Magnetic-activated Cell Sorting Research Articles

An integrated pipeline for comprehensive analysis of immune cells in human brain tumor clinical samples.

Human tissue samples represent an invaluable source of information for the analysis of disease-specific cellular alterations and their variation between different pathologies. In cancer research, advancing a comprehensive understanding of the unique characteristics of individual tumor types and their microenvironment is of considerable importance for clinical translation. However, investigating human brain tumor tissue is challenging due to the often-limited availability of surgical specimens. Here we describe a multimodule integrated pipeline for the processing of freshly resected human brain tumor tissue and matched blood that enables analysis of the tumor microenvironment, with a particular focus on the tumor immune microenvironment (TIME). The protocol maximizes the information yield from limited tissue and includes both the preservation of bulk tissue, which can be performed within 1 h following surgical resection, as well as tissue dissociation for an in-depth characterization of individual TIME cell populations, which typically takes several hours depending on tissue quantity and further downstream processing. We also describe integrated modules for immunofluorescent staining of sectioned tissue, bulk tissue genomic analysis and fluorescence- or magnetic-activated cell sorting of digested tissue for subsequent culture or transcriptomic analysis by RNA sequencing. Applying this pipeline, we have previously described the overall TIME landscape across different human brain malignancies, and were able to delineate disease-specific alterations of tissue-resident versus recruited macrophage populations. This protocol will enable researchers to use this pipeline to address further research questions regarding the tumor microenvironment.

Application and utility of alternative methods in isolation of pure cells from forensic biological mixtures in modern-day: a review

Development of genetic profiles from the biological mixtures has remained challenging, although modern-day technologies may help forensic scientists to attain a reliable genetic profile in the identification of the accused. In the case of rape, vaginal swab exhibits usually contain epithelial cells of victims and sperm cells of accused, such samples are more challenging when there is more than one contributor. In such cases, separation of distinct cells from a mixture that includes blood cells, epithelial cells and sperm cells for their single genetic profile is important. In the last ten decades several new techniques were developed and invented for the separation of single cell from the biological mixture that includes differential lysis, laser micro-dissection, cell sorting (FACS), sieve-based filtration, (vi) micro-fluidic devices or immunomagnetic beads cell separation of fresh samples, and the magnetic activated cell sorting (MACS). Out of them, some techniques have been commonly applied for cell separation in forensic biology. Each technique has its own limitation. Some recent studies showed, magnetic activated cell sorting (MACS), laser capture microdissection (LCM), DEPArray technology and fluorescence activated cell sorting (FACS) has proved to be effective in separation of single cell from cell mixtures. Therefore, in this review we have evaluated these four alternative methods and their potential application in the modern-day over the others for the separation of a single cell from the mixture. In this review we also discuss the advantage of these methods and their modern–day applicability and acceptance in the forensic world.

Differentiation Potential of Cultured Extracellular DEAD-Box Helicase 4+ Oogonial Stem Cells from Adult Human Ovaries into Somatic Lineages

The transdifferentiation potential of human oogonial stem cells (hOSCs) isolated using the antibody against extracellular DEAD-Box Helicase 4 (ecDDX4) remains undetermined. Hence, this study isolated OSCs from ovarian cortical pieces of premenopausal women using ecDDX4 antibody by magnetic activated cell sorting and expanded these cells under embryonic stem cell (ESC)-like culture conditions to inves­tigate their transdifferentiation potential. The number of ecDDX4+ cells obtained was variable in each isolation. When cultured on inactivated mouse embryonic fibroblast feeder layer with human leukemia inhibitory factor (hLIF) and basic fibroblast growth factor (bFGF) in Minimum Essential Medium, the hOSCs aggregated, forming ESC-like colonies. The average size of these cells was around 10 μm. hOSCs in culture were positive for alkaline phosphatase and further formed embryoid bodies (EBs) when grown on low attachment plates containing Essential 6 Medium without hLIF and bFGF. Subsequently, EBs differentiated into 3 germ layers, which were confirmed by staining with beta-III tubulin (TUJ1) for ectoderm, alpha-fetoprotein (AFP) for endoderm, and smooth muscle actin (SMA) for mesoderm. Further, using appropriate induction media, the EBs derived from ecDDX4+ hOSCs were differentiated into somatic lineages such as adipocytes, osteoblasts, cardiomyocytes, and neuronal precursor-like cells, which were confirmed by immunofluorescence using antibodies against specific markers for each cell type. This study corroborated the previous findings that ovaries of adult women possess germ cell progenitors that can be isolated using ecDDX4, and these cells can be manipulated as pluripotent stem cells by culturing them under ESC-like culture conditions akin to their male counterparts, the spermatogonial stem cells. Further, these cells could differentiate into somatic lineages under specific signalling environments.

