An Alternative Application of Magnetic-Activated Cell Sorting: CD45 and CD235a Based Purification of Semen and Testicular Tissue Samples.

Magnetic activated cell sorting (MACS) is a well-known sperm selection technique, which is able to remove apoptotic spermatozoa from semen samples using the classic annexinV based method. Leukocytes and erythrocytes in semen samples or in testicular tissue processed for in vitro fertilization (IVF) could exert detrimental effects on sperm. In the current study, we rethought the aforementioned technique and used magnetic microbeads conjugated with anti-CD45/CD235a antibodies to eliminate contaminating leukocytes and erythrocytes from leukocytospermic semen samples and testicular tissue samples gained via testicular sperm extraction (TESE). With this technique, a 15.7- and a 30.8-fold reduction could be achieved in the ratio of leukocytes in semen and in the number of erythrocytes in TESE samples, respectively. Our results show that MACS is a method worth to reconsider, with more potential alternative applications. Investigations to find molecules labeling high-quality sperm population and the development of positive selection procedures based on these might be a direction of future research.

Methods to Isolate Muscle Stem Cells for Cell-Based Cultured Meat Production: A Review.

Cultured meat production relies on various cell types, including muscle stem cells (MuSCs), embryonic stem cell lines, induced pluripotent cell lines, and naturally immortalized cell lines. MuSCs possess superior muscle differentiation capabilities compared to the other three cell lines, making them key for cultured meat development. Therefore, to produce cultured meat using MuSCs, they must first be effectively separated from muscles. At present, the methods used to isolate MuSCs from muscles include (1) the pre-plating method, using the ability of cells to adhere differently, which is a biological characteristic of MuSCs; (2) the density gradient centrifugation method, using the intrinsic density difference of cells, which is a physical characteristic of MuSCs; and (3) fluorescence- and magnetic-activated cell sorting methods, using the surface marker protein on the cell surface of MuSCs, which is a molecular characteristic of MuSCs. Further efficient and valuable methods for separating MuSCs are expected to be required as the cell-based cultured meat industry develops. Thus, we take a closer look at the four methods currently in use and discuss future development directions in this review.

Microfluidics-assisted Tumor Cell Separation Approaches for Clinical Applications: An Overview on Emerging Devices.

Recent advances in science and technology have led to revolutions in many different scientific and industrial fields. The term lab on a chip, or in other word, performing a variety of complex analyzes in just a short time and in a very small space, is a term that has become very common in recent years, and what used to be a dream has now come to life in practice. In this paper, we has been tried to investigate a specific type of lab technology on a chip, which is of course one of the most common, namely the knowledge and technology of cell separation by using microfluidic technique that can be separated based on size and deformation, adhesion and electrical properties. The tissue of the human body is degraded due to injury or aging, which is often tried to treat this tissue disorder by using drugs, but they are not always enough. Stem cell-based medicine is a novel form of medicine which promises the restoration or regeneration of tissues and functioning organs. Although many models of microfluidic systems have been designed for cell separation, the choice of appropriate device to achieve a reliable result is presented as a challenge. So, in this study, Fluorescence Activated Cell Sorting (FACS), Dielectrophoresis (DEP), Magnetic Activated Cell Sorting (MACS) and Acoustic microfluidic system are four distinct categories of active microfluidic systems explored. Also, the advantages, disadvantages and the current status of the mentioned devices in these methods, are reviewed.

Laser-Guided Sperm Selection: Optimizing the Reproductive Success Rate in Assisted Reproductive Technology.

Assisted reproductive technologies (ART) enable these patient's spermatozoa to fertilize the oocyte and create viable and healthy offspring, but the effectiveness of the various procedures still has room to increase. In the field of assisted reproductive technology, the need to improve fertility results has led to the development of novel sperm selection strategies. Laser-assisted selection of immotile sperm (LAISS) appears to be a promising strategy, harnessing the power of modern optical instruments to better the selection process and, ultimately, maximize the probability of successful fertilization. This technology takes advantage of sperm cells' distinctive features, such as shape, form, and motility patterns, that can be sensitively changed by laser forces. Using precision laser manipulation, spermatozoa with desirable features can be precisely targeted, improving the overall quality and viability of the sperm population. The existence of an elevated percentage of DNA-damaged sperm in a patient's ejaculation may be one of the key factors decreasing ART outcomes. As a result, one of the most difficult tasks in reproductive medicine is ensuring the best quality of spermatozoa utilized in ART, particularly with regard to genetic integrity. The most recent approaches for preparing and selecting human spermatozoa by LAISS techniques are covered here, with an emphasis on those that have been shown to improve.

