Size Determination and Phenotypic Analysis of Urinary Extracellular Vesicles using Flow Cytometry

Summary

This protocol describes a method for the isolation of urinary extracellular vesicles, uEVs, from healthy human donors and their phenotypic characterization by the size and surface marker expression using flow cytometry.

Abstract

Extracellular vesicles, EVs, are a heterogeneous complex of lipidic membranes, secreted by any cell type, in any fluid such as urine. EVs can be of different sizes ranging from 40-100 nm in diameter such as in exosomes to 100-1000 nm in microvesicles. They can also contain different molecules that can be used as biomarkers for the prognosis and diagnosis of many diseases. Many techniques have been developed to characterize these vesicles. One of these is flow cytometry. However, there are no existing reports to show how to quantify the concentration of EVs and differentiate them by size, along with biomarker detection. This work aims to describe a procedure for the isolation, quantification, and phenotypification of urinary extracellular vesicles, uEVs, using a conventional cytometer for the analysis without any modification to its configuration. The method's limitations include staining a maximum of four different biomarkers per sample. The method is also limited by the amount of EVs available in the sample. Despite these limitations, with this protocol and its subsequent analysis, we can obtain more information on the enrichment of EVs markers and the abundance of these vesicles present in urine samples, in diseases involving kidney and brain damage.

Introduction

In mammals, blood is filtered by passing through the kidneys 250 – 300 times; during this time, urine is formed. Production of this biofluid is the result of a series of processes, including glomerular filtration, tubular reabsorption, and secretion. Metabolic waste products and electrolytes are the main components of urine. Also, other byproducts such as peptides, functional proteins, and extracellular vesicles (EVs) are excreted^1,2,3,4,5,6. Initially, urinary extracellular vesicles (uEVs) were identified in urine samples from patients suffering from water-balance disorders. These patients showed the presence of molecules such as aquaporin-2 (AQP2), which was then used as a biomarker for this disease⁷. Several subsequent studies focused on identifying the cellular origin of uEVs, describing that these structures can be secreted by kidney cells (glomerulus, podocytes, etc.) and other cell types of endothelial or leukocytic lineages. Moreover, the number and molecule-enrichment in uEVs can correlate with the status of many diseases and disorders^{8,9,10,11,12,13,14}.

Altogether, EVs make up a highly heterogeneous family of particles enclosed by lipid bilayers and released by cells through passive or active mechanisms into different fluids. Depending on their origin, EVs can be classified as endosome originated exosomes or plasma membrane-derived microvesicles/microparticles. However, this classification criterion can only be applied when the biogenesis of the particles is directly observed. Therefore, other non-trivial criteria, including physical, biochemical, and cellular origin, have been endorsed by several researchers in the field^15,16,17. Depending on the nature of the isolate analyzed, different analytical techniques were suggested for EVs characterization. For example, based on the enrichment of big (≥100 nm) or small (≤100 nm) EVs, quantification via flow cytometry or nanoparticle tracking is suggested, respectively¹⁸.

Nowadays, the use of EVs as biomarkers for many diseases has become relevant, so the search for different sources are been investigated. One of the most promising sources is the urine as it can be obtained in an easy and non-invasive manner. Therefore, this protocol describes a procedure for the isolation of uEVs by differential centrifugation, processing with fluorochrome-conjugated antibodies, and downstream analysis using a conventional 2-lasers/4-colors cytometer.

Protocol

The human urine samples were obtained from healthy volunteers who had signed donor-informed consent. These procedures were also approved by the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán Research Ethics Committee. 1. Isolation of urinary extracellular vesicles NOTE: The isolation protocol of uEVs is modified from ref.19. Figure 1 depicts the representation of the protocol to …

Representative Results

There are several checkpoints through the protocol, and before the staining of uEVs. Therefore, it is essential to first verify the amount of protein present in the extract of uEVs. All the research groups that work with extracellular vesicles quantify the protein, as indicated in step 2.1. Supplementary Figure 2 shows a representative 96 well plate containing uEVs fraction in wells 4E, 5E, and 6E. Wells 1A, 2A, and 3A consist of blanks, but if there are no uEVs purified, the wells will take similar colo…

Discussion

Nowadays, the use of extracellular vesicles as biomarkers for several diseases has augmented, especially for those that can be isolated from non-invasive sources such as urine^{5,21,22,23,24}. It has been proved that the isolation of uEVs is a vital resource to know the status of a healthy individual, and the diagnosis/prognosis of patients suffering several dise…

Materials

APC anti human CD156c (ADAM10) antibody	BioLegend	352706	Add 5 µL to the 20 µL of uEVs in PBS
APC anti human TSPAN33 (BAAM) antibody	BioLegend	395406	Add 5 µL to the 20 µL of uEVs in PBS
Avanti centrifuge with JA-25.5O fixed angle rotor	Beckamn Coulter	J-26S XPI
BD Accuri C6 Flow Cytometer	BD Biosciences
β-mercaptoethanol	SIGMA-Aldrich	M3148
Benchtop centrifuge with A-4-44 rotor	Eppendorf	5804
BLUEstain 2 protein ladder	GOLDBIO	P008
CD9 (C-4) mouse monoclonal antibody	Santa Cruz Biotechnology	sc-13118
CD63 (MX-49.129.5) mouse monoclonal antibody	Santa Cruz Biotechnology	sc-5275
Cell Trace CFSE cell proliferation kit for flow cytometry	Thermo Scientific	C34554
Chemidoc XRS+ system	BIORAD	5837
FITC anti human CD9 antibody	BioLegend	312104	Add 5 µL to the 20 µL of uEVs in PBS
FITC anti human CD37 antibody	BioLegend	356304	Add 5 µL to the 20 µL of uEVs in PBS
Fluorescent yellow particles	Spherotech	FP-0252-2
Fluorescent yellow particles	Spherotech	FP-0552-2
Fluorescent yellow particles	Spherotech	FP-1552-2
FlowJo Software	Becton, Dickinson and Company
Goat anti-mouse immunoglobulins/HRP	Dako	P0447
Halt protease inhibitor cocktail	Thermo Scientific	78429
Immun-Blot PVDF membrane 0.22µm	BIORAD	1620177
Megamix-Plus FSC beads	COSMO BIO CO.LTD	7802
NuPAGE LDS sample buffer 4X	Thermo Scientific	NP0007
Optima ultracentrifuge with rotor 90Ti fixed angle 355530	Beckamn Coulter	XPN100
Page Blue protein staining solution	Thermo Scientific	24620
PE anti human CD53 antibody	BioLegend	325406	Add 5 µL to the 20 µL of uEVs in PBS
Pierce BCA Protein assay kit	Thermo Scientific	23227
Pierce RIPA buffer	Thermo Scientific	89900
Polycarbonate thick wall centrifuge tubes	Beckamn Coulter	355630
Spherotech 8-Peak validation beads (FL1-FL3)	BD Accuri	653144
Spherotech 6-Peak validation beads (FL4)	BD Accuri	653145
Sucrose	SIGMA-Aldrich	59378
Triethanolamine	SIGMA-Aldrich	90279