Relative Centrifugal Force Research Articles

Regenerative Capacity of Leukocyte-rich and Platelet-rich Fibrin in Indirect Sinus Elevation Procedure May be Dependent on Model-Specific Modification of the Centrifugation Cycle.

Context:To compare optical density (OD) and fibrinogen content of leukocyte-rich and platelet-rich fibrin (L-PRF) generated by standard protocol (2700 rotations per minute [RPM] for 12 min) versus relative centrifugal force (RCF)-adjusted protocol across two widely used laboratory centrifuges with swing-out rotors.Aims:Centrifuges for PRF production generate forces in excess of 800 g. The study aimed to evaluate OD, fibrinogen content and effectiveness in bone-added osteotome sinus floor elevation (BAOSFE) of leukocyte-rich and platelet-rich fibrin (L-PRF) generated by the standard protocol (2700 RPM for 12 min) versus a RCF-adjusted protocol to generate precisely 400 g of force across two centrifuges with swing-out rotors. The outcomes were compared to a standard centrifuge configured to generate L-PRF as per the original Choukroun guidelines.Settings and Design:Sample size for the present study was calculated using proportional power calculation. A minimum sample size of 8 per group was needed to detect a bone height difference of 2 mm when the power of the test is 0.80 at a significance level of 0.05.Subjects and Methods:Based on the centrifuge and protocol used to generate L-PRF, 10 participants were assigned to each of the following groups are as follows: D group, fixed angle centrifuge (DUO Quattro®) at default setting. R-O group: Swing-out centrifuge (Remi 8C®) + standard protocol. R-A group: Remi 8C® centrifuge + RCF-adjusted protocol. C-O group: Swing-out centrifuge (Remi C854®) + standard protocol. and C-A group: Remi C854® + RCF-adjusted protocol. OD, fibrinogen content, and gain in bone fill and bone height after BAOSFE were the evaluated outcomes.Statistical Analysis Used:Data were analyzed using GraphPad Prism® Software version 6.0 (GraphPad Software Inc., La Jolla, USA) and SAS Software® version 9.3 versions (SAS, New Delhi, India). Data were summarized by mean ± standard deviation for continuous data and median ± inter-quartile range for the score data. The comparison between different time points was done by analysis of one-way repeated measures test, followed by post hoc test for score data. The comparison between two groups for repeated data was made by analysis of two-way repeated measures test and followed by post hoc test. Spearman's Rho correlation test was used to test the correlation between prognosis and the other variables.Results:L-PRF from the Remi C854® centrifuge with RCF-adjusted protocol showed OD (P = 0.152) and fibrinogen content (P = 0.232) identical to those from the DUO Quattro® centrifuge. L-PRF from Remi 8C® centrifuge with the RCF-adjusted protocol resulted in maximum postoperative bone height gain (7.01 ± 1.44 mm) and bone fill (13.50 ± 4.51 mm2) which was higher than that of the outcomes from the DUO Quattro® centrifuge (6.82 ± 2.92 mm and 12.32 ± 5.31 mm2).Conclusions:A reduction in RCF resulted in a less dense clot and had a positive influence on the regenerative potential of L-PRF in BAOSFE procedure.

Computational modelling of water flow inside laboratory Knelson concentrator bowl

ABSTRACTEnhanced gravity concentrators such as Knelson concentrator (KC) are extensively used in the mineral processing industry. The complexities of KC bowl geometry and variation of feed characteristics have forced process engineers to design empirically new units using laboratory and pilot-scale Knelson concentrators. However, numerical modelling methods such as computational fluid dynamics (CFD) and discrete element method (DEM) provide a better insight of flow behaviour of fluid and particulate solid phases inside these processing units. This article reports findings of CFD simulations for single-phase water flow inside the laboratory KC. An available standard 7.5-cm laboratory KC bowl was numerically simulated using realisable k-ε turbulence model to resolve the turbulence dispersion of existing transitional flow regime. The effects of relative centrifugal force (RCF) intensity and bed fluidisation water flow rate on the water velocity and pressure distributions were studied. Simulations confirmed the swirling flow pattern governing inside the bowl. The results revealed that the impact of RCF intensity on the water field values is greater than that of bed fluidisation water flow rate. Both velocity and pressure variations inside the bowl rings followed a linear trend.

