CN105368781A - Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells - Google Patents
Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells Download PDFInfo
- Publication number
- CN105368781A CN105368781A CN201510929780.2A CN201510929780A CN105368781A CN 105368781 A CN105368781 A CN 105368781A CN 201510929780 A CN201510929780 A CN 201510929780A CN 105368781 A CN105368781 A CN 105368781A
- Authority
- CN
- China
- Prior art keywords
- hematopoietic stem
- placenta
- solution
- enzymolysis
- stem cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a lavage fluid, an enzymolysis solution and a method for separating placenta hematopoietic stem cells. The lavage fluid comprises a penicillin-streptomycin double antibody, phentolamine, vitamin C and PBS. The enzymolysis solution comprises a basic culture medium, collagenase I, collagenase II, heparin sodium, phentolamine, vitamin C and Z-VAD-FMK. The method for separating the placenta hematopoietic stem cells comprises the following steps: washing the blood in the placenta with a lavage fluid; injecting enzymolysis liquid into placenta for enzymolysis at room temperature; collecting the enzymolysis liquid, centrifuging and taking cell precipitate; resuspending the cell precipitate with PBS, adding hydroxyethyl starch solution, standing, centrifuging the supernatant, and removing the supernatant; resuspending the cell pellet with erythrocyte lysate, lysing the residual erythrocytes, centrifuging and discarding the supernatant; and (4) resuspending the cell sediment by using a culture medium, centrifuging and then discarding the supernatant to obtain the placenta hematopoietic stem cells. The invention obviously improves the separation and extraction efficiency, and the obtained cells have large quantity, high content of hematopoietic stem cells, high survival rate and extremely low cell pollution rate.
Description
Technical field
The present invention relates to stem cell field, particularly relate to a kind of irrigating solution, enzymolysis solution and the method that are separated placental hematopoietic stem cell.
Background technology
Hemopoietic stem cell (HemopoieticStemcell, HSC) is a group primitive hematopoietic cell be present in hemopoietic tissue, and it is not organize fixed cell, can be present in hemopoietic tissue and blood.Hemopoietic stem cell is the origin of all hematopoietic cells and immunocyte.
Along with day by day going deep into of research, HSC transplants the effective means having become the many Malignant hematologic diseases for the treatment of and other tumours.At present, the main source of HSC is marrow, peripheral blood and bleeding of the umbilicus, and marrow and peripheral blood HSC breed and differentiation capability declines, and is subject to the impact of donor state of health, and bleeding of the umbilicus HSC limited amount, the HSC clinical application demand that these limiting factors make it not meet day by day to increase.In recent years, Chinese scholars research finds, containing a large amount of early stage stem cells in placenta, comprises the hemopoietic stem cell that quantity is abundant.These stem cells exercise the function of hematopoiesis in placenta.Hemopoietic stem cell contained in the placenta peeled off after fetal birth is 8 ~ 10 times of Cord blood, can be personal several times for it, even can be supplied to the treatment of multiple adult patient.
In addition, the hemopoietic stem cell extracted in placenta not only has rich content, transplant after rejection low, without advantages such as ethical hindrances, in addition following advantage: the hemopoietic stem cell in placenta is more early stage, and multiplication capacity is strong; The hemopoietic stem cell that placenta is numerous also can be used to prepare red corpuscle and megalokaryocyte/thrombocyte on a large scale, is used for substituting blood transfusion.In view of these advantages of placental hematopoietic stem cell, carrying out of placenta tissue hemopoietic stem cell bank technology is that one of stem-cell research field is in progress greatly, when effectively will solve transplanting marrow or after mobilizing derived from peripheral blood not enough, Cord blood quantity waits technical barrier not, is expected to replace marrow, mobilizes rear peripheral blood and Cord blood for Hemopoietic Stem Cell Transplantation Malignant hematologic diseases.
At present, the separating and extracting method of placenta HSC mainly adopts mechanical enzymolysis process.Although mechanical enzymolysis process can obtain the cell of abundant amount, heteroproteose cell content is high, hemopoietic stem cell content is lower, and hemopoietic stem cell motility rate is low.In addition, mainly carry out enzymolysis, digestion to Chorionic villi of placenta position in prior art, digestion tissue volume is large, and need with a large amount of enzymolysis solutions, production cost is high; Whole leaching process operation length complicated, consuming time, the cell quantity fluctuation of extraction is comparatively large, pollution probability increases greatly, is unfavorable for industrialization.
Summary of the invention
In view of this, be necessary for above-mentioned problem, a kind of irrigating solution, enzymolysis solution and the method that are separated placental hematopoietic stem cell are provided.
Be separated an irrigating solution for placental hematopoietic stem cell, comprise damping fluid, dual anti-, vasodilator and antioxidant.
Preferably, described irrigating solution comprise that PBS, green grass or young crops-Streptomycin sulphate are dual anti-, phentolamine and vitamins C.
More preferably, in described irrigating solution, the content of each component in PBS is: 1 × ~ 3 × green grass or young crops-Streptomycin sulphate is dual anti-, 0.01 ~ 0.1mg/mL phentolamine, 1 ~ 5mg/mL vitamins C.
Be separated an enzymolysis solution for placental hematopoietic stem cell, comprise basic medium, Collagenase I, collagenase II, heparin sodium, vasodilator, antioxidant and anti-apoptotic reagent.
Preferably, described enzymolysis solution comprises RPMI-1640 substratum, Collagenase I, collagenase II, heparin sodium, phentolamine, vitamins C and Z-VAD-FMK (Caspase inhibitor).
