CN115992091A - A method for isolating and culturing rat bone marrow mesenchymal stem cells - Google Patents

Abstract

The invention belongs to the field of biotechnology, and discloses a method for isolating and culturing rat bone marrow mesenchymal stem cells, comprising: obtaining bilateral femurs and tibias of rats, removing the muscles on the surface, and repeatedly rinsing them with a cleaning solution; Under sterile conditions, cut off the two ends of the bilateral femur and tibia, and use the cleaning solution to wash the fractures at both ends of the femur and tibia several times; wash the bone marrow into the culture solution, and inoculate the cell suspension directly in the culture bottle for cultivation. After the cells are 80% confluent, trypsinize and subculture; take the second generation of healthy growing rat MSCs to make a cell suspension, transfer it to a centrifuge tube, and centrifuge; discard the supernatant, and adjust the cell concentration with the cleaning solution; add rabbit anti-mouse CD45, CD44, CD90 monoclonal antibodies were incubated at room temperature to obtain rat bone marrow mesenchymal stem cells. The invention provides a method for isolating, cultivating and identifying rat bone marrow mesenchymal stem cells, which has simple operation and high cell yield and survival rate.

Description

A method for isolating and culturing rat bone marrow mesenchymal stem cells

technical field

The invention belongs to the field of biotechnology, in particular to a method for separating and culturing rat bone marrow mesenchymal stem cells.

Background technique

At present, the existing sources of primary mesenchymal stem cells are limited, and experimental research needs to use a large number of 3-5 passage cultured cells. The existing primary mesenchymal stem cell extraction and isolation methods are complicated to operate, and the cell yield and survival rate are low. The Fangming method provides a rich source of materials for the research and application of mesenchymal stem cells. Mesenchymal stem cells (MSCs) widely exist in bone marrow, umbilical cord blood, fat and other tissues, and have strong proliferation ability, multidirectional Differentiation potential, immunoregulatory function, convenient source, no immune rejection, no ethical issues, etc., including pretreatment and expansion of MSCs, conditioned media (CM) of MSCs, paracrine factors, secretome, extracellular MSCs derivatives including exosomes and extracellular vesicles (Extracellular vesicles, EVs) have the characteristics of lower immunogenicity, stable properties, and no risk of tumor formation and thrombosis. Cell therapy may be the best solution to treat various complex diseases. The success of MSCs and their derivatives as innovative therapies for disease will likely depend on a better understanding of their mechanisms of action, as well as the identification of optimal strategies for their use in clinical settings, thus requiring large numbers of primary MSCs for experimental studies .

Based on the above analysis, the existing problems and defects of the prior art are: the existing method for in vitro isolation and culture of rat bone marrow mesenchymal stem cells is complicated to operate, and the cell yield and survival rate are low.

Contents of the invention

Aiming at the problems existing in the prior art, the present invention provides a method for isolating and culturing rat bone marrow mesenchymal stem cells.

The present invention realizes the separation and cultivation of a large number of primary bone marrow mesenchymal stem cells by extracting the mesenchymal stem cells present in the long bone marrow of rats, and provides a method for isolating and culturing rat bone marrow mesenchymal stem cells. The isolation and culture methods of bone marrow mesenchymal stem cells include:

Step 1, obtain the isolated bilateral femur and tibia of the rat, and remove the muscles on the surface of the bilateral femur and tibia, and use the cleaning solution to repeatedly rinse; (by obtaining the long bones of the limbs of the rat, the original The generation of mesenchymal stem cells, removal of the bone surface and cleaning can exclude muscle, vascular endothelial, fat and other cells that interfere with primary cell extraction)

Step 2, under aseptic conditions, cut off the two ends of the bilateral femur and tibia, and use the cleaning solution to rinse the fractures at both ends of the femur and tibia several times; Further eliminate interference and improve the purity of primary bone marrow mesenchymal stem cells)

Step 3, the bone marrow is washed into the culture medium, and the cell suspension is directly inoculated in the culture flask for cultivation, and after the cells are 80% confluent, trypsinizes and subcultures; (the cell suspension is inoculated into the culture flask, and the cell growth rate is faster , allowing rapid amplification.)

Step 4, take the second-generation rat MSCs of healthy growth to make a cell suspension, move it to a centrifuge tube, and centrifuge; discard the supernatant, and adjust the cell concentration with the cleaning solution; (adjust the cell concentration to an appropriate concentration, and the yield and speed increase)

Step five, adding rabbit anti-mouse CD45, CD44, CD90 monoclonal antibodies, incubating at room temperature, to obtain rat bone marrow mesenchymal stem cells. (Because rat bone marrow mesenchymal stem cells are characterized by CD44(-), CD90(-), CD45(+), bone marrow mesenchymal stem cells can be screened by rabbit anti-mouse CD45, CD44, CD90 monoclonal antibodies)

Further, the cleaning solution is PBS solution.

