CN110551686A - method for separating peripheral blood mononuclear cells - Google Patents
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Abstract
The invention provides a separation method of peripheral blood mononuclear cells, and relates to the field of regenerative medicine. The separation method of the invention comprises the following steps: pretreatment: centrifuging the collected peripheral blood, separating upper layer blood and lower layer blood cells, mixing the lower layer blood cells with a hydroxyethyl starch solution, uniformly mixing, standing for sedimentation, removing supernatant after sedimentation, and keeping the lower layer sediment; separation: adding erythrocyte lysate into the lower layer sediment, mixing uniformly, centrifuging, taking cell sediment, adding PBS for resuspension, adding cell suspension into lymphocyte separation liquid, centrifuging, removing supernatant, retaining the cell sediment, adding PBS, heparin and human serum albumin, mixing, centrifuging to obtain PBMC cell sediment; culturing: resuspending the PBMC cell pellet with the culture medium, and subpackaging into culture flasks for culture. By adopting the method, the separated peripheral blood mononuclear cells have the advantages of large quantity, high purity and high activity.
Description
Technical Field
The invention relates to the field of regenerative medicine, in particular to a separation method and application of peripheral blood mononuclear cells.
Background
peripheral Blood Mononuclear Cells (PBMC), which are cells with a Mononuclear nucleus in Peripheral Blood, mainly include lymphocytes (T lymphocytes and B lymphocytes), monocytes, phagocytic cells, dendritic cells and other small Cell types, which are important cells constituting the immune response function of the body. In recent years, peripheral blood mononuclear cells are studied more and more deeply, and the research shows that the peripheral blood mononuclear cells can proliferate and differentiate directionally in vitro. Compared with the method for extracting immune cells from bone marrow, the method for extracting peripheral blood mononuclear cells from peripheral blood is easier to obtain and basically causes pain to human bodies.
in 2010, a study was carried out to show that CD80 in combination with PBMC activated by CD28/CpG ODN has a killing effect on gastric cancer cells. Another study in 2014 explored, from clinical and experimental aspects, the effect of uterine cavity perfusion of peripheral blood mononuclear cells on embryo implantation and the mechanism thereof. Therefore, the peripheral blood mononuclear cells are expected to become a new disease treatment means.
At present, a plurality of methods for separating peripheral blood mononuclear cells exist, but the prepared mononuclear cells have low yield and low activity, and are not beneficial to the application. Therefore, there is a need to develop a new method for isolating peripheral blood mononuclear cells, which improves the isolation efficiency and activity thereof.
disclosure of Invention
In view of this, it is necessary to provide a method for separating peripheral blood mononuclear cells, which is directed to the problems of low yield and low activity of the conventional methods for separating peripheral blood mononuclear cells.
A method for separating peripheral blood mononuclear cells, comprising the following steps:
Pretreatment: centrifuging the collected peripheral blood, separating upper layer blood and lower layer blood cells, mixing the lower layer blood cells with a hydroxyethyl starch solution, uniformly mixing, standing for sedimentation, removing supernatant after sedimentation, and keeping the lower layer sediment;
Separation: adding erythrocyte lysate into the lower layer sediment, mixing uniformly, centrifuging, taking cell sediment, adding PBS for resuspension, adding cell suspension into lymphocyte separation liquid, centrifuging, removing supernatant, retaining the cell sediment, adding PBS, heparin and human serum albumin, mixing, centrifuging to obtain PBMC cell sediment;
Culturing: resuspending the PBMC cell pellet with the culture medium, and subpackaging into culture flasks for culture.
the separation method adopts hydroxyethyl starch solution to settle blood cells, adopts erythrocyte lysate to lyse the erythrocytes, adopts lymphocyte separation solution to separate mononuclear cells, adds heparin to inhibit the adhesion and aggregation of platelets, is favorable for removing the remaining platelets in cell suspension, adds human serum albumin to maintain the osmotic pressure and the transport function of blood, and combines the steps to ensure that the separated peripheral blood mononuclear cells have large quantity and high activity.
In one embodiment, the pre-processing step comprises: the centrifugal speed is 1700-1900 rpm, and the centrifugal time is 8-12 min.
In one embodiment, the volume ratio of the lower layer blood cells to the hydroxyethyl starch solution is (3-5): 1; the settling time is 50-70 min.
