Methods and systems for determining phenotypes using exosomes

A phenotypic, exosome-based technology, applied in 25 fields, can solve the problems of expensive, lack of specificity, lack of sensitivity of readout, etc.

Inactive Publication Date: 2011-12-28
CARIS LIFE SCI LUXEMBOURG HLDG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Methods for obtaining tissue of interest for analysis are often invasive, expensive, and put patients at risk of complications
In addition, the use of bodily fluids to isolate or detect biomarkers often dilutes the biomarkers significantly such that the readout lacks the necessary sensitivity
Furthermore, in addition to diseased tissues, normal tissues produce low or moderate amounts of most biomarkers, so this lack of specificity is also problematic

Method used

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  • Methods and systems for determining phenotypes using exosomes
  • Methods and systems for determining phenotypes using exosomes
  • Methods and systems for determining phenotypes using exosomes

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0905] Example 1: Purification of exosomes from prostate cancer cell lines

[0906] Prostate cancer cell lines were cultured for 3-4 days in medium containing 20% ​​FBS (fetal bovine serum) and 1% P / S / G. The cells were then pre-centrifuged at 400xg for 10 minutes at 4°C. Save the supernatant and centrifuge at 2000 x g for 20 min at 4 °C. The supernatant containing exosomes can be centrifuged using a Millipore Centricon Plus-70 (Catalog #UFC710008 Fisher).

[0907] The centrifuge ultrafiltration tubes were pre-washed with 30 ml of PBS at 1000 xg for 3 minutes at room temperature. Then, pour 15-70 ml of the pre-centrifuged cell culture supernatant into a Concentrate Cup and centrifuge in a Swing Bucket Adapter (Fisher #75-008-144) at room temperature at 1000xg for 30 minute.

[0908] Pour off fluid that passes through the Collection Cup. Using any other supernatant, bring the volume in the concentrate cup to 60ml. Centrifuge the concentration cup at 1000 xg for 30 minute...

Embodiment 2

[0911] Example 2: Purification of exosomes from VCaP and 22Rv1

[0912] Cone bodies from the prostate (VCaP) and 22Rv1, a human prostate cancer cell line derived from a human prostate cancer xenograft (CWR22R), were harvested by ultracentrifugation by first diluting the plasma with an equal volume of PBS (1 ml) Cancerous exosomes. Transfer the diluted fluid to a 15 ml Falcon centrifuge tube and centrifuge at 2000 x g for 30 min at 4 °C. The supernatant (-2ml) was transferred to an ultracentrifuge tube 5.0ml PA thin wall tube (Sorvall #03127) and centrifuged at 12000xg for 45 minutes at 4°C.

[0913] The supernatant (-2ml) was transferred to a new 5.0ml ultracentrifuge tube and filled to maximum volume with the addition of 2.5ml PBS, then centrifuged at 110000 xg for 90 minutes at 4°C. The supernatant was decanted without disturbing the pellet and the pellet was resuspended in 1 ml PBS. The tubes were filled to maximum volume with the addition of 4.5 ml PBS and centrifuged...

Embodiment 3

[0915] Example 3: Collection of plasma and purification of exosomes

[0916] Blood was collected by standard puncture into 7ml K2-EDTA tubes. Plasma was separated from blood cells by centrifuging the samples at 400 g for 10 minutes in a centrifuge (SORVALL Legend RT+ centrifuge) at 4°C. By careful pipetting, the supernatant (plasma) was transferred to a 15ml Falcon centrifuge tube. Plasma was centrifuged at 2000g for 20 minutes and the supernatant collected.

[0917] For storage, approximately 1 ml of plasma (supernatant) was aliquoted into cryovials, placed in dry ice to freeze them, and stored at -80°C. Before purifying exosomes, if samples were stored at -80 °C, cool down in a cold water bath for 5 min. The samples were mixed by hand by inversion so that undissolved material disappeared.

[0918] In the first precentrifugation, the plasma is diluted with an equal volume of PBS (for example, 2 ml of PBS is used to dilute approximately 2 ml of plasma). Transfer the dil...

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Abstract

Exosomes can be used for detecting biomarkers for diagnostic, therapy-related or prognostic methods to identify phenotypes, such as a condition or disease, for example, the stage or progression of a disease. Cell-of-origin exosomes can be used in profiling of physiological states or determining phenotypes. Biomarkers or markers from cell-of-origin specific exosomes can be used to determine treatment regimens for diseases, conditions, disease stages, and stages of a condition, and can also be used to determine treatment efficacy. Markers from cell-of-origin specific exosomes can also be used to identify conditions of diseases of unknown origin.

Description

[0001] cross reference [0002] This application claims Serial No. 61 / 114,045 filed November 12, 2008, Serial No. 61 / 114,058 filed November 12, 2008, Serial No. 61 / 114,065 filed November 13, 2008, February 2009 No. 61 / 151,183 filed on October 9, No. 61 / 278,049 filed on October 2, 2009, No. 61 / 250,454 filed on October 9, 2009, and No. 61 filed on October 19, 2009 / 253,027, each of which is hereby incorporated by reference in its entirety. Additionally, this application is a related application of U.S. Application No. 12 / 609,847, filed October 30, 2009, which claims U.S. Provisional Application No. 61 / 109,742, filed October 30, 2008, No. 61 / 112,571 filed on November 7, No. 61 / 114,045 filed on November 12, 2008, No. 61 / 114,058 filed on November 12, 2008, No. 61 filed on November 13, 2008 / 114,065, No. 61 / 151,183 filed February 9, 2009, No. 61 / 278,049 filed October 2, 2009, No. 61 / 250,454 filed October 9, 2009, and October 19, 2009 Priority to Ser. No. 61 / 253,027 filed on , each ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68G01N33/53G01N33/574
CPCG01N33/6848G01N33/57484G01N33/54326G01N33/57419G01N33/57434C12Q2600/112G01N33/5023C12Q1/6886C12Q2600/178C12Q2600/16G01N33/574C12Q1/6811G01N2800/50G01N2800/52C12Q2525/205C12Q2525/207G01N33/57488G01N33/68
Inventor C·库斯利驰G·波斯特M·克拉斯
Owner CARIS LIFE SCI LUXEMBOURG HLDG
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