CN105754938A - Kit and method for separating mononuclear cells from umbilical cord blood - Google Patents
Kit and method for separating mononuclear cells from umbilical cord blood Download PDFInfo
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Abstract
The present invention discloses a kit and a method for separating mononuclear cells from umbilical cord blood. The kit for separating the mononuclear cells from the umbilical cord blood comprises the following components: a dilution solution which is a sodium chloride solution with the mass percentage of 0.1% to 5% or a phosphate buffer with pH of 7 to 7.5; a precipitating agent which is a hydroxyethyl starch solution with the mass percentage of 2.4%-12%, a hydroxymethyl starch solution with the mass percentage of 2.4%-12% or a methyl cellulose solution with the mass percentage of 2.4%-12%; a resuspension solution which is an albumin solution with the concentration of 5 to 10% or human AB blood plasma with the concentration of 10-30%; and a separation liquid which is a silica gel particle suspension treated by polyvinylpyrrolidone, and the separation liquid has a density of 1.072-1.086g / ml. The kit and the method can effectively separate and save the mononuclear cells, and have good application prospect. The effect is better than that of commercially available kits.
Description
Technical field
The present invention relates to a kind of from the test kit separating mononuclearcell from Cord blood and method.
Background technology
Cord blood (UmbilicalCordBloodStemCells) is after delivery of fetus, residue in the blood in ligation umbilical cord and Placenta Hominis, all the components containing normal blood, such as erythrocyte, leukocyte, platelet and blood plasma, possibly together with cord blood stem cell (UmbilicalCordBloodStemCells), endothelial precursor cell etc..
Cord blood has the cell colony of individual cells core, mainly include with hematopoietic stem cell, mescenchymal stem cell, endothelial progenitor cell be representative ancestral cells and lymphocyte, mononuclear cell.Wherein, mononuclear cell and lymphocyte in leukocyte feed back in the patient in vitro after treatment, have the effect of broad-spectrum anti-tumor, can be used for the treatment of kinds of tumors different phase.Cord blood stem cell (UmbilicalCordBloodStemCells), umbilical cord stem cells includes hematopoietic stem/progenitor, also includes mescenchymal stem cell, endothelial precursor cell etc..Hematopoietic stem/progenitor can be divided into various blood cell, such as erythrocyte, leukocyte, platelet etc..Compared with bone marrow and peripheral blood hematopoietic stem cells, it is low that cord blood stem cell has immunogenicity, and allosome rejection is little, regeneration capacity is higher compared with the above two and speed faster, the feature such as the risk that is infected by the virus is low.At present, hematopoietic stem/progenitor is mainly used in treatment disease in the blood system (aplastic anemia, thalassemia etc.), malignant disease (acute lymphoblastic leukemia, acute myelogenous leukemia, multiple myeloma etc.), congenital metabolic disease (alba adrenal gland's muscular dystrophy, amyloidosis, dyskeratosis congenita etc.), autoimmune disease (multiple sclerosis, rheumatic arthritis, lupus erythematosus etc.).It addition, mesenchymal stem cells in umbilical cord blood is for the treatment of damaging Regeneration and Repair, endothelium ancestral cells is used for treating ischemic diseases, has all achieved prominent curative effect clinically.Thus, once it was considered the living resources that the Cord blood of Biohazard Waste has nowadays become important.
At present, world medical circle has been accepted as cord blood stem cell contained in human umbilical cord blood mononuclear cell and lymphocyte etc. and has the potential treating multiple disease, but how to separate from Cord blood, purification obtains clinical grade cell, and how as far as possible the damage in separating and preserving of the reduction cell is still the hot issue of research.The main purpose separating Cord blood is in that all to get rid of erythrocyte therein, platelet and blood plasma, and will wherein be enriched with at interior nucleated cell containing cord blood stem cell and lymphocyte etc..By processes such as separation, concentration, purification, the concentration of human umbilical cord blood mononuclear cell is promoted, in order to meet the requirement of clinical practice.
Conventional Cord blood separation method includes ammonium chloride cracking process, the hetastarch sedimentation method, density－gradient centrifuga－tion method, immunological magnetic bead sorting method and monoclonal antibody selected by flow cytometry apoptosis method etc..Wherein, density－gradient centrifuga－tion method is simple effectively the most commonly used because of it, at present, also has many commercialization mononuclearcell separating kits, but separating effect is still not fully up to expectations, it is necessary to improve further.
