NIH/3T3 mouse fibroblast (ATCC® CRL-1658) cells were cultured in 5 mL of RPMI 1640 medium, pH 7.4 (Gibco, Grand Island, USA), with 10% fetal bovine serum supplementation (Cultilab, Campinas, Brazil), 2g / L sodium bicarbonate (NaHCO3, Sigma-Aldrich, Brazil) and 1% penicillin and streptomycin solution (10000 U/ 10000 µg/mL, Gibco, Carlsbad, USA) in sterile plastic cell culture flasks with 25cm2 of culture area (Corning, Tewksbury, USA) kept in incubators in a controlled atmosphere and temperature (95% O2, 5% CO2, 37ºC), up to an estimated confluence of 60 to 70%. Subculture took place after depletion of the culture medium used, washing the adherent cells with 5 mL of PBS containing 5 mM EDTA (Sigma-Aldrich, Brazil). After separating the bottles, the cells were suspended in RPMI 1640 medium, prepared as described and centrifuged at 1500 rpm for 5 minutes in 15 mL conical tubes (Corning, Tewksbury, USA). Only a viable fraction, specified by incorporation of 0.4% trypan blue vital dye (Life Technologies, Carlsbad, USA) was used for subculture or experimentation.
Production of paramagnetic iron nanoparticles (PION’s) for magnetic aggregation cell cultures
Magnetite nanoparticle suspensions (Fe3O4) were produced as described previously (Bonfim et al. 2019), but using another energy source to reduce free Fe2+ ions. Concentrations of iron (II) sulfate heptahydrate (Fe2SO4.7H2O, CAS 7782-63-0) and glycine (NH2CH2COOH, CAS 56-40-6) were diluted in ultrapure water free of O2 at pH 12 obtained by titration with sodium hydroxide (NaOH). Volumes of this solution were irradiated by 15kGy in the treadmill electron accelerator of the IPEN/CNEN-SP Radiation Technology Center . Black precipitates were formed, and were separated from the liquid phase by magnetic attraction and washed with ultrapure water. Subsequently, 15 mL of acetic acid were added and the solution was kept under ultrasonic agitation for 5 minutes. After this process, the acetic acid was removed and a solution of poly-lysine bromide (D-Lys-(D-Lys)n-D-Lys. xHBr) in ultrapure water at pH 7 (0.02 mg / mL) was added and kept in ultrasound for another 5 minutes. Poly-lysine carries protonable amine groups at physiological pH, which conferred a positive charge on the particles and made possible the electrostatic interaction with the cells, leading to the adsorption of the particles by the cell membranes. The final precipitates of nanoparticles were washed again with sterile ultrapure water and stored at 4 °C until the moment of application to cell culture for subsequent formation of spheroids. Magnetite formation was confirmed by X-Ray diffraction analysis (XRD) in a Orion diffractometer, model RKS-400SV-R at the Multi-user Center of the Chemistry and Environment Center (CEQMA), IPEN / CNEN-SP. Peak patterns were compared to COD database using the QualX software (Altomare et al. 2015). The crystallite size was calculated using the Debye-Scherrer equation, D = 0.9λ/βcosθ, where D is the crystal size in nm, and λ is the X-ray wavelength, β is the half-width of the peak in rad, and θ is the corresponding diffraction angle. Dilutions of nanoparticles in ultrapure water were used to evaluate the dinamic light scattering (DLS) zeta potential of the particles in a Particle Analyzer Litesizer 500 (Anton Paar) at the Center of Radiation Technology (CETER), IPEN / CNEN-SP.
Cells were associated with produced particles as described. The formation of spheroids consisted of exposing cell cultures to amounts of nanoparticles for 24 hours. After this period, trypsinization was performed and cells were centrifuged and resuspended with the nanoparticles in the calculated volume of culture medium for plating, 100 µL of cell suspension. Cells with nanoparticles were seeded (104 cells/well) in 96-well plates made of cell-repellent plastic (Cell Repellent, GreinerBioOne), and a plate with 96 magnets (Magnetic drive, GreinerBioOne) was kept below the wells in an incubator as described for cell aggregation. After obtaining cohesive spheroids, the magnet plate was removed and the plate was incubated as described .
