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Method for treating premature ovarian failure by virtue of placenta mesenchymal stem cells and cell preparation

A cell preparation, stem cell technology, applied in the fields of biotechnology and biomedicine

Active Publication Date: 2019-03-19
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods are subject to further improvement in terms of extract purity and / or recovery

Method used

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  • Method for treating premature ovarian failure by virtue of placenta mesenchymal stem cells and cell preparation
  • Method for treating premature ovarian failure by virtue of placenta mesenchymal stem cells and cell preparation
  • Method for treating premature ovarian failure by virtue of placenta mesenchymal stem cells and cell preparation

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Embodiment 1, placental whole cell processing:

[0157] 1. Pre-preparation of the mixed synthase digestion solution: pipette 22ml of HBSS (Hank's Balanced Salt Solution) containing calcium and magnesium ions, 0.4ml of Roche Liberase MNP-S enzyme (for example, purchased from Xibao Biology, article number: 5578582001), 0.7ml Put ml of DNA type I enzyme in a 50ml centrifuge tube, then add zinc chloride (addition concentration is 0.2g / L, 0.25g / L, or 0.3g / L), mix well, and preheat at 37°C for more than 20min. The Hank's balanced salt solution consists of: 8.0g / L of NaCl, 0.4g / L of KCl, 0.1g / L of MgSO4 7H2O, 0.1g / L of MgCl2 6H2O, 0.06g / L of Na2HPO4 2H2O, 0.06g / L of KH2PO4, 1.0g / L of glucose, 0.14g / L of CaCl2, 0.35g / L of NaHCO3, 0.2g / L of phenol red, hydrochloric acid or sodium hydroxide to adjust the pH to 7.4.

[0158] 2. Preparation of placenta lobule: take out the placenta from the collection bag and place it in a white porcelain plate, rinse with tissue cleaning soluti...

Embodiment 2

[0175] Embodiment 2, primary cell recovery and subculture

[0176] 1. Cell recovery:

[0177] Take 2 tubes of frozen cells, thaw them quickly at 37°C, transfer the cells to a 15ml centrifuge tube, add 8ml of complete medium to recover by dripping; unless otherwise stated, the complete medium used in this article is DMEM-F12 culture containing 10% FBS base;

[0178] Then centrifuge at 1200rpm for 5min (acceleration 9, deceleration 7), remove the supernatant, add 5ml of complete medium to resuspend; each tube of cells is inoculated in a T75 culture flask, supplement the complete medium to 30ml, and place in a CO2 incubator ( Culture at 37°C, 5% CO2, saturated humidity); replace the medium with complete medium every 3-4 days, count according to the colony formation after 12 days of recovery, until the cell density is not less than 3000 cells / cm 2 Subsequent passages can be carried out;

[0179] 2. Cell subculture: Take the revived P0 generation cells and wash them with PBS, ...

Embodiment 3

[0193] Embodiment 3, biological characteristic identification of placental MSC

[0194] With reference to the methods [0062] to [0089] of the authorized patent CN102676451A, the biological characteristics of placental mesenchymal stem cells were identified. The ability of cell differentiation proves that the MSC obtained by the method of the present invention has stem cell characteristics.

[0195] For example, exemplarily, the induction differentiation test is performed on the P5 generation cells, and the results show that these cells have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Typical micrographs of adipogenic, osteogenic, and chondrogenic differentiation are shown in Image 6 .

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Abstract

The invention relates to a method for treating premature ovarian failure by virtue of placenta mesenchymal stem cells and a cell preparation and particularly relates to the cell preparation on one hand. The cell preparation is cell suspension prepared by mixing mesenchymal stem cells such as placenta mesenchymal stem cells into a 0.9% sodium chloride solution and is particularly prepared by the steps of transferring mesenchymal stem cells obtained through cell passage into a centrifuge tube, carrying out centrifuging to remove supernatant, adding the 0.9% sodium chloride solution, and carryingout resuspension, so as to obtain the cell preparation. The mesenchymal stem cells are prepared by the steps of processing placental lobules, carrying out digestion and termination on mixed enzyme, collecting primary cells, carrying out cryopreservation on the primary cells, carrying out thawing, passage, detection and cryopreservation on the cells, and relating with a database. The cell preparation prepared by virtue of the method presents excellent biological effect in the treatment of the premature ovarian failure.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedicine, and relates to a method for using stem cells to treat premature ovarian failure (POF) and the cell preparation used. Specifically, the present invention relates to the isolation of stem cells from the placenta, in particular to the isolation of mesenchymal stem cells from the placenta, and the use of such placental mesenchymal stem cells to treat premature ovarian failure, and more particularly to a method using the unique The digestive enzyme composition formulated so as to isolate mesenchymal stem cells from placental tissue and culture them into mesenchymal stem cells, and then use the placental mesenchymal stem cells to treat premature ovarian failure. Using the method of the present invention can effectively improve the efficiency of isolating mesenchymal stem cells from the placenta, and further can effectively improve the effect of using the placental mesenchymal stem cells to t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K35/28A61P15/00C12N5/0775
CPCA61K35/28A61P15/00C12N5/0668C12N2509/00
Inventor 许晓椿李容肖海蓉刘冰
Owner BOYALIFE
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