Method for rapidly and efficiently separating monkey PBMC

Abstract

The invention discloses a method for quickly and efficiently separating monkey PBMC (peripheral blood monkey monkey). According to the method, the red blood cells are split by using sterile water and a sterile NaCl solution with the mass fraction of 1.7%-2.2%, the red blood cells can be effectively split, the PBMC is not influenced, and the separated PBMC is high in purity and motility rate; monkey serum is used for preparing 17%-22% DMSO as a cell cryopreservation solution, and a cryopreservation mode of precooling and dropwise adding is adopted, so that sufficient nutrients can be provided for cells, the damage to the cells in the cryopreservation cooling process is greatly reduced, and the motility rate is increased; a serum-containing RPMI-1640 culture medium is added before cell counting, so that sufficient nutrients can be provided for cells, cell death can be reduced, and residual lymphocyte separation liquid can be effectively cleaned and removed. According to the method, the number of extracted PBMC cells can be remarkably increased, the cell recovery rate is increased, and the application prospect is wide.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly and efficiently separating monkey PBMC. Background Art

[0002] Non-human primates are closest to humans in terms of kinship and are highly similar to humans in terms of tissue structure, pathophysiology, immune regulation and energy metabolism. Therefore, the research value of non-human primates such as crab-eating macaques is significantly better than that of other species of experimental animals.

[0003] PBMC, or peripheral blood mononuclear cells, mainly include two categories: lymphocytes and monocytes. They are an important part of the immune system and play a major role in both specific and nonspecific immunity. PBMC isolation technology is a basic experimental technology for immune system research.

[0004] The currently commonly used PBMC separation is mainly based on the density gradient centrifugation method. During the separation process, red blood cells are easily absorbed. Red blood cells not only affect the purity of the product, but also confuse the cell count, resulting in an inaccurate number of frozen cells, which affects the freezing effect and subsequent recovery culture. The currently commonly used method to exclude red blood cells is to add red blood cell lysis solution, which has the disadvantages of incomplete lysis of residual red blood cells or excessive lysis that damages PBMC. In addition, the formula of red blood cell lysis solution is complicated, and if the cleaning is not thorough during the operation, residual reagents may remain.

[0005] Commonly used PBMC cryopreservation solutions are commercially available cell cryopreservation solutions, such as Biolife Solutions' Cryostor, etc. These commercially available cell cryopreservation solutions cannot effectively maintain the activity of PBMC cells, and the viability of frozen PBMC cells after recovery is not high.

[0006] In summary, the cell recovery rate of PBMC obtained by the commonly used extraction method is generally only 80%-90%, and 5 mL of whole blood can generally only extract 0.5×10 7 Therefore, there is an urgent need to provide a monkey PBMC separation and freezing method that can significantly increase the PBMC extraction efficiency and improve the PBMC recovery rate. Summary of the invention

[0007] The purpose of the present invention is to provide a method for rapidly and efficiently isolating monkey PBMC. By optimizing the separation and freezing methods, the recovery rate of the isolated PBMC cells can reach 95%-99%, and 1-3×10 7 cells, significantly better than existing technologies.

[0008] To achieve the above-mentioned purpose, the present invention adopts the following technical solutions:

[0009] A method for rapidly and efficiently isolating monkey PBMCs comprises the following steps:

[0010] (1) Fresh blood was collected from experimental monkeys and anticoagulated with sodium heparin;

[0011] (2) Mixing equal volumes of whole blood and D-Hank's solution to obtain mixed blood;

[0012] (3) superimposing the mixed blood on the lymphocyte separation solution and centrifuging;

[0013] (4) Pipette the middle PBMC layer into a centrifuge tube, add D-Hank's solution to mix, centrifuge, and discard the supernatant;

[0014] (5) Add sterile water to lyse the red blood cells, gently pipette and mix thoroughly, then add 1.7%-2.2% sterile NaCl solution, mix well, centrifuge, and discard the supernatant;

[0015] (6) Add D-Hank's solution to wash again and retain the precipitate;

[0016] (7) adding serum-containing RPMI-1640 medium to the precipitate of step (6), mixing to obtain a cell suspension and counting the cells;

[0017] (8) Centrifuging the cell suspension, discarding the supernatant, adding monkey serum to adjust the cell concentration, and then dispensing into cryopreservation tubes;

[0018] (9) adding an equal volume of pre-cooled cell freezing solution dropwise into the cryopreservation tube on wet ice and mixing; the cell freezing solution is composed of 78%-83% monkey serum and 17%-22% DMSO;

[0019] (10) Place the cells in a programmed cooling box and store them in an ultra-low temperature refrigerator overnight. The next day, transfer them to liquid nitrogen for storage.

