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Objective The purpose of this study was to evaluate the yield, viability, clinical safety, and efficacy of the stromal vascular fraction (SVF) separated with a new protocol with all clinical-grade drugs. Materials and Methods SVF cells were isolated from lipoaspirate obtained from 13 participants aged from 30 to 56 years by using a new clinical protocol and the laboratory protocol. The cell yield, viability, morphology, mesenchymal stem cell (MSC) surface marker expression, and differentiation abilities of the SVF cells harvested from the two protocols were compared. Furthermore, three related clinical trials were conducted to verify the safety and efficiency of SVF cells isolated by the new clinical protocol. Results There were no significant differences in the yield, viability, morphology, and differentiation potential of the SVFs isolated with the clinical protocol and laboratory protocol. Adipose-derived mesenchymal stem cell (ASC) surface marker expression, including that of CD14, CD31, CD44, CD90, CD105, and CD133, was consistent between the two protocols. Clinical trials have demonstrated the effectiveness of the SVF isolated with the new clinical protocol in improving skin grafting, promoting mechanical stretch-induced skin regeneration and improving facial skin texture. No complications occurred. Conclusion SVF isolated by the new clinical protocol had a noninferior yield and viability to that of the SVF separated by the laboratory protocol. SVFs obtained by the new protocol can be safely and effectively applied to improve skin grafting, promote mechanical stretch-induced skin regeneration, and improve facial skin texture. Trial Registration The trials were registered with the ClinicalTrials.gov ( NCT03189628 ), the Chinese Clinical Trial Registry ( ChiCTR2000039317 ), and the ClinicalTrials.gov ( NCT02546882 ). All the three trials were not patient-funded trials. 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 .

PurposeTo evaluate the efficacy of magnetic-activated cell sorting (MACS) or testicular sperm aspiration (TESA) to improve reproductive outcomes in cases with elevated sperm DNA fragmentation undergoing assisted reproduction.MethodsThis randomized controlled trial included couples with failed IVF cycles and sperm DNA fragmentation > 30%. Sperm DNA fragmentation was assessed using the sperm chromatin structure assay (SCSA) method. Participants were randomly assigned to either the MACS or TESA group. Testicular sperm retrieval was performed for the TESA group, while MACS involved sperm selection using magnetic beads. Extended blastocyst culture, freeze all policy of blastocysts by vitrification, and frozen embryo transfer were undertaken as per clinic’s standard operating protocols. Blastocyst formation rate, implantation rate, miscarriage rate, multiple pregnancy rate, and live birth rate were analyzed and compared between MACS and TESA groups.ResultsThere were no significant differences in female age, male age, or sperm DNA fragmentation index (DFI) between the MACS and TESA groups. The blastocyst conversion rate was slightly higher in the TESA group (39%) compared to the MACS group (32%). However, the MACS group had a higher implantation rate (50%) than the TESA group (35%). Miscarriage rates, multiple pregnancy rates, and live birth rates did not show statistically significant differences between the groups. A chi-squared test was conducted to compare categorical variables, and t-tests were done to compare continuous variables.ConclusionIn cases with raised sperm DNA fragmentation, sperm selection by MACS or TESA seems to offer comparable reproductive outcomes. There seems no superiority of one intervention over the other in cases with raised sperm DNA fragmentation undergoing assisted reproduction. Both interventions seem to be beneficial for couples seeking assisted reproduction with raised sperm DNA fragmentation.

PurposeTo know which sperm selection technique, physiological intracytoplasmic sperm injection (PICSI) or magnetic-activated cell sorting (MACS), is better for the selection of sperm with abnormal sperm DNA fragmentation (SDF) in patients undergoing intracytoplasmic sperm injection (ICSI).MethodsA prospective randomized trial included 413 ICSI cases with abnormal SDF (> 20.3%) by TUNEL assay. Patients with at least 1 million total progressive motile sperm count were randomized to PICSI or MACS groups on the day of ICSI. PICSI depends on the hyaluronan binding of better SDF sperm where individual sperm was selected, while MACS selects non-apoptotic sperm population using Annexin V magnetic beads. All pre-implantation embryogenic parameters were observed and the main outcome was the ongoing pregnancy rate.ResultsThere were no significant differences between patients allocated to PICSI and MACS in the studied parameters including pre-implantation embryological data, implantation, clinical pregnancy, and ongoing pregnancy rates. Meanwhile, sub-analysis according to the female age has shown that female patients with less than 30 years of age in the MACS group had significantly higher good-quality blastocyst, clinical pregnancy, and ongoing pregnancy rates than the PICSI group. However, the higher implantation (p = 0.051), clinical pregnancy (p = 0.078), and ongoing pregnancy (p = 0.097) rates observed in females between 30 and 35 years of age in the PICSI group did not reach significance level.ConclusionsPICSI and MACS are efficient techniques for sperm selection in cases with abnormal sperm DNA fragmentation. However, MACS is preferred when the females are younger than 30 years, while PICSI is preferred in older females.Clinical trial registration numberNCT03398317 (retrospectively registered)