Preferably, the content of each component of described enzymolysis solution in RPMI-1640 substratum is: 0.01 ~ 0.05mg/mL Collagenase I, 0.01 ~ 0.05mg/mL collagenase II, 10 ~ 50U/mL heparin sodium, 0.01 ~ 0.1mg/mL phentolamine, 1 ~ 5mg/mL vitamins C, 5 ~ 20ng/mLZ-VAD-FMK.
Preferably, the content of each component of described enzymolysis solution in RPMI-1640 substratum is: 0.05mg/mL Collagenase I, 0.01mg/mL collagenase II, 50U/mL heparin sodium, 0.05mg/mL phentolamine, 3mg/mL vitamins C, 10ng/mLZ-VAD-FMK.
Be separated a method for placental hematopoietic stem cell, comprise the following steps:
S1, with irrigating solution cleaning placenta inner blood;
S2, in placenta, pour into enzymolysis solution, room temperature enzymolysis 0.5 ~ 1.5h;
Enzymolysis solution after S3, collection enzymolysis, centrifuging and taking cell precipitation;
S4, use PBS re-suspended cell precipitation, then add the hydroxyethyl starch solution of 1 ~ 3 times of volume, left at room temperature 20 ~ 40min, get the centrifugal rear supernatant discarded of supernatant liquid; By erythrocyte cracked liquid re-suspended cell precipitation, red corpuscle 5 ~ 15min that under room temperature, cracking is residual, abandons supernatant after centrifugal;
S5, use RPMI-1640 substratum re-suspended cell precipitation, abandon supernatant after centrifugal, gained cell precipitation is placental hematopoietic stem cell.
Preferably, the concentration of hydroxyethyl starch solution is 6m/v%.
Preferably, after the enzymolysis solution of described step S3 after collecting enzymolysis, with irrigating solution irrigation placenta, collect irrigating solution and merge into mixed solution with the enzymolysis solution after the enzymolysis collected, after mixed solution is centrifugal, getting cell precipitation.
Preferably, by PBS re-suspended cell precipitation in described step S4, then the hydroxyethyl starch solution of 1 ~ 3 times of volume is added, left at room temperature 20 ~ 40min; Get the centrifugal 5 ~ 10min of supernatant liquid 300 ~ 800g; Centrifugal rear supernatant discarded; By 1 × erythrocyte cracked liquid re-suspended cell precipitation, the volume ratio of cell precipitation and 1 × erythrocyte cracked liquid is 1:5 ~ 1:10, vortex oscillation, the red corpuscle 10min that under room temperature, cracking is residual, and the centrifugal 5 ~ 10min of 200 ~ 500g, abandons supernatant after centrifugal;
Preferably, by 20 ~ 40mLRPMI-1640 substratum re-suspended cell precipitation in described step S5, the centrifugal 5 ~ 10min of 200 ~ 500g, abandons supernatant after centrifugal, and gained cell precipitation is placental hematopoietic stem cell.
Preferably, placenta is immersed in placenta conserving liquid and preserves transport under 0 ~ 4 DEG C of condition, is separated placental hematopoietic stem cell in 6 ~ 12 hours; Described placenta conserving liquid comprises that green grass or young crops-Streptomycin sulphate is dual anti-, heparin sodium, low molecular dextran and PBS, and the content of each component in PBS is: 1 × ~ 3 × green grass or young crops-Streptomycin sulphate is dual anti-, 10 ~ 50U/mL heparin sodium, 1m/v% low molecular dextran.
The present inventor is found by research: the key that perfusion enzymolysis process extracts placental hematopoietic stem cell is the survival rate improving the extraction quantity of hemopoietic stem cell, ensure hemopoietic stem cell.Therefore, the moiety of enzymolysis solution becomes vital factor.Compared with prior art, the present invention has following beneficial effect:
(1) enzymolysis solution component is simple, except substratum, with the addition of anti-apoptotic reagent C aspase inhibitor, vasodilator phentolamine, antioxidant vitamin C and enzyme.RPMI-1640 substratum, as solvent, is cells with nutrient supply, ensure that the motility rate of cell in enzymolysis process.Caspase inhibitor inhibits cell to start apoptosis program greatly, improves the motility rate of cell.Enzymolysis solution of the present invention is gentleer, use Collagenase I and collagenase II simultaneously, not only reduce the damage to cell, better can also digest placenta tissue at short notice, discharge the hemopoietic stem cell of tissue the inside, improve the quantity and motility rate that are separated the placental hematopoietic stem cell obtained.Antioxidant vitamin C can strengthen the resistance of oxidation of cell, improves its anti-adversity ability, and then improves the motility rate of cell.Enzymolysis solution medium vessels expander phentolamine has good vasorelaxation action, utilizes the perfusion operation in experiment.Heparin sodium then reduces the blood coagulation phenomenon in blood vessel, is beneficial to the eliminating of blood.In addition, phentolamine and heparin sodium also help enzymolysis solution and infiltrate into tail vein, fully digest placenta tissue.
(2) irrigating solution component of the present invention is simple, except dual anti-, PBS, containing phentolamine, vitamins C.Green grass or young crops-Streptomycin sulphate is dual anti-has anti-microbial effect, preventing pollution.PBS is for maintaining intraor extracellular osmotic pressure.Phentolamine and ascorbic effect are identical with its effect in enzymolysis solution, and vitamins C, for strengthening the resistance of oxidation of cell, improves the motility rate of cell; Phentolamine plays vasorelaxation action, utilizes the perfusion operation in experiment.
