Isolation of Extracellular Vesicles Using Formulas to Adapt Centrifugation to Different Centrifuges.

Extracellular vesicles (EVs) are small lipid bilayer vesicles released by cells to facilitate cell-to-cell communication. To study their biological roles and functions, they need to be isolated and purified, which can be achieved through a variety of methods. Here, we describe different methods for isolating and purifying EVs, with a focus on calculating the required g-force and centrifugation time with different centrifuges and rotors. We have compiled key formulas and tested predicted parameters for EV acquisitions to provide a comprehensive guide for EV isolation.

The Impact of Centrifugal Force on Isolation of Bone Marrow Mononuclear Cells Using Density Gradient Centrifugation.

BACKGROUND: Bone marrow mononuclear cells (BMMNCs) have great potential in bone regenerative therapy. The main method used today to obtain BMMNCs is Ficoll density gradient centrifugation. However, the centrifugal force for this isolation method is still suboptimal. OBJECTIVES: To determine the optimal centrifugal force in Ficoll density gradient centrifugation of bone marrow (BM) to achieve high stem/progenitor cell content BMMNCs for regenerative therapy. METHODS: BM was aspirated from nine minipigs and divided into three groups according to different centrifugal forces (200 g, 300 g and 400 g). Immediately after BMMNCs were obtained from each group by Ficoll density gradient centrifugation, residual red blood cell (RBC) level, nucleated cell counting, viability and flow cytometric analyses of apoptosis and reactive oxygen species (ROS) generation were measured. The phenotypic CD90 and colony formation analyses of BMMNCs of each group were performed as well. Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested at passage 2, then morphology, cell phenotype, proliferation, adipogenic, chondrogenic and osteogenic lineage differentiation potential of BMSCs from each group were compared. RESULTS: The 300 g centrifugal force was able to isolate BMMNCs from BM with the same efficiency as 400 g and provided significantly higher yields of CD90+ BMSCs and fibroblastic colony-forming units of BMSC (CFU-f(BMSC)), which is more crucial for the regenerative efficacy of BMMNCs. Meanwhile, 200 g hosted the most RBC contamination and minimum CFU-f (BMSC) yield, which will be disadvantageous for BMMNC-based cell therapy. As for in vitro cultured BMSCs which were isolated from BMMNCs by different centrifugal forces, no significant differences were found on morphology, cell proliferation rate, phenotypic marker, adipogenic, chondrogenic and osteogenic differentiation potential. CONCLUSIONS: 300 g may be the optimal centrifugal force when using Ficoll density gradient centrifugation to isolate BMMNCs for bone regenerative therapy. 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.

A Comparative Study of Different Protocols for Isolation of Murine Neutrophils from Bone Marrow and Spleen.

Neutrophils are considered as the main player in innate immunity. In the last few years, it has been shown that they are involved in different physiological conditions and diseases. However, progress in the field of neutrophil biology is relatively slow due to existing difficulties in neutrophil isolation and maintenance in culture. Here we compare four protocols based on density-gradient and immunomagnetic methods for isolation of murine neutrophils from bone marrow and spleen. Neutrophil isolation was performed using Ficoll 1.077/1.119 g/mL density gradient, Ficoll 1.083/1.090/1.110 g/mL density gradient and immunomagnetic method of negative and positive selection. The different protocols were compared with respect to sample purity, cell viability, yield, and cost. The functionality of isolated neutrophils was checked by NETosis analysis and neutrophil oxidative burst test. Obtained data revealed that given purity/yield/viability/cost ratio the protocol based on cell centrifugation on Ficoll 1.077/1.119 g/mL density gradient is recommended for isolation of neutrophils from bone marrow, whereas immunomagnetic method of positive selection using Dynabeads is recommended for isolation of splenic neutrophils.

Protocol to dissociate and isolate wide-diversity single cells by density gradient centrifugation from human hepatoblastoma tissue.

Single-cell transcriptome sequencing can characterize various cell types in human liver tissue and facilitate understanding of hepatoblastoma heterogeneity. Here, we present a protocol for isolating hepatocytes and immune cells from human hepatoblastoma samples with high viability. We describe steps for tissue processing, enzymatic digestion, Percoll density gradient separation, cell lysis, cell suspension quality control, and scRNA library construction. We then detail sequencing and data analysis. This protocol is applicable to preparing single-cell suspensions from other human liver tissue samples.

