Extracellular Vesicle Isolation via Two-Phase Centrifugation

The patent divides the isolation process into multiple sequential centrifugation steps with increasing speeds. First, low-speed centrifugation separates major cellular components (RBCs, WBCs, platelets). Then, high-speed centrifugation isolates extracellular vesicles from the plasma fraction. This segmented approach allows each centrifugation step to target specific components, achieving both effective separation and concentration of extracellular vesicles.

The patent applies different centrifugation conditions to different fractions of the blood/marrow sample. The plasma fraction, which contains the extracellular vesicles, is subjected to high-speed centrifugation (e.g., 10,000-20,000 g) while the cellular fraction is handled separately. This localized application of appropriate centrifugation intensity to each fraction optimizes the isolation of extracellular vesicles without damaging other components.

The patent segments the isolation process into distinct phases: (1) initial low-speed centrifugation to separate cellular components from plasma, (2) collection of the plasma fraction which contains extracellular vesicles, and (3) high-speed centrifugation of the plasma to pellet the extracellular vesicles. This segmentation ensures that extracellular vesicles are effectively partitioned into a separate concentrate fraction while maintaining high concentration of therapeutic agents.

The patent performs preliminary low-speed centrifugation to remove cellular components before isolating extracellular vesicles. This preliminary action clears the sample of interfering elements (RBCs, WBCs, platelets) so that subsequent high-speed centrifugation can effectively concentrate extracellular vesicles without contamination from cellular debris, improving both concentration and partitioning accuracy.

The patent extracts the plasma fraction containing extracellular vesicles from the whole blood or bone marrow sample through low-speed centrifugation. By removing the plasma fraction for separate processing, the system simplifies the subsequent isolation step and avoids the need for complex filtration or ultra-centrifugation equipment. The extracellular vesicles are then concentrated from this extracted plasma using straightforward centrifugation.

The patent replaces complex mechanical filtration systems or ultra-centrifugation equipment with a simpler two-step centrifugation approach. Instead of using filters with specific pore sizes or ultra-high-speed centrifuges, the system uses sequentially applied centrifugal forces that are easier to implement with standard equipment, reducing device complexity while maintaining isolation purity.