Umbilical cord blood mononuclear cell separation and extraction method

Abstract

The invention provides an umbilical cord blood mononuclear cell separation and extraction method, and belongs to the technical field of cells. The invention provides an umbilical cord blood mononuclear cell separation and extraction method, which comprises the following steps: mixing umbilical cord blood and red blood cell sedimentation liquid, carrying out natural sedimentation, and collecting supernatant to obtain pretreated umbilical cord blood; and adding the pretreated umbilical cord blood into the upper layer of a sample density separation medium, centrifuging, and collecting an albuginea layer to obtain the mononuclear cells of the umbilical cord blood. According to the method, the cell recovery rate is effectively increased to 93% or above. Meanwhile, the vitrified cryopreservation liquid and the cryopreservation bag are optimized for cryopreservation of the mononuclear cells, the cell recovery rate can be effectively increased, the cryopreservation process is simplified, and the method is suitable for wide application and popularization.

Description

Technical Field

[0001] The invention belongs to the field of cell technology, and in particular relates to a method for separating and extracting mononuclear cells from umbilical cord blood. Background Art

[0002] Mononuclear cells in umbilical cord blood can be used to treat a variety of malignant blood tumors, acquired or inherited bone marrow failure syndromes, and non-malignant diseases (such as spinal cord injury, liver cirrhosis, diabetes, myocardial injury, and autoimmune diseases). Because umbilical cord blood has the advantages of sufficient sources and weak immunogenicity, it provides patients with more treatment options. Mononuclear cells in umbilical cord blood include lymphocytes and monocytes, which are important tools for studying the development and function of the immune system.

[0003] At present, the separation and extraction methods of umbilical cord blood mononuclear cells mainly include sedimentation separation and density gradient centrifugation separation. At present, the separation of umbilical cord blood mononuclear cells has the problem of low cell recovery rate. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a method for separating and extracting mononuclear cells from umbilical cord blood, which combines sedimentation method with density gradient centrifugation method to greatly improve the recovery rate of mononuclear cells.

[0005] The present invention provides a method for separating and extracting mononuclear cells from umbilical cord blood, comprising the following steps:

[0006] The umbilical cord blood and the red blood cell sedimentation fluid are mixed and then naturally precipitated, and the upper layer fluid is collected to obtain the pre-treated umbilical cord blood;

[0007] The pretreated umbilical cord blood is added to the upper layer of the sample density separation solution, centrifuged, and the buffy coat layer is collected to obtain umbilical cord blood mononuclear cells.

[0008] Preferably, the volume ratio of the umbilical cord blood and the erythrocyte sedimentation fluid is (0.8-1.2):(0.8-1.2).

[0009] Preferably, the natural sedimentation time is 20 to 50 minutes.

[0010] Preferably, after collecting the supernatant, the method further comprises mixing the supernatant with physiological saline to obtain pretreated umbilical cord blood.

[0011] Preferably, the volume ratio of the upper layer liquid to the physiological saline is (0.8-1.2):(0.8-1.2).

[0012] Preferably, the volume ratio of the pretreated umbilical cord blood and the sample density separation fluid is (0.8-1.2): (0.8-1.2).

[0013] Preferably, the centrifugation procedure is 800g, 20min.

[0014] Preferably, after obtaining the umbilical cord blood mononuclear cells, the method further includes cryopreservation of the umbilical cord blood mononuclear cells.

[0015] Preferably, the cryopreservation solution of the umbilical cord blood mononuclear cells comprises a vitrification cryopreservation solution;

[0016] The cryopreservation concentration of the umbilical cord blood mononuclear cells is 1×10 7 / ml~1.5×10 7 / ml.

[0017] Preferably, the cryopreservation container for the umbilical cord blood mononuclear cells comprises a cryopreservation bag.