Note: Rotor design optimization for vacuum chamber-free ultracentrifuge.

This paper proposes a fixed-angle rotor design for a vacuum chamber-free ultracentrifuge using a rigid shaft and magnetic bearings. The rotor is designed for a stable operation in the ultracentrifugation region with air friction. And the optimal design parameters and optimized connection method of the rotor and rigid shaft are proposed. The proposed rotor structure can withstand a maximum rotation speed of 100 000 rpm and achieves a maximum relative centrifugal force of 600 000 g. To verify the performance of the proposed rotor design with a rigid shaft, finite-element analysis, an impact test, and a drive operating experiment were conducted.

Sea ice centrifugal desalination based on microwave heating

Desalinating sea ice to obtain fresh water is a new way to solve the water shortage. Centrifugal desalination (CD) is an important method for desalination of sea ice, and it is of great significance to improve desalination efficiency. Microwave heating has a significant effect on improving salt-water separation efficiency, but the effect on centrifugal desalination is still unclear. In this paper, sea ice centrifugal desalination by microwave heating (CDBMH) was designed. The four factors of the particle size of ice, initial ice temperature, relative centrifugal force and centrifugation time were selected from the key parameters of centrifugal technology and sea ice material properties, and the impact of each factor on the salinity of the melted water, the desalination rate, and the water yield rate was explored. The results showed that with the decrease of the particle size of ice, the increase of initial ice temperature, relative centrifugal force and centrifugation time, the desalination rate showed an upward trend, while the water yield rate and the salinity of the melted water gradually decreased. As a result of the CDBMH process, the desalination rate was 93.3%, and the water yield rate was 72.4%, and the salinity of the melted water was 0.4‰.

Modified Two-Step Method to Prepare Long-Term Stable CNT Nanofluids for Heat Transfer Applications

CNT nanofluids are getting attention in heat transfer applications due to their very high thermal conductivity in comparison with conventional fluids. For commercial exploitation of CNT nanofluids as heat transfer media, they must have long-term stability. In this study, the two-step method was modified to prepare dynamically stable CNT nanofluids by utilizing commercial grade multiwalled carbon nanotubes and SDBS as a surfactant. The modified technique consists of separation of coarse agglomerates of CNT from the CNT nanofluids by applying centrifugal action after its preparation. The effect of relative centrifugal force was also studied for the very first time on the stable concentration of CNT nanofluids. The stability of CNT nanofluids was analyzed by measurement of their CNT concentration and Zeta potential. Results showed that CNT nanofluids possess good stability and remain stable for more than 15 months. In addition to stability, thermo-physical properties such as thermal conductivity, density, and viscosity of CNT nanofluids were also measured. The results of this study elucidated the effect of RCF on the stable concentration of CNT nanofluids. It is expected that the results obtained in this study may significantly contribute to the proper tailoring of CNT nanofluids, by providing long-term stable CNT nanofluids which are suitable for industrial heat transfer applications.

Effective surface-enhanced Raman scattering of randomly branched gold nano-urchins with Rhodamine 6G as Raman reporters

Gold nano-urchins (AuNUs) can be effectively used in surface-enhanced Raman scattering (SERS) because of the sharp tips that grow randomly on the surface. However, it is difficult to control the multiple tips of AuNUs given the variation in their number, size, and distance from the core. Furthermore, it is difficult to achieve effective adsorption of Raman probe molecules on the rough and uneven surfaces of AuNUs. Because of these problems, it is doubtful whether they can be effective SERS substrates. Herein, we present a method for effective SERS using branched AuNUs and Rhodamine 6G (R6G) probe molecules at 785nm, close to their localized surface plasmon resonance wavelength. We investigated the effect of several factors on the enhancement of SERS, including binding time, centrifugation speed and time, ratio of AuNUs and probe molecules, and stirring time. The enhancement of SERS was effectively achieved with a set of conditions that included a AuNUs:R6G concentration ratio of 8:1, a binding time of 6–8h, centrifuging for 40min at a relative centrifugal force of 2400, and no stirring during sample preparation. The results provide important information on effective adsorption of R6G Raman probe molecules on the branched and uneven surfaces of AuNUs for effective enhancement of SERS.

