CN106350489A - Human marrow, umbilical cord blood and peripheral blood stem cell isolation kit and isolation method thereof - Google Patents
Human marrow, umbilical cord blood and peripheral blood stem cell isolation kit and isolation method thereof Download PDFInfo
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Abstract
The invention provides a human marrow, umbilical cord blood and peripheral blood stem cell isolation kit and an isolation method thereof. The isolation kit comprises a reagent A, a reagent B and a reagent C, wherein the reagent A is sodium chloride solution, the reagent B is sodium chloride solution with hydroxyethyl starch and a mixed antibody, and the reagent C is iodixanol operating fluid or lymphocyte separating medium. In the reagent B, a mass concentration of the hydroxyethyl starch is 5-10%, and a concentration of the mixed antibody is 4-12microgram/ml. The isolation method is characterized in that a negative selection collection method is adopted for selectively removing red blood cells, blood platelets, all plasma substances, mature blood cells and lymphocytes; all lin negative stem/progenitor cells can be collected; adoption of a mobilizing agent and plasma residuals are avoided; high-capacity isolation of marrow, umbilical cord blood and peripheral blood can be realized, and cell recovery rate reaches 85%. The isolation kit and the isolation method are especially suitable for treatment of marrow, umbilical cord blood and peripheral blood samples extracted clinically and in umbilical cord blood banks.
Description
Technical field
The present invention relates to a kind of people's bone marrow, Cord blood and peripheral hematopoietic stem cells separating kit and its separation method, specifically
It is related to the negative sharp separation Large Copacity people's bone marrow selecting method principle to make, Cord blood, the test kit of peripheral hematopoietic stem cells, by
Diluent, separating liquid and cleaning mixture composition, to adapt to needed for clinical stem-cell therapy.
Background technology
Step into the end of the year within 2007, life sciences circle are filled with great waves again.Japan and Liang Ge seminar of the U.S. in succession use and turn
Gene technology, imports oct4, the gene such as nanog, sox2 in the fibroblast of ordinary people, finds fibroblast reprogramming
Possesses the characteristic of hESC.This achievement in research causes whole world vibrations.U.S. government redoubles one's efforts, and puts into
3000000000 dollars of funds, continue deeper into the research of this problem.This achievement of stem-cell research, substantially reduces human embryonic stem
Cell applies to the distance of clinic.
Always well known, stem cell is that a class has self-renewal capacity, can be divided into other cell types, itself is in
The cell of undifferentiated state.Stem cell can be divided into all-round, multipotency and three kinds of committed stem cell at present according to its differentiation capability.Root
According to its source, then can be divided into embryonic stem cell and adult stem cell.Our mankind are exactly to pass through this myeloid-lymphoid stem cell of germ cell
Constantly differentiate.
1998, the seminar of the Thomson professor leader of University of Wisconsin-Madison pungent Madison university was successfully set up the mankind
Embryonic stem cell line, this achievement is described as the milestone of embryo cleavage, and hereafter, the research of stem cell field becomes always
Forward position for life science and focus.Core Ji medical college digestion through effort for many years, successfully established in 2006
Three plants of human embryonic stem cell lines, have started simultaneously at the exploration that adult stem cell applies to liver disease therapy.Stem cell
Research, is becoming the Neoma Foam of human disease treatment.
(1) treatment of hESC's research and clinical disease
Fetal hepatocytes, are the cells with versatility, in vitro under suitable inductive condition, can successfully induce
Various cells.Doctor Zhang Suchun of U.S. uw, makes a breakthrough in the induction of motor neuron cell.And winconsin
The scientist of primatess institute, then induce the nerve cells transplantation of gained to wait mould to parkinson by monkey embryonic stem cell
In type, whole experiment lasts 10 years, finally succeeds, transplanted cells have cured sufferer monkey completely.And University of Minnesota
Red Kaufman doctor overcome technical obstacle, obtain in inducing embryo hepatocyte and can kill the immunocyte of tumor
On make a breakthrough.Doctor Kuai little Ling that Ren Ji hospital digests institute's stem cell laboratory uses fetal hepatocytes induced liver cell,
Achieve successfully.In terms of the induction of blood cell, the U.S., Britain, the top laboratory in Canadian various countries, all put into a large amount of people
Power, material resources, wish hammer and tongs to obtain the hematopoietic cell being available for transplanting.The mexims doctor of U.S. uw is named as with one kind
The mouse sertoli cells of op9, co-culture hESC, obtain substantial amounts of hematopoietic stem cell.Mick Sabeer Bhatia professor leads
The Canadian mcmaster university tumor led and stem-cell research institute unsuccessful movement hESC, establish stable body
Outer culture and induction system, successfully obtain the hematopoietic stem cell with further differentiation potential from embryonic stem cell.Core is helped
, on the basis of the top greatly laboratory of summary two, establishing can be extensive from hESC for the scientist of medical college
Obtain the external evoked system (see Fig. 1) of the hematopoietic stem cell being available for transplanting.
