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Virus purification by cesium chloride (CsCl) density gradient, which generally requires an expensive ultracentrifuge, is an essential technique in virology. Here, we optimized virus purification by CsCl density gradient using general centrifugation (40,000 × g , 2 h, 4 °C), which showed almost the same purification ability as conventional CsCl density gradient ultracentrifugation (100,000 × g , 1 h, 4 °C) using phages S13′ and φEF24C. Moreover, adenovirus strain JM1/1 was also successfully purified by this method. We suggest that general centrifugation can become a less costly alternative to ultracentrifugation for virus purification by CsCl densiy gradient and will thus encourage research in virology.

PurposeTo compare the effect of two different sperm preparation techniques, including swim-up and gradient methods on sperm deoxyribonucleic acid (DNA) fragmentation status of semen samples from unexplained and mild male factor subfertile patients undergoing intrauterine insemination (IUI).DesignA prospective randomized study was conducted in 65 subfertile patients, including 34 unexplained and 31 male factor infertility to compare basal and post-procedure DNA fragmentation rates in swim-up and gradient techniques. Sperm DNA fragmentation rates were evaluated by a sperm chromatin dispersion (SCD) test in two portions of each sample of semen that was prepared with either swim-up or gradient techniques. Sperm motility and morphology were also assessed based on WHO 2010 criteria.ResultsSwim-up but not gradient method yielded a statistically significant reduction in the DNA fragmented sperm rate after preparation as compared to basal rates, in the semen samples of both unexplained (41.85 ± 22.04 vs. 28.58 ± 21.93, p < 0.001 for swim-up; and 41.85 ± 22.04 vs. 38.79 ± 22.30, p = 0.160 for gradient) and mild male factor (46.61 ± 19.38 vs. 30.32 ± 18.20, p < 0.001 for swim-up and 46.61 ± 19.38 vs. 44.03 ± 20.87, p = 0.470 for gradient) subgroups.ConclusionsSwim-up method significantly reduces sperm DNA fragmentation rates and may have some prognostic value on intrauterine insemination in patients with decreased sperm DNA integrity.

We present a miniaturized centrifugal platform that uses density centrifugation for separation and analysis of biological components in small volume samples (~5 μL). We demonstrate the ability to enrich leukocytes for on-disk visualization via microscopy, as well as recovery of viable cells from each of the gradient partitions. In addition, we simplified the traditional Modified Wright-Giemsa staining by decreasing the time, volume, and expertise involved in the procedure. From a whole blood sample, we were able to extract 95.15% of leukocytes while excluding 99.8% of red blood cells. This platform has great potential in both medical diagnostics and research applications as it offers a simpler, automated, and inexpensive method for biological sample separation, analysis, and downstream culturing.

Summary This work examines the effects of subsequent cycles of freezing–thawing on giant panda (Ailuropoda melanoleuca) sperm morphometry and function, and assesses whether density‐gradient centrifugation (DGC) can increase the number of freezing–thawing cycles this sperm can withstand. A sperm sample was collected by electroejaculation from a mature giant panda and subjected to five freezing–thawing cycles. Although repeated freezing–thawing negatively affected (P < 0.05) sperm motility and membrane integrity, in both nonselected and DCG‐selected sperm samples, >60% of the sperm cells in both treatments showed acrosome integrity even after the fifth freezing cycle. In fresh semen, the sperm head length was 4.7 μm, the head width 3.6 μm, area 14.3 μm2 and perimeter length 14.1 μm. The present results suggest that giant panda sperm trends to be resistant to repeated freezing–thawing, even without DGC selection.