A low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo

Platelet-rich fibrin (PRF) is generated from the patients’ own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors’ concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.

Controversies related to scientific report describing g-forces from studies on platelet-rich fibrin: Necessity for standardization of relative centrifugal force values

Leukocyte and platelet-rich fibrin (PRF), a second-generation platelet concentrate has been the focus of intensive research endeavors over the last 2 decades. Over the years, numerous reports have however failed to accurately report g-force values which have caused considerable confusion in the field. These values have since been re-transcribed incorrectly in many studies moving forward, and this article aims to address this topic to avoid further confusion in the field. We address several reports in which PRF centrifugal g-forces have been calculated at the PRF clot (referred to as relative centrifugal force [RCF]-clot) as opposed to the international standard method described at the bottom of centrifugation tubes (RCF-max). We further highlight how RCF-clot is not only a deviation from the standard international method used to report g-force values, but one subject to significant error owing to centrifugation time, patient hematocrit levels, initial volume of blood collected, and other factors. For these reasons and those further reported throughout this article, we address this controversy in detail to avoid further confusion regarding the report of g-force values in future studies. Furthermore, we propose a standardization regarding the accurate report of g-force values in future studies investigating PRF at the RCF-max.

Preanalytics of urine sediment examination: effect of relative centrifugal force, tube type, volume of sample and supernatant removal.

IntroductionLaboratories often modify procedures recommended by the European Urinalysis Guidelines for urine sediment analysis. The aim of this study was to compare the recommended protocol with our routine laboratory procedure and to evaluate the possible impact of modifications in the relative centrifugal force, type of tube, method of supernatant aspiration and urine volume on patient’s results.Material and methodsFirstly, relative centrifugal force was investigated using 20 pairs of samples examined after centrifugation at 400xg and 1358xg. In phase two, 110 samples were examined, paired as: round bottom vs conical tube (N = 46), decanting vs suction of supernatant (N = 100) and 10 mL vs 5 mL of urine sample (N = 101).ResultsThe number of erythrocytes, leukocytes and squamous epithelial cells was significantly lower after centrifugation at 400xg (P = 0.001, 0.002 and 0.004, respectively). The number of leukocytes was significantly lower in conical tubes (P = 0.010), after the suction of supernatant (P = 0.045) and in 5 mL urine (P < 0.001). The number of squamous epithelial cells was significantly lower after the suction of supernatant (P < 0.001) and in 5 mL urine (P < 0.001). The number of erythrocytes (P < 0.001), total non-hyaline casts (P < 0.001) and the frequency of granular casts (P = 0.039) was significantly lower in 5 mL urine.ConclusionLower results of leukocytes, erythrocytes, squamous cells and non-hyaline casts were recorded in recommended procedures (centrifugation at 400xg, suction of supernatant, conical tube, 5 mL of sample) than in routine procedure (centrifugation at 1358xg, decanting of supernatant, round bottom tube, 10 mL) used in our laboratory.

Reduction of the relative centrifugal force influences cell number and growth factor release within injectable PRF-based matrices.

Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60-966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-β1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-β1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system.

A simple method for removing low-density granulocytes to purify T lymphocytes from peripheral blood mononuclear cells.

Low-density granulocytes (LDGs) can form neutrophil extracellular traps (NETs) spontaneously and excessively. When peripheral blood mononuclear cells (PBMCs) are used for studying T lymphocytes, LDGs contained in the PBMCs may decrease the threshold of activating T lymphocytes by forming NETs. This study focused on the profiles of LDGs in common autoimmune diseases and methods for removing LDGs from PBMCs. The percentages of LDGs in PBMCs from 55 patients with dermatomyositis (DM), 15 with polymyositis (PM), 42 with rheumatoid arthritis (RA), 25 with systemic lupus erythematosus (SLE), and 19 healthy controls were determined by flow cytometry. Three methods of removing LDGs were explored and compared. After removal, PBMCs from six patients with positive T-SPOT.TB were tested again to find out if LDGs contained in the PBMCs could influence T lymphocyte reactions. Significantly higher LDG percentages were found in PBMCs from patients with DM ((8.41±10.87)%, P<0.0001), PM ((8.41±10.39)%, P<0.0001), RA ((4.05±6.97)%, P=0.0249), and SLE ((7.53±11.52)%, P=0.0006), compared with the controls ((1.28±0.73)%). The T-SPOT.TB values significantly decreased after LDGs were removed. Increasing relative centrifugal force (RCF) within a limited range can decrease the LDG percentage from an initial high level, but not markedly increase the LDG clearance rate. Compared with the whole blood sediment method, the PBMC adherence method can significantly remove LDGs yet scarcely influence the T lymphocyte percentage in PBMCs. The LDG percentage in PBMCs is significantly increased in patients with SLE, DM, PM, and RA. The influence of LDGs on T lymphocytes cannot be ignored in PBMC cultures. The adherence method is a simple and easy-to-use method for removing LDGs and purifying T lymphocytes from PBMCs.

