Method for efficiently and conveniently separating bone marrow mononuclear cells
Through bone perforation and the use of sodium chloride solution, combined with the separation steps of the centrifuge, the problem of low yield and operation dependence when separating bone marrow mononuclear cells was solved, achieving efficient and convenient cell separation, with higher yields and no differences in cell quality.
Patent Information
- Application Number
- CN202411716504.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-03-04
AI Technical Summary
The existing density gradient centrifugation method has limited yields (less than 30%) when isolating bone marrow mononuclear cells and is greatly affected by artificial operations, resulting in severe loss of BMSC (up to 90%), affecting the therapeutic effect, and requires the use of lymphocyte isolation fluid that may cause safety problems.
Bone marrow blood was extracted by bone penetration, and 0.3% sodium chloride solution was prepared, and the bone marrow blood and sodium chloride solution were mixed in a ratio of 1:5 for cell lysis. Then the cells were isolated using a centrifuge, and the supernatant was discarded and the bottom precipitation was retained to extract bone marrow mononuclear cells.
A higher cell yield was achieved, and there was no statistical difference between the residual red blood cell level, cell viability and colony formation ability from traditional methods. It was simple, convenient and efficient in operation.
Smart Images
Figure CN119552814A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of clinical medicine, and in particular to a method for separating bone marrow mononuclear cells efficiently and conveniently. Background Art
[0002] In recent years, bone marrow mononuclear cells (BMMNCs) have been introduced into the fields of wound healing, fat transplantation, and most importantly, craniofacial bone regeneration. BMMNCs are a group of cells isolated from the bone marrow that are rich in a variety of regenerative cells, including bone marrow mesenchymal stem cells (BMSCs). As an "off-the-shelf" product, BMMNCs are a promising new therapy that can avoid the cost and risk of in vitro culture better than BMSCs. Currently, density gradient centrifugation (DGC) is mainly used to separate BMMNCs during surgery. However, the BMMNCs yield of this classic method is still limited (less than 30%) and is greatly affected by human manipulation. It has also been reported that BMSCs are severely lost during centrifugation (up to 90%), which may affect the therapeutic effect. In addition, density gradient centrifugation also requires the use of lymphocyte separation fluids such as ficoll, which not only increases the separation cost but also may cause safety issues. Therefore, it is imperative to invent an efficient and convenient method for isolating bone marrow mononuclear cells to obtain high-quality BMMNCs for clinical use. Summary of the invention
[0003] The purpose of the present invention is to solve the defects in the prior art and to propose an efficient and convenient method for separating bone marrow mononuclear cells.
[0004] In order to achieve the above object, the present invention adopts the following technical solutions:
[0005] An efficient and convenient method for separating bone marrow mononuclear cells, the specific steps of the separation method are as follows:
[0006] Ⅰ. Extract bone marrow blood through bone marrow puncture;
[0007] Ⅱ. Prepare 0.3% sodium chloride solution;
[0008] III. Mix bone marrow blood with 0.3% sodium chloride solution in a ratio of 1:5 to perform cell lysis;
[0009] IV. Use a centrifuge to separate the lysed cells;
[0010] V. Discard the supernatant and retain the bottom pellet to extract bone marrow mononuclear cells.
[0011] As a further solution of the present invention, the specific steps of extracting bone marrow blood in step I are as follows:
[0012] S1.1: Select the bone marrow puncture site, prepare a sterile bone puncture needle, syringe, sterile gauze, iodine or alcohol, gloves and disinfectants, disinfect the area around the bone marrow puncture site with iodine or alcohol and disinfectants, and then inject local anesthetic around the selected bone marrow puncture site;
[0013] S1.2: Insert the sterile bone puncture needle vertically into the skin along the anesthesia point, keep the needle vertical, and slowly puncture the bone cortex by rotating and pushing until it enters the bone marrow cavity. After verifying that the needle is in the correct position, remove the needle core and connect the sterile syringe.
