Method for separating total nucleated cells from mononuclear cells from umbilical cord blood

A technology of nucleated cells and umbilical cord blood, applied in the medical field, can solve the problems of low viability of nucleated cells, and achieve the effects of low reagent cost, low permeability damage, and high separation purity

Active Publication Date: 2019-10-18
SHANDONG QILU STEM CELL ENG
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the low viability of nucleated cells obtained from frozen cord blood, the present invention provides a method for efficiently recovering total nucleated cells and mononuclear cells from frozen cord blood

Method used

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  • Method for separating total nucleated cells from mononuclear cells from umbilical cord blood
  • Method for separating total nucleated cells from mononuclear cells from umbilical cord blood
  • Method for separating total nucleated cells from mononuclear cells from umbilical cord blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Isolation of total nucleated cells in cord blood

[0028] The cryopreserved cord blood was taken from the Cord Blood Hematopoietic Stem Cell Bank of Shandong Province.

[0029] The centrifuge adopts Thermo Fisher Scientific Sorvall ST 16R high-speed refrigerated centrifuge.

[0030] Composition of PBS buffer:

[0031] The formulation of the PBS buffer solution is 137mM sodium chloride, 2.7mM potassium chloride, 2mM potassium dihydrogen phosphate and 10mM disodium hydrogen phosphate, and the pH is about 7.3-7.5.

[0032] (1) Cord blood resuscitation: quickly take out a frozen cord blood from the liquid nitrogen tank, place it in a 37°C water bath, and shake the cryopreservation bag while incubating for rapid recovery;

[0033] (2) Separate TNCs: Transfer 30mL of recovered cord blood into a 250mL centrifuge tube, add 4℃ pre-cooled PBS buffer containing 5% dextran and 2% human serum albumin to 150mL, mix well , Centrifuge at 1500 rpm for 10 min at 4°C, increase speed 9, de...

Embodiment 2

[0037] Example 2 Yield and viability of TNCs

[0038] 1. Recovery rate

[0039] The TNCs precipitate obtained in Example 1 and Comparative Example 1 was resuspended again in 100 mL of PBS buffer containing 5% dextran and 2% human serum albumin, and 20 μL was drawn for cell counting. The total number of cells was calculated with a hemocytometer, and The recovery rate of TNCs is calculated as follows:

[0040] Recovery rate of TNCs (%) = number of TNC precipitated cells / number of cord blood TNC before cryopreservation

[0041] The result is figure 1 Shown: the recovery rate of TNCs in Comparative Example 1 was 75.34%±10.41%; significantly lower than the recovery rate of TNCs in Example 1 of 96.38%±2.40% (t-test).

[0042] 2. Recovery rate

[0043] The cell viability of the TNCs recovered in Example 1 was measured by flow cytometry, and the TNCs cell suspension was diluted to 1×10 with PBS buffer containing 5% dextran and 2% human serum albumin 6 / mL, take 4 flow-style loading tubes, add 2...

Embodiment 3

[0044] Example 3 Isolation of mononuclear cells in cord blood

[0045] After resuspending the TNCs precipitate in Example 1 with 100 mL of PBS buffer containing 5% dextran and 2% human serum albumin, 50 mL of the cell suspension was taken and slowly added to an equal volume of Ficoll lymphocyte separation solution. The density gradient was centrifuged. Centrifuge at 2000rpm / min for 30min, speed up 1, speed down 1. Collect the albuginea layer of mononuclear cells, centrifuge at 1000rpm / min for 5 minutes at 4℃, speed up 9, speed down 7, centrifuge, discard the supernatant, and obtain MNCs precipitation.

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Abstract

The invention provides a method for separating total nucleated cells from mononuclear cells from umbilical cord blood. The method comprises the steps that firstly, TNCs is separated, wherein a PBS buffer solution containing 5% dextran and 2% human serum albumin is added into the umbilical cord blood, centrifugation is conducted to discard supernate, and TNCs precipitate I is obtained; after precipitating and washing, TNCs precipitate is obtained; the TNCs precipitate is subjected to density gradient centrifugation by using a Ficoll lymphocyte separation liquid, a mononuclear cell albuginea layer is collected, and mononuclear cell precipitate is obtained after centrifugation. The method is efficient, convenient to use, capable of saving time and high in repeatability, rich umbilical cord blood resources in an umbilical blood bank can be utilized, and the separation efficiency of derived stem cells and immune cells in the cryopreserved umbilical cord blood is improved.

Description

Technical field [0001] The invention belongs to the medical field, relates to a method for obtaining nucleated cells, and relates to a method for separating total nucleated cells and mononuclear cells from umbilical cord blood. Background technique [0002] Umbilical cord blood (UCB) has become an important source of stem cells in addition to bone marrow. According to incomplete statistics, a number of standardized and standardized cord blood banks have been established worldwide, with hundreds of thousands of frozen The stored cord blood is increasing rapidly at a rate of nearly a thousand cases per day, and it is intended to be used for clinical treatment in the future. Cord blood is rich in a variety of stem / progenitor cells and a variety of immune cells, including hematopoietic stem cells and endothelial progenitor cells, which are useful for the treatment of a variety of malignant hematological tumors, acquired or inherited bone marrow failure syndromes, and non-malignant di...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 付亚茹孙阳阳刘小盾曲廷瑜
Owner SHANDONG QILU STEM CELL ENG
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