TABLE 1.
Studies on Trypanosoma cruzi EVs.
| Reference | Vesicle cellular origin | Cell amounts (parasites/mL) | Vesiculation general conditions | Cell viability at time of EV harvest | Pre‐isolation/cell debris remotion | EV isolation and/or purification | EV characterization | EVs yield per batch (size/concentration) | In vitro/in vivo experiments using EVs | Markers |
|---|---|---|---|---|---|---|---|---|---|---|
| da Silveira et al., 1979 | EVs from epimastigotes (Y strain) | 1‐5 × 109 par/mL |
37°C, 1 h, LIT medium |
NR |
C: 4000 × g, 10 min, 4°C UC: 100,000 × g, 1 h, 4°C Purification at SEC |
UC: 105,000 × g, no time reported | Radioactivity determination and EM | NR | pH (base/acid) | NR |
| Abuin et al., 1996a, 1989, 1996b | EVs observed from trypomastigotes of the Y and YuYu strains |
5 × 107par./mL |
Cells cultured in LIT, TAU or DMEM with 5% FCS |
NR |
Fixation: 60 min in glutaraldehyde/4% formaldehyde, 0.1 M phosphate buffer, pH 7.2, Blocking: Incubation in NH4Cl H1A10 MAb (anti‐gp85) |
Treatment with formvar and carbon‐coated gold grids 0.1% polylysine | TEM | NR | Detection of parasite antigens | GP85 |
| Gonçalves et al., 1991 |
Exosomes secreted by trypomastigotes from Y, CA1, RA and YuYu strains obtained from infected LLC‐MK2 cells |
5 × 107par./mL |
Strains cultured for 4 h, 37°C, in DMEM w/5 % dialyzed FCS |
PBS with 500 μCi Na131I, 20 μg iodo‐gen, 4°C, 10 min |
C: 2500 × g, 5 min Filtration: 0.22 μm |
SEC | EM |
20–80 nm Conc.: NR |
IP | GP85 |
| Bayer‐Santos et al., 2013 | EVs obtained from Dm28c clone of epimastigotes and metacyclic trypomastigote forms |
1 × 08par./mL |
Strain cultured in LIT and TAU3AAG at 28°C | FC: propidium iodide |
C: 3000 × g, 10 min, 4°C → F: 0.45 μm |
UC: 100,000 × g, 2 h, 4°C 100,000 × g, 16 h, 4°C |
NTA, TEM, proteomics (2D LC−MS/MS) |
74–143 nm (mean) Conc.: NR |
WB; IIF |
FCaBP, GP82, GP35/50 (35‐50 kDa mucins) |
| Garcia‐Silva et al., 2014 | EVs obtained from Dm28c clone epimastigotes and trypomastigotes | 1 × 1011 par./mL | LIT and RPMI 1640 with 10% FBS | FC: propidium iodide | C: 2000 × g, 15 min → 2000 × g, 30 min 15,000 × g, 30 min |
UC: 110,000 × g, 4°C 70 min → 110,000 × g, 1 h, 4°C |
TEM | NR | Exchange of tsRNAs; TcPIWItryp protein Small RNA deep sequencing; electroporation sequencing | NR |
| Fernandez‐Calero et al., 2015 | EVs obtained from the Dm28c clone epimastigotes | 1 × 1010 par./mL | LITd with 10% FBS at 28°C | FC | C ‐ 2000 × g for 15 min/C ‐ 15,000 × g at 4°C for 30 min | UC at 110,000 × g at 4°C for 70 min. The pellet was washed twice in PBS and UC at 110,000 × g for 1 h. | TEM and Bradford | 1.2 μg per 1 × 1010 parasites | RNA Analyse | NR |
| De Pablos et al., 2016 | Trypomastigotes EVs from mice infected with trypomastigotes of PAN4 strain | 5 × 107 par./mL |
RPMI 1640 + 10% IFCS UC: 100,000 × g, 1 h |
NR | C: 3000 × g, 15 min, → 17,000 × g, 20 min → Filtration: 0.2 μm | UC: 100,000 × g, 1 h, 2 × | TEM |
90–300 nm (mean: 154 nm) |
IEM | Clathrin |
| Díaz Lozano et al., 2017 | Trypomastigotes (PAN4 strain) was isolated from a patient; cultured amastigotes and trypomastigote forms were obtained from infected Vero cells culture | NR |
RPMI 1640 with 25 mM HEPES pH 7.4 and 10% EV‐free IFCS |
NR |
C: 3000 × g, 15 min → 17,000 × g, 20 min → F: 0.2 μm |
UC: 100,000 × g, 1 h (twice) |
SEM TEM CM |
50–100 nm | WB and ELISA | NR |
| Lovo‐Martins et al., 2018 |
EVs obtained from Y strain trypomastigotes |
1 × 108 par./mL | Incubation in RPMI without FBS for 2 h at 37°C | NR | C ‐ 2600 × g, 10 min | Filtration (0.45 μm); total exosome isolation from cell‐culture media reagent | NTA | 136.33 nm | Experimental model | NR |
| Ribeiro et al., 2018 | EVs from Y and YuYu strains trypomastigotes | 1 × 109 par./mL | DMEM 2% glucose at 37°C, 5% CO2 | NR | C at 1000 × g for 10 min; filtration | SEC and UC–100,000 × g for 16 h at 4°C |
SEM NTA |