Potency is a quantitative measure of the desired biological function of an advanced therapy medicinal product (ATMP) and is a prerequisite for market approval application (MAA). To assess the potency of human skeletal muscle-derived cells (SMDCs), which are currently investigated in clinical trials for the regeneration of skeletal muscle defects, we evaluated acetylcholinesterase (AChE), which is expressed in skeletal muscle and nervous tissue of all mammals. CD56+ SMDCs were separated from CD56- SMDCs by magnetic activated cell sorting (MACS) and both differentiated in skeletal muscle differentiation medium. AChE activity of in vitro differentiated SMDCs was correlated with CD56 expression, fusion index, cell number, cell doubling numbers, differentiation markers and compared to the clinical efficacy in patients treated with SMDCs against fecal incontinence. CD56- SMDCs did not form multinucleated myotubes and remained low in AChE activity during differentiation. CD56+ SMDCs generated myotubes and increased in AChE activity during differentiation. AChE activity was found to accurately reflect the number of CD56+ SMDCs in culture, their fusion competence, and cell doubling number. In patients with fecal incontinence responding to SMDCs treatment, the improvement of clinical symptoms was positively linked with the AChE activity of the SMDCs injected. AChE activity was found to truly reflect the in vitro differentiation status of SMDCs and to be superior to the mere use of surface markers as it reflects not only the number of myogenic SMDCs in culture but also their fusion competence and population doubling number, thus combining cell quality and quantification of the expected mode of action (MoA) of SMDCs. Moreover, the successful in vitro validation of the assay proves its suitability for routine use. Most convincingly, our results demonstrate a link between clinical efficacy and the AChE activity of the SMDCs preparations used for the treatment of fecal incontinence. Thus, we recommend using AChE activity of in vitro differentiated SMDCs as a potency measure in end stage (phase III) clinical trials using SMDCs for skeletal muscle regeneration and subsequent market approval application (MAA).

The ability to isolate rare live cells within a heterogeneous population based solely on visual criteria remains technically challenging, due largely to limitations imposed by existing sorting technologies. Here, we present a new method that permits labeling cells of interest by attaching streptavidin-coated magnetic beads to their membranes using the lasers of a confocal microscope. A simple magnet allows highly specific isolation of the labeled cells, which then remain viable and proliferate normally. As proof of principle, we tagged, isolated, and expanded individual cells based on three biologically relevant visual characteristics: i) presence of multiple nuclei, ii) accumulation of lipid vesicles, and iii) ability to resolve ionizing radiation-induced DNA damage foci. Our method constitutes a rapid, efficient, and cost-effective approach for isolation and subsequent characterization of rare cells based on observable traits such as movement, shape, or location, which in turn can generate novel mechanistic insights into important biological processes. When scientists use microscopes to look at cells, they often want to then isolate certain cells based on how these look like. For example, researchers may want to select cells with specific shapes, movements or division rates, because these visual clues give important information about how the cells may be behaving in the body. However, it remains difficult to precisely pick a few live cells within a bigger sample. To address this problem, Binan et al. created a new approach, called single cell magneto-optical capture (scMOCa), to set aside specific cells within a larger population. The technique uses the lasers present on confocal microscopes to attach tiny metallic beads to the surface of chosen cell. Then, a magnetic field is applied to gently pull the cell to a new location. The method is cheap – it relies on commonly available research tools – and it works on a broad variety of cells. In the future, scMOCa could be used to capture and then grow cells that can only be recognized by how they look or behave, which will help to study them in greater details.

