Journal: International Journal of Molecular Sciences

Article Title: Development of Novel High-Resolution Size-Guided Turbidimetry-Enabled Particle Purification Liquid Chromatography (PPLC): Extracellular Vesicles and Membraneless Condensates in Focus

doi: 10.3390/ijms21155361

Figure Lengend Snippet: Columns packed with gradient size exclusion chromatography (gSEC) beads achieve higher resolution as compared to monosize bead columns. ( a , b ) Schematic describing that monosize bead separation ( a ) would end up with only two peaks, whereas separation with gSEC beads ( b ) may achieve a multi-peak resolution. ( c ) Schematic of the workflow for the clarification of crude extracellular vesicle (EV) mixtures from body fluids and cell culture supernatants by differential centrifugation before column separation. ( d ) Seminal plasma from 6 healthy men were pooled and clarified, and equal volumes (1 mL) were run on 50 cm × 0.5 cm G-10, G-100, or gSEC Sephadex bead-packed columns. Elution was carried out with phosphate-buffered saline (PBS), fractions were collected in 96-well plates, and the UV-Vis absorbance measurements were recorded using a plate reader. The profiles shown correspond to the 280-nm wavelength. ( e ) Gradient separation profiles of seminal plasma on columns of different lengths. Aliquots (1.5 mL) from the same pool of clarified seminal plasma (healthy, n = 6) were loaded on different-length Econo ® columns (20, 50, and 100 cm) packed with multi-size beads, with the elution and collection carried out under the same conditions. ( f – j ) Samples were purified on a 100 × 1 cm gSEC column. Fractions were eluted with PBS, collected in 96-well plates, and the UV-Vis absorbance measurements were recorded using a plate reader. ( f ) Seminal plasma separation profiles (1.5 mL) from 4 individual healthy donors. ( g ) Blood plasma and serum separation profiles. Blood was collected from 4 donors in different collection tubes, and clarified plasmas and serums were pooled by tube type. Three-hundred microliters of each pool were used for separation. ( h ) Forty milliliters of a first-void clean-catch urine sample was collected from a healthy male, clarified, and concentrated 10 times to 4 mL (Amicon™ ultra centrifugal filter unit, 3000 Da)—of which, 1.5 mL was used for purification. ( i ) Commercial cow milk separation profiles. Whole, 2% fat, and fat-free cow milk were purchased from Walmart, clarified by centrifugation at 10,000× g for 30 min, and 1 mL of each clarified sample was used. ( j ) U1, 293T, and MDA-MB-231 cells were cultured in 150 × 20 mm dishes in media supplemented with 10% exosome-depleted fetal bovine serum (FBS) for 3 days, and 10 mL of supernatants were clarified by differential centrifugation and concentrated (Amicon™ ultra-centrifugal filter unit, 3000 Da) to 1 mL before purification. All profiles shown correspond to the 280-nm wavelength. RT: room temperature.

Article Snippet: All available size-exclusion dextran-based Sephadex TM beads were purchased from Cytiva (formely GE Healthcare, Marlborough, MA, USA) and were as follows: G-10 (17-0010-01), G-15 (170020-01), G-25 fine (17-0032-01), G-50 fine (17-0042-01), G-75 (17-0050-01), and G-100 (17-0060-01). gSEC column was prepared by layering the beads from the smallest to the largest in different Econo-Columns ® (Bio-Rad, Hercules, CA, USA) at room temperature by gravity.

Techniques: Size-exclusion Chromatography, Clarification Assay, Cell Culture, Centrifugation, Purification