CN112251404A - Separation method of peripheral blood mononuclear cells - Google Patents
Separation method of peripheral blood mononuclear cells Download PDFInfo
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- 238000000926 separation method Methods 0.000 title claims abstract description 25
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 title claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 35
- 210000004027 cell Anatomy 0.000 claims abstract description 32
- 210000004369 blood Anatomy 0.000 claims abstract description 28
- 239000008280 blood Substances 0.000 claims abstract description 28
- 229920001612 Hydroxyethyl starch Polymers 0.000 claims abstract description 11
- 229940050526 hydroxyethylstarch Drugs 0.000 claims abstract description 11
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims description 19
- 238000007664 blowing Methods 0.000 claims description 18
- 238000005119 centrifugation Methods 0.000 claims description 18
- 238000002156 mixing Methods 0.000 claims description 15
- 210000005259 peripheral blood Anatomy 0.000 claims description 15
- 239000011886 peripheral blood Substances 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 9
- 238000007710 freezing Methods 0.000 claims description 9
- 239000013049 sediment Substances 0.000 claims description 9
- 239000008354 sodium chloride injection Substances 0.000 claims description 9
- 238000004113 cell culture Methods 0.000 claims description 8
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 244000005700 microbiome Species 0.000 claims description 6
- 238000005070 sampling Methods 0.000 claims description 6
- 238000005406 washing Methods 0.000 claims description 6
- 238000010009 beating Methods 0.000 claims description 5
- 238000002955 isolation Methods 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 4
- 239000012895 dilution Substances 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 230000003833 cell viability Effects 0.000 claims description 3
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 238000007865 diluting Methods 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 230000007480 spreading Effects 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 13
- 229920001917 Ficoll Polymers 0.000 abstract description 7
- 238000000432 density-gradient centrifugation Methods 0.000 abstract description 7
- 210000003743 erythrocyte Anatomy 0.000 abstract description 7
- 238000001556 precipitation Methods 0.000 abstract description 6
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- 238000002659 cell therapy Methods 0.000 description 4
- 210000003714 granulocyte Anatomy 0.000 description 4
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
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Abstract
The invention discloses a separation method of peripheral blood mononuclear cells, which aims at the separation characteristic of a large-capacity blood sample, firstly uses a hydroxyethyl starch precipitation method to remove most of red blood cells, then naturally settles in the air, and then uses a Ficoll density gradient centrifugation method to further purify the mononuclear cells, thereby obtaining PBMC with the purity of more than 95 percent and the vitality of more than 90 percent. Compared with a method without natural sedimentation in air, the obtained cell has higher activity and purity.
Description
Technical Field
The invention relates to the technical field of cell separation, in particular to a separation method of peripheral blood mononuclear cells.
Background
Cell therapy is a new disease treatment technology which is emerging in recent years, and means that certain cells with specific functions are obtained by a bioengineering method and/or are treated by in vitro amplification, special culture and the like, so that the cells have treatment effects of enhancing immunity, killing pathogens and tumor cells, promoting regeneration of tissues and organs, recovering organisms and the like, and the purpose of treating diseases is achieved.
The cell therapy has the unique advantages of good curative effect, small side effect, individuation and the like, and provides a choice, sometimes even the last choice, for the treatment of some intractable diseases. In the current and long-term history stage in the future, cell therapy plays an important role in clinical treatment, and the twenty-first century is an age in which cell therapy plays an important role.
In the immunocytotherapy, Peripheral Blood Mononuclear Cells (PBMCs) are generally isolated by hydroxyethyl starch (HES) centrifugation or Ficoll density gradient centrifugation. Although the hydroxyethyl starch centrifugal precipitation method is simple and convenient in operation, the cell separation effect is poor, the purity of the obtained cells is difficult to meet the requirement, and the cells have more platelets, red blood cells, granulocytes and the like. The Ficoll density gradient centrifugation method is adopted, the specific gravity of red blood cells and granulocytes is high, and the red blood cells and the granulocytes sink to the bottom of a tube after centrifugation; the specific gravity of the lymphocyte and the monocyte is less than or equal to that of the separating liquid, the lymphocyte and the monocyte float on the liquid surface of the separating liquid after centrifugation, or a small part of the lymphocyte is suspended in the separating liquid, and the monocyte can be separated from the peripheral blood by sucking the white membrane layer cell on the liquid surface of the separating liquid. The purity of the cells obtained by the method is good, but the requirement on the sample volume is limited to a certain extent, and if the sample volume exceeds 50ml, the separation effect is greatly reduced; moreover, the requirements for operation are relatively high, and if the operation is not careful enough, the separated cells have a large influence on the quantity and purity. In addition, different centrifugal forces and centrifugal times have great influence on the cell separation effect, and the purity and the activity of the cells separated by adopting different centrifugal parameters are greatly different.