Toxoplasma gondii (RH strain) is routinely maintained at the Protozoology Laboratory of the Instituto de Medicina Tropical de São Paulo of the Medicine School of University of São Paulo (IMTSP-FMUSP). Tachyzoites were collected up to 96 h after intraperitoneal inoculation in Swiss mice using sterile intraperitoneal washes with cell culture medium. After 96 hours of spheroid formation, tachyzoites in medium were inoculated in a concentration of 104 tachyzoites / well (spheroid).
Polimerase chain reaction
Spheroids were removed from the culture plate still intact and placed in 1.5 mL microtubes together with 200 µL of PBS buffered saline solution. Each sample was subjected to genomic DNA extraction using the QIAmpMini Kit (Qiagen® GmbH) DNA kit according to the manufacturer's instructions. Samples were analyzed using NanoDrop 2000 spectrophotometer (ThermoScientific, Middlesex) and showed concentrations of 0.9 and 1.6 µg/µL. A region of the B1 gene was amplified using using the forward primer (TOXO1: 5″- GGAACTGCATCCGTTCATGAG-3″) and the reverse primer (TOXO2: 5″- TCTTTAAAGGGTTCGTGGTC-3") in a Step One PlusTM thermocycler (Applied Biosystems, Foster City, CA) using the following cyclic profile: initial denaturation at 94°C for 4 min, followed by 35 cycles of added denaturation at 94°C for 1 min, annealing at 62°C for 30s and an extension at 72°C for 1 min, followed by a final extension for 10 min at 72 °C. As a positive control, a total DNA extract of T. gondii, strain RH purified as described, was used. The amplified products were resolved on 1% agarose gels in Tris-Borate EDTA buffer (TBE) stained with ethidium bromide solution (10 𝜇g / mL) (Veronesi et al. 2017).
Transmission electron microscopy (TEM)
Samples were stored in glass tubes containing glutaraldehyde (C5H8O2) and processed at the laboratory of the Department of Pathology - FMUSP. The samples were centrifuged for 3 min at 6000 rpm and washed twice with 0.1M cacodylate buffer solution (pH 7.4) with sucrose (0.1M) and centrifuged again for 3 min. After centrifugation, fixed spheroids were placed in colidine buffer (osmium tetroxide), homogenized by vortexing and kept in the solution for 30 min, and were washed with cacodylate buffer and kept in alcoholic uranyl acetate (1%) for 5 min. After 5 min, samples were washed twice with 70% ethanol and then received 2,2-dimethoxypropane 5 min. The 2,2-dimethoxypropane was removed and acetone was added for 5 min. After removing the alcohol, the prepared resin 1:1 was added for 30 min. The resin was removed again, to add the pure resin and kept for 10 min. The pellet in the tube was suspended with the aid of forceps for better penetration of the resin and left in an oven at 40°C for 10 min. The material was poured into molds in an oven at 100°C for 18 hours. After solidification of the resin, the block was ground for partial exposure of the sample. Thick slices of 0.5 µm (semifine) and 0.008 µm (MET) were performed. Staining for semifine was done with toluidine blue. A diamond blade was used to cut the material and deionized water for deposition of the cut material. The samples were stored in 3mm copper and 200µm mesh grids. These grids were dried with 11 cm filter paper after incorporation of the material. For staining, the material was left in saturated (aqueous) uranyl acetate for 15 min, then washed in deionized water and left in the solution of lead citrate and sodium citrate for 8 min. After this period, the solution was discarded in an appropriate place and the material was washed again with deionized water and stored in glasses after drying. The tomes were observed under the microscope JEM 1011 (JEOL) operated by LIM59 – Laboratory of Cell Biology, Faculty of Medicine, University of São Paulo.
NIH/3T3 spheroids (3x103 cells) were infected 1x106 by tachyzoites 72 hours after the start of magnet aggregation. At 24, 48 and 72h of inoculation, 80μL (80%) of the culture medium from each well were removed, then stained for 0.5h with 2µg / mL of Rhodamine 123 (ThermoFisher Scientific, R302), and incubated for 30 min at 37°C. After two washes with PBS, the samples were centrifuged at 10,000 rpm for 10 min and resuspended in 200 μL of PBS containing 10 μL of latex fluorescent microspheres (AccuCheck Counting Beads - Thermo-Fisher Scientific, PBC100). These spheres have an average diameter of 6μm and appear in two well-discriminated populations in FSC x SSC plots and in FL-2 (585/40 nm) by excitation at 488nm in the analysis by flow cytometry. Rhodamine 123 was excited by the equipment's laser (488nm) and its emission captured by the FL-1 channel (530/30nm). Each sample was composed by adding the volume of culture medium collected from wells in quadruplicate. The amounts of tachyzoites and beads were determined and variations in the amount of tachyzoites were expressed as the proportion between their number and the number of beads acquired (T/B ratio, or ratio). 180μL were captured per sample. The relevant events (tachyzoites and microspheres) were selected in the plots generated by the equipment using the strategy shown in Supplemental Material.
Fluorescence Microscopy (INCell Analyzer)
After extraction by peritoneal washes from infected mice, suspensions with Toxoplasma gondii tachyzoites were maintained in culture medium at 4°C. The parasites were concentrated by centrifugation (200 x G / 10min and then at 1000 x G / 10min, room temperature). The resulting pellet was re suspended with culture medium containing Rhodamine 123 as described. Parasite viability was determined by the trypan blue incorporation test. Spheroids of NIH/3T3 cells (5 x 103 cells per well) were formed by magnetic aggregation in repellent plastic plates as described. After 72h in culture were carefully transferred to wells of appropriate plates for evaluation by inverted microscopy (μClear®, Greiner BioOne) and received 80µL of culture medium containing 5µg / mL of Hoechst 33342 (Sigma-Aldrich, CAS 875756-97-1), which has affinity for nuclear DNA and marked nuclei of the murine cells, and were incubated for 30 minutes in an incubator. Then, each spheroid received 20µL of the tachyzoite suspension, containing 2x106 viable parasites/mL. The spheroids were previewed using a Nikon Ts-100 coupled to an excitation LED module (Lumencar® Mira Light Engine 4-NII-FA) to confirm the fluorescence and subsequently evaluated by fluorescence microscopy immediately after the inoculum (0h) and 0.5, 2, 4 and 24 hours later, using the INCell Analyzer HS 2500 equipment (Cytiva). Stacks of images were generated spaced on the z axis by 4μm and one stack for each wavelength evaluated – “Green” channel evaluated the green fluorescence, emitted by tachyzoites marked by Rhodamine 123; “Blue” channel captured the fluorescence of Hoechst 33342 in the nuclei of NIH/3T3 spheroids cells and “Brightfield” channel captured the brightfield images. The equipment was adjusted to perform a 2D image acquisition every 4µm of the spheroids depth, starting from the inside to surface. A 10X magnification was used applying the 3D Convolution algorithm for further analysis of the three-dimensional structures. The stacks acquired in the “Blue” channel were analyzed using the IN Carta 1.14 package (Cytiva). The parameters were evaluated: number of NIH/3T3 cores; spheroid cross-sectional area and spheroid volume calculation. To find tachyzoites in the extracellular environment, the stacks acquired in the “Green” channel were also analyzed using the same software. For selection of only relevant events, the program parameters were adjusted to consider only events with volumes between 10 and 30μm3, according to high resolution microscopy data of tachyzoite volumes acquired under culture conditions (Firdaus et al. 2020).
Orthogonal views of acquisitions were created from data from stacks of relevant channels and deconvolution algoritm. Three-dimensional point spreading functions (3D-PSF) for 450 (blue) and 530 (green) nanometers were estimated using PSF Generator ImageJ plugin (Kirshner et al. 2013), choosing the Born & Wolf method (Born and Wolf 1999). Stacks were finally deconvoluted using the Tikhonov-Miller method (number of iterations: 10) using the DeconvolutionLab2 plugin (Sage et al. 2017), and submitted to background correction (rolling ball radius: 50px). Events in stacks of green fluorescence were also analyzed using the “3D Object Counter” ImageJ plugin (Universitaire 2006). After initial observations, only objects with volumes between 8 and 32 voxels were included in analyses. Projections were generated using the “Extended Depth of Field” plugin (Forster et al. 2004), and results were binarized and objects were protruded in 2 dimensions by the command “Find Edges” for three consecutive iterations.
Statistical Tests and Graphical Analysis
Results were compiled in plot using Prism® 8 software (GraphPad). Statistical analysis were performed applying ANOVA test, followed by Bonferroni post-tests.