CN111172109A - Immune cell culture method and application thereof - Google Patents
Immune cell culture method and application thereof Download PDFInfo
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- 210000002865 immune cell Anatomy 0.000 title claims abstract description 37
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- 239000008280 blood Substances 0.000 claims abstract description 24
- 210000003954 umbilical cord Anatomy 0.000 claims abstract description 24
- 210000005087 mononuclear cell Anatomy 0.000 claims abstract description 20
- 210000003606 umbilical vein Anatomy 0.000 claims abstract description 18
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- 229920002307 Dextran Polymers 0.000 claims abstract description 12
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- 238000005189 flocculation Methods 0.000 claims abstract description 9
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- 230000001954 sterilising effect Effects 0.000 claims abstract description 9
- 238000012360 testing method Methods 0.000 claims abstract description 5
- 238000012258 culturing Methods 0.000 claims description 40
- 210000004027 cell Anatomy 0.000 claims description 33
- 239000001963 growth medium Substances 0.000 claims description 24
- 239000000243 solution Substances 0.000 claims description 19
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 16
- 244000309466 calf Species 0.000 claims description 16
- 210000002966 serum Anatomy 0.000 claims description 16
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- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 8
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- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 claims description 3
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- 238000007865 diluting Methods 0.000 claims description 3
- 210000003714 granulocyte Anatomy 0.000 claims description 3
- 229910052740 iodine Inorganic materials 0.000 claims description 3
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- MIKKOBKEXMRYFQ-WZTVWXICSA-N meglumine amidotrizoate Chemical compound C[NH2+]C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO.CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C([O-])=O)=C1I MIKKOBKEXMRYFQ-WZTVWXICSA-N 0.000 claims description 3
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses an immune cell culture method and application thereof, comprising the following steps: step a: clamping and cutting the umbilical cord at a position of 10-15 cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: mononuclear cells in cord blood were isolated by dextran diatrizoate density gradient centrifugation. The immune cell culture method has simple steps and high culture efficiency, is favorable for carrying out a large number of immune cell application tests or clinical use of immune cells clinically, has high growth speed and high proliferation rate of the immune cells in the culture process, forms larger bacterial colonies, improves the culture efficiency of the immune cells, and has good application prospect clinically.
Description
Technical Field
The invention relates to the technical field of immune cells, in particular to an immune cell culture method and application thereof.
Background
Natural killer cells are important immune cells of the body, are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in hypersensitivity reaction and autoimmune disease occurrence under certain conditions, can recognize target cells and killing mediators, and although NK cells perform important immune monitoring functions, over-activation of NK cells can cause unnecessary tissue damage and even endanger life in some cases, so that the body needs to maintain the activity of NK cells in a controllable range by some means so as to maintain the tolerance of NK cells to themselves.
Compared with the immune tolerance of T, B cells, the research for maintaining the NK cell to self tolerance is less, and at present, the research can be realized by an inhibitory receptor mediated mode or apoptosis induced after activation, while the inhibitory receptor plays an important regulation and control function in the interaction process between the NK cell and the helper cell, in the liver rich in the NK cell, the interaction between the NK cell and the helper cell is an important link of NK cell mediated natural immune response, and the research on the regulation and control function of the inhibitory receptor in the NK cell mediated liver natural immune response and the influence of pathological change on the latter on the regulation and control function of the inhibitory receptor in the NK cell mediated liver natural immune response is helpful for understanding the mechanism of the NK cell to maintain self tolerance.
Disclosure of Invention
The invention aims to provide an immune cell culture method and application thereof, which have the advantage of convenient use of the immune cell culture method and solve the problem of inconvenient immune cell culture.
In order to achieve the purpose, the invention provides the following technical scheme: an immune cell culture method comprising the steps of:
step a: clamping and cutting the umbilical cord at a position of 10-15 cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor;
step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished;
step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method;
step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 50% -70% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 multiplied by 10;
step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Preferably, the immune cell culture method is an application in preparing immune cell products.
