Method for efficiently separating and culturing mesenchymal stem cells of primarily-cultured rabbit

A bone marrow mesenchymal, separation and culture technology, applied in the field of efficient separation and culture of primary rabbit bone marrow mesenchymal stem cells, to achieve the effect of simple and effective separation and culture, high efficiency separation and culture

Inactive Publication Date: 2011-07-13
TONGJI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

As we all know, the density and vitality of stem cells in the bone marrow have a great relationship with age. The younger the animal, the greater the density and vitali

Method used

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  • Method for efficiently separating and culturing mesenchymal stem cells of primarily-cultured rabbit
  • Method for efficiently separating and culturing mesenchymal stem cells of primarily-cultured rabbit

Examples

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Embodiment 1

[0017] Example 1: A 1-month-old New Zealand white rabbit was killed by injecting air into the ear vein. Soak and disinfect in 75% alcohol for 15-20 minutes, aseptically remove the bones of the limbs on the ultra-clean workbench, try to remove the soft tissues such as muscles and fascia attached to the bones, wash them in PBS and DMEM culture medium containers respectively, and cut the bones At both ends, the bone marrow cavity was flushed with DMEM solution containing an appropriate amount of heparin (625U / ml) to obtain about 30 ml of bone marrow fluid. Single-cell suspensions were prepared by filtering with syringe needles of different calibers, and the bone marrow fluid was divided into 6 tubes, injected into centrifuge tubes containing an equal volume of Percoll (1.073g / L) separation solution, centrifuged at 2500r / min for 20min, and collected For the cloudy cell layer in the middle, wash the cells with LG-DMEM solution, centrifuge at 1000r / min for 5min, and repeat twice to ...

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Abstract

The invention relates to a method for efficiently separating and culturing mesenchymal stem cells of a primarily-cultured rabbit, which comprises the specific steps of: executing an experimental animal by pumping air embolism and then soaking for 15-20min with alcohol for sterilizing, placing in a clean bench for separating limb bones, shearing two ends of the bones after repeatedly washing with a PBS (Phosphate Buffer) and DMEM (Dulbecco's Modified Eagle Medium) culture solution, and washing marrow cavities with the DMEM solution containing heparin with concentration of 625U/ml; respectively filtering marrow liquids obtained from the step 1 by using syringes with different diameters, preparing a monoplast suspension and then slowly injecting into an eccentric pipe containing isovolumetric Percol 1 for centrifuging for 20min, collecting an intermediate cloud cell layer, washing cells with an LG-DMEM solution, centrifuging for 5min, repeatedly washing for 2-3 times, adding the washed cells to the LG-DMEM solution containing a proper proportion of double antibodies (1%) and comprising 10% fetal calf serum, blowing and beating to form a cell suspension, counting and regulating the cell density to 1*10<8>/L, inoculating in a 25cm<2> plastic culture bottle, and placing in a culturing box with conditions of a temperature of 37 DEG C and a humidity of 5 percent for culturing. The invention can be effectively, conveniently and efficiently separating and culturing the mesenchymal stem cells of the primarily-cultured rabbit and is prepared for further basic experiments and clinical research.

Description

technical field [0001] The invention relates to a method for separating and culturing primary cells, in particular to a method for efficiently separating and culturing primary rabbit bone marrow mesenchymal stem cells. Background technique [0002] As we all know, embryonic stem cells (ES) and mesenchymal stem cells (MSCs) have been the most commonly used seed cells for tissue engineering. However, due to the controversy of medical ethics in the isolation and acquisition of ES, the research has been limited to a certain extent. MSCs are a type of stem cells with multi-directional differentiation potential, which can be derived from various tissues such as bone marrow, fat, placenta, umbilical cord blood, umbilical vein subendothelium, peripheral blood and muscle. Under certain conditions, it can transform into mesenchymal tissue cells such as bone, cartilage, cardiac muscle, fat, and vascular endothelium. It has the advantages of wide source, relatively easy acquisition, s...

Claims

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Application Information

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IPC IPC(8): C12N5/0775
Inventor 徐红珍苏俭生
Owner TONGJI UNIV
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