(3) method that the present invention is separated placental hematopoietic stem cell is confined to be separated the hemopoietic stem cell in chorion tissue, also by the hemopoietic stem cell of the comprehensive extraction placenta of enzymolysis perfusion incessantly.Whole operating process is simply effective, and the experimental implementation time is short, significantly improves separation and Extraction efficiency, and the cell quantity of acquisition is large, and the content of hemopoietic stem cell improves greatly, and cell quantity is more stable; Optimize the condition of extraction and isolation simultaneously, improve the survival rate of hemopoietic stem cell as much as possible, cell contamination rate is extremely low.
Accompanying drawing explanation
Fig. 1 is the flow cytometer detection result that HSC that in the embodiment of the present invention 1 prepared by method does not add antibody control group.Wherein, Fig. 1-A and Fig. 1-B is the control group not adding antibody.Fig. 1-A is that namely the flow cytometer detection figure detecting mononuclearcell sum, institute collar region P1 represent mononuclearcell sum percentage.Fig. 1-B is the flow cytometer detection figure detecting CD34 positive cell number, and right side per-cent is the per-cent of CD34 positive cell in mononuclearcell.
Fig. 2 is the flow cytometer detection result of the HSC interpolation antibody test group that in the embodiment of the present invention 1 prepared by method.Wherein, Fig. 2-A and Fig. 2-B is the control group not adding antibody.Fig. 2-A is that namely the flow cytometer detection figure detecting mononuclearcell sum, institute collar region P1 represent mononuclearcell sum percentage.Fig. 2-B is the flow cytometer detection figure detecting CD34 positive cell number, and right side per-cent is the per-cent of CD34 positive cell in mononuclearcell.
Embodiment
In order to better the present invention is described, be described further below in conjunction with the drawings and specific embodiments.
In the present invention, agents useful for same or instrument all can be buied by market.Hydroxyethyl starch solution uses hydroxyethyl starch injection liquid, and purchased from Guangzhou Pharmaceuticals Ltd, 500mL/ wraps, and wherein the content of hydroxyethylamyle (HES) is 6m/v%; 1 × erythrocyte cracked liquid is described as biotechnology Instrument Ltd. purchased from Guangzhou.Of the present invention 1 × ~ 3 × dual anti-be the reagent concentration method for expressing of this area routine, general green grass or young crops-the Streptomycin sulphate bought is dual anti-is high power mother liquor, as the dual anti-majority of green grass or young crops-Streptomycin sulphate bought be 100 times of concentration, can dilute in use and be configured to low multiple, as 1 times, 1 × dual anti-concentration is penicillin 100U/mL, Streptomycin sulphate 100 μ g/mL.When preparing placenta conserving liquid, low molecule dextrose directly uses low molecular dextran glucose injection (purchased from Guangzhou pharmaceuticals), and its concentration containing low molecular dextran-40 is 6m/v%, and the concentration containing glucose is 5m/v%.
The present invention's placenta used is provided by certain obstetrics and gynecology hospital, chooses the transmissible disease such as hepatitis, syphilis, acquired immune deficiency syndrome (AIDS) and detects negative and without the placenta of obstetric complication, preoperative warp obtains puerpera's informed consent and signs Informed Consent Form.Preoperative Method sterile polystyrene placenta collecting cassette is as conveying containers, and in-built placenta conserving liquid, delivers to preparation place and be separated preparation placental hematopoietic stem cell in 6 ~ 12 hours under 0 ~ 4 DEG C of condition after gathering.Preferably, described placenta conserving liquid comprises umbilical cord blood plasma, glucose, green grass or young crops-Streptomycin sulphate, low molecular dextran-40 and PBS.More preferably, described placenta conserving liquid comprises 5v/v% umbilical cord blood plasma, 5m/v% glucose, 1 × green grass or young crops-Streptomycin sulphate, 1m/v% low molecular dextran-40, PBS surplus.The present invention selects the placenta of healthy childbirth, adopts low temperature placenta conserving liquid to preserve placenta, enters experiment process after a few hours, extract active high hemopoietic stem cell at short notice as much as possible.Containing nutritive substances such as umbilical cord blood plasmas in placenta conserving liquid, ensure that the nutrition supply of placenta tissue.
Before using the method for separation placental hematopoietic stem cell of the present invention to be separated placental hematopoietic stem cell, need to carry out pre-treatment to placenta, this measure belongs to known in this field.Pre-treatment comprises and carries out disinfection to placenta, removes the step such as placenta amnion and washing.Before isolating hematopoietic stem cells, those skilled in the art all know these pre-treatment step and can select concrete pre-treatment step according to practical situation.
Embodiment 1
The placenta that childbirth obtains is soaked in placenta conserving liquid, preserves transport for 4 DEG C.The formula of placenta conserving liquid is:, 25U/mL heparin sodium, 1m/v% low molecular dextran-40 dual anti-containing 1 × green grass or young crops-Streptomycin sulphate in PBS.Divide the separation carrying out placental hematopoietic stem cell in 6 hours puerperiums as follows:
S1, denuded amniotic membrane, tentatively clean placenta with 0 ~ 4 DEG C of irrigating solution.The concrete grammar of cleaning placenta is: draw irrigating solution with disposable syringe and pour into into placenta from two placental artery, lavation is cleaned up to placenta inner blood, till flowing out transparent irrigating solution.
The formula of irrigating solution is:, 0.05mg/mL phentolamine dual anti-containing 1 × green grass or young crops-Streptomycin sulphate in PBS, 3mg/mL vitamins C.
S2, draw enzymolysis solution pour into in placenta from two placental artery with disposable syringe, when being pink enzymolysis solution to Venous flow liquid out, ligation arteriovenous, is placed in room temperature enzymolysis 1h.
