METHOD FOR ISOLATION AND HARVESTING MICROVESICLES

Abstract

A method for isolation and culturing of T Lymphocytes from whole blood and harvesting exosomes. The method comprises isolating peripheral blood mononuclear cells from human blood, culturing the isolated peripheral blood mononuclear cells, treating the cultured peripheral blood mononuclear cells with cold atmospheric plasma for secretion of microvesicles, and harvesting microvesicles from the CAP-treated peripheral blood mononuclear cells.

Claims

1. A method for isolation and culturing of microvesicles, comprising: isolating peripheral blood mononuclear cells from human blood; culturing the isolated peripheral blood mononuclear cells; treating the cultured peripheral blood mononuclear cells with cold atmospheric plasma for secretion of microvesicles; and harvesting microvesicles from the CAP-treated peripheral blood mononuclear cells.

2. A method according to claim 1, wherein the harvested microvesicles comprise T lymphocyte exosomes.

3. A method according to claim 1, wherein the step of isolating peripheral blood mononuclear cells from human blood comprises: adding human blood to a polymorph density medium in a first flask; centrifuging the first flask of blood and polymorph density medium to separate the peripheral blood mononuclear cells from other blood components; collecting the peripheral blood mononuclear cells from the first flask and transferring the peripheral blood mononuclear cells to a second flask; centrifuging the second flask of peripheral blood mononuclear cells; and washing the peripheral blood mononuclear cells in the second flask.

4. A method according to claim 3, wherein the step of culturing the peripheral blood mononuclear cells comprises: culturing the washed peripheral blood mononuclear cells in selective media; removing the peripheral blood mononuclear cells from the selective media and transferring them to a third flask; centrifuging the peripheral blood mononuclear cells in the third flask; transferring the centrifuged peripheral blood mononuclear cells from the third flask to a fourth flask; centrifuging the peripheral blood mononuclear cells in the fourth flask; culturing the centrifuged peripheral blood mononuclear cells in the fourth flask in selective media containing IL-2 or IL-15; and replacing the selective media with exosome free fetal bovine serum.

5. A method according to claim 4, wherein the step of harvesting microvesicles from the CAP-treated peripheral blood mononuclear cells comprises an isolation method.

6. A method according to claim 5, wherein the isolation method comprises: incubating the CAP-treated peripheral blood mononuclear cells; differentially centrifuging the incubated CAP-treated peripheral blood mononuclear cells; mixing the centrifuged incubated CAP-treated peripheral blood mononuclear cells with an exosome isolation solution; incubating the exosome isolation solution and centrifuged incubated CAP-treated peripheral blood mononuclear cells; centrifuging the incubated exosome isolation solution and centrifuged incubated CAP-treated peripheral blood mononuclear cells; and collecting microvesicle pellets from the centrifuged incubated exosome isolation solution and centrifuged incubated CAP-treated peripheral blood mononuclear cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] For a more complete understanding of the present invention and the advantages thereof, reference is now made to the following description and the accompanying drawings, in which:

[0013] FIG. 1 is a flow chart of a protocol for isolation and culturing Of T Lymphocytes from whole blood and harvesting exosomes in accordance with a preferred embodiment of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention is described with reference to the drawing. A preferred embodiment of the present invention for isolating and harvesting microvesicles is described with reference to FIG. 1. Human blood obtained from a donor (102) is added to a polymorph density medium (104). The blood and polymorph solution is then centrifuged (106), for 45 minutes in the example shown in FIG. 1. The peripheral blood mononuclear cells (PBMC), which now will have separated from the other blood components, are removed, centrifuged and washed (108). The PMBC is then cultured in a selective media for 24 hours (202). The lymphocytes are then removed with the media (204) and transferred to a new flask and the centrifuge step is repeated (206). The lymphocytes are then centrifuged, transferred to a new flask, and the step if repeated (208). The lymphocytes are then cultured in selective media containing IL-2 or IL-15 for 2 days (210). The media is then replaced with exosome free FBS (212). The T lymphocytes are then treated with cold atmospheric plasma (302). Following CAP treatment, the CAP-treated lymphocytes are incubated for 24 hours (304). The T lymphocyte exosomes are then harvested using an isolation method (402). For example, the conditioned media can be collected and differentially centrifuged at 300×g, 1500×g, 4500×g and 10,000×g. The supernatant collected and the final centrifuged step are mixed with exosome isolation solution (Total Exosome Isolation Reagent, Thermo) and incubated overnight at 4 C. The next day the conditioned media is centrifuged at 10,000×g and the exosome pellets are collected and resuspended in appropriate vol of PBS and stored at −80 C until used. The application of CAP to the process of isolating and harvesting exosomes dramatically increased the exosome production by as much as seven times.

Example

[0015] A more specific method in accordance with the present invention to isolate and harvest T lymphocyte exosomes has four phases: (1) isolation of T Lymphocytes; (2) culture of T Lymphocytes; (3) CAP treatment for secretion of exosomes from Human T Lymphocytes; and (4) harvesting exosomes from Human T Lymphocytes.