In one embodiment, the concentration of the hydroxyethyl starch solution is 20-40 g/mL.
in one embodiment, in the pretreatment step, when peripheral blood is collected, the peripheral blood is placed in a heparin sodium blood collection tube, and after the peripheral blood is collected, the heparin sodium blood collection tube is placed in a sealed sterile transport bottle for refrigerated transport, wherein the refrigerated temperature is 4-8 ℃.
in one embodiment, the separating step specifically comprises: adding erythrocyte lysate into the lower-layer sediment, wherein the volume ratio of the erythrocyte lysate to the lower-layer sediment is (0.8-1.2): 1, mixing uniformly, placing at 4 +/-1 ℃ for storage for 2-4 min, centrifuging at the rotating speed of 1700-1900 rpm for 8-12 min, taking the cell sediment, adding PBS for resuspension, adding the cell suspension into lymphocyte separation liquid, centrifuging at the rotating speed of 600-1000 rpm for 18-22 min, removing supernatant, retaining the cell sediment, adding PBS, heparin and human serum albumin, mixing uniformly, placing at 4 +/-1 ℃ for storage for 2-4 min, centrifuging at the rotating speed of 1700-1900 rpm for 8-12 min, and obtaining PBMC cell sediment.
In one embodiment, the working concentration of the heparin is 0.2-1U/mL, and the working concentration of the human serum albumin is 10-30 g/mL.
In one embodiment, the lymphocyte separation medium is a Ficoll lymphocyte separation medium.
In one embodiment, the volume ratio of the cell suspension to the Ficoll lymphocyte separation solution is 5 (2-4).
In one embodiment, the culture flask is 75cm 2, and the humidity is saturated humidity, the temperature is 37 + -0.5 deg.C, and the concentration of CO 2 is 5.0% + -0.5%.
Compared with the prior art, the invention has the following beneficial effects:
The separation method adopts hydroxyethyl starch solution to settle blood cells, adopts erythrocyte lysate to crack the erythrocytes, adopts lymphocyte separation solution to separate mononuclear cells, adds heparin to inhibit the adhesion and aggregation of platelets, is favorable for removing the remaining platelets in cell suspension, and adds human serum albumin to maintain the osmotic pressure and the transport function of blood.
Drawings
FIG. 1 is a photograph of peripheral blood mononuclear cells isolated in the example.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the following description. The following is a description of preferred embodiments of the invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
example 1
A method for separating peripheral blood mononuclear cells, comprising the following steps:
1) Collecting peripheral blood: drawing 70mL of human peripheral blood, placing the blood in a heparin sodium blood collection tube, and after collection is finished, placing the heparin sodium blood collection tube in a sealed sterile transport bottle for cold storage and transport, wherein the cold storage temperature is 4-8 ℃;
2) pretreatment: after the peripheral blood is transported to a laboratory, operating by an experimenter in a sterile environment, placing the heparin sodium blood collection tube in a centrifuge for centrifugation, wherein the centrifugation speed is 1800rpm, the time is 10min, the ascending speed is 7, and the descending speed is 1, and separating upper-layer blood and lower-layer blood cells; extracting upper layer plasma for preservation, transferring lower layer blood cells into a 100mL transfer bag, adding hydroxyethyl starch, uniformly mixing the lower layer blood cells and hydroxyethyl starch solution in a volume ratio of 4:1, settling for 60min, removing supernatant after settling, and retaining lower layer precipitate;
3) Separation: adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 4 deg.C for 3min, taking out, centrifuging at 1800rpm for 10min to obtain PBMC cell precipitate;
4) and (3) suspending the PBMC cell sediment by using 10mL of culture medium, subpackaging the PBMC cell sediment into 75cm 2 culture bottles for culture, wherein the humidity is saturated humidity, the temperature is 37 +/-1 ℃, the concentration of CO 2 is 5.0% +/-1%, and subculture is carried out as required.