Further, in the step three, the culture medium is: DMEM culture medium.

Further, in the third step, inoculating the cell suspension directly into the culture flask for culturing includes: changing the medium halfway after 24 hours. (The half-change solution can not only supplement the nutrients for cell growth, but also retain exosomes, extracellular vesicles, microRNA and other substances secreted during the growth of mesenchymal stem cells, and the cell growth environment is suitable)

Further, in the third step, the concentration of trypsin is 2.5g/L.

Further, in step 4, the centrifugation includes: centrifugation at 1000 r/min for 5 minutes. (The centrifugation rate is higher than the conventional 800r/min, which increases the amount of cells obtained after centrifugation)

Further, in the step 4, adjusting the cell concentration includes: adjusting the cell concentration to 1×10 ⁶ .

Further, the adding of rabbit anti-mouse CD45, CD44, and CD90 monoclonal antibodies includes: adding 5 μL each of rabbit anti-mouse CD45, CD44, and CD90 monoclonal antibodies.

Further, in the step five, the incubation time at room temperature is 15 minutes.

Further, after the incubation at room temperature, it is necessary to carry out: putting the incubation product into a flow cytometer for identification.

Combining the above-mentioned technical solutions and technical problems to be solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, closely combine the technical solution to be protected in the present invention and the results and data in the research and development process, etc., to analyze in detail how the technical solution of the present invention solves it Technical problems, some creative technical effects brought about after solving the problems. The specific description is as follows:

The invention provides a method for isolating, cultivating and identifying rat bone marrow mesenchymal stem cells, which has simple operation and high cell yield and survival rate.

Second, regarding the technical solution as a whole or from the perspective of a product, the technical effects and advantages of the technical solution to be protected by the present invention are specifically described as follows:

The method for isolating and culturing rat bone marrow mesenchymal stem cells of the present invention is an ideal culture method for rat bone marrow mesenchymal stem cells, which can meet the requirements of various physiological and biochemical experiments.

Third, as an auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects: the expected income and commercial value after the transformation of the technical solution of the present invention are: compared with the existing separation and cultivation methods, the The obtained rat bone marrow MSCs have high purity and strong vitality; and the time required for culturing cells in the present invention is short, and the cells can grow to the rapid expansion stage after the primary cells are cultured for 7-8 days; there is no damage to the isolated cells. It has the requirement of stable quantity and high-quality expansion and culture of rat bone marrow MSCs, and can efficiently meet the requirements of various physiological and biochemical experiments.

Description of drawings

Fig. 1 is a flowchart of the method for isolating and culturing rat bone marrow mesenchymal stem cells provided by the embodiment of the present invention;

Fig. 2 is a schematic diagram of the paracrine function of radiation on bone marrow mesenchymal stem cells provided by the embodiment of the present invention;

Fig. 3 is a schematic diagram of the experimental results provided by the embodiment of the present invention.

Detailed ways

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

1. Explain the embodiment. In order to make those skilled in the art fully understand how to implement the present invention, this part is an explanatory embodiment for explaining the technical solution of the claims.

As shown in Figure 1, the method for isolating and culturing rat bone marrow mesenchymal stem cells provided by the embodiment of the present invention includes:

S101, obtaining the isolated bilateral femur and tibia of the rat, removing the muscles on the surface of the bilateral femur and tibia, and repeatedly washing them with PBS;

S102, under aseptic conditions, cut off the two ends of the bilateral femur and tibia, and use PBS solution to wash the fracture at both ends of the femur and tibia multiple times;

S103, wash the bone marrow into the DMEM culture medium, inoculate the cell suspension directly into the culture flask for culture, change the medium after 24 hours, and digest and subculture with 2.5g/L trypsin after the cells are 80% confluent;

S104, take the second-generation rat MSCs of healthy growth to make a cell suspension, transfer it to a centrifuge tube, and centrifuge at 1000r/min for 5min; discard the supernatant, and adjust the cell concentration to 1×10 ⁶ with the cleaning solution;

S105, add 5 μL each of rabbit anti-mouse CD45, CD44, and CD90 monoclonal antibodies, and incubate at room temperature for 15 minutes to obtain rat bone marrow mesenchymal stem cells.