Optimisation of a double-centrifugation method for preparation of canine platelet-rich plasma

BackgroundPlatelet-rich plasma (PRP) has been expected for regenerative medicine because of its growth factors. However, there is considerable variability in the recovery and yield of platelets and the concentration of growth factors in PRP preparations. The aim of this study was to identify optimal relative centrifugal force and spin time for the preparation of PRP from canine blood using a double-centrifugation tube method.MethodsWhole blood samples were collected in citrate blood collection tubes from 12 healthy beagles. For the first centrifugation step, 10 different run conditions were compared to determine which condition produced optimal recovery of platelets. Once the optimal condition was identified, platelet-containing plasma prepared using that condition was subjected to a second centrifugation to pellet platelets. For the second centrifugation, 12 different run conditions were compared to identify the centrifugal force and spin time to produce maximal pellet recovery and concentration increase. Growth factor levels were estimated by using ELISA to measure platelet-derived growth factor-BB (PDGF-BB) concentrations in optimised CaCl2-activated platelet fractions.ResultsThe highest platelet recovery rate and yield were obtained by first centrifuging whole blood at 1000 g for 5 min and then centrifuging the recovered platelet-enriched plasma at 1500 g for 15 min. This protocol recovered 80% of platelets from whole blood and increased platelet concentration six-fold and produced the highest concentration of PDGF-BB in activated fractions.ConclusionsWe have described an optimised double-centrifugation tube method for the preparation of PRP from canine blood. This optimised method does not require particularly expensive equipment or high technical ability and can readily be carried out in a veterinary clinical setting.

A facile method for preparation of emulsion using the high gravity technique

This study demonstrates a new process for preparation of oil-in-water (O/W) emulsion using the high gravity technique. This involves a mixture of oil and water that are passed through a rotating packed bed, under a high shear force, from which oil is emulsified into tiny droplets and subsequently dispersed in water. The process is cycled in order to break the droplets repeatedly and achieve an emulsion with small size and low polydispersity index (PDI). The advantage of the high gravity technique is that the emulsions with a desired size and polydispersity can be rapidly obtained by tuning experimental parameters, such as relative centrifugal force (RCF), cycle times (CT), liquid flow rate (VL) and surfactant concentration (Csurfactant). The size of emulsions is shown to decrease with increasing RCF, CT, VL and Csurfactant. The PDI of emulsion prepared by high gravity technique is also much improved in comparison to that prepared by conventional sonication, which is further confirmed with dynamic light scattering and confocal imaging characterization. To provide an additional perspective on the high gravity technique as a tool to make O/W emulsions, uniformly distributed liquid crystal droplets were prepared by using the high gravity technique, which have been well studied for their in situ chemical and biological detection. In short, the high gravity technique for preparing emulsions is facile, fast and can be potentially applied for large scale industry applications.