Embryonic stem cell just will really apply to clinic, also remote road, and wherein, topmost difficult point has at 2 points, its
One: how to solve the problems, such as immunologic rejection.For this problem, have different thinkings it is thus proposed that, nuclear transplantation can be used
Method, the somatic cell enucleation of patient is transplanted in the ovum of enucleation, then hybrid cell is pushed to the blastaea of embryo again
Stage, therefrom Isolation of Embryonic Stem Cell.The success of clone monkey in the recent period is that the realization of this thinking brings hope.Scholar is separately had to carry
Go out to obtain the embryo of the mankind using gynecogenic mode.The achievement of this respect is still needed and is inquired into further.And above-mentioned from skin
Cell obtains hESC, is a brand-new thinking, but the means obtaining quiding gene during cell are related to
To virus, whether can really apply to clinical treatment patient for this cell future, need the further depth of subsequent experimental
Enter.Fetal hepatocytes are that stem cell itself forms teratomatous characteristic in place of applying to another melancholy people of clinical disease treatment.Though
So the cell of differentiation gained not yet finds into the phenomenon of tumor in transplanting, but during embryonic stem cell is expanded in vitro whether
There is the change of the epigenetic of similar tumor, be the proposition of a significant.
(2) adult stem cell research and clinical disease treatment
Adult stem cell applies to the treatment of clinical disease, such as bone marrow stem cell, and umbilical blood or autologous peripheral blood stemcell transplant are controlled
Treat hematopathy it has been reported that a lot.The non-disease of hematopoietic system of alone bone marrow stem cells, is then increasingly subject to weight in recent years
Depending on.Developing rapidly and safety raising with clinical research, the utilization of this technology has extended to other every field, bag
Include treatment Parkinson's disease, senile dementia, fracture, femur head necrosiss, osteoporosis, cardiovascular diseasess, Acute myocardial obstructs
Plug, i patients with type Ⅰ DM, myotrophy is lacked of proper care, diabetic foot, thromboangiitis obliterans, and hepatitis, after the treatment of liver cirrhosis and tumor, chemotherapy
Supporting treatment, be directed to stem cell application.For example, substantial amounts of document report: due to chronic diabetes people cause chronic
Limb ischemia, after have passed through the transplantation treatment of bone marrow stem cell, ischemic ulcer or bad in the limbs of patient of nearly half
The improvement of cellulitis, points out these limb therapeutic successes.And implant a kind of bone marrow stem cell of entitled cd133, whole latter stage chronic ischemia
The cardiac function of property Mutation of Patients with Cardiomyopathy is then obviously improved.Comprehensively our experience and observation, bone marrow stem cell is in liver
Dirty regeneration in the Fibrotic treatment of regulating liver-QI equally has effect.The scientist of the many Sai Duofu of Germany finds bone marrow stem cell for ginseng
Play certain effect with liver regeneration, the proliferation rate of the liver for the treatment of group, after stem cell transplantation, faster than matched group
2.5 times, this new Therapeutic Method is pointed out clinically to have the potential improving and promoting liver regeneration.
In sum, stem cell is current study hotspot in life science.In transplantation medicine field, due to same
The critical shortage of kind of donor, needs the patient of transplantation treatment cannot give treatment in time in a large number, therefore, heterogenous animal and stem cell
Application just becomes the hope of people.But many reasons cause xenotransplantation to fail to make a breakthrough, and the research of stem cell
Then bring dawn to various Diseases.Bone Marrow Stem Cells Transplantation has applied to clinical treatment patient, and stem-cell research
Deeply so that generating the multiple tool functionals tissues being available for transplanting with stem cell and organ is possibly realized.Cell therapy
Various cell injury diseases can be treated at present and have become as common recognition.The research carrying out novel stem cells separating kit has
Wide market application foreground.
It is known that the simply and effectively method of traditional slender karyon of the separation mankind is initially to be existed by arne boyum
Nineteen sixty-eight report, the lymphocyte separation medium (lymphoprep of commercialization over 25 yearstmAlso known as ficoll-isopaque)
It is widely used for monocytic separation.It rationale here is that mononuclear cell (mononuclear cell and lymphocyte) drenches with multirow core
Bar cell (granular cell) is compared with erythrocyte has lighter buoyant density.Most of monocytic buoyant densities are less than
1.077g/ml.These cells can carry out separating in the isosmotic solution of 1.077g/ml by a kind of density.By centrifugation, shape
Become density gradient, erythrocyte and multirow karyolymph cell precipitation, and mononuclear cell concentrates at the isopycnic interface of separating liquid.