Disclosure of Invention
The main object of the present invention is to improve the PBMC separation method in combination with hydroxyethyl starch precipitation and Ficoll density gradient centrifugation, especially for large volume (>50ml) blood sample separation.
In order to achieve the above object, the present invention provides a method for separating peripheral blood mononuclear cells, comprising the steps of:
s10: peripheral blood separation:
s101: disinfecting the surface of a blood collection tube or a blood collection bag by using 75% alcohol, and then transferring the blood collection tube or the blood collection bag into a normally operated purification workbench;
s102: preparing a T75 cell culture bottle, opening the bottle cap, sucking peripheral blood in a blood collection tube by using a pipette, transferring the peripheral blood into a T75 cell culture bottle, and blowing, beating and uniformly mixing;
s103: taking 1.5ml of the limit of the detected microorganisms and counting the whole blood, adding 20ml of hydroxyethyl starch into 60ml to 100ml of the detected microorganisms according to the transfer volume, and uniformly mixing the mixture by blowing;
s104: marking the separation starting time for 30 minutes, sucking the separated supernatant, transferring the supernatant into two 50ml centrifuge tubes, and recording the sucked volume;
s105: centrifugation was performed at room temperature.
Preferably, the method further comprises the following steps:
s20: plasma subpackaging:
s201: the centrifuged supernatant (namely the plasma) of the peripheral blood is gently sucked by a pipette and transferred into a 50mL centrifuge tube and marked;
s202: taking 20mL of plasma, subpackaging into 5 freezing tubes with 4.5mL, each tube with 4mL, and discarding the surplus;
s203: sealing, labeling, transferring to a freezing storage box, and temporarily storing the freezing storage box in a refrigerator at 4 ℃;
s204: transferring the plasma temporarily stored in the refrigerator to a refrigerator with the temperature of-80 ℃ or a liquid nitrogen temporary storage tank for temporary storage.
Preferably, the method further comprises the following steps:
s30: isolation of mononuclear cells:
s301: diluting each tube of cell sediment after centrifugation by using 0.9% sodium chloride injection, fixing the volume to 30mL, and blowing, beating and uniformly mixing;
s302: slowly spreading the diluted cell sediment into 2 separation liquid tubes, adding 30mL of the cell sediment into each tube, keeping the interface between the separation liquid and the diluted blood clear, and marking;
s303: the centrifuge was slowed down and then centrifuged.
Preferably, the method further comprises the following steps:
s40: first centrifugal washing:
s401: after the centrifugation is finished, sucking the mononuclear cell layer into 2 50mL centrifuge tubes, using 0.9% sodium chloride injection for each centrifuge tube to fix the volume to 50mL, blowing, uniformly mixing and marking;
s402: centrifugation was performed at room temperature.
Preferably, the method further comprises the following steps:
s50: second centrifugal washing and sampling:
and after the centrifugation is finished, discarding the supernatant, merging the cell precipitates into 1 centrifuge tube, metering the volume to 50mL by using 0.9% sodium chloride injection, blowing and uniformly mixing, sampling 0.5mL, detecting the cell number and the cell viability, and marking.
Preferably, in S105, the centrifugal force of the centrifuge is 900g, and the centrifugal time is 15 min.
Preferably, in S203, the plasma is temporarily stored in the refrigerator for 12 hours.
Preferably, in S301, the dilution ratio is such that the ratio of the whole blood volume to the diluted volume is equal to 1: 1.
Preferably, in S303, the centrifuge is set to 1 st, 600g, and centrifuged for 15min at room temperature;
preferably, in S402, the centrifugal force of the centrifuge is set to 600g, and the centrifugal time is 10 min.
The invention discloses a method for separating peripheral blood mononuclear cells, which aims at the separation characteristic of a large-capacity blood sample, firstly uses a hydroxyethyl starch precipitation method to remove most of red blood cells, then naturally settles in the air, and then uses a Ficoll density gradient centrifugation method to further purify the mononuclear cells, thereby obtaining PBMC with the purity of more than 95 percent and the vitality of more than 90 percent. Compared with a method without natural sedimentation in air, the obtained cell has higher activity and purity.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the structures shown in the drawings without creative efforts.