Characteristics of multilineage-differentiating stress-enduring cell clusters in different culture conditions.

OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.

Reprogramming Cancer Cells to Antigen-presenting Cells.

Cancer cells evade the immune system by downregulating antigen presentation. Although immune checkpoint inhibitors (ICI) and adoptive T-cell therapies revolutionized cancer treatment, their efficacy relies on the intrinsic immunogenicity of tumor cells and antigen presentation by dendritic cells. Here, we describe a protocol to directly reprogram murine and human cancer cells into tumor-antigen-presenting cells (tumor-APCs), using the type 1 conventional dendritic cell (cDC1) transcription factors PU.1, IRF8, and BATF3 delivered by a lentiviral vector. Tumor-APCs acquire a cDC1 cell-like phenotype, transcriptional and epigenetic programs, and function within nine days (Zimmermannova et al., 2023). Tumor-APCs express the hematopoietic marker CD45 and acquire the antigen presentation complexes MHC class I and II as well as co-stimulatory molecules required for antigen presentation to T cells, but do not express high levels of negative immune checkpoint regulators. Enriched tumor-APCs present antigens to Naïve CD8+ and CD4+ T cells, are targeted by activated cytotoxic T lymphocytes, and elicit anti-tumor responses in vivo. The tumor-APC reprogramming protocol described here provides a simple and robust method to revert tumor evasion mechanisms by increasing antigen presentation in cancer cells. This platform has the potential to prime antigen-specific T-cell expansion, which can be leveraged for developing new cancer vaccines, neoantigen discovery, and expansion of tumor-infiltrating lymphocytes. Key features • This protocol describes the generation of antigen-presenting cells from cancer cells by direct reprogramming using lineage-instructive transcription factors of conventional dendritic cells type I. • Verification of reprogramming efficiency by flow cytometry and functional assessment of tumor-APCs by antigen presentation assays.

Human Schwann Cells in vitro II. Passaging, Purification, Banking, and Labeling of Established Cultures.

This manuscript describes step-by-step procedures to establish and manage fresh and cryopreserved cultures of nerve-derived human Schwann cells (hSCs) at the desired scale. Adaptable protocols are provided to propagate hSC cultures through serial passaging and perform routine manipulations such as enzymatic dissociation, purification, cryogenic preservation, live-cell labeling, and gene delivery. Expanded hSCs cultures are metabolically active, proliferative, and phenotypically stable for at least three consecutive passages. Cell yields are expected to be variable as determined by the rate of growth of individual batches and the rounds of subculture. The purity, however, can be maintained high at >95% hSC regardless of passage. The cells obtained in this manner are suitable for various applications, including small drug screens, in vitro modeling of neurodevelopmental processes, and cell transplantation. One caveat of this protocol is that continued expansion of same-batch hSC populations is eventually restricted due to senescence-linked growth arrest.

The CD44std and CD44v9 subpopulations in non-tumorigenic invasive SNU-423 cells present different features of cancer stem cells.

Hepatocellular carcinoma (HCC) is a type of liver cancer, in which CD44 isoforms have been proposed as markers to identify cancer stem cells (CSCs). However, it is unclear what characteristics are associated with CSCs that exclusively express CD44 isoforms. The objective of the present study was to determine the expression of CD44 isoforms and their properties in CSCs. Analysis of transcriptomic data from HCC patient samples identified CD44v8-10 as a potential marker in HCC. In SNU-423 cells, CD44 expression was detected in over 99% of cells, and two CD44 isoforms, namely, CD44std and CD44v9, were identified in this cell line. CD44 subpopulations, including both CD44v9+ (CD44v9) and CD44v9- (CD44std) cells, were obtained by purification using a magnetic cell separation kit for human CD44v9+ cancer stem cells. CD44v9 cells showed greater potential for colony and spheroid formation, whereas CD44std cells demonstrated significant migration and invasion capabilities. These findings suggested that CD44std and CD44v9 may be used to identify features in CSC populations and provide insights into their roles in HCC.

Effects of sperm processing techniques on IVF pregnancy rates: a mini-review.