The density of common lymphocyte separation medium is 1.077g/ml and contains a kind of composition of entitled polysaccharide, permissible
Promote erythrocyte aggregation and layering.But this also brings two unfavorable places: (1) polysaccharide can be in conjunction with pouring
Bar cell surface, the division of impact cell；(2) because erythrocyte aggregation, the agglomerate of erythrocyte to be processed further just
Seem very difficult.With lymphocyte separation medium separation mononuclear cell, need carefully to add the blood sample of dilution very much
In separating liquid, and between blood sample to be kept and separating liquid, there is clearly interface.Accomplish this step, need good instruction
Practice and be time-consuming for substantial amounts of sample.After loaded down with trivial details separation, by mononuclear cell through immunomagnetic beadses
Partition method or fluidic cell partition method carrying out the separation of stem cell.Whole process takes costliness, and loss cell is serious, lives
Power is significantly affected.
Content of the invention
For defect of the prior art, it is an object of the invention to provide a kind of people's bone marrow, Cord blood, peripheral hematopoietic stem cells
Separating kit and its separation method.The technical program application is negative to select collecting method, can optionally remove erythrocyte, blood is little
Plate, whole plasma substance, ripe hemocyte and lymphocyte；The negative ancestral cells of all of lin can be collected；It is not required to
Mobilization agent to be adopted, no plasma residence；Large Copacity bone marrow, Cord blood and peripheral blood can be separated, cell recoveries reach
85%.It is particularly suitable and process in Cord Blood Bank and the clinical bone marrow extracting, Cord blood and peripheral blood sample.
The test kit of the present invention has been abandoned and has been added to this kind of separating liquid of similar lymphocyte separation medium by collecting blood sample, and leads to
Cross density gradient centrifugation collect stem cell thinking, also abandoned explore a kind of efficient quick go for large sample
Bone marrow, Cord blood and peripheral hematopoietic stem cells isolate the Perfected process of hematopoietic cell, but with hetastarch (hes
Hydroxyethyl starch 130/0.4) it is main body, it is used in combination up-to-date " roseleaf antibody complex " technology, adopt
Collecting method removal is selected to include erythrocyte and most of non-stem cell cell colony with negative.
So-called " roseleaf antibody complex " (as shown in Figure 2) refers to by antibody complex by erythrocyte and not
The nucleated cell needing couples together.Specifically it is simply that nucleated cell is combined by antibody (a), anti-glycophorina antibody
B () combines erythrocyte, the third antibody (c) in complex can be with the fc part of binding antibody (a) and antibody (b).So, one
Just have the process that a lot of nucleated cell and erythrocyte are bonded to each other around individual erythrocyte, constitute erythrocyte-antibody a- antibody
The polymer of c- antibody b- target cell (specific mononuclear cell).Because this polymer is in roseleaf sample, so crying " Flos Rosae Rugosae
Petal antibody complex ".The mass ratio of antibody is (a): (b): (c)=1:3:4.In the present invention, the antibody (a) that we adopt
It is a kind of combination formula of cocktail type, because stem cell is inhomogenous cell colony, lack direct morphological feature, do
Cell is different due to respective growth stage, and its surface antigenicity is different.According to the difference of surface antigenicity, stem cell is divided into
Numerous species, such as: cd34, cd133, sca-1, c-kit, lin feminine gender etc..In cell therapy procedures, which kind of stem cell is rising
Main Function, the plasticity of which kind of stem cell is the most active, and the scientist that needs studies further and confirms.So this reagent
The design philosophy of box is the stem/progenitor cells retaining all kinds.
In the present invention, we do not adopt traditional density gradient separation liquid to separate erythrocyte with mononuclear cell, but
Using hetastarch (hes), rapidly erythrocyte is separated with hematopoietic cell, because the concentration bar in suitable hetastarch
Under part, hetastarch and bone marrow, Cord blood or peripheral blood are mixed, static 30 minutes, you can to efficiently separate mononuclear cell.
But, if being added without roseleaf antibody complex, be separated to is mononuclear cell.
Therefore, the present invention is used in combination two schemes, so, has both quickly rapidly removed a large amount of blood from Large Copacity blood thin
Born of the same parents, the pure non-stem cell constituents of comparison of getting back.Because the reagent in the present invention will not make erythrocyte in bulk condense, and ethoxy
Starch is exactly medically inherently the product expanding as blood volume obtaining Ministry of Public Health lot number, has in clinical practice
There is safety.Therefore, treated erythrocyte can also feed back to patient.This has the advantages that very big in clinic.
The composition liquid c purpose of the last design of the present invention is by density gradient centrifugation to remove the compositions such as platelet.
The pure cell mass covering various stem cell of comparison may finally be obtained through three step process.
More specifically, the purpose of the present invention is achieved by the following technical solution:
In a first aspect, the present invention provides a kind of people's bone marrow, Cord blood, peripheral hematopoietic stem cells separating kit, described separation
Test kit includes following reagent: reagent a, reagent b, reagent c；
Reagent a: sodium chloride solution；
Reagent b: the sodium chloride solution containing hetastarch, mixed antibody；
Reagent c: lymphocyte separation medium or iodixanol working solution.