Preferably, in the step a, the umbilical cord is quickly cleaned from the umbilical cord broken end to the placenta direction by using sterile gauze, and the iodine disinfection frequency is 2 times.
Preferably, in the step b, the blood bag is gently shaken when the cord blood flows into the blood bag, so that the cord blood is fully mixed with the anticoagulant.
Preferably, the method for separating mononuclear cells in cord blood by dextran diatrizoate density gradient centrifugation in the step c comprises the following steps:
step 1: 340g/L of diatrizoate meglumine is added into a Ficoll solution to prepare a layering solution with proper density;
step 2: during separation, firstly placing the layering solution on the bottom layer of a test tube, then diluting heparinized whole blood properly with Hanks solution or PBS solution, and slightly superposing the heparinized whole blood on the layering solution to ensure that the heparinized whole blood and the layering solution form a clear interface;
and step 3: after horizontal centrifugation, a plurality of liquid and cell zones of different layers appear in the centrifugal tube, the density of red blood cells and granulocytes is higher than that of the layering liquid, the red blood cells are aggregated into a string of money shape due to the fact that the red blood cells meet Ficoll and are deposited at the bottom of the tube, platelets are suspended in plasma due to low density, single nuclear cells with the density equivalent to that of the layering liquid are densely distributed in the interface of the plasma layer and the layering liquid and are in a white membrane shape, and the cells in the layer are sucked and delivered to be washed and then suspended in the high heart.
Compared with the prior art, the invention has the following beneficial effects: the immune cell culture method has simple steps and high culture efficiency, is favorable for carrying out a large number of immune cell application tests or clinical use of immune cells clinically, has high growth speed and high proliferation rate of the immune cells in the culture process, forms larger bacterial colonies, improves the culture efficiency of the immune cells, and has good application prospect clinically.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all 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.
An immune cell culture method comprising the steps of:
step a: clamping and cutting the umbilical cord at a position of 10-15 cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor;
step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished;
step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method;
step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 50% -70% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 multiplied by 10;
step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Example 1
An immune cell culture method comprising the steps of: step a: clamping and cutting off the umbilical cord at a position of 10cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method; step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding the cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 50% and 5% at 37 ℃ after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 multiplied by 10; step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Example 2
In example 1, the following additional steps were added:
the immune cell culture method is an application in preparing immune cell products.
An immune cell culture method comprising the steps of: step a: clamping and cutting off the umbilical cord at a position 15cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method; step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 70% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 x 10; step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Example 3
In example 2, the following steps were added:
in the step a, the umbilical cord is quickly cleaned from the umbilical cord broken end to the placenta direction by using sterile gauze, and the iodine disinfection frequency is 2 times.
An immune cell culture method comprising the steps of: step a: clamping and cutting off the umbilical cord at a position 11cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method; step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 60% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 x 10; step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Example 4
In example 3, the following steps were added:
and c, slowly shaking the blood bag when the cord blood flows into the blood bag in the step b to fully mix the cord blood and the anticoagulant.
An immune cell culture method comprising the steps of: step a: clamping and cutting off the umbilical cord at a position 14cm away from the end of the neonate by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method; step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 55% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 1 multiplied by 10; step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Example 5
In example 4, the following steps were added:
the method for separating the mononuclear cells in the cord blood by the dextran diatrizoate density gradient centrifugation method in the step c comprises the following steps: step 1: 340g/L of diatrizoate meglumine is added into a Ficoll solution to prepare a layering solution with proper density; step 2: during separation, firstly placing the layering solution on the bottom layer of a test tube, then diluting heparinized whole blood properly with Hanks solution or PBS solution, and slightly superposing the heparinized whole blood on the layering solution to ensure that the heparinized whole blood and the layering solution form a clear interface; and step 3: after horizontal centrifugation, a plurality of liquid and cell zones of different layers appear in the centrifugal tube, the density of red blood cells and granulocytes is higher than that of the layering liquid, the red blood cells are aggregated into a string of money shape due to the fact that the red blood cells meet Ficoll and are deposited at the bottom of the tube, platelets are suspended in plasma due to low density, single nuclear cells with the density equivalent to that of the layering liquid are densely distributed in the interface of the plasma layer and the layering liquid and are in a white membrane shape, and the cells in the layer are sucked and delivered to be washed and then suspended in the high heart.
