CN113717938A - Synchronous separation kit for components of umbilical cord blood and use method thereof - Google Patents
Synchronous separation kit for components of umbilical cord blood and use method thereof Download PDFInfo
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Abstract
The invention discloses a synchronous separation kit for various components of umbilical cord blood, which comprises No. A diluent, No. B1 separating medium, No. B2 separating medium, No. C washing solution and No. D lysate; the A dilution is 0.9% normal saline, the B1 separation solution and the B2 separation solution are respectively a mixed solution of 9% sucrose solution and 34% compound diatrizoate solution according to the volume ratio of 50:19.11 to 50:23.28, and the C washing solution is 0.9% normal saline or pH 7.4 +/-0.1 and does not contain Ca2+And Mg2+PB of (1)The S solution and the D lysate are erythrocyte lysates. The invention provides a technology and a kit for synchronously separating components such as platelets, red blood cells, plasma, exosomes and neutrophils from umbilical cord blood, which can be industrialized, can solve the technical problem that the components except hematopoietic stem cells in the umbilical cord blood are not synchronously separated, and can greatly promote the full utilization of the components in the umbilical cord blood.
Description
Technical Field
The invention relates to a kit and a using method thereof, in particular to a synchronous separation kit for all components of umbilical cord blood and a using method thereof, which are a kit capable of synchronously separating platelets, red blood cells, plasma, exosomes, neutrophils, monocytes, endothelial progenitor cells, stem cells and lymphocytes of the umbilical cord blood, and belong to the technical field of umbilical cord blood separation.
Background
Since the first case of umbilical cord blood is successfully transplanted in 1988, the application and research of the umbilical cord blood have made great progress, the application of the umbilical cord blood in the world is over 8 thousands, and the application of the umbilical cord blood in China is up to 1.6 thousands. Cord blood has been used internationally to treat over 100 diseases, both hematological and non-hematological. However, in comparison with the prior art, the application of umbilical cord blood in China is mainly based on blood diseases, and the application range needs to be expanded. The most mature of the current stem cell therapy applications are hematopoietic stem cells, an important source of which is cord blood. The 7 provincial normal cord blood banks approved by the Weijian committee of China to be constructed store a large amount of cord blood resources. The umbilical cord blood contains other types of stem cells, immune cells and the like besides hematopoietic stem cells, and the hematopoietic stem cells in the umbilical cord blood are mainly used for treating blood diseases in China at present in clinical treatment, while the umbilical cord blood is used for treating more than 100 diseases internationally, including blood diseases and non-blood diseases.
The cord blood has immature T lymphocyte development and low immune function, but the expressed antigen and costimulatory molecule have no obvious difference compared with the adult periphery, and can be activated to proliferate by proper stimulation, and the proliferation time and process are slightly different from the periphery. Based on this, the lymphocytes of the umbilical cord blood can be used for the culture preparation of tumor immune cells (CAR-T, NKT, DC cells, etc.).
The plasma of umbilical cord blood is also a good resource, and the research on the plasma of umbilical cord blood is gradually increased in recent years. The umbilical cord blood plasma contains factor GDF11 with anti-aging effect. In the aspect of cell culture, umbilical cord blood serum can be used for replacing fetal calf serum, and a very satisfactory effect is achieved. The cord blood plasma can also be used for preparing cell cryopreservation liquid, and the cryopreservation effect is very ideal.
Exosomes carry some biological information molecules of the source cell, and have been studied as important vectors for in vivo information exchange and transmission. In fact, exosomes mediate a variety of biological processes, and both exosomes secreted by immune cells and exosomes secreted by non-immune cells play an important role in immune regulation, and are abundant in umbilical cord blood plasma.
Platelet-rich plasma (PRP) is Platelet-rich plasma prepared from blood. Since platelets in the human body can produce proteins having a cell adhesion function in a high concentration state and can promote a large amount of platelets to secrete a variety of growth factors that promote wound healing, tissue healing, and cell regeneration, PRP is also called a growth factor-rich plasma. PRP has the functions of rapidly stopping bleeding, relieving pain and accelerating wound healing, can greatly reduce the formation of postoperative scars, is widely applied to various surgical operations, cardiac operations and plastic operations from the middle of the nineties of the last century, and is also widely applied to the aspect of medical cosmetology at present. The PRP in cord blood has stronger capability of secreting various factors and stronger growth factors rich in plasma of cord blood, and the PRP in cord blood of the same blood type has the functions of stopping bleeding, relieving pain and accelerating wound healing and can be slowly embodied in the aspects of medical cosmetology.
The content of hemoglobin (Hb) in umbilical cord blood is 20-50% higher than that in adult venous blood, and the umbilical cord blood is rich in erythropoietin, so that the infusion amount can be reduced. Therefore, umbilical cord blood is also used more and more clinically for transfusion therapy. Especially for easy-allergic groups, the umbilical cord blood has no allergen, and the complicated step that adult blood is used for washing red blood cells for many times is avoided.
At present, after umbilical cord blood is collected in seven umbilical cord blood hematopoietic stem cell banks in the whole country, the umbilical cord blood hematopoietic stem cells are extracted and stored, other components in the umbilical cord blood are not fully utilized, which is a great waste of umbilical cord blood resources, and the main reason for the extraction is that when the umbilical cord blood hematopoietic stem cells are separated, components such as umbilical cord blood platelets, red blood cells, plasma, exosomes and neutrophils are synchronously separated without a mature technology. In addition, the use of cord blood instead is a considerable solution in the face of the generally low availability of blood donors and the lack of plasma resources. In the aspect of cord blood PRP, with the increase of age (especially for the old), the activity of platelets and hematopoietic stem cells in peripheral blood is increasingly poor, the functional availability of various released growth factors is low, the activity of cord blood PRP extracted from cord blood is strong, the functional availability of various released growth factors is high, and the effects of treating cartilage injury, relieving pain and recovering functions are obvious, especially the effect of treating senile osteoarthritis is obvious; meanwhile, the curative effect of facial beauty, diabetic foot and wound repair difficult to heal and striae gravidarum repair treatment is superior to that of autologous blood PRP.
Disclosure of Invention
The invention aims to solve the technical problem of the prior art and provides a synchronous separation kit for each component of umbilical cord blood and a use method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention firstly provides a synchronous separation kit for various components of umbilical cord blood, which comprises No. A diluent, No. B1 separating medium, No. B2 separating medium, No. C washing solution and No. D lysis solution.
In the above technical scheme, the kit further comprises consumables, wherein the consumables comprise 10 of 50ml centrifuge tubes, 20 of 15ml centrifuge tubes, 10 of 10ml pipettes, 5 of 3ml pasteur pipettes and 175cm of straws23 cell culture flasks.
In the technical scheme, the diluent A is 0.9% of normal saline.
In the technical scheme, the A diluent is preferably added with human serum albumin, and the mass concentration of the human serum albumin added into the A diluent is 0.5-1%.
In the technical scheme, the B1 separating medium is prepared by the following method: preparing 40% of polysucrose liquid into 9% of polysucrose liquid by Hank's liquid; then preparing the 60 percent compound meglumine diatrizoate injection into a compound meglumine diatrizoate solution with the mass concentration of 34 percent by using Hank's solution; finally, 9 percent of the polysucrose solution and 34 percent of the compound diatrizoate meglumine solution are mixed according to the volume ratio of 50:19.11, and the mixture is filtered and sterilized by a filter membrane with the diameter of 0.22 mu m to prepare a separation solution B1.
In the technical scheme, the mass concentration of the separation liquid B1 is 1.075 +/-0.001 g/ml.
In the technical scheme, the B2 separating medium is prepared by the following method: preparing powdery Fi-coll-400 into 9% of polysucrose liquid by using Hank's liquid; then, 60 percent of compound meglumine diatrizoate injection is prepared into 34 percent of compound meglumine diatrizoate solution by Hank's solution, and finally 9 percent of sucrose solution and 34 percent of compound meglumine diatrizoate solution are mixed according to the volume ratio of 50:23.28, and the mixture is filtered and sterilized by a 0.22 mu m filter membrane to prepare separation solution B2.
In the technical scheme, the mass concentration of the B2 separating medium is 1.082 +/-0.001 g/ml.
In the technical scheme, the No. C washing solution is 0.9% of normal saline or does not contain Ca at the pH value of 7.4 +/-0.12+And Mg2+In PBS.
In the above technical scheme, the lysate D is an erythrocyte lysate, and is prepared by the following method: mixing 8.02g NH4Cl, 1g KHCO3 and 37.2mg Na2Dissolving EDTA in small amount of distilled water, adjusting pH to 7.2-7.4, adding distilled water to 1000ml, and filtering with 0.22 μm filter membrane for sterilization to obtain # D lysate.
The invention also provides a method for synchronously separating all components in the cord blood by using the synchronous separation kit for all components in the cord blood, which comprises the following steps:
firstly, separating plasma at one time: subpackaging the umbilical cord blood into a centrifuge tube a, centrifuging at 2000-;
separating primary mononuclear cells and neutrophilic granulocytes: adding equal volume of the liquid A into the centrifuge tube a in the step I, fully and uniformly mixing, then carefully adding 2-4ml of the separation liquid B1, 2-4ml of the separation liquid B2 and the uniformly mixed liquid in the centrifuge tube a into a 15ml centrifuge tube along the tube wall, centrifuging at 2000-2500rpm for 20-30 minutes, and dividing into five layers: sucking the first layer into a centrifuge tube c by using a pasteur pipette, sucking the second layer (annular milky white cell layer) of mononuclear cell layer into a centrifuge tube d, sucking the third layer (annular milky white cell layer) of neutrophilic granulocyte layer into a centrifuge tube f, discarding the fourth layer, leaving the remaining fifth layer of cells as a red blood cell layer, and leaving the layer of sediment in a 15ml centrifuge tube;
③ secondary purification of blood plasma: centrifuging the centrifuge tube b in the step two at the speed of 2000-;
concentration of platelets: centrifuging the centrifuge tube c in the step II at 2000-;
separating various cells: suspending the mononuclear cells in the centrifuge tube d in the step II by using 50-100ml of serum-free cell culture solution, then placing the mononuclear cells into a cell culture bottle, and then placing the cell culture bottle into a carbon dioxide incubator for adherent culture, wherein the mononuclear cells adhere to the wall within 2-4 hours, the endothelial cells and stem cells adhere to the wall within 10-24 hours, and the lymphocytes do not adhere to the wall;
extraction of plasma exosomes: centrifuging the secondary purified plasma obtained in the step (c) at 4 ℃ low temperature 3000-3500rpm for 5min, taking the supernatant, centrifuging the supernatant at 4 ℃ low temperature 8000-10000rpm for 30min, taking the supernatant after centrifugation, filtering the supernatant through a 0.22 mu m filter membrane, ultracentrifuging the supernatant at 4 ℃ low temperature 44000rpm for 2h, absorbing the supernatant after ultracentrifugation to obtain plasma, freezing the plasma in a refrigerator at-20 ℃, resuspending the precipitate obtained after ultracentrifugation with 20-50ml of C number solution, abandoning the ultracentrifugation at 4 ℃ low temperature 44000rpm for 2h after resuspension, taking the supernatant, re-suspending the precipitate with 5-10ml of C number solution to obtain exosomes, and subpackaging the exosomes in the refrigerator at-80 ℃ for later use.
In the technical scheme, in the step II, the volume ratio of the separation liquid B1 to the separation liquid B2 added into the centrifuge tube B is 3: 2.
In the technical scheme, in the second step, if mononuclear cells in the centrifuge tube D or neutrophils in the centrifuge tube f are mixed with red blood cells, 5-8 times of volume of lysis solution D is added into the centrifuge tube D or the centrifuge tube f to lyse the red blood cells in the centrifuge tube D or the centrifuge tube f, and the lysis time is 2 minutes; after the lysis is finished, uniformly mixing products in the centrifuge tube d or the centrifuge tube f by using 20-50ml of C-washing solution, centrifuging for 5-10 minutes at 1800 plus 2000rpm, removing supernatant and leaving precipitates, uniformly mixing the precipitates by using 20-50ml of C-washing solution again, repeatedly washing to obtain umbilical cord blood mononuclear cells and neutrophils, and then culturing the mononuclear cells.
In the technical scheme, in the step IV, the precipitates in the centrifugal tube b and the centrifugal tube c are combined and then suspended by using a proper amount of blood plasma obtained in the step III, and the total amount of the precipitates and the volume ratio of the total amount of the precipitates to the blood plasma are 1: (20-30).
In the technical scheme, in the fifth step, the mononuclear cells in the centrifugal tube d are regulated to 3 multiplied by 10 by using GTT551 serum-free cell culture solution to adjust the cell density6After each ml, put into a container of 175cm2In a cell culture flask.
The invention designs a kit capable of synchronously separating all components of umbilical cord blood and realizing industrialized production, and the kit can synchronously separate platelets, red blood cells, plasma, exosomes, neutrophils, monocytes, endothelial progenitor cells, stem cells and lymphocytes of the umbilical cord blood, thereby filling the domestic blank. The cord blood components separated by the kit can be clinically applied to: the mononuclear cell and the lymphocyte can be used for the culture and preparation of tumor immune cells; ② the lymphocyte can be used for allogeneic lymphocyte immunotherapy habitual abortion; ③ the monocyte and the lymphocyte can be used as immune cells for storage; the endothelial progenitor cells can be used for researching and treating ischemic diseases; platelet can prepare PRP and PRF and can be applied to knee osteoarthritis, wound repair of difficult healing, facial cosmetology, etc.; sixthly, applying the blood plasma to cell culture and preparing a cell freezing solution; seventhly, the umbilical cord red blood cells can replace adult red blood cells; the umbilical cord blood plasma exosome can be applied to relevant researches.
The invention provides a technology and a kit for synchronously separating components such as platelets, red blood cells, plasma, exosomes and neutrophils from umbilical cord blood, which can be industrialized, can solve the technical problem that the components except hematopoietic stem cells in the umbilical cord blood are not synchronously separated, and can greatly promote the full utilization of the components in the umbilical cord blood.
Detailed Description
The following detailed description of the embodiments of the present invention is provided, but the present invention is not limited to the following descriptions:
example 1 synchronous separation kit for various components of umbilical cord blood
Comprises No. A diluent, No. B1 separating medium, No. B2 separating medium, No. C washing liquid and No. D cracking liquid; also comprises consumables, which comprise 10 50ml centrifuge tubes, 20 15ml centrifuge tubes, 10ml pipettes, 5 pasteur pipettes and 175cm23 cell culture flasks;
the No. A diluent is 0.9% physiological saline, wherein human serum albumin is added, and the mass concentration of the human serum albumin added into the No. A diluent is 0.75%;
the B1 separating medium is prepared by the following method: preparing 40% of polysucrose liquid into 9% of polysucrose liquid by Hank's liquid; then preparing the 60 percent compound meglumine diatrizoate injection into a compound meglumine diatrizoate solution with the mass concentration of 34 percent by using Hank's solution; finally, 9 percent of the sucrose solution and 34 percent of the compound diatrizoate meglumine solution are mixed according to the volume ratio of 50:19.11, and the mixture is filtered and sterilized by a filter membrane of 0.22 mu m to prepare separation liquid B1 with the mass concentration of 1.075 +/-0.001 g/ml;
the B2 separating medium is prepared by the following method: preparing powdery Fi-coll-400 into 9% of polysucrose liquid by using Hank's liquid; then preparing the 60 percent compound meglumine diatrizoate injection into a compound meglumine diatrizoate solution with the mass concentration of 34 percent by Hank's solution, finally mixing 9 percent of sucrose solution and 34 percent of compound meglumine diatrizoate solution according to the volume ratio of 50:23.28, and filtering and sterilizing by a 0.22 mu m filter membrane to prepare a separation solution B2; the mass concentration of the B2 separating medium is 1.082 +/-0.001 g/ml;
the No. C washing solution is 0.9% of normal saline;
the lysate D is erythrocyte lysate, and is prepared by the following method: mixing 8.02g NH4Cl, 1g KHCO3 and 37.2mg Na2EDTA dissolved in a small amount of distilled waterAdjusting pH to about 7.3 in water, adding distilled water to 1000ml, filtering with 0.22 μm filter membrane, and sterilizing to obtain No. D lysate;
example 2 synchronous separation kit for various components of umbilical cord blood
Essentially the same as example 1, except that the diluent a is 0.9% physiological saline; the No. C washing solution is Ca-free with pH of about 7.42+And Mg2+In PBS.
Example 3 method for synchronously separating Components in cord blood with the kit for synchronously separating Components of cord blood according to example 1
Subpackaging 100ml of umbilical cord blood donated from a volunteer into two 50ml centrifuge tubes a, centrifuging at 2500rpm for 10 minutes, and then sucking an upper plasma layer into a centrifuge tube b;
adding equal volume of the solution A into the separated centrifuge tube a, fully and uniformly mixing, carefully adding 3ml of the solution B1, 3ml of the solution B2 and the uniformly mixed solution in the centrifuge tube a into a 15ml centrifuge tube along the wall, centrifuging at 2000rpm for 25 minutes, and dividing into five layers: sucking the first layer into a centrifuge tube c by using a pasteur pipette, sucking the second layer (annular milky white cell layer) of mononuclear cell layer into a centrifuge tube d, sucking the third layer (annular milky white cell layer) of neutrophilic granulosa cell layer into a centrifuge tube f, discarding the fourth layer, and leaving the remaining fifth layer of cells as an erythrocyte layer in a 15ml centrifuge tube;
thirdly, centrifuging the centrifuge tube b with the plasma at 2000rpm for 8 minutes, absorbing supernatant in the centrifuge tube b to obtain secondary purified plasma, and storing the secondary purified plasma in a refrigerator at 4 ℃;
fourthly, centrifuging the centrifugal tube c for 5 to 10 minutes at the speed of 2000-;
suspending the mononuclear cells in the centrifugal tube d by using 50ml of serum-free cell culture solution, then placing the mononuclear cells in a cell culture bottle, then placing the mononuclear cells in a carbon dioxide incubator for adherent culture, collecting the mononuclear cells adherent for 3 hours, collecting endothelial cells and stem cells adherent for 18 hours, and collecting the lymphocytes not adherent after 18 hours;
sixthly, centrifuging the obtained secondary purified plasma at the low temperature of 4 ℃ for 5min by 3000g, taking the supernatant, centrifuging the supernatant at the low temperature of 4 ℃ for 30min by 8000g, filtering the supernatant by a filter membrane of 0.22 mu m, ultracentrifuging the supernatant at the low temperature of 4 ℃ at the rotating speed of 44000rpm for 2h, absorbing the supernatant after the centrifugation to obtain the plasma, freezing and storing the plasma in a refrigerator at the temperature of-20 ℃, resuspending the ultracentrifuged precipitate by using a solution of No. 20mlC, ultracentrifuging the precipitate at the low temperature of 4 ℃ at the speed of 44000rpm for 2h, discarding the supernatant, re-suspending the supernatant by using a solution of No. 10mlC to obtain exosomes, subpackaging and placing the exosomes in the refrigerator at the temperature of-80 ℃ for later use.
The kit provided by the invention can synchronously separate components such as platelets, red blood cells, plasma, exosomes and neutrophils of the umbilical cord blood, can be industrialized, can solve the technical problem that the components in the umbilical cord blood except hematopoietic stem cells are not synchronously separated, and can greatly promote the full utilization of the components in the umbilical cord blood.
The above examples are only for illustrating the technical concept and features of the present invention, and are not intended to limit the scope of the present invention. All equivalent changes or modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
1. A synchronous separation kit for components of umbilical cord blood is characterized in that: comprises No. A diluent, No. B1 separating medium, No. B2 separating medium, No. C washing liquid and No. D lysis liquid.
2. The kit of claim 1, wherein: the kit also comprises consumables, wherein the consumables comprise 10 50ml centrifuge tubes, 20ml centrifuge tubes, 10ml pipettes, 5 pasteur pipettes and 175cm23 cell culture flasks.
3. The kit of claim 1, wherein: the diluent A is 0.9 percent of normal saline; the A-type diluent is added with human serum albumin, and the mass concentration of the human serum albumin added into the A-type diluent is 0.5-1%.
4. The kit of claim 1, wherein: the B1 separating medium is prepared by the following method: preparing 40% of polysucrose liquid into 9% of polysucrose liquid by Hank's liquid; then preparing the 60 percent compound meglumine diatrizoate injection into a compound meglumine diatrizoate solution with the mass concentration of 34 percent by using Hank's solution; finally, 9 percent of the polysucrose solution and 34 percent of the compound diatrizoate meglumine solution are mixed according to the volume ratio of 50:19.11, and the mixture is filtered and sterilized by a filter membrane of 0.22 mu m to prepare a separation solution B1; the mass concentration of the B1 separating medium is 1.075 +/-0.001 g/ml.
5. The kit of claim 1, wherein: the B2 separating medium is prepared by the following method: preparing powdery Fi-coll-400 into 9% of polysucrose liquid by using Hank's liquid; then preparing the 60 percent compound meglumine diatrizoate injection into a compound meglumine diatrizoate solution with the mass concentration of 34 percent by Hank's solution, finally mixing 9 percent of sucrose solution and 34 percent of compound meglumine diatrizoate solution according to the volume ratio of 50:23.28, and filtering and sterilizing by a 0.22 mu m filter membrane to prepare a separation solution B2; the mass concentration of the B2 separating medium is 1.082 +/-0.001 g/ml.
6. The kit of claim 1, wherein: the No. C washing solution is 0.9% physiological saline or pH 7.4 +/-0.1 and does not contain Ca2+And Mg2+The PBS solution of (1); the lysate D is erythrocyte lysate, and is prepared by the following method: mixing 8.02g NH4Cl, 1g KHCO3 and 37.2mg Na2Dissolving EDTA in small amount of distilled water, adjusting pH to 7.2-7.4, adding distilled water to 1000ml, and filtering with 0.22 μm filter membrane for sterilization to obtain # D lysate.
7. A method for synchronously separating components in cord blood by using the synchronous separation kit for components in cord blood according to any one of claims 1 to 6, which is characterized by comprising the following steps:
firstly, separating plasma at one time: subpackaging the umbilical cord blood into a centrifuge tube a, centrifuging at 2000-;
separating primary mononuclear cells and neutrophilic granulocytes: adding the equal volume of the solution A into the centrifuge tube a in the step I, fully and uniformly mixing, then carefully adding the separation solution No. 2-4mlB1, the separation solution No. 2-4ml B2 and the uniformly mixed solution in the centrifuge tube a into a 15ml centrifuge tube along the tube wall, centrifuging at 2000-2500rpm for 20-30 minutes, and dividing into five layers: sucking the first layer into a centrifuge tube c by using a pasteur pipette, sucking the second layer (annular milky white cell layer) of mononuclear cell layer into a centrifuge tube d, sucking the third layer (annular milky white cell layer) of neutrophilic granulocyte layer into a centrifuge tube f, discarding the fourth layer, leaving the remaining fifth layer of cells as a red blood cell layer, and leaving the layer of sediment in a 15ml centrifuge tube;
③ secondary purification of blood plasma: centrifuging the centrifuge tube b in the step two at the speed of 2000-;
concentration of platelets: centrifuging the centrifuge tube c in the step II at 2000-;
separating various cells: suspending the mononuclear cells in the centrifuge tube d in the step II by using 50-100ml of serum-free cell culture solution, then placing the mononuclear cells into a cell culture bottle, and then placing the cell culture bottle into a carbon dioxide incubator for adherent culture, wherein the mononuclear cells adhere to the wall within 2-4 hours, the endothelial cells and stem cells adhere to the wall within 10-24 hours, and the lymphocytes do not adhere to the wall;
extraction of plasma exosomes: centrifuging the secondary purified plasma obtained in the step (c) at 4 ℃ low temperature 3000-3500rpm for 5min, taking the supernatant, centrifuging the supernatant at 4 ℃ low temperature 8000-10000rpm for 30min, taking the supernatant after centrifugation, filtering the supernatant through a 0.22 mu m filter membrane, ultracentrifuging the supernatant at 4 ℃ low temperature 44000rpm for 2h, sucking the supernatant after ultracentrifuging to obtain plasma, freezing the plasma in a refrigerator at-20 ℃, resuspending the precipitate obtained after ultracentrifuging with 20-50ml of C number solution, ultracentrifuging the precipitate at 4 ℃ low temperature 44000rpm for 2h after resuspension, discarding the supernatant, resuspending the precipitate with 5-10ml of C number solution again to obtain exosomes, and subpackaging the exosomes in the refrigerator at-80 ℃ for later use.
8. The method of claim 7, wherein in the second step, the volume ratio of the separation solution B1 to the separation solution B2 added to the centrifuge tube B is 3: 2.
9. The method according to claim 7, wherein in the step (iv), the precipitates in the centrifuge tube b and the centrifuge tube c are combined and then suspended by using a proper amount of plasma obtained in the step (iii), and the total amount of the precipitates and the volume ratio of the precipitates to the plasma are 1: (20-30).
10. The method of claim 7, wherein in step five, the mononuclear cells in the centrifuge tube d are adjusted to 3 x 10 cell density by GTT551 serum-free cell culture solution6After each ml, put into a container of 175cm2In a cell culture flask.
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