A method for storing a tumor cell vesicle preparation

A technology of tumor cells and vesicles, which is applied in the storage field of tumor cell vesicle preparations, can solve the problems of wasting manpower and preparation material costs, which is not conducive to large-scale preparation, storage, and ready-to-use, so as to maintain biological functions, take with convenient effect

Active Publication Date: 2019-01-15
HUBEI SOUNDNY BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It is very unfavorable for large-scale preparation, storage and a...

Method used

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  • A method for storing a tumor cell vesicle preparation
  • A method for storing a tumor cell vesicle preparation
  • A method for storing a tumor cell vesicle preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1: Preparation, cryopreservation and thawing of H22 cell-derived vesicles

[0029] 1. Experimental materials and instruments

[0030] H22 mouse hepatoma cells (H22 cells), RPMI 1640 medium, 0.9% saline, methotrexate, high-speed centrifuge, ultraviolet device (conventional cell ultra-clean workbench comes with).

[0031] 2. Experimental steps

[0032] (1) Resuspend H22 mouse hepatoma cells in serum-free RPMI 1640 medium to make the cell concentration reach 1×10 7 / mL, a total of 17ml. UV irradiation was performed for 1 h, and then the chemotherapeutic drug methotrexate was added to make the drug concentration in the culture medium reach 1 mg / mL, and incubated in an incubator for 18-20 h.

[0033] (2) Gradual centrifugation to obtain vesicles derived from H22 mouse hepatoma cells:

[0034] Centrifuge at 1500 rpm for 8 min and discard the precipitate; centrifuge at 5000 rpm for 8 min and discard the precipitate; centrifuge at 14,000 g for 1 min and discard the ...

Embodiment 2

[0037] Example 2: Counting and particle size detection of H22 cell-derived vesicles

[0038] 1. Experimental materials

[0039] H22 mouse liver cancer cells, RPMI 1640 medium, 0.9% saline, methotrexate, ultraviolet device (conventional cell ultra-clean workbench comes with), high-speed centrifuge, flow cytometer, laser particle size analyzer.

[0040] 2. Experimental steps

[0041] (1) The vesicles derived from H22 cells were prepared, isolated, extracted, cryopreserved, and thawed according to the method of Example 1.

[0042] (2) Wash the flow tube, add 500 μL of ultrapure water; add 10 μL of commercial 3 μm particle size polystyrene microsphere solution and 10 μL of vesicles, shake well, and measure the vesicle concentration by flow cytometry. The calculation formula is: :

[0043] Vesicle concentration = vesicle percentage / microsphere percentage × 6.77 × 10 7 / mL

[0044] (3) Dilute H22 vesicles to 1×10 6 / mL, use a laser particle size analyzer (DLS) to detect the pa...

Embodiment 3

[0048]Example 3: Detection of Chemotherapy Drug (Methotrexate) Content Encapsulated in H22 Cell-derived Vesicles

[0049] 1. Experimental materials and instruments

[0050] H22 mouse liver cancer cells, RPMI 1640 medium, 0.9% saline, methotrexate, UV device (conventional cell ultra-clean workbench comes with), cell lysis solution, acetonitrile, chloroform, high-speed centrifuge, ultrasonic cell grinder ,High performance liquid chromatography.

[0051] 2. Experimental steps

[0052] (1) The vesicles derived from H22 cells were prepared, isolated, extracted, cryopreserved, and thawed according to the method of Example 1.

[0053] (2) The H22 cell-derived vesicles were centrifuged at 14,000 g for 1 h, and the supernatant was removed to leave the precipitate. Add 500 μL of cell lysate, pipette evenly, and incubate on ice for 20 min.

[0054] (3) Use an ultrasonic cell pulverizer to pulverize for 1 min to destroy the membrane structure of the vesicles to release the drug. After...

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Abstract

The invention provides a method for storing a tumor cell vesicle preparation, belonging to the field of pharmaceutical preparation preservation. The cell vesicle preparation is a cell vesicle derivedfrom an apoptotic tumor cell. As in that prior art, the tumor cell vesicle preparation is refrigerated at 4 DEG C and can only remain stable in function for 48 hours. The method of the invention can freeze the tumor vesicle preparation for a long time at -20 DEG C only by lowering the temperature gradient and keep its biological function. The method solves the problem of storage timeliness of tumor cell vesicle preparation, facilitates mass production and large-scale cryopreservation, thereby greatly reduces production cost and improves production efficiency.

Description

technical field [0001] The invention belongs to the field of pharmaceutical preparations, in particular to a storage method for tumor cell vesicle preparations. Background technique [0002] It is well known that cells are composed of cell membranes encapsulating cell contents, and cell membranes are composed of protein molecules embedded in the middle of phospholipid bilayers. be maintained. When cells are stimulated to undergo apoptosis by external factors (such as ultraviolet rays, etc.), part of the protein filaments attached to the cell membrane of the cytoskeleton break or lose their attachment, and the centripetal pulling force suddenly disappears, making the local cell membrane structure under the action of outward pulling force , swells outward, protrudes and wraps the cell contents in the form of vesicles and is released into the sub-hierarchical structure between cells and molecules outside the cell, which can form a kind of basically nano-scale microparticles ca...

Claims

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Application Information

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IPC IPC(8): A61K9/50A61K47/46A61K31/519A61P35/00
CPCA61K9/5068A61K31/519
Inventor 陈彬张一唐科
Owner HUBEI SOUNDNY BIOLOGICAL TECH
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