Preferably, described sodium chloride solution is normal saline.
Preferably, in reagent b, mass concentration in sodium chloride solution for the described hetastarch is 5-10%, described mixed
Conjunction concentration in sodium chloride solution for the antibody is 4-12 mcg/ml.
Preferably, described reagent b is to be made by the steps and obtains:
Take mixed antibody to be added in the hetastarch normal saline solution for 5-10% for the mass concentration, obtain reagent b.
Preferably, in reagent b, the preparation of mixed antibody concentrated solution is to be made by the steps and obtains:
S1, weigh 1 milligram of antibody (anti-cd2, cd3, cd14, cd16, cd19, cd24, cd56 and cd66b etc.)；
S2,3 milligrams of anti-glycophorin a (glycophorina) antibody (anti-erythrocyte) of addition, are mixed with antibody in s1；
S3, add 4.0 milligrams of p9 antibody or 2.72 milligrams of p9f (ab ')2Antibody fragment, 37 DEG C of overnight incubation, make three
Person obtains crosslinking.
In use, mixed antibody is dense is diluted in 100 milliliters of normal saline with 1:4, obtains mixed antibody solution,
The concentration of mixed antibody is made to reach 4-12 mcg/ml.The final concentration of the antibody of so every kind of nucleated cell is in cell suspension
It is 1.0-3.0 mcg/ml.
Preferably, in reagent c, iodixanol working solution includes composition 1 and composition 2；
Composition 1 forms: cell suspending culture solution ph7.4,0.85% (w/v) nacl (sodium chloride) and 10 mMs every liter
Tricine-naoh (n- tri- (methylol) methylglycine-sodium hydroxide)；
Composition 2 forms: Iodixanol Injection 40%, 0.85% (w/v) nacl (sodium chloride) and 30 mMs every liter
Tricine-naoh (n- tri- (methylol) methylglycine-sodium hydroxide).
Preferably, 10mmol/l tricine-naoh and 30mmol/l tricine-naoh is to be made by the steps
And obtain:
A1, tricine is made into 100mmol/l storing liquid, 4 DEG C of preservations；
A2,0.85 gram of sodium chloride of dissolving are in 50 milliliters of water, plus 10 milliliters of tricine storing liquid, use 1mol/l hydrogen-oxygen
Change sodium regulation system ph to 7.4, be settled to 100 milliliters with ultra-pure water, prepare to obtain 10mmol/l tricine- in composition 1
naoh；
0.85 gram of sodium chloride of dissolving is in 50 milliliters of water, plus 30 milliliters of tricine storing liquid, uses 1mol/l sodium hydroxide
Regulation system ph, to 7.4, is settled to 100 milliliters with ultra-pure water, prepares to obtain 30mmol/l tricine-naoh in composition 2.
Preferably, each test kit includes:
1 bottle of 500ml of reagent a,
1 bottle of 100ml of reagent b,
1 bottle of 100ml of reagent c.
In reagent b therein select antibody and reagent c all adopt import material (c be axisshield poc as public affairs
Department's product).
Reagent a in this test kit, reagent c are colourless transparent liquid；Reagent b is for slightly adhesive clear liquid sometimes
Aobvious slightly floating light；Such as muddiness in tri- bottles of reagent of a, b, c, precipitate or have floccule then can not use.
Second aspect, the present invention provides a kind of people's bone marrow, Cord blood, the separation side of peripheral hematopoietic stem cells separating kit
Method, comprises the following steps:
B1, aseptic by transferring to containing one or two bone marrow, Cord blood or peripheral blood in sodium citrate, anticoagulant heparin
In container, reagent a is added to mix；The volume ratio of described bone marrow, Cord blood or peripheral blood and reagent a is 1-3:0.5-2；Described nothing
Bacterium volume of a container is 500 milliliters, determines the size of container also dependent on detached bone marrow, Cord blood or peripheral blood volume.
B2, step b1 is mixed after mixed liquor mix by the volume ratio of 1-5:0.5-3 with reagent b, abundant mixing, room temperature
Standing, collects upper liquid a；Wherein, stand about 20-30 minute, container must not be shaken again during standing, erythrocyte sedimentation is under
Layer, is moved to upper liquid a (accounting for the 3/4 of full capacity) in 50 milliliters of sterile centrifugation tube with Sterile pipette, is drawn to liquid level and hands over
At boundary, do not inhale red blood cell layer as far as possible；
B3, by the centrifugation of upper liquid a (centrifuge can be passed through, abandon supernatant), collect lower floor thing b and (collect centrifugation
The cell of bottom of the tube)；
B4, with reagent a rinse lower floor thing b (with reagent a by each centrifuge tube cell rinse, move to a centrifuge tube
Interior), move to and in centrifugal device, carry out solid-liquid separation again, collect lower floor thing c；
B5, will in lower floor thing c add 10-30 milliliter reagent a in, be added to after mixing in 20-35 milliliter reagent c, centrifugation；
When being added in reagent c, need to be added slowly in the centrifuge tube equipped with reagent c along centrifugation tube wall, note adding speed slow, protect
Demonstrate,prove two liquid boundary clear；
In the middle of drawing after b6, centrifugation, vaporific thin layer adds in new centrifuge tube, is washed twice with reagent a, centrifugation, collects
Cell is standby.