An immune cell culture method comprising the steps of: step a: clamping and cutting off the umbilical cord at a position of 10cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor; step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished; step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method; step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding the cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 65% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 multiplied by 10; step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (5)
1. An immune cell culture method, comprising: the method comprises the following steps:
step a: clamping and cutting the umbilical cord at a position of 10-15 cm away from the end of the newborn by using hemostatic forceps, cleaning the umbilical cord by using sterile gauze, and then sterilizing by using iodophor;
step b: the needle head penetrates through the umbilical vein, and umbilical cord blood is collected through a blood bag filled with an anti-flocculation agent, when the umbilical vein gradually becomes white, the collection of the umbilical cord blood is finished;
step c: separating mononuclear cells in cord blood by a dextran diatrizoate density gradient centrifugation method;
step d: inoculating the separated mononuclear cells into a culture bottle, coating antibodies of the culture bottle, adding 10 ml of PBS and 1 ml of ErbB2 antibody (2.4 mg/ml) into the culture bottle, incubating for 1h, gently washing for 2 times along the side wall of the culture bottle by using 10 ml of PBS, finally adding a culture medium into the culture bottle coated with the ErbB2 antibody, adding 4 ml of PBS9 (-) and 1 ml of ErbB2 stock solution into the culture bottle with 75 square centimeters, respectively adding cell suspensions into the culture bottle after uniformly mixing, and culturing in a carbon dioxide culture box with the humidity of 50% -70% and the temperature of 37 ℃ and the humidity of 5% after observing under an inverted microscope, wherein the inoculated cell concentration is about 7 th power cell/ml of 1 multiplied by 10;
step e: after culturing for 36h, adding calf serum, adding the calf serum to the final concentration of 20ng/ml, culturing for 48h again, adding 500 ml of ALys505NK-AC culture medium, IL-51000u/ml and 5 ml of fresh plasma, culturing for 36h again, adding 300 ml of ALys505NK-AC culture medium and IL-51000u/ml, and finally culturing for 24h, and then harvesting the cells.
2. An immune cell culture method according to claim 1, wherein: the immune cell culture method is an application in preparing immune cell products.
3. An immune cell culture method according to claim 1, wherein: and (b) rapidly cleaning the umbilical cord from the umbilical cord broken end to the placenta in the step a by using sterile gauze, wherein the iodine disinfection frequency is 2 times.
4. An immune cell culture method according to claim 1, wherein: and c, slowly shaking the blood bag when the cord blood flows into the blood bag in the step b to fully mix the cord blood and the anticoagulant.
5. An immune cell culture method according to claim 1, wherein: the method for separating the mononuclear cells in the cord blood by the dextran diatrizoate density gradient centrifugation method in the step c comprises the following steps:
step 1: 340g/L of diatrizoate meglumine is added into a Ficoll solution to prepare a layering solution with proper density;
step 2: during separation, firstly placing the layering solution on the bottom layer of a test tube, then diluting heparinized whole blood properly with Hanks solution or PBS solution, and slightly superposing the heparinized whole blood on the layering solution to ensure that the heparinized whole blood and the layering solution form a clear interface;
and step 3: after horizontal centrifugation, a plurality of liquid and cell zones of different layers appear in the centrifugal tube, the density of red blood cells and granulocytes is higher than that of the layering liquid, the red blood cells are aggregated into a string of money shape due to the fact that the red blood cells meet Ficoll and are deposited at the bottom of the tube, platelets are suspended in plasma due to low density, single nuclear cells with the density equivalent to that of the layering liquid are densely distributed in the interface of the plasma layer and the layering liquid and are in a white membrane shape, and the cells in the layer are sucked and delivered to be washed and then suspended in the high heart.
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