Many factors associated with assisted reproductive technologies significantly influence the success of pregnancy after in vitro fertilization (IVF) either directly or indirectly. These factors include sperm processing techniques, egg retrieval, intrauterine artificial insemination, intracytoplasmic sperm injection, and embryo transfer. Among these technologies, sperm quality is one of the most critical factors for a successful IVF pregnancy. The method used for sperm processing plays a crucial role in determining the quality of sperm. Several widely used sorting techniques, such as conventional swim-up, density gradient centrifugation, magnetic activated cell sorting, and hyaluronic acid, have been extensively compared in various studies. Previous studies have shown that each sperm processing method causes varying degrees of sperm damage, particularly in sperm motility, concentration, morphological features, viability, and DNA integrity. However, sperm processing techniques have been developed slowly, and the impact of these methods on pregnancy rates is still unclear. Further exploration is needed. In this review, we aim to compare the results of different sperm processing techniques concerning sperm quality and IVF pregnancy rates. We will also discuss possible clinical approaches, such as microfluidics and integrated approaches, for testing and improving sperm quality.

Semen processing using magnetic-activated cell sorting before ICSI is deemed safe for obstetric and perinatal outcomes: a retrospective multicentre study.

RESEARCH QUESTION: Is magnetic-activated cell sorting (MACS) a safe semen sample processing technique for newborns and mothers when used for semen processing prior to intracytoplasmic sperm injection (ICSI) cycles? DESIGN: This retrospective multicentre cohort study involved patients undergoing ICSI cycles with either donor or autologous oocytes from January 2008 to February 2020. They were divided into two groups: those who underwent standard semen preparation (reference group) and those who had an added MACS procedure (MACS group). A total of 25,356 deliveries were assessed in the case of cycles using donor oocytes, and 19,703 deliveries from cycles using autologous oocytes. Of these, 20,439 and 15,917, respectively, were singleton deliveries. Obstetric and perinatal outcomes were retrospectively assessed. All means, rates and incidences were computed per live newborn in each study group. RESULTS: There were no significant differences between the main obstetric and perinatal morbidities affecting the mothers' and newborns' well-being between groups using either donated or autologous oocytes. There was a significant increase in the incidence of gestational anaemia in both subpopulations (donor oocytes P = 0.01; autologous oocytes P < 0.001). However, this incidence was within the estimated prevalence for gestational anaemia in the general population. There was a statistically significant decrease in preterm (P = 0.02) and very preterm (P = 0.01) birth rates in the MACS group in cycles using donor oocytes. CONCLUSIONS: The use of MACS during semen preparation before ICSI using either donor or autologous oocytes appears to be safe for the mothers' and newborns' well-being during pregnancy and birth. Nevertheless, a close follow-up of these parameters in the future is advised, especially concerning anaemia, in order to detect even smaller effect sizes.

Advanced Technologies and Automation in mES Cell Workflow.

Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. To streamline and increase the efficiency in our ES cell pipeline and decrease the timeline for mouse models produced via ES cells, automation is introduced in the pipeline. Below, we describe a novel and effective approach utilizing ddPCR, dPCR, automated DNA purification, MultiMACS, and adenovirus recombinase combined screening workflow that reduces the time between therapeutic target identification and experimental validation.

Analyzing the Differential Impact of Semen Preparation Methods on the Outcomes of Assisted Reproductive Techniques.

Sperm separation plays a critical role in assisted reproductive technology. Based on migration, density gradient centrifugation and filtration, a properly selected sperm could help in increasing assisted reproductive outcomes in teratozoospermia (TZs). The current study aimed to assess the prognostic value of four sperm selection techniques: density gradient centrifugation (DGC), swim-up (SU), DGC-SU and DGC followed by magnetic-activated cell sorting (DGC-MACS). These were evaluated using spermatozoa functional parameters. A total of 385 infertile couples underwent the procedure of intracytoplasmic sperm injection (ICSI), with an isolated teratozoospermia in the male partner. Semen samples were prepared by using one of the mentioned sperm preparation techniques. The improvements in the percentage of normal mature spermatozoa, rate of fertilization, cleavage, pregnancy and the number of live births were assessed. The normal morphology, spermatozoa DNA fragmentation (SDF) and chromatin maturity checked by using chromomycin A3 (CMA3) with DGC-MACS preparation were better compared to the other three methods. Embryo cleavage, clinical pregnancy and implantation were better improved in the DGC-MACS than in the other tested techniques. The DGC-MACS technique helped in the selection of an increased percentage of normal viable and mature sperm with intact chromatin integrity in patients with teratozoospermia.

Identification and Purification of Human T Cell Precursors.

During their development, human T cells undergo similar genomic changes and pass through the same developmental checkpoints as developing thymocytes in the mouse. The difference between both species, however, is that some of these developmental stages are characterized by different phenotypic markers, and as a result, evidence emerges that the molecular regulation of human T cell development subtly differs from the mouse (Taghon et al., Curr Top Microbiol Immunol 360:75-97, 2021; Haddad et al., Immunity 24:217-230, 2006; Hao et al., Blood 111:1318-1326, 2008; Taghon and Rothenberg, Semin Immunopathol 30:383-398, 2008). In this chapter, we describe in detail how the different stages of human T cell development can be characterized and isolated using specific surface markers.

