Methods of measuring exosomes using intrinsic fluorescence
Described herein are novel rapid and reliable methods of detection of extracellular vesicles and quantifying extracellular vesicle concentrations and absolute number from various sources, including raw cell harvest. The methods described herein comprise detection of intrinsic fluorescence of extracellular vesicles in biological samples. Extracellular vesicles analyzed by the methods of this application have a stereotypical elution profile distinct from known contaminants. The methods described herein are a significant improvement over the state of the art and fulfills an unmet need in the field of extracellular vesicle manufacturing and quality control.
1. A method of detecting exosomes in a sample, comprising determining an intrinsic fluorescence emission signal from the sample, wherein the intrinsic fluorescence emission signal is indicative of the presence of the exosomes within the sample, wherein the intrinsic fluorescence emission signal is determined at an emission wavelength of 550 nm to 590 nm and an excitation wavelength ranging from about 500 nm to about 600 nm.
2. The method of claim 1 , wherein the intrinsic fluorescence emission signal is determined at an excitation wavelength of 530 nm to 570 nm.
3. The method of claim 1 , comprising separating the sample into fractions prior to determining the intrinsic fluorescence emission signal.
4. The method of claim 3 , wherein the separating comprises one or more column chromatography separation steps.
5. The method of claim 4 , wherein the one or more column chromatography separation steps comprise a size-exclusion chromatography, ion-exchange chromatography, or both.
6. The method of claim 5 , wherein the ion-exchange chromatography comprises an anion exchange chromatography.
7. The method of claim 1 , wherein the intrinsic fluorescence emission signal is determined using a flow cell.
8. The method of claim 1 , further comprising subjecting the sample to: (i) a filtration step prior to determining the intrinsic fluorescence emission signal; (ii) a centrifugation separation step prior to determining the intrinsic fluorescence emission signal; (iii) a density gradient step prior to determining the intrinsic fluorescence emission signal; or (iv) any combination of (i) to (iii).
9. The method of claim 1 , wherein the sample is derived from: (i) a cell culture, (ii) a body fluid of an animal, or (iii) both.
10. The method of claim 1 , further comprising quantifying the amount of the exosomes within the sample, wherein the quantifying: (i) comprises comparing the intrinsic fluorescence emission signal to the intrinsic fluorescence emission signal of a standard; (ii) is based upon the area of the curve of a chromatogram comprising the intrinsic fluorescence emission signal; (iii) comprises comparing the intrinsic fluorescence emission signal to the luminescence signal of a standard; or (iv) any combination of (i) to (iii).
11. The method of claim 10 , wherein the luminescence signal of a standard:
is measured using a luminescence proximity assay; (ii) is calculated based on the relative abundance of one or more extracellular vesicle-associated proteins; or (iii) both (i) and (ii).
12. The method of claim 11 , wherein the one or more extracellular vesicle-associated proteins are selected from the group consisting of CD9, CD81, CD63, and combinations thereof.
13. The method of claim 1 , wherein the exosome comprises a therapeutic payload.
14. The method of claim 1 , wherein the intrinsic fluorescence emission signal is determined at an excitation wavelength of about 556 nm and at an emission wavelength of about 573 nm.