Isolation of Mammalian Peroxisomes by Density Gradient Centrifugation.

Sophisticated organelle fractionation strategies were the workhorse of early peroxisome research and led to the characterization of the principal functions of the organelle. However, even in the era of molecular biology and "omics" technologies, they are still of importance to unravel peroxisome-specific proteomes, confirm the localization of still uncharacterized proteins, analyze peroxisome metabolism or lipid composition, or study their protein import mechanism. To isolate and analyze peroxisomes for these purposes, density gradient centrifugation still represents a highly reliable and reproducible technique. This article describes two protocols to purify peroxisomes from either liver tissue or the HepG2 hepatoma cell line. The protocol for liver enables purification of peroxisome fractions with high purity (95%) and is therefore suitable to study low-abundant peroxisomal proteins or analyze their lipid composition, for example. The protocol presented for HepG2 cells is not suitable to gain highly pure peroxisomal fractions but is intended to be used for gradient profiling experiments and allows easier manipulation of the peroxisomal compartment, e.g., by gene knockdown or protein overexpression for functional studies. Both purification methods therefore represent complementary tools to be used to analyze different aspects of peroxisome physiology. Please note that this is an updated version of a protocol, which has been published in a former volume of Methods in Molecular Biology.

The influence of Percoll® density gradient centrifugation before cryopreservation on the quality of frozen wisent (Bison bonasus) epididymal spermatozoa.

BACKGROUND: The wisent (Bison bonasus) is a species that has undergone a population bottleneck. Homozygosity is prevalent within the population and may have a negative impact on semen quality in wisent bulls. Semen samples containing a large amount of functionally and morphologically impaired or dead spermatozoa have lower tolerance for cryopreservation process. Such samples are prone to involve damage acrosomes, to produce and release reactive oxygen which negatively affects proper function of spermatozoas. It is a good practice to select intact and viable gametes before subjecting the sample to cryopreservation to improve the efficiency of this process. The aim of this study was to assess the ability of Percoll® density gradient centrifugation in order to improve the quality of wisent spermatozoa after cryopreservation. Spermatozoa samples were analysed with computer-assisted semen analysis system and flow cytometry. RESULTS: Percoll® density gradient centrifugation resulted in increased percentage of motile spermatozoa, higher proportion of spermatozoa with normal morphology and proper functionality but also in a significant reduction of the total number of gametes. Nevertheless, the concentration of frozen spermatoza was still sufficient for obtaining a few complete insemination doses suggested for cattle from each epididymis. CONCLUSIONS: While creating a high-quality genetic reserve, for in vitro fertilisation purposes, eliminating detritus and improving the overall quality of samples is more important than total number of spermatozoa. For these reasons, the achievement of higher post thaw quality of spermatozoa justifies the purification of samples by centrifugation in a Percoll® density gradient prior to the cryopreservation process.

A Comprehensive Analysis of Human Endogenous Retroviruses HERV-K (HML.2) from Teratocarcinoma Cell Lines and Detection of Viral Cargo in Microvesicles.

About 8% of our genome is composed of sequences from Human Endogenous Retroviruses (HERVs). The HERV-K (HML.2) family, here abbreviated HML.2, is able to produce virus particles that were detected in cell lines, malignant tumors and in autoimmune diseases. Parameters and properties of HML.2 released from teratocarcinoma cell lines GH and Tera-1 were investigated in detail. In most experiments, analyzed viruses were purified by density gradient centrifugation. HML.2 structural proteins, reverse transcriptase (RT) activity, viral RNA (vRNA) and particle morphology were analyzed. The HML.2 markers were predominantly detected in fractions with a buoyant density of 1.16 g/cm3. Deglycosylation of TM revealed truncated forms of transmembrane (TM) protein. Free virions and extracellular vesicles (presumably microvesicles-MVs) with HML.2 elements, including budding intermediates, were detected by electron microscopy. Viral elements and assembled virions captured and exported by MVs can boost specific immune responses and trigger immunomodulation in recipient cells. Sequencing of cDNA clones demonstrated exclusive presence of HERV-K108 env in HML.2 from Tera-1 cells. Not counting two recombinant variants, four known env sequences were found in HML.2 from GH cells. Obtained results shed light on parameters and morphology of HML.2. A possible mechanism of HML.2-induced diseases is discussed.

