Placenta mesenchymal stem cell preparation and application of placental mesenchymal stem cell preparation in treating sclerosis

A technology of cell preparation and mesenchymal stem cells, applied in the field of biotechnology and biomedicine

Active Publication Date: 2019-04-26
BOYALIFE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These methods are subject to further improvem

Method used

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  • Placenta mesenchymal stem cell preparation and application of placental mesenchymal stem cell preparation in treating sclerosis
  • Placenta mesenchymal stem cell preparation and application of placental mesenchymal stem cell preparation in treating sclerosis
  • Placenta mesenchymal stem cell preparation and application of placental mesenchymal stem cell preparation in treating sclerosis

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Experimental program
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Embodiment 1

[0162] Embodiment 1, placental whole cell processing:

[0163] 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.

[0164] 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

[0181] Embodiment 2, primary cell recovery and subculture

[0182] 1. Cell recovery:

[0183] 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;

[0184] 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;

[0185] 2. Cell subculture: Take the revived P0 generation cells and wash them with PBS, ...

Embodiment 3

[0199] Embodiment 3, biological characteristic identification of placental MSC

[0200] 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.

[0201] 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 Figure 6 .

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Abstract

The invention relates to a placenta mesenchymal stem cell preparation and application of the placenta mesenchymal stem cell preparation in treating sclerosis. Specifically, the invention relates to the cell preparation. The cell preparation is a cell suspension prepared by mixing and suspending mesenchymal stem cells such as placenta mesenchymal stem cells in a 0.9% sodium chloride solution and isspecifically prepared through a method which comprises the following steps of transferring the mesenchymal stem cells obtained through cell passage to a centrifuge tube, carrying out centrifuging, discarding a supernatant, and adding the 0.9% sodium chloride solution for re-suspending so as to prepare the cell preparation. The invention further relates to the application of the cell preparation in preparation of drugs for treating and/or preventing the systemic sclerosis. The invention further relates to a method for preparing the cell preparation and the placenta mesenchymal stem cells usedby the cell preparation and mixed enzyme digestive juice used in the method. The cell preparation has an excellent biological effect in the aspect of treating the sclerosis.

Description

technical field [0001] The invention belongs to the fields of biotechnology and biomedicine, and relates to a cell preparation using stem cells to treat systemic sclerosis. Specifically, the present invention relates to a method for isolating stem cells from the placenta, in particular to a method for isolating mesenchymal stem cells from the placenta, and more particularly relates to a digestive enzyme composition using the unique formula of the present invention to extract from the placenta A method for isolating mesenchymal stem cells from tissues and culturing them into mesenchymal stem cells. Using the method of the invention can effectively improve the efficiency of isolating mesenchymal stem cells from placenta. Further, the present invention relates to using the above-mentioned placental mesenchymal stem cells to prepare cell preparations, and then use the cell preparations to treat systemic sclerosis. Background technique [0002] Systemic sclerosis (SSc), also kn...

Claims

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Application Information

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IPC IPC(8): A61K35/28A61K9/10A61K47/24A61K47/26A61P17/00A61P37/02C12N5/0775
CPCA61K47/24A61K47/26A61P17/00A61P37/02A61K9/10A61K35/28C12N5/0668C12N2509/00
Inventor 许晓椿李容肖海蓉刘冰
Owner BOYALIFE
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