The formula of enzymolysis solution is: containing 0.05mg/mL Collagenase I, 0.01mg/mL collagenase II, 50U/mL heparin sodium, 0.05mg/mL phentolamine in RPMI-1640 substratum, 3mg/mL vitamins C, 10ng/mLZ-VAD-FMK.
S3, unclamp arteriovenous, collect enzymolysis solution, and draw irrigating solution with disposable syringe and pour into in placenta from two placental artery, collect irrigating solution, the enzymolysis solution of collection and irrigating solution are merged into mixed solution, the centrifugal mixed solution 10min of 500g.
S4, supernatant discarded; By PBS re-suspended cell precipitation, then add the hydroxyethyl starch injection liquid of 1 times of volume, left at room temperature 30min; Draw supernatant liquid in 50mL centrifuge tube, the centrifugal 10min of 500g, centrifugal rear supernatant discarded; By 1 × erythrocyte cracked liquid re-suspended cell precipitation, the volume ratio of cell precipitation and 1 × erythrocyte cracked liquid is 1:10, vortex oscillation 5s, and the red corpuscle 10min that under room temperature, cracking is residual, the centrifugal 10min of 500g, abandons supernatant after centrifugal.
S5, use 40mLRPMI-1640 substratum re-suspended cell precipitation, the centrifugal 10min of 500g, abandons supernatant after centrifugal, and gained cell precipitation is placental hematopoietic stem cell.
Embodiment 2
The placenta that childbirth obtains is soaked in placenta conserving liquid, preserves transport for 4 DEG C.The formula of placenta conserving liquid is: in PBS containing 2 × dual anti-, 25U/mL heparin sodium, 1m/v% low molecular dextran-40.Divide the separation carrying out placental hematopoietic stem cell in 12 hours puerperiums as follows.
S1, denuded amniotic membrane, tentatively clean placenta with 0 ~ 4 DEG C of irrigating solution.The concrete grammar of cleaning placenta is: draw irrigating solution with disposable syringe and pour into into placenta from two placental artery, lavation is cleaned up to placenta inner blood, till flowing out transparent irrigating solution.
The formula of irrigating solution is: containing 2 × dual anti-, 0.05mg/mL phentolamine in PBS, 3mg/mL vitamins C.
S2, draw enzymolysis solution pour into in placenta from two placental artery with disposable syringe, when being pink enzymolysis solution to Venous flow liquid out, ligation arteriovenous, is placed in room temperature 0.5 ~ 1.5h.
The formula of enzymolysis solution is: containing 0.05mg/mL Collagenase I, 0.05mg/mL collagenase II, 25U/mL heparin sodium, 0.01mg/mL phentolamine in RPMI-1640 substratum, 5mg/mL vitamins C, 5ng/mLZ-VAD-FMK.
S3, unclamp arteriovenous, collect enzymolysis solution, and draw irrigating solution with disposable syringe and pour into in placenta from two placental artery, collect irrigating solution, the enzymolysis solution of collection and irrigating solution are merged into mixed solution, the centrifugal mixed solution 5min of 300g.
S4, supernatant discarded; By PBS re-suspended cell precipitation, then add the hydroxyethyl starch injection liquid of 1 times of volume, left at room temperature 20min; Draw supernatant liquid in 50mL centrifuge tube, the centrifugal 5min of 300g, centrifugal rear supernatant discarded; By 1 × erythrocyte cracked liquid re-suspended cell precipitation, the volume ratio of cell precipitation and 1 × erythrocyte cracked liquid is 1:10, vortex oscillation 5s, and the red corpuscle 10min that under room temperature, cracking is residual, the centrifugal 5min of 200g, abandons supernatant after centrifugal.
S5, use 20mLRPMI-1640 substratum re-suspended cell precipitation, the centrifugal 5min of 200g, abandons supernatant after centrifugal, and gained cell precipitation is placental hematopoietic stem cell.
Embodiment 3
The placenta that childbirth obtains is soaked in placenta conserving liquid, preserves transport for 4 DEG C.The formula of placenta conserving liquid is: in PBS containing 3 × dual anti-, 25U/mL heparin sodium, 1m/v% low molecular dextran-40.Divide the separation carrying out placental hematopoietic stem cell in 6 hours puerperiums as follows.
S1, denuded amniotic membrane, tentatively clean placenta with 0 ~ 4 DEG C of irrigating solution.The concrete grammar of cleaning placenta is: draw irrigating solution with disposable syringe and pour into into placenta from two placental artery, lavation is cleaned up to placenta inner blood, till flowing out transparent irrigating solution.
The formula of irrigating solution is: containing 3 × dual anti-, 0.05mg/mL phentolamine in PBS, 3mg/mL vitamins C.
S2, draw enzymolysis solution pour into in placenta from two placental artery with disposable syringe, when being pink enzymolysis solution to Venous flow liquid out, ligation arteriovenous, is placed in room temperature 0.5 ~ 1.5h.
The formula of enzymolysis solution is: containing 0.01mg/mL Collagenase I, 0.05mg/mL collagenase II, 10U/mL heparin sodium, 0.1mg/mL phentolamine in RPMI-1640 substratum, 1mg/mL vitamins C, 20ng/mLZ-VAD-FMK.
S3, unclamp arteriovenous, collect enzymolysis solution, and draw irrigating solution with disposable syringe and pour into in placenta from two placental artery, collect irrigating solution, the enzymolysis solution of collection and irrigating solution are merged into mixed solution, the centrifugal mixed solution 5 ~ 10min of 300 ~ 800g.
