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A preparation method of placenta-derived maternal mesenchymal stem cells

A stem cell, placenta-derived technology, applied in the field of culture and separation of mesenchymal stem cells, can solve the problems of undetected and complex placental tissue, and achieve the effect of small cell damage and simple method.

Active Publication Date: 2015-10-14
BEIJING HEALTH & BIOTECH (H&B) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the multi-step multi-enzyme digestion method is commonly used to obtain mesenchymal stem cells from placenta and umbilical cord, such as: Lu Guohui et al. Experimental study. Southern Medical University Journal, 2011; 31 (2): 262-265 "Using double-enzyme digestion and density gradient centrifugation to isolate clonal-like adherent growth cells from term placental decidua, expressing typical MSCs surface antigens, but because the placental tissue is more complex and there are both maternal and fetal cells, Lu Guohui et al. did not detect that the obtained cells were maternal cells

Method used

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  • A preparation method of placenta-derived maternal mesenchymal stem cells
  • A preparation method of placenta-derived maternal mesenchymal stem cells
  • A preparation method of placenta-derived maternal mesenchymal stem cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Isolation, culture and cryopreservation of human placenta-derived maternal mesenchymal stem cells

[0027] (1) After washing the placenta repeatedly with phosphate buffered solution (PBS), carefully separate the decidual part of the placenta, then rinse repeatedly with PBS to remove residual blood, and cut it into small pieces smaller than 0.5-1 square centimeters;

[0028] (2) Add 1 times the volume of trypLE according to the total volume of the tissue block TM (Invitrogen, USA) The mixed enzyme solution digested the tissue block at 37°C for 0.3 hours, and stirred with a rotor during the digestion process; then washed and removed the digestive fluid, and then the residual tissue block after the first step was digested, and then 2 times the volume of the tissue block trypLE TM The enzyme solution digested the tissue block at 37°C for 0.5 hours to obtain the digested digestive juice,

[0029] (3) Filter the digestive juice obtained in step (2) with a sieve, s...

Embodiment 2

[0036] Example 2 Expansion and Preparation of Human Placenta-derived Maternal Mesenchymal Stem Cells

[0037] (1) From the qualified primary subculture cell bank obtained in Example 1, a cell containing 3×10 6 Cell cryopreservation tubes, thaw the cells at 37°C;

[0038] (2) press 2×10 with serum-free medium (LONZA) 4 / cm 2 Inoculate into culture flasks at a density of 37°C, saturated humidity, and 5% CO2 incubator for culture. When the cells reach 80-90% confluence, they are passaged at a ratio of 1:3, and after 3 passages;

[0039] (3) When the confluence of the third passage cells reaches 90%, use trypLE TM Digest the cells, count them, resuspend the cells with the prepared cell protection solution at an appropriate concentration, divide them into sterile containers, seal them, and store them in a suitable environment.

[0040] (4) Take part of the cell resuspension preparation and use the methods well-known to professionals in the field to conduct pathogenic microorgan...

Embodiment 3

[0042] Example 3 Adipogenic and osteogenic differentiation of human placenta-derived maternal mesenchymal stem cells

[0043] (1) Osteogenic differentiation detection: The 4th passage of placental decidual mesenchymal stem cells were selected, routinely digested and centrifuged to make a single cell suspension, and weighed 2×10 4 Each well was seeded in a 24-well plate, and the osteogenic medium (Hyclone Company) was replaced when the cell confluence reached 70%, and then the medium was changed every 3 days, and the osteogenesis was detected by alizarin red staining after 21 days.

[0044] (2) Detection of adipogenic differentiation: select the 4th passage of placental decidual mesenchymal stem cells, routinely digest and centrifuge to make a single cell suspension, press 2×10 4 Each well was seeded in a 24-well plate, and the adipogenic medium (Hyclone Company) was replaced when the cell fusion reached 70%. Then the medium was changed every 3 days, and oil red O staining was...

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Abstract

The invention provides a preparation method of a placenta source matrix mesenchymal stem cell. The preparation method comprises the following steps: (1) separating the placenta source matrix mesenchymal stem cell; (2) cultivating the placenta source matrix mesenchymal stem cell, wherein a trypLE<TM> enzyme solution is utilized to process a placenta tissue in two steps in the step (1), and a serum-free medium is utilized in the step (2). By adopting the method provided by the invention, the placenta source matrix mesenchymal stem cell is a complete matrix source by short tandem repeat sequence (STR) atlas analysis, and is qualified by chromosome karyotype examination, pathogenic microorganism examination of bacteria, funguses and viruses, cell purity identification and cell biology function identification. Therefore, the placenta source matrix mesenchymal stem cell obtained by the method provided by the invention does not contain foreign protein, can meet the requirements of clinical use, is especially suitable for use by mothers, and also provides a technical support for building an excellent placenta source matrix mesenchymal stem cell bank.

Description

technical field [0001] The invention relates to a method for isolating and culturing mesenchymal stem cells, in particular to a method for isolating and preparing placenta-derived maternal mesenchymal stem cells. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from mesoderm and ectoderm in early development. They were originally found in bone marrow because of their following characteristics: 1) Hematopoietic support. As the main cellular component of the hematopoietic microenvironment—the stem / progenitor cells of the stromal cell line, co-transplantation of MSCs and hematopoietic stem cells can promote the engraftment of hematopoietic stem and progenitor cells. 2) Immune regulation. MSCs do not express CD80, CD86, HLA-DR and other co-stimulatory molecules, and can inhibit mixed lymphocyte reaction in vitro, which is of great significance for the prevention and treatment of graft-versus-host disease caused by allogene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/0775A61K35/28A61P37/00A61L27/38
Inventor 韩之海王涛
Owner BEIJING HEALTH & BIOTECH (H&B) CO LTD
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