[0018] The present invention provides a method for separating and extracting mononuclear cells from umbilical cord blood, comprising the following steps: mixing umbilical cord blood and red blood cell sedimentation fluid and subjecting them to natural sedimentation, collecting the upper layer of fluid to obtain pretreated umbilical cord blood; adding the pretreated umbilical cord blood to the upper layer of the sample density separation fluid, centrifuging, collecting the buffy coat layer, and obtaining umbilical cord blood mononuclear cells. The present invention combines sedimentation method with density gradient centrifugation method to separate and extract mononuclear cells from umbilical cord blood, which can greatly improve the recovery rate of cells. Experiments show that the cell recovery rate of the treatment group using only sedimentation method or density gradient centrifugation method is 17% to 20%, while the cell recovery rate of the treatment group using sedimentation method combined with density gradient centrifugation method is 29.4% to 35.1%, which can significantly improve the recovery rate of mononuclear cells. It can be seen that the separation and extraction method provided by the present invention can solve the problem of low recovery rate of mononuclear cells at present, and lay a foundation for the research and application of mononuclear cells.

[0019] The present invention further limits the cryopreservation of umbilical cord blood mononuclear cells, and the cell cryopreservation liquid limits a specific vitrified cryopreservation liquid, and the cryopreservation container is replaced by a cryopreservation bag. The vitrified cryopreservation liquid does not contain DMSO, which reduces the toxicity to the cells and further protects the cells. The vitrified cryopreservation liquid is a finished product, and there is no need to configure the cryopreservation liquid, which reduces the operating procedures. The use of the vitrified cryopreservation liquid does not require a program cooling box, and can be directly placed in a -80 refrigerator, which simplifies the cryopreservation process. In addition, the use of cryopreservation bags for cryopreservation can effectively simplify the operating procedures that require the cryopreservation tubes to be packaged, simplifying the operation. Experiments have shown that when the mononuclear cells obtained by the same separation and extraction method are cryopreserved, the viability of the mononuclear cells after cryopreservation can be significantly improved after the vitrified cryopreservation liquid replaces the cell culture containing DMSO, and the viability of the cryopreservation can be further improved after the cryopreservation bag replaces the cryopreservation tube. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of the umbilical cord blood sedimentation in Example 1 are as follows;

[0021] Figure 2This is the result after the umbilical cord blood pre-treated in Example 1 was added to the sample density separation solution. DETAILED DESCRIPTION

[0022] The present invention provides a method for separating and extracting mononuclear cells from umbilical cord blood, comprising the following steps:

[0023] The umbilical cord blood and the red blood cell sedimentation fluid are mixed and then naturally precipitated, and the upper layer fluid is collected to obtain the pre-treated umbilical cord blood;

[0024] The pretreated umbilical cord blood is added to the upper layer of the sample density separation solution, centrifuged, and the buffy coat layer is collected to obtain umbilical cord blood mononuclear cells.

[0025] The present invention mixes umbilical cord blood and red blood cell sedimentation fluid, performs natural sedimentation, collects the upper layer fluid, and obtains pretreated umbilical cord blood.

[0026] In the present invention, before the umbilical cord blood is mixed, the blood collection bag containing the umbilical cord blood is preferably disinfected. The method of disinfection preferably uses 75% alcohol for surface wiping. The volume ratio of the umbilical cord blood and the red blood cell sedimentation fluid is preferably (0.8-1.2): (0.8-1.2), and can be 1:1. The time for natural sedimentation is preferably 20-50 minutes, can be 25-40 minutes, and can also be 30 minutes. The temperature of natural sedimentation is preferably 20-27°C, and can be 25°C. The natural sedimentation causes the red blood cells in the umbilical cord blood to settle to the bottom layer of the solution, and the red blood cells in the umbilical cord blood are preliminarily removed.

[0027] In the present invention, after collecting the upper layer liquid, preferably the upper layer liquid is mixed with physiological saline to obtain pretreated umbilical cord blood. The volume ratio of the upper layer liquid to the physiological saline is preferably (0.8-1.2): (0.8-1.2), and can be 1:1.