S4, supernatant discarded; By PBS re-suspended cell precipitation, then add the hydroxyethyl starch injection liquid of 1 times of volume, left at room temperature 40min; Draw supernatant liquid in 50mL centrifuge tube, the centrifugal 10min of 800g, centrifugal rear supernatant discarded; By 1 × erythrocyte cracked liquid re-suspended cell precipitation, the volume ratio of cell precipitation and 1 × erythrocyte cracked liquid is 1:10, vortex oscillation 5s, and the red corpuscle 10min that under room temperature, cracking is residual, the centrifugal 10min of 300g, abandons supernatant after centrifugal.
S5, use 30mLRPMI-1640 substratum re-suspended cell precipitation, the centrifugal 10min of 300g, abandons supernatant after centrifugal, and gained cell precipitation is placental hematopoietic stem cell.
Comparative example 1
Placental hematopoietic stem cell is separated according to the method for publication number disclosed in the Chinese invention patent application of CN104774806A described in embodiment 1.Concrete grammar is summarized as follows:
In aseptic super clean bench, get 1 ~ 2mL placenta collection liquid and do Sterility testing, then by ablatio placentae amnion, tentatively clean placenta with 0 ~ 4 DEG C of irrigating solution, draw irrigating solution with disposable syringe to pour into into the artery of two in placenta, until serum is cleaned up in placenta; Irrigating solution: comprise the PBS damping fluid that 1 × green grass or young crops-Streptomycin sulphate is dual anti-, the pH value of 0.02%EDTA, 1 × citrate buffered soln, the Papaverine of 0.05mg/mL and the N-acetylcystein of 1.0mmol/L is 7.0.
Drawing mobilization agent with disposable syringe pours into into the artery of two in placenta, and when Venous flow liquid is out mobilization agent, ligation arteriovenous is placed on room temperature 2 ~ 5h; Mobilization agent: comprise that 1.0mg/LAMD3100,1 × citrate buffered soln, 1 × green grass or young crops-Streptomycin sulphate are dual anti-, the DMEM height glycosyl basal culture medium of the Z-VAD-FMK of the N-acetylcystein of the Papaverine of 0.05mg/mL, 1.0mmol/L, 0.3mg/mL vitamins C and 50ng/mL.
Unclamp arteriovenous, disposable syringe is drawn and is poured into into the artery of two in placenta with the aforementioned mobilization agent with measuring, and collects whole liquid.Centrifugal 5 ~ the 10min of liquid 300 ~ 800g collected, supernatant discarded; In cell precipitation: the ratio of hydroxyethyl starch injection liquid (V:V)=1:2, by hydroxyethyl starch injection liquid re-suspended cell precipitation, left at room temperature 20 ~ 40min, draws supernatant in 50mL centrifuge tube, the centrifugal 5 ~ 10min of 300 ~ 800g; Centrifugal rear supernatant discarded; In cell precipitation: the ratio of 1 × erythrocyte cracked liquid (V:V)=1:8, by 1 × erythrocyte cracked liquid re-suspended cell precipitation, vortex oscillation 5s, the red corpuscle 10min that under room temperature, cracking is residual, the centrifugal 5 ~ 10min of 200 ~ 500g; Abandon supernatant after centrifugal, obtain the mononuclearcell comprising hemopoietic stem cell, add 20 ~ 40mLDMEM high glucose medium, re-suspended cell precipitates, centrifugal 5 ~ the 10min of 200 ~ 500g, precipitation DMEM height glycosyl basal culture medium re-suspended cell, obtains described placental hematopoietic stem cell.
Comparative example 2
Placental hematopoietic stem cell is separated according to the method for publication number disclosed in the Chinese invention patent application of CN104711226A described in embodiment 1.Concrete grammar is summarized as follows:
In aseptic super clean bench, get 1 ~ 2mL placenta collection liquid and do Sterility testing, then by ablatio placentae amnion, tentatively clean placenta with 0 ~ 4 DEG C of irrigating solution, draw irrigating solution with disposable syringe to pour into into the artery of two in placenta, until serum is cleaned up in placenta; Irrigating solution: comprise the PBS damping fluid that 1 × green grass or young crops-Streptomycin sulphate is dual anti-, the pH value of 0.02%EDTA, 1 × citrate buffered soln, the Papaverine of 0.05mg/mL and the N-acetylcystein of 1.0mmol/L is 7.0.
Drawing enzymolysis solution with disposable syringe pours into into the artery of two in placenta, and when Venous flow liquid is out enzymolysis solution, ligation arteriovenous is placed on room temperature 2 ~ 5h; The NTx enzyme of enzymolysis solution: 0.05mg/mL, the Unidasa of 0.02mg/mL, the trypsinase of 0.001% (mass percent), 1 × citrate buffered soln, 1 × green grass or young crops-Streptomycin sulphate are dual anti-, the DMEM height glycosyl basal culture medium of the Papaverine of 0.05mg/mL, the N-acetylcystein of 1.0mmol/L.
Unclamp arteriovenous, disposable syringe is drawn irrigating solution and is poured into into the artery of two in placenta, collects whole enzymolysis liquid.Centrifugal 5 ~ the 10min of liquid 300 ~ 800g collected, supernatant discarded; In cell precipitation: the ratio of hydroxyethyl starch injection liquid (V:V)=1:2, by hydroxyethyl starch injection liquid re-suspended cell precipitation, left at room temperature 20 ~ 40min, draws supernatant in 50mL centrifuge tube, the centrifugal 5 ~ 10min of 300 ~ 800g; Centrifugal rear supernatant discarded; In cell precipitation: the ratio of 1 × erythrocyte cracked liquid (V:V)=1:8, by 1 × erythrocyte cracked liquid re-suspended cell precipitation, vortex oscillation 5s, the red corpuscle 10min that under room temperature, cracking is residual, the centrifugal 5 ~ 10min of 200 ~ 500g; Abandon supernatant after centrifugal, obtain the mononuclearcell comprising hemopoietic stem cell, add 20 ~ 40mLDMEM high glucose medium, re-suspended cell precipitates, centrifugal 5 ~ the 10min of 200 ~ 500g, precipitation DMEM height glycosyl basal culture medium re-suspended cell, obtains described placental hematopoietic stem cell.
