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Anti-inflammatory exosomes from inflamed cells or tissues

An exosome and tissue technology, applied in artificial cell constructs, medical raw materials derived from mammals, anti-inflammatory agents, etc., can solve problems such as increasing serious side effects

Inactive Publication Date: 2019-07-30
VBC HLDG LLC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Anti-inflammatory drugs and pain relievers sometimes have limited long-term effectiveness, and short- and long-term use of these drugs increases the risk of potentially serious side effects

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Isolate cells from inflamed tissue

[0092] Biopsy samples of tissue exhibiting characteristic metaplasia were obtained from the esophagus of a patient diagnosed with Barrett's esophagus carcinoma. Cells were isolated essentially as described in Secunda R, 2015, Cytotechnology 67(5):793-807, doi: 10.1007 / s10616-014-9718-z. Biopsy samples were minced and digested with 0.075% collagenase type I for 30 min at 37°C, followed by neutralization of collagenase type I activity by the addition of α-MEM containing 20% ​​heat-inactivated fetal calf serum.

[0093] After neutralizing the enzyme, samples were centrifuged at 600 x g for 10 min and filtered through a 70 μm cell strainer. Freshly isolated cells (1.5×10 6 ) Eagle's minimum basal medium (containing 10% fetal bovine serum, 10mL / L Pen / Strep solution, 2mM Ala-Gln solution, 10ng / ml epidermal growth factor, 10μg / ml insulin solution, 100μM 2-fosfo-L-ascorbic acid trisodium salt and 0.01 μM dexamethasone).

[0094] The medi...

Embodiment 2

[0096] Culture of isolated cells under inflammatory conditions with activating compositions

[0097] For example, the cells are grown by inoculating the medium at about 30,000 to about 50,000 cells per milliliter.

[0098] The cells obtained in Example 1 were incubated in a suitable medium under inflammatory conditions. Inflammatory conditions are provided by adding one or more pro-inflammatory cytokines and / or fluid extracted from inflamed tissue to the culture medium at 37°C. After culturing for a period of time sufficient to accumulate useful levels of anti-inflammatory exosomes, eg, about 2 days to about 4 days, the cultured cells are separated from the culture medium, and the culture medium is collected.

Embodiment 3

[0100] Isolation of anti-inflammatory exosomes

[0101] By polymer precipitation (ExoQuick from SBI, Palo Alto, CA TM ), immunoaffinity capture (Greening et al. 2015, Methods in Molecular Biology, impact factor: 1.29) exosomes were purified and isolated from the medium collected in Example 2.

[0102] By combining with magnetic beads (Exo-FLOW TM , SBI) combined to further purify the exosomes isolated by polymer precipitation. Magnetic beads [9.1 μm, 1.6 × 107 beads / ml] were coupled with anti-CD9 or anti-CD63 or anti-CD81 biotinylated antibodies for 2 h on ice, then incubated with exosomes overnight at 4 °C on a rotating rack to implement the capture. Beads were coated with three different antibodies and then mixed to capture exosomes.

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Abstract

The invention provides anti-inflammatory exosomes that, when administered locally or systemically to a subject diagnosed with an inflammatory disease or disorder, will downregulate the inflammatory process in the subject.

Description

technical field [0001] This document relates generally to anti-inflammatory exosomes, methods of obtaining or producing anti-inflammatory exosomes, and treating subjects exhibiting or caused by an inflammatory process by administering anti-inflammatory exosomes to a subject in need of such treatment The method of causing a disease or condition. Background technique [0002] The Background Art includes information useful for understanding the present invention. It is not an admission that the information presented herein is prior art or relevant to the present invention, or that publications cited directly or indirectly are prior art. [0003] Inflammation is the normal response of the immune system to various types of injury, infection and / or other damage to living tissue. Typically, inflammation results from an acute injury or disease process, and symptoms of inflammation (eg, pain, heat, redness, swelling, and loss of function) are limited in scope and duration. The inf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071A61K35/12C12N5/0786A61P1/00A61P29/00A61P19/02
CPCA61K35/38C12N5/0645C12N5/0679A61P29/00C12N2501/25C12N2501/2301C12N2501/2306C12N2501/2312C12N2501/24C12N2506/115
Inventor K·尼亚齐F·柯西奥
Owner VBC HLDG LLC
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