FIG. 1 is a flow chart of a method for separating peripheral blood mononuclear cells according to an embodiment of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all the directional indicators (such as up, down, left, right, front, and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
In the embodiment of the present invention, referring to fig. 1, the method for separating peripheral blood mononuclear cells includes the following steps:
s10: peripheral blood separation:
s101: disinfecting the surface of a blood collection tube or a blood collection bag by using 75% alcohol, and then transferring the blood collection tube or the blood collection bag into a normally operated purification workbench;
s102: preparing a T75 cell culture bottle, opening the bottle cap, sucking peripheral blood in a blood collection tube by using a pipette, transferring the peripheral blood into a T75 cell culture bottle, and blowing and mixing the peripheral blood.
When in use, the pipette is cleaned and naturally drained, the pipette is rinsed for 2-3 times with the solution to be measured, and the place above the marked line of the neck is pinched by the thumb and middle finger of the right hand. The solution was gently sucked up by holding a ball of rubber with the left hand and the eyes noticed the rising liquid level. The T75 cell culture bottle is inclined by 30 degrees, the pipette is upright, the lower end of the tube is tightly close to the inner wall of the T75 cell culture bottle, the forefinger is slightly loosened to allow the solution to slowly flow down along the bottle wall, and after all the solution flows out, the pipette is taken out after waiting for 15s so as to allow part of peripheral blood attached to the tube wall to flow out.
When the air blowing is carried out, the culture medium for air blowing cannot be too small, so that bubbles are avoided, and the phenomenon that the air blowing is rapidly and repeatedly carried out during passage is easy to generate foams which are unfavorable for cells is avoided as much as possible. And the liquid is sucked by the suction pipe after the liquid is pumped out by the suction pipe at each time.
S103: taking 1.5ml of the limit of the detected microorganisms and counting the whole blood, adding 20ml of hydroxyethyl starch into 60ml to 100ml of the detected microorganisms according to the transfer volume, and uniformly mixing the mixture by blowing; when the cell is blown, the liquid is blown out along the wall as much as possible, the operation is soft, and the generation of foam can be avoided to the greatest extent.
S104: marking the separation starting time for 30 minutes to improve the activity of the plasma, then sucking the separated supernatant and transferring the supernatant into two 50ml centrifuge tubes, and recording the sucked volume;
s105: centrifuging at room temperature with a centrifuge force of 900g for 15 min.
Further, the method also comprises the following steps:
s20: plasma subpackaging:
s201: the centrifuged supernatant (namely the plasma) of the peripheral blood is gently sucked by a pipette and transferred into a 50mL centrifuge tube and marked;
s202: taking 20mL of plasma, subpackaging into 5 freezing tubes with 4.5mL, each tube with 4mL, and discarding the surplus;
s203: sealing, labeling, transferring to a freezing storage box, and temporarily storing the freezing storage box in a refrigerator at 4 ℃ for 12 hours.
S204: transferring the plasma temporarily stored in the refrigerator to a refrigerator with the temperature of-80 ℃ or a liquid nitrogen temporary storage tank for temporary storage.
In this embodiment, one of the keys to improving the PBMC isolation effect is to reduce the proportion of mixed cells such as platelets, erythrocytes and granulocytes and improve the purity of PBMC cells while ensuring the viability and quantity of the PBMC cells obtained by isolation. Firstly, hydroxyethyl starch is used for precipitation, after natural sedimentation in the air, the separation method of Ficoll density gradient centrifugation is adopted, and PBMC with high purity and activity is obtained.
Further, the method also comprises the following steps:
s30: isolation of mononuclear cells:
s301: diluting each tube of cell sediment after centrifugation by using 0.9% sodium chloride injection, fixing the volume to 30mL (the dilution ratio is that the volume ratio of whole blood to the volume after dilution is equal to 1: 1.), and blowing, beating and uniformly mixing;
s302: slowly spreading the diluted cell sediment into 2 separation liquid tubes, adding 30mL of the cell sediment into each tube, keeping the interface between the separation liquid and the diluted blood clear, and marking;
s303: the centrifuge was slowed down and then centrifuged. The centrifuge is set to 1 st, 600g, and centrifuged for 15min at room temperature.
Further, the method also comprises the following steps:
s40: first centrifugal washing:
s401: after the centrifugation is finished, sucking the mononuclear cell layer into 2 50mL centrifuge tubes, using 0.9% sodium chloride injection for each centrifuge tube to fix the volume to 50mL, blowing, uniformly mixing and marking;
s402: centrifugation was carried out at room temperature with a centrifuge set at 600g for 10 min.