Robust sequential biophysical fractionation of blood plasma to study variations in the biomolecular landscape of systemically circulating extracellular vesicles across clinical conditions.

Separating extracellular vesicles (EV) from blood plasma is challenging and complicates their biological understanding and biomarker development. In this study, we fractionate blood plasma by combining size-exclusion chromatography (SEC) and OptiPrep density gradient centrifugation to study clinical context-dependent and time-dependent variations in the biomolecular landscape of systemically circulating EV. Using pooled blood plasma samples from breast cancer patients, we first demonstrate the technical repeatability of blood plasma fractionation. Using serial blood plasma samples from HIV and ovarian cancer patients (n = 10) we next show that EV carry a clinical context-dependent and/or time-dependent protein and small RNA composition, including miRNA and tRNA. In addition, differential analysis of blood plasma fractions provides a catalogue of putative proteins not associated with systemically circulating EV. In conclusion, the implementation of blood plasma fractionation allows to advance the biological understanding and biomarker development of systemically circulating EV.

Purification of Functional Platelet Mitochondria Using a Discontinuous Percoll Gradient.

The isolation of mitochondria is gaining importance in experimental and clinical laboratory settings. Of interest, mitochondria and mitochondrial components (i.e., circular mitochondrial DNA, N-formylated peptides, cardiolipin) have been involved in several human inflammatory pathologies, such as cancer, Alzheimer's disease, Parkinson's disease, and rheumatoid arthritis. While several mitochondrial isolation methods have been previously published, these techniques are aimed at yielding mitochondria from cell types other than platelets. In addition, little information is known on the number of platelet-derived microvesicles that can contaminate the mitochondrial preparation or even the overall quality as well as functional and structural integrity of mitochondria. Here we describe a purification method, using a discontinuous Percoll gradient, yielding mitochondria of high purity and integrity from human platelets.

Isolation and characterization of extracellular vesicle subpopulations from tissues.

Extracellular vesicles (EVs) are lipid bilayered membrane structures released by all cells. Most EV studies have been performed by using cell lines or body fluids, but the number of studies on tissue-derived EVs is still limited. Here, we present a protocol to isolate up to six different EV subpopulations directly from tissues. The approach includes enzymatic treatment of dissociated tissues followed by differential ultracentrifugation and density separation. The isolated EV subpopulations are characterized by electron microscopy and RNA profiling. In addition, their protein cargo can be determined with mass spectrometry, western blot and ExoView. Tissue-EV isolation can be performed in 22 h, but a simplified version can be completed in 8 h. Most experiments with the protocol have used human melanoma metastases, but the protocol can be applied to other cancer and non-cancer tissues. The procedure can be adopted by researchers experienced with cell culture and EV isolation.

Analysis of Tumor-Derived Exosomes by Nanoscale Flow Cytometry.

The study of tumor exosomes has gained relevance in the last decades due to their potential use for therapeutic and diagnostic application. Although there is extensive knowledge of exosome biology, some biological samples like tumor-derived exosomes have been difficult to characterize due to their complexity and heterogeneity. This distinctive feature makes difficult the identification of specific exosome subpopulations with a shared molecular signature that could allow for targeting of exosomes with therapeutic and diagnostic potential use in cancer patients. Nanoscale flow cytometry has lately emerged as an alternative tool that can be adapted to the study of nanoparticles, such as exosomes. However, the physicochemical properties of these particles are an important issue to consider as nanoparticles need the application of specific settings which differ from those used in conventional flow cytometry of cells. Therefore, in the last few years, one of the main aims has been the optimization of technical and experimental protocols to improve exosome analysis. In this chapter, we discuss several aspects of cytometric systems with a special emphasis in technical considerations of samples and equipment.

Assessment of Cell Cycle in Primitive Chronic Myeloid Leukemia Cells by Flow Cytometry After Coculture with Endothelial Cells.