Summary of the invention
The invention provides the density－gradient centrifuga－tion method of a kind of improvement and supporting test kit thereof, for separating mononuclearcell from Cord blood.
The present invention separates the test kit of mononuclearcell from Cord blood, and it includes following component:
Diluent: mass percent be 0.1%～5% sodium chloride solution or pH be the phosphate buffer of 7～7.5；
Precipitant: hydroxymethyl starch solution or the methocel solution of mass percent 2.4%～12% that hydroxyethyl starch solution that mass percent is 2.4%～12%, mass percent are 2.4%～12%；
Re-suspension liquid: concentration is the albumin solution of 5～10% or people's AB blood plasma that concentration is 10-30%；
Separating medium: density is the silica gel particle suspension that 1.072～1.086g/ml polyvinylpyrrolidone processes.
Preferably, described diluent is mass percent is 0.9% sodium chloride solution.
Preferably, described precipitant is mass percent is the hydroxyethyl starch solution of 1.2%.
Preferably, described re-suspension liquid is the albumin solution of concentration 7% or people's AB blood plasma that concentration is 20%.
Preferably, described separating medium to be density be silica gel particle suspension that 1.078g/ml polyvinylpyrrolidone processes.
The present invention adopts the method that aforementioned agents box separates mononuclearcell from Cord blood, and it comprises the steps:
(1) take Cord blood, use isopyknic diluted, add the precipitant of Cord blood 1/2 volume, mixing, sedimentation, Aspirate supernatant；
(2) centrifugal, obtain precipitation, wherein, centrifugal force is 600～900g, and centrifugation time is 5～10min；
(3) by re-suspension liquid resuspended step (2) gained precipitation, solution is obtained；
(4) noting in separating medium by step (3) gained solution, centrifugal, centrifugal force is 500～800g, and centrifugation time is 10～30min, draws cloud and mist layer；
(5) with the resuspended cloud and mist layer of re-suspension liquid, obtaining solution, centrifugal, centrifugal force is 600～900g, and centrifugation time is 5～10min, obtains precipitation；
(6) by re-suspension liquid resuspended step (5) gained precipitation, obtaining solution, centrifugal, centrifugal force is 600～900g, and centrifugation time is 5～10min, is repeated once, and obtains precipitation, mononuclearcell；
(7) with the resuspended mononuclearcell of re-suspension liquid, preserve.
In step (1), described sedimentation is natural subsidence or centrifugal sedimentation, and described natural subsidence is standing 30～50min；Described centrifugal sedimentation is centrifugal 5～10min under 50g centrifugal force.
In step (2), centrifugal force is 800g, and centrifugation time is 5min；
In step (3), the volume of gained solution is equal with the volume of Cord blood in step (1)；
In step (4), time centrifugal, centrifugal force is 550g, and centrifugation time is that 25min, raising speed and reduction of speed are set to 0；
In step (5) and step (6), centrifugal centrifugal force is 800g, and centrifugation time is 5min.
In step (5), the volume of gained solution is in step (1) 2 times of the volume of Cord blood；
In step (6), the volume of gained solution is in step (1) the 1/2 of the volume of Cord blood.
The present inventor finds in long-term practice, existing density－gradient centrifuga－tion method, all adopt the resuspended precipitation of normal saline, cause that the mononuclear cell response rate is low, and inventor has been found that the employing resuspended precipitation of re-suspension liquid of the present invention, in combination with special process of the present invention, but can be effectively improved the monocytic response rate, achieve wholly unexpected technique effect.
To sum up, adopting test kit of the present invention, the efficiency separating purification of individual nucleus according to the inventive method from Cord blood is high, and the response rate of mononuclearcell is 2.1～3.9 times that existing commercial reagent box is compared；Meanwhile, adopting re-suspension liquid in test kit of the present invention to preserve the excellent effect of mononuclearcell as preservation liquid, application prospect is good.
Below by detailed description of the invention, the present invention is described in further details, but it is not limitation of the present invention, foregoing according to the present invention, ordinary technical knowledge and customary means according to this area, without departing under the above-mentioned basic fundamental thought premise of the present invention, it is also possible to make the amendment of other various ways, replacement or change.