[0020] Preferably, in step (3), the volume ratio of the mixed blood to the lymphocyte separation fluid is 2:1.

[0021] Preferably, in step (3), the centrifugation is performed at 1000 x g for 30 min, the speed is adjusted to 0, and the centrifugation temperature is 20°C.

[0022] Preferably, in step (4), the volume ratio of the D-Hank's solution to the PBMC layer is 5:1.

[0023] Preferably, in steps (4), (5) and (8), the centrifugation is performed at 1500 rpm for 10 min at a centrifugation temperature of 20°C.

[0024] Preferably, in step (5), the reagents used to lyse red blood cells are sterile water and sterile NaCl solution, with a volume ratio of 1:1, and the order of addition is sterile water first, and then sterile NaCl solution 1 minute later, and the mass fraction of sterile NaCl solution is 1.7%-2.2%.

[0025] The principle of the method for lysing red blood cells and eliminating red blood cell interference in step (5) of the present invention is: using sterile water to place the cells in a hypotonic environment, red blood cells are easy to rupture within 1 minute, while PBMCs are not easy to rupture, and then a sodium chloride solution with a concentration of about 2 times that of normal saline is added in time to make the environment in which the cells are located become isotonic, thereby terminating the lysis.

[0026] Preferably, in step (8), the cell concentration is adjusted to 2×10 7 cells / mL.

[0027] Preferably, in step (9), the precooling of the cell freezing solution is to precool the cell freezing solution at 4°C.

[0028] Preferably, in step (10), the overnight storage in an ultra-low temperature refrigerator is overnight storage at -80°C.

[0029] The second object of the present invention is to provide a cell freezing solution, which is composed of 78%-83% monkey serum and 17%-22% DMSO.

[0030] The third object of the present invention is to provide the use of the above-mentioned cell freezing solution in the preparation of products of cryopreserved monkey PBMC.

[0031] Preferably, the product comprises a kit.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. The present invention uses sterile water + sodium chloride solution with a mass fraction of 1.7%-2.2%, which can effectively lyse red blood cells and has no effect on PBMC. The separated PBMC has high purity and high viability.

[0034] 2. The cell freezing solution of the present invention is composed of 78%-83% monkey serum and 17%-22% DMSO, and has the following advantages: serum of the same animal species can provide sufficient nutrients to cells, greatly reduce the damage to cells during the freezing and cooling process, and improve the survival rate.

[0035] 3. The present invention optimizes the steps of adding the freezing solution, adopts a pre-cooling method and a drop-by-drop addition method, and operates on wet ice to reduce the damage to cells caused by the large amount of heat generated when DMSO is diluted.

[0036] 4. The present invention adds serum-containing RPMI-1640 culture medium before cell counting, which has the following advantages: serum-containing RPMI-1640 culture medium has sufficient nutrients. The cell counting process may take a long time, and the culture medium can provide cells with sufficient nutrients to reduce cell death. Moreover, adding RPMI-1640 culture medium once is equivalent to washing the cells once more, which can effectively wash and remove the residual lymphocyte separation fluid.

[0037] 5. The present invention optimizes the separation and freezing methods, and the recovery rate of isolated PBMC cells can reach 95%-99%, and 1-3×10 7 cells, significantly better than existing technologies. DETAILED DESCRIPTION

[0038] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0039] Example 1

[0040] A method for rapidly and efficiently isolating monkey PBMCs comprises the following steps:

[0041] (1) Fresh blood was collected from experimental monkeys, sodium heparin was added for anticoagulation, and the laboratory temperature was adjusted to 20-25°C.

[0042] (2) 2 mL whole blood + 2 mL D-Hank's solution (restored to room temperature) and mix well.

[0043] (3) The mixed blood was superimposed on 2 mL of lymphocyte separation solution (restored to room temperature), and centrifuged at 1000 x g for 30 min. The speed was adjusted to 0 and the centrifugation temperature was 20°C.

[0044] (4) Pipette the middle PBMC layer (the white flocculent layer floating in the middle) into a 15 mL centrifuge tube, add 5 times more D-Hank's solution and mix well, centrifuge at 1500 rpm for 10 min at 20°C, and discard the supernatant after centrifugation.

[0045] (5) Add 1 mL of sterile water to lyse the red blood cells, gently pipette and mix thoroughly, add 1 mL of 1.7%-2.2% sterile NaCl solution within 1 min, mix well, centrifuge at 1500 rpm for 10 min at 20°C, and discard the supernatant after centrifugation.

[0046] (6) Add D-Hank's solution to wash again and retain the precipitate.

[0047] (7) Preparation of cell freezing solution: Use monkey serum to prepare DMSO with a volume fraction of 17%-22%, and precool it together with the freezing tube at 4°C.

[0048] (8) Add 10 mL of RPMI-1640 culture medium (containing serum) to the precipitate in step (6) and mix well to obtain a cell suspension.

[0049] (9) The cell suspension was stained with 0.4% trypan blue at a volume ratio of 1:1, and the total number of cells and the viability were calculated.

[0050] (10) The cell suspension from step (8) was centrifuged at 1500 rpm for 10 min at 20°C and the supernatant was discarded.

[0051] (11) Calculate the amount of monkey serum to be added based on the total number of cells and use monkey serum to adjust the cell concentration to 2×10 7 cells / mL and dispensed into cryopreservation tubes.

[0052] (12) Add an equal volume of the cell freezing solution prepared in step (7) dropwise and mix well (note that it should be shaken while adding and the operation should be done on wet ice). The final cell concentration is 1×10 7 cells / mL.

[0053] (13) Place the cells in a programmed cooling box at -80°C overnight and then transfer to liquid nitrogen for long-term storage.

[0054] Example 2

[0055] The effects of the method of the present invention (Example 1) and the commonly used PBMC isolation method on the total number of PBMC cells obtained from 5 mL of whole blood and the cell viability were compared. Among them, the commonly used PBMC isolation method was performed according to the instructions of the commercially available lymphocyte isolation kit A and the commercially available lymphocyte isolation kit B, respectively, and the results are shown in Table 1.

[0056] Table 1 Comparison with commonly used PBMC isolation methods

[0057] Blood sample (5 mL whole blood) Total number of PBMC cells Live rate Blood sample 1 (using commercially available lymphocyte isolation kit A) <![CDATA[0.52×10 7 ]]> 83.2％ Blood sample 2 (using commercially available lymphocyte isolation kit A) <![CDATA[0.56×10 7 ]]> 85.6％ Blood sample 3 (using commercially available lymphocyte isolation kit B) <![CDATA[0.61×10 7 ]]> 82.0％ Blood sample 4 (using commercially available lymphocyte isolation kit B) <![CDATA[0.50×10 7 ]]> 84.7％ Blood sample 5 <![CDATA[0.8×10 7 ]]> 99.60％ Blood sample 6 <![CDATA[1.5×10 7 ]]> 97.70％ Blood sample 7 <![CDATA[1.5×10 7 ]]> 99.40％ Blood sample 8 <![CDATA[1.2×10 7 ]]> 98.10％ Blood sample 9 <![CDATA[0.8×10 7 ]]> 99.60％ Blood sample 10 <![CDATA[1×10 7 ]]> 97.70％ Blood sample 11 <![CDATA[1.4×10 7 ]]> 99.60％ Blood sample 12 <![CDATA[2.7×10 7 ]]> 97.60％ Blood sample 13 <![CDATA[2.0×10 7 ]]> 99.60％ Blood sample 14 <![CDATA[2.3×10 7 ]]> 99.30％ Blood sample 15 <![CDATA[1.9×10 7 ]]> 99.40％ Blood sample 16 <![CDATA[1.1×10 7 ]]> 99.70％ Blood sample 17 <![CDATA[1.9×10 7 ]]> 98.40％

[0058] Note: Lymphocyte isolation kit A was purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., catalog number LTS1092;

[0059] Lymphocyte isolation kit B was purchased from Hangzhou Bioer Technology Co., Ltd., Cat# BSA07M1.

[0060] As shown in Table 1, the total number of cells extracted from 5 mL of whole blood was approximately 0.5 × 10 7 The cell viability was 82%-86%. However, the method of Example 1 of the present invention was used to separate and extract PBMC (blood samples 5-17). 5 mL of whole blood could extract 1-3×107 cells, and the cell viability reached 97%-99%.

[0061] Example 3

[0062] The effects of the method of the present invention (Example 1) and conventional commercially available cryopreservation solutions on the recovery rate of PBMC after cryopreservation and thawing, and the cell thawing viability were compared. The results are shown in Table 2.

[0063] Table 2 Comparison with commercially available cryopreservation solutions

[0064] Group Recovery rate after cryopreservation and resuscitation Resuscitation rate Commercially available cryopreservation solution A 85.6％ 85.1％ Commercially available cryopreservation solution B 84.2％ 84.6％ Cryopreservation method of the present invention 89.3％ 97.7％

[0065] Note: Commercially available cryopreservation solution A: Biolife Solutions, LOT#23231;

[0066] Commercially available freezing solution B: Biosharp, Cat# BL203B.

[0067] As shown in Table 2, when PBMCs are cryopreserved using conventional commercially available cryopreservation solutions, the cell recovery rate is only 84%-85%, while when the cell cryopreservation solution and cryopreservation method provided by the present invention are used, the cell recovery rate can reach 97.7%.

[0068] The above are only preferred embodiments of the present invention. It should be noted that the above preferred embodiments should not be regarded as limiting the present invention, and the protection scope of the present invention should be based on the scope defined by the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the spirit and scope of the present invention, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims

1. A method for rapidly and efficiently isolating monkey PBMC, characterized in that: The following steps are involved: (1) Fresh blood was collected from experimental monkeys and anticoagulated with sodium heparin; (2) Mixing equal volumes of whole blood and D-Hank's solution to obtain mixed blood; (3) superimposing the mixed blood on the lymphocyte separation solution and centrifuging; (4) Pipette the middle PBMC layer into a centrifuge tube, add D-Hank's solution to mix, centrifuge, and discard the supernatant; (5) Add sterile water to lyse the red blood cells, gently pipette and mix thoroughly, then add 1.7%-2.2% sterile NaCl solution, mix well, centrifuge, and discard the supernatant; (6) Add D-Hank's solution to wash again and retain the precipitate; (7) adding serum-containing RPMI-1640 medium to the precipitate of step (6), mixing to obtain a cell suspension and counting the cells; (8) Centrifuging the cell suspension, discarding the supernatant, adding monkey serum to adjust the cell concentration, and then dispensing into cryopreservation tubes; (9) adding an equal volume of pre-cooled cell freezing solution dropwise into the cryopreservation tube on wet ice and mixing; the cell freezing solution is composed of 78%-83% monkey serum and 17%-22% DMSO; (10) Place the cells in a programmed cooling box and store them in an ultra-low temperature refrigerator overnight. The next day, transfer them to liquid nitrogen for storage.

2. The method according to claim 1, characterized in that In step (3), the volume ratio of the mixed blood to the lymphocyte separation fluid is 2:

1.

3. The method according to claim 1, characterized in that In step (3), the centrifugation is performed at 1000 x g for 30 min, the speed is adjusted to 0, and the centrifugation temperature is 20°C.

4. The method according to claim 1, characterized in that: In step (4), the volume ratio of the D-Hank's solution to the PBMC layer is 5:

1.

5. The method according to claim 1, characterized in that: In steps (4), (5) and (8), the centrifugation is performed at 1500 rpm for 10 min at a centrifugal temperature of 20°C.

6. The method according to claim 1, characterized in that In step (5), the reagents used to lyse red blood cells are sterile water and sterile NaCl solution, with a volume ratio of 1:1, and the order of addition is to add sterile water first and then add sterile NaCl solution 1 minute later.

7. The method according to claim 1, characterized in that In step (8), the cell concentration is adjusted to 2×10 7 cells / mL.

8. A cell freezing solution, characterized in that: It is composed of 78%-83% monkey serum and 17%-22% DMSO.

9. Use of the cell freezing solution according to claim 8 in preparing a product of cryopreserved monkey PBMC.

10. The use according to claim 9, characterized in that: The products include kits.