed provides the means for use of small volumes (10-200 µL) of frozen plasma as a starting material for continuous isopycnic lipoprotein separation within a carbon- and pH-stable analyte matrix, which, following post-separation fraction clean up, created samples suitable for highly accurate 14C/12C isotope ratio determinations by AMS. Manual aspiration achieved 99.2 ± 0.41% recovery of [5-14CH3]-(2R, 4'R, 8'R)-α-tocopherol contained within 25 µL plasma recovered in triacylglycerol rich lipoproteins (TRL = Chylomicrons + VLDL), LDL, HDL, and infranatant (INF) from each of 10 different sampling times for one male and one female subject, n = 20 total samples. Small sample volumes of previously frozen plasma and high analyte recoveries make this an attractive method for AMS studies using newer, smaller footprint AMS equipment to develop genuine tracer analyses of lipophilic nutrients or compounds in all human age ranges.

Operator impact assessment on qualitative and quantitative parameters of canine platelet-rich plasma.

Platelet-rich plasma (PRP) has emerged as a cornerstone in veterinary regenerative medicine. The present study evaluated the impact of the operator on the qualitative and quantitative features of non-activated PRP derived from canine whole blood. Blood was collected in anticoagulant acid citrate dextrose from twelve healthy adult dogs and PRP was prepared according to the double-spin method. Both operators followed an identical protocol and utilized the same equipment for PRP preparation from the pooled blood samples. The resulting PRP underwent characterization, classification and coding based on minimum reporting standards. The consistency and internal reliability of different parameters were also assessed using the intraclass correlation coefficient and Cronbach's alpha values. Variables such as white blood cell (WBC) concentration, relative WBC composition and mean platelet volume (MPV) showed poor reliability, and WBC concentration and MPV also had unacceptable internal consistency. Significant differences were observed in several qualitative and quantitative parameters of the prepared PRP, highlighting the influence of the operator even when the same protocol and equipment were used. Our study has direct implications to regenerative medicine, reinforcing the urgency to set minimum requirements for reporting PRP in research studies.

Swim-up method is superior to density gradient centrifugation for preserving sperm DNA integrity during sperm processing.

Purpose: This study aimed to evaluate the effects of swim-up and density gradient centrifugation methods on sperm DNA fragmentation. Methods: Nineteen normozoospermic patient samples with ≥100 × 106 motile sperms were included in this study. Sperm DNA fragmentation, progressive motility, and progressive motile sperm number were measured before and after the swim-up method or density gradient centrifugation. Results: Sperm DNA fragmentation was not statistically different between swim-up-(14.4 ± 2.1%, p = 0.32) and density gradient centrifugation-processed (25.0 ± 3.0%, p = 0.20) and unprocessed semen samples (19.2 ± 1.9%). Sperm DNA fragmentation was significantly lower in swim-up-than in density gradient centrifugation-processed samples (p < 0.05). Sperm progressive motility was significantly higher (p < 0.05) in swim-up-(92.9 ± 1.0%) and density gradient centrifugation-processed (81.3 ± 2.0%) samples, with the former being higher, than in unprocessed semen samples (53.1 ± 3.7%). The recovery rate of progressive motile sperms was significantly lower in swim-up-(9.7 ± 1.4%) than in density gradient centrifugation-processed samples (17.2 ± 1.8%, p < 0.05). Conclusions: The swim-up method is superior to density gradient centrifugation, evidenced by less sperm DNA fragmentation and higher sperm progressive motility. The recovery rate of progressive motile sperms was better after density gradient centrifugation than after swim-up.