Comparative example 3
Placental hematopoietic stem cell is separated according to the method in publication number embodiment 1 disclosed in the Chinese invention patent application of CN103789262A described in enzymic digestion method A.Concrete grammar is summarized as follows:
1, normal natural labor or c-section person's placenta is gathered under aseptic condition, carry out pre-treatment immediately, then two Umbilical artery and a umbilical vein is isolated, the puncture needle or children scalp that connect syringe or constant flow pump are needled into umbilical vein, with 300mL, containing the dual anti-DEME nutrient solution of penicillin, Streptomycin sulphate, (antibiotic concentration is 200mg/L, wherein the additional proportion of penicillin, Streptomycin sulphate is 1:1) as scavenging solution perfusion placental villi blood vessel, collect the scavenging solution after perfusion;
2, enzymic digestion method A: the Digestive system perfusion placental villi blood vessel being DEME nutrient solution through 0.1% collagenase I of 37 DEG C of preheatings and the mixing of 0.1% collagenase II equal proportion, solvent with 150mL, then vises two Umbilical artery and umbilical vein, puts 37 DEG C of digestion 12min;
3, two Umbilical artery are placed in collection tube, then decontrol clamping down on of two Umbilical artery and a umbilical vein, pour into from umbilical vein as collection liquid with 300mlDMEM nutrient solution, collect the liquid after perfusion;
4, by the liquid that obtains in step 1 and step 3 below 10 DEG C, 1500r/min, centrifugal 15min, abandons supernatant, adds DEME nutrient solution, piping and druming mixing.As required, can repeated centrifugation wash several times, to remove unnecessary fragment of tissue and red corpuscle;
5, hydroxyethylamyle sedimentation is separated with lymphocyte separation medium two-step separation: 6m/v% hydroxyethylamyle mixes by 1:4 volume ratio with cell suspension, and less than 10 DEG C leave standstill 30min, get supernatant liquor, less than 10 DEG C, the centrifugal 10min of 1000r/min, remove supernatant liquor; After PBS re-suspended cell precipitation, it slowly added to containing on equal-volume Ficoll parting liquid, less than 10 DEG C, the centrifugal 20min of 2000r/min, gets middle tunica albuginea layer, uses PBS resuspended after washing 2 times with PBS with the centrifugal 5min of 1000r/min.
Effect example 1, cell quantity calculate and survival rate test
With the cell that the resuspended the various embodiments described above of RPMI-1640 substratum, comparative example are separated, adjustment cell density is 1 × 10
6cells/mL.According to cell suspension: the ratio of 0.4% trypan blue (V:V)=1:3, the cell suspension that takes a morsel carries out cell quantity calculating and survival rate test.The placental hematopoietic stem cell that embodiment 1 ~ 3 is separated is experimental group 1 ~ 3, and the placental hematopoietic stem cell that comparative example 1 ~ 3 is separated is control group 1 ~ 3.Result is as shown in table 1.
Table 1, cell quantity and survival rate test result
| Group | Mononuclearcell viable count | The total cell count of mononuclearcell | Cell viability |
| Experimental group 1 | 6.697×10 8cells | 6.820×10 8cells | 98.2％ |
| Experimental group 2 | 6.730×10 8cells | 6.974×10 8cells | 96.5％ |
| Experimental group 3 | 6.430×10 8cells | 6.712×10 8cells | 95.8％ |
| Control group 1 | 6.61×10 8cells | 7.14×10 8cells | 92.58％ |
| Control group 2 | 8.45×10 8cells | 9.87×10 8cells | 85.65％ |
| Control group 3 | 5.79×10 7cells | 8.86×10 7cells | 65.34％ |
As known from Table 1, the inventive method is used to extract 6.712 × 10 from placenta
8~ 6.974 × 10
8individual mononuclearcell, slightly lower than the mononuclearcell that control group 1 ~ 3 obtains, but the mononuclearcell viable cell quantity using the present invention to obtain is 6.430 × 10
8~ 6.730 × 10
8individual cell, Cell viability up to 95.8% ~ 98.2%, apparently higher than the Cell viability of HSC that control group is separated.Therefore, the Cell viability that puts forward of perfusion enzymolysis process of the present invention is high, vigor good.
Effect example 2, flow cytometer detection
Get the cell each 1 × 10 of each embodiment, comparative example separation
6~ 3 × 10
6cells carries out flow cytometer detection.The PBS of cell suspension containing 10%FBS cleans twice; Be divided into two tube cells, resuspended with the PBS of 10%FBS, wherein a pipe (sample sets) adds the antibody of 2.5 μ LCD34, abundant mixing, another pipe (control group) does not add antibody, closes the border simultaneously and hatches 30min, after washing twice with the PBS of 10%FBS, resuspended with 1640, upflowing instrument detects CD34
+cell content.The placental hematopoietic stem cell that embodiment 1 ~ 3 is separated is experimental group 1 ~ 3, and the placental hematopoietic stem cell that comparative example 1 ~ 3 is separated is control group 1 ~ 3.Result is as shown in Fig. 1-2 and table 2.