In step b3, step b4, step b6, centrifugal condition is 2000-3000rpm, is centrifuged 3-10 minute；In step b5
Centrifugal condition is 1000-1500rpm, is centrifuged 10-30 minute.
In step b2, after room temperature standing, lower floor is red blood cell layer, with can reuse after brine erythrocyte.
Compared with prior art, the present invention has a following beneficial effect:
1st, during cell sorting, only need to reagent b and the bone marrow through reagent a dilution, Cord blood and peripheral blood straight
Connect mixing it is possible to remove almost all of non-stem cell composition immediately, operate and its simple, be particularly suitable for transplantation experiments institute
Need.
2nd, after final program process, no any reagent residue is left, and has for the safety after in input body body
Absolute guarantee.
3rd, farthest isolate the various stem cell of human marrow, Cord blood and peripheral blood, greatly reduce dry thin
The separation costs of born of the same parents.
4th, 60% aseptic iodixanol aqueous solution is made cell separation medium-iodixanol separating liquid, constitute examination
Agent c.This complex, for the toxicity of cell, well below the first pantothenic acid of the first generation, particularly with stem cell, cell after separating
Survival rate is high, and after transplanting, effect is good.
Brief description
The detailed description with reference to the following drawings, non-limiting example made by reading, the further feature of the present invention,
Objects and advantages will become more apparent upon:
Fig. 1 is the diagram inducing the blood stem cell being available for transplanting from hESC；
Fig. 2 is the schematic diagram of roseleaf antibody complex；
Fig. 3 is the structural formula of iodixanol iodixanol；
Fig. 4 is the structural formula of first generation metrizoic acid metrizoic acid.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following examples will be helpful to the technology of this area
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this area
For personnel, without departing from the inventive concept of the premise, some deformation can also be made and improve.These broadly fall into the present invention
Protection domain.
Test kit of the present invention is used exclusively for separating from human marrow, Cord blood, peripheral blood, purification nucleated cell, no
Can be used for separating other histiocytes.Operating process requires to complete in the superclean bench of ten thousand grades of Clean Operating Labs, to ensure
The sterile working of whole separation process.Must assure that other containers and all directly contacts bone marrow/umbilical cord of operating process use
The article of blood/peripheral blood are aseptic.
Each test kit designs separable purification bone marrow, Cord blood or 200 milliliters of peripheral blood.Must once make after corkage
With it is impossible to be used for multiple times.
Embodiment
A kind of bone marrow, Cord blood, peripheral hematopoietic stem cells separating kit include following reagent: reagent a, reagent b, reagent c；
Described reagent a is sodium chloride solution；Described reagent b is the sodium chloride solution containing hetastarch, mixed antibody；Described reagent c
For iodixanol working solution or lymphocyte separation medium.Wherein, in reagent b, the mass concentration of described hetastarch is 5-
10%；The concentration of described mixed antibody is 4-12 mcg/ml.
The preparation process of described reagent b is as follows: taking 100 microlitres of mixed antibody solution to be added to mass concentration is 5-10%'s
In hetastarch normal saline solution, obtain reagent b.
In reagent b, mixed antibody is to be prepared by the following method:
S1, weigh 1 milligram of antibody (anti-cd2, cd3, cd14, cd16, cd19, cd24, cd56 and cd66b etc.)；
S2,3 milligrams of anti-glycophorin a (glycophorina) antibody (anti-erythrocyte) of addition, are mixed with antibody in s1
Uniformly；
S3, add 4.0 milligrams of p9 antibody or 2.72 milligrams of p9f (ab ') 2 antibody fragments, 37 DEG C of overnight incubation, make three
Person obtains crosslinking.
Mixed antibody is dissolved in 100 milliliters of normal saline, obtains mixed antibody solution, and the concentration of mixed antibody reaches 4-12
Mcg/ml.During use, mixed antibody is diluted with 1:4, and the final concentration of the antibody of so every kind of nucleated cell hangs in cell
It is 1.0-3.0 mcg/ml in liquid.
In described reagent c, iodixanol working solution includes composition 1 and composition 2；
Composition 1 forms: cell suspending culture solution ph7.4,0.85% (w/v) nacl (sodium chloride) and 10 mMs every liter
Tricine-naoh (n- tri- (methylol) methylglycine-sodium hydroxide)；
Composition 2 forms: Iodixanol Injection 40%, 0.85% (w/v) nacl (sodium chloride) and 30 mMs every liter
Tricine-naoh (n- tri- (methylol) methylglycine-sodium hydroxide).