BackgroundDespite the advantages of autologous fat transfer to the breast, there are many complications after the surgery, such as oil cysts, calcification and palpable breast nodules. The fat purification process is a key step to reduce those complications, but there is currently no standard processing method. This study was designed to compare the incidence of fat necrosis after autologous fat grafting to the breast with low-speed centrifugation and sedimentation.MethodsThis study analyzed 100 patients (167 breasts) who underwent autologous fat grafts to the breasts from January, 2015 to March, 2017. Patients were divided into two groups randomly, low-speed centrifugation (800 r/min) and sedimentation (15 min). Postoperative fat necrosis such as oil cysts and palpable breast nodules was measured using physical examination and breast ultrasound 3 months after the surgery. The number and the diameter of the fat necroses were detected.ResultsA total of 100 patients (167 breasts) were included this research. There were 21 breasts with clinically palpable nodules (12.57%); fifteen (19.48%) were in the low-speed centrifugation group and six (6.67%) were in the sedimentation group (p < 0.05). According to postoperative breast ultrasounds, there were 83 breasts with hypoechoic cyst formations (49.7%); forty-five (58.44%) in the low-speed centrifugation group and 38 (42.22%) in the sedimentation group (p < 0.05). A positive correlation between the number of operative sessions and fat necrosis was found out.ConclusionAlthough low-speed centrifugation could achieve higher fat purification efficacy when compared with sedimentation, it causes more fat necrosis than sedimentation, such as clinically palpable nodules and subclinical nodules, especially in patients who had a history of breast surgery and those who needed more than one fat grafting session.Level of Evidence IIIThis 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.

Lipoprotein lipase (LPL), the enzyme that hydrolyzes triglycerides in plasma lipoproteins, is assumed to be active only as a homodimer. In support of this idea, several groups have reported that the size of LPL, as measured by density gradient ultracentrifugation, is ∼110 kDa, twice the size of LPL monomers (∼55 kDa). Of note, however, in those studies the LPL had been incubated with heparin, a polyanionic substance that binds and stabilizes LPL. Here we revisited the assumption that LPL is active only as a homodimer. When freshly secreted human LPL (or purified preparations of LPL) was subjected to density gradient ultracentrifugation (in the absence of heparin), LPL mass and activity peaks exhibited the size expected of monomers (near the 66-kDa albumin standard). GPIHBP1-bound LPL also exhibited the size expected for a monomer. In the presence of heparin, LPL size increased, overlapping with a 97.2-kDa standard. We also used density gradient ultracentrifugation to characterize the LPL within the high-salt and low-salt peaks from a heparin-Sepharose column. The catalytically active LPL within the high-salt peak exhibited the size of monomers, whereas most of the inactive LPL in the low-salt peak was at the bottom of the tube (in aggregates). Consistent with those findings, the LPL in the low-salt peak, but not that in the high-salt peak, was easily detectable with single mAb sandwich ELISAs, in which LPL is captured and detected with the same antibody. We conclude that catalytically active LPL can exist in a monomeric state.

We present a microchannel-based adaptation of mass density-gradient centrifugation for the mass density measurement of cells or microparticles. The workflow consists of three basic steps: Microchannel filling, centrifugation, and microscopy. Microchannel filling and subsequent centrifugation of two liquids with different densities allows the instantaneous, precise, and repeatable generation of a one-dimensional mass-density gradient and the simultaneous sedimentation of cells or particles of interest to a point where their mass density is equal to the gradient’s mass density. We introduce two different methods, calibration particles and tracer molecules, for microscopic mass density readout. We demonstrate the measurement principle by measuring the mass density of yeast cells and show that the method allows the mass density measurement of single cells with a position dependent median uncertainty of 3.3   with a throughput of approximately 16000 cells per hour. This measurement precision is in the range of the best single-cell methods currently in use but with a higher throughput. The method is technically straightforward, robust, and affordable, making single-cell mass density measurements widely available.

