Method for isolating cells from bone marrow and inducing generation of endothelial progenitor cells

Abstract

The invention provides a method for separating cells from bone marrow and inducing to generate endothelial progenitor cells, which comprises the following steps of: firstly, killing an SD (Sprague Dawley) rat by using a cervical vertebra dislocation method, and disinfecting the surface of the rat by using 75% ethanol; thirdly, cutting open the lower limbs of the rat to take out the femur and the tibia, and cutting off two ends of the bone to expose a marrow cavity; then, preparing an ECM culture medium containing 5% of fetal calf serum, 1% of ECGS and 1% of PS, and flushing the marrow cavity with the ECM culture medium until the bone becomes white; then, collecting the flushing fluid, centrifuging to remove supernate, and filtering by using a 200-mesh cell sieve to remove impurities; then, inoculating the cell precipitate into a six-well plate, and putting the six-well plate into a constant-temperature incubator with the temperature of 37 DEG C and the CO2 content of 5% for culturing; after 72 hours, changing the liquid to remove non-adherent cells, and then changing the liquid once every two days until the cells need passage; and finally, when the fusion degree of the cells reaches about 80%, digesting the cells by using 0.25% trypsin, and inoculating the cells into a T25 culture bottle for further culture. The method is simple to operate, and the cell purity and the differentiation efficiency of the endothelial progenitor cells are effectively improved.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, in particular to a method for separating cells from bone marrow and inducing the generation of endothelial progenitor cells. Background Art

[0002] Endothelial progenitor cells are precursor cells of vascular endothelial cells, also known as angioblasts. They are a group of cells that have the potential to differentiate into mature endothelial cells and participate in the formation of new blood vessels. Studies have shown that cardiovascular function and angiogenesis are significantly regulated by endothelial progenitor cells originating from the bone marrow, which increase angiogenesis, improve vascular repair and improve endothelial function. Therefore, endothelial progenitor cells have broad application prospects in ischemic diseases such as myocardial infarction, atherosclerosis, and diabetic peripheral vascular disease.

[0003] Bone marrow is the body's hematopoietic tissue, located in many bones of the body. There are two types of bone marrow in adult animals: red bone marrow and yellow bone marrow. Red bone marrow can produce red blood cells, platelets and various white blood cells. Platelets have a hemostatic effect, white blood cells can kill and inhibit various pathogens, including bacteria, viruses, etc.; some lymphocytes can produce antibodies. Therefore, bone marrow is not only a hematopoietic organ, it is also an important immune organ. Bone marrow is a spongy tissue that exists in the medullary cavity of long bones (such as humerus and femur) and the mesh between the loose bone of flat bones (such as ilium). The bone marrow that can produce blood cells is slightly red and is called red bone marrow. At birth, red bone marrow fills the bone marrow cavity of the whole body. As age increases, fat cells increase, and a considerable part of the red bone marrow is replaced by yellow bone marrow. In the end, there is almost only red bone marrow in the medullary cavity of flat bones.

[0004] Studies have found that endothelial progenitor cells are mainly present in bone marrow, umbilical cord blood and peripheral blood, but a small amount of endothelial progenitor cells have also been found in adipose tissue, spleen, heart, blood vessels, and skeletal muscle. The endothelial progenitor cells contained in peripheral blood are extremely low, and it is difficult to obtain umbilical cord blood, so bone marrow is used to separate endothelial progenitor cells. There are five main methods for separating bone marrow cells, among which density gradient centrifugation, adhesion screening method, and immunomagnetic bead method are more commonly used. Although the immunomagnetic bead method obtains a higher purity of cells, the operation is complicated and the cost is expensive, so it is rarely used at present. Although the density gradient centrifugation method is very economical, its cell specificity of sorting is limited, and the density gradient preparation requirements are relatively high. The direct adhesion screening method is a simple and easy method that uses the difference in cell adhesion and adhesion firmness to gradually remove non-adherent cells and impurity cells. This method does not require density gradient preparation, and the method is fast, simple, low-cost and reproducible. Summary of the invention

[0005] The object of the present invention is to provide a method for isolating cells from bone marrow and inducing the generation of endothelial progenitor cells, so as to solve the problems raised by the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for isolating cells from bone marrow and inducing the generation of endothelial progenitor cells, comprising the following steps:

[0007] Step 1: SD rats were killed by cervical dislocation and soaked in 75% ethanol;