[0016] The T Lymphocytes are isolated through the following steps: [0017] Obtain human blood from a healthy donor. Allow the blood to cool to room temperature (˜30 min) before proceeding to the next step. [0018] Gently pipette 3 mL of room temperature Polymorph density gradient media into an 8 mL round-bottom polystyrene tube. Gently add 3 mL of whole blood on top of the Polymorph media. It is important to avoid mixing of the two reagents. [0019] Centrifuge the tubes at 500×g for 45 minutes at room temperature. [0020] Following the centrifugation, the peripheral blood mononuclear cells (PBMC) have now separated from other blood components into the top cell layer. The PBMC layer appears, from the top down, as the first cloudy band. [0021] Carefully remove the clear yellow-colored upper phase of the blood, above the PBMC layer, and then use a P1000 micropipette to transfer the PBMC layer to a 15 mL or 50 mL conical tube. [0022] Wash the PBMC twice with PBS, centrifuging cells at 500×g for 5 minutes each time. The supernatant will be somewhat cloudy after each wash.

[0023] The T Lymphocytes are cultured through the following steps: [0024] Using a pipette, transfer the PBMC to a T-75 culture flask in 20 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/mL phytohemagglutinin (PHA). [0025] Incubate at 37° C. and 5% CO.sub.2 for at least 1 hour, and up to 24 hours. This step allows monocytes, which will be adherent to the flask surface, to be separated from the lymphocytes that remain in suspension. If a short incubation (1 hour) is used at this step, it is acceptable to use RPMI 1640 media containing 10% FBS and 1% penicillin/streptomycin without supplementing with PHA as specified in step 2.1. [0026] Carefully remove all of the media from the flask, add it to a 50 mL conical tube, and centrifuge at 500×g for 5 minutes. [0027] Resuspend the cell pellet, which now primarily contains lymphocytes, and transfer the cells to a new T-75 flask containing 25 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 1 μg/mL PHA. [0028] Incubate at 37° C. for 3 days (2 days if the initial incubation of PBMC was overnight). After 24 hours of growth, it may be necessary to add 15-20 mL of fresh media and transfer to a larger T-175 flask. [0029] After 3 days, use a pipette to remove the media and suspended lymphocytes from the flask and transfer to a 50 mL conical tube. Centrifuge at 500×g for 5 minutes. [0030] Resuspend the cell pellet and transfer cells to a new T-75 or T-175 flask containing 25 mL (T-75) or 50 mL (T-175) RPMI 1640 with 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL human IL-2 or IL-15. [0031] Grow lymphocytes for 4-7 days. If starting with a T-75 flask, the culture will need to be expanded and transferred to a T-175 flask after 1-2 days.

[0032] CAP treatment for secretion of exosomes from Human T Lymphocytes is performed through the following steps: [0033] Transfer 100K T lymphocytes to each well on a 12 wells culture plate in 1 mL RPMI 1640 media containing 10% FBS, 1% penicillin/streptomycin, and 20 ng/mL human IL-2 or IL-15. [0034] Culture for 2 days and replace the media to RPMI 1640 media containing 10% NU serum, 1% penicillin/streptomycin, and 20 ng/mL human IL-2 or IL-15. [0035] After hour incubation, carryout Canady Cold Atmospheric Plasma (CAP) treatment at 120p for 3 minutes at 3 L Helium supply. [0036] Incubate the plates at 37° C. and 5% CO.sub.2 for the required period.

[0037] Harvesting exosomes from Human T Lymphocytes is performed through the following steps: [0038] 1. After incubation, Collect the media and centrifuge at 300×g for 10 mins at 4 C, collect the supernatant. [0039] Centrifuge the supernatant at 1500×g for 10 mins at 4 C, collect the supernatant. [0040] Centrifuge the supernatant at 4500×g for 10 mins at 4 C, collect the supernatant [0041] Transfer the required volume of cell-free culture media to a new tube and add 1:1 volume of JCRI Exosome Isolation reagent and mix well. [0042] Mix the culture media/reagent mixture well by vortexing, or pipetting up and down until there is a homogenous solution [0043] Incubate samples at 2° C. to 8° C. overnight. [0044] After incubation, centrifuge the samples at 10,000×g for 1 hour at 2° C. to 8° C. [0045] Carefully aspirate and discard the supernatant. Exosomes are contained in the pellet at the bottom of the tube (not visible in most cases). [0046] Resuspend the pellet in a convenient volume of 1×PBS or culture media. [0047] Keep isolated exosomes at 2° C. to 8° C. for up to 1 week, or at <80° C. for long-term storage.

[0048] The foregoing description of the preferred embodiment of the invention has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of the invention. The embodiment was chosen and described in order to explain the principles of the invention and its practical application to enable one skilled in the art to utilize the invention in various embodiments as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims appended hereto, and their equivalents. The entirety of each of the aforementioned documents is incorporated by reference herein.