From the knowledge that hematopoiesis does not occur randomly in the bone marrow but is regulated by the different components of the microenvironment, the use of in vitro coculture systems has been used as a powerful tool in the analysis of different processes that are involved in the maintenance of blood cells. In this chapter, we describe a methodological strategy to perform a coculture between primitive hematopoietic cells and endothelial cells to evaluate cell cycle, an aspect of relevant importance in the permanence of primitive leukemic cells.

Circulating tumor cells with FGFR2 expression might be useful to identify patients with existing FGFR2-overexpressing tumor.

Fibroblast growth factor receptor (FGFR) is associated with proliferation, migration, and angiogenesis of carcinomas, and FGFR signaling inhibitors are considered a key drug for the treatment of solid tumors with FGFR overexpression. Amplification of FGFR2 is reportedly identified in 3%-10% of gastric cancers (GCs). The aim of this study is to clarify whether the identification of the circulating tumor cells (CTCs) with FGFR2 overexpression is useful to detect patients with FGFR2-overexpressing GC. One hundred GC patients who underwent gastrectomy were enrolled. A total volume of 8 mL of peripheral blood was collected from each patient just before gastrectomy, and mononuclear cells were enriched by Ficol density gradient centrifugation. These cells were immunostained with PI/CD45/EpCAM/FGFR2. The number of CTCs with FGFR2 expression in each sample was enumerated by FACScan. The FGFR2 expression level of the resected primary tumor was assessed by immunohistochemistry. The number of FGFR2-positive CTCs in the GC patients' peripheral blood was significantly correlated with the FGFR2 expression level of the primary GC. The relapse-free survival of the patients with FGFR2-positive CTCs (≥5 cells/10 mL blood) was significantly poorer (P = .018, log-rank) than that of the patients without FGFR2-positive CTCs (<5 cell/10 mL blood). These findings suggested that the determination of FGFR2-positive CTCs might help identify an existing tumor with FGFR2 overexpression.

Purification of Recombinant Adeno-Associated Viruses (rAAVs) by Cesium Chloride Gradient Sedimentation.

Centrifugation to equilibrium in cesium chloride gradients has been used for more than 40 yr to purify viruses. The application of high G-forces for a long period of time to a solution of CsCl generates a density gradient that allows separation of empty, partially packaged, and fully packaged viral particles from cellular debris, proteins, and nucleic acids in the crude viral lysate on the basis of their buoyant densities. This protocol describes the use of CsCl gradients to purify AAV vectors from crude viral lysates.

Characterization and internalization of small extracellular vesicles released by human primary macrophages derived from circulating monocytes.

Extracellular vesicles (EVs) are small membrane-limited structures derived from outward budding of the plasma membrane or endosomal system that participate in cellular communication processes through the transport of bioactive molecules to recipient cells. To date, there are no published methodological works showing step-by-step the isolation, characterization and internalization of small EVs secreted by human primary macrophages derived from circulating monocytes (MDM-derived sEVs). Thus, here we aimed to provide an alternative protocol based on differential ultracentrifugation (dUC) to describe small EVs (sEVs) from these cells. Monocyte-derived macrophages were cultured in EV-free medium during 24, 48 or 72 h and, then, EVs were isolated from culture supernatants by (dUC). Macrophages secreted a large amount of sEVs in the first 24 h, with size ranging from 40-150 nm, peaking at 105 nm, as evaluated by nanoparticle tracking analysis and scanning electron microscopy. The markers Alix, CD63 and CD81 were detected by immunoblotting in EV samples, and the co-localization of CD63 and CD81 after sucrose density gradient ultracentrifugation (S-DGUC) indicated the presence of sEVs from late endosomal origin. Confocal fluorescence revealed that the sEVs were internalized by primary macrophages after three hours of co-culture. The methodology here applied aims to contribute for enhancing reproducibility between the limited number of available protocols for the isolation and characterization of MDM-derived sEVs, thus providing basic knowledge in the area of EV methods that can be useful for those investigators working with sEVs released by human primary macrophages derived from circulating monocytes.

Cushioned centrifugation during sperm selection increases the fertilization and cleavage rates of cattle embryos produced in vitro.