Accompanying drawing explanation
Fig. 1 be in embodiment 3 mononuclearcell preserving liquid 1, preserve liquid 2 and preserve in liquid 37 Amino Actinomycin dyeing, flow cytomery cell survival rate after 24 hours；
Fig. 2 be in embodiment 3 mononuclearcell preserving liquid 1, preserve liquid 2 and preserve in liquid 37 Amino Actinomycin dyeing, flow cytomery cell survival rate after 48 hours；
Fig. 3 be in embodiment 3 mononuclearcell preserving liquid 1, preserve liquid 2 and preserve in liquid 37 Amino Actinomycin dyeing, flow cytomery cell survival rate after 72 hours.
Detailed description of the invention
Material:；
0.9% sodium chloride solution, Kelun Pharm Ind Co., Ltd., Sichuan；
Hetastarch 130/0.4 sodium chloride injection, the bright medical joint-stock company of Switzerland shellfish；
The silica gel particle suspension that polyvinylpyrrolidone processes, GE company；
People's AB blood plasma, LifeTechnologies company；
20% human albumin, Chengdu Rong Sheng pharmaceutcal corporation, Ltd；
5810R centrifuge, Eppendorf company.
Embodiment 1 test kit of the present invention and separation method
One, test kit composition
Diluent: mass percent is 0.9% sodium chloride solution.
Precipitant: mass percent is the hydroxyethyl starch solution of 6%.
Separating medium: density is the silica gel particle suspension that 1.078g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is people's AB blood plasma of 20%.
Two, separation method
1, collecting and be stored in anticoagulant blood bag by Human Umbilical Cord's blood in an aseptic environment, be loaded in 50ml centrifuge tube by Human Umbilical Cord's blood system of collection in super-clean bench, often 20ml Cord blood inserted by pipe；
2, injecting equal-volume diluent in the centrifuge tube of the above-mentioned 20ml of filling Cord blood to be diluted, add 10ml precipitant, gentle inversion mixes, and stands 30～50 minutes；
3, the supernatant that above-mentioned sedimentation obtains is placed in a new centrifuge tube；
4, the centrifuge tube that the 3rd step obtains is placed in centrifuge, centrifuge 800g is set centrifugal 5 minutes, draws and preserve supernatant autologous plasma in another new centrifuge tube；
5, by the resuspended precipitation of re-suspension liquid, it is settled to 20ml, slowly notes on separating medium；
6, being placed in centrifuge by the 5th step centrifuge tube, arrange centrifuge raising speed and reduction of speed is set to 0, centrifugal force is 550g, and centrifugation time is 25 minutes；
7, cloud and mist layer is drawn to a new 50ml centrifuge tube.
8, with the resuspended cloud and mist layer of re-suspension liquid, it is settled to 40ml, is placed in centrifuge, centrifuge 800g is set centrifugal 5 minutes, centrifugal；
9, abandon supernatant, with the resuspended cloud and mist layer of re-suspension liquid, be settled to 10ml, 800g centrifugal 5 minutes, be repeated once, obtain mononuclearcell；
10, resuspended by re-suspension liquid, it is settled to 10ml, preserves.
Embodiment 2 test kit of the present invention and separation method
One, test kit composition
Diluent: mass percent is 0.1% sodium chloride solution dilution.
Precipitant: mass percent is the hydroxyethyl starch solution of 2.4%.
Separating medium: density is the silica gel particle suspension that 1.072g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is people's AB blood plasma of 10%.
Two, separation method
1, collecting and be stored in anticoagulant blood bag by Human Umbilical Cord's blood in an aseptic environment, be loaded in 50ml centrifuge tube by Human Umbilical Cord's blood system of collection in super-clean bench, often 20ml Cord blood inserted by pipe；
2, injecting equal-volume diluent in the centrifuge tube of the above-mentioned 20ml of filling Cord blood to be diluted, add 10ml precipitant, gentle inversion mixes, centrifugal 5～10min under 50g centrifugal force；
3, the supernatant that above-mentioned sedimentation obtains is placed in a new centrifuge tube；
4, the centrifuge tube that the 3rd step obtains is placed in centrifuge, centrifuge 600g is set centrifugal 10 minutes, draws and preserve supernatant autologous plasma in another new centrifuge tube；
5, by the resuspended precipitation of re-suspension liquid, it is settled to 20ml, slowly notes on separating medium；
6, being placed in centrifuge by the 5th step centrifuge tube, arrange centrifuge raising speed and reduction of speed is set to 0, centrifugal force is 500g, and centrifugation time is 30 minutes；
7, cloud and mist layer is drawn to a new 50ml centrifuge tube.
