We present a protocol for the isolation and freeze-fracturing of extracellular vesicles (EVs) originating from cancerous urothelial cells. The freeze-fracture technique revealed the EVs’ diameter and shape and-as a unique feature-the internal organization of the EV membranes. These are of immense importance in understanding how EVs interact with the recipient membranes.
Extracellular vesicles (EVs) are membrane-limited structures released from the cells into the extracellular space and are implicated in intercellular communication. EVs consist of three populations of vesicles, namely microvesicles (MVs), exosomes, and apoptotic bodies. The limiting membrane of EVs is crucially involved in the interactions with the recipient cells, which could lead to the transfer of biologically active molecules to the recipient cells and, consequently, affect their behavior. The freeze-fracture electron microscopy technique is used to study the internal organization of biological membranes. Here, we present a protocol for MV isolation from cultured cancerous urothelial cells and the freeze-fracture of MVs in the steps of rapid freezing, fracturing, making and cleaning the replicas, and analyzing them with transmission electron microscopy. The results show that the protocol for isolation yields a homogenous population of EVs, which correspond to the shape and size of MVs. Intramembrane particles are found mainly in the protoplasmic face of the limiting membrane. Hence, freeze-fracture is the technique of choice to characterize the MVs’ diameter, shape, and distribution of membrane proteins. The presented protocol is applicable to other populations of EVs.
Extracellular vesicles (EVs) are membrane-limited vesicles released from cells into the extracellular space. The three main populations of EVs are exosomes, microvesicles (MVs), and apoptotic bodies, which differ in their origin, size, and molecular composition1,2,3. The composition of EVs reflects the molecular profile of the donor cell and its physiological status (i.e., healthy or diseased)4,5. This gives EVs immense potential in the diagnosis, prognosis, and therapy of human diseases, and they have promising medical applications for the use in personalized medicine6,7,8.
EVs are mediators of intercellular communication. They contain biologically active proteins, lipids, and RNAs, which interfere with biological processes in the recipient cell and can change its behavior9,10. However, the composition of the EV limiting membrane is crucial for the interaction with the recipient cell membrane.
The sources of EVs are body fluids and conditioned culture media. To isolate an EV population, a suitable isolation technique must be used. For example, centrifugation at 10,000 × g yields a fraction enriched in MVs, whereas centrifugal forces of ≥100,000 × g yield a fraction enriched in exosomes11,12. The isolated fraction of EVs must be validated in terms of purity, size, and shape. For that purpose, the International Society for Extracellular Vesicles 2018 recommended three classes of high-resolution imaging techniques: electron microscopy, atomic force microscopy, and light microscopy-based super-resolution microscopy13. None of these techniques can provide information on the EV membrane interior.
Freeze-fracture electron microscopy is a technique of breaking frozen specimens to reveal their internal structures, particularly giving a view of the membrane interior. The steps of sample preparation are (1) rapid freezing, (2) fracturing, (3) making the replica, and (4) cleaning the replica14. In step 1, the sample is (optionally) chemically fixed, cryoprotected with glycerol, and frozen in liquid freon. In step 2, the frozen specimen is fractured in a freeze-fracture unit, which exposes the interior of the membrane bilayer. In step 3, the exposed fractured faces are shadowed with platinum (Pt) and carbon (C) to produce replicas. In step 4, the organic material is removed. The replica is analyzed in the transmission electron microscope (TEM). For accurate interpretation of the micrographs, one must follow guidelines for their proper orientation14,15. Briefly, the direction of shadows in the micrograph is a reference to orientate the micrograph (i.e., to determine the direction of Pt shadowing) and, consequently, to determine convex and concave shapes (Figure 1). Two interior views termed fractured faces of the membrane bilayer can be seen as a result of splitting the membrane by freeze-fracturing: the protoplasmic face (P-face) and the exoplasmic face (E-face). The P-face represents the membrane leaflet adjacent to the cell protoplasm, while the E-face represents the membrane leaflet adjacent to the extracellular space. Integral membrane proteins and their associations are seen as protruding intramembrane particles14,15.
Here, the goal is to apply the freeze-fracture technique to characterize MVs in terms of size, shape, and the structure of their limiting membrane. Here, we present a protocol for the isolation and freeze-fracturing of MVs originating from human invasive bladder cancerous urothelial cells.
1. Culturing cancerous urothelial cells and isolation of EVs
NOTE: A protocol to obtain EVs from a human invasive bladder cancer urothelial (T24) cell line is presented. However, culturing conditions have to be optimized to use other cell types.
2. Freezing of EVs
NOTE: Before freezing, first cryoprotect the samples with glycerol.
3. Fracturing of EVs and making the replicas
NOTE: Prepare the freeze-fracturing unit according to the manufacturer's operating instructions. (Figure 4A). Clean the chamber of the unit. Position platinum (Pt/C) and carbon (C) guns at angles of 45° and 90°, respectively (Figure 4B).
4. Cleaning the replicas and replica analysis
MVs were isolated from the conditioned medium of cancer urothelial T24 cells after differential centrifugations. Following the protocol, the EV fraction was first detected after centrifugation at 10,000 × g when it was seen as a white pellet (Figure 2G).
Next, the EVs were processed according to the above protocol and examined under TEM. Pt/C shadowing produced (Figure 4L) relatively large replicas that frequently broke up into smaller fragments during the cleaning step. Large replicas and their smaller fragments were picked on the TEM grid and compared under the microscope. The results showed no differences in the quality of the replica surfaces regardless of their size, and they can, therefore, all be used. Low magnification examinations showed regions of the replica with i) a homogeneous surface (background), ii) concave and convex spherical profiles (vesicles), and iii) regions of damaged surface (artifacts; Figure 5A). Typical artifacts were seen as ruptures, folds, and irregular dimmer shadows (i.e., replicas of ice-crystal deposits on the specimen). It is also important to note that no cell debris or cellular organelles were seen, confirming the purity of the sample (Figure 5B).
The isolated vesicles were commonly gathered either in clusters of three or more or were individually distributed (Figure 5B). The vesicles were spherical, which points to good preservation of the ultrastructure during isolation, fixation, and freezing (Figure 5C). "Flat-ball" and elongated vesicles (Figure 5C), which were seen occasionally, were presumably artifacts of preparation and were not included in the subsequent measurements of vesicle diameter. The diameter of the visible profiles was 238.5 nm (±8.0 nm, n = 190), which, taking into account the correction factor proposed by Hallett et al.17, corresponds to the mean vesicle diameter of 304 nm (±10 nm; Figure 5D). The size correlates to a diameter range of MVs between 100 nm and 1000 nm and proves the effectiveness of the used isolation protocol. The images of the vesicles together with diameter determination unequivocally confirmed that the EVs in the isolate were enriched with MVs.
The analysis of the replicas revealed the organization of P-face and E-face of the MV limiting membranes. MVs were observed as concave and convex round shapes (Figure 5C,E,F), reflecting the fracture plane. The convex shapes represent E-faces (i.e., fractured exoplasmic leaflet of the membranes; Figure 5E). The exoplasmic faces of the EVs had a smooth, uniform appearance. The concave shapes correspond to P-faces (Figure 5F). In the P-faces, a few protruding intramembrane particles were seen within smooth membranes (Figure 5C,F). This implies that MVs isolated from cancer urothelial T24 cells contain only a low amount of membrane proteins.
Figure 1: Schematic presentation of membrane determination after freeze-fracturing. (A) Microvesicles bud from the plasma membrane into the extracellular space. (B) Microvesicle is limited with P- and E- membrane leaflet until freeze-fractioning, which splits the leaflets and exposes the interior views of leaflets, termed fractured faces. (C) After Pt/C shadowing, two fractured faces are discernible: the protoplasmic face (P-face) with a convex shape facing the cytoplasm (protoplasm) and an exoplasmic face (E-face) with a concave shape facing the extracellular space. Figure 1 was created using Biorender.com. Please click here to view a larger version of this figure.
Figure 2: Isolation of EVs. (A) T24 cells grown in a CO2 incubator are examined with (B) a light microscope to confirm their viability and confluence before MV isolation. (C) The cell culture medium is collected and (D) consecutively centrifuged at 300 × g and at 2,000 × g (E) each time the supernatant is collected. (F,G) After the centrifugation at 10,000 × g, a white patch indicating a pellet is visible and marked. The supernatant is removed and (H) fixative is carefully added to the pellet without resuspending it. Please click here to view a larger version of this figure.
Figure 3: Freezing of EVs. (A) Air-dried clean copper carriers with a central pit are (B) marked before processing. Microvesicles are resuspended in glycerol to get a homogenous sample and (C) added to a central pit of a cooper carrier under a stereomicroscope. (D) One has to add a volume of the sample such that it forms a convex drop in the central pit. (E) Immediately before freezing, mix LN2-cooled freon with a metal rod to liquefy the freon. (F) Freeze the sample by submerging it in freon, and then (G) transfer it into LN2. (H) Carriers with the frozen sample can be collected into cryovials and stored in an LN2 Dewar container. Please click here to view a larger version of this figure.
Figure 4: Freeze-fracturing and making a replica. (A) Freeze-fracturing unit. Inside the unit chamber (B) is a platinum (Pt) and carbon (C) electron gun, a knife, and the sample table. (C) To start fracturing, carriers with the sample are transferred to the sample table, (D) the freeze-fracturing unit is cooled, and a vacuum is established. (E) Sectioning is done by motorized (F) movement of the knife, but fracturing is preferably done manually. (G) Sample before sectioning and (H) after fracturing. (I) Immediately after fracturing, the replica is made. (J) During this process, Pt is shadowed on the sample. Platinum shadowing is seen as a bright light (sparks) in the chamber. (K) Sample carriers are collected into a porcelain well filled with water. (L) Replica (arrow) floating in the sodium hypochlorite solution during the cleaning step. Please click here to view a larger version of this figure.
Figure 5: Electron micrographs of freeze-fractured and Pt/C shadowed MVs. (A) An overview of the freeze-fractured and shadowed EV pellet (i) at lower magnification. The homogeneous surface is background (ii), bright areas are ruptures in the replicas (iii), darker areas are folds of replicas (asterisk), and irregular dimmer shadows are due to ice crystals (two asterisks). (B) Cluster of round-shaped EVs with concave and convex surfaces. (C) High magnification of EVs. In convex fractures, which exhibit P-face of the EV membranes, intramembrane particles (arrows) and patches of a smooth surface (arrowheads) are seen. Extracellular vesicles with elongated (star) and flat-ball shapes (two stars) are found. (D) The mean diameter of isolated urothelial MVs is 304 nm ± 10 nm according to the size measurements as proposed by Hallett et al.17. Data are presented as mean ± SEM. (E,F) E-face and P-face of MVs. Legend: arrows = intramembrane particles in P-face, encircled arrows = the direction of Pt/C shadowing. Scale bars: A = 10 µm, B-F = 400 nm. Please click here to view a larger version of this figure.
The characterization of MVs, or any other population of isolated EVs, is of prime importance to begin with before starting downstream analyses like "omics" studies or functional studies11,18. Herein, EVs from human invasive bladder cancer urothelial T24 cells were isolated by centrifugation, and following the provided protocol for analysis by freeze-fracture electron microscopy, we demonstrated that the isolated fraction was enriched in MVs11,13. The isolate of MVs was devoid of cell debris or organelles, confirming a successful isolation and purification protocol.
The combination of chemical fixation and freezing, which are critical steps of the protocol, retained the spherical shape of the EVs12. However, precautions are needed when assessing EV diameter by freeze-fracturing17. Since the fracture passes through the sample randomly, MV membranes are split into their equatorial and non-equatorial planes. To provide a rigorous method for analyzing the images of freeze-fractured and shadowed specimens, Hallett et al. showed that the mean vesicle diameter is 4/π times the actual size of the vesicular diameter on the image17. Accounting for that, EVs from T24 cells were calculated to have a diameter of 304 nm, which fits in the MV's theoretical size distribution range of 100-1000 nm19.
Freeze-fracturing can supplement negative staining, the most extensively used TEM technique to visualize EVs. By negative staining, the sample is commonly chemically fixed, dried and attached to a TEM grid, and contrasted with uranyl solution. Without supporting media, EVs tend to collapse, which gives them a cup-shaped appearance. By freeze-fracturing, we show that MVs are spheres, which reflects their shape in extracellular spaces and body fluids12. By that, our results are also in agreement with observations of EVs in cryo-ultrathin sections20.
A crucial advantage of the freeze-fracture technique is its power to resolve the internal organization of the limiting membrane, which is a key factor in understanding how EVs are targeted to and interact with recipient membranes. Here, we analyzed the membranes of MVs, yet freeze-fracturing could reveal the membrane organization of any other population of EVs. MVs are formed by plasma membrane budding; therefore, plasma membrane proteins and protein clusters are expected to be found in the MV membrane. Our results supported that the MVs from T24 cells contained intramembrane particles, presenting integral membrane proteins. Based on particle distribution between the E-face and P-face, it is reasonable to expect that the particles observed in MVs are transmembrane proteins uroplakins, which are urothelial cell specific21,24. The observed particles were sparse, which is in accordance with previous studies reporting a reduction in uroplakins during urothelial carcinogenesis21,22,23. However, to further investigate the protein composition of MV membranes, the use of the freeze-fracture replica immune-labeling (FRIL) technique is recommended. FRIL is an upgrade of the presented freeze-fracture technique and is dedicated to revealing the identity of proteins in replicas by antibody recognition24,25. To sum up, the freeze-fracture technique is an electron microscopy technique suitable for the characterization of the EV limiting membrane, as well as the shape, size, and purity of the isolated EV fractions. The presented protocol can be used also for the assessment of other populations of isolated EVs; therefore, the freeze-fracturing technique merits inclusion in the International Society for Extracellular Vesicles guidelines for studies exploring the organization of EV limiting membranes.
The authors have nothing to disclose.
This research was funded by the Slovenian Research Agency (research core funding no. P3-0108 and project J7-2594) and the MRIC UL IP-0510 Infrastructure program. This work contributes to the COST Action CA17116 International Network for Translating Research on Perinatal Derivatives into Therapeutic Approaches (SPRINT), supported by COST (European Cooperation in Science and Technology). The authors would like to thank Linda Štrus, Sanja Čabraja, Nada Pavlica Dubarič, and Sabina Železnik for technical help with cell culturing and preparing the samples and Marko Vogrinc, Ota Širca Roš, and Nejc Debevec for technical help preparing the video.
A-DMEM | ThermoFisher Scientific, USA | 12491015 | |
Balzers freeze-fracture device | Balzers AG, Liechtenstein | Balzers BAF 200 | |
Centrifuge | Eppendorf, Germany | model 5810R | |
CO2 incubator HeraCell | Heraues, Germany | ||
Copper carriers | Balzers | BB 113 142-2 | 100+ mesh |
Copper grids | SPI, United States | 2010C | |
Culture flask T75 | TPP, Switzerland | growing surface 75 cm2 | |
F12 (HAM) | Sigma-Aldrich, Germany | 21765029 | |
FBS | Gibco, Life Technologies, USA | 10500064 | |
Glazed Porcelain Plate 6 Well | Fischer Sientific, United States | 50-949-072 | |
Glycerol | Kemika, Croatia | 711901 | final concentration 30 % (v/v) |
Glutaradehyde | Serva, Germany | 23114.01 | final concentration 2.5 % (v/v) |
GlutaMAX | Gibco, Life Technologies | 35050-079 | final concentration 4 mM |
Penicillin | Gibco, Life Technologies | 15140163 | final concentration 100 U/ml |
Phase-contast inverted microscope | Nikon, Japan | model Eclipse | |
Rotor | Eppendorf, Germany | A 4-44 | |
Rotor | Eppendorf, Germany | F-34-6-38 | |
Serological pipetts | TPP, Switzerland | 50mL volume | |
Sodium hypochloride solution in water | Carlo Erba, Italy | 481181 | |
Stereomicroscope | Leica, Germany | ||
Streptomycin | Gibco, Life Technologies | 35050038 | final concentration 100 mg/mL |
Transmission electron microscope | Philips, The Netherlands | model CM100 | working at 80 kV |
Tweezers | SPI, United States |