Example 2
A method for separating peripheral blood mononuclear cells, comprising the following steps:
1) Collecting peripheral blood: drawing 70mL of human peripheral blood, placing the blood in a heparin sodium blood collection tube, and after collection is finished, placing the heparin sodium blood collection tube in a sealed sterile transport bottle for cold storage and transport, wherein the cold storage temperature is 4-8 ℃;
2) Pretreatment: after the peripheral blood is transported to a laboratory, operating by an experimenter in a sterile environment, placing the heparin sodium blood collection tube in a centrifuge for centrifugation at 1700rpm for 12min, and separating upper-layer blood and lower-layer blood cells; extracting upper layer plasma for preservation, transferring lower layer blood cells into a 100mL transfer bag, adding hydroxyethyl starch, uniformly mixing the lower layer blood cells and hydroxyethyl starch solution in a volume ratio of 3:1, settling for 65min, removing supernatant after settling, and retaining lower layer precipitate;
3) Separation: adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1700rpm for 12 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging is carried out at the rotating speed of 700rpm for 21 min; removing supernatant, sucking the leukocyte layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 4 deg.C for 3min, taking out, centrifuging at 1700rpm for 12min to obtain PBMC cell precipitate;
4) And (3) suspending the PBMC cell sediment by using 10mL of culture medium, subpackaging the PBMC cell sediment into 75cm 2 culture bottles for culture, wherein the humidity is saturated humidity, the temperature is 37 +/-1 ℃, the concentration of CO 2 is 5.0% +/-1%, and subculture is carried out as required.
example 3
A method for separating peripheral blood mononuclear cells, comprising the following steps:
1) Collecting peripheral blood: drawing 70mL of human peripheral blood, placing the blood in a heparin sodium blood collection tube, and after collection is finished, placing the heparin sodium blood collection tube in a sealed sterile transport bottle for cold storage and transport, wherein the cold storage temperature is 4-8 ℃;
2) Pretreatment: after the peripheral blood is transported to a laboratory, operating by an experimenter in a sterile environment, placing the heparin sodium blood collection tube in a centrifuge for centrifugation at 1900rpm for 8min, and separating upper-layer blood and lower-layer blood cells; extracting upper layer plasma for preservation, transferring lower layer blood cells into a 100mL transfer bag, adding hydroxyethyl starch, uniformly mixing the lower layer blood cells and hydroxyethyl starch solution in a volume ratio of 3:1, settling for 65min, removing supernatant after settling, and retaining lower layer precipitate;
3) Separation: adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1900rpm for 8 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging is carried out at the rotating speed of 600rpm for 22 min; removing supernatant, sucking the leukocyte layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 4 deg.C for 3min, taking out, centrifuging at 1900rpm for 8min to obtain PBMC cell precipitate;
4) And (3) suspending the PBMC cell sediment by using 10mL of culture medium, subpackaging the PBMC cell sediment into 75cm 2 culture bottles for culture, wherein the humidity is saturated humidity, the temperature is 37 +/-1 ℃, the concentration of CO 2 is 5.0% +/-1%, and subculture is carried out as required.
Comparative example 1
A method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
Adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte membrane layer with pipette gun, adding PBS to dilute to 100mL, adding 1mL human serum albumin, mixing, storing at 4 deg.C for 3min, taking out, centrifuging at 1800rpm for 10min to obtain PBMC cell precipitate
Comparative example 2
A method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
Adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leucocyte layer with a pipette, adding PBS to dilute to 100mL, adding 0.5U/mL heparin, mixing uniformly, storing at 4 ℃ for 3min, taking out, centrifuging at 1800rpm for 10min to obtain PBMC cell precipitate.
Comparative example 3
a method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
Adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte membrane layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 1 deg.C for 3min, taking out, centrifuging at 800rpm for 10min to obtain PBMC cell precipitate.
comparative example 4
a method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
Adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1700rpm for 12 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte membrane layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 25 deg.C for 3min, taking out, centrifuging at 1700rpm for 12min to obtain PBMC cell precipitate.
Comparative example 5
A method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte membrane layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 5 deg.C for 3min, taking out, centrifuging at 1000rpm for 12min to obtain PBMC cell precipitate.
Comparative example 6
a method for separating peripheral blood mononuclear cells, which is substantially the same as in example 1 except that the step 3) is:
Adding erythrocyte lysate into the lower layer precipitate, mixing, storing at 4 deg.C for 3min, centrifuging at 1800rpm for 10 min; removing supernatant, adding 25mL of PBS into cell sediment for resuspension, transferring the cell suspension into the Ficoll lymphocyte separation liquid along the wall of a centrifugal tube, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation liquid is 5:3, the Ficoll lymphocyte separation liquid is vertical to a horizontal plane, and centrifuging at the rotating speed of 800rpm for 20 min; removing supernatant, sucking the leukocyte membrane layer with a pipette gun, adding PBS to dilute to 100mL, adding 0.5U/mL heparin and 1mL human serum albumin, mixing, storing at 5 deg.C for 3min, taking out, centrifuging at 2500rpm for 8min to obtain PBMC cell precipitate.
