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Marrow umbilical cord blood stem cell in vitro separating kit and application method thereof

A technology of umbilical cord blood stem cells and a kit, which is applied in the field of in vitro separation kits for bone marrow umbilical cord blood stem cells, can solve the problems of high cost, difficulty in clinical promotion and application, and inability to directly obtain stem cells, and achieve the effect of a simple method

Inactive Publication Date: 2008-03-19
NINGXIA ZHONGLIANDA BIOPHYSICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The common problems of these methods are: firstly, all types of stem cells cannot be effectively preserved; secondly, the cost is high; thirdly, the technical requirements are relatively high; there is a certain distance from industrial production, and there is a certain difficulty in clinical application.
[0009] The patent application "a kit for in vitro separation of nucleated cells and its application method" (application number: 200610106875.5) provides an in vitro separation kit for isolating nucleated cells from human bone marrow and umbilical cord blood. This technology has the advantages of simple method, The cost is low, it can be produced industrially, and it is easy to promote. However, this technology retains all nucleated cells except plasma, red blood cells, platelets, plasma proteins and ions, that is, mature monocytes, lymphocytes, multinucleated cells, all Stem cells, progenitor cells, mesenchymal stem cells, etc., cannot directly obtain stem cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Kit composition:

[0028] 1. Lin antibody erythrocyte precipitant: Hang the Lin antibody on the macromolecular hydroxyethyl starch or methyl cellulose. The molecular weight of hydroxyethyl starch and methyl cellulose is required to be between 20,000 and 160,000. The weight of the substance is calculated as a 4-8% aqueous solution;

[0029] 2. Sodium diatrizoate-polysucrose 400 # : Sodium Diatrizoate 0.3% and Ficoll 400 8% # The aqueous solution formed has a density of 1.077g / ml and a pH value of 7.

[0030] 3. Cell maintenance solution: phosphate buffered saline (PBS);

[0031] After the preparation of the above three liquids, they were sterilized at 121°C for 35 minutes, and the endotoxin content was tested to be ≤0.5 EU / ml, and then stored in the kits respectively.

[0032] Take 100ml of bone marrow or umbilical cord blood, add 100ml of phosphate buffered solution (PBS) to dilute, add 20ml of Lin antibody erythrocyte precipitant, mix well and let stand for 8-20 mi...

Embodiment 2

[0035] Kit composition:

[0036] 1. Lin antibody erythrocyte precipitant: hang the Lin antibody on the macromolecular hydroxyethyl starch or methyl cellulose. The molecular weight of hydroxyethyl starch and methyl cellulose is required to be between 20,000 and 160,000. The weight of the substance is calculated as a 4-8% aqueous solution;

[0037] 2. HISTOPAQUE1077 (commercially available product);

[0038] 3. Cell maintenance solution: 0.9% normal saline;

[0039] After the preparation of the above three liquids, they were sterilized at 121°C for 35 minutes, and the endotoxin content was tested to be ≤0.5 EU / ml, and then stored in the kits respectively.

[0040] Concrete application method is the same as embodiment 1.

[0041] The above concentrations are mass concentrations.

Embodiment 3

[0043] Kit composition:

[0044] 1. Lin antibody erythrocyte precipitant: hang the Lin antibody on the macromolecular hydroxyethyl starch or methyl cellulose. The molecular weight of hydroxyethyl starch and methyl cellulose is required to be between 20,000 and 160,000. The weight of the substance is calculated as a 4-8% aqueous solution;

[0045] 2. Ficoll (commercially available product);

[0046] 3. Cell maintenance solution: phosphate buffered saline (PBS);

[0047] After the preparation of the above three liquids, they were sterilized at 121°C for 35 minutes, and the endotoxin content was tested to be ≤0.5 EU / ml, and then stored in the kits respectively.

[0048] Concrete application method is the same as embodiment 1.

[0049] The above concentrations are mass concentrations.

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Abstract

The present invention relates to a reagent kit of the isolation of a bone marrow umbilical cord blood stem cell in vitro and the application method thereof. The present invention is characterized in that Lin antibody red blood cell precipitant cell preserving solution, and sodium iothalamate-polysucrose 400 # or HISTOPAQUE1077 or Ficoll are contained in the reagent kit, and each reagent solution is preserved dividually. The present invention adopts the method which combines a negative collection method and a density method, thus the surface of the stem cell which is collected and abstracted has no any label, the cells which are isolated and abstracted are Lin antigenic negative cells, the method is simple, and the present invention can be directly used to isolate and abstract the stem cells clinically. The reagent kit of the present invention can operate the industrialized production, to realize the promotion of the isolation and purification technology of the human hematopoietic stem cell, leading the doctor to conveniently cure the patient with the stem cells like the medicine usage.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a kit for in vitro separation of bone marrow and umbilical cord blood stem cells and an application method thereof. Background technique [0002] The components of bone marrow and umbilical cord blood are composed of plasma, red blood cells, platelets, monocytes, lymphocytes, and stem cells, progenitor cells, and mesenchymal stem cells that have developed to different stages. Stem cells are divided into CDs due to their surface antigens 34 、CD 38 、CD 17 、CD 133 、CD 55 , SCA-1, Lin - etc., have discovered stem cells and progenitor cells with more than 180 different surface antigens. Lin antibody is the boundary between mature cells and primitive cells (stem cells). Lin antibody positive indicates mature cells, and Lin antibody negative indicates both primitive cells and stem cells. [0003] Lin is a lineage surface antigen. When stem cells differentiate into progenitor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/074
Inventor 王怀林
Owner NINGXIA ZHONGLIANDA BIOPHYSICS
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