Background Interstitial lung disease (ILD) can be a severe extra-articular disease manifestation in Rheumatoid Arthritis (RA). A potential role of fibrocytes in RA associated ILD (RA-ILD) has not previously been described. We present a modified faster method for measuring circulating fibrocytes, without intracellular staining. The results are compared to the traditional culture method, where the number of monocytes that differentiate into mature fibrocytes in vitro are counted. The results are following compared to disease activity in patients with severe asthma, ILD, RA (without diagnosed ILD) and RA with verified ILD (RA-ILD). Method CD45 + CD34 + CD11b + (7-AAD − CD3 − CD19 − CD294 − ) cells were isolated by cell sorting and stained for pro-collagen type 1. Thirty-nine patients (10 RA, 9 ILD and 10 with severe asthma, 10 with RA-ILD) and 10 healthy controls (HC) were included. Current medication, disease activity, pulmonary function test and radiographic data were collected. Circulating fibrocytes were quantified by flow cytometry. Peripheral blood mononuclear cells were isolated and cultured for 5 days and the numbers of mature fibrocytes were counted. Results 90.2% (mean, SD = 1.5%) of the sorted cells were pro-collagen type 1 positive and thereby fulfilled the criteria for being circulating fibrocytes. The ILD and RA-ILD groups had increased levels of circulating fibrocytes compared to HC ( p  < 0.05). Levels of circulating fibrocytes correlated overall to number of monocytes that subsequently in vitro differentiated to mature fibrocytes ( r  = 0.81, p  < 0.001). RA patients with pathologically reduced diffusion capacity for carbon monoxide adjusted for hemoglobin (DLCO c ) in both the RA and in the combined RA + RA-ILD group, had significantly higher levels of both circulating and number of cultured mature fibrocytes (both p  < 0.05). In both groups, the level of circulating fibrocytes and number of mature fibrocytes in culture also correlated to a reduction in DLCO c ( r  = −0.61 an r  = −0.58 both p  < 0.05). Conclusions We presented a fast and valid method for measuring circulating fibrocytes using flow cytometry on lysed peripheral blood. Further, we showed for the first time, that the level of circulating fibrocytes correlated with the number of peripheral blood mononuclear cells, that differentiated into mature fibrocytes in vitro. Reduced DLCO c was correlated with high levels of circulating and mature fibrocytes in RA, which have not been reported previously. In such, this study suggests that fibrocytes may exhibit an important role in the pathogenesis of RA-ILD, which requires further clarification in future studies. Trial registration ClinicalTrials.gov : NCT02711657 , registered 13/3–2016, retrospectively registered.

Purpose The purpose of this study is to assess outcomes after magnetic-activated cell sorting (MACS) technology on obstetric and perinatal outcomes compared with those achieved after swim up from randomized controlled trial. Methods This is a two-arm, unicentric, prospective, randomized, and triple-blinded trial and has a total of 237 infertile couples, between October 2010 and January 2013. A total of 65 and 66 newborns from MACS and control group, respectively, were described. Results MACS had no clinically relevant adverse effects on obstetric and perinatal outcomes. No differences were found for obstetric problems including premature rupture of membranes 6.1% (CI95% 0–12.8) vs. 5.9% (CI95% 0–12.4), 1st trimester bleeding 28.6% (CI95% 15.9–41.2) vs. 23.5% (CI95% 11.9–35.1), invasive procedures as amniocentesis 2.0% (CI95% 0–5.9) vs. 3.9% (CI95% 0–9.2), diabetes 14.3% (CI95% 4.5–24.1) vs. 9.8% (CI95% 1.6–17.9), anemia 6.1% (CI95% 0–12.8) vs. 5.9%(CI95% 0–12.4), 2nd and 3rd trimesters 10.2% (CI95% 1.7–18.7) vs. 5.9% (CI95% 0–12.4), urinary tract infection 8.2% (CI95% 0.5–15.9) vs. 3.9% (CI95% 0–9.2), pregnancy-induced hypertension 6.1% (CI95% 0–12.8) vs. 15.7% (CI95% 5.7–25.7), birth weight (g) 2684.10 (CI95% 2499.48–2868.72) vs. 2676.12 (CI95% 2499.02–2852.21), neonatal height (cm) 48.3 (CI95% 47.1–49.4) vs. 46.5 (CI95% 44.6–48.4), and gestational cholestasis 0%(CI95% 0–0) vs. 3.9% (CI95% 0–9.2), respectively, in MACS group compared with control group. Conclusions Our data suggest that MACS technology does not increase or decrease Powered by Editorial Manager ® and ProduXion Manager ® from Aries Systems Corporation adverse obstetric and perinatal outcomes in children conceived when this technology was performed, being the largest randomized control trial with live birth reported results with MACS.