8, with the resuspended cloud and mist layer of re-suspension liquid, it is settled to 50ml, is placed in centrifuge, centrifuge 600g is set centrifugal 10 minutes, centrifugal；
9, abandon supernatant, with the resuspended cloud and mist layer of re-suspension liquid, be settled to 10ml, 600g centrifugal 10 minutes, be repeated once, obtain mononuclearcell；
10, resuspended by re-suspension liquid, it is settled to 10ml, preserves.
Embodiment 3 test kit of the present invention and separation method
One, test kit composition
Diluent: mass percent is 5% sodium chloride solution dilution.
Precipitant: mass percent is the hydroxyethyl starch solution of 12%.
Separating medium: density is the silica gel particle suspension that 1.086g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is people's AB blood plasma of 30%.
Two, separation method
1, collecting and be stored in anticoagulant blood bag by Human Umbilical Cord's blood in an aseptic environment, be loaded in 50ml centrifuge tube by Human Umbilical Cord's blood system of collection in super-clean bench, often 20ml Cord blood inserted by pipe；
2, injecting equal-volume diluent in the centrifuge tube of the above-mentioned 20ml of filling Cord blood to be diluted, add 10ml precipitant, gentle inversion mixes, and stands 30～50 minutes；
3, the supernatant that above-mentioned sedimentation obtains is placed in a new centrifuge tube；
4, the centrifuge tube that the 3rd step obtains is placed in centrifuge, centrifuge 900g is set centrifugal 6 minutes, draws and preserve supernatant autologous plasma in another new centrifuge tube；
5, by the resuspended precipitation of re-suspension liquid, it is settled to 20ml, slowly notes on separating medium；
6, being placed in centrifuge by the 5th step centrifuge tube, arrange centrifuge raising speed and reduction of speed is set to 0, centrifugal force is 800g, and centrifugation time is 10 minutes；
7, cloud and mist layer is drawn to a new 50ml centrifuge tube.
8, with the resuspended cloud and mist layer of re-suspension liquid, it is settled to 40ml, is placed in centrifuge, centrifuge 900g is set centrifugal 6 minutes, centrifugal；
9, abandon supernatant, with the resuspended cloud and mist layer of re-suspension liquid, be settled to 10ml, 900g centrifugal 6 minutes, be repeated once, obtain mononuclearcell；
10, resuspended by re-suspension liquid, it is settled to 10ml, preserves.
Embodiment 4 test kit of the present invention and separation method
One, test kit composition
Diluent: pH is the phosphate buffer of 7.
Precipitant: mass percent is the hydroxymethyl starch solution of 2.4%.
Separating medium: to be density be silica gel particle suspension that 1.078g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is the albumin solution of 7%.
Two, separation method
With embodiment 1.
Embodiment 5 test kit of the present invention and separation method
One, test kit composition
Diluent: pH is the phosphate buffer of 7.5.
Precipitant: mass percent is the hydroxymethyl starch solution of 12%.
Separating medium: to be density be silica gel particle suspension that 1.072g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is the albumin solution of 5%.
Two, separation method
With embodiment 1.
Embodiment 6 test kit of the present invention and separation method
One, test kit composition
Diluent: mass percent is the sodium chloride solution of 0.9%.
Precipitant: mass percent is the methocel solution of 2.4%.
Separating medium: to be density be silica gel particle suspension that 1.078g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is the albumin solution of 10%.
Two, separation method
With embodiment 1.
Embodiment 7 test kit of the present invention and separation method
One, test kit composition
Diluent: mass percent is the sodium chloride solution of 5%.
Precipitant: mass percent is the methocel solution of 12%.
Separating medium: to be density be silica gel particle suspension that 1.086g/ml polyvinylpyrrolidone processes.
