Patent EA-003235-B1 (EA)
Title: Method for preparing membrane vesicles
Inventor: DHELLIN OLIVER
IPC: A61K35/12
Filed: 2000-01-19
Abstract: 1. Method for preparing membrane vesicles (MV) from a biological sample comprising separation of vesicles from biological contaminants, characterized in that the vesicles are separated by anion-exchange chromatography (AEC). 2. Method according to claim 1, characterized in that comprises at least one step of anion-exchange chromatography in which a strong anion exchanger is used. 3. Method according to claim 1 or 2, characterized in that comprises at least one stage of anion-exchange chromatography and gel permeation chromatography (GPC). 4. Method according to any of claims 1 to 3, characterized in that a biological sample is a sample selected from the group including biological fluid, culture supernatant, cellular lysate and a prepurified solution. 5. Method for preparing the MV from a biological sample, characterized in that comprises at least a stage of enriching the sample by MV, and a stage of treating the sample by AEC and/or by GPC. 6. Method according to claim 5, characterized in that comprises culturing population of cells producing MV (e.g. exosomes), under conditions that cause release of MV, a stage of step of enriching the sample by MV, and a stage of treating the sample by AEC and/or by GPC. 7. Method according to any of claims 5 or 6, characterized in that the stage of enriching comprises a step of clarification and a subsequent stage of concentration. 8. Method according to any of claims 5 to 7, characterized in that the step of enriching comprises a stage of affinity chromatography (AC), preferably by a colorant. 9. Method according to any of claims 7 or 8, characterized in that the step of enriching comprises a stage of centrifugation at a low rate and/or filtration. 10. Method according to any of claims 7 to 9, characterized in that the step of enriching comprises at least one stage of ultrafiltration, in particular, tangential ultrafiltration. 11. Method for preparing membrane vesicles, characterized in that comprising culturing population of cells producing MV exosomes under conditions that cause release of MV, treating the culture supernatant to produce a biological sample enriched in MV exosomes by at least one stage of ultrafiltration or AC, wherein vesicles are separated from biological contaminants by AEC and/or by GPC. 12. Method according to claim 11, characterized in that comprises a stage of filtration of the material produced. 13. Method according to any preceding claim, characterized in that MV have the diameter between 60 to 90 nm. 14. Method according to any preceding claim, characterized in that MV are vesicles produced by antigen presenting cells, in particular, dendritic cells, B-lymphcytes, macrophages or mastocytes. 15. Method according to claim 14, characterized in that MV are vesicles produced by dentritic, in particular human cells. 16. Method according to any preceding claim 1 to 13, characterized in that MV are vesicles produced by tumor, especially human cells. 17. Method for preparing MV from a biological sample comprising separation of vesicles from biological contaminants, characterized in that comprises obtaining a population of dendritic cells, culturing dendritic cells under conditions permitting to produce MV, treating the culture supernatant to produce a biological sample enriched in MV by at least one stage of ultrafiltration or AC, wherein vesicles are separated from biological contaminants by AEC and/or by GPC. 18. Method for preparing MV from a biological sample comprising separation of vesicles from biological contaminants, characterized in that comprises obtaining a population of dendritic cells, culturing dendritic cells under conditions permitting to produce MV, treating the culture supernatant to produce a biological sample enriched in MV by at least one stage of ultrafiltration or AC, wherein vesicles are separated from biological contaminants by AEC and/or by GPC. 19. Method according to claim 17 or 18, characterized in that dendritic cells are obtained from a biological sample of a subject, for example osseous marrow or peripheral blood. 20. Method according to any of claims 17 to 19, characterized in that dendritic cells are immature. 21. Method according to any of claims 17 to 20, characterized in that dendritic cells are sensibilyzed by an antigen till producing MV. 22. Method according to any of claims 17 to 21, characterized in that at the stage of culturing dendritic cells are cultured under conditions stimulating producing MV.