Mesenchyme stem cell preserving fluid and use thereof

A mesenchymal stem cell and preservation solution technology, applied in the field of mesenchymal stem cell preservation solution, to achieve the effect of reducing adhesion, high survival rate and increasing range

Active Publication Date: 2008-07-02
TIANJIN AMCELLGENE ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, no one has used albumin and heparin in the mesenchymal stem cell preservation solution at the same time.

Method used

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  • Mesenchyme stem cell preserving fluid and use thereof
  • Mesenchyme stem cell preserving fluid and use thereof
  • Mesenchyme stem cell preserving fluid and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Isolation, cultivation and phenotypic identification of human umbilical cord cells

[0025] (1) In vitro, the human umbilical cord was washed repeatedly with phosphate buffered solution (PBS) to remove residual blood, and chopped to about 1mm 3 Add 0.1% collagenase by weight of raw materials, and digest at 37°C for 45 minutes; (2) Add trypsin (Sigma, USA) with 0.125% by weight of raw materials to digest for 45 minutes at 37°C, stir with a rotor during the digestion process, add An appropriate amount of serum terminates the action of pancreatic enzymes; (3) Filter the digestive juice with a 100-mesh and 200-mesh filter successively to remove undigested tissue; (4) Dilute the filtered digestive juice with phosphate buffer , the ratio of digestive solution to phosphate buffer is 1:10; (5) centrifuge to obtain cells, and the speed of centrifuge is 1500rpm / min to obtain cells; (6) cells are obtained by 1~2×10 1 / cm 2 Inoculate in a plastic culture bottle, use a m...

Embodiment 2

[0028] Solution medium selection in embodiment 2 preservation solution preparation method

[0029] 1.1 Operation steps

[0030] 1.1.1 Preparation of TC199 containing 1% human serum albumin and physiological saline containing 1% human serum albumin.

[0031]1.1.2 Use TC199 containing 1% human serum albumin and saline containing 1% human serum albumin to prepare umbilical cord mesenchymal stem cell suspension, 5×10 5 / ml / ml. Pack into 6 tubes (3 parallel tubes for each preservation solution), 2.5ml / tube.

[0032] 1.1.3 The umbilical cord mesenchymal stem cell suspension was transferred to vials respectively, placed at 4°C, stored for 8 hours, and the cell viability was counted by trypan blue staining.

[0033] Judgment of results: Dead cells can be stained with trypan blue, dark blue cells can be seen under the microscope, live cells are not stained, and appear colorless and transparent under the microscope.

[0034] result:

[0035] TC199 group

Embodiment 3

[0036] The selection of embodiment 3 human serum albumin concentrations

[0037] 1.1 Operation steps

[0038] 1.1.1 Preparation of TC199 containing 5%, 2%, 1% and 0.5% human serum albumin.

[0039] 1.1.2 Preparation of umbilical cord mesenchymal stem cell suspension, 5×10 5 / ml / ml. Pack into 12 tubes (3 parallel tubes for each preservation solution), 2.5ml / tube.

[0040] 1.1.3 After centrifuging at 800rpm for 5min, discard the supernatant. Add 2.5ml of each prepared preservation solution respectively.

[0041] 1.1.4 Cells were mixed and transferred to 12 vials respectively, placed at 4°C, stored for 8 hours, and stained with trypan blue to count the cell viability.

[0042] Judgment of results: Dead cells can be stained with trypan blue, dark blue cells can be seen under the microscope, live cells are not stained, and appear colorless and transparent under the microscope.

[0043] result:

[0044]

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PUM

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Abstract

The invention discloses a preservation solution for mesenchymal stem cells and application thereof. The cell preservation solution contains human albumin and heparin as the main components, and other auxiliary reagents such as human cytokine, phosphate ion, metal ions or monosaccharide are contained in a buffer solution for preserving human mesenchymal stem cells. The preservation solution can keep high survival rate of human mesenchymal stem cells in transportation process, reduce adhesion between cells and between the cell and the inner wall of a container, and reduce the possible occurrence of cell mass embolism in blood vessel while clinically infusing human mesenchymal stem cells. The mesenchymal stem cells can be maintained in a state of single-cell suspension at an environment temperature of 4 to 15 DEG C for 24 h, thus greatly enlarging the clinic application range of the human mesenchymal stem cells. The components used in the solution accord with the clinic application, and can meet the requirement for clinic use of the human mesenchymal stem cells.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a mesenchymal stem cell preservation solution and its application. Background technique [0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells derived from the mesoderm and ectoderm in the early stages of development. They were originally found in the bone marrow and have attracted increasing attention because of their following characteristics: 1) Hematopoietic support and promote implantation. As the main cell component of the hematopoietic microenvironment—the stem / progenitor cells of the stromal cell line, MSCs realize the fine regulation of hematopoiesis and support the development of hematopoietic stem and progenitor cells through direct cell-to-cell, secretion of extracellular matrix and various cytokines. grow. Co-transplantation with hematopoietic stem cells can promote the implantation of hematopoietic stem and progenitor cells, which is of great sign...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A01N1/02
Inventor 韩忠朝刘拥军韩之波龚伟池颖
Owner TIANJIN AMCELLGENE ENG
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