[0028] After obtaining the pretreated umbilical cord blood, the present invention adds the pretreated umbilical cord blood to the upper layer of the sample density separation solution, centrifuges, collects the buffy coat layer, and obtains umbilical cord blood mononuclear cells.

[0029] In the present invention, the volume ratio of the pretreated umbilical cord blood and the sample density separation liquid is preferably (0.8-1.2): (0.8-1.2), which can be 1:1. When the pretreated umbilical cord blood is added, be careful to inject it gently to avoid the pretreated umbilical cord blood from destroying the liquid surface of the sample density separation liquid and preventing the two liquids from mixing. The centrifugation procedure is preferably 800g, 20min. After the centrifugation, the centrifugal system is preferably layered, from top to bottom, including a physiological saline layer, a white film layer (mononuclear cell layer), a separation liquid layer, and a red blood cell layer. When collecting the white film layer, it is preferred to aspirate the physiological saline layer, then slowly aspirate the white film layer, and transfer it to a new centrifuge tube. After collecting the white film layer, it is preferably washed with physiological saline.

[0030] In the present invention, after obtaining the umbilical cord blood mononuclear cells, it is preferred that the umbilical cord blood mononuclear cells are cryopreserved. The cryopreservation solution of the umbilical cord blood mononuclear cells preferably includes a vitrified cryopreservation solution. The cryopreservation concentration of the umbilical cord blood mononuclear cells is preferably 1×10 7 / ml~1.5×10 7 / ml. The cryopreservation container of the umbilical cord blood mononuclear cells preferably comprises a cryopreservation bag.

[0031] In the examples of the present invention, experiments were carried out to separate and extract mononuclear cells using only red blood cell sedimentation fluid sedimentation method, only sample density gradient fluid for density gradient centrifugation method, and a combination of the two methods. The results showed that compared with separation and extraction using only one method (Comparative Example 1 and Comparative Example 2), separation and extraction of mononuclear cells using a combination of the two methods (Example 1) can greatly improve the cell recovery rate, which is increased by more than 25%.

[0032] In another embodiment of the present invention, an optimization experiment was conducted on the cryopreservation method of isolated mononuclear cells, and a cell culture medium containing 10% DMSO, a vitrification cryopreservation solution, a cryopreservation tube, and a cryopreservation bag were used to carry out cryopreservation experiments. The results showed that compared with Comparative Example 5 (using cryopreservation tubes), the cryopreservation method in Example 1 (using cryopreservation bags) had a significantly improved resuscitation rate. Compared with Comparative Example 3, the cryopreservation method in Example 1 had a significantly improved resuscitation rate.

[0033] The following is a detailed description of the method for separating and extracting mononuclear cells from umbilical cord blood provided by the present invention in conjunction with the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1

[0035] A method for separating and extracting mononuclear cells from umbilical cord blood

[0036] 1. Instruments and Reagents

[0037] 1.1. Reagents: Sample density separation solution (prepared by FICOLL), brand Tianjin Haoyang, item number: LDS1075, packaging specification: 200ml;

[0038] Vitrification cryopreservation solution: purchased from Youkang Biotechnology (Beijing) Co., Ltd., specification 100mL / bottle; catalog number NC1011;

[0039] Erythrocyte sedimentation fluid (6% hydroxyethyl starch 550) was purchased from Wak-chemie, with the catalog number Lot: 234657641 Exp: 2025.10;

[0040] Umbilical cord blood-derived blood samples 1, 2, 3 and 4 were obtained from Changsha Maternal and Child Health Hospital.

[0041] 1.2. Equipment: The cryobags were purchased from OriGen, product model: CS50 Lot: W23167 Exp: 2027-02-01.

[0042] 1.3. Instruments and Equipment

[0043] 1.3.1 Biological safety cabinet, clean bench, centrifuge.

[0044] 2. Operation environment: It should be carried out in a Class A clean bench or biological safety cabinet in the Class B clean area of ​​the cell preparation department.

