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Optimized karyote in-vitro separation kit and application method thereof

A nucleated cell and kit technology, applied in the field of biomedicine, can solve the problems of inability to effectively retain nucleated cells or stem cells, technical conditions that do not meet clinical use, and inability to effectively retain cell types, so as to reduce the probability of cell loss, The effect of shortening the time of in vitro separation and shortening the time of cell separation and extraction

Active Publication Date: 2012-08-22
NINGXIA ZHONGLIANDA BIOPHYSICS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The common problems in these methods are: first, all types of nucleated cells or stem cells cannot be effectively retained; second, the isolated and extracted cell types are single, and all cell types cannot be effectively retained; third, the cost is too expensive; fourth, technical conditions Not suitable for clinical use, only suitable for scientific research methods
[0011] After more than 4 years of clinical use of the bone marrow and umbilical cord blood cell in vitro separation kit produced according to this patent, we have carefully studied the production, packaging, storage, transportation, and clinical operation of the kit, summed up the experience and found that there are still needs room for improvement and optimization

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Kit composition:

[0036] 1. Reagent 1 prepares an aqueous solution of 1.0-6.0% hydroxyethyl starch and 0.45% sodium chloride, adjusts the pH value to 7.4±0.1 with 10% sodium hydroxide and 1% dilute hydrochloric acid, and uses 0.22 μm Fill in separate bottles after filter sterilization, or autoclave at 121°C after bottled.

[0037] 2. Reagent 2 prepares an aqueous solution whose concentration is 10% sodium diatrizoate and 3% polysucrose 400# (trade name HISTOPAQUE1077 of SIGMA company product), the main technical indicators are density at 1.076-1.090, pH value 7.2-7.3, use 0.22 Fill in separate bottles after μm filter sterilization, or autoclave at 121°C after bottled.

[0038] The above two reagents have passed the sterility test, and the endotoxin content test is ≤0.5EU / ml. One bottle of reagent 1 and reagent 2 is put into the kit, and the qualified products leave the factory.

[0039] How to use the kit:

[0040] Take one of the above-mentioned qualified factory kit...

Embodiment 2

[0047] Kit composition:

[0048] 1. Reagent 1 prepares an aqueous solution of 1.0-6.0% hydroxyethyl starch and 0.45% potassium chloride, adjusts the pH value to 7.4±0.1 with 10% sodium hydroxide and 1% dilute hydrochloric acid, and uses 0.22 μm Fill in separate bottles after filter sterilization, or autoclave at 121°C after bottled.

[0049] 2. Reagent 2 prepares an aqueous solution with a concentration of 10% sodium diatrizoate and 3% dextran. The main technical indicators are that the density is 1.076-1.090 and the pH value is 7.2-7.4. It is filtered and sterilized with 0.22 μm and then bottled or bottled. Then autoclave at 121°C.

[0050] The above two reagents have passed the sterility test, and the endotoxin content test is ≤0.5EU / ml. One bottle of reagent 1 and reagent 2 is put into the kit, and the qualified products leave the factory.

[0051] The usage method and detection method of the kit are the same as in Example 1.

Embodiment 3

[0053] Kit composition:

[0054] 1. Reagent 1 prepares an aqueous solution of 1.0-6.0% hydroxyethyl starch and 0.20% sodium chloride, adjusts the pH value to 7.4±0.1 with 10% sodium hydroxide and 1% dilute hydrochloric acid, and uses 0.22 μm Fill in separate bottles after filter sterilization, or autoclave at 121°C after bottled.

[0055] 2. Reagent 2 prepares an aqueous solution with a concentration of 6-8% Percoll (Percoll silica polyvinylpyrrolidone colloid). bottle filling, or autoclave at 121°C after bottle filling.

[0056] The above two reagents have passed the sterility test, and the endotoxin content test is ≤0.5EU / ml. One bottle of reagent 1 and reagent 2 is put into the kit, and the qualified products leave the factory.

[0057] The usage method and detection method of the kit are the same as in Example 1.

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Abstract

An optimized karyote in-vitro separation kit comprises two reagents. The reagent 1 is composed of erythrocyte precipitant, sodium chloride or potassium chloride. The reagent 2 is composed of natrii diatrizoas and polysucrose No.400, natrii diatrizoas and polysucrose dextranum, or percoll, and has density of 1.076-1.090. An application method of the optimized karyote in-vitro separation kit includes directly adding 100ml of human marrow blood, umbilical cord blood or peripheral blood into 200ml of solution of the reagent 1, allowing for standing for 10-30 minutes, collecting supernate, centrifuging at 2000rpm (1100xg) for 5 minutes, discarding the supernate, adding remnant a centrifugal tube containing solution of the reagent 2, performing density gradient centrifugation for 30 minutes, controlling the rotation speed at 1500rpm, absorbing a karyote layer, performing centrifugal cleaning for once to three times, using 50ml of cell preservative solution for diluting, and taking 100 microliters for testing or self-testing.

Description

technical field [0001] The invention relates to the technical field of biomedicine, and belongs to a kit technology for separating and extracting nucleated cells from human peripheral blood, umbilical cord blood and bone marrow blood, in particular to an optimized in vitro separation kit for nucleated cells and a method of use. Background technique [0002] Human peripheral blood, umbilical cord blood, and bone marrow blood all belong to the blood tissues of the human body. Their common feature is that they contain red blood cells, white blood cells, platelets, and plasma substances; the shape, diameter, and quality of the same type of cells are the same. In addition to lymphocytes and granulocytes, white blood cells also contain adult stem cells such as hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, multipotential adult progenitor cells, and a small amount of adipose stem cells. Stem cells of each line can be divided into more than 100 types because...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078C12N5/0786C12N5/0789
CPCC12P7/04
Inventor 王怀林
Owner NINGXIA ZHONGLIANDA BIOPHYSICS
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