Examples of the experiments
1ml of cell suspensions of examples and comparative examples were taken, counted by an XS-1000i type full-automatic hemocytometer and a Countstar automatic cytometer, and the number of erythrocytes, platelets and the total number of mononuclear cells in the obtained peripheral blood mononuclear cells were measured, and the recovery rate and the survival rate of mononuclear cells were calculated, and the results are shown in Table 1:
TABLE 1 cell purity assay
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
the above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. A method for separating peripheral blood mononuclear cells, comprising the steps of:
pretreatment: centrifuging the collected peripheral blood, separating upper layer blood and lower layer blood cells, mixing the lower layer blood cells with a hydroxyethyl starch solution, uniformly mixing, standing for sedimentation, removing supernatant after sedimentation, and keeping the lower layer sediment;
Separation: adding erythrocyte lysate into the lower layer sediment, mixing uniformly, centrifuging, taking cell sediment, adding PBS for resuspension, adding cell suspension into lymphocyte separation liquid, centrifuging, removing supernatant, retaining the cell sediment, adding PBS, heparin and human serum albumin, mixing, centrifuging to obtain PBMC cell sediment;
Culturing: resuspending the PBMC cell pellet with the culture medium, and subpackaging into culture flasks for culture.
2. The method for separating peripheral blood mononuclear cells according to claim 1, wherein the pretreatment step comprises: the centrifugal speed is 1700-1900 rpm, and the centrifugal time is 8-12 min.
3. The method for separating peripheral blood mononuclear cells according to claim 2, wherein the volume ratio of the lower layer blood cells to the hydroxyethyl starch solution is (3-5): 1; the settling time is 50-70 min.
4. The method for separating peripheral blood mononuclear cells according to claim 3, wherein the concentration of the hydroxyethyl starch solution is 20 to 40 g/mL.
5. The method for separating peripheral blood mononuclear cells according to claim 1, wherein in the pretreatment step, the peripheral blood is collected by placing the peripheral blood in a heparin sodium blood collection tube, and after the collection is completed, the heparin sodium blood collection tube is placed in a sealed sterile transport bottle for refrigerated transport at a refrigerated temperature of 4 to 8 ℃.
6. The method for separating peripheral blood mononuclear cells according to claim 1, wherein the separation step is specifically: adding erythrocyte lysate into the lower-layer sediment, wherein the volume ratio of the erythrocyte lysate to the lower-layer sediment is (0.8-1.2): 1, mixing uniformly, placing at 4 +/-1 ℃ for storage for 2-4 min, centrifuging at the rotating speed of 1700-1900 rpm for 8-12 min, taking the cell sediment, adding PBS for resuspension, adding the cell suspension into lymphocyte separation liquid, centrifuging at the rotating speed of 600-1000 rpm for 18-22 min, removing supernatant, retaining the cell sediment, adding PBS, heparin and human serum albumin, mixing uniformly, placing at 4 +/-1 ℃ for storage for 2-4 min, centrifuging at the rotating speed of 1700-1900 rpm for 8-12 min, and obtaining PBMC cell sediment.
7. the method for separating peripheral blood mononuclear cells according to claim 6, wherein the working concentration of heparin is 0.2 to 1U/mL, and the working concentration of human serum albumin is 10 to 30 g/mL.
8. The method for separating peripheral blood mononuclear cells according to claim 6, wherein the lymphocyte separation medium is a Ficoll lymphocyte separation medium.
9. the method for separating peripheral blood mononuclear cells according to claim 8, wherein the volume ratio of the cell suspension to the Ficoll lymphocyte separation medium is 5 (2-4).
10. The method for separating peripheral blood mononuclear cells according to claim 1, wherein in the culturing step, the culture flask is a 75cm 2 culture flask, and the humidity in the culture environment is saturated humidity, the temperature is 37 ± 0.5 ℃, and the concentration of CO 2 is 5.0% ± 0.5%.
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Application publication date: 20191210 |