Method for extracting exosome from amniotic mesenchymal stem cells and application of exosome

A technology of amniotic mesenchyme and extraction method, applied in the field of exosome extraction, can solve problems such as insufficient vascularization, and achieve the effects of high extraction efficiency, high safety factor and high purity

Pending Publication Date: 2020-03-24
AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although bone allografts may have advantages over autologous bone grafts in orthopedic surgery, there is a problem with insufficient vascularization in the early post-implantation period

Method used

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  • Method for extracting exosome from amniotic mesenchymal stem cells and application of exosome
  • Method for extracting exosome from amniotic mesenchymal stem cells and application of exosome
  • Method for extracting exosome from amniotic mesenchymal stem cells and application of exosome

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Experimental program
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Effect test

Embodiment 1

[0038] (1) Human amniotic membrane-derived mesenchymal stem cells were selected for isolation, culture and passage:

[0039] A1. The healthy amniotic membrane is mechanically separated from the placenta and washed to remove blood and debris;

[0040] In step A1, the number of weeks of the placenta is 38 ± 1 week, and the time for obtaining the placenta is within 4 hours after cesarean section; the washing is to repeatedly wash the amniotic membrane in a PBS container containing double antibodies;

[0041] B1. Soak the washed amniotic membrane in mixed solution 1 for 0.5-1 hour, wash it with tris buffer solution, gently scrape the amniotic membrane surface with a cell scraper to remove epithelial cells, rinse with PBS for 3-5 times, and shred it mechanically; Wherein, the mixed solution 1 is tris buffer containing 0.03-0.05% (w / v) sodium lauryl sulfate and 0.1-0.5% (w / v) ethylenediaminetetraacetic acid;

[0042] In step B1, the sodium lauryl sulfate concentration is 0.03% (w / v...

Embodiment 2

[0054] An exosome hydrogel complex, comprising a hydrogel based on chitosan-β-sodium glycerophosphate accounting for a mass volume fraction (W / V) of the complex of 1-20%, 150-200 μg / mL temperature Sensitive hydrogel additive (that is, the exosomes prepared in the above-mentioned Example 1) and the rest of physiological saline.

Embodiment 3

[0056] An aqueous solution of exosomes, which uses sterile PBS solution as a solvent, and the concentration of exosomes in the solvent ranges from 150 to 200 μg / mL.

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Abstract

The invention discloses a method for extracting exosome from amniotic mesenchymal stem cells and application of the exosome. The exosome is obtained through the steps of amnion separation, washing, epithelial cell removal, shearing, digestion, centrifugation, second-layer suspension taking, resuspension, inoculated culture, subsequent extraction preparation and the like. When the exosome is applied to a medicine for promoting angiogenesis and osteogenesis, vascular endothelial cell aggregation and angiogenesis can be achieved, a microcirculation is reconstructed, blood perfusion is increased,lost of other functions is not caused, and the application prospect is promising. According to the method, the cell extraction process is optimized, the amniotic stem cell extraction efficiency is higher, the purity is higher, and on the premise that more cells are obtained, it can be guaranteed that more exosomes are obtained.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for extracting exosomes derived from amniotic mesenchymal stem cells and an application thereof. Background technique [0002] Repair of bone defects caused by trauma, infection, or tumor resection remains a major challenge in jaw defects. The use of autologous bone grafts, most commonly from the iliac crest, is considered the gold standard. Drawbacks are donor site morbidity and graft size limitations. The process of harvesting an autograft from healthy bone increases surgical time and may be associated with potential blood loss and infection risks. In addition, patient age and metabolic disturbances may also affect the quality of autografts. Inconsistent or low concentrations of endogenous mesenchymal stem cells (MSCs) can significantly reduce the efficacy of autologous transplantation. Therefore, bone tissue engineering methods that can help ove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775A61K35/28A61P19/08
CPCC12N5/0668A61K35/28A61P19/08C12N2509/00
Inventor 王羽立袁华杜一飞马晓杰卞一峰
Owner AFFILIATED STOMATOLOGICAL HOSPITAL OF NANJING MEDICAL UNIV
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