HansaBioMed Life Sciences — Pioneering the Extracellular Vesicle Field

Extracellular vesicles (EVs) are cell-derived nanoparticles, differing in their cellular origin, biogenesis mechanism, size and molecular content. Even though the nomenclature of EVs is still lacking consensus, the most prominent types of EVs are Exosomes, shedding Microvesicles (MVs) and Apoptotic Bodies. Recently, EVs have started to emerge as an important mean of intercellular communication and attract more attention as diagnostic, prognostic and therapeutic tool, due to their biomarker potential, virtue to represent physiological status of parent cell and ability to modulate functions of recipient cells.

Lyophilized Small Extracellular Vesicles (s-EVs)/Exosomes

Purified s-EVs are obtained from a variety of biological sources: cell culture supernatant, human plasma, serum, urine. s-Evs are purified following a combination of tangential flow filtration (TFF) and size exclusion chromatography (SEC). Subsequently, isolated vesicles are quantified and validated for overall protein content, size distribution, concentration and EV specific marker expression.

Plant-EVs

Plant EVs, called also plant EV-like nanoparticles (ELNs), are attracting interest from researchers in academic sector, but also from the nutraceutical and the cosmetic industry. Indeed, plant EVs, such as the EV produced by mammalian cells, reflect the properties of the parental cells, can be purified from the plant leaves, stems, or juice, easily sterilized by filtration, and long-term stored.

HansaBioMed Life Sciences provides purified ELNs from different plant extracts, ELNs are purified by tangential flow filtration and characterized by particle size distribution and concentration, expression of TET8 marker (corresponding to mammalian CD63). A certificate of analysis reports the origin of the material.

FLuoEVs: Purified EVs Expressing Fluorescent Proteins

FLuoEVs are stably-fluorescent EVs expressing the fluorescent protein EGFP (green) BFP (blue) or mCherry (Red).

FLuoEVs are extracellular vesicles purified by a combination of Tangential Flow Filtration (TFF) and Size Exclusion Chromatography (SEC) from cells engineered in order to express the fluorescent proteins as fusion protein with the tetraspanin CD9, CD63 or CD81.

FLuoEVs are currently available from HEK293 cells, expressing EGFP. On request, they can be produced from other cell lines and with fluorescent proteins mCherry (red) and BFP (Blue).

FLuo-EVs demonstrate high stability of the fluorophores and several advantages over the lipidic dyes, widely used for EV labelling, including higher percentage of fluorescent particles. Moreover, all the fluorescent particles are originated from the parental cells, avoiding artifacts derived from the presence of dye micelles or non-specifically labelled lipidic particles (lipoproteins).

Application: FLuoEVs can be used for cell spike-in experiment and in vitro tracking studies, and as positive control for EV analysis, being compatible with the most common method of analysis (Fluorescence NTA, Flow Cytometry, Microplate Fluorescence Reading) and analyzers (Particle Metrix instruments, Nanoanlyzer NanoFCM, etc).

TFF-Easy: Tangential Flow Filter for EV Concentration

Tangential flow filtration (TFF) is a rapid and efficient method, usually used for separation and purification of biomolecules. TFF can be also used to concentrate and desalt sample solutions, and is emerging as a new technique for easily isolating EVs, if coupled with SEC.

HansaBioMed’s TFF-Easy is a filter cartridge containing hollow fibers made of polysulfone suitable for the concentration and the removal of small proteins and molecules from diluted matrices (cell conditioned media, urine, etc.), prior to the EV purification.

TFF-Easy can be used manually with syringes of different size, for concentrating small volumes of fluids, or can be connected to a mechanical pump for concentrating larger volumes.

TFF-MV: Filter for Large EV Separation and Purification

TFF-MV is a filter able to separate large microvesicles (MVs) by size, avoiding the separation by centrifugation at 10000 g, which often causes the loss of part of small EVs. TFF-MV retains vesicles larger than 150-200 nm, whereas small EVs and circulating molecules pass in the permeate. Retained MVs can be recovered with a syringe in PBS buffer, without additional purification steps.

TFF-EVs: Fast EV Purification

TFF-EVs is our next-generation filter which allows a rapid, reproducible and scalable purification of EVs. It can be used on the lab bench for purifying small sample volumes (min 5 ml) or connected with a mechanical system for purifying larger volumes.

TFF-EVs is a filter cartridge made of polyethersulfone hollow fibers with pores of 50 nm size (cut off 300 kDa). The filter allows the purification of EVs and particles > 50 nm.

