Method for separating mononuclear cells of umbilical cord blood
By pre-treating umbilical cord blood samples and performing multiple centrifugation operations, the problem of low efficiency in the separation of mononuclear cells from umbilical cord blood in existing technologies has been solved, realizing an efficient and simple cell separation method that improves cell recovery rate and maintains cell viability.
Patent Information
- Application Number
- CN202511279987.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-09
- Publication Date
- 2025-11-21
AI Technical Summary
In existing technologies, the recovery rate of umbilical cord blood mononuclear cells separated by Ficoll density gradient centrifugation is low, and there is a need to improve the separation efficiency to meet the needs of clinical applications.
The umbilical cord blood samples were pretreated by adding an anticoagulant, then pressurized and oxygenated in a closed environment and shaken on a shaker. Next, they were mixed with Ficoll separation solution at low temperature and allowed to warm up. Then, they were centrifuged multiple times to separate the mononuclear cell layer, ensuring that the cell viability was not damaged.
It significantly improves the isolation efficiency and recovery rate of mononuclear cells while maintaining cell viability, and optimizes the ease of operation and effectiveness of the isolation process.
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Figure CN120988993A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of separation of mononuclear cells, in particular to a method for separating mononuclear cells from umbilical cord blood. BACKGROUND
[0002] The umbilical cord blood contains a large number of mononuclear cells, which can stimulate the generation of synapses, complete different repairs, and be used for treating various malignant hematological tumors, acquired or genetic bone marrow failure syndromes, and non-malignant diseases (such as spinal cord injury, cirrhosis, diabetes, myocardial injury, and autoimmune diseases).
[0003] At present, the separation of mononuclear cells from umbilical cord blood mainly adopts Ficoll density gradient centrifugation method, AXP umbilical cord blood automatic separation system two-step method, magnetic bead method, flow cytometry, and gelatin precipitation method, but generally, the Ficoll density gradient centrifugation method is widely used in consideration of cost and operation simplicity.
[0004] The recovery rate of mononuclear cells separated by the Ficoll density gradient centrifugation method is low, and it is an important research direction at the present stage to improve the separation yield of mononuclear cells from umbilical cord blood. SUMMARY
[0005] In view of the deficiencies in the prior art, the present application provides a method for separating mononuclear cells from umbilical cord blood, which effectively improves the separation efficiency of mononuclear cells from umbilical cord blood and improves the final yield of mononuclear cells without affecting the cell activity.
[0006] To achieve the above object, the present application is realized by the following technical scheme: A method for separating mononuclear cells from umbilical cord blood, comprising the following steps: S1, adding an anticoagulant to the umbilical cord blood and mixing uniformly, then centrifuging to separate the plasma layer to obtain pretreated umbilical cord blood for standby; S2, diluting the pretreated umbilical cord blood with a diluent and then placing it in a closed environment to adjust the oxygenation pressure to 0.3-0.5Mpa, and shaking for 10-15min to obtain a diluted blood sample for standby; S3, slowly injecting the diluted blood sample into the upper layer of a pre-cooled separation liquid at 10-12℃, and standing at room temperature until it returns to room temperature to obtain a blood sample mixed with the separation liquid; S4, centrifuging the blood sample mixed with the separation liquid, and after centrifugation, sucking the mononuclear cell layer from the second layer from top to bottom for standby; S5, mixing the mononuclear cell layer with a diluent and continuing to centrifuge, discarding the supernatant, and the lower layer of the precipitate is the separated mononuclear cells from umbilical cord blood.
[0007] Preferably, the anticoagulant in step S1 is sodium heparin.
[0008] Preferably, the specific way of centrifuging the plasma layer is centrifuging the umbilical cord blood at a speed of 1800-2000 r / min for 10 min, and the centrifuging speed-up time is controlled to be 9 min and the centrifuging speed-down time is controlled to be 4 min.
[0009] Preferably, the diluent in steps S2 and S5 is 0.9% sodium chloride injection, and the diluent and the pretreated umbilical cord blood are mixed in step S2 according to an equal volume ratio.
[0010] Preferably, the speed of the shaking table in step S2 is 40-60 r / min.
[0011] Preferably, the separation liquid in step S3 is Ficoll separation liquid, and the volume ratio of the separation liquid to the diluted blood sample is 1:1-2.
[0012] Preferably, the speed of the centrifuging in step S4 is 1800-2800 r / min, and the centrifuging time is 25-30 min; the centrifuging speed-up time is controlled to be 6 min and the centrifuging speed-down time is controlled to be 2 min.
[0013] Preferably, the speed of the centrifuging in step S5 is 1800-2000 r / min, and the centrifuging time is 8-15 min; the centrifuging speed-up time is controlled to be 9 min and the centrifuging speed-down time is controlled to be 5 min.
