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Exosome secretion inducer, exosome secretion inducing culture medium, exosome production method using exosome secretion inducing culture medium and application of exosome

A technology for inducing culture medium and exosomes, which is applied in the application field of medicine, can solve problems such as limited production, and achieve the effects of promoting secretion, simple ingredients, and increasing production

Active Publication Date: 2021-06-08
GUIDON PHARM INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing exosome acquisition method is mainly cell autocrine, but its output is very limited

Method used

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  • Exosome secretion inducer, exosome secretion inducing culture medium, exosome production method using exosome secretion inducing culture medium and application of exosome
  • Exosome secretion inducer, exosome secretion inducing culture medium, exosome production method using exosome secretion inducing culture medium and application of exosome
  • Exosome secretion inducer, exosome secretion inducing culture medium, exosome production method using exosome secretion inducing culture medium and application of exosome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Exosome secretion induction medium and its use method

[0037] In order to adapt to the production of high-yield exosomes, this example provides an exosome secretion induction medium for high-efficiency production of exosomes.

[0038]Prepare the exosome secretion induction medium (named as GDEV medium) for the efficient production of exosomes from stem cells (ESC or iPSC, etc.) in the present invention, and its components include: DMEM / F12 (Gibco 11330032 ) and exosome secretion inducers. The exosome secretion inducer is composed of the following concentrations: L-Ascorbic acid 2phosphate magnesium salt (L-ascorbic acid diphosphate magnesium salt, Sigma-AldrichA8960) with a concentration of 64mg / L, sodium selenium ( Sodium selenium, Sigma-Aldrich S5261), NaHCO with a concentration of 543mg / L 3 (Sigma-Aldrich S5761), Insulin (insulin, Puxitan I10022) with a concentration of 19.4 mg / L.

[0039] As a control, its E8 medium was prepared according to the metho...

Embodiment 2

[0061] Example 2: Exosome secretion inducer

[0062] In order to adapt to the production of high-yield exosomes, this example provides an exosome secretion inducer for efficient production of exosomes.

[0063] According to the following Table 2, the exosome secretion inducers provided by the present invention for the efficient production of exosomes (named GD1, GD2 and GD3 respectively, wherein the concentration of each component is the concentration in the basal medium DMEM / F12) were prepared according to the present invention. , each exosome secretion inducer and basal medium DMEM / F12 were induced to culture iPSCs according to the steps 1.1 to 1.3 in the above-mentioned Example 1, and then the exosomes in the cell culture supernatant were collected, and the exosome content was measured respectively. The test was carried out, and the results are shown in Table 3 below.

[0064] Table 2: Formulations of exosome secretion inducers

[0065]

[0066] Table 3: Exosome produc...

Embodiment 3

[0070] Example 3: Application of exosomes

[0071] In this example, the exosome solution obtained from GDEV medium-induced cultured iPSCs in the above-mentioned Example 1 is used to verify its effect on stroke neuron models (glucose and oxygen deprivation test, OGD), improving the condition of senescent cells, and stroke neuron models (glucose and oxygen deprivation test, OGD). effect in the oxygen deprivation test).

[0072] 3.1. Uptake of exosomes

[0073] 1) Label the exosome solution with PKH26 according to the technical manual of the PKH26 Red Fluorescent Cell Linker Mini Kit (purchased from Sigma, Cat. No. MINI26), using Exosome Spin Columns (MW3000) (purchased from Invitrogen, Cat. No. 4484449) kit The technical manual removes excess dye to obtain a solution of labeled exosomes.

[0074] 2) Add the labeled exosome solution to human skin fibroblasts, umbilical cord mesenchymal stem cells, and neuron cells at a ratio of exosomes:cells of 10,000:1 (products of Guodian (B...

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Abstract

The invention discloses an exosome secretion inducer, an exosome secretion inducing culture medium, an exosome production method using the exosome secretion inducing culture medium and application of the exosome, and belongs to the technical field of biology. The exosome secretion inducing culture medium provided by the invention only contains the following components: a basic culture medium, and four additives, wherein the additives are L-ascorbic acid or salt thereof, selenium or salt thereof, NaHCO<3> and Insulin. Although the components are simple, the exosome secretion inducing culture medium is capable of remarkably increasing yield of exosome secreted by stem cells, so that the production cost of the exosome can be significantly reduced; and moreover, the produced exosome can more effectively protect neurons from being damaged, as well as improve condition of senescent cells.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an exosome secretion inducer, an induction medium, and a production method and application of exosomes using the same, especially relates to the preparation of exosomes to improve the condition of aging cells and / or protect nerves. The application of the element in medicine. Background technique [0002] Exosomes are small membrane vesicles (30-150nm) specifically secreted containing complex RNA and proteins, which participate in intercellular communication and regulate the normal function and damage repair of various tissues and organs. [0003] Since exosomes derived from different tissues have their specific protein molecules and key molecules that perform their functions, exosomes derived from stem cells have an important therapeutic effect of stem cells and are safer, and can effectively transport drugs with therapeutic effects on diseases. Biologically active substa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/074C12N5/0735A61K35/28A61P39/06
CPCC12N5/0606C12N5/0696A61K35/28A61P39/06C12N2500/38C12N2500/25C12N2500/05
Inventor 张建民王靓曲典林埕宇
Owner GUIDON PHARM INC
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