Although ultracentrifugation is still widely used, it is not anymore considered the gold standard methodology for Extracellular Vesicles isolation, since it does not isolate EVs efficiently, tends to alter the vesicle shape and functionality, requires expensive equipment and is time-consuming. HansaBioMed has developed and optimized methods and tools for a fast, scalable and reproducible EV purification, for addressing the EV heterogeneity and high throughput solutions for biomarker discovery:

• Ultrafilters - For EV size separation and medium concentration
• Size Exclusion Chromatography Columns - For EV purification and removal of contaminants
• Immunoaffinity Isolation - For biomarker screening and enrichment of EV subpopulations
• Chemical Isolation - For biomarker screening and enrichment of EV subpopulations

EV-Spinner: Ultrafiltration Concentrator

EV-Spinner is a 2.5 ml non-stick ultrafiltration (UF) concentrator to enhance the recovery of extracellular vesicle (EVs) during the concentration or buffer exchange step. The ultrafiltration works in the opposite direction to the centrifugal force, providing higher particle recovery. The low protein binding membrane (polyethersulphone) reduces the EV loss, compared to V-shape concentrators, and the reverse design of the EV-spinner ensures that the filter does not clog.

PURE-EVs Size Exclusion Chromatography Columns

Size Exclusion Chromatography (SEC) is an efficient method for isolating and purifying Extracellular Vesicles (EVs) from different fluids, not affecting the original shape and functionality of the vesicles. We have developed a set of SEC columns which allow the EV purification from small (100 μl) and large volumes (up to 20 ml) of fluids. The EV purification process with PURE-EV columns is very fast, taking approximately 15 minutes of time.

Immunoaffinity Isolation of Extracellular Vesicles

Addressing the EV heterogeneity is becoming an important issue, in particular for the different roles that Extracellular Vesicles have in pathological processes as cancer, infection, or neurodegenerative diseases. HBM-LS provides pre-coated ELISA Immunoplates and Latex or Magnetic Immunobeads for the capture and enrichment of total or specific EV-subpopulations.

Immunoplates for EV Capture and Isolation

HBM-LS Immunoplates are 96 multiwell plates covalently pre-coated with specific EV-binding antibodies allowing the capture and isolation of vesicles from different sources (cell supernatant, human plasma, serum, urine and saliva). There are different types of plates for capturing the total or for enriching specific EV subpopulations (tumoral, neural, glial derived). Plates are blocked and stabilized for long-term storage.

Immunobeads for Total EV Isolation

Latex or Magnetic Immunobeads are covalently coupled with antibodies against common EV surface antigens (CD9, CD63). They allow capturing EV from human biofluids (tested for plasma, serum, and urine) and cell culture media without the necessity of pre-purification steps. The kit includes Beads, Elution Buffer, for detaching captured EVs from antibodies, and Regeneration Buffer. Immunobeads can be regenerated with the Regeneration Buffer and reused. Beads are sold in package of 10 reactions and are available in 2 sizes (0.4- and 1-micron diameter).

EXO-Prep: One Step EV Isolation Reagent

EXO-Prep is a fast and convenient method for extracellular vesicle isolation from biofluids, cell culture supernatants and plant extracts. Isolation with EXO-Prep is based on chemical precipitation. Samples are incubated with EXO-Prep solution on ice so that EVs will precipitate following centrifugation. The obtained pellet can be resuspended in 1x PBS or deionized water. The protocol is user-friendly, time-saving (around 1 hour), and does not require capital-intensive laboratory equipment. Isolated vesicles are in particular suitable for isolation of nucleic acid associated to EVs.

EVs research needs reliable, affordable and optimized tools for quantification and characterization of vesicles in complex biological samples as well as in cellular models. HBM-LS offers the ExoTEST™ and the Exo-FACS kits for quantification and characterization of EV markers by ELISA and FACS, respectively, and the new Enolase Activity Kit, which allows to characterize the different EVs sources by the determination of the enolase activity.

ExoTEST™: ELISA Ready-to-Use Quantification Kit

ExoTEST is a patented double sandwich ELISA assay for quantitative and qualitative analysis of vesicles. In particular, ExoTEST is a successful platform for exosome quantification and characterization from small amount of human biological fluids or cell conditioned media. ExoTEST enables reliable and precise quantitative measurement and comparison among samples and individual experiments and provides increased sensitivity in detection of EV markers with respect to other analytical methods (i.e., FACS, WB).