[0014] The present application provides a method for separating umbilical cord blood mononuclear cells, which has the following advantages compared with the prior art: The present application improves the activity of cells and promotes the subsequent separation of mononuclear cells by increasing the pressure and oxygen of the blood sample in the early stage, and the mononuclear cells are effectively separated by pre-cooling the separation liquid at a low temperature, standing the mixed blood sample, re-warming, and then centrifuging at a certain speed, thereby improving the yield of subsequent mononuclear cells and comprehensively improving the separation efficiency. BRIEF DESCRIPTION OF DRAWINGS
[0015] Figure 1 FIG. 1 is a schematic diagram of the Ficoll separation liquid layer after centrifugation in step (3) of Example 1 of the present application; Figure 2 FIG. 3 is a schematic diagram of the final separated mononuclear cells in Experimental Group 3 of the present application; Figure 3 FIG. 4 is a schematic diagram of the final separated mononuclear cells in Example 2 of the present application. DETAILED DESCRIPTION
[0016] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are some but not all of the embodiments of the present application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.
[0017] Embodiment 1 Isolation of umbilical cord blood mononuclear cells: (1) Add 1200 IU of heparin sodium solution (heparin sodium mixed with normal saline to prepare a solution of 1000 IU / mL) into a blood collection bag, then inject 50 mL of fresh umbilical cord blood into the blood collection bag, shake the blood collection bag gently to mix thoroughly to obtain anticoagulated whole blood, then pour the anticoagulated whole blood into a centrifuge tube and place the centrifuge tube in a centrifuge, control the centrifuge to increase the speed to 1800 r / min in 9 min, then centrifuge for 10 min, then decrease the speed to static state in 2 min, take out the centrifuge tube, separate the upper plasma layer, and obtain pretreated umbilical cord blood for standby; (2) Mix and dilute the pretreated umbilical cord blood with an equal volume of 0.9% sodium chloride injection, then place the mixture on a shaking bed, adjust the speed to 40 r / min, and fill oxygen to adjust the pressure to 0.3 Mpa, then shake for 15 min, and then take out the diluted blood sample for standby; (3) Pre-cool 25 mL of Ficoll separation medium in a centrifuge tube at 10°C, then slowly add the diluted blood sample to the upper surface of the Ficoll separation medium at a volume ratio of 1:1, and after complete addition, wait until the liquid in the centrifuge tube returns to room temperature, then place the centrifuge tube in a centrifuge, control the speed to increase to 1800 r / min in 6 min, then centrifuge for 30 min, and then decrease the speed to static state in 2 min, after centrifugation, the first layer is a plasma layer, the second layer is a mononuclear cell layer, the third layer is a sample density separation medium layer, and the fourth layer is a red blood cell layer (as shown in Figure 1 ); (4) Suck and discard the upper plasma layer, slowly suck the second layer of mononuclear cell layer into a new 50 ml centrifuge tube, and supplement 0.9% sodium chloride injection to 45 ml; then place the centrifuge tube in a centrifuge, control the speed to increase to 1800 r / min in 9 min, centrifuge for 15 min, then decrease the speed to static state in 5 min, remove the supernatant, and the lower layer of precipitate is the separated mononuclear cells, which are resuspended with 0.9% sodium chloride injection.
[0018] Embodiment 2 Isolation of umbilical cord blood mononuclear cells: (1) In the blood bag, add 1200 IU of heparin sodium solution (heparin sodium mixed with normal saline to prepare a solution of 1000 IU / mL), then inject 50 mL of fresh umbilical cord blood into the blood collection bag, shake the blood collection bag gently to mix thoroughly, then pour into a centrifuge tube and place in a centrifuge. Control the centrifuge to increase the speed to 2000 r / min in 9 min, then centrifuge for 10 min, then reduce the speed to static state in 2 min, take out the centrifuge tube, separate the upper plasma layer, and obtain the pretreated umbilical cord blood for standby; (2) Mix and dilute the pretreated umbilical cord blood with 0.9% sodium chloride injection in equal volume, then place on a shaking bed to adjust the speed to 60 r / min, and fill with oxygen to adjust the pressure to 0.3 Mpa, then shake for 10 min, then take out the diluted blood sample for standby; (3) Place 25 mL of Ficoll separation medium in a centrifuge tube and pre-cool at 10°C, then slowly add the diluted blood sample to the upper surface of the Ficoll separation medium at a volume ratio of 1:2, and after complete addition, let stand until the liquid in the centrifuge tube returns to room temperature, then place the centrifuge tube in a centrifuge and control the speed to increase to 2800 r / min in 6 min, then centrifuge for 25 min, then reduce the speed to static state in 2 min. After centrifugation, the first layer is the plasma layer, the second layer is the mononuclear cell layer, the third layer is the sample density separation medium layer, and the fourth layer is the red blood cell layer; (4) Absorb the upper plasma layer, slowly absorb the second layer of mononuclear cell layer into a new 50 ml centrifuge tube, and supplement with 0.9% sodium chloride injection to 45 ml; then place in a centrifuge, increase the speed to 2000 r / min in 9 min, centrifuge for 8 min, then reduce the speed to static state in 5 min, remove the supernatant, and the lower layer of sediment is the separated mononuclear cells, which are resuspended with 0.9% sodium chloride injection.
[0019] Comparative Example 1: Isolation of umbilical cord blood mononuclear cells: Referring to the separation method of Example 1, only step (3) is different: (3) Place 25 mL of Ficoll separation medium at room temperature in a centrifuge tube, then slowly add the diluted blood sample to the upper surface of the Ficoll separation medium at a volume ratio of 1:1, and after complete addition, place the centrifuge tube in a centrifuge and control the speed to increase to 1800 r / min in 6 min, then centrifuge for 30 min, then reduce the speed to static state in 2 min. After centrifugation, the first layer is the plasma layer, the second layer is the mononuclear cell layer, the third layer is the sample density separation medium layer, and the fourth layer is the red blood cell layer; Comparative Example 2: Isolation of umbilical cord blood mononuclear cells: Referring to the separation method of Example 1, only steps (2) and (3) are different: (2) Dilute the pretreated cord blood with 0.9% sodium chloride injection solution by equal volume to prepare diluted blood sample; (3) Put 25 mL of Ficoll separation solution at room temperature into a centrifuge tube, then slowly add the diluted blood sample to the upper surface of the Ficoll separation solution at a volume ratio of 1:1, and then put the centrifuge tube into a centrifuge, control the speed to 1800 r / min for 6 min, then centrifuge for 30 min, and then reduce the speed to stand for 2 min. After centrifugation, the first layer is plasma layer, the second layer is mononuclear cell layer, the third layer is sample density separation solution layer, and the fourth layer is red blood cell layer; Detection: Referring to the separation method of Example 1, only change the pressure adjustment of oxygen filling in step (2) to separate the mononuclear cells of the cord blood, and refer to Table 1 below for details: Table 1
[0020] Wherein " / " represents that no oxygen is filled for pressure adjustment operation; The anticoagulated whole blood samples used in experimental groups 1-6 and Example 2, and Comparative Example 1 were counted (before separation), and the obtained 0.9% sodium chloride injection solution was resuspended mononuclear cells (after separation) were counted, and the final yield of mononuclear cells (based on 50 mL of cord blood volume) was calculated. The specific results are shown in Table 2 below: Table 2
[0021] As can be seen from the above table, experimental groups 3-5 and Example 2 have higher yields of mononuclear cells.
[0022] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present application.
Claims
1. A method for isolating mononuclear cells from umbilical cord blood, characterized in that, The separation method includes the following steps: S1. Add the anticoagulant to the umbilical cord blood and mix well. Then centrifuge to separate the plasma layer to obtain pretreated umbilical cord blood for later use. S2. Dilute the pretreated umbilical cord blood with diluent, then place it in a closed environment and adjust the oxygenation and pressurization to 0.3-0.5 MPa. Shake the blood on a shaker for 10-15 minutes to obtain a diluted blood sample for later use. S3. Slowly inject the diluted blood sample into the upper layer of the pre-cooled separation solution at 10-12℃, and let it stand at room temperature until it returns to room temperature to obtain the mixed separation solution of blood. S4. Centrifuge the blood in the mixed separation solution. After centrifugation, collect the second layer of mononuclear cells from the top for later use. S5. After mixing the mononuclear cell layer with diluent, continue centrifugation, discard the supernatant, and the lower precipitate is the separated umbilical cord blood mononuclear cells.
2. The separation method according to claim 1, characterized in that: The anticoagulant in step S1 is sodium heparin.
3. The separation method according to claim 1, characterized in that: The specific method for centrifuging and separating the plasma layer is to centrifuge the umbilical cord blood at a speed of 1800-2000 r / min for 10 min, and control the centrifugation acceleration time to be 9 min and the centrifugation deceleration time to be 4 min.
4. The separation method according to claim 1, characterized in that: The diluent in steps S2 and S5 is 0.9% sodium chloride injection, and in step S2, the diluent is mixed with the pretreated umbilical cord blood in an equal volume ratio.
5. The separation method according to claim 1, characterized in that: In step S2, the rotational speed of the shaking table is 40-60 r / min.
6. The separation method according to claim 1, characterized in that: In step S3, the separation solution is Ficoll separation solution, and the volume ratio of the separation solution to the diluted blood sample is 1:1-2.
7. The separation method according to claim 1, characterized in that: In step S4, the centrifugation speed is 1800-2800 r / min, and the centrifugation time is 25-30 min; the centrifugation acceleration time is controlled at 6 min, and the deceleration time is controlled at 2 min.
8. The separation method according to claim 1, characterized in that: In step S5, the centrifugation speed is 1800-2000 r / min, and the centrifugation time is 8-15 min. The centrifugation acceleration time is controlled at 9 min, and the deceleration time is controlled at 5 min.