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Method for separating mesenchymal stem cells from placentas and digestive enzyme composition adopted in method

A technology of mesenchymal stem cells and digestive enzymes, applied in the field of separating stem cells

Active Publication Date: 2017-10-10
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 improvement in terms of extract purity and / or recovery

Method used

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  • Method for separating mesenchymal stem cells from placentas and digestive enzyme composition adopted in method
  • Method for separating mesenchymal stem cells from placentas and digestive enzyme composition adopted in method
  • Method for separating mesenchymal stem cells from placentas and digestive enzyme composition adopted in method

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Effect test

Embodiment 1

[0097] Embodiment 1, the separation of placental MSC

[0098] Within four hours after delivery, the placenta leaflets were cut off under aseptic conditions, and the placenta leaflets were fully washed with PBS buffer solution containing 10% volume FBS to remove residual blood in the placenta leaflets. First cut the placenta leaflet into 1cm 3 Tissue pieces of the same size were added to PBS buffer containing 0.1mg / mL dispase, 0.25mg / mL trypsin, 0.25mg / mL DNase I, 1mg / mL collagenase IV, 1mg / mL hyaluronidase for digestion at 37°C After 15 minutes, an appropriate amount of FBS was added to terminate the digestion. Then filter the tissue block and cell suspension together with a 200-mesh copper mesh and grind the tissue block with a syringe plunger, collect the filtered cell suspension and add it to a centrifuge tube for 10 minutes at 1000rpm, pour off the supernatant, and wash with 10% FBS Resuspend cells in PBS buffer. Then use density gradient centrifugation to separate an...

Embodiment 2

[0099] Embodiment 2, subculture of placental MSC and cryopreservation thereof

[0100] After about 10 days, after the scattered adherent cells formed colonies, they were digested with 0.05% trypsin / 2mM EDTA. 2 Carry out subculture. Afterwards, subculture was digested when the cells reached about 70% confluency. Take 3×10 after digestion 6 The cells were added to 1ml of cell freezing solution (containing 50% low-sugar DMEM culture medium, 40% FBS, 10% dimethyl sulfoxide), and were cooled by a program, and finally entered into a liquid nitrogen tube for freezing.

Embodiment 3

[0101] Embodiment 3, biological characteristic identification of placental MSC

[0102] 1. Cell growth and morphological characteristics

[0103] By the isolation culture of embodiment 1 and embodiment 2, the placental mononuclear cells can be clearly seen under the microscope after the placental mononuclear cells are cultured for 72 hours. Spindle-shaped adherent cells ( figure 1 A), about 10 days will form turbine-shaped cell clones ( figure 1 B. figure 1 D), after digestion and passage, about 80% of the fusion adherent layer will be formed ( figure 1 C. figure 1 E. figure 1 F). During the culture process, it was found that the cell shape was relatively uniform, the proliferation speed was fast, the adhesion speed was fast, and it was easily digested by trypsin. After passage to more than 15 generations, its shape and growth characteristics did not change significantly.

[0104] 2. Identification of MSC surface markers by flow cytometry

[0105] The 3rd, 6...

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Abstract

The invention relates to a method for separating mesenchymal stem cells from placentas and a digestive enzyme composition adopted in the method. The method includes: (a) completely flushing placental lobules with PBS buffer solution to remove residual blood in the placentas; (b) cutting the placental lobules into blocks, adding into tissue digestive enzyme containing PBS buffer solution, and performing incubation digestion at 37 DEG C; (c) filtering the tissue blocks through a copper screen, and grinding to promote filtration in necessity; (d) centrifuging collected filtrate, separating mononuclear cells, adopting an MSC culture medium for suspension of the obtained cells, and culturing in a 5% CO2 incubator at 37 DEG C; (e) after cloning of dispersed cells is formed, picking each clone cell, respectively culturing with the MSC culture medium, and after cell fusion, performing trypsinization passage to obtain the mesenchymal stem cells. By adoption of the method, high-purity mesenchymal stem cells can be obtained, andn low cost and high cell yield are realized.

Description

technical field [0001] The present invention relates to a method for isolating stem cells from placenta, in particular to a method for isolating mesenchymal stem cells from placenta, and more particularly relates to a method for isolating mesenchymal stem cells from placenta using the digestive enzyme composition of the unique formula of the present invention Stem Cell Approach. Using the method of the invention can effectively improve the efficiency of isolating mesenchymal stem cells from placenta. Background technique [0002] Mesenchymal stem cells (mesenchymal stem cells, MSCs) such as human mesenchymal stem cells were first isolated from bone marrow, a type of tissue stem cells derived from mesoderm with multi-lineage differentiation potential and self-renewal ability, in vivo and Under specific conditions in vitro, it has the ability to differentiate into various adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, ...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/0735
Inventor 许晓椿陆晗燕朱业峰
Owner BOYALIFE
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