This study was conducted to evaluate the effect of utilization of an iodixanol-based solution as a cushioning method during the sperm selection utilizing discontinuous Percoll gradient centrifugation in in vitro production (IVP) of cattle embryos. In Experiment I, all aliquots of thawed semen were subjected to sperm selection using the same discontinuous Percoll® gradients, except for the following four conditions: presence of cushioning solution (Cushion Fluid, Minitube) during the first centrifugation process (C1), presence of cushioning solution during the second centrifugation process (C2), inclusion of cushioning solution in both centrifugation steps (C1-2), and no addiction of cushioning solution (C; control group). Recovery rates, sperm kinetics, and reactive oxygen species (ROS) production were evaluated. In Experiment II, sperm cells were processed using sperm selection conditions C and C1, and fertilization rates and embryonic development kinetics were compared between experimental groups. With use of condition C1, there was improvement in fertilization and cleavage rates when compared to use of condition C (56.4% compared with 45.5% and 80.0% compared 64.7%, respectively). In conclusion, results indicate the use of a cushioning solution during sperm selection positively affects the developmental potential of embryos.

Establishing a density-based method to separate proliferating and senescent cells from bone marrow stromal cells.

To assist in the study of cellular aging, we established a new method of enriching physiologically aged bone marrow stromal cells (BMSCs) in animals of any age using a Percoll density gradient centrifugation technique. BMSCs from mice 2 months of age were isolated, and their cellular age determined (over 80% Scal-1+ CD29+ CD11b- CD45- CD105- and able to differentiate into osteoblasts, adipocytes, and chondrocytes). As proof -of principle, cells were aged in vitro and confirmed by low bromodeoxyuridine (BrdU) incorporation and senescence-associated ß-galactosidase (SA-ß-gal) staining. Proliferating cells were enriched in high-density gradient layers, and senescent cells were enriched in low-density gradient layers. We confirmed that over 80% of cells from the low-density gradient layer were senescent with SA-ß-gal staining and telomere dysfunction-induced foci (TIF) assay. This density-based method, which can separate proliferating and senescent BMSCs, could be used to study mechanisms of physiologic cell aging and may have implications for the use of BMSCs in clinical transplant applications.

Polysome Profiling and Metabolic Labeling Methods to Measure Translation in Trypanosoma brucei.

The amount of a protein that is made in a cell is determined not only by the corresponding mRNA level but also by the efficiency with which the mRNA is translated. Very powerful transcriptome-wide methods are available to analyze both the density of ribosomes on each mRNA and the rate at which polypeptides are elongated. However, for many research questions, simpler, less expensive methods are more suitable. Here we describe two methods to assess the general translation status of cells: polysome profiling by sucrose density gradient centrifugation and metabolic labeling using radioactive amino acids. Both methods can also be used to examine translation of individual mRNAs.

A Scalable Purification Method for Mitochondria from Trypanosoma brucei.

Due to its unique biology the mitochondrion of Trypanosoma brucei has attracted a lot of interest since many decades, making it arguably the best studied mitochondrion outside yeast and mammals. Here we describe a method allowing purification of mitochondria from procyclic trypanosomes that yields highly enriched and functional organelles. The method is based on isotonic lysis of cells by nitrogen cavitation, DNase I digestion, differential centrifugation and Nycodenz gradient centrifugation. The method is scalable and can be adapted to culture volumes a small as 100 mL or as large as 24 L.

Isolation and Characterization of Acidocalcisomes from Trypanosomatids.

Acidocalcisomes are membrane-bounded, electron-dense, acidic organelles, rich in calcium and polyphosphate. These organelles were first described in trypanosomatids and later found from bacteria to human cells. Some of the functions of the acidocalcisome are the storage of cations and phosphorus, participation in pyrophosphate (PPi) and polyphosphate (polyP) metabolism, calcium signaling, maintenance of intracellular pH homeostasis, autophagy, and osmoregulation. Isolation of acidocalcisomes is an important technique for understanding their composition and function. Here, we provide detailed subcellular fractionation protocols using iodixanol gradient centrifugations to isolate high-quality acidocalcisomes from Trypanosoma brucei, which are subsequently validated by electron microscopy, and enzymatic and immunoblot assays with organellar markers.