Purification of human iPSC-derived cells at large scale using microRNA switch and magnetic-activated cell sorting.

For regenerative cell therapies using pluripotent stem cell (PSC)-derived cells, large quantities of purified cells are required. Magnetic-activated cell sorting (MACS) is a powerful approach to collect target antigen-positive cells; however, it remains a challenge to purify various cell types efficiently at large scale without using antibodies specific to the desired cell type. Here we develop a technology that combines microRNA (miRNA)-responsive mRNA switch (miR-switch) with MACS (miR-switch-MACS) to purify large amounts of PSC-derived cells rapidly and effectively. We designed miR-switches that detect specific miRNAs expressed in target cells and controlled the translation of a CD4-coding transgene as a selection marker for MACS. For the large-scale purification of induced PSC-derived cardiomyocytes (iPSC-CMs), we transferred miR-208a-CD4 switch-MACS and obtained purified iPSC-CMs efficiently. Moreover, miR-375-CD4 switch-MACS highly purified pancreatic insulin-producing cells and their progenitors expressing Chromogranin A. Overall, the miR-switch-MACS method can efficiently purify target PSC-derived cells for cell replacement therapy.

Magnetic-Activated Cell Sorting as a Method to Improve Necrozoospermia-Related Asthenozoospermic Samples.

According to some statistics, absolute asthenozoospermia affects every 1 in 5000 men. Although this incidence rate does not appear to be too high, it is extremely important to address the phenomenon because it can drastically reduce the chances of pregnancy, even with assisted reproduction. The biggest problem with absolute asthenozoospermia is that it is difficult to distinguish between live and dead sperm cells, and fertilization with non-viable spermatozoa may contribute to the failure of an assisted reproduction cycle. Nowadays, DNA fragmentation (DF) is a crucial parameter of semen analysis, and in this paper, we provide evidence of the correlation between DF and vitality. For this purpose, the main semen parameters were investigated by a CASA system (concentration, motility, progressive motility, vitality and DF). In the necrozoospermic group (vitality < 58%), all the measured parameters showed significant differences compared to normal vitality. Concentration (30.1 M mL−1 vs. 13.6 M mL−1), motility (31.9% vs. 18.3%), and progressive motility (24.3% vs. 12.7%) were significantly decreased, while DF was significantly increased (17.4% vs. 23.7%). Based on the connection between vitality decrement and DF increment, DF lowering methods, such as magnetic-activated cell sorting, have been hypothesized as novel methods for the elimination of dead spermatozoa.

High-Efficiency Bovine Sperm Sexing Used Magnetic-Activated Cell Sorting by Coupling scFv Antibodies Specific to Y-Chromosome-Bearing Sperm on Magnetic Microbeads.

Sperm sexing technique is favored in the dairy industry. This research focuses on the efficiency of bovine sperm sexing using magnetic-activated cell sorting (MACS) by scFv antibody against Y-chromosome-bearing sperm (Y-scFv) coupled to magnetic microbeads and its effects on kinematic variables, sperm quality, and X/Y-sperm ratio. In this study, the optimal concentration of Y-scFv antibody coupling to the surface of magnetic microbeads was 2-4 mg/mL. PY-microbeads revealed significantly enriched Y-chromosome-bearing sperm (Y-sperm) in the eluted fraction (78.01-81.43%) and X-chromosome-bearing sperm (X-sperm) in the supernatant fraction (79.04-82.65%). The quality of frozen-thawed sexed sperm was analyzed by CASA and imaging flow cytometer, which showed that PY-microbeads did not have a negative effect on X-sperm motility, viability, or acrosome integrity. However, sexed Y-sperm had significantly decreased motility and viability. The X/Y-sperm ratio was determined using an imaging flow cytometer and real-time PCR. PY-microbeads produced sperm with up to 82.65% X-sperm in the X-enriched fraction and up to 81.43% Y-sperm in the Y-enriched fraction. Bovine sperm sexing by PY-microbeads showed high efficiency in separating Y-sperm from X-sperm and acceptable sperm quality. This initial technique is feasible for bovine sperm sexing, which increases the number of heifers in dairy herds while lowering production expenses.

Laminin-221-derived recombinant fragment facilitates isolation of cultured skeletal myoblasts.