Extraction of soil solution by drainage centrifugation\u2014effects of centrifugal force and time of centrifugation on soil moisture recovery and solute concentration in soil moisture of loess subsoils

The solute concentration in the subsoil beneath the root zone is an important parameter for leaching assessment. Drainage centrifugation is considered a simple and straightforward method of determining soil solution chemistry. Although several studies have been carried out to determine whether this method is robust, hardly any results are available for loess subsoils. To study the effect of centrifugation conditions on soil moisture recovery and solute concentration, we sampled the subsoil (1.5–3.0 m depth) at commercial farms in the loess region of the Netherlands. The effect of time (20, 35, 60, 120 and 240 min) on recovery was studied at two levels of the relative centrifugal force (733 and 6597g). The effect of force on recovery was studied by centrifugation for 35 min at 117, 264, 733, 2932, 6597 and 14,191g. All soil moisture samples were chemically analysed. This study shows that drainage centrifugation offers a robust, reproducible and standardised way for determining solute concentrations in mobile soil moisture in silt loam subsoils. The centrifugal force, rather than centrifugation time, has a major effect on recovery. The maximum recovery for silt loams at field capacity is about 40%. Concentrations of most solutes are fairly constant with an increasing recovery, as most solutes, including nitrate, did not show a change in concentration with an increasing recovery.

Centrifugal Microfluidic Integrated Optical System for Sensing Pesticide Residues

We proposed and demonstrated an effective and non-destructive centrifugal microfluidic sensor (CMS) for analysis of organophosphorus pesticides (OPPs). The CMS was designed to have a sequential sample injection with two capillary valves. The actuation mechanism of CMS was controlled by centrifugal force, i.e. relative centrifugal force (×g). The OPPs analyte in CMS could be successfully derived into a mixer at 34 g and then spined into a detection reservoir at 537 g. The experiment results indicated that limit of detection for OPPs using CMS was 0.05 ppm, a linear relationship between OPPs logarithmic concentration (ppm) and inhibition rate (%) was 0.9215. Furthermore, series fresh vegetables were extracted and assayed for OPPs residue detection by using CMS. It was effective for OPPs detection. A non-destructive, rapid, reliable, and sensitive approach to OPPs analysis was demonstrated. This in turn opens up opportunities for healthcare applications, notably environmental monitoring, foods safety and other fields.

Unbound fraction of fluconazole and linezolid in human plasma as determined by ultrafiltration: Impact of membrane type.

Ultrafiltration is a rapid and convenient method to determine the free concentrations of drugs in plasma. Several ultrafiltration devices based on Eppendorf cups are commercially available, but are not validated for such use by the manufacturer. Plasma pH, temperature and relative centrifugal force as well as membrane type can influence the results. In the present work, we developed an ultrafiltration method in order to determine the free concentrations of linezolid or fluconazole, both neutral and moderately lipophilic antiinfective drugs for parenteral as well as oral administration, in plasma of patients. Whereas both substances behaved relatively insensitive in human plasma regarding variations in pH (7.0-8.5), temperature (5-37°C) or relative centrifugal force (1000-10.000xg), losses of linezolid were observed with the Nanosep Omega device due to adsorption onto the polyethersulfone membrane (unbound fraction 75% at 100mg/L and 45% at 0.1mg/L, respectively). No losses were observed with Vivacon which is equipped with a membrane of regenerated cellulose. With fluconazole no differences between Nanosep and Vivacon were observed. Applying standard conditions (pH 7.4/37°C/1000xg/20min), the mean unbound fraction of linezolid in pooled plasma from healthy volunteers was 81.5±2.8% using Vivacon, that of fluconazole was 87.9±3.5% using Nanosep or 89.4±3.3% using Vivacon. The unbound fraction of linezolid was 85.4±3.7% in plasma samples from surgical patients and 92.1±6.2% in ICU patients, respectively. The unbound fraction of fluconazole was 93.9±3.3% in plasma samples from ICU patients.

Cationic gemini surfactant with cleavable spacer: Emulsion stability

In this study, a cationic gemini surfactant with cleavable spacer and two monomeric single chain counterparts were synthesized and used. The critical micelle concentration (CMC) was measured by tensiometry for these surfactants. The emulsion stability of (Toluene 30:70 Water) emulsions in the presence of these surfactants and at various concentrations was determined by two dispersion testers (i.e. Turbiscan® under gravity force and Lumisizer® analytical centrifuge). The surfactant critical concentration for the emulsion stability was found to be at 2×CMC. The emulsion stability in the presence of these surfactants followed the same trend and remained almost constant at surfactant concentration (2–6×CMC). The gemini surfactant with lower CMC value is far better an emulsion stabilizer on concentration basis than the monomeric surfactants. The phase separation velocities of the emulsions as a function of the relative centrifugal force were fitted with linear and power correlations. The results showed that the estimated separation velocities at earth’s gravity (1×g) are larger than the measured values by Turbiscan for all of the surfactants. While the accelerated phase separation by Lumisizer is a fast and easy method for testing the emulsion stability and comparing the predicted shelf lives, the extrapolation and estimation of the real emulsion stability should be considered cautiously.

