Human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicle preparation and preparation method

A stem cell factor and stem cell technology, which is applied in the field of preparation and preparation of human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicles, can solve the problem of limited total amount of microvesicles, influence of product uniformity, and influence on the preparation of microvesicles on cell state. and other problems, to avoid medical ethics problems, to achieve good product uniformity, and to facilitate large-scale production.

Active Publication Date: 2021-08-31
武汉五州润达生物医药科技有限公司
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  • Summary
  • Abstract
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AI Technical Summary

Problems solved by technology

[0004] (1) Most of the current production of human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicles uses medium containing animal serum to ensure cell viability, but it will cause animal-derived blood components to mix into the suspension and cause human tissue allergies, which will affect the clinical application of the product. A small number of serum-free media are used because the cell viability is affected, the efficiency of obtaining microvesicles is low, and the product uniformity is affected, making it impossible to apply large-scale production
[0005] (2) In order to avoid contamination in the prior art, antibiotics are used in the medium during the preparation of microvesicles, which may affect the state of cells and the preparation of microvesicles
[0006] (3) At present, the preparation of stem cell factor microvesicles derived from human umbilical cord mesenchymal stem cells is usually prepared by P3 cells, and the total amount of microvesicles produced is limited, which is not conducive to large-scale production and product uniformity control
Cells are subcultured using trypsin, which may cause exogenous protein contamination and affect the quality of subsequent microvesicle production
[0007] (4) The microvesicles prepared by the prior art are all stored in the form of a suspension. However, the microvesicles and the active ingredients in the vesicles are easily degraded under normal temperature storage. The active ingredients in the vesicles and vesicles will still degrade, therefore, the problem that the microvesicle suspension cannot be stored for a long time limits its application range

Method used

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  • Human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicle preparation and preparation method
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  • Human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicle preparation and preparation method

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Embodiment 1

[0053] 1. Obtain primary human umbilical cord mesenchymal stem cells

[0054] (1) Aseptically obtain the umbilical cord of a full-term infant, place it in a sterile bag filled with sterile injection-grade normal saline at 4°C, in which the sterile injection-grade normal saline contains 50 μg / ml gentamicin, 2.5 μg / ml ml amphotericin B, 100 U / ml penicillin and 100 μg / ml streptomycin. Store the aseptic bag in a sterile storage cylinder at 4°C, and quickly transport it to a million-level cell laboratory for separation and preparation;

[0055] (2) Put the umbilical cord in a petri dish and place it in the cell operation table, remove 5cm lengths at both ends of the umbilical cord with sterile surgical instruments, wash the residual blood in the umbilical cord tissue with 4°C normal saline, and then place the umbilical cord at 4°C for medical use. Sterile injection-grade normal saline was cut longitudinally, the umbilical vessels were removed, and the amniotic tissue was completel...

Embodiment 2

[0075] Embodiment 2: Stability test of freeze-dried powder

[0076] Dissolve the lyophilized powder after being stored in a refrigerator at 4°C for half a year with sterile physiological saline at a concentration of 0.22mg / mL, and the total amount of sterile saline is 1ml; at the same time, the prepared lyophilized powder is immediately dissolved in sterile physiological saline The solution formed by saline (concentration is 0.25mg / mL, total amount is 1ml) was used as a control, and the expression levels of three representative human umbilical cord mesenchymal stem cell factors were detected by ELISA, as shown in Table 1.

[0077] Table 1 The concentration of representative cytokines VEGF, GEF, and FGF secreted by human umbilical cord mesenchymal stem cells detected by ELISA

[0078]

[0079] Note: In this embodiment, sterile physiological saline was used as the solvent each time the freeze-dried powder was dissolved to prepare the freeze-dried powder detection reagent, and...

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Abstract

The invention relates to a human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicle preparation and a preparation method. The method uses the umbilical cord tissue discarded by clinical pregnant women after childbirth as a material to obtain human umbilical cord mesenchymal stem cells and human umbilical cord mesenchymal stem cells with medical value Stem cell factor microvesicles derived from stem cells have good product uniformity and high production efficiency, which is conducive to large-scale production and avoids medical ethics issues. This method strictly operates in the process of culturing and expanding human umbilical cord mesenchymal stem cells. The whole process is carried out in a million-level cell laboratory without using antibiotics, which avoids possible changes in stem cell properties and damage to human umbilical cord mesenchymal stem cells. The influence of the microvesicle quality of the source stem cell factor can trigger tissue allergic reactions; use optimized serum-free human umbilical cord mesenchymal stem cell culture medium to avoid animal-derived blood components mixed into the suspension and cause human tissue allergy; the frozen microvesicle suspension prepared Dry powder is easy to store and provides convenience for subsequent product production.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicle preparation and a preparation method. Background technique [0002] Human umbilical cord mesenchymal stem cells have biological functions such as tissue repair and immune regulation, but because external use of human umbilical cord mesenchymal stem cells may cause blockage of skin pores and sebaceous glands, local skin inflammation and even skin allergies, as well as long-term abnormal differentiation and tumor formation, etc. Risk, resulting in its limited clinical application in plastic surgery, especially in skin repair. Human umbilical cord mesenchymal stem cell-derived stem cell factor microvesicles are produced by human umbilical cord mesenchymal stem cells under a special induction state, which can effectively simulate the functional membranous spherical structure of human umbilical cord mesenchym...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K9/10A61K9/19A61K47/36A61K47/18A61P17/00
CPCA61K9/10A61K9/19A61K35/28A61K47/183A61K47/36A61P17/00C12N5/0668C12N2509/00
Inventor 刘韦成朱霞朱子豪
Owner 武汉五州润达生物医药科技有限公司
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