YuYu:1.0 × 107– 9.7 × 109 particles/mL; Y: 2.7 × 106– 8.9 × 108 particles/mL |
ELISA. WB, and PA, invasion assay | Mucin, GP85 and TS |
| Paranaiba et al., 2019 | EVs obtained from epimastigotes | 1 × 105 par./mL | LIT incubation at 28°C for 2 h | NR | NR | Filtration (0.22 μm) and ultracentrifugati on (100,000 × g, 2 h, 4°C) |
NTA TEM |
223.1 nm, (D10 = 143.6; D50 = 245.5 and D90 = 264.7); 6.84 × 107 particles/mL | Functional studies | NR |
| Retana Moreira et al., 2019 | EVs from trypomastigotes forms from Pan4 strain derived from Vero and 3T3 cells cultures | NR | RPMI medium buffered with 25 mM HEPES at 7.2 and supplemented with 10% exosome‐free IFBS at 37°C | Trypan blue |
Centrifugation: 3500 × g for 15 min 17,000 × g for 30 min at 4°C Filtration through a 0.22 μm pore filter |
UC‐100,000 × g for 16–18 h. |
NTA TEM DLS |
70.7 nm/5 × 1010 p/mL | WB; invasion assays | NR |
| Cronemberger‐Andrade et al., 2020 |
EVs from THP‐1 cells infected and with trypomastigotes from Y strain |
NR | Trypomastigotes EVs were obtained by incubation of parasites for 2 h in DMEM containing 2% glucose at 37°C. THP‐1 EVs were obtained incubating trypomastigotes for 4 h at 37°C/5% CO2 in RPMI | NR | C—10 min at 500 × g, 10 min at 3000 × g, 15 min at 8000 × g | UC‐ 100,000 × g for 1 h |
NTA SEM |
50–200 nm/105‐ 106 particles/mL |
WB; RT‐PCR |
Mucin, GP85 and TS |
| Retana Moreira et al., 2021 | EVs from epimastigotes and trypomastigotes forms from Pan4 strain derived from Vero cell cultures | 5 × 107 |
MEM with 10% FBS maintained at 37°C LIT with 10% FBS serum, at 28°C. |
Trypan blue |
UC ‐ 100,000 × g 16–18 h C‐3500 × g for 15 min. Filtration ‐ 0.22 μm |
UC‐ 100,000 × g 16–18 h |
NTA TEM DLS |
143–259 nm | PA, WB | NR |
| Vasconcelos et al., 2021 |
EVs obtained from Y strain trypomastigotes |
1 × 107 | DMEM with FBS or 5% glucose, temperatures and pH levels, and/or in the presence of metabolic inhibitors and nitroxidative compounds (NaN3 and NaNO2) and methylβcyclodextrin (CBD). | PrestoBlue | C ‐ 1000 × g for 15 min | C‐ 10,000 × g for 15 min. Then, the supernatant was filtered (0.22 μm) and ultracentrifuged at 100,000 × g, for 1 h, at 4°C |
SEM NTA |
150 nm |
EV labeling (Fluorescent cell dye), macrophage interaction, gene expression by qRT‐PCR |
NR |
| Cestari et al., 2012 |
EVs from epimastigote, MT and tissue culture trypomastigote |
5 × 106 |
Cells were incubated in RPMI at 37°C for 1 h and then 5 min on ice |
NR | NR |
DC (5 min, 1603 × g; 2 × 3 30 min, 4000 × g; and 90 min, 100,000 × g) |
TEM | NR | Invasion assay, cleavage assay, kinetic studies, WB, ELISA, in vivo model | NR |
| Trocoli Torrecilhas et al., 2009 | EVs from trypomastigotes | NR | Cells were incubated in RPMI at 37°C for 2 h | NR | C ‐ 2500 × g for 5 min and filtration 0.22 μm | SEC | EM | NR |
WB In vivo model |
TS/gp85 Cruzipain and mucin |
| Cortes‐Serra et al., 2020 | Plasma‐derived EVs from chronic Chagas disease patients | 1 mL of plasma from blood collected in sodium citrate | NR | NR | 2000 × g for 10 min at RT (2×) | SEC (iZON columns) | BBA, BCA, NTA, proteomic analysis | For proteomic analysis: 100 mL of SEC fractions (710) pooled | Identification of potential biomarkers of chronic Chagas disease in plasma‐derived EVs |
CD5L CD9 CD63 |
| Madeira et al., 2021, 2022 | Plasma‐derived EVs from chronic Chagas disease patients | 3 mL of plasma from blood collected in sodium citrate/EDTA or serum | NR | NR | 2000 × g for 10 min at RT |
UC SEC |
NTA BCA |
NR | Identification of potential biomarkers of chronic Chagas disease in plasma‐derived EVs | CD9,CD81, TS and total parasites antigens |
Abbreviations: NR, not reported; C, centrifugation; UC, ultracentrifugation; FC, flow cytometry; DLS, dynamic light scattering; NTA, Nanoparticle tracking analysis; PA, proteomic analysis; SEM, scanning electronic microscopy; TEM, transmission electronic microscopy.