Wherein, 10mmol/l tricine-naoh and 30mmol/l tricine-naoh be to be made by the steps and
:
1) tricine is made into 100mmol/l storing liquid, 4 DEG C of preservations；
2) dissolve 0.85 gram of sodium chloride in 50 milliliters of water, plus 10 milliliters of tricine storing liquid, use 1mol/l hydroxide
Sodium regulation system ph, to 7.4, is settled to 100 milliliters with ultra-pure water, prepares to obtain 10mmol/l tricine-naoh in composition 1；
0.85 gram of sodium chloride of dissolving is in 50 milliliters of water, plus 30 milliliters of tricine storing liquid, uses 1mol/l sodium hydroxide
Regulation system ph, to 7.4, is settled to 100 milliliters with ultra-pure water, prepares to obtain 30mmol/l tricine-naoh in composition 2.
Each bone marrow, Cord blood, peripheral hematopoietic stem cells separating kit include:
1 bottle of 500ml of reagent a；
1 bottle of 100ml of reagent b；
1 bottle of 100ml of reagent c.
In reagent b select antibody and reagent c all adopt import material (c be axisshield poc as company product
Product).Reagent a in this test kit, reagent c are colourless transparent liquid；Reagent b is that slightly adhesive clear liquid shows light sometimes
Micro- floating light；Such as muddiness in tri- bottles of reagent of a, b, c, precipitate or have floccule then can not use.
Based on above-mentioned people's bone marrow, Cord blood, peripheral hematopoietic stem cells separating kit separation method, comprise the following steps:
Step 1,500 milliliters will be transferred to containing a kind of bone marrow in sodium citrate, anticoagulant heparin, Cord blood or peripheral blood
Sterile chamber in (can according to detached bone marrow, Cord blood or peripheral blood volume determine container size), bone marrow/Cord blood/
Peripheral blood and reagent a are mixed in the ratio of 1-3:0.5-2；
Liquid volume after step 2, the 1st step and reagent b are added in the ratio of 1-5:0.5-3, fully mixed after container closure
Even, room temperature stands (must not shake container during standing again), about 20-30 minute；
After step 3,20-30 minute, erythrocyte sedimentation to orlop, with Sterile pipette, supernatant liquid (is accounted for entirely holding
The 3/4 of amount) move in 50 milliliters of sterile centrifugation tube, it is drawn to liquid level intersection, do not inhale red blood cell layer as far as possible；
Step 4, abandon red blood cell layer (if needed can by erythrocyte brine, feed back patient)；
Step 5, the centrifuge tube in centrifuge tube will be collected, in centrifuge, 2000-3000rpm, it is centrifuged 3-10 minute；
Step 6, take the centrifuge tube after the 5th step centrifugation, abandon supernatant, collect the cell of centrifugation bottom of the tube；
Step 7, with reagent a by each centrifuge tube cell rinse, move in a centrifuge tube, add reagent a,
2000-3000rpm, is centrifuged 3-10 minute；
Step 8, take the centrifuge tube of new 50 milliliters, add 20-35 milliliter reagent c；
Step 9, abandon the 7th step centrifugation supernatant, add reagent a 10-30 milliliter, by cell mix tailing edge centrifugation tube wall
It is added slowly in the centrifuge tube equipped with 20-35 milliliter reagent c, note adding speed to want slow it is ensured that two liquid boundary are clear；
The centrifuge tube 1000-1500rpm of the 9th step, is centrifuged 10-30 minute；
In the middle of drawing after step 10, centrifugation, vaporific thin layer adds in new centrifuge tube, washes twice (centrifugation with reagent a
Condition is with the 7th step), collect cell standby.
Result:
1st, separating resulting
The response rate >=85% of stem/progenitor cells；
Stem/progenitor cells survival rate >=96% of separation and Extraction；
Separation, purge process can be effectively retained the ancestral cells of all categories；
Test kit can effectively remove whole erythrocyte, platelet, plasma substance, can remove the white of overwhelming majority maturation
Cell, lymphocyte；
The sum of the stem/progenitor cells isolated varies with each individual；
The obtainable total cellular score of 4 × 50 milliliters of bone marrow is typically in (0.5-5.0) × 109；
The obtainable total cellular score of 4 × 50 milliliters of Cord blood is in (0.2-2.0) × 109；
The obtainable total cellular score of 4 × 50 milliliters of peripheral bloods is in (0.2-2.0) × 109.
2nd, test kit preservation condition: preservation condition 2-8 DEG C, 24 months effect duration, 20 DEG C of room temperature preservation are valid for one year.
