Dog MSC exosome preparation and preparing method thereof

A technology of exosomes and systems, applied in the field of bioengineering, can solve the problems of scar formation, affect the appearance, and easy secondary infection, etc., achieve the effect of stable dosage and guaranteed therapeutic effect

Inactive Publication Date: 2018-06-12
FOSHAN UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As the most common type of injury, skin injury has many pathogenic factors such as burns, mechanical injuries, especially today when wars occur frequently and car accidents continue, the repair of skin injuries is particularly important, and the current treatment methods are prone to secondary Infection, the wound takes a long time to heal, and more scars are formed after healing, without hair follicle regeneration, which affects the appearance

Method used

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  • Dog MSC exosome preparation and preparing method thereof
  • Dog MSC exosome preparation and preparing method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1, the preparation of canine UC-MSC

[0020] 1) Primary culture of canine UC-MSC

[0021] Take the umbilical cord tissue of newborn dogs, remove the membranous tissue and vascular tissue in the ultra-clean workbench, clean the remaining glial tissue and cut it to 1mm with sterile dissecting scissors 2 Then transfer the tissue to a 15mL centrifuge tube, add 0.1mg / mL type Ⅰ collagenase, tighten the bottle cap and place it in a 37°C water bath for digestion for 4 hours. After the tissue block is basically digested, filter it with a 100-mesh cell sieve. Centrifuge the filtrate at 1200rpm for 5min, discard the supernatant, resuspend the pellet with complete DMEM medium (88% DMEM basal medium + 1% double antibody solution + 1% L-glutamine), and inoculate the suspension into a cell culture dish , and then placed at 37°C, 5% CO 2 Cultivated in an incubator;

[0022] 2) Subculture of canine UC-MSC

[0023] When the cells were cultured for 24 hours, the original cu...

Embodiment 2

[0029] Example 2, Preparation of Canine UC-MSC-derived Exosome Preparation

[0030] 1) Isolation of canine UC-MSC-derived exosomes

[0031] 1-1) Preparation of exosome-free fetal bovine serum

[0032] Use a 0.22um filter membrane to filter fetal bovine serum to remove particles larger than 220nm, collect the filtrate, place it in an ultrahigh-speed centrifuge tube, and centrifuge at 120000g for 90min at 4°C, carefully collect the supernatant, and then use 0.22 um membrane filter and sterilize for later use;

[0033] 1-2) Isolation of exosomes derived from canine UC-MSCs

[0034] The P3-P5 generation UC-MSCs were cultured with a complete medium containing 10% exosome-free fetal bovine serum, 1% double antibody, 1% L-glutamine and 88% DMEM basal medium, and the supernatant was collected in Centrifuge at 300g for 10min at 4°C to remove dead cells; collect the supernatant and centrifuge at 16,500g for 20min at 4°C to remove cell debris; filter the supernatant with a 0.22um filt...

Embodiment 3

[0041] Example 3, Canine UC-MSC-derived exosome preparations for the treatment of canine skin damage

[0042] 1) Establishment of canine skin injury model

[0043] Twelve healthy puppies with the same breed, age, and growth status were randomly divided into two groups, with 6 repetitions in each group. After the dog was anesthetized, local hair removal, disinfection, and draping were performed. Under aseptic operation, a 4×4cm square full-thickness skin injury model was made in the middle of the dog’s back and below the shoulder blade. The whole operation process was performed by the same experimenter to avoid too much error.

[0044] 2) Local injection therapy of canine UC-MSC-derived exosome preparation

[0045] After the wound model was established, drugs were injected subcutaneously at 4 points around the wound, each point was 1 cm away from the incision, and the direction of needle insertion was towards the wound to avoid the needle tip from penetrating the cortex. The ...

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Abstract

The invention provides a dog MSC exosome preparation and a preparing method thereof. The preparing method includes: preparing exosome-free fetal calf serum, and separating dog MSC exosome. The preparing method has the advantages that the extracted exosome of the dog MSC exosome preparation prepared by the method is small in size, can easily achieve in-vivo circulation and can easily reach woundedtissue; in-vivo dosage stability can be guaranteed by directly using the exosome, the treatment effect is guaranteed to a certain degree, the exosome is free of toxic and side effects, and a foundation is laid for large-scale clinical application; the prepared preparation is applicable to dog experiment models, the dog MSC exosome preparation for treating skin injury is prepared, a dog skin woundcan be repaired by injecting the preparation to the periphery of the wound or directly applying the preparation to the wound surface, inflammation can be inhibited, healing can be accelerated, and scar formation can be reduced.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a canine MSC exosome preparation that can be used to treat canine skin damage, and a preparation method thereof. Background technique [0002] Mesenchymal Stem Cells (MSCs) are adult stem cells developed from mesoderm and present in various tissues of the body, which have the ability of self-renewal and multidirectional differentiation. As an emerging treatment for difficult diseases, treatment has set off a research boom in pet clinics and even human clinics in recent years. Many research results have shown that it has unexpected effects in the treatment of various diseases, but people have always attributed its effect to the rapid proliferation and differentiation of MSCs into damaged cells after reaching the damaged tissue, gradually replacing the original damaged tissue. In later studies, it was found that the use of MSC-derived exosomes to treat tissue damage had the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61P17/02A61P29/00C12N5/0775
Inventor 王丙云詹小舒罗东章李东升陈志胜陈胜锋罗兆益
Owner FOSHAN UNIVERSITY
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