[0008] Step 2: Then cut open the rat's leg and take out the femur and tibia of the rat's lower limbs;

[0009] Step 3: Use ophthalmic scissors to cut off both ends of the femur and tibia to expose the medullary cavity;

[0010] Step 4: Prepare complete endothelial progenitor cell culture medium, using ECM culture medium containing 5% fetal bovine serum, 1% ECGS, and 1% PS;

[0011] Step 5: Use a syringe to repeatedly flush the bone marrow cavity with complete culture medium until the bone turns white;

[0012] Step 6: Collect bone marrow fluid and remove supernatant by centrifugation;

[0013] Step 7: Add appropriate amount of culture medium to inoculate in a six-well plate and place in a constant temperature incubator at 37°C and 5% CO2 for culture;

[0014] Step 8: After 72 hours, change the medium to remove non-adherent cells, and then change the medium every 2 days until subculture is required;

[0015] Step 9: Cell subculture: after the cell confluence reaches about 80%, the cells are collected by digestion using 0.25% mass percent trypsin and inoculated into a T25 culture flask for culture.

[0016] As a preferred technical solution of the present invention, in step 2, the removed bilateral femurs and tibiae are immersed in sterile 1×PBS containing dual antibodies, and washed three times with 1×PBS containing dual antibodies.

[0017] As a preferred technical solution of the present invention, in step six, the collected bone marrow fluid is filtered through a 200-mesh cell sieve to remove a large amount of impurities.

[0018] As a preferred technical solution of the present invention, in step eight, the medium is replaced after 72 hours to remove non-adherent cells because the cells in the bone marrow have strong differentiation ability. The cells that can induce the generation of endothelial progenitor cells are the cells that adhere to the bone marrow after 72 hours of culture after separation.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the method and culture of endothelial progenitor cells obtained by the direct adhesion screening induction method adopted by the present invention are simple, low-cost, good cell activity, high purity, and high concentration of final harvested cells. The endothelial progenitor cells can be quickly and efficiently separated and induced. After about 48 hours of culture, the cells can be seen to adhere to the wall and grow with obvious cell morphology, and proliferate rapidly. The acquisition rate of primary cells is significantly improved, the final harvested cell concentration is high, and the entire endothelial progenitor cell culture process can be standardized, programmed, and normalized. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attached Figure 1 These are pictures of the femur and tibia of the bilateral lower limbs of rats;

[0021] Attached Figure 2 This is the picture after collecting bone marrow fluid by centrifugation;

[0022] Attached Figure 3 0 h after plating, observed under a microscope at 100x magnification;

[0023] Attached Figure 4 Observation under a microscope at 100x magnification 48 h after plating;

[0024] Attached Figure 5 Observation under a microscope at 100x magnification 72 h after plating;

[0025] Attached Figure 6 Observation under a microscope at 100x magnification 4 days after plating;

[0026] Attached Figure 7 Observation under a microscope at 100x magnification 6 days after plating;

[0027] Attached Figure 8 This is a picture of the P1 cell morphology, observed under a microscope at 40 times magnification. DETAILED DESCRIPTION

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1: Isolation of cells from bone marrow and induction of endothelial progenitor cells

[0030] Experimental materials and reagents

[0031] Experimental animals: SD rats, weighing about 200-250 g.

[0032] Culture medium: ECM culture medium (containing 5% fetal bovine serum, 1% ECGS, 1% PS).

[0033] Other reagents: 75% ethanol, 1× PBS (containing double antibody), trypsin.

[0034] Experimental procedures

[0035] Experimental animals were killed: SD rats were killed by cervical dislocation, and the surface of the rats was immediately disinfected by soaking in 75% ethanol.

[0036] Obtaining bone marrow: Use sterile scissors to cut open the lower limbs of the rat and expose the femur and tibia.

[0037] Use ophthalmic scissors to cut off the ends of the femur and tibia to expose the medullary cavity.

[0038] Preparation of complete endothelial progenitor cell culture medium

[0039] Prepare ECM culture medium containing 5% fetal bovine serum, 1% ECGS, and 1% PS.

[0040] Flush the bone marrow cavity: Use a syringe loaded with complete culture medium to repeatedly flush the bone marrow cavity until the bone turns white.

[0041] Collect bone marrow fluid: collect the flushing fluid, centrifuge to remove the supernatant, and leave the precipitate.