Introduction: Laminin is a major component of the basement membrane, containing multiple domains that bind integrin, collagen, nidogen, dystroglycan, and heparan sulfate. Laminin-221, expressed in skeletal and cardiac muscles, has strong affinity for the cell-surface receptor, integrin α7X2ß1. The E8 domain of laminin-221, which is essential for cell integrin binding, is commercially available as a purified recombinant protein fragment. In this study, recombinant E8 fragment was used to purify primary rodent myoblasts. We established a facile and inexpensive method for primary myoblast culture exploiting the high affinity binding of integrin α7X2ß1 to laminin-221. Methods: Total cell populations from dissociated muscle tissue were enzymatically digested and seeded onto laminin-221 E8 fragment-coated dishes. The culture medium containing non-adherent floating cells was removed after 2-hour culture at 37 °C. The adherent cells were subjected to immunofluorescence staining of desmin, differentiation experiments, and gene expression analysis. Results: The cells obtained were 70.3 ± 5.49% (n = 5) desmin positive in mouse and 67.7 ± 1.65% (n = 3) in rat. Immunofluorescent staining and gene expression analyses of cultured cells showed phenotypic traits of myoblasts. Conclusion: This study reports a novel facile method for primary culture of myoblasts obtained from mouse and rat skeletal muscle by exploiting the high affinity of integrin α7X2ß1 to laminin-221.

Functional Characterization of Endothelial Cells Differentiated from Porcine Epiblast Stem Cells.

Endothelial cells (ECs), lining blood vessels' lumen, play an essential role in regulating vascular functions. As multifunctional components of vascular structures, pluripotent stem cells (PSCs) are the promising source for potential therapeutic applications in various vascular diseases. Our laboratory has previously established an approach for differentiating porcine epiblast stem cells (pEpiSCs) into ECs, representing an alternative and potentially superior cell source. However, the condition of pEpiSCs-derived ECs growth has yet to be determined, and whether pEpiSCs differentiate into functional ECs remained unclear. Changes in morphology, proliferation and functional endothelial marker were assessed in pEpiSCs-derived ECs in vitro. pEpiSCs-derived ECs were subjected to magnetic-activated cell sorting (MACS) to collect CD-31+ of ECs. We found that sorted ECs showed the highest proliferation rate in differentiation media in primary culture and M199 media in the subculture. Next, sorted ECs were examined for their ability to act as typical vascular ECs through capillary-like structure formation assay, Dil-acetylated low-density lipoprotein (Dil-Ac-LDL) uptake, and three-dimensional spheroid sprouting. Consequently, pEpiSCs-derived ECs function as typical vascular ECs, indicating that pEpiSC-derived ECs might be used to develop cell therapeutics for vascular disease.

Rapid and Highly Efficient Isolation and Purification of Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (iPSCs) hold great promise for biomedical applications. However, establishment of new iPSC lines still presents many challenges. Here we describe a simple yet highly efficient two-step protocol for the isolation and purification of human iPSC lines. The first step adapts iPSCs to single cell culture and passaging, promoting survival and self-renewal; the second step enables the isolation and purification of bona fide iPSCs from a mixed population using column-based positive selection of cells expressing pluripotency markers such as TRA-1-60. Both steps utilize commercially available reagents. Using this protocol, iPSCs can be purified from cell preparations containing differentiated or unreprogrammed cells, or even be isolated directly from reprogramming vessels. The protocol could be adopted for high throughput isolation and expansion of iPSC lines and facilitate the widespread use of iPSCs in future applications.

A Rapid Protocol for Direct Isolation of Osteoclast Lineage Cells from Mouse Bone Marrow.

Osteoclast lineage cells (OLCs), including osteoclast precursors (OCPs) and mature osteoclasts (MOCs), participate in bone remodeling and mediate pathologic bone loss. Thus, it is essential to obtain OLCs for exploring their molecular features in both physiological and pathological conditions in vivo. However, the conventional protocols for obtaining OLCs ex vivo are not only time-consuming, but also unable to capture the cellular status of OLCs in vivo. In addition, the current antibody-based isolation approaches, such as fluorescence-/ magnetic-activated cell sorting, are not able to obtain pure osteoclasts because no unique surface antigen for osteoclasts has been identified. Here, we develop a rapid protocol for directly isolating OLCs from mouse bone marrow through magnetic-activated cell sorting (MACS). This protocol can rapidly enrich OCPs and MOCs, respectively, depending on the expression of the distinctive surface markers at their differentiation stages. It is optimized to isolate OLCs from four mice concurrently, of which sorting procedure could be completed within ~5 h.