Research Highlights

Research Highlights

Heterogeneous response of cancer-associated fibroblasts to the glucose deprivation through mitochondrial calcium uniporter

Cancer-associated fibroblasts (CAFs) are an abundant component of the tumor microenvironment and have distinct features from normal fibroblasts (NFs). However, the discriminative nature of heterogeneous CAFs under glucose starvation remains unknown. In this study, we investigated the changes in the mitochondrial calcium concentration and relevant intracellular machinery in CAFs under glucose-deficient conditions. Xenografted tumor masses were dissected into multiple pieces and subjected to the CAF isolation using magnetically activated cell sorting (MACS). NFs were separated from the normal lung and skin. Under glucose starvation, CAFs from the tumor mass exhibited heterogeneity in cell proliferation, ATP production and calcium concentration. Compared to NFs, mitochondrial calcium concentration was significantly higher in glucose-starved CAFs with upregulation of mitochondrial calcium uniporter (MCU) that led to enhancement of ATP production and cell growth. Intriguingly, treatment of glucose-starved CAFs with oligomycin increased apoptosis by disrupted calcium homeostasis following overactivation of the mPTP. Moreover, oligomycin-induced apoptosis was mitigated by calcium chelation. This study demonstrated that the discriminative calcium influx to mitochondria through MCU coordinated cell growth and apoptosis in glucose-starved CAFs but not in NFs.

P–091 Magnetic Activated Cell Sorting (MACS) improves euploid blastocysts rate in pre-implantation genetic testing cycles with high levels of sperm DNA fragmentation and advanced paternal age

Abstract Study question Can MACS increase euploid blastocyst rate in Pre-implantation Genetic Testing (PGT) cycles for AMA-APA (Advanced Maternal-Paternal Age) in patients with high sperm DNA fragmentation (SDF)? Summary answer A slight increase in euploid blastocyst rate was found using MACS in infertile patients with high SDF undergoing PGT cycles compared to the control group. What is known already Many authors have shown a close correlation between the presence of apoptotic markers on spermatozoa and the failure of assisted reproduction treatments. In normal physiological conditions, apoptotic spermatozoa with phosphatidylserine (PS) residues externalized on the plasma membrane, are eliminated along female genital tract, preventing oocyte fertilization. MACS eliminates apoptotic sperm whit PS residues using superparamagnetic microbeads conjugated with annexin V. This technique reduces the proportion of sperm with high rates of SDF and can be used to maximize ART procedures results. MACS application improves sperm quality, fertilization, cleavage and pregnancy rates reducing miscarriage rate. Study design, size, duration From June to November 2020, 10 couples in which MACS was applied to select non-apoptotic spermatozoa, were randomly enrolled in our study (MACS group) and 8 couples without MACS were considered as controls (No-MACS Group). All couples in both groups underwent a PGT cycle and had high sperm DNA Fragmentation (> 20%). A higher rate of euploid and diploid-euploid mosaic blastocysts were obtained in the MACS group compared to the control group. Participants/materials, setting, methods Patients with severe oligoastenoteratozoospermia were excluded. MACS protocol was performed as follows: semen sample was analyzed (WHO 2010) and washed with buffered medium; pellet was removed and a swim-up was performed. Retrieved spermatozoa were washed with a binding buffer (Miltenyi Biotec), centrifuged (400 g x 4 minutes) and supernatant discarded. Pellet was covered with Annexin-V and re-suspended. After 15 minutes incubation at room temperature, the sample was eluted through the column and collected for ICSI. Main results and the role of chance In MACS group, female and male mean age ± SD were 41.6 ± 2.1 and 43.5 ± 7.3, respectively. Female and male mean age ± SD were 41.7 ± 2.8 and 44.6 ± 8.1 in the No-MACS group, respectively. In MACS and No-MACS groups, injected oocytes were 44 and 35, fertilized oocytes were 32 (72.3%) and 27 (77.1%) (NS), blastocyst formation rates were 71.8% (23/32) and 48.1% (13/27) (NS), respectively. In No-MACS group, only 1 euploid and 1 diploid-euploid mosaic blastocysts were obtained (1/13 = 8%) (NS). In MACS group, 4 euploid blastocysts were formed (4/23 = 17.4%) whereas mosaic diploid-euploid blastocysts were 3/23 (13.0%) (NS). Aneuploid blastocysts were 16/23 (69.6%) in MACS group and 11/13 (84.6%) in No-MACS group (NS). Limitations, reasons for caution AMA and APA of couples enrolled should be considered as a limit of the study. A larger number of patients and biopsied blastocysts are needed to analyze clinical results and perform a robust statistical analysis establishing if MACS is useful to improve transferable blastocyst rate in patients with high SDF. Wider implications of the findings: MACS is useful to select non apoptotic sperms; although fertilization, cleavage and blastocyst rates are not improved, aneuploid blastocysts rate slightly decreases using MACS. It I possible that, selecting spermatozoa free from PS residues, MACS allows to choose spermatozoa with a better DNA packaging, thus affecting the embryo ploidy. Trial registration number non applicable

