CN103756965A - Method for lavaging hematopoietic stem cells from placenta - Google Patents
Method for lavaging hematopoietic stem cells from placenta Download PDFInfo
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- CN103756965A CN103756965A CN201410039328.4A CN201410039328A CN103756965A CN 103756965 A CN103756965 A CN 103756965A CN 201410039328 A CN201410039328 A CN 201410039328A CN 103756965 A CN103756965 A CN 103756965A
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Abstract
The invention provides a method for lavaging hematopoietic stem cells from a placenta. The method comprises the following six steps: collecting the placenta, initially detecting the placenta, disinfecting the placenta in advance, detecting the placenta and maternal blood, lavaging the placenta hematopoietic stem cells, and concentrating and purifying the hematopoietic stem cells. The method and devices used are simple and convenient to operate; the eluted hematopoietic stem cells are low in damage, large in amount, high in concentration and strong in activity, can be applied for autologous or xenogenous hematopoietic stem cell transplantation, and are helpful to repair damaged tissues.
Description
Technical field
The present invention relates to a kind of from placenta the method for lavation hemopoietic stem cell.
Background technology
The method of the hematopoietic stem cell transplantation of mainly taking in the treatment of international disease in the blood system at present, according to the source of cell, can be divided three classes: marrow hemopoietic stem cells is transplanted (BMT), mobilized Peripheral blood stem cell transplantation (MPST) and cord blood stem cell to transplant (UCBT).The above two source of human stem cell are abundant, and general number of nucleated cells can reach 5-10 × 10
8/ Kg, CD34+ cell (a kind of surface markers of hematopoietic stem/progenitor) can reach 1-5 × 10
6/ Kg, owing to needing HLA Strict Compliance between donor and acceptor, the success that guarantee is transplanted, otherwise donor hemopoietic stem cell is difficult for, in patient body's interplantation work, even if survive, more serious graft versus host disease (GVH disease) (GVHD) also can occurring.In the children of one family, the probability that only has 1/4 HLA to conform to, and in the unrelated donor of non-blood relationship, this probability only has one of percentage, has limited greatly BMT or MPST widespread use clinically.
Umbilical cord blood hematopoietic stem cell is implanted in clinically the development that successfully application has promoted that umbilical cord blood hematopoietic stem cell is studied in the recent decade.Cord blood comes from placenta, conventionally discarded after Delivery, to find now to contain abundant hemopoietic stem cell in Cord blood, concentration and the marrow of its CD34+ cell are similar, account for the 0.1-0.5% of total cell count, and more early stage hemopoietic stem cell CD 34-also higher compared with marrow concentration.As a kind of source of hematopoietic cell, utilize now Umbilical Cord Blood Transplantation operation to increase gradually.Compared with BMT, the advantage of UCBT has been to reduce serious graft-vs-host reaction, has effectively expanded the possibility that lacks suitable human leukocyte antigen matching family or irrelevant donor transplanting for those.Umbilical Cord Blood Transplantation is main is limited in the hemopoietic stem cell limited amount in bleeding of the umbilicus.This restriction impels larger adult being help others of clinical middle use 2 multiple dose unit's umbilical cord blood transplantations, another kind method is to carry out the cultivation of Hematopoiesis in Vitro expansion of stem cells, but amplification in vitro needs spended time, expense is also high, when the more important thing is amplification, hemopoietic stem cell also breaks up, and the result of clinical application shows that cord blood cells amplification front and back are to transplanting without much difference.
There is a large amount of hemopoietic stem cells in the mature placenta of the mankind, than Cord blood, has more hemopoietic stem cell, and these placenta hemopoietic stem cells can be separated before and after refrigerated storage.The activity of placenta hemopoietic stem cell colony-forming unit (CFU) determines, the transplantation experiments in Immune deficient mice has proved the potentiality of placenta cells in transplanting.These results effectively show, the mature placenta of the mankind likely becomes a kind of novel source for the hemopoietic stem cell transplanted.And in placenta, there is a large amount of hemopoietic stem cells, placental blood stem cell is comparatively early stage stem cell, can be divided in vivo various cells, placental blood contains abundant early stage hemopoietic stem cell of various stages, its content is approximately tens times of Cord blood, hemopoietic stem cell in a placenta can be satisfied with two grownups' demand completely, if can be applied to patient together with cord blood cell, greatly increased undoubtedly the content of hemopoietic stem cell, made this hemopoietic stem cell can be applied to all applicable crowds completely.
Patent CN01131190.8 discloses and a kind ofly by the method that shreds digestion, has obtained the method for hemopoietic stem cell, method complexity, and the hemopoietic stem cell quantity of acquisition is few, purity is low, and the easy microbiological contamination of cell.
Summary of the invention
Technical problem to be solved by this invention be to provide a kind of from healthy puerpera's mature placenta the method for lavation hemopoietic stem cell.
Described method comprises the collection of placenta, the initial survey of placenta, pre-sterilization, placenta and female blood examination survey of placenta, the lavation of placenta hemopoietic stem cell, concentrated and purified six steps of hemopoietic stem cell, and each step is specially:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 ℃, sends to laboratory in 24 hours;
(2) initial survey of placenta: check in placenta accumulating box box, whether temperature, label, the date of service, accumulating box have seepage, whether have female blood blood sample;
(3) the pre-sterilization of placenta: the fetus face of placenta is launched to be positioned over to the bottom of placenta flushing box, with normal saline flushing placenta surface, open the water vent of placenta flushing box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation;
(4) placenta and female blood examination are surveyed: got placenta and female blood sample are originally detected, and the project of detection comprises that hepatitis virus detects, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM);
(5) lavation of placenta hemopoietic stem cell: placenta is washed to blood clot and the hematocele on placenta by stroke-physiological saline solution under aseptic condition, adopt after sterilizing agent sterilization, perfusion liquid syringe needle is inserted in placenta Umbilical artery, placenta perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, slowly open constant flow pump, perfusion liquid, through sebific duct, switch, peristaltic pump, syringe needle, placenta artery, placenta, placenta vein, placenta perfusion returnable bottle syringe needle, finally receives irrigating solution in irrigating solution returnable bottle;
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, in whizzer with the centrifugal 15-20 minute of 1500-2000 rpm, remove supernatant liquor, the liquid of collecting precipitation and lower floor, the liquid of the precipitation of collecting and lower floor and physiological saline are mixed to obtain to mixed solution in the ratio of 2:1-1:2, then mixed solution and lymphocyte separation medium are joined respectively in centrifuge tube in the ratio of 2:1-1:2, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add again mixed solution, in adition process, note keeping the liquid level of lymphocyte separation medium smooth, after adding with the centrifugal 20-25 minute of 2200-2500 rpm, when centrifugal, slow acceleration slowed down slowly, after centrifugal end, in the middle of collecting, tunica albuginea layer is in new centrifuge tube, with the ratio of 2:1-1:2, adding physiological saline mixes, the centrifugal 10-15 minute of 1200-1500 rpm, after centrifugal end, remove supernatant liquor, add 10-20 mL physiological saline piping and druming precipitation, the centrifugal 10-15 minute of 1200-1500 rpm, after centrifugal end, removes supernatant liquor again, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out to cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), active (Trypan Blue), the hemopoietic stem cell qualitative detection (CFU-GM) of detecting of hemopoietic stem cell.
The lavation step of the hemopoietic stem cell described in aforesaid method is:
The irrigating solution using while starting lavation is in every 500 mL DMEM substratum, to add 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions; Close constant flow pump, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(5 mg/ bottle) irrigating solution of 0.5 bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions; Before preparation lavation DMEM substratum used, 10% FBS are used, be first positioned in the water-bath of 37 ℃, be preheating in advance 37 ℃.The lavation step of the hemopoietic stem cell described in aforesaid method can also be:
The irrigating solution using while starting lavation is in every 500 mL DMEM substratum, to add 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions; Close constant flow pump, changing irrigating solution into 50 mL is the DMEM substratum of 100 mg/L containing the concentration of AMD3100, and with the speed lavation 5 minutes of drip irrigation washing lotion p.s.s 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(5 mg/ bottle) irrigating solution of 0.5 bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions, reclaim irrigating solution.
Hemopoietic stem cell described in aforesaid method is concentrated and purified can also be: by lavation irrigating solution out, centrifugal, centrifugal condition is: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 20 min, after centrifugal end, returnable bottle is transferred in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid, stay hemocyte precipitation, toward the physiological saline that adds 10-20 mL in hemocyte precipitation, blown and beaten gently precipitation, get 50 mL centrifuge tubes, every pipe adds the lymphocyte separation medium of 20 mL, tilt containing the centrifuge tube of lymphocyte separation medium, with liquid-transfering gun absorption, blowing and beating the hemocyte getting up slowly joins on lymphocyte separation medium along centrifuge tube tube wall, the process adding is wanted slowly, keep lymphocyte separation medium liquid level smooth, every pipe adds hemocyte 25 mL, after moving liquid and completing, centrifugal, centrifugal condition: rotating speed 2200 rpm, pick-up period 600 s, deceleration times 600 s, centrifugation time 20 min, after centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, every pipe 20 mL transfer in 50 new mL centrifuge tubes, and then every pipe adds the physiological saline of 20 mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 15 min, after centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, every pipe adds the physiological saline piping and druming precipitation of 20 mL, then centrifugal, centrifugal condition: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 15 min, after centrifugal end, remove supernatant liquor, with the DMEM substratum re-suspended cell of 10% FBS of 20 mL, collect hemopoietic stem cell group, cell mass is carried out to cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), active (Trypan Blue), the hemopoietic stem cell qualitative detection (CFU-GM) of detecting of hemopoietic stem cell.
In hematopoietic stem cell transplantation, the content of placenta hemopoietic stem cell is very important, adopts the placenta hemopoietic stem cell of present method extraction after lymphocyte separation medium separates, and uses cell counter counting, has 8 × 10
7-1.5 × 10
8mononuclearcell number, although individual difference is larger, also can meet the sick human needs of all transplanting.Wherein approximately having 1.5-3.6% is CD34 positive cell, and external granulocyte colony is cultivated (CFU-GM), the visible huge cell mass of colony.
The placenta hemopoietic stem cell obtaining by method lavation of the present invention can be applied to various malignant hematologic diseases, as leukemia; Heredopathia, as congenital hemolytic anemia; Blood hypoplasia disease, as aplastic anemia; Radiation disease, as radioactive rays surprisingly etc.Methods for the treatment of is to adopt the various means such as chemotherapy or radiotherapy to destroy ill medullary cell and the immunity system of patient self, then input healthy hemopoietic stem cell, the hemopoietic stem cell obtaining from placenta lavation by the inventive method can well be used for the reconstruction of chemotherapy or the postoperative hematopoietic cell of radiotherapy, graft-vs-host reaction is little, has improved greatly surviving rate.
The application approach of placenta stem-cell prepared by the present invention, can be intravenous injection, intra-arterial injection, also can local injection, generally with vein, input to patient, and do not get rid of the application approach that adopts other modes.
The present invention's reagent used or pharmacy composition, receive injection liquid and AMD3100 etc. as physiological saline, DMEM substratum, nitroglycerin injection, foetal calf serum (FBS), heparin, is all aseptic, apyrogenic, all applicable to the patient of clinical transplantation.
All operations step of the present invention, it is all the routine operation method that pharmacopeia allows, comprise and adopt aseptic technique, stroke-physiological saline solution washing, sterilizing, aseptic cell concentration and purifying etc., applied reagent or pharmacy composition, as physiological saline, DMEM substratum, nitroglycerin injection, foetal calf serum, heparin, receiving injection liquid and AMD3100 etc. is all the requirements that meet China health organ, after end of operation, do conventional virus, cytoscopy and bacterium, mycotic culture, the placenta hemopoietic stem cell only meeting the requirements just can be applied to clinical.
The present invention preparation method's required for protection advantage:
(1) equipment of the inventive method and use is simple, easy to operate, and efficiency is high, greatly shorten from being prepared into the cycle of application, and placenta source is abundant, donor no pain;
(2) the inventive method lavation hematopoietic stem cell injuries out from placenta is little, and quantity is many, and purity is high, active high, can be applied to the transplanting of the hemopoietic stem cell of autologous or allosome, and contribute to the reparation of damaged tissue, greatly improve patient's surviving rate;
(3) irrigation system of the inventive method is the system of sealing, does not contact with air is extraneous, avoids microbiological contamination;
(4) the inventive method can keep the integrity of placenta, convenient other cells that extract from placenta;
(5) reagent that the inventive method is used is few, effectively economizes on resources and cost.
accompanying drawing explanation
The flow cytometer detected result of Fig. 1: the embodiment mono-method hemopoietic stem cell that lavation obtains from placenta.
Fig. 2: the colony of the hemopoietic stem cell that lavation obtains from placenta is cultivated (CFU-GM) culture experiment result.
Fig. 3: separate active (Trypan Blue) detected result that detects of the hemopoietic stem cell obtaining from placenta.
The flow cytometer detected result of Fig. 4: the embodiment bis-methods hemopoietic stem cell that lavation obtains from placenta.
In figure, Fig. 1 refers to: the flow cytometer detected result of the embodiment mono-method hemopoietic stem cell that lavation obtains from placenta, visible cell a considerable number of, wherein the ratio of CD34 positive cell is 1.65%, the a considerable number of of hemopoietic stem cell that placenta lavation obtains is described, ratio is very high, very high to clinical using value.
Fig. 2 refers to: the placenta hemopoietic stem cell obtaining is cultivated with methylcellulose gum semisolid medium, cultivate after two time-of-weeks, can observe and in methylcellulose gum semisolid medium, have obvious colony, the activity of the hemopoietic stem cell that this explanation obtains and multiplication capacity are all higher.
Fig. 3 refers to: placenta hemopoietic stem cell is carried out to activity and detect, do not observe hemopoietic stem cell and be colored, illustrate that the activity of hemopoietic stem cell is better, and the form of cell is fine.
Fig. 4 refers to: the flow cytometer detected result of the embodiment bis-methods hemopoietic stem cell that lavation obtains from placenta, visible cell a considerable number of, wherein the ratio of CD34 positive cell is 3.2%, the a considerable number of of hemopoietic stem cell that placenta lavation obtains is described, ratio is very high, very high to clinical using value.
embodiment
Below in conjunction with specific embodiment, the invention will be further described, but not as a limitation of the invention.
The technical scheme the present invention relates to for provide a kind of from placenta the method for lavation hemopoietic stem cell.
Of the present invention from placenta the method for lavation hemopoietic stem cell comprise the collection of placenta, the initial survey of placenta, pre-sterilization, placenta and female blood examination survey of placenta, the lavation of placenta hemopoietic stem cell, the concentrated and purified step of hemopoietic stem cell, it is respectively:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 ℃, sends to laboratory in 24 hours;
(2) initial survey of placenta: open placenta accumulating box, check thermometer scales in box, require temperature in box to remain between 4-10 ℃; Label checks, on placenta accumulating box, labelling should be complete, clear, and the date of service before the placenta accumulating box Expiration Date, otherwise be considered as defectively, will not receive; Check that whether placenta accumulating box has seepage, if any seepage, will not receive; Check whether there is female blood blood sample, and indicate completely, if without mother's blood sample, will not receive;
(3) the pre-sterilization of placenta: by careful placenta taking out with tweezers are overall, being positioned over gently the placenta of having disinfected in advance rinses in box, launch placenta, fetus face is launched to be positioned over to the bottom of placenta flushing box, with normal saline flushing placenta surface, open the water vent that placenta rinses box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation, placental calcification is due to due to the focal infarct of pregnant woman's placentogenesis in late period, infarct is more, occur that calcification point is just more, what under B ultrasonic, show is just more compared with high light spot, when detecting, B ultrasonic can be divided into three degree according to the placental calcification spot large young pathbreaker's calcification degree that distributes, it is I degree, II degree, III degree, the calcification situation of diagnosing under B ultrasonic not necessarily conforms to actual, and make a definite diagnosis and must conclude according to postpartum examination placental calcification area, according to placenta Surface calcification situation, determine calcification degree I degree, II degree, III degree, keeping records,
(4) placenta and female blood examination are surveyed: got placenta and female blood sample are originally detected, and the project of detection comprises that hepatitis virus detects, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM);
(5) lavation of placenta hemopoietic stem cell: placenta is cleaned by stroke-physiological saline solution under aseptic condition, wash blood clot and hematocele on placenta, adopt after sterilizing agent sterilization, by the current face of placenta, fetus face and umbilical cord cross section mug, then perfusion liquid syringe needle is inserted in placenta Umbilical artery, in umbilical cord, there are two Umbilical artery, only insert wherein one, mosquito forceps steps up to insert the syringe needle of Umbilical artery, due to the existence of venous sinus, irrigating solution can not flow out from another Umbilical artery, placenta perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps the syringe needle that inserts umbilical vein, irrigating solution is placed on 32 ℃ of constant temperature electric heating sheets, the box of depositing placenta is positioned in the constant temperature electric heating sheet of 32 ℃, after temperature-stable, slowly open the switch of constant flow pump and (before intubate, will open constant flow pump, by ducted Bas Discharged, make to be full of in sebific duct irrigating solution), by speed setting to middling speed (with the speed of 3 drip irrigation washing lotions p.s.), guarantee to rinse path smoothness, the for some time of rinsing beginning will be noted the take-off rate of receiving end irrigating solution the moment, if the speed that cut flows out or flows out obviously declines, close constant flow pump, open vein stanch pincers, take out returnable bottle syringe needle, look into and see if there is sludged blood obstruction syringe needle, and then pin, clamp mosquito forceps, open constant flow pump, in the process of rinsing, also will note the gathering speed of liquid in returnable bottle the moment, perfusion liquid, through sebific duct, switch, peristaltic pump, syringe needle, placenta artery, placenta, placenta vein, placenta perfusion returnable bottle syringe needle, finally receives irrigating solution in irrigating solution returnable bottle,
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, in whizzer with 1500 rpm centrifugal 20 minutes, remove supernatant liquor, the liquid of collecting precipitation and lower floor, the liquid of the precipitation of collecting and lower floor and physiological saline are mixed to obtain to mixed solution in the ratio of 1: 1, then mixed solution and lymphocyte separation medium are joined respectively in centrifuge tube in the ratio of 1: 1, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add again mixed solution, in adition process, note keeping the liquid level of lymphocyte separation medium smooth, after adding, with 2200, leave the heart 20 minutes, when centrifugal, slow acceleration slowed down slowly, after centrifugal end, centrifuge tube from top to bottom solution is divided into four layers, the first layer is plasma layer, the second layer is ring-type oyster white buffy coat, the 3rd layer is transparent parting liquid, the 4th layer is red corpuscle layer.Collect second layer tunica albuginea layer in new centrifuge tube, add 10-20mL physiological saline and mix, 1500 leave the heart 15 minutes; After centrifugal end, remove supernatant liquor, physiological saline piping and druming precipitation, 1500 leave the heart 15 minutes again, after centrifugal end, remove supernatant liquor, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out to cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), active (Trypan Blue), the hemopoietic stem cell qualitative detection (CFU-GM) of detecting of hemopoietic stem cell.
the method of embodiment mono-lavation hemopoietic stem cell from placenta
Placenta comes from local obstetrics and gynecology hospital.First, obtain pregnant woman or its family members agree to contribute after placenta, consult all audit reports of pregnant woman, confirm without the relevant virus infection of any virus, syphilis and blood, then inquire pregnant woman's family's medical history, infection and infection medical history.After determining that all are normally, gather female blood 5 mL, placenta splendid attire together with female blood is entered to placenta accumulating box, post label, bar code.In box in placenta storage and transport process, temperature remains on 4-10 ℃, in 24 hours, is transported to laboratory.
Behind laboratory, bar code and label are all inputted in computer, by careful placenta taking out with tweezers are overall, being positioned over gently the placenta of having disinfected in advance rinses in box, launch placenta, fetus face is launched to be positioned over to the bottom of placenta flushing box, with normal saline flushing placenta surface, open the water vent of placenta flushing box bottom surface, remove the physiological saline of flushing.Check placenta Surface calcification situation.Got female blood and placental samples are detected, and the project of detection comprises that hepatitis virus detects, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM).
Placenta is cleaned by stroke-physiological saline solution under aseptic condition, wash blood clot and hematocele on placenta, adopt after thimerosal sterilization, by the current face of placenta, fetus face and umbilical cord cross section mug.Then perfusion liquid syringe needle is inserted in placenta Umbilical artery, in umbilical cord, there are two Umbilical artery, only insert wherein one, mosquito forceps steps up, due to the existence of venous sinus, irrigating solution can be from wherein not flowing out an artery, placenta perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, open perfusion system, the irrigating solution using while starting lavation is to add 10% FBS 50 mL in every 500 mL DMEM substratum, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions, close constant flow pump, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(A5602,5 mg/ bottles) irrigating solution of 0.5 bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions, at receiving end, receive irrigating solution.
By the irrigating solution being recovered in 250 mL returnable bottles, trim, centrifugal, centrifugal condition is: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 20 min; After centrifugal end, returnable bottle is transferred in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid and pours in waste liquid cylinder, stays hemocyte precipitation, adds the physiological saline of 20 mL according to hemocyte precipitation, has blown and beaten gently precipitation.Get 50 mL centrifuge tubes, every pipe adds the lymphocyte separation medium of 20 mL, tilt containing the centrifuge tube of lymphocyte separation medium, with liquid-transfering gun, draw and blow and beat the hemocyte of getting up, along centrifuge tube tube wall, slowly join on lymphocyte separation medium, the process adding must blunting of attention, keeps lymphocyte separation medium liquid level smooth, and every pipe adds hemocyte 25 mL; After moving liquid and completing, centrifugal, centrifugal condition: rotating speed 2200 rpm, pick-up period 600 s, deceleration times 600 s, centrifugation time 20 min; After centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, every pipe 20 mL transfer in 50 new mL centrifuge tubes, and then every pipe adds the physiological saline of 20 mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 15 min; After centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, every pipe adds the physiological saline of 20 mL, piping and druming precipitation, then centrifugal, centrifugal condition: rotating speed 1500rpm, pick-up period 45s, deceleration times 45 s, