CN114410580A - A kind of extraction method of PBMC - Google Patents

Abstract

The invention relates to the technical field of PBMC detection, in particular to a PBMC extraction method, which comprises the following steps of S1: adding a sample diluted by whole blood into a centrifuge tube, adding an HBSS solution, and performing 1: 1, diluting; s2: slowly adding the diluted blood sample into a centrifuge tube filled with lymphocyte separation liquid to ensure that the blood sample is on the upper layer of the lymphocyte separation liquid; s3: placing the solution obtained in the step S2 at room temperature, and performing horizontal centrifugal separation to separate the solution into layers; s4: taking a white film layer, adding a cleaning solution, and cleaning; s5: performing centrifugal separation on the mixed solution obtained in the step S4, and re-suspending cells to obtain a cell suspension; s6: taking the cell suspension, and carrying out cell counting and activity detection. The whole blood sample is divided into a plurality of parts, and the parts are respectively extracted and then combined, so that the influence of operation on the extraction result is reduced. Compared with the prior art, the extraction method has higher cell yield and repeatability by controlling the experimental conditions in the extraction method.

Description

A kind of extraction method of PBMC

【Technical field】

The invention relates to the technical field of PBMC detection, in particular to a method for extracting PBMC.

【Background technique】

Peripheral blood mononuclear cells (PBMC) include lymphocytes and monocytes, while lymphocytes include T cells, B cells and natural killer (NK) cells. PBMC is the most commonly used cell model in the study of immunological functions, such as cell proliferation, cytotoxicity, cytokine secretion, etc. For example, in the field of cancer immunotherapy, PBMCs need to be isolated from the whole blood of patients, and the isolated PBMCs can be further expanded or subjected to different functional tests.

At present, for the extraction of PBMC, the method of density gradient centrifugation is mostly used. However, in clinical trials, due to different experimental conditions such as separation liquid, centrifugal force, and centrifugation time, the total number of cells obtained varies greatly.

[Content of the invention]

The purpose of the present invention is to provide a method for extracting PBMC, which is suitable for the process of extracting PBMC from whole blood, and overcomes the problem of low cell acquisition rate.

To achieve the above object, the present invention has adopted the following technical solutions:

A kind of extraction method of PBMC, it is characterized in that: comprise the steps,

S1: Take a whole blood sample and add it to a centrifuge tube, add HBSS solution for 1:1 dilution;

S2: Slowly add the diluted blood sample into the centrifuge tube containing the lymphocyte separation solution, so that the blood sample is on the upper layer of the lymphocyte separation solution;

S3: Place the solution obtained in step S2 at room temperature, and perform horizontal centrifugation to separate the solution into layers, which are the red blood cell layer, the separation liquid layer, the buffy coat layer (mononuclear cell layer) and the diluted plasma layer in order from bottom to top ;

S4: Take the buffy coat layer, add cleaning solution, and clean;

S5: the mixed solution obtained in step S4 is centrifuged, the supernatant is aspirated and discarded, the washing solution is added, and the cells are resuspended to obtain a cell suspension;

S6: Take the cell suspension for cell counting and viability detection.

As a further improvement of the present invention, in the step S1, the diluted samples of whole blood are divided into at least two parts, which are extracted separately, and the finally obtained extracts are combined.

As a further improvement of the present invention, the lymphocyte separation solution is Ficoll Paque solution.

As a further improvement of the present invention, the conditions for the horizontal centrifugation in step S3 are canceling the braking mode, rotating at 790 g, and centrifuging for 20 minutes.

As a further improvement of the present invention, the cleaning solution in step S4 is HBSS solution.

As a further improvement of the present invention, the conditions for centrifugation in step S5 are to set the maximum acceleration and braking mode, at room temperature of 290 g, and centrifuge for 10 minutes.

Compared with the prior art, the present invention has the beneficial effect of dividing the whole blood sample into at least two parts, which are extracted separately and then merged after being lifted, thereby reducing the influence of the operation on the extraction result. The control of the centrifugation conditions in step S2 makes it easier for the mixed solution to be stratified between solutions of different densities and avoids excessive loss of lymphocytes. The control of centrifugal force in step S5 avoids the risk of cell lysis. Compared with the existing method, the extraction method of the present invention has higher cell acquisition rate and repeatability.

【Description of Attachment】

FIG. 1 is a graph comparing the total number of PBMC cells in the example of the present invention and the comparative example.

Fig. 2 is a graph comparing the PBMC viability of the embodiment of the present invention and the comparative example.

【Detailed ways】

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some, but not all, embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

The reagents used in the following examples are as follows:

reagent factory article number Storage conditions Ficoll Paque GE Healthcare 17-5442-02 2-8℃ HBSS gibco 14170-112 2-8℃ AO/PI Stain* CountStar RE010212 2°C-8°C

Example 1

A kind of extraction method of PBMC, specifically comprises the steps:

a) Prepare three 15mL centrifuge tubes for each sample in advance, and mark the sample information.

b) In the biological safety cabinet, add 5ml of Ficoll Paque solution to 2 15ml centrifuge tubes corresponding to each sample with a serological pipette.

c) Use an electronic pipette to slowly add the diluted blood sample to the two 15mL centrifuge tubes containing Ficoll Paque solution along the tube wall. Clear layered interface.

d) Put the centrifuge tube into the horizontal rotor of the centrifuge, cancel the braking mode, and centrifuge at 790g at room temperature for 20 minutes.

e) After centrifugation, observe the layering effect. The red blood cells should sink to the bottom layer, followed by the separation liquid layer, the buffy coat layer (mononuclear cell layer) and the diluted plasma layer.

f) First suck up the upper diluted plasma layer and discard it until it is 2-3mm away from the buffy coat layer.

g) Slowly aspirate the buffy coat cells with a sterile tube, being careful not to aspirate other layers, transfer the buffy coat layer to a new 15mL centrifuge tube, and finally make up to 15mL with 1×HBSS and mix well.

h) Set the maximum acceleration and braking mode, and centrifuge at 290g for 10 minutes at room temperature.

i) Discard the supernatant, flick the bottom cell pellet, add 1×HBSS again to 15mL and mix by gentle inversion to resuspend the cells.

j) Set the maximum acceleration and braking mode, and centrifuge at 290g for 10 minutes at room temperature.

k) After discarding the supernatant, add 4 mL of 1× HBSS, flick the bottom of the test tube to mix well, and resuspend the cells.

l) Combine the resuspended cell solutions, conduct cell count and viability detection and record in the table.

Example 2

Comparative ratio

a) Prepare 2 50mL and 2 15mL centrifuge tubes for each sample in advance, and mark the sample information.

b) Add 15 mL of Ficoll Paque solution to a 50 mL centrifuge tube corresponding to each sample for use.

c) Use an electronic pipette and a serological pipette (sterile) to draw 1x HBSS to rinse the blood collection tube in turn, and continue to transfer it into the corresponding 50mL centrifuge tube above. The final volume obtained was twice the volume of the Ficoll Paque solution, ie 30 mL.

d) Use an electronic pipette and a serological pipette (sterile) to slowly add the diluted blood sample to the aforementioned 50mL centrifuge tube containing Ficoll Paque solution along the tube wall to ensure that all the blood samples are on the upper layer of the separation solution and form a clear layered interface.

e) Put the centrifuge tube into the horizontal rotor of the centrifuge, set the maximum acceleration and cancel the braking mode, and centrifuge at 490g at room temperature for 40 minutes.

f) After centrifugation, observe the layering effect. The red blood cells should sink to the bottom layer, followed by the separation liquid layer, the buffy coat layer (mononuclear cell layer) and the diluted plasma layer.

g) First suck up the upper diluted plasma layer and discard it until it is 2-3mm away from the buffy coat layer.

h) Slowly aspirate the buffy coat cells with a sterile pipette, being careful not to aspirate other layers, and transfer the buffy coat layer to a new 15mL centrifuge tube.

i) Set the maximum acceleration and braking mode, and centrifuge at 280g for 10 minutes at room temperature.

j) Transfer the supernatant to a new 15mL centrifuge tube to avoid cell loss as much as possible.

k) Add 1 mL of 1x HBSS and mix by flicking the bottom of the tube to resuspend the cells.

l) Carry out cell count and viability test and record in the table.

Example 3

Cell counting and viability assays

a) Take an EP tube for each sample and mark the sample information.

b) Add 12 μL of AO/PI staining solution and an equal volume of sample to the EP tube and mix well.

c) Take out the cell counting plate and mark it, and add 20 μL of sample mixture to each empty slot.

d) Insert the sample plate into the inlet of the cell counter, select "AOPI cell viability", enter the sample information, and click "Start" to perform automatic measurement.

According to the above process, the cell solutions obtained in Example 1 and Example 2 were respectively counted and tested for viability, and the number of cells obtained and cell viability of PBMC were counted. The statistical results are shown in Table 1 below.

Table 1: Cell Counting and Viability Test Results of Example 1 and Example 2

The statistical results showed that the total number and concentration of PBMC cells obtained by the method of Example 1 were significantly higher than the total number and concentration of PBMC cells obtained by the method of Example 2, and both methods had higher cell viability.

In the extraction method of the present invention, the whole blood sample is divided into at least two parts, which are respectively subjected to extraction processing and then combined after being lifted, thereby reducing the influence of the operation on the extraction result. The control of the centrifugation conditions in step S2 makes it easier for the mixed solution to be stratified between solutions of different densities and avoids excessive loss of lymphocytes. The control of centrifugal force in step S5 avoids the risk of cell lysis. Compared with the existing method, the present invention has higher cell acquisition rate and repeatability.

Claims (6)

1. A PBMC extraction method is characterized in that: comprises the following steps of (a) carrying out,

s1: adding a whole blood sample into a centrifuge tube, adding an HBSS solution, and performing 1: 1, diluting;

s2: slowly adding the diluted blood sample into a centrifuge tube filled with lymphocyte separation liquid to ensure that the blood sample is on the upper layer of the lymphocyte separation liquid;

s3: placing the solution obtained in the step S2 at room temperature, performing horizontal centrifugal separation to layer the solution, and sequentially including a red blood cell layer, a separation liquid layer, a leukocyte layer (mononuclear cell layer), and a diluted plasma layer from bottom to top;

s4: taking a white film layer, adding a cleaning solution, and cleaning;

s5: centrifuging the mixed solution obtained in the step S4, removing supernatant, adding a cleaning solution, and resuspending cells to obtain a cell suspension;

s6: taking the cell suspension, and carrying out cell counting and activity detection.

2. The method for extracting PBMCs according to claim 1, wherein: in step S1, the sample after dilution of whole blood is divided into at least 2 parts, and extraction processes are respectively performed, and finally obtained extraction solutions are combined.

3. The method for extracting PBMCs according to claim 1, wherein: the lymphocyte separation solution is a Ficoll-Paque solution.

4. The method for extracting PBMCs according to claim 1, wherein: the conditions of the horizontal centrifugation in step S3 are to cancel the braking mode, rotate at 790g, and centrifuge for 20 minutes.

5. The method for extracting PBMCs according to claim 1, wherein: the cleaning solution in step S4 is HBSS solution.

6. The method for extracting PBMCs according to claim 1, wherein: the conditions of the centrifugation in the step S5 are to set the maximum acceleration and braking mode, the room temperature is 290g, and the centrifugation is performed for 10 minutes.

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