Table 2, flow cytometer detection result
| Group | CD34 +Cell quantity |
| Experimental group 1 | 29.2％ |
| Experimental group 2 | 30.1％ |
| Experimental group 3 | 28.4％ |
| Control group 1 | 30.2％ |
| Control group 2 | 29.8％ |
| Control group 3 | 31.0％ |
As shown in Table 2, the inventive method is separated CD34 in the hemopoietic stem cell obtained
+the percentage composition of cell is 28.4% ~ 30.1%, suitable with prior art.
The present invention adopt the simple irrigating solution of component, enzymolysis solution to be separated the quantity of the placental hematopoietic stem cell obtained, motility rate, flow cytometer detection result and use component is more complicated, nutrition is abundanter irrigating solution, index of correlation that enzymolysis solution is separated the placental hematopoietic stem cell obtained quite or more excellent, but the irrigating solution used because of the present invention, enzymolysis solution component are simple, and not only preparation is convenient but also cost is lower.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. be separated an irrigating solution for placental hematopoietic stem cell, it is characterized in that, comprise damping fluid, dual anti-, vasodilator and antioxidant.
2. the irrigating solution of separation placental hematopoietic stem cell according to claim 1, is characterized in that, in described irrigating solution, the content of each component in PBS is: 1 × ~ 3 × green grass or young crops-Streptomycin sulphate is dual anti-, 0.01 ~ 0.1mg/mL phentolamine, 1 ~ 5mg/mL vitamins C.
3. be separated an enzymolysis solution for placental hematopoietic stem cell, it is characterized in that, comprise basic medium, Collagenase I, collagenase II, heparin sodium, vasodilator, antioxidant and anti-apoptotic reagent.
4. the enzymolysis solution of separation placental hematopoietic stem cell according to claim 3, is characterized in that, described enzymolysis solution comprises RPMI-1640 substratum, Collagenase I, collagenase II, heparin sodium, phentolamine, vitamins C and Z-VAD-FMK.
5. the enzymolysis solution of separation placental hematopoietic stem cell according to claim 4, it is characterized in that, the content of each component of described enzymolysis solution in RPMI-1640 substratum is: 0.01 ~ 0.05mg/mL Collagenase I, 0.01 ~ 0.05mg/mL collagenase II, 10 ~ 50U/mL heparin sodium, 0.01 ~ 0.1mg/mL phentolamine, 1 ~ 5mg/mL vitamins C, 5 ~ 20ng/mLZ-VAD-FMK.
6. the enzymolysis solution of separation placental hematopoietic stem cell according to claim 5, it is characterized in that, the content of each component of described enzymolysis solution in RPMI-1640 substratum is: 0.05mg/mL Collagenase I, 0.01mg/mL collagenase II, 50U/mL heparin sodium, 0.05mg/mL phentolamine, 3mg/mL vitamins C, 10ng/mLZ-VAD-FMK.
7. be separated a method for placental hematopoietic stem cell, it is characterized in that, comprise the following steps:
S1, with irrigating solution cleaning placenta inner blood;
S2, in placenta, pour into enzymolysis solution, room temperature enzymolysis 0.5 ~ 1.5h;
Enzymolysis solution after S3, collection enzymolysis, centrifuging and taking cell precipitation;
S4, use PBS re-suspended cell precipitation, then add the hydroxyethyl starch solution of 1 ~ 3 times of volume, left at room temperature 20 ~ 40min, get the centrifugal rear supernatant discarded of supernatant liquid; By erythrocyte cracked liquid re-suspended cell precipitation, red corpuscle 5 ~ 15min that under room temperature, cracking is residual, abandons supernatant after centrifugal;
S5, use RPMI-1640 substratum re-suspended cell precipitation, abandon supernatant after centrifugal, gained cell precipitation is placental hematopoietic stem cell.
8. the method for separation placental hematopoietic stem cell according to claim 7, it is characterized in that, after the enzymolysis solution of described step S3 after collecting enzymolysis, with irrigating solution irrigation placenta, collect irrigating solution and merge into mixed solution with the enzymolysis solution after the enzymolysis collected, after mixed solution is centrifugal, getting cell precipitation.
9. the method for separation placental hematopoietic stem cell according to claim 7, is characterized in that, by PBS re-suspended cell precipitation in described step S4, then adds the hydroxyethyl starch solution of 1 ~ 3 times of volume, left at room temperature 20 ~ 40min; Get the centrifugal 5 ~ 10min of supernatant liquid 300 ~ 800g; Centrifugal rear supernatant discarded; By 1 × erythrocyte cracked liquid re-suspended cell precipitation, the volume ratio of cell precipitation and 1 × erythrocyte cracked liquid is 1:5 ~ 1:10, vortex oscillation, the red corpuscle 10min that under room temperature, cracking is residual, and the centrifugal 5 ~ 10min of 200 ~ 500g, abandons supernatant after centrifugal;
By 20 ~ 40mLRPMI-1640 substratum re-suspended cell precipitation in described step S5, the centrifugal 5 ~ 10min of 200 ~ 500g, abandons supernatant after centrifugal, and gained cell precipitation is placental hematopoietic stem cell.