[0042] Filter the bone marrow fluid: Use a 200-mesh cell sieve to filter the bone marrow fluid to remove impurities.

[0043] Cultivate cells: Add appropriate amount of culture medium to the collected cell pellet and inoculate into a six-well plate.

[0044] The six-well plate was placed in a constant temperature incubator at 37°C and 5% CO2 for culture.

[0045] Initial medium change: After 72 hours, change the medium to remove non-adherent cells.

[0046] The medium was changed every 2 days until the cells needed to be passaged.

[0047] Cell subculture: When the cell confluence reaches about 80%, use 0.25% trypsin to digest the cells.

[0048] The digested cells were inoculated into T25 culture flasks for further culture.

[0049] Example 2: Optimization steps

[0050] Bone Marrow Processing Optimization

[0051] In step 2, the removed femur and tibia were immersed in sterile 1× PBS containing double antibodies, and washed three times with 1× PBS containing double antibodies to ensure a sterile environment.

[0052] Cell filtration optimization

[0053] In step six, the bone marrow fluid is filtered using a 200-mesh cell sieve, which can effectively remove a large amount of impurities and increase cell purity.

[0054] Cell culture optimization

[0055] In step eight, the medium is changed after 72 hours to remove non-adherent cells. This is because cells in the bone marrow have strong differentiation ability and adherent cells are more conducive to inducing the generation of endothelial progenitor cells.

[0056] Advantages and effects

[0057] Compared with the prior art, the present invention has the following advantages:

[0058] Simple operation: The method and steps are clear and easy to operate.

[0059] High cell purity: Double antibody treatment and cell filtration can effectively improve cell purity.

[0060] Efficient differentiation: The medium is changed every 72 hours to remove non-adherent cells, which improves the differentiation efficiency of endothelial progenitor cells.

[0061] The above embodiments are only used to illustrate the method of the present invention, and are not intended to limit the present invention. After reading the specification, those skilled in the art may make various modifications and changes to the present invention, and these equivalent changes and improvements should all be within the scope of protection of the present invention.

[0062] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in the field. Although the present invention has been described in detail with reference to the aforementioned embodiments, it is still possible for those skilled in the art to modify the technical solutions described in the aforementioned embodiments, or to make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for isolating cells from bone marrow and inducing the generation of endothelial progenitor cells, characterized in that: The following steps are involved: Step 1: SD rats were killed by cervical dislocation and soaked in 75% ethanol; Step 2: Then cut open the rat's leg and take out the femur and tibia of the rat's lower limbs; Step 3: Use ophthalmic scissors to cut off both ends of the femur and tibia to expose the medullary cavity; Step 4: Prepare complete endothelial progenitor cell culture medium, using ECM culture medium containing 5% fetal bovine serum, 1% ECGS, and 1% PS; Step 5: Use a syringe to repeatedly flush the bone marrow cavity with complete culture medium until the bone turns white; Step 6: Collect bone marrow fluid and remove supernatant by centrifugation; Step 7: Add appropriate amount of culture medium to inoculate in a six-well plate and place in a constant temperature incubator at 37°C and 5% CO2 for culture; Step 8: After 72 hours, change the medium to remove non-adherent cells, and then change the medium every 2 days until subculture is required; Step 9: Cell subculture: after the cell confluence reaches about 80%, the cells are collected by digestion using 0.25% mass percent trypsin and inoculated into a T25 culture flask for culture.

2. The method for isolating bone marrow cells and inducing the generation of endothelial progenitor cells as claimed in claim 1, characterized in that: In step 2, the removed bilateral femurs and tibiae were immersed in sterile 1×PBS containing double antibodies, and then washed three times with 1×PBS containing double antibodies.

3. The method for isolating bone marrow cells and inducing the generation of endothelial progenitor cells as claimed in claim 1, characterized in that: In step six, the collected bone marrow fluid is filtered through a 200-mesh cell sieve to remove a large amount of impurities.

4. The method for isolating bone marrow cells and inducing the generation of endothelial progenitor cells as claimed in claim 1, characterized in that: In step eight, the medium is changed after 72 hours to remove non-adherent cells because the cells in the bone marrow have a strong differentiation ability. The cells that can induce the generation of endothelial progenitor cells are the cells that adhere to the wall after 72 hours of culture after the bone marrow is isolated.

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