Lipid nanoparticles (LNPs) have been widely utilized as carriers for nucleic acid drug delivery; however, their inherent heterogeneity impedes the accurate characterization of physicochemical and biological properties. Conventional analytical methods are inherently limited in resolving such heterogeneity. This study employed sucrose density gradient centrifugation (S-DGC) to separate LNP subpopulations of varying densities. It investigated the effects of lipid formulation parameters—including nitrogen-to-phosphorus (N/P) ratio, lipid composition, polyethylene glycol (PEG) concentration—and microfluidic preparation conditions (flow rate) on LNP heterogeneity and biological functionality. Formulation stability was assessed via freeze–thaw testing. The results demonstrated that S-DGC could effectively separate LNP subpopulations with divergent densities and physicochemical characteristics. Changes in the N/P ratio and lipid composition significantly modulate subphase distribution and properties. When cholesterol (Chol) and distearoylphosphatidylcholine (DSPC) are absent from the formulation, LNPs aggregate in the low-density layer (0–10% sucrose density layer). The concentration of PEGylated lipids serves as a critical regulatory factor. When the concentration increased from 0.5% to 2.5%, the LNP particle size decreased from approximately 202 nm to 118.7 nm. Furthermore, the S-DGC profile indicates that LNP transitions from an aggregated low-density distribution to a uniformly dense subpopulation concentrated within the 0–20% sucrose layer, where transfection efficiency is optimal. In freeze–thaw stability assessment, unprotected LNP exhibited a drastic decline in encapsulation efficiency to 5.3% after three freeze–thaw cycles at −80 °C. The S-DGC diagram revealed aggregation in the 20–30% high-density region. However, adding 5% sucrose maintained encapsulation efficiency above 96%. This study confirms that the S-DGC analytical platform serves as a potent tool for resolving LNP heterogeneity and correlating formulation structure with function. Based on these findings, this study contends that during the early prescription development of LNP-based nucleic acid therapeutics, formulation screening should not be confined to meeting overall particle size and encapsulation rate targets. Instead, S-DGC can be employed to proactively identify and minimize ineffective subpopulations (such as particles distributed in extremely high or low density zones), thereby enhancing product quality uniformity and predictability from the outset of R&D.

Abstract PurposeDeveloping optimized techniques for the isolation of human spermatozoa possessing low levels of DNA damage is an important objective for the ART industry. The purpose of this study was to compare a novel electrophoretic system (Felix™) of sperm isolation with a conventional method involving density gradient centrifugation (DGC).MethodsFive international ART Centres in Australia, India, Sweden, the USA, and China have collaborated in order to compare the quality of the sperm populations isolated by Felix™ and DGC in terms of processing time, sperm concentration, motility, vitality, and DNA integrity as assessed by 3 methods: SCSA, Halo, and TUNEL.ResultsAcross all centers, 112 comparisons were performed. Although significant differences were noted between centers in terms of the quality of the semen samples subjected for analysis, overall, both methods were equally capable of isolating populations of spermatozoa exhibiting high levels of vitality and progressive motility. The absolute numbers of spermatozoa recovered were significantly (p < 0.001) lower with the Felix™ device although sperm quality was higher with 4/5 centers reporting a significant improvement in DNA integrity relative to DGC (p < 0.01–p < 0.001). In practical terms, the Felix™ device featured a standardized 6 min preparation time whereas clinical DGC protocols varied from center to center but generally took around 40 min to complete.ConclusionsThe Felix™ device is a positive technical development capable of isolating suspensions of highly motile spermatozoa exhibiting low levels of DNA damage in a fraction of the time taken by conventional procedures such as DGC.

Does sperm preparation using the FERTILE PLUS™ Sperm Sorting Chip improve fertilization rates, blastocyst formation, utilization, and euploidy rates in patients undergoing intracytoplasmic sperm injection (ICSI), compared with density gradient centrifugation (DGC)? A single-cohort, retrospective data review including data from 53 couples who underwent ICSI cycles within a 12-month period. For each couple, the two closest, consecutive cycles were identified, where one used the standard technique of sperm preparation (DGC) and the subsequent used FERTILE PLUS™, therefore, couples acted as their own controls. Paired samples t -test was used to compare means for the outcomes (fertilization, blastocyst formation, utilization, and euploidy rates). Binary logistic regression analysis assessed the relationship between female age, the presence of male factor infertility, and euploidy rates. Blastocyst, utilization, and euploidy rates were significantly higher for cycles using FERTILE PLUS™ compared to DGC (76% vs 56%, p  = 0.002; 60% vs 41%, p  = 0.005, and 40% vs 20%, p  = 0.001, respectively). Although there was an increase in fertilization rates for cycles using FERTILE PLUS™, this was not significant (72% vs 68%, p  = 0.449). The euploidy rates of females ≤ 35 years were significantly increased when the FERTILE PLUS™ sperm preparation method was used, compared to the older age group (OR 2.31, p  = 0.007). No significant association was found between the presence or absence of male factor infertility and euploidy rates between the two cycles. This study provides tentative evidence that the FERTILE PLUS™ microfluidic sorting device for sperm selection can improve blastocyst formation, utilization, and euploidy rates following ICSI in comparison to the DGC method.