10. the method for separation placental hematopoietic stem cell according to claim 7, is characterized in that, placenta is immersed in placenta conserving liquid and preserves transport under 0 ~ 4 DEG C of condition, is separated placental hematopoietic stem cell in 6 ~ 12 hours; , described placenta conserving liquid comprises that green grass or young crops-Streptomycin sulphate is dual anti-, heparin sodium, low molecular dextran and PBS, and the content of each component in PBS is: 1 × ~ 3 × green grass or young crops-Streptomycin sulphate is dual anti-, 10 ~ 50U/mL heparin sodium, 1m/v% low molecular dextran.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510929780.2A CN105368781B (en) | 2015-12-14 | 2015-12-14 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510929780.2A CN105368781B (en) | 2015-12-14 | 2015-12-14 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105368781A true CN105368781A (en) | 2016-03-02 |
| CN105368781B CN105368781B (en) | 2019-06-21 |
Family
ID=55371416
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510929780.2A Active CN105368781B (en) | 2015-12-14 | 2015-12-14 | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105368781B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695302A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Filling and acquiring devices for extracting hematopoietic stem cells from placenta |
| CN105969721A (en) * | 2016-07-19 | 2016-09-28 | 安徽惠恩生物科技股份有限公司 | Extraction preparation method of placenta hematopoietic stem cells |
| CN106967675A (en) * | 2017-05-31 | 2017-07-21 | 东莞市保莱生物科技有限公司 | A kind of method of the separation and Extraction candidate stem cell from placenta |
| CN107058224A (en) * | 2017-02-10 | 2017-08-18 | 广东唯泰生物科技有限公司 | A kind of candidate stem cell using placenta as source is extracted and cryopreservation methods |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100041009A1 (en) * | 2007-03-08 | 2010-02-18 | Hemacell Perfusion, Inc. | Method for isolation of afterbirth derived cells |
| CN104711226A (en) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method of placenta hematopoietic stem cells |
-
2015
- 2015-12-14 CN CN201510929780.2A patent/CN105368781B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100041009A1 (en) * | 2007-03-08 | 2010-02-18 | Hemacell Perfusion, Inc. | Method for isolation of afterbirth derived cells |
| CN104711226A (en) * | 2015-04-09 | 2015-06-17 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method of placenta hematopoietic stem cells |
Non-Patent Citations (2)
| Title |
|---|
| ELISA PFEIFFER等: "Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells", 《EXPERIMENTAL BIOLOGY AND MEDICINE》 * |
| 陆 琰等: "人胎盘间充质干细胞4种消化分离方法的效果比较", 《中国组织工程研究与临床康复》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695302A (en) * | 2016-03-14 | 2016-06-22 | 广州赛莱拉干细胞科技股份有限公司 | Filling and acquiring devices for extracting hematopoietic stem cells from placenta |
| CN105695302B (en) * | 2016-03-14 | 2018-07-24 | 广州赛莱拉干细胞科技股份有限公司 | A kind of preparation method of in vitro placental hematopoietic stem cell |
| CN105969721A (en) * | 2016-07-19 | 2016-09-28 | 安徽惠恩生物科技股份有限公司 | Extraction preparation method of placenta hematopoietic stem cells |
| CN107058224A (en) * | 2017-02-10 | 2017-08-18 | 广东唯泰生物科技有限公司 | A kind of candidate stem cell using placenta as source is extracted and cryopreservation methods |
| CN106967675A (en) * | 2017-05-31 | 2017-07-21 | 东莞市保莱生物科技有限公司 | A kind of method of the separation and Extraction candidate stem cell from placenta |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105368781B (en) | 2019-06-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105368780A (en) | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells | |
| CN105861430B (en) | A kind of excretion body, the preparation method of excretion body and its application in preparation treatment medication for treating pyemia or preparation | |
| CN104711226A (en) | Preparation method of placenta hematopoietic stem cells | |
| CN107058224B (en) | Method for extracting and cryopreserving hematopoietic stem cells from placenta | |
| CN103789262A (en) | Preparation method and preservation method of clinical application-level placental hematopoietic stem cells | |
| CN106214702A (en) | Cord blood purposes in treatment premature labor complication | |
| CN105368781A (en) | Lavage fluid, enzymatic hydrolysate and method for separating placenta hematopoietic stem cells | |
| CN101919378B (en) | Mesenchymal stem cell cryopreserving liquid directly applied to veins | |
| CN104152405B (en) | The method of separation and Extraction hematopoietic stem cell from Placenta Hominis | |
| CN106367388A (en) | Method for protecting umbilical cord mesenchymal stem cell culture supernatant at 4 DEG C | |
| CN104774806A (en) | Preparation method for placenta hemopoietic stem cell | |
| CN108148804A (en) | One kind meets clinic bleeding of the umbilicus multipotential stem cell production purification process | |
| CN101919379A (en) | Frozen stock solution used for freezing and storing umbilical mesenchymal stem cells for long time | |
| CN103756965B (en) | A kind of method of lavation hemopoietic stem cell from placenta | |
| CN109337871A (en) | A kind of placental hematopoietic stem cell preparation method | |
| CN109652372A (en) | A kind of quick separating of human placenta source candidate stem cell, preparation method | |
| CN106236776A (en) | The application in preparation treatment hepatic fibrosis medicine of a kind of endometrial stem cells | |
| CN103756957B (en) | A kind of method of wash-out endothelial progenitor cells from placenta | |
| CN103484427A (en) | Preparation technology of lecithin-treated adipose stromal vascular components | |
| CN112481205A (en) | Bone marrow collection and nucleated cell separation technology | |
| CN105695302A (en) | Filling and acquiring devices for extracting hematopoietic stem cells from placenta | |
| CN207713738U (en) | Blood sampling separation bag | |
| CN103087987A (en) | In-vitro separation kit of cells used for tissue engineering, and application method thereof | |
| CN221759811U (en) | Immune cell separation trigeminy bag | |
| CN114540296B (en) | Preparation method of composite exosome and application of composite exosome in directional enhancement of angiogenesis capacity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |