Method for simultaneous isolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells

A technology of hematopoietic stem cells and bone marrow mesenchyme, which is applied in the direction of bone/connective tissue cells, animal cells, vertebrate cells, etc., can solve the problems of cumbersome operation steps, large cell damage, and many human factors, so as to overcome the screening steps, The effect of uniform cell shape and abundant material sources

Active Publication Date: 2014-11-19
YUNNAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

[0003] Traditional methods for isolating BMMSCs and HSCs mainly include density gradient centrifugation, flow cytometry separation, magnetic bead sorting, etc., but are limited by the cumbersome operation steps, long time, great damage to cells, and many human factors. , and the in vitro culture of HSCs needs to add various growth factors and induction factors

Method used

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Examples

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Embodiment 1

[0013] Example 1, step 1, in vitro separation of BMMSCs and HSCs: routinely anesthetized experimental dogs, skin preparation, disinfection, and draping of the posterior superior iliac spine, and a bone marrow puncture needle containing 1 ml of heparin sodium was used to extract a total of about 10 ml of bone marrow at 2 to 3 points , mix well to prevent coagulation, quickly transfer to a 50ml sterilized centrifuge tube, dilute and mix with an equal amount of DMEM / F12; take 10 centrifuge tubes with 5ml lymphocyte separation solution (1.077 g / ml) added to the bottom, and add carefully to the upper layer (paste Wall inflow) equal volume of bone marrow diluent, seal. Centrifuge at 2000 r / min at 4°C for 20 minutes to remove the upper fat cells, and carefully absorb the buffy coat cells at the middle junction. Mix and wash the interstitial cells with 10ml DMEM / F12 at 4°C, centrifuge at 2000r / min for 20min, remove the supernatant, and repeat the washing once; 5ml of DMEM / F12 medium ...

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Abstract

The invention relates to a canine stem cell isolated culture technology, and especially relates to a method for in vitro simultaneous insolated culture of canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells. The method is characterized in that adherent culture of primary mesenchymal stem cells is carried out based on the separation of bone marrow mononuclear cells, half medium change is adopted, and a mononuclear cell culture suspension is added, that is, hematopoiesis microenvironment provided by the adherent-growth mesenchymal stem cells is used to culture the suspending-growth multifunctional hematopoietic stem cells, so the achieve the simultaneous isolated culture of the canine bone marrow mesenchymal stem cells and the multifunctional hematopoietic stem cells is realized.

Description

technical field [0001] The invention relates to a technique for isolating and culturing canine stem cells, in particular to a technique for simultaneously isolating and culturing canine bone marrow mesenchymal stem cells and multifunctional hematopoietic stem cells in vitro. Background technique [0002] Bone marrow mesenchymal stem cells (BMMSCs) are non-hematopoietic stem cells that exist in the bone marrow. They have convenient sources, no immune rejection, multilineage differentiation and high proliferation potential. The field of repair and disease treatment presents an extremely attractive prospect; multifunctional hematopoietic stem cells (HSCs) are a type of primitive cells in hematopoietic tissues such as bone marrow, with self-renewal and directed differentiation (that is, differentiation into various mature blood cells) Biological functions such as blood circulation and homing (that is, enter the medullary sinus through the peripheral blood circulation after intra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/0789
Inventor 严玉霖汪登如曹景锋陈玲严梓丹
Owner YUNNAN AGRICULTURAL UNIVERSITY
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