P–062 Annexin-V MACS method could be a good choice for sperm selection with high PLCZ1 expression and high blastocyst rate in high DFI male factor

Abstract Study question What effect does Annexin-V MACS method have on sperm selection by pLCZ1 expression and blastocyst rate in male DNA fragmentation? Summary answer AnnexinV-MACS method could be a good choice for sperm selection with high pLCZ1 expression and high blastocyst rate in high DNA fragmentation male factor. What is known already Sperm selection based on morphology and motility in ART techniques, is not enough for choosing the best sperm especially in male factor patients. In Annexin-V magnetic activated cell sorting (MACS) technique, apoptotic sperm are separated from non-apoptotic one by negative selection. So, this method can improved quality of compaction rate in embryo. PLCζ is oocyte activating factors that it starts oscillations of calcium ca2+ in oocyte and it has a significant effect on fertilization and implantation. Study design, size, duration Semen samples from 30 male factor infertile couples with high DFI (DFI>30%) were selected and divided into two group (control and experimental) in each patient. Participants/materials, setting, methods Control was washed with DGC and experimental one was selected by MACS-DGC. Retrieved eggs in each patient, were divided in 2. Both group were injected by DGC and MACS respectively. Semen parameters and DFI (SCD test) were analyzed before and after processing. After ICSI, rate of fertilization, embryo development and blastocyst formation were evaluated. Real time PCR evaluate expression of PLCζ. Comparison between results of two groups was assessed by SPSS analysis. Main results and the role of chance Results showed that, sperm motility and morphology after MACS method (45%, 1.7%) was significantly higher than DGC method (40%, 1.1%) and before washing (35%, 0.9%). Percent of DFI in MACS group (36%) was significantly decreased compared to DGC (45%) and primitive group (55%). The number of oocytes were injected in DGC group was 93 and in MACS group was 111. Fertilization rate in both groups was almost the same (72.07% in MACS vs 73.11 in DGC). Rate of day 3 embryo with good grade in MACS group (72. 5%) was significantly higher than DGC (51.47%) (P < 0.05). The blastocyst rate in MACS-DGC group (69.69%) was significantly increased compared to DGC group (48%). PLCζ gene expression in MACS-DGC was significantly higher than DGC group (p-value=0.046). Limitations, reasons for caution This experiment was performed invivo. Wider implications of the findings: Sperm selection by MACS-DGC method can improve sperm motility, morphology and reduce sperm DFI. No significant difference was observed in fertilization rate, but percent of high-quality embryo on days 3 and 5 was significantly higher by this method,also it can be suggested as a good choice for patients with highDF. Trial registration number Not applicable

P–005 Magnetic-activated cell sorting in couples undergoing preimplantation genetic testing for aneuploidies (PGT-A) using autologous oocytes shows slightly lower aneuploidy rates compared to standard semen processing