The method for isolating and culturing rat bone marrow mesenchymal stem cells provided in the embodiment of the present invention includes:

SD rats were intraperitoneally anesthetized with 3% pentobarbital sodium at a dose of 1 mL/kg. The bilateral femur and tibia were taken under aseptic operation, the superficial muscles were removed, and washed with PBS solution repeatedly. Aseptically cut off the femur and tibia, rinse the fractures of both ends of the femur and tibia with PBS solution several times, pour the bone marrow into the DMEM culture medium, inoculate the cell suspension directly into the culture bottle, change the medium after 24 hours, and wait until the cells are 80% confluent Digest and subculture with 2.5g/L trypsin. The second generation of healthy rat MSCs was taken to make a cell suspension, transferred to a centrifuge tube, and centrifuged at 1000r/min for 5min. Discard the supernatant, and adjust the cell concentration to 1×10 ⁶ with PBS. Add 5 μL each of rabbit anti-mouse CD45, CD44, and CD90 monoclonal antibodies, incubate at room temperature for 15 min, and put them into a flow cytometer for identification.

2. Application example: In the study of the effect of radiation on the paracrine function of bone marrow mesenchymal stem cells, the method can be used to extract the primary culture for 4 days, and it can be observed that the cells adhere to the wall and spread, showing a spindle shape, a large number, and a large number of local cells. scattered clusters of cells. as shown in picture 2,

In order to prove the creativity and technical value of the technical solution of the present invention, this part is the application example of the claimed technical solution on specific products or related technologies.

3. Evidence of the relevant effects of the embodiment. This method begins to form typical, uniformly distributed MSC cluster proliferation foci on the 4th to 6th day, the number of cells increases, and the cell shape is long spindle-shaped and closely arranged. increased, the scope expanded. On the 7th to 9th day, the cells can grow to reach 80% to 90% confluence, showing a long spindle shape, closely arranged like a whirlpool. 4 hours after inoculation, the subcultured cells began to adhere to the wall, and 12 hours after the living cells basically completed the wall, proliferated rapidly on the 3rd day, and reached 80% to 90% confluence on the 7th day, showing a more uniform long spindle shape than the primary one. The results of flow cytometry showed that among the identified cells, CD44(-), CD90(-), and CD45(+), indicating that the cultured cells were indeed MSCs. PerCP-PE labeled CD90 fluorescence; C: PerCP-FITC (fluorescein isothiocyanate) labeled CD45 fluorescence; D: PerCP-FITC labeled CD44 fluorescence. As shown in Figure 3.

The embodiment of the present invention has achieved some positive effects in the process of research and development or use, and indeed has great advantages compared with the prior art. The following content is described in conjunction with the data and charts of the test process.

The above is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Anyone familiar with the technical field within the technical scope disclosed in the present invention, whoever is within the spirit and principles of the present invention Any modifications, equivalent replacements and improvements made within shall fall within the protection scope of the present invention.

Claims (10)

1. The method for separating and culturing the rat bone marrow mesenchymal stem cells is characterized by comprising the following steps:

firstly, obtaining isolated bilateral femur and tibia of a rat, removing muscles on the surfaces of the bilateral femur and tibia, and repeatedly washing the muscles by using a cleaning solution;

step two, under the aseptic condition, cutting off two ends of the femur and tibia on two sides, and flushing fracture at two ends of the femur and tibia for multiple times by using cleaning liquid;

step three, the marrow is flushed into a culture solution, the cell suspension is directly inoculated into a culture flask for culture, and after 80% of cells are converged, the cells are subjected to trypsin digestion for subculture;

step four, taking MSC of the healthy growth generation 2 rat to prepare cell suspension, transferring the cell suspension into a centrifuge tube, and centrifuging; discarding the supernatant, and regulating the cell concentration by using a cleaning solution;

and fifthly, adding rabbit anti-mouse CD45, CD44 and CD90 monoclonal antibodies, and incubating at room temperature to obtain the rat bone marrow mesenchymal stem cells.

2. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein the washing liquid is PBS liquid.

3. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the third step, the culture solution is: DMEM broth.

4. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the third step, the cell suspension is directly inoculated into a culture flask for culturing, comprising: half liquid change is carried out after 24 hours.

5. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the third step, the concentration of trypsin is 2.5g/L.

6. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the fourth step, the centrifugation comprises: centrifuging at 1000r/min for 5min.

7. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the fourth step, the step of adjusting the cell concentration comprises: cell concentration was adjusted to 1X 10 ⁶ 。

8. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein the adding rabbit anti-mouse CD45, CD44, CD90 monoclonal antibodies comprises: mu.L of each of the rabbit anti-mouse CD45, CD44, CD90 monoclonal antibodies was added.

9. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein in the fifth step, the incubation time at room temperature is 15min.

10. The method for isolating and culturing mesenchymal stem cells of rat according to claim 1, wherein the incubation at room temperature is further performed by: the incubation products were identified by placing them into a flow cytometer.

Images