Further, the method also comprises the following steps:
s50: second centrifugal washing and sampling:
and after the centrifugation is finished, discarding the supernatant, merging the cell precipitates into 1 centrifuge tube, metering the volume to 50mL by using 0.9% sodium chloride injection, blowing and uniformly mixing, sampling 0.5mL, detecting the cell number and the cell viability, and marking.
The characteristic of the invention for separating large-capacity blood samples is that most of red blood cells are removed by hydroxyethyl starch precipitation method, then naturally settled in the air, and then mononuclear cells are further purified by Ficoll density gradient centrifugation method, thus obtaining PBMC with purity over 95% and vitality over 90%. Compared with a method without natural sedimentation in air, the obtained cell has higher activity and purity.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents of the present invention, which are made by the contents of the present specification and the accompanying drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A method for separating peripheral blood mononuclear cells, comprising the steps of:
s10: peripheral blood separation:
s101: disinfecting the surface of a blood collection tube or a blood collection bag by using 75% alcohol, and then transferring the blood collection tube or the blood collection bag into a normally operated purification workbench;
s102: preparing a T75 cell culture bottle, opening the bottle cap, sucking peripheral blood in a blood collection tube by using a pipette, transferring the peripheral blood into a T75 cell culture bottle, and blowing, beating and uniformly mixing;
s103: taking 1.5ml of the limit of the detected microorganisms and counting the whole blood, adding 20ml of hydroxyethyl starch into 60ml to 100ml of the detected microorganisms according to the transfer volume, and uniformly mixing the mixture by blowing;
s104: marking the separation starting time for 30 minutes, sucking the separated supernatant, transferring the supernatant into two 50ml centrifuge tubes, and recording the sucked volume;
s105: centrifugation was performed at room temperature.
2. The method of claim 1, further comprising the steps of:
s20: plasma subpackaging:
s201: the centrifuged supernatant (namely the plasma) of the peripheral blood is gently sucked by a pipette and transferred into a 50mL centrifuge tube and marked;
s202: taking 20mL of plasma, subpackaging into 5 freezing tubes with 4.5mL, each tube with 4mL, and discarding the surplus;
s203: sealing, labeling, transferring to a freezing storage box, and temporarily storing the freezing storage box in a refrigerator at 4 ℃;
s204: transferring the plasma temporarily stored in the refrigerator to a refrigerator with the temperature of-80 ℃ or a liquid nitrogen temporary storage tank for temporary storage.
3. The method of claim 1, further comprising the steps of:
s30: isolation of mononuclear cells:
s301: diluting each tube of cell sediment after centrifugation by using 0.9% sodium chloride injection, fixing the volume to 30mL, and blowing, beating and uniformly mixing;
s302: slowly spreading the diluted cell sediment into 2 separation liquid tubes, adding 30mL of the cell sediment into each tube, keeping the interface between the separation liquid and the diluted blood clear, and marking;
s303: the centrifuge was slowed down and then centrifuged.
4. The method of claim 1, further comprising the steps of:
s40: first centrifugal washing:
s401: after the centrifugation is finished, sucking the mononuclear cell layer into 2 50mL centrifuge tubes, using 0.9% sodium chloride injection for each centrifuge tube to fix the volume to 50mL, blowing, uniformly mixing and marking;
s402: centrifugation was performed at room temperature.
5. The method of claim 1, further comprising the steps of:
s50: second centrifugal washing and sampling:
and after the centrifugation is finished, discarding the supernatant, merging the cell precipitates into 1 centrifuge tube, metering the volume to 50mL by using 0.9% sodium chloride injection, blowing and uniformly mixing, sampling 0.5mL, detecting the cell number and the cell viability, and marking.
6. The method according to claim 1, wherein the centrifuge has a centrifugal force of 900g for 15min in S105.
7. The method of claim 2, wherein the plasma is kept in the refrigerator for 12 hours in step S203.
8. The method for separating peripheral blood mononuclear cells according to claim 3, wherein in S301, the dilution ratio is equal to 1:1 of the whole blood volume to the diluted volume.
9. The method according to claim 3, wherein in step S303, the centrifuge is set to 1 st, 600g, and centrifuged at room temperature for 15 min.
10. The method according to claim 4, wherein in S402, the centrifugal force of the centrifuge is set to 600g, and the centrifugation time is 10 min.
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Cited By (2)
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| CN115261314A (en) * | 2022-06-28 | 2022-11-01 | 吉林省拓华生物科技有限公司 | Method for preparing mononuclear cells and platelets |
| CN115521910A (en) * | 2022-09-30 | 2022-12-27 | 深圳市北科生物科技有限公司 | Method for separating peripheral blood mononuclear cells |
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