Abstract Study question Does the selection of non-apoptotic sperm via magnetic-activated cell sorting (MACS) reduce the aneuploidy rate of embryos from couples undergoing ICSI cycles with PGT-A using the patients’ own oocytes? Summary answer It does. The aneuploidy rate in the MACS group was 4.34% lower than the one obtained using semen samples processed according to standard clinical practice. What is known already MACS is a successful tool in eliminating proapoptotic sperm from a semen sample. However, the true effect of this technique on reproductive outcomes and the quality of the resulting embryos are a matter of controversy. Some studies report that its use improves the percentage of good quality blastocysts in women older than 30 years old compared to standard ICSI. Randomized clinical trials that compare MACS to a control sample consider parameters of embryo quality such as morphology at day 3 or day 5, symmetry of the blastomeres, blastocysts’ stage of expansion, but they do not consider embryo ploidy. Study design, size, duration Retrospective, multicentre, observational cohort study. 14,145 patients and 18,710 cycles were evaluated in the reference group. In the MACS group, 615 patients and 974 cycles were considered. Data were exported from cycles performed in Spanish IVIRMA clinics between January 2008 and February 2020. Participants/materials, setting, methods Unselected males in couples undergoing PGT-A cycles, then subdivided into male factor (MF) - total progressive motile sperm count lower than 5 million - and non-male factor (NMF) infertility. Statistical analysis performed using R v.4.0.0. Means were calculated and compared using two-tailed paired t-test, while proportions were compared using Fisher’s exact test and the chi-squared test and the appropriate correction for multiple comparisons. The aneuploidy rates for each group were compared using Fisher’s exact test. Main results and the role of chance In the control group 73,228 biopsied embryos, from which 71,439 were informative in the PGT-A. In the MACS group 3,919 biopsied embryos, from which 3,843 were informative. The aneuploidy rate, computed per informative embryo, was 68.87% (68.40%, 69.34%) in the reference group and 64.53% (62.43%, 66.64%) in the MACS group. Both comparisons were statistically significant (p-value ˂0.00001). According to these results, an embryo in the PGT-A programme using non-apoptotic sperm selected through MACS and autologous oocytes had a 5% less chance of being aneuploid than those embryos fertilised with standardly selected sperm (relative risk of 0.95 (0.91–0.98) p = 0.006769). Embryos conceived from NMF patients whose semen had been processed using MACS had a 4.27% lower aneuploidy rate than the reference (65.52% (63.16%, 67.88%) vs 69.79% (69.20%, 70.37%) respectively). This difference was statistically significant. Those embryos conceived using semen from patients with MF using MACS also showed a lower aneuploidy rate than the reference with MF (0.28% (55.48%, 65.08%) vs (64.94% (63.35%, 66.23%) respectively), although this difference was not statistically significant. Thus, the decrease in aneuploidy rate observed when comparing MACS and reference groups undergoing PGT-A cycles using autologous oocytes remained approximately the same in both MF and NMF semen samples. Limitations, reasons for caution The retrospective nature of the study subjects the data to biases or inaccuracies in their annotation in the clinics’ informatic platform from which they were exported. However, the statistical analysis aimed at controlling these biases as much as possible. Wider implications of the findings: The vast amount of data compiled for this study confirms that the selection of non-apoptotic sperm through MACS slightly decreases the aneuploidy rate of embryos compared to semen samples processed according to the clinics’ standards. This would be interesting for patients who are considering undergoing PGT-A cycles in the future. Trial registration number Not applicable

P–081 Microfluidic sorting does not improve clinical outcomes compared to magnetic activated cell sorting (MACS) in Assisted Reproduction

Abstract Study question Does microfluidic sorting improve clinical outcomes over magnetic activated cell sorting (MACS) in ovum donation cycles? Summary answer Performing microfluidic sorting does not seem to improve clinical outcomes compared to MACS in ovum donation cycles. What is known already Novel sperm selection techniques, such as magnetic activated cell sorting (MACS), have been described as useful procedures to increase reproductive outcomes when male factor is present. Because of centrifugation steps associated to swim-up or density-gradient can induce sperm DNA fragmentation, microfluidic sperm sorters are being used to isolate motile human spermatozoa based on fluid dynamics and avoiding sample manipulation. This new technology has been shown to reduce the level of sperm DNA damage, especially double strand breaks, but an improvement of clinical outcome by using this technique remain unclear. Study design, size, duration Prospective and observational study to evaluate the efficacy of a sperm sorting technique based on microfluidic technology versus a technique based on the removal of apoptotic spermatozoa by MACS. The study was performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. All men attending for an ovum donation cycle during the aforementioned study period were included. The exclusion criteria were sperm concentration <5 mill/mL and <15% of progressive motile spermatozoa. Participants/materials, setting, methods Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. Subsequently, each of the processed sperm samples was used to microinject half of the oocytes obtained during oocyte retrieval. In all case, a single-embryo transfer was performed. Variables were expressed as mean values and standard deviations. Statistical analysis was performed by ANOVA and Chi-squared where applicable; significance established under 0.05. Main results and the role of chance We included 48 couples in the study; of these, n = 31 transferred an embryo derived from a MACS processed sperm sample, while n = 17 received an embryo from the microfluidic one. Groups were homogeneous in terms of number of transferred embryos and frozen embryos, usable blastocyst rate and fertilization rate; results were as follows for MACS and microfluidic processing respectively: number of transferred embryos (1.0±0.0 vs. 1.0±0.0, p = 0.978); number of frozen embryos (1.6±0.5 vs. 1.4±0.7 p = 0.168); usable blastocyst rate (40.7% vs. 43.1%, p = 0.384); and fertilization rate (80.4% vs. 75.3%, p = 0.075). However, according clinical outcomes, we observed significant differences in implantation rate (74.2% vs. 58.8%, p < 0.001) and in clinical pregnancy rate (74.2% vs. 58.8%, p < 0.001); finally, the miscarriage rate was similar between the two groups of study (6.4% vs. 5.8%, p = 0.876). Limitations, reasons for caution This study has not considered the indication in male factor couples due to a high degree of double-strand DNA fragmentation. Therefore, more specific studies are required to determine in which patients, microfluidics sorter selection would significantly improve clinical outcomes. Wider implications of the findings: In an unselected population, magnetic activated cell sorting significantly improves clinical outcomes compared to a microfluidic technique, so this latter method should not be recommended without a male factor indication associated with sperm DNA damage. The proposed microfluidic technology does not seem to offer a flow-free approach to select spermatozoa. Trial registration number NCT04061486