[0014] S1.3: Gently draw back the syringe to absorb the required amount of bone marrow blood. After the extraction is completed, quickly remove the bone marrow needle, apply local pressure to stop the bleeding, cover the puncture site with sterile gauze and fix it, and quickly transfer the obtained bone marrow blood to a pre-prepared container for storage.
[0015] As a further solution of the present invention, the specific steps of preparing the 0.3% sodium chloride solution in step II are as follows:
[0016] S2.1: Prepare 0.9% sodium chloride solution and distilled water. According to the target concentration of 0.3%, accurately measure the required volume of 0.9% sodium chloride solution and distilled water in a ratio of 1:2 using a measuring cylinder or pipette;
[0017] S2.2: Pour the measured 0.9% sodium chloride solution into a clean container, add distilled water, and stir the solution with a glass stirring rod to fully mix the 0.9% sodium chloride solution and distilled water until the solution is uniform and transparent;
[0018] S2.3: Confirm whether the solution concentration meets the standard of 0.3% sodium chloride solution through specific gravity test, and then transfer the prepared 0.3% sodium chloride solution to a clean reagent bottle.
[0019] Cover the bottle tightly, then label the reagent bottle, indicating the solution name, concentration, preparation date and expiration date.
[0020] As a further embodiment of the present invention, the specific steps of cell lysis in step 1 are as follows:
[0021] S3.1: Use a pipette or a straw to slowly draw the bone marrow blood and inject it into a sterile test tube. Then add 0.3% sodium chloride solution in a ratio of 1:5 between the bone marrow blood and 0.3% sodium chloride solution. Use a vortex mixer to gently mix the mixture in the test tube. If there is no vortex mixer, gently flip the test tube up and down to ensure that the bone marrow blood is in full contact with the 0.3% sodium chloride solution.
[0022] S3.2: After mixing, let the mixture stand at room temperature for 10 minutes. After standing, observe whether the liquid in the test tube turns into transparent red. If the expected effect is not achieved, extend the lysis time and pay attention to the lysis situation in real time.
[0023] As a further embodiment of the present invention, the specific steps of separating and lysing the cells in step IV are as follows:
[0024] S4.1: Take the mixture after lysis treatment and make sure that the sample is mixed evenly without precipitation. Slowly transfer the mixture after lysis to a centrifuge tube to avoid sample overflow or bubble generation. If the sample volume is insufficient, add an equal amount of clean water or an empty tube to the opposite centrifuge tube for balance.
[0025] S4.2: At room temperature, set the centrifuge to a speed of 300 g and a centrifugation time of 5 minutes. After closing the centrifuge cover, start the device and monitor its operating status. After the centrifugation is completed, open the centrifuge cover after the device completely stops running, and take out the centrifuge tube;
[0026] S4.3: Inspect the sample after centrifugation. If no clear stratification is observed, adjust the centrifugation parameters and repeat the operation.
[0027] As a further embodiment of the present invention, the specific steps of extracting bone marrow mononuclear cells in step V are as follows:
[0028] S5.1: Prepare a sterile pipette or straw, and a clean centrifuge tube or sample container. Take out the sample after centrifugation. Keep the centrifuge tube vertical and use a sterile pipette or straw to aspirate the supernatant. Avoid contact between the sterile pipette and the bottom sediment. After completely discarding the supernatant, check whether the bottom sediment is intact and not lost.
[0029] S5.2: After the examination, take part of the bone marrow mononuclear cell pellet and resuspend the cells with PBS or culture medium, and ensure that the cells are evenly distributed. Then use a blood cell counting plate or an automatic cell counter to take 10 μL of the cell suspension for counting. Repeat the counting 2 to 3 times and calculate the average value. Calculate the cell yield based on the total collected volume and the number of cells per unit volume.
