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Reagent box for separating karyocyte in vitro and application method thereof

A nucleated cell and kit technology, applied in the field of biomedicine, can solve the problems of high cost, high technical requirements, and inability to effectively retain stem cells.

Active Publication Date: 2007-02-28
NINGXIA ZHONGLIANDA BIOPHYSICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 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.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] Kit composition:

[0017] 1. Erythrocyte precipitant: an aqueous solution composed of 0.4% carboxymethyl starch and 0.4% calcium gluconate, the pH value of the solution is 7.4;

[0018] 2. Sodium diatrizoate-polysucrose 400 # : Sodium Diatrizoate 0.3% and Ficoll 400 8% # The aqueous solution has a pH value of 7.

[0019] 3. Cell maintenance solution: phosphate buffered saline (PBS);

[0020] 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.

[0021] Take 100ml of bone marrow or umbilical cord blood, add 100ml of cell maintenance solution to dilute, add 20ml of erythrocyte precipitant, mix well, let it stand for 8-20 minutes, collect the supernatant, and concentrate it by centrifugation at 1200 rpm / 8 minutes. Aspirate the concentrate and add to the solution containing sodium diatrizoate-polysucrose 400 # In a centrifuge tube,...

Embodiment 2

[0025] Kit composition:

[0026] 1. Erythrocyte precipitant: a solution obtained by dissolving 1.2% hydroxyethyl starch and 0.2% calcium gluconate in water, with a pH value of 8;

[0027] 2. HISTOPAQUE1077;

[0028] 3. Cell maintenance solution: normal saline;

[0029] 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.

[0030] Concrete application method is the same as embodiment 1.

[0031] The above concentrations are mass concentrations.

Embodiment 3

[0033] Kit composition:

[0034] 1. Erythrocyte precipitant: 0.8% methylcellulose and 0.3% calcium gluconate dissolved in water, pH value 8;

[0035] 2. Sodium diatrizoate-polysucrose 400 # ;

[0036] 3. Cell maintenance solution: phosphate buffered saline (PBS);

[0037] 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.

[0038] Concrete application method is the same as embodiment 1.

[0039] The above concentrations are mass concentrations.

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Abstract

The invention relates the separating agent box and application. The agent box comprises erythrocyte precipitating agent, hypaque sodium- ficoll 400# or HISTOPAQUE1077 and cell maintenance liquid. The agent box can separate bone marrow and umbilical cord blood, on the surface of cell there is no any mark, and it doesn't change biological activity of cell. The invention can commercially manufacture, and the application is wide.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a kit for in vitro separation of nucleated cells in human bone marrow or umbilical cord blood and an application method thereof. Background technique [0002] Bone marrow and umbilical cord blood are human hematopoietic tissues, which are rich in hematopoietic stem cells / progenitor cells of various lines and a large number of mature cells, such as red blood cells, white blood cells, platelets, etc., and because of the different antigenicity contained on the surface of each line of stem cells, they can Divided into more than one hundred kinds, such as CD34, CD117, CD10, SCA-1, Lin-, etc. All lines of stem cells and mature white blood cells are nucleated cells. Nucleated cells include monocytes, multinucleated cells, lymphocytes, stem cells / progenitor cells of various lines, mesenchymal cells, etc. [0003] At present, there are many methods for separating and extracting stem...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/0786
Inventor 王怀林
Owner NINGXIA ZHONGLIANDA BIOPHYSICS
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