Re-suspension liquid: concentration is the albumin solution of 7%.
Two, separation method
With embodiment 1.
By the mode of experimental example, beneficial effects of the present invention is described below:
The comparison of experimental example 1 separation method of the present invention and existing separation method
One, experimental technique
Same cord blood is equally divided into 3 parts, is easily separated by following 3 kinds of reagent and method；
1, reagent and method 1: be easily separated according to the embodiment of the present invention 1 test kit and separation method；
2, reagent and method 2: the method on the reagent provided according to Tianjin virtue Si Tanmu cell processing kit and description is easily separated, specific as follows:
(1) by the 20ml Cord blood containing sodium citrate or anticoagulant heparin and A liquid equal-volume；
(2) being added by Cord blood equal-volume B liquid in the liquid of the 1st step, fully mix, room temperature stands 20～30 minutes；
(3) supernatant is moved in new 50ml centrifuge tube, do not draw red blood cell layer as far as possible；
(4) the 3rd step is collected the liquid in centrifuge tube, be placed in centrifuge, arrange 2500 and leave the heart 5 minutes；
(5) take the centrifuge tube after the 4th step is centrifuged, abandon supernatant, be settled to 20ml with normal saline is resuspended；
(6) take new 50ml centrifuge tube, add 20mlC liquid, the cell suspension obtained of the 5th step is slowly added on C liquid liquid level along centrifugal tube wall；
(7) centrifuge tube of the 6th step is placed in centrifuge, arranges 1500 and leave the heart 25 minutes, draw cloud and mist layer after centrifugal to new centrifuge tube, add normal saline and be settled to 40ml；
(8) centrifuge tube of the 7th step is placed in centrifuge, arranges 2500 and leave the heart 5 minutes, abandon supernatant, after repeating above-mentioned washing centrifugation step, be settled to 10ml with normal saline.
3, reagent and method 3: according to Shenyang Si Taimu Sail sample rate separating medium, the method on the reagent provided and description is easily separated, specific as follows:
(1) the 20ml Cord blood containing sodium citrate or heparin sodium anticoagulant is transferred in 50ml centrifuge tube, mixes with A liquid equal-volume；
(2) being added by Cord blood equal-volume B liquid in the liquid of the 1st step, fully mix after container closure, room temperature stands 20～30 minutes；
(3) supernatant is moved in new 50ml centrifuge tube, do not pipette precipitation as far as possible；
(4) being placed in centrifuge by the cell suspension that the 3rd step is collected, 2500 leave the heart 5 minutes, abandon supernatant, and normal saline is resuspended is settled to 20ml；
(5) the 4th step cell suspension being layered on the centrifuge tube upper strata equipped with 20mlC liquid, be placed in centrifuge and arrange 650g centrifugal 25 minutes, arrange centrifuge lifting speed is 0 simultaneously；
(6) draw in the new centrifuge tube of cloud and mist stratification, add normal saline and mix to 45ml, be placed in centrifuge 800g centrifugal 5 minutes；
(7) abandon supernatant, after repeated washing centrifugation step, be settled to 10ml with normal saline is resuspended.
Detection: reagent and method 1, reagent and method 2 and reagent are separated after the cell suspension obtained draws 200 μ l respectively with method 3, detect the absolute value of erythrocyte, leukocyte, lymphocyte, mononuclear cell and mononuclearcell in each cell suspension sample with Japan's Sysmex blood cell analysis machine, and calculate every cell recoveries；
Total cellular score × 100% before cell number/separation that the response rate=separation obtains
Two, experimental result
The present embodiment experiment repeats more than 3 times, and often group results averaged is as described in Table 1:
Table 1: Cord blood separates, with method 3, the response rate obtaining leukocyte, lymphocyte, mononuclear cell and mononuclearcell with method 2 and reagent with method 1, reagent at reagent
| The present invention (reagent and method 1) | Reagent and method 2 | Reagent and method 3 | |
| Erythrocyte residual rate (%) | 0.01 | 0.01 | 0.01 |
| Leukocyte recovery (%) | 24.65 | 6.41 | 11.81 |
| The lymphocyte response rate (%) | 57.23 | 19.29 | 35.03 |
| The mononuclear cell response rate (%) | 76.55 | 8.60 | 17.30 |
| The mononuclearcell response rate (%) | 62.76 | 16.11 | 29.71 |
From table 1 it follows that adopt test kit of the present invention, the response rate separating purification of individual nucleus according to the inventive method from Cord blood significantly improves, and is 2.1～3.9 times of commercial reagent box.
Experimental result illustrates, adopts the inventive method can efficiently separate mononuclearcell, and separating effect is significantly better than commercially available test kit.
The survival rate that experimental example 2 Human Umbilical Cord's blood mononuclear cell preserves in liquid in difference compares
1, after the 10ml cell suspension that embodiment 1 obtains being equally divided into three parts, it is placed in centrifuge, 800g is set centrifugal 5 minutes；
2, abandon supernatant, preserve liquid re-suspended cell with following three kinds respectively:
Preserve liquid 1 (present invention): concentration is people's AB blood plasma of 20%；
Preserve liquid 2: normal saline；
Preserving liquid 3: Cell protective solutions, formula is table 2 below such as,
Table 2: cytoprotective formula of liquid
| Mass percent is 2% bovine serum albumin | Percent by volume 10% |
| Mass percent is 1% ethylenediaminetetraacetic acid | Percent by volume 0.02% |
| Mass percent is 10% glucose | Percent by volume 5% |
| 5000AxaIU/ml calciparine | Percent by volume 1.5% |
| Sterilized water | Percent by volume 36.5% |
3, obtain cell after 24 hours, three kinds preserve liquid respectively take 1 × 105Individual cell, after 2ml brine, abandons supernatant after arranging centrifugal force 800g centrifugal 5 minutes, resuspended with 50 μ l normal saline；
4, cell gravity treatment liquid add respectively the CD45 streaming antibody of FITC labelling, the CD34 streaming antibody of PE labelling and each 10 μ l of 7 Amino Actinomycin, fully mixes with cell sample；
5, the cell suspension of mixing is hatched 20 minutes or 4 DEG C of lucifuges hatch 30 minutes in room temperature lucifuge；
6, in cell suspension, add 2ml normal saline respectively, abandon supernatant after centrifugal force 800g is set centrifugal 5 minutes, and precipitate with 500 μ l normal saline re-suspended cells；
7, with Beckman Ku Erte FC500 flow cytomery cell survival rate.Within 24 hours, often organize result as shown in Figure 1；
8, little constantly 48 hours and 72, repeat embodiment 3-7 step, to detect the survival rate of 48 hours and 72 hour cells, within 48 hours and 72 hours, often organize result as shown in Figures 2 and 3；
9, embodiment 3 experiment repeats more than 3 times, and often group results averaged is as described in Table 3.
Table 3: each group preserves the cell survival rate 24 hours, 48 hours and 72 hours in liquid
| Preserve liquid 1 (present invention) | Preserve liquid 2 | Preserve liquid 3 | |
| 24 hours | 94.17% | 90.37% | 92.18% |
| 48 hours | 93.89% | 87.50% | 91.78% |
| 72 hours | 93.38% | 83.59% | 91.37% |
As can be seen from Table 3; mononuclearcell preserves the survival rate in liquid apparently higher than normal saline as the preservation liquid 3 preserving liquid 2 and cytoprotective formula of liquid in the present invention; illustrate that the present invention preserves liquid and can effectively protect mononuclearcell, effect to be substantially better than normal saline and preservation liquid conventional at present.
To sum up, adopt test kit of the present invention, mononuclearcell can be efficiently separated according to the inventive method, the response rate of mononuclearcell is high, is significantly better than commercially available test kit, simultaneously, adopting re-suspension liquid in test kit of the present invention to preserve the excellent effect of mononuclearcell as preservation liquid, application prospect is good.
Claims (12)
1. the test kit separating mononuclearcell from Cord blood, it is characterised in that: it includes following component:
Diluent: mass percent be 0.1%～5% sodium chloride solution or pH be the phosphate buffer of 7～7.5；
Precipitant: hydroxymethyl starch solution or the methocel solution of mass percent 2.4%～12% that hydroxyethyl starch solution that mass percent is 2.4%～12%, mass percent are 2.4%～12%；
Re-suspension liquid: concentration is the albumin solution of 5～10% or people's AB blood plasma that concentration is 10-30%；
Separating medium: density is the silica gel particle suspension that 1.072～1.086g/ml polyvinylpyrrolidone processes.
2. test kit according to claim 1, it is characterised in that: described diluent is mass percent is 0.9% sodium chloride solution dilution.
3. test kit according to claim 1, it is characterised in that: described precipitant is mass percent is the hydroxyethyl starch solution of 6%.
4. test kit according to claim 1, it is characterised in that: described re-suspension liquid is the albumin solution of concentration 7% or people's AB blood plasma that concentration is 20%.
5. test kit according to claim 1, it is characterised in that: described separating medium is density be 1.078g/ml polyvinylpyrrolidone process silica gel particle suspension.
6. one kind adopts test kit described in Claims 1 to 5 any one to separate the method preserving mononuclearcell, it is characterised in that: it comprises the steps:
(1) take Cord blood, use isopyknic diluted, add the precipitant of Cord blood 1/2 volume, mixing, sedimentation, Aspirate supernatant；
(2) centrifugal, obtain precipitation, wherein, centrifugal force is 600～900g, and centrifugation time is 5～10min；
(3) by re-suspension liquid resuspended step (2) gained precipitation, solution is obtained；
(4) noting in separating medium by step (3) gained solution, centrifugal, centrifugal force is 500～800g, and centrifugation time is 10～30min, draws cloud and mist layer；
(5) with the resuspended cloud and mist layer of re-suspension liquid, obtaining solution, centrifugal, centrifugal force is 600～900g, and centrifugation time is 5～10min, obtains precipitation；
(6) by re-suspension liquid resuspended step (5) gained precipitation, obtaining solution, centrifugal, centrifugal force is 600～900g, and centrifugation time is 5～10min, is repeated once, and obtains precipitation, mononuclearcell；
(7) with the resuspended mononuclearcell of re-suspension liquid, preserve.
7. method according to claim 6, it is characterised in that: in step (1), described sedimentation is natural subsidence or centrifugal sedimentation, and described natural subsidence is standing 30～50min；Described centrifugal sedimentation is centrifugal 5～10min under 50g centrifugal force.
8. method according to claim 6, it is characterised in that: in step (2), centrifugal force is 800g, and centrifugation time is 5min.
9. method according to claim 6, it is characterised in that: in step (3), the volume of gained solution is equal with the volume of Cord blood in step (1).
10. method according to claim 6, it is characterised in that: in step (4), time centrifugal, centrifugal force is 550g, and centrifugation time is that 25min, raising speed and reduction of speed are set to 0.
11. method according to claim 6, it is characterised in that: in step (5), centrifugal centrifugal force is 800g, and centrifugation time is 5min；The volume of gained solution is in step (1) 2～2.5 times of the volume of Cord blood.
12. method according to claim 6, it is characterised in that: in step (6), centrifugal centrifugal force is 800g, and centrifugation time is 5min；The volume of gained solution is in step (1) the 1/2 of the volume of Cord blood.
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| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| CN109576223A (en) * | 2017-09-29 | 2019-04-05 | 重庆金时代生物技术有限公司 | A kind of cultural method of placental blood candidate stem cell |
| CN113061577A (en) * | 2017-09-05 | 2021-07-02 | 四川新生命干细胞科技股份有限公司 | Isolated culture method of high-purity NK cells |
| CN116429734A (en) * | 2022-01-04 | 2023-07-14 | 南京医科大学 | Method for separating human peripheral blood leucocyte and detecting active oxygen |
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| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| CN106754714B (en) * | 2016-11-11 | 2020-05-19 | 北正赛欧（北京）生物科技有限公司 | Umbilical cord blood sample diluent, kit and method for processing umbilical cord blood to obtain stem cells |
| CN113061577A (en) * | 2017-09-05 | 2021-07-02 | 四川新生命干细胞科技股份有限公司 | Isolated culture method of high-purity NK cells |
| CN109576223A (en) * | 2017-09-29 | 2019-04-05 | 重庆金时代生物技术有限公司 | A kind of cultural method of placental blood candidate stem cell |
| CN116429734A (en) * | 2022-01-04 | 2023-07-14 | 南京医科大学 | Method for separating human peripheral blood leucocyte and detecting active oxygen |
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