In fluorescence-activated cell sorting, characteristic target features are labeled with a specific fluorophore, and cells displaying different fluorophores are sorted. Ota et al. describe a technique called ghost cytometry that allows cell sorting based on the morphology of the cytoplasm, labeled with a single-color fluorophore. The motion of cells relative to a patterned optical structure provides spatial information that is compressed into temporal signals, which are sequentially measured by a single-pixel detector. Images can be reconstructed from this spatial and temporal information, but this is computationally costly. Instead, using machine learning, cells are classified directly from the compressed signals, without reconstructing an image. The method was able to separate morphologically similar cell types in an ultrahigh-speed fluorescence imaging–activated cell sorter. Science , this issue p. 1246 Morphology-based cell classification and sorting is achieved at high accuracy and throughput without obtaining images. Ghost imaging is a technique used to produce an object’s image without using a spatially resolving detector. Here we develop a technique we term “ghost cytometry,” an image-free ultrafast fluorescence “imaging” cytometry based on a single-pixel detector. Spatial information obtained from the motion of cells relative to a static randomly patterned optical structure is compressively converted into signals that arrive sequentially at a single-pixel detector. Combinatorial use of the temporal waveform with the intensity distribution of the random pattern allows us to computationally reconstruct cell morphology. More importantly, we show that applying machine-learning methods directly on the compressed waveforms without image reconstruction enables efficient image-free morphology-based cytometry. Despite a compact and inexpensive instrumentation, image-free ghost cytometry achieves accurate and high-throughput cell classification and selective sorting on the basis of cell morphology without a specific biomarker, both of which have been challenging to accomplish using conventional flow cytometers.

Significance We have developed a unique approach for the separation of particles and biological cells through standing surface acoustic waves oriented at an optimum angle to the fluid flow direction in a microfluidic device. This experimental setup, optimized by systematic analyses, has been used to demonstrate effective separation based on size, compressibility, and mechanical properties of particles and cells. The potential of this method for biological–biomedical applications was demonstrated through the example of isolating MCF-7 breast cancer cells from white blood cells. The method offers a possible route for label-free particle or cell separation for many applications in research, disease diagnosis, and drug-efficacy assessment.

Circulating tumor cells (CTCs) are important targets for cancer biology studies. To further elucidate the role of CTCs in cancer metastasis and prognosis, effective methods for isolating extremely rare tumor cells from peripheral blood must be developed. Acoustic-based methods, which are known to preserve the integrity, functionality, and viability of biological cells using label-free and contact-free sorting, have thus far not been successfully developed to isolate rare CTCs using clinical samples from cancer patients owing to technical constraints, insufficient throughput, and lack of long-term device stability. In this work, we demonstrate the development of an acoustic-based microfluidic device that is capable of high-throughput separation of CTCs from peripheral blood samples obtained from cancer patients. Our method uses tilted-angle standing surface acoustic waves. Parametric numerical simulations were performed to design optimum device geometry, tilt angle, and cell throughput that is more than 20 times higher than previously possible for such devices. We first validated the capability of this device by successfully separating low concentrations (∼100 cells/mL) of a variety of cancer cells from cell culture lines from WBCs with a recovery rate better than 83%. We then demonstrated the isolation of CTCs in blood samples obtained from patients with breast cancer. Our acoustic-based separation method thus offers the potential to serve as an invaluable supplemental tool in cancer research, diagnostics, drug efficacy assessment, and therapeutics owing to its excellent biocompatibility, simple design, and label-free automated operation while offering the capability to isolate rare CTCs in a viable state. Significance The separation and analysis of circulating tumor cells (CTCs) provides physicians a minimally invasive way to monitor the response of cancer patients to various treatments. Among the existing cell-separation methods, acoustic-based approaches provide significant potential to preserve the phenotypic and genotypic characteristics of sorted cells, owing to their safe, label-free, and contactless nature. In this work, we report the development of an acoustic-based device that successfully demonstrates the isolation of rare CTCs from the clinical blood samples of cancer patients. Our work thus provides a unique means to obtain viable and undamaged CTCs, which can subsequently be cultured. The results presented here offer unique pathways for better cancer diagnosis, prognosis, therapy monitoring, and metastasis research.