Method for sorting human marrow very small embryonic-like stem cells

An embryo-like stem cell and bone marrow technology, which can be applied to non-embryonic pluripotent stem cells, artificially induced pluripotent cells, animal cells, etc., can solve the problems of restricting the research and application of stem cells, backwardness, etc. The effect of high extraction rate

Inactive Publication Date: 2013-09-11
杨跃进 +1
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Problems solved by technology

The backward sorting of VSELs seriously restricts the research and application of stem cells

Method used

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  • Method for sorting human marrow very small embryonic-like stem cells

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Embodiment Construction

[0020] The step of this method is to carry out under the condition of constant temperature 4 ℃, and step is as follows:

[0021] (1) 10 mL of human bone marrow anticoagulated with heparin saline, add 30 mL of erythrocyte lysate 3 times its volume, mix well, lyse at 4°C, centrifuge for 10 minutes, discard the supernatant, and add to the precipitate Red blood cell lysate 20mL, which accounts for 2 times its volume, is mixed well, lysed at 4°C, centrifuged for 10 minutes, and then the supernatant is discarded to obtain a secondary precipitate, which is washed with EDTA buffer at 4°C to obtain cells suspension;

[0022] (2) Filter the cell suspension with a 30μM mesh, centrifuge the filtrate at 4°C for 5 minutes, discard the supernatant, add EDTA buffer to the precipitate to 300μL-310μL to obtain a resuspended cell solution ;

[0023] (3) For negative selection, take 300 μL of resuspended cells, add 100 μL of Miltenyi 130-092-211 solution, mix well, place at 4°C for 10 minutes, ...

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Abstract

The invention belongs to a method for sorting human marrow very small embryonic-like stem cells, which comprises the following steps: under a constant temperature of 4 DEGC, adding 30 milliliters of erythrocyte lysing solution into 10 milliliters of human marrow which is subjected to anticoagulation treatment by heparin saline, uniformly mixing, performing centrifugal separation for 10 minutes, removing supernate, adding 20 milliliters of erythrocyte lysing solution into 10 milliliters of sediment, uniformly mixing, performing centrifugal separation for 10 minutes, removing supernate, obtaining secondary sediment, washing with ethylene diamine tetraacetic acid (EDTA) buffer solution under a condition of 4 DEG C, and obtaining cell suspension; and filtering the cell suspension with 30-micrometer meshes, subjecting the filtrate to centrifugal separation at 4 DEG C for 5 minutes, removing supernate, adding the sediment into the EDTA buffer solution till the volume is 300 to 310 microliter, and obtaining re-suspended cell solution; and performing negative sorting and positive sorting. The method has the advantages of protecting the integrity of very small embryonic-like stem cells, simplifying sorting flow, reducing sorting time, achieving high recovery rate, along with simplicity, practicality and low extraction cost.

Description

technical field [0001] The invention belongs to a method for sorting human bone marrow minimal embryonic stem cells. Background technique [0002] Stem cell research has brought hope for the regeneration of human organs and the treatment of organ failure. "Very small embryonic-like stem cells" (VSELs) have been found in the bone marrow and umbilical cord blood of mice and humans in recent years. Discovered adult stem cells that have not been identified in other species. The diameter of VSELs is small (<10μM); the markers are Sca-1(+) / Lin(-) / CD45(-) / CXCR4(+) in mice and Oct-4(+)CXCR4(+)CD133( +)Lin(-)CD45(-); VSELs express pluripotency marker proteins of embryonic stem cells: Oct4, Nanog, SSEA, etc.; typical embryonic stem cell morphological characteristics can be seen under electron microscope; typical embryoid bodies can be seen in VSELs culture, which can promote differentiation properly Under these conditions, it can differentiate into cells derived from the three ge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/074
Inventor 杨跃进王红
Owner 杨跃进
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