P–235 Are morphokinetics parameters and profitability of an assisted reproduction cycle related to sperm selection technique? Microfluidic sperm sorter chip versus magnetic activated cell sorting

Abstract Study question Does microfluidic sperm sorter offer any biological improvement over magnetic activated cell sorting (MACS)? Summary answer Microfluidic approach for selecting high profile spermatozoa is as good as magnetic activated cell sorting in terms of morphokinetic, fertilization and good quality blastocyst rate. What is known already Microfluidic sperm sorter chip is a method for select non-fragmented DNA sperm. We know that the use of these non-fragmented DNA sperms can improve the clinical results and the useful blastocyst rate. As several studies have shown previously, embryos morphokinetics parameters are affected by culture medium, ovarian stimulation, oxygen tension, origin of the oocytes, or the age of the patient, this is why we wanted to compare if the cleavage times using time lapse technology, are different depending on the sperm selection method used to select sperm with the non-fragmented DNA. Study design, size, duration Prospective and observational study performed between May 2019 to January 2021 in IVI Madrid and IVI Sevilla. Seminal samples from couples participating in the study were divided into two aliquots; each of them was processed according to one of the study methods. 53 couples were included in the study. Half of the oocyte from each donor were microinjected with sperm selected through MACS (n = 281) and the other half through a microfluidic device (n = 275). Participants/materials, setting, methods These oocytes were microinjected with both types of sperm samples and incubated in EmbryoScope. Cellular events studied in this study included cellular divisions until blastocyst stage, appearance and fading of some cellular structures and the duration of the first, second and third cellular cycle (cc1, cc2 and cc3) as well as their synchrony (S1, S2, S3). Data were exported from the EmbryoViewer data base. We perform an ANOVA statistical analysis to analyze the data. Main results and the role of chance No significant differences between both sperm selection methods were found regarding the time of cell division (from T2 to Tblstocyst), the cellular cycles duration (cc1, cc2 and cc3) or the synchrony of the cellular cycles divisions (s1, s2 and s3). However, a clear trend towards statistical significance has been found in both duration of cc2 (p = 0.052) being longer in MACS embryos than in microfluidic sperm sorting embryos, and in the expansion of the blastocyst, which occurs earlier in embryos that come from MACS than in those that come from microfluidic sperm sorting (p = 0.097). These two events could indicate a better embryo cleavage dynamic in the case of MACS embryos, with a better blastocyst expandability and the necessary time to carry out all the biological events that must occur in the cc2. However, significant difference was found in the direct cleavage from 1 to 3 cells embryo stage, which is one of the adverse events that more affects embryo implantation, being higher in microfluidic sperm sorting group (p = 0,037). Finally, the fertilization rate (73.1% vs 76.9%) and the good quality blastocyst rate (53.7% vs 56.5%) were higher in MACS embryos than in microfluidic sperm sorting embryos, although no significant differences were found. Limitations, reasons for caution This study has been performed in donated oocytes, so these results may not be extrapolated to other groups of assisted reproduction patients. However, more data are needed to draw firm conclusions. Furthermore, it´s crucial to increase the sample size to check if the trends founded reach statistical significance. Wider implications of the findings: Microfluidic sorting of unprocessed semen in un unselected population is as efficacious as magnetic activated cell sorting according embryo morphokinetic, fertilization rate and useful blastocyst rate. Microfluidic sperm sorting does not show clinical advantage over MACS considering this data collection. Trial registration number NCT04061484