ExoTEST consists of ELISA plates pre-coated with proprietary pan-exosome antibodies enabling specific capture of exosomes from different biological samples. Quantification and characterization of exosomal proteins is subsequently performed using appropriate detection antibodies against exosome surface antigens. Lyophilized Exosome Standards, characterized for protein content and particle number (NTA) allow the quantification of unknown sample by a standard calibration curve.

Exo-FACS: Ready-to-Use Kit for EV FACS Analysis

The kit consists of EXO-Prep reagent for exosome isolation, 4 µm beads used for the overall capture of pre-isolated exosomes, lyophilized exosomes from cell culture supernatants or human biological fluids as positive control. The characterization of exosomal proteins (membrane-expressed or internal) is subsequently performed using appropriate detection antibodies against exosome associated antigens.

HBM-LS offers different Exo-FACS kits for staining of EV markers from human biofluids (plasma, urine, serum, saliva) and from cell culture supernatants. Exo- FACS contains reagents for 20 reactions (lyophilized exosomes, beads, antibodies and buffers). The primary antibody included in the kit is against a common exosomal marker (CD9 or CD63) and can be used as a positive control for protein profiling via FACS analysis.

EV-Enolase: Enolase Activity on EVs

Enolase (EC 4.2.1.11), also called 2-phospho-D-glycerate hydrolase or 2-phosphoglycerate dehydratase, is a key enzyme in glycolysis. It converts 2-phosphoglycerate to phosphoenolpyruvate (PEP) and also catalyzes the reverse reaction, PEP to 2-phosphoglycerate under anabolic conditions during gluconeogenesis. This enzyme exists in all organisms, which can undergo glycolysis. Enolase activity is easily detectable in Extracellular Vesicles (EVs) derived from eukaryotic cells and it could be used for evaluating functionality and stability of EVs. Moreover, its increased activity is associated with tumorigenesis and therefore precise measurement of enolase activity may be of great interest for EV-based tumor diagnosis.

Tissue biopsies are invasive and non-feasible for longitudinal monitoring of patients; moreover, they do not capture the heterogeneity of the tissue or a disease of interest. Liquid biopsies are non-invasive and enable real time snapshot of the tissue homeostasis or alterations by detection of relevant biomarkers in the bloodstream. Tissue or disease specific exosomes and extracellular vesicles (EVs) can be isolated and analyzed from routine blood or urine samples featuring the ideal platform for biomarker discovery and clinical diagnostic development.

THE CHALLENGE: The major challenge and opportunity in using EVs based liquid biopsy lies in the tremendous complexity of biofluid samples, heterogeneity of exosomes and extracellular vesicles and the low abundance of specific tissue or disease markers.

THE SOLUTION: HansaBioMed Life Sciences provides a range of pre-analytical solutions, empowered by Exosomics s.p.a, to isolate either overall EVs or selectively enrich for specific subpopulations (i.e., tumor derived-EVs) from biofluids with high yield and purity, and further extract their DNA and RNA content. The technologies are efficient in both biomarker discovery and confirmation studies, and comply with clinical grade analytical platforms. In clinical settings, our solutions enable ultrasensitive detection of tumor associated mutations and RNA molecules, enabling next generation of tumor screening, monitoring, staging and monitoring tests.

• HansaBioMed Catalog: Pioneering the Extracellular Vesicle Field
• Antibodies for Exosome Research
• evGAG: Fast Extracellular Vesicle Purification for Biomarker Discovery
• EV-Spinner Ultrafiltration Concentrator
• Exo-FACS: Exosome Isolation and FACS Analysis of Exosome Markers
• Exosomes One Step Isolation from a Wide Range of Biofluids or Cell Culture Media
• ELISA Exosome Quantification
• Immunoaffinity Capture of Extracellular Vesicles
• Lyophilized Exosome Standards
• Purified and Lyophilized Exosomes and Microvesicles
• PURE-EVs: SEC Columns for EV Isolation
• Exosome RNA Extraction Kits
• HansaBioMed Services: Extracellular Vesicle Purification and Characterization
• TFF-Easy: Tangential Flow Filter for EV Concentration
• TFF-EVs: Tangential Flow Filter for EV Purification
• TFF-MV: Tangential Flow Filter for Large EV Separation

Application Notes

• Double Tangential Flow Filtration for Purification of Urine EVs Provides High Particle Yield and Removal of THP
• Tangential Flow Filtration for Efficient Depletion of Nanoparticles Contained in Fetal Bovine Serum