[0030] S5.3: Mix 50 μL of bone marrow mononuclear cell suspension with an equal volume of stain, let stand for a few minutes, drop the mixture on a glass slide, cover it with a coverslip and observe it under a microscope, and count the number of residual red blood cells and bone marrow mononuclear cells in the field of view, and calculate the residual red blood cell level determination based on the statistical results;
[0031] S5.4: Use 0.4% trypan blue dye as a viability detection reagent, take 10 μL of bone marrow mononuclear cell suspension, add 10 μL trypan blue dye, mix well and let stand for 1 to 2 minutes, count the number of stained and unstained cells under a microscope, and calculate the corresponding cell viability based on the statistical cell data;
[0032] S5.5: Prepare colony-forming medium, inoculate the remaining bone marrow mononuclear cells in a culture dish at a density of 1000-2000 cells / dish, and culture the culture dish in an incubator at 37°C, 5% CO2 for 10 to 14 days. After the culture period, observe the colonies in the culture dish under a microscope, count the colonies based on their morphology and size, distinguish different types, record and calculate the colony-forming unit density of each sample.
[0033] Compared with the prior art, the present invention has the following beneficial effects:
[0034] The efficient and convenient method for separating bone marrow mononuclear cells comprises selecting a bone marrow puncture site, extracting bone marrow blood through bone marrow puncture, preparing 0.9% sodium chloride solution and distilled water, and accurately measuring the required volume of 0.9% sodium chloride solution and distilled water in a ratio of 1:2 using a measuring cylinder or a pipette according to a target concentration of 0.3%, pouring the measured 0.9% sodium chloride solution into a clean container, adding distilled water, stirring the solution with a glass stirring rod to fully mix the 0.9% sodium chloride solution and distilled water, slowly drawing bone marrow blood with a pipette or a straw, and injecting it into a sterile test tube, and then mixing the bone marrow blood and 0.3% sodium chloride solution in a ratio of 1:2. 0.3% sodium chloride solution is added to 5% sodium chloride solution in a ratio of 1:5 to mix, and the mixture in the test tube is gently mixed. After mixing, the mixture is allowed to stand at room temperature for 10 minutes. After standing, the mixture is centrifuged at room temperature at 300g for 5 minutes by a centrifuge, and the supernatant of the mixture after centrifugation is removed to obtain bone marrow mononuclear cells. Compared with the density gradient centrifugation method, the present invention has a higher cell yield, no statistical difference in the residual red blood cell level, cell viability and colony forming ability, simple, convenient and efficient operation, has advantages over traditional density gradient centrifugation, and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.
[0036] Figure 1 A flowchart of an efficient and convenient method for separating bone marrow mononuclear cells proposed by the present invention;
[0037] Figure 2A bar graph comparing the cell yield determination of an efficient and convenient method for isolating bone marrow mononuclear cells proposed by the present invention;
[0038] Figure 3 A comparative bar graph showing the residual red blood cell level determination of an efficient and convenient method for separating bone marrow mononuclear cells proposed by the present invention;
[0039] Figure 4 A bar graph showing a comparison of cell viability measurements in a method for isolating bone marrow mononuclear cells that is efficient and convenient and is proposed by the present invention;
[0040] Figure 5 This is a colony number observation diagram of an efficient and convenient method for isolating bone marrow mononuclear cells proposed by the present invention;
[0041] Figure 6 A bar graph comparing colony numbers of an efficient and convenient method for isolating bone marrow mononuclear cells proposed in the present invention. DETAILED DESCRIPTION
[0042] Reference Figure 1-6 , an efficient and convenient method for separating bone marrow mononuclear cells, the specific steps of the separation method are as follows:
[0043] Bone marrow blood is drawn during a bone marrow biopsy.
