2.4. Isolation of Exosomes

64 semen samples from four clinical groups (n = 16/group) were liquefied at room temperature for 30 min and subsequently centrifuged at 10,000× g for 30 min to remove cellular debris and large vesicles. Clarified seminal plasmas were transferred to new tubes. For Nano Tracking Analysis (NTA) experiments, six pools of samples in each group, each pool from 2 participants (100 µL/sample), were used. Samples were pooled to obtain sufficient volume needed for efficient separation and analysis. For the rest of the experiments, 4 pooled samples (n = 16, 50 µL/sample) per clinical group were used. Exosomes were purified by size exclusion chromatography (SEC), where clarified seminal plasma was loaded onto Sephadex G-50 fine beads (GE-Healthcare, Pittsburgh, PA, USA) packed in a 22 cm × 1 cm Econo-column (Bio-Rad, Hercules, CA, USA). Elution was achieved by gravity using Phosphate Buffered Saline (PBS, Corning, NY, USA). Fractions of 200 µL were collected, and elution profiles were determined by absorbance measurements at 280 nm and 600 nm. The first peak which corresponds to semen exosomes (SE) was collected, and the protein content was measured by the Bradford Assay (Bio-Rad, Hercules, CA, USA). Of note, HIV could not be efficiently separated from semen exosomes using the Optiprep (Iodixanol)-based density gradient centrifugation method. While a good gradient prior to centrifugation was obtained, a satisfactory purification was not achieved due to the fact that the gold-standard exosomal marker AChE, as well as the exosomal markers CD9, CD63, and HSP70, along with the viral protein reverse transcriptase (RT) were found across the gradients. This is not surprising since HIV and exosomes overlap in size, density, and charge, and HIV is known to incorporate exosomal markers such as CD9, CD81 [58], and CD63 [59], while exosomes in turn also contain viral proteins [60] and RNA [61]. Immunocapture purification could not be used either because this mechanism depends on the use of antibodies against either host or viral proteins which are present in exosomes and HIV. Moreover, the “release” mechanism of exosomes trapped on the antibody-bead complex was inefficient. Thus, the inclusion of exosomal proteins in HIV and HIV proteins in exosomes hindered separation of these vesicles but also highlighted the need to assess the vesicles in their near-native state to understand their effect on host cells.