O-212 MACS vs TESA for raised sperm DNA fragmentation index – a RCT

Abstract Study question In Individuals with raised Sperm DNA Fragmentation Index (SDF), will sperm selection by magnetic activated cell sorting (MACS) or surgical retrieval of testicular sperms (TESA) optimize the reproductive outcomes? Summary answer Couples with failed implantation raised SDF, TESA /MACS offer similar results. This RCT doesn’t prove superiority or added benefit with any of the above interventions. What is known already It is evident that raised SDF negatively affects the reproductive outcomes. Management for raised SDF to optimize reproductive outcomes is still elusive. Study design, size, duration This was a Randomized Control Trial (RCT) with prior approval from institutional Ethical Committee and trial registration. Couples undergoing stimulation with raised SDF were randomized to MACS (n = 75) and TESA (n = 75) for sperm selection between April2019 & February2020. Participants/materials, setting, methods Couples with history of one failed IVF had SDF testing and SDF>30% were recruited. SDF test done with SCSA method and randomized using software. ICSI was the method of insemination. Extended embryo culture till blastocyst was done and freeze all policy was opted. Two Blastocysts that showed 100% survival were transferred in a Frozen Embryo transfer (FET) cycle. Embryonic and Reproductive outcomes were compared between both groups. Live birth and Miscarriage were the primary outcomes. Main results and the role of chance Reproductive Outcomes of MACS Vs TESA were: Average Blastocyst conversion - 32% Vs 39% (RR 1.22, CI1.00 to 1.50) Implantation rate (IR) - 50% Vs 35% (RR - 0.71, CI 0.51 to 0.98) Miscarriage rate (MR) - 5.3% Vs 11% (RR1.6333, CI 0.5227 to 5.1039) Multiple Pregnancy rate (MPR) - 8% Vs 4% Live birth Rate (LBR) per Intention to treat (ITT) - 41.3% Vs 44% (RR 0.95, 95% CI 0.72 to 1.26) LBR per ET cycle - 63% Vs 56% (RR 1.23, 95% CI 0.77 to 1.94) Our preliminary results suggest that despite greater availability of blastocysts for transfer in the TESA group, no difference in ART outcomes was observed between the groups. Though the IR was statistically low with TESA, our primary outcomes LBR and MR were comparable. TESA or MACS seem to offer similar outcomes. Considering the invasiveness with TESA, MACS can be offered for better sperm selection for couples with raised sperm DFI & failed implantation. Limitations, reasons for caution Small sample size. TESA is a surgical intervention Wider implications of the findings Optimal intervention for management of SDF still needs further research. Trial registration number CTRI/2019/07/020140

P–103 Novel sperm preparation techniques compared with conventional preparation method

Abstract Study question Which sperm preparation technique separate the best quality sperm? Summary answer Microfluidic method improved the sperm parameters and decreased sperm DNA damage. What is known already About 40% of infertility issues are due to male factor. One of the known causes of male infertility is associated with low sperm parameters and high level DNA fragmentation. Sperm preparation techniques in ICSI procedures is used in order to obtain the best-quality sperm. Study design, size, duration The present study was designed to compare Microfluidic, Zeta potential, Magnetic Activated Cell Sorting (MACS) and Swim-up methods for sperm preparation and the effect of these methods on semen parameters and sperm DNA integrity in infertile men (n = 25) with a mean age of 38. Participants/materials, setting, methods In this study, each sample was divided into 4 groups, one part for preparing by Microfluidic method, one of them for preparing by Swim -up method, the other one was prepared by MACS and the last one was prepared by zeta potential. Then sperm count, viability, motility and morphology were assessed according to WHO 2010. DNA damage were assessed by Sperm DNA Fragmentation assay and sperm chromatin packaging assessed by CMA3 staining test Main results and the role of chance Sperm parameters including viability, motility, and morphology in the Microfluidic method were significantly improved and sperm DNA damage were significantly lower than three other methods (P-value <0.05). The sperm parameters and sperm DNA damage after preparation by MACS and Zeta potential methods were not significantly different however in the Swim-up method sperm parameters were lower than three other methods (P-value <0.05). Limitations, reasons for caution The fertilization and pregnancy rate of the resulting embryos are not available. Wider implications of the findings: Our results showed that Microfluidic can be an effective way to improve sperm quality of infertile male compared to conventional preparation methods. We also found instead of the MACS method, we can use the Zeta potential method according to their costs, for sperm preparation during ART cycle. Trial registration number *

The effect of Acellularized Wharton's Jelly-derived exosomes on myeloid differentiation of umbilical cord blood-derived CD34+ hematopoietic stem cells

PurposeHematopoietic stem cell transplantation (HSCT) has been introduced as an effective treatment for hematological malignancies. Extracellular vesicles including exosomes and microvesicles, have recently attracted researcher's attention as the main factor in proliferation and differentiation of hematopoietic stem cells (HSCs) to accelerate the transplantation. Accordingly, this study aimed to evaluate the effect of exosomes derived from acellular Wharton’'s Jelly (WJ) on myeloid differentiation of umbilical cord blood (UCB)-derived CD34+ HSCs. MethodsAfter isolation of CD34+ cells from human UCB via magnetic-activated cell sorting, these cells were cultured and incubated for three weeks. Then, UC-derived WJ was acellularized and used for exosome isolation. Finally, these exosomes were co-cultured with CD34+ cells and their differentiation to myeloid lineage was assessed by flow cytometry and Real-Time PCR. ResultsThe expression of CD33 was higher in the exosome group than that of the control group on day 0 (P < 0.001), however, the change in expression of CD33, in the presence of exosome was not significant in comparison with the control group on day 3 (P ≥ 0.05). Also, the expression of myeloid-specific gene (RUNX1) and was reduced in the exosome group after three days (P < 0.05) and the expression of another myeloid-specific gene(C/EBP–α) had no significant difference with the control group (P ≥ 0.05). ConclusionOverall, these observations indicated that AWJ-exosomes may have no potential impact on myeloid differentiation of CD34+ HSCs derived from UCB.

Sperm Selection Technology in ART.

Intracytoplasmic sperm injection (ICSI) is a commonly used in vitro fertilization technique. Selection of sperm for injection is currently done using subjective assessment of morphology, which may not accurately identify the best-quality sperm. Hyaluronic acid receptors on sperm plasma membranes are a marker of maturity, and sperms which are capable of binding immobilized hyaluronic acid in vitro are of higher quality. This can be used as an advanced sperm selection technique to choose sperm for ICSI, termed physiological ICSI (PICSI). Several studies reported improved fertility treatment outcomes when using PICSI compared with conventional ICSI; however, the majority of studies are underpowered. Recently, a large, multicenter, randomized controlled trial, known as the Hyaluronic Acid Binding Sperm Selection (HABSelect) trial, found a significant reduction in miscarriage rates with PICSI, but no significant effect on live birth rate. There are still many avenues through which PICSI may provide an advantage, subject to confirmation by future research, such as improved long-term health of offspring. Other advanced sperm selection techniques include intracytoplasmic morphologically selected sperm injection, magnetic-activated cell sorting, and Zeta potential sperm selection; however, the most recent Cochrane review concluded that there is currently insufficient evidence to ascertain whether these techniques improve clinical outcomes, such as live birth rates.

Progress and prospects of magnetic iron oxide nanoparticles in biomedical applications: A review.

Nanoscience has been considered as one of the most substantial research in modern science. The utilization of nanoparticle (NP) materials provides numerous advantages in biomedical applications due to their unique properties. Among various types of nanoparticles, the magnetic nanoparticles (MNPs) of iron oxide possess intrinsic features, which have been efficiently exploited for biomedical purposes including drug delivery, magnetic resonance imaging, Magnetic-activated cell sorting, nanobiosensors, hyperthermia, and tissue engineering and regenerative medicine. The size and shape of nanostructures are the main factors affecting the physicochemical features of superparamagnetic iron oxide nanoparticles, which play an important role in the improvement of MNP properties, and can be controlled by appropriate synthesis strategies. On the other hand, the proper modification and functionalization of the surface of iron oxide nanoparticles have significant effects on the improvement of physicochemical and mechanical features, biocompatibility, stability, and surface activity of MNPs. This review focuses on popular methods of fabrication, beneficial surface coatings with regard to the main required features for their biomedical use, as well as new applications.

An efficient and cost-effective method for the purification of human basophils.

Human basophils are terminally differentiated granulocytes that are least abundant in the peripheral blood but play important roles in allergic diseases. Studies on human basophils are limited by the high cost on the isolation of human basophils by magnetic-activated cell sorting (MACS) for negative depletion of non-basophils, followed by CD123-based positive selection of basophils. Moreover, such CD123-based purification of basophils may be limited by blocking of the binding of IL-3/anti-CD123 to the surface CD123. Here we identified SSClow CD4- CD127- HLA-DR- CRTH2high as unique markers for the identification of human basophils through stringent flow cytometric analysis of leukocytes from buffy coat. We established an efficient and cost-effective method for isolating human basophils from buffy coat based on positive magnetic selection of CRTH2+ cells followed by flow cytometric sorting of SSClow CD4- CD127- HLA-DR- CRTH2high cells. Approximately 1 to 1.5 million basophils were isolated from one buffy coat with a purity of >97%. Basophils purified by this method were viable and efficiently responded to key regulators of basophils including IL-3 and anti-IgE. This method can be used for purifying human basophils for subsequent functional studies.

PrPC Regulates the Cancer Stem Cell Properties via Interaction With c-Met in Colorectal Cancer Cells.

Studies have reported that the expression of c-Met and PrPC improves tumor progression. However, not much is known about their relationship. We hypothesized that c-Met and PrPC interact with each other, and enhance cancer stem cell (CSC) characteristics. Magnetic activated cell sorting was used to examine the interaction between c-Met and PrPC The effects of the interaction on downstream signals, stem cell marker expression, and sphere formation of colorectal cancer (CRC) cells were investigated. We demonstrated the increased expression and binding levels of c-Met and PrPC in CRC cells compared to normal colon epithelial cells. We revealed that the c-Met and PrPC interaction induced the ERK activation and Oct4 upregulation. The inhibition of c-Met by crizotinib reduced ERK activation and Oct4 expression and suppressed CSC properties. c-Met and PrPC interact with each other, and targeting c-Met using crizotinib could be a powerful strategy for CRC therapy.

Label-free quantitative lymphocyte activation profiling using microfluidic impedance cytometry

Circulating lymphocytes are integral components of our adaptive immunity with emerging clinical applications in immune status monitoring in infectious diseases and cell-mediated cancer immunotherapies. Herein we present a novel impedance-based microfluidic assay for label-free lymphocyte activation profiling based on native or antigen-specific T-lymphocyte biophysical responses. Single cell impedance profiling of T-lymphocytes first revealed distinct biophysical differences in cell size and membrane electrical impedance of healthy, activated (CD3/CD28) and dead lymphocyte populations. Impedance characterization of peripheral blood mononuclear cells (PBMCs) stimulated with mitogen phytohemagglutinin (PHA) or Tuberculin Purified Protein Derivative antigen (PPD) after 24 h also showed an increase in lymphocyte cell size (∼8 to 10 μm) which corresponded to activated lymphocytes (CD69+CD137+). We next developed a spiral inertial microfluidics cell sorter integrated with coplanar electrodes for direct impedance quantification of activated lymphocytes. By removing non-activated smaller lymphocytes (< 8 μm) and employing hydrodynamic-based single stream particle focusing, we demonstrated significant enrichment of activated lymphocytes (∼11.7-fold) to electrically detect low levels of lymphocyte activation (< 5%). Finally, the developed biochip is coupled with magnetic activated cell sorting (MACS) to quantify CD4+ T-lymphocytes response in PBMCs stimulated with PPD. A differential impedance cell count ratio (stimulated/unstimulated) was defined to distinguish activated T-lymphocytes, which showed better sensitivity as compared to immunophenotyping by flow cytometry. Taken together, the integrated impedance biosensor can be further developed as a rapid multiplexed screening assay to detect antigen-specific T-lymphocyte responses to characterize host immunity and diagnosis of infectious diseases (e.g tuberculosis, dengue and COVID-19).

Magnetic-activated cell sorting improves high-quality spermatozoa in bovine semen

Teresinha Inês de Assumpção, Neimar Correa Severo, João Pedro Brandão Zandonaide and Gustavo Guerino Macedo. Journal of Animal Reproduction and Biotechnology 2021;36:91-8. https://doi.org/10.12750/JARB.36.2.91

Is cervical swab an efficient method for developing a new noninvasive prenatal diagnostic test for numerical and structural chromosome anomalies?

Background/aim Prenatal diagnosis is vital to obtain healthy generation for risky pregnancies. There have been several approaches, some of which are routinely applied in clinics to evaluate the possible prenatal deficiencies and/or diseases. In the present study, we aimed to isolate the fetal cells from endocervical samples and try to identify possible anomalies which were proved by Amniocentesis (AS) and chorionic villus sampling (CVS) methods.Materials and methodsEndoservical specimens were collected from 100 pregnant women. Cells were separated in parallel by fluorescence-activated cell sorting (FACS) and magnetic-activated cell sorting (MACS) using human leukocyte antigen (HLA) G233 and placental alkaline phosphatase (PLAP) antibodies. CMA (comprehensive meta-analysis) were carried out and male fetuses were confirmed with Sex determining region Y (SRY) amplification.Results The percent of HLA G233 and placental and placental alkaline phosphatase (PLAP) positive cells were 4.55% and 84.59%, respectively. The percent of cells positive for both markers was 14.75%. CMA analyses were not informative. (SRY) was amplified in 67% of the samples.Conclusion However, the success rate of the both cell sorting and scanning of DNA anomalies by aCGH and/or RT-PCR was limited, preventing the applicability of this proposal in the clinics. Still, the success of the proposed method depends on the development of the novel fetal cell-specific antibodies and the improvements in the sorting systems.