Development of a 3D circular microfluidic centrifuge for the separation of mixed particles by using their different centrifuge times

This paper presents a circular microfluidic centrifuge with two inlets and two outlets to separate mixed microparticles with a specially designed sample injection hole. To separate the mixed particles, it uses a rotational flow, generated in a chamber by counter primary flows in the microchannels. The shape and sizes of the circular microfluidic centrifuge have been designed through numerical evaluation to have a large relative centrifugal force. The difference of centrifuge times of the mixed particles of 1 μm and 6 μm was determined to be 8.2 s at an inlet Reynolds number of 500 and a sample Reynolds number of 20. In the experiment, this was measured to be about 10 s. From the separation of the two polymer particles analogous to the representative sizes of platelets and red blood cells, the circular microfluidic centrifuge shows a potential to separate human blood cells size-selectively by using the difference of centrifuge times.

An optimized method for high-titer lentivirus preparations without ultracentrifugation.

Lentiviral technology has proven to be a powerful tool to express exogenous genes in dividing and non-dividing cells. Currently, most protocols for generating high-titer lentivirus require ultracentrifugation, which can be an instrumental barrier for routine operations in a laboratory. In this study, the effect of relative centrifugal force (RCF) on the concentration efficiency of the lentivirus was systematically explored, and it was found that sucrose gradient centrifugation with a relatively low speed (≤10,000 g) robustly produces a high-titer virus (up to 2 × 108 TU/ml). The optimal sucrose concentration is 10%, and the recovery rate of the functional virus is greater than 80%. The infection efficiency of both concentrated and un-concentrated lentivirus decreases rapidly when the viruses are stored at 4 °C (τ ≈ 1.3 days) or subjected to multiple freeze-thaw cycles (τ = 1.1 rounds). In summary, we describe an efficient and easy-to-handle protocol for high-titer lentivirus purification.

Optimization and development of stable w/o/w cosmetic multiple emulsions by means of the Quality by Design approach.

The aim of our present work was to develop stable water-in-oil-in-water (w/o/w) cosmetic multiple emulsions that are proper for cosmetic use and can also be applied on the skin as pharmaceutical vehicles by means of Quality by Design (QbD) concept. This product design concept consists of a risk assessment step and also the 'predetermination' of the critical material attributes and process parameters of a stable multiple emulsion system. We have set up the hypothesis that the stability of multiple emulsions can be improved by the development based on such systematic planning - making a map of critical product parameters - so their industrial usage can be increased. The risk assessment and the determination of critical physical-chemical stability parameters of w/o/w multiple emulsions to define critical control points were performed by means of quality tools and the leanqbd(™) (QbD Works LLC, Fremont, CA, U.S.A.) software. Critical materials and process parameters: Based on the results of preformulation experiments, three factors, namely entrapped active agent, preparation methodology and shear rate, were found to be highly critical factors for critical quality attributes (CQAs) and for stability, whereas the nature of oil was found a medium level risk factor. The results of the risk assessment are the following: (i) droplet structure and size distribution should be evaluated together to be able to predict the stability issues, (ii) the presence of entrapped active agents had a great impact on droplet structure, (iii) the viscosity curves represent the structural changes during storage, if the decrease in relative viscosity is >15% the emulsion disintegrates, and (iv) it is enough to use the shear rate between 34g and 116g relative centrifugal force (RCF). CQAs: By risk assessment, we discovered that four factors should be considered to be high-risk variables as compared to others: droplet size, droplet structure, viscosity and multiple character were found to be highly critical attributes. The preformulation experiment is the part of a development plan. On the basis of these results, the control strategy can be defined and a stable multiple emulsion can be ensured that meets the relevant stakeholders' quality expectations.