[0044] Specifically, select a bone marrow puncture site, prepare a sterile bone puncture needle, syringe, sterile gauze, iodine or alcohol, gloves and disinfectants, disinfect the area around the bone marrow puncture site with iodine or alcohol and disinfectants, then inject local anesthetics around the selected bone marrow puncture site, insert the sterile bone puncture needle vertically into the skin along the anesthesia point, keep the needle vertical, and slowly puncture the bone cortex using a rotary propulsion method until it enters the bone marrow cavity, then verify that the needle is in the correct position, pull out the needle core, connect the sterile syringe, gently draw back the syringe, and draw out the required amount of bone marrow blood. After the extraction is completed, quickly pull out the bone puncture needle, apply local pressure to stop bleeding, cover the puncture site with sterile gauze and fix it, and quickly transfer the obtained bone marrow blood to a pre-prepared container for storage.
[0045] Prepare 0.3% sodium chloride solution.
[0046] Specifically, prepare 0.9% sodium chloride solution and distilled water. According to the target concentration of 0.3%, accurately measure the required volume of 0.9% sodium chloride solution and distilled water in a ratio of 1:2 using a measuring cylinder or a pipette. Pour the measured 0.9% sodium chloride solution into a clean container, add distilled water, and stir the solution with a glass stirring rod to fully mix the 0.9% sodium chloride solution and distilled water until the solution is uniform and transparent. Confirm whether the solution concentration meets the standard of 0.3% sodium chloride solution through specific gravity detection, and then transfer the prepared 0.3% sodium chloride solution to a clean reagent bottle and cover the bottle mouth. Then label the reagent bottle with the solution name, concentration, preparation date and expiration date.
[0047] Bone marrow blood was mixed with 0.3% sodium chloride solution at a ratio of 1:5 for cell lysis.
[0048] Specifically, use a pipette or a straw to slowly draw out the bone marrow blood and inject it into a sterile test tube, then add 0.3% sodium chloride solution at a ratio of 1:5 between the bone marrow blood and the 0.3% sodium chloride solution to mix, use a vortex mixer to gently mix the mixture in the test tube. If there is no vortex mixer, gently turn the test tube up and down to ensure that the bone marrow blood is in full contact with the 0.3% sodium chloride solution. After mixing, let the mixture stand at room temperature for 10 minutes. After standing, observe whether the liquid in the test tube turns into transparent red. If the expected effect is not achieved, extend the lysis time and pay real-time attention to the lysis situation.
[0049] Use a centrifuge to separate the lysed cells.
[0050] Specifically, take the mixture that has completed the lysis treatment, and confirm that the sample is mixed evenly without precipitation, and slowly transfer the lysis mixture to a centrifuge tube to avoid sample overflow or bubble generation. If the sample volume is insufficient, add an equal amount of clean water or an empty tube to the opposite centrifuge tube for balance. At room temperature, set the centrifuge to a speed of 300g, set the centrifugation time to 5 minutes, close the centrifuge cover, start the device and monitor its operating status. After centrifugation is completed, open the centrifuge cover after the device completely stops running, take out the centrifuge tube, check the centrifuged sample, and if no clear stratification is observed, adjust the centrifugation parameters and repeat the operation.
[0051] The supernatant was discarded and the bottom pellet was retained to extract bone marrow mononuclear cells.
[0052] Specifically, prepare a sterile pipette or straw, and prepare a clean centrifuge tube or sample container, take out the sample after centrifugation, keep the centrifuge tube vertical, use a sterile pipette or straw to absorb the supernatant, and avoid the sterile pipette or straw from touching the bottom sediment. After completely discarding the supernatant, check whether the bottom sediment is intact and not lost. After the inspection is completed, take part of the bone marrow mononuclear cell sediment, resuspend the cells with PBS or culture medium, and ensure that the cells are evenly distributed, then use a hemocytometer or automatic cell counter to take 10μL of the cell suspension for counting, repeat the counting 2 to 3 times, calculate the average value, calculate the cell yield based on the total collection volume and the number of cells per unit volume, mix 50μL of the bone marrow mononuclear cell suspension with an equal volume of the dye, let it stand for a few minutes, drop the mixture on a glass slide, cover it with a coverslip and observe it under a microscope, and summarize it. Count the number of residual red blood cells and bone marrow mononuclear cells in the field of view, and calculate the residual red blood cell level by statistical results. Use 0.4% trypan blue dye as a viability detection reagent, take 10 μL of bone marrow mononuclear cell suspension, add 10 μL of trypan blue dye, mix well and let stand for 1 to 2 minutes, count the number of stained and unstained cells under a microscope, and calculate the corresponding cell viability based on the statistical cell data, prepare colony formation medium, inoculate the remaining bone marrow mononuclear cells in a culture dish at a density of 1000 to 2000 cells / dish, and place the culture dish in an incubator at 37°C and 5% CO2 for 10 to 14 days. After the culture period, observe the colonies in the culture dish under a microscope, count the number of colonies according to morphology and size, distinguish different types, record and count the data, and calculate the colony forming unit density of each sample.
[0053] In addition, it should be noted that the reference Figure 2-Figure 6 It can be seen that, by comparing the cell yield of bone marrow mononuclear cells obtained by 0.3% sodium chloride lysis method and density gradient centrifugation method, the level of residual red blood cells in bone marrow mononuclear cells, the cell viability and colony forming ability of bone marrow mononuclear cells, the cell yield of sodium chloride lysis method is higher, there is no statistical difference in the level of residual red blood cells in the cells, there is no statistical difference in cell viability, and there is no statistical difference in colony forming ability.
Claims
1. A method for separating bone marrow mononuclear cells efficiently and conveniently, characterized in that: The specific steps of the separation method are as follows: Ⅰ. Extract bone marrow blood through bone marrow puncture; Ⅱ. Prepare 0.3% sodium chloride solution; III. Mix bone marrow blood with 0.3% sodium chloride solution in a ratio of 1:5 to perform cell lysis; IV. Use a centrifuge to separate the lysed cells; V. Discard the supernatant and retain the bottom pellet to extract bone marrow mononuclear cells.
2. A method for separating bone marrow mononuclear cells efficiently and conveniently according to claim 1, characterized in that: The specific steps of extracting bone marrow blood in step Ⅰ are as follows: S1.1: Select the bone marrow puncture site, prepare a sterile bone puncture needle, syringe, sterile gauze, iodine or alcohol, gloves and disinfectants, disinfect the area around the bone marrow puncture site with iodine or alcohol and disinfectants, and then inject local anesthetic around the selected bone marrow puncture site; S1.2: Insert the sterile bone puncture needle vertically into the skin along the anesthesia point, keep the needle vertical, and slowly puncture the bone cortex by rotating and pushing until it enters the bone marrow cavity. After verifying that the needle is in the correct position, remove the needle core and connect the sterile syringe. S1.3: Gently draw back the syringe to absorb the required amount of bone marrow blood. After the extraction is completed, quickly remove the bone marrow needle, apply local pressure to stop the bleeding, cover the puncture site with sterile gauze and fix it, and quickly transfer the obtained bone marrow blood to a pre-prepared container for storage.
3. The method for separating bone marrow mononuclear cells efficiently and conveniently according to claim 1, characterized in that: The specific steps for preparing 0.3% sodium chloride solution in step II are as follows: S2.1: Prepare 0.9% sodium chloride solution and distilled water. According to the target concentration of 0.3%, accurately measure the required volume of 0.9% sodium chloride solution and distilled water in a ratio of 1:2 using a measuring cylinder or pipette; S2.2: Pour the measured 0.9% sodium chloride solution into a clean container, add distilled water, and stir the solution with a glass stirring rod to fully mix the 0.9% sodium chloride solution and distilled water until the solution is uniform and transparent; S2.3: Confirm whether the solution concentration meets the standard of 0.3% sodium chloride solution through specific gravity test, then transfer the prepared 0.3% sodium chloride solution to a clean reagent bottle and cover the bottle mouth tightly. Then label the reagent bottle with the solution name, concentration, preparation date and expiration date.
4. The method for separating bone marrow mononuclear cells efficiently and conveniently according to claim 1, characterized in that: The specific steps of cell lysis in step 1 are as follows: S3.1: Use a pipette or a straw to slowly draw the bone marrow blood and inject it into a sterile test tube. Then add 0.3% sodium chloride solution in a ratio of 1:5 between the bone marrow blood and 0.3% sodium chloride solution. Use a vortex mixer to gently mix the mixture in the test tube. If there is no vortex mixer, gently flip the test tube up and down to ensure that the bone marrow blood is in full contact with the 0.3% sodium chloride solution. S3.2: After mixing, let the mixture stand at room temperature for 10 minutes. After standing, observe whether the liquid in the test tube turns into transparent red. If the expected effect is not achieved, extend the lysis time and pay attention to the lysis situation in real time.
5. A method for separating bone marrow mononuclear cells efficiently and conveniently according to claim 4, characterized in that: The specific steps of separating and lysing the cells in step IV are as follows: S4.1: Take the mixture after lysis treatment and make sure that the sample is mixed evenly without precipitation. Slowly transfer the mixture after lysis to a centrifuge tube to avoid sample overflow or bubble generation. If the sample volume is insufficient, add an equal amount of clean water or an empty tube to the opposite centrifuge tube for balance. S4.2: At room temperature, set the centrifuge to a speed of 300 g and a centrifugation time of 5 minutes. After closing the centrifuge cover, start the device and monitor its operating status. After the centrifugation is completed, open the centrifuge cover after the device completely stops running, and take out the centrifuge tube; S4.3: Inspect the sample after centrifugation. If no clear stratification is observed, adjust the centrifugation parameters and repeat the operation.
6. The method for separating bone marrow mononuclear cells efficiently and conveniently according to claim 5, characterized in that: The specific steps of extracting bone marrow mononuclear cells in step V are as follows: S5.1: Prepare a sterile pipette or straw, and a clean centrifuge tube or sample container. Take out the sample after centrifugation. Keep the centrifuge tube vertical and use a sterile pipette or straw to aspirate the supernatant. Avoid contact between the sterile pipette and the bottom sediment. After completely discarding the supernatant, check whether the bottom sediment is intact and not lost. S5.2: After the examination, take part of the bone marrow mononuclear cell pellet and resuspend the cells with PBS or culture medium, and ensure that the cells are evenly distributed. Then use a blood cell counting plate or an automatic cell counter to take 10 μL of the cell suspension for counting. Repeat the counting 2 to 3 times and calculate the average value. Calculate the cell yield based on the total collected volume and the number of cells per unit volume. S5.3: Mix 50 μL of bone marrow mononuclear cell suspension with an equal volume of stain, let stand for a few minutes, drop the mixture on a glass slide, cover it with a coverslip and observe it under a microscope, and count the number of residual red blood cells and bone marrow mononuclear cells in the field of view, and calculate the residual red blood cell level determination based on the statistical results; S5.4: Use 0.4% trypan blue dye as a viability detection reagent, take 10 μL of bone marrow mononuclear cell suspension, add 10 μL trypan blue dye, mix well and let stand for 1 to 2 minutes, count the number of stained and unstained cells under a microscope, and calculate the corresponding cell viability based on the statistical cell data; S5.5: Prepare colony-forming medium, inoculate the remaining bone marrow mononuclear cells in a culture dish at a density of 1000-2000 cells / dish, and culture the culture dish in an incubator at 37°C, 5% CO2 for 10 to 14 days. After the culture period, observe the colonies in the culture dish under a microscope, count the colonies based on their morphology and size, distinguish different types, record and calculate the colony-forming unit density of each sample.