3rd, the requirement to equipment and environment for the test kit:
Stem cell separates, purification requirements are carried out in 10,000 grades of Clean Operating Labs, and bone marrow, Cord blood, peripheral blood and reagent move
Take and require to operate in locally 100 grades of environment；
Require a large capacity refrigerated centrifuge, in 4000 revs/min of rotating speed；
Reagent pipetting volume and bone marrow, Cord blood, peripheral blood are pipetted and are pipetted using the pipettor with filter membrane as far as possible
Used by separation, purification, centrifuge tube, pipet must use disposable sterilized product.
4th, separate, stem cell after purification detects
The detection of cell survival rate: using Trypan Blue.
The calculating of extracted total cellular score: cell counting count board counting method, blood counting instrument counting method
Stem cell qualitative determination: culture TRAP, flow cytomery method, with measuring after known antigenic mark.
cd34+、cd117、cd133、c-kit、sca-1.
The present invention first using negative collection mixing method, compared with traditional several stem cell partition method, has at home
Following characteristics (are shown in Table 1):
Table 1
In sum, the innovative point of the present invention is:
(1) application innovation point: easy, easy to operate.
Core technology principle of the present invention is: will represent the known antibodies of non-stem cell, and the composition in bone marrow and blood plasma mixes
Close so that the proportion of non-stem cell is changed due to cell agglutination, the method then adopting natural subsidence, remove red first
Cell, platelet, whole plasma substance, and the leukocyte of maturation, lymphocyte.Retain lin- cell.Initiate at home
Antibody complex is merged using separation material, achieves important breakthrough in the detached key technology area of stem cell:
1st, during cell sorting, only need to reagent b and the bone marrow through reagent a dilution, Cord blood and peripheral blood straight
Connect mixing it is possible to remove almost all of non-stem cell composition immediately, operate and its simple, be particularly suitable for transplantation experiments institute
Need.
2nd, after final program process, no any reagent residue is left, and has for the safety after in input body body
Absolute guarantee.
3rd, farthest isolate the various stem cell of human marrow, Cord blood and peripheral blood, greatly reduce dry thin
The separation costs of born of the same parents.
The Beads enrichment of international popular at present, needs expensive Beads enrichment instrument, and Magnetic Isolation post.Cell passes through
Have very big loss during detached dowel, part cell receive extruding and dead.And, at present, Beads enrichment post does not conform to and is suitable to
The separation of bone marrow prepare.Because containing substantial amounts of fragment in bone marrow, fats dope, at this moment, how and magnetic cell
Pearls knot closed post, and cell is often bonded on pillar, thus leading to sorting purification rate very low.Former approach and the latter's method phase
The former is more simple, low cost, safety, and cell yield and purification ratio are higher for ratio.Traditional stem cell isolation techniques: stem cell
Separate traditionally it is impossible to rapidly by erythrocyte, blood plasma, platelet and non-stem cell constituents one step are separated.Need multiple rings
Section is progressively processed, and time-consuming, involved expensive reagents, easily causes pollution, and the cell after separating needs a period of time to recover
Cytoactive.Particularly traditional magnetic bead crosses post method, can only remove all unlabelled cells, therefore, is only available for selecting spy
Fixed cd34, cd133 cell etc., it substantially lost the stem cell population of much not yet understandings at present.And, entirely separately generation
High price is expensive.Although separated several specific cells purity can reach 99%, in most cases, for micro presence
Stem cell, the whole separation effect of magnetic bead is poor.Particularly with bone marrow prepare, because bone marrow contains a little fiber, bone marrow piece,
Impurity, therefore, applies traditional magnetic bead to cross post method and can not obtain optimal separation effect.Therefore, every new technique in this test kit
Invention so that the rapid human stem cells/CFU-GM that separates on a large scale is possibly realized, be particularly suitable for bone marrow, Cord blood and
Periphery blood specimen.
(2) structure innovation point:
Iodixanol iodixanol is the non-ionic iodinated complex of latest generation, and its structure is shown in Fig. 3；First acute pyogenic infection of nails is general
The structure of shadow acid metrizoic acid is shown in Fig. 4.
60% aseptic iodixanol aqueous solution is made cell separation medium-iodixanol separating liquid, constitutes reagent
c.Through inspection, this complex, for the toxicity of cell, well below the first pantothenic acid of the first generation, particularly with stem cell, after separating
Cell survival rate is high, and after transplanting, effect is good.Meanwhile, these materials will not and albumen, blood plasma of the such as mankind etc. combine, according to not
Same proportional concentration, can form a continuous density, and relatively low osmotic pressure is so that the damaged membrane of cell is permissible
It is preferably minimized, and its viscosity is then compared with percoll, ficoll is then much lower, therefore, in the test kit of present invention synthesis
In it may be determined that the density of cell mass to be separated, then the cell removing will be needed, with the method for magnetic bead antibodies,
Increase its density so that it may perfect cell separation can be obtained through sedimentation.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned
Particular implementation, those skilled in the art can make various modifications or modification within the scope of the claims, this not shadow
Ring the flesh and blood of the present invention.
Claims (11)
1. a kind of people's bone marrow, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that wrap in described separating kit
Include following reagent:
Reagent a: sodium chloride solution；
Reagent b: the sodium chloride solution containing hetastarch, mixed antibody；
Reagent c: lymphocyte separation medium or iodixanol working solution.
2. people's bone marrow according to claim 1, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that described
Sodium chloride solution is normal saline.
3. people's bone marrow according to claim 1, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that reagent
In b, mass concentration in sodium chloride solution for the described hetastarch is 5-10%, and described mixed antibody is in sodium chloride solution
Concentration be 4-12 mcg/ml.
4. people's bone marrow according to claim 3, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that described
Reagent b is to be made by the steps and obtains: takes mixed antibody to be added in hetastarch normal saline solution, obtains reagent
b.
5. people's bone marrow according to claim 4, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that described
Mixed antibody is to be made by the steps and obtains:
S1, by the antibody for 1:3 for the mass ratio and anti-glycophorin a antibody mix homogeneously；
S2, add p9 antibody or p9f (ab ')2Antibody fragment, 37 DEG C of overnight incubation, make three obtain crosslinking；Described p9 resists
Body is 4:1 with the mass ratio of antibody, described p9f (ab ')2Antibody fragment is 2.72:1 with the mass ratio of antibody.
6. people's bone marrow according to claim 5, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that step
In s1, described antibody be one of anti-cd2, anti-cd3, anti-cd14, anti-cd16, anti-cd19, anti-cd24, anti-cd5, anti-cd66 or
Several.
7. people's bone marrow according to claim 1, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that reagent
In c, iodixanol working solution includes composition 1 and composition 2；
Composition 1 forms: cell suspending culture solution ph7.4,0.85%w/v sodium chloride and 10mmol/l tricine-naoh；
Composition 2 forms: Iodixanol Injection 40%, 0.85%w/v sodium chloride and 30mmol/l tricine-naoh.
8. people's bone marrow according to claim 7, Cord blood, peripheral hematopoietic stem cells separating kit are it is characterised in that described
Tricine-naoh is to be made by the steps and obtains:
A1, tricine is made into 100mmol/l storing liquid, 4 DEG C of preservations；
A2,0.85 gram of sodium chloride of dissolving are in water, plus 10 milliliters of tricine storing liquid, with sodium hydroxide regulation system ph extremely
7.4, it is settled to 100 milliliters with ultra-pure water, prepare to obtain 10mmol/l tricine-naoh in composition 1；
0.85 gram of sodium chloride of dissolving is in water, plus 30 milliliters of tricine storing liquid, with sodium hydroxide regulation system ph to 7.4,
It is settled to 100 milliliters with ultra-pure water, prepare to obtain 30mmol/l tricine-naoh in composition 2.
9. the people's bone marrow as described in a kind of any one as claim 1-8, Cord blood, the separation of peripheral hematopoietic stem cells separating kit
Method is it is characterised in that comprise the following steps:
B1, sterile chamber will be transferred to containing one or two bone marrow, Cord blood or peripheral blood in sodium citrate, anticoagulant heparin
Interior, add reagent a to mix；The volume ratio of described bone marrow, Cord blood or peripheral blood and reagent a is 1-3:0.5-2；
B2, step b1 is mixed after mixed liquor mix by the volume ratio of 1-5:0.5-3 with reagent b, abundant mixing, room temperature is quiet
Put, collect upper liquid a；
B3, by the centrifugation of upper liquid a, collect lower floor thing b；
B4, with reagent a rinse lower floor thing b, move to and in centrifugal device, carry out solid-liquid separation again, collect lower floor thing c；
B5, will in lower floor thing c add 10-30 milliliter reagent a in, be added to after mixing in 20-35 milliliter reagent c, centrifugation；
In the middle of drawing after b6, centrifugation, vaporific thin layer, is washed with reagent a, centrifugation, collects cell standby.
10. people's bone marrow according to claim 9, Cord blood, the separation method of peripheral hematopoietic stem cells separating kit, it is special
Levy and be, in step b3, step b4, step b6, centrifugal condition is 2000-3000rpm, be centrifuged 3-10 minute；In step b5 from
Heart condition is 1000-1500rpm, is centrifuged 10-30 minute.
11. people's bone marrow according to claim 9, Cord blood, the separation method of peripheral hematopoietic stem cells separating kit, it is special
Levy and be, in step b2, after room temperature standing, lower floor is red blood cell layer, with can reuse after brine erythrocyte.
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| CN112481205A (en) * | 2020-12-04 | 2021-03-12 | 上海本梵医疗技术有限公司 | Bone marrow collection and nucleated cell separation technology |
| CN113980894A (en) * | 2021-12-27 | 2022-01-28 | 深圳博雅感知医疗科技有限公司 | Method for preparing bone marrow condensed cells and application thereof in treating premature ovarian failure |
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