Composition for tissue regeneration and wound healing comprising induced exosomes

A technology of skin regeneration and exosomes, applied in the field of exosomes, can solve the problems of low yield of exosomes, and achieve the effect of high yield, high efficiency, and complete wound healing

Pending Publication Date: 2020-04-28
金贤锡
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in terms of related technologies including the therapeutic composition using exosomes in the above-mentioned published patents, the yield of exosomes is low, and most of them use stem cells that are harsh in isolation and expansion.

Method used

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  • Composition for tissue regeneration and wound healing comprising induced exosomes
  • Composition for tissue regeneration and wound healing comprising induced exosomes
  • Composition for tissue regeneration and wound healing comprising induced exosomes

Examples

Experimental program
Comparison scheme
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preparation example Construction

[0048] According to an embodiment of the present invention, the preparation method of exosomes with skin regeneration and wound healing effects includes: the step of directly / indirectly providing ultrasonic stimulation to the cells; the step of cultivating the mixture of the above cells and the medium for a specified time; The step of isolating exosomes in the above mixture, wherein directly providing stimulation refers to applying ultrasonic stimulation to the medium containing cells, and indirectly providing stimulation refers to mixing the above-mentioned medium and the above-mentioned cells after applying ultrasonic stimulation to the medium not containing cells.

[0049] Wherein, the above-mentioned step of directly / indirectly providing ultrasonic stimulation to the cells is performed by any one of the following methods: after mixing the cells and the medium, providing the above-mentioned mixture with ultrasonic stimulation, or after providing the medium with ultrasonic sti...

Embodiment 1

[0067] Example 1. Preparation of exosomes with skin regeneration and wound healing effects

[0068] In order to obtain exosomes with skin regeneration and wound healing effects, 1 × 10 6 Personal dermal fibroblasts (HDF) applied 20KHz, 1.0W / cm 2 Ultrasonic stimulation for 5 seconds. In a 35-mm Petri dish, place 2 × 10 5 UHDF with ultrasonic treatment (20KHz, 5.0W / cm 2 , 10 minutes) of embryonic stem cell culture medium ((DMEM) / F12, 15% fetal bovine serum (FBS), 2mM GlutaMAX, 0.1% non-essential amino acids (NEAA), 0.1% penicillin / streptomycin, 0.1mM β-mercapto Ethanol, 1000unit / ml Leukemia Inhibitory Factor (LIF)) were cultured together for one day. Exosomes were isolated from culture medium of sonicated HDF (UHDF) as follows: after centrifugation of the medium at 3000×g for 20 minutes to remove cell debris and dead cells, exosomes were separated with a 0.22-mm filter( Syringe Filter, Sartorius, Goettingen, Germany) filtered the supernatant. Put the medium that passed...

experiment example 1

[0069] Experimental example 1. Exosome analysis experiment with skin regeneration and wound healing effects

[0070] The results of analyzing the cells (UHDF) cultured according to the method for preparing exosomes with skin regeneration and wound healing effects according to Example 1 by immunofluorescent staining based on CD63 exosome markers (at this time, the control staining For nuclear staining agent 4',6-diamidino-2-phenylindole (DAPI)), compared with the control group, the CD63 signal intensity was very high, which confirmed that a large number of exosomes were being produced ( figure 1 ). For the RNA-seq results of the prepared exosomes, the exosomes (iExo) prepared according to Example 1 were compared to the exosomes (nExo) isolated from cells cultured without ultrasonic stimulation, especially Higher expression of genes related to wound healing, angiogenesis, cell growth, cell movement, cell proliferation, inflammatory response, regeneration, secretion, tissue re...

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Abstract

Disclosed is a method for producing exosomes having effects of skin regeneration and wound healing, comprising the steps of: providing ultrasonic stimulation directly or indirectly to cells; culturinga mixture of the cells and a medium for a predetermined amount of time; and separating exosomes from the mixture, wherein direct provision of stimulation is the application of ultrasonic stimulationto a medium comprising cells, and indirect provision of stimulation is the application of ultrasonic stimulation to a medium that does not comprise cells and then mixing the medium with the cells. Theexosome production method enables exosomes, having effects of skin regeneration and wound healing, to be obtained in a high yield and short amount of time by means of a simple process called sonication from stem cells, which are difficult to be handled, and also from somatic cells, which can be easily obtained and maintained. The exosomes obtained thereby also have a high amount and number of various included factors, the exosomes and compositions comprising same have increased various factors, which can induce skin regeneration and wound healing, and the exosomes have a phospholipid-based membrane structure so as to facilitate skin penetration and allow high efficiency of substance delivery, thereby enabling skin regeneration and wound healing to be effectively induced, and are safe andhave no side effects caused by the use of synthetic drugs.

Description

technical field [0001] The present invention relates to an exosome with skin regeneration and wound healing effects, a preparation method thereof and a composition containing the exosome. Background technique [0002] As the largest tissue in the human body, the skin serves as the basic first line of defense to provide a protective film against disease and water evaporation, in addition to receiving sensations, synthesizing vitamin D, participating in body temperature regulation, and immune surveillance. play an important role. Skin wound healing is a complex process involving four steps of hemostasis, inflammation, proliferation and remodeling. In the first step, which occurs within hours of wound formation, the steps of blood clotting and the formation of a provisional wound matrix, the inflammatory step activated by the first step, by The recruitment of neutrophils, the participation and degeneration of monocytes remove pathogens and necrotic tissue. In the proliferati...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N13/00C12N5/071A61K35/36A61P17/00A61P17/02
CPCC12N13/00C12N5/0625A61K35/36A61P17/00A61P17/02C12N2509/00A61K35/33A61K35/28C12M35/04C12N5/0656C12N2521/10C12N2320/30
Inventor 李镕丞
Owner 金贤锡
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