[0045] 3. Preparation

[0046] Before the operation begins, preparatory work should be carried out in accordance with YP-CP-SOP-037 "Standard Operating Procedure for Pre-Operation Inspection" and the completion of the preparatory work should be checked.

[0047] 4. Preparation Steps

[0048] 4.1 Umbilical cord blood pretreatment:

[0049] Gently invert the blood collection bag 3 times to mix it, wipe the opening of the blood collection bag catheter with gauze soaked in 75% alcohol, clamp the bottom of the catheter opening with hemostats, and cut the catheter opening with scissors. Prepare a 50ml centrifuge tube according to the volume of blood collected, open it for later use, and pour the umbilical cord blood in the blood collection bag into the centrifuge tube. Measure the initial white blood cell count (WBC) using a blood cell analyzer.

[0050] According to the blood volume, red blood cell sedimentation fluid (6% hydroxyethyl starch 550) was added at a volume ratio of 1:1 and allowed to stand at room temperature for 30 minutes.

[0051] There are two layers from top to bottom in the centrifuge tube, which are the ring-shaped milky white blood cell layer and the red blood cell layer. Collect the white blood cell layer, add the corresponding volume of physiological saline in a 1:1 ratio, mix thoroughly and wash, and obtain the pretreated umbilical cord blood (see Figure 1 ).

[0052] 4.2 Isolation and recovery of umbilical cord blood-derived mononuclear cells (UCB-MNCs)

[0053] The sample density separation solution (prepared by FICOLL) was dispensed into new 50 ml centrifuge tubes, 15 ml per tube.

[0054] Use a 10ml pipette to draw pre-treated umbilical cord blood and slowly add it along the tube wall. Be careful not to break the interface between the blood and the separation solution. Each tube should not exceed 30ml of diluted and mixed blood. After the layering is completed, carefully cover the centrifuge tube and take it out. After balancing, put it into the centrifuge. The whole process must be gentle to prevent the blood sample from mixing with the separation solution. Centrifuge at room temperature at 800g for 20 minutes.

[0055] After centrifugation, carefully take out the centrifuge tube and put it on the operating table. You can clearly see that the centrifuge tube contains a saline layer, a white film layer (mononuclear cell layer), a separation liquid layer, and a red blood cell layer from top to bottom. Use a pipette to suck up the saline layer and discard it (be careful not to suck up the mononuclear cell layer). Then pipette the white film layer into a new 50ml centrifuge tube, add an equal volume of saline and mix well, then add saline to make up to 45ml, pipette and mix well, centrifuge at room temperature at 500g for 10 minutes, collect the cells, and see Figure 2 After centrifugation, the supernatant was removed, the cell pellet was resuspended with saline, and the pellet was transferred into a centrifuge tube. The volume was then fixed to 45 ml with saline. The number of white blood cells was measured by a hematology analyzer to calculate the cell recovery rate and to serve as the basis for subsequent adjustment of the frozen cell concentration.

[0056] The cell recovery rate was calculated based on the cell counting results.

[0057] Cell recovery rate (%) = (number of mononuclear cells after separation) / (number of mononuclear cells in whole blood) × 100% Formula II.

[0058] 4.3 Cell washing and pre-cryopreservation treatment

[0059] The mononuclear cells resuspended in physiological saline were centrifuged at 400 g for 12 min at room temperature to collect the cell pellet. The volume of the cryopreservation solution was calculated according to the number of white blood cells in the counting results according to Formula III, and the vitrification cryopreservation solution was added to the cell pellet to ensure that the cryopreservation density of white blood cells was about 1×10 7 / ml~1.5×10 7 / ml to obtain cell suspension.

[0060] Cryopreservation fluid volume = total number of white blood cells / cryopreservation density Formula III.

[0061] 4.4 Transfer to cryopreservation bag

[0062] Wipe the surface of the cryopreservation bag with gauze and place it in the biosafety cabinet, then wipe the opening of the cryopreservation bag catheter with 75% alcohol gauze.

[0063] Remove the Luer connector from the tubing of the cryobag.

[0064] Use a syringe to draw the cell suspension from the centrifuge tube, remove the needle of the syringe, insert the syringe into the opening of the cryopreservation bag, open the hemostatic clamp, inject the cell suspension into the cryopreservation bag, and repeat the above steps until all the cell suspension is injected into the cryopreservation bag.

[0065] Then use a syringe to suck out the air in the freezing bag, clamp the hemostat, and pull out the syringe.

[0066] Heat seal the cryopreservation bag tubing near the bag end and at the tee of the tubing, leaving 2 small braids of about 5 to 6 cm for cell counting and viability testing during recovery.

[0067] 4.5 Mark the preparation number on the cryopreservation bag with a marker pen, pass it out of the clean area through the transfer window, and then use a marking machine to mark the cell preparation number on the freezing box in the outer packaging room, and then put the cryopreservation bag directly into the -80 refrigerator. Transfer the cells to -196℃ liquid nitrogen as soon as possible within 24 hours for long-term storage.

[0068] 4.6 Cell Recovery

[0069] Open the thermostatic water bath in advance, set the temperature of the thermostatic water bath to 40℃, and make the temperature of the thermostatic water bath reach 40℃ and stabilize. Confirm whether the number of the removed cells / cell preparations is consistent with the number of the cells / cell preparations in the approved outbound application; quickly put the cells / cell preparations taken out of the liquid nitrogen tank into a thermostatic water bath heated to 40℃, and shake them from time to time to make them melt quickly and completely, and the time should not exceed 3 minutes. After the cells / cell preparations are completely melted, clean their surfaces. Use a 50ml syringe to collect the cells in the cryopreservation bag into a 50ml centrifuge tube, place the centrifuge tube in the centrifuge, balance all centrifuge tubes, set the centrifugation parameters to: 680×g, 9 up and 9 down, centrifuge for 10 minutes, and discard the supernatant after the centrifugation. Resuspend the cells with 0.9% saline containing 0.1% albumin, pipette evenly, and then dilute to 40ml with 0.9% saline containing 0.1% albumin. Place the centrifuge tubes in the centrifuge, balance all the centrifuge tubes, set the centrifugation parameters to: 680×g, centrifuge for 10min, discard the supernatant after the centrifugation, and repeat this step once. After diluting to 40ml for the second time, take 500μL of the cell suspension and detect the cell number and recovery viability.

[0070] The calculation formula for the resuscitation survival rate is shown in Formula IV.

[0071] Cell recovery rate = (number of viable cells after recovery) / (total number of cells)×100% Formula IV.

[0072] Comparative Example 1

[0073] The blood sample in Example 1 was used to separate and extract umbilical cord blood mononuclear cells. Compared with the method in Example 1, the only difference was that the process of mixing the sample density separation solution for density gradient centrifugation was omitted, and the vitrification freezing solution was replaced with a cell culture medium containing 10% DMSO during freezing (MSC-T4 culture medium produced by Beso, product number ot: ​​03112401Exp: 2025.03.10, the same below), and the other steps were the same as the method described in Example 1.

[0074] Comparative Example 2

[0075] The blood sample in Example 1 was used to separate and extract umbilical cord blood mononuclear cells. Compared with the method in Example 1, the only difference was the process of red blood cell sedimentation in red blood cell sedimentation fluid (pretreatment process) and the replacement of the vitrification freezing fluid with a cell culture medium containing 10% DMSO during freezing. The other steps were the same as those described in Example 1.

[0076] Comparative Example 3

[0077] The blood sample in Example 1 was used to separate and extract umbilical cord blood mononuclear cells. Compared with the method in Example 1, the only difference was that the vitrification freezing solution was replaced with a cell culture medium containing 10% DMSO during freezing, and the other steps were the same as those described in Example 1.

[0078] Comparative Example 4

[0079] The blood sample in Example 1 was used to separate and extract umbilical cord blood mononuclear cells. Compared with the method in Example 1, the only difference was that the vitrification freezing solution was replaced with a cell culture medium containing 10% DMSO during freezing, and the freezing bag was replaced with a freezing tube. The other steps were the same as those described in Example 1.

[0080] Comparative Example 5

[0081] The blood sample in Example 1 was used to separate and extract umbilical cord blood mononuclear cells. Compared with the method in Example 1, the only difference was that the cryopreservation bag was replaced with a cryopreservation tube during freezing, and the other steps were the same as those described in Example 1.

[0082] The separation, extraction and cryopreservation effects of Example 1 and Comparative Examples 1 to 5 are shown in Table 1.

[0083] Table 1 Separation, extraction and cryopreservation effects of Example 1 and Comparative Examples 1 to 5

[0084]

[0085]

[0086]

[0087] As shown in Table 1, from the results of blood samples from four groups of different sources, it can be seen that the umbilical cord blood is respectively subjected to sedimentation pretreatment with red blood cell sedimentation fluid and density gradient centrifugation with sample density gradient fluid, which can greatly improve the recovery rate of mononuclear cells compared with only a single separation method, which is 29.4% to 35.1%. At the same time, the present invention also optimizes the recovery of frozen mononuclear cells. The results show that the use of cell culture medium containing 10% DMSO to recover cells is not conducive to the improvement of recovery survival rate, while the present invention uses vitrification freezing fluid to recover cells, which can greatly improve the recovery survival rate of cells. At the same time, the use of freezing bags to replace freezing tubes for cell freezing can further recover the survival rate. The experiment shows that the freezing and recovery of mononuclear cells using the technical solution of the present invention can greatly improve the freezing survival rate, reaching 93.4% to 95.3%.

[0088] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for separating and extracting mononuclear cells from umbilical cord blood, characterized in that: The following steps are involved: The umbilical cord blood and the red blood cell sedimentation fluid are mixed and then naturally precipitated, and the upper layer fluid is collected to obtain the pre-treated umbilical cord blood; The pretreated umbilical cord blood is added to the upper layer of the sample density separation solution, centrifuged, and the buffy coat layer is collected to obtain umbilical cord blood mononuclear cells.

2. The method for separating and extracting mononuclear cells from cord blood according to claim 1, characterized in that: The volume ratio of the umbilical cord blood to the red blood cell sedimentation fluid is (0.8-1.2): (0.8-1.2).

3. The method for separating and extracting mononuclear cells from cord blood according to claim 1, characterized in that: The natural sedimentation time is 20 to 50 minutes.

4. The method for separating and extracting mononuclear cells from cord blood according to claim 1, characterized in that: After collecting the upper layer fluid, the method further includes mixing the upper layer fluid with physiological saline to obtain pretreated umbilical cord blood.

5. The method for separating and extracting mononuclear cells from cord blood according to claim 4, characterized in that: The volume ratio of the upper layer liquid to the physiological saline is (0.8-1.2):(0.8-1.2).

6. The method for separating and extracting mononuclear cells from cord blood according to claim 1, characterized in that: The volume ratio of the pretreated umbilical cord blood to the sample density separation fluid is (0.8-1.2): (0.8-1.2).

7. The method for separating and extracting mononuclear cells from cord blood according to claim 1, characterized in that: The centrifugation procedure was 800 g, 20 min.

8. The method for separating and extracting mononuclear cells from umbilical cord blood according to any one of claims 1 to 7, characterized in that: After obtaining the umbilical cord blood mononuclear cells, the umbilical cord blood mononuclear cells are also cryopreserved.

9. The method for separating and extracting mononuclear cells from cord blood according to claim 8, characterized in that: The cryopreservation solution of the umbilical cord blood mononuclear cells includes a vitrification cryopreservation solution; The cryopreservation concentration of the umbilical cord blood mononuclear cells is 1×10 7 / ml~1.5×10 7 / ml.

10. The method for separating and extracting mononuclear cells from cord blood according to claim 8, characterized in that: The cryopreservation container of the umbilical cord blood mononuclear cells comprises a cryopreservation bag.

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