Umbilical cord blood mesenchymal stem cell separation liquid and separation flow

A technology of mesenchymal stem cells and separation fluid, applied in the field of improved umbilical cord blood mesenchymal stem cell separation fluid and optimized separation process, can solve the problems of no objective and scientific standards, and achieve good cell state, high vitality, and good stability Effect

Inactive Publication Date: 2012-07-25
刘爱兵 +1
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Various experimental conditions in the process of stem cell separation, such as: centrifugal force, centrifugation time, ratio of separation liquid to

Method used

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  • Umbilical cord blood mesenchymal stem cell separation liquid and separation flow
  • Umbilical cord blood mesenchymal stem cell separation liquid and separation flow
  • Umbilical cord blood mesenchymal stem cell separation liquid and separation flow

Examples

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Effect test

Embodiment 1

[0078] Embodiment 1: Separation liquid of human umbilical cord blood mesenchymal stem cells (density 1.073±0.001g / L). The preparation method is as follows: pharmaceutical grade polysucrose dry powder, molecular weight 400,000, is prepared into 40±1% (w / v) aqueous solution with sterilized non-pyrogenic distilled water, and set aside. For application, prepare a 9% solution with distilled water. 60% Meglumine Diatrizoate Solution (Medical Contrast Media). When applying, take 20ml of 60% meglumine diatrizoate stock solution, add 15.38ml of double distilled water to get 33.9% meglumine diatrizoate. At room temperature (18-20°C), take 26.88 parts of 9% polysucrose and 10 parts of 33.9% meglumine diatrizoate and mix well, measure the specific gravity of the mixture with a Baume specific gravity meter or adjust the volumes of the two properly to obtain 1.073± 0.001g / L umbilical cord blood mesenchymal stem cell separation fluid. Divide into 100ml / bottle, sterilize by filtration, and...

Embodiment 2

[0079] Example 2: Two-step method for isolating umbilical cord blood mesenchymal stem cells. The first step: erythrocytes were precipitated with hydroxyethyl starch. Take 80-100ml of sodium citrate anticoagulated umbilical cord blood, put it into a 500ml round sedimentation bottle aseptically, add PBS with 2 times the volume of umbilical cord blood, and mix well. Add medical 6% medium molecule hydroxyethyl starch 130 / 0.4 sodium chloride injection twice the volume of umbilical cord blood, mix well, cover (do not cover tightly), and let stand at 20°C for 60 minutes.

[0080] The second step: the mesenchymal stem cells were separated by density gradient centrifugation using a separation medium with a density of 1.073 g / L. The supernatant after the erythrocytes were precipitated with hydroxyethyl starch was sucked, and slowly added to the liquid surface of the separation liquid (supernatant: separation liquid: 1:1). Put it on a horizontal centrifuge at 4°C, centrifugal force 700...

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Abstract

The invention discloses umbilical cord blood mesenchymal stem cell separation liquid and an umbilical cord blood mesenchymal stem cell separation flow. The separation liquid is prepared by mixing saccharosan of which the concentration is 9 percent and meglumine diatrizoate of which the concentration is 33.9 percent in the ratio of 26.88:10, wherein the concentration of the separation liquid is 1.073+/-0.01g/L. A two-step method for separating umbilical cord blood mesenchymal stem cells comprises the following steps of: 1, precipitating red cells by a hydroxyethyl starch; and 2, separating the mesenchymal stem cells by the separation liquid of which the concentration is1.073g/L. The mesenchymal stem cell with the highest proportion in umbilical cord blood can be obtained by the separation liquid and the separation flow, the proportions of lymphocytes and mononuclear cells are very low, and the obtained cells are good in state and high in activity. The used materials and reagents are medical products, are non-toxic, do not have heat sources, can be industrially produced and are convenient to store. The used method is an optimized flow, is high in stability and repeatability, can be also used for separating human marrow and peripheral blood mesenchymal stem cells, is the best method for separating and extracting the mesenchymal stem cells in the current density gradient centrifugation and is better than other products and methods.

Description

technical field [0001] The invention relates to an improved umbilical cord blood mesenchymal stem cell separation liquid and an optimized separation process. Background technique [0002] Domestic and foreign methods for isolating umbilical cord blood stem cells often use magnetic beads / flow cytometry, umbilical cord blood bank instruments / hydroxyethyl starch precipitation and density gradient centrifugation. Due to different purposes, each method has its own characteristics. The magnetic bead method or flow cytometry method uses the corresponding monoclonal antibody to separate and obtain a relatively pure cell population, but the cell harvest rate is low, which is suitable for analysis and scientific research applications. Hydroxyethyl starch precipitation and instrumental centrifugation are commonly used to isolate stem cells from cord blood banks. The separated cells are large in volume and have many components but mixed with a large number of red blood cells, which are...

Claims

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Application Information

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IPC IPC(8): C12N5/0775C12N5/0789
Inventor 刘爱兵沈炳谦戴成祥郭继强王东平张蕊张姗姗王黎明沈丹
Owner 刘爱兵
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