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Volume centrifugal separation method of umbilical cord blood stem cells

A cord blood stem cell and centrifugation technology, applied in blood/immune system cells, animal cells, vertebrate cells, etc., can solve the problems of low purity and complex cell population components, and achieve good repeatability, good cell status, high vitality effect

Inactive Publication Date: 2015-07-08
HUBEI SHENGMINGYUAN STEM CELL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Density gradient centrifugation mostly uses Percoll and Ficoll separation medium to separate cells to obtain mononuclear cell populations, which have complex components and low purity[8-9]

Method used

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  • Volume centrifugal separation method of umbilical cord blood stem cells

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Volume Centrifugation of Cord Blood Stem Cells

[0018] Take 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, mix well, add umbilical cord blood, 1.5 times the volume of medical 6% middle molecule Hydroxyethyl starch 130 / 0.4 sodium chloride injection, mix well, cover, 18°C, let stand for 60min.

[0019] Absorb the supernatant after the erythrocytes were precipitated with hydroxyethyl starch, slowly add it to the liquid surface of the separation liquid, put it on a horizontal centrifuge, 4°C, centrifugal force 700g, centrifuge for 30min, after centrifugation, use a pipette to reach the mononuclear cell layer, gently Gently aspirate, add to PBS solution, wash cells 3 times at 4°C, and then concentrate.

[0020] A layer of Pt wire filter is arranged on the inner surface of the horizontal centrifuge, and the pore size of the Pt wire filter is 20 ...

Embodiment 2

[0022] Volume Centrifugation of Cord Blood Stem Cells

[0023] Take sodium citrate anticoagulated umbilical cord blood 80, put it into a 500ml round sedimentation bottle aseptically, add 3 times the volume of umbilical cord blood PBS, mix well, add umbilical cord blood, 1.5 times the volume of medical 6% middle molecule Hydroxyethyl starch 130 / 0.4 sodium chloride injection, mix well, cover, 18°C, let stand for 60min.

[0024] Add 6% hydroxyethyl starch into the collected blood bag according to 1 / 5 of the cord blood volume; after mixing evenly, use sterile sealing glue to seal the puncture point, and stand it upside down and in a low temperature environment of 12°C for 8 Hours; after the separation, release the red blood cells in the lower layer of cord blood slowly, take a 50ml centrifuge tube and put it in another 50ml centrifuge tube; put the centrifuge tube containing red blood cells at room temperature, 600r / min Centrifuge for 8 minutes, suck out the upper layer of plasma...

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Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a volume centrifugal separation method of umbilical cord blood stem cells. The volume centrifugal separation method comprises the following steps of: (1), precipitating erythrocyte by utilizing hydroxyethyl starch; and (2), carrying out volume centrifugal separation of a mononuclear cell by using a centrifugal machine with a built-in Pt mesh filter net. By means of separation liquid and a separation process, mesenchymal stem cells having the maximum ratio in umbilical cord blood can be obtained by separation; the obtained cells are good in state and high in activity and can be directly applied to clinical treatment or further cultivation and amplification; the obtained cells also can be used for separating and extracting peripheral blood and mesenchymal stem cells; the method is an optimized process, which is simple and feasible and good in stability and repeatability; and therefore, the method can be widely popularized and applied.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a volume centrifugal separation method of umbilical cord blood stem cells. 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 num...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
Inventor 王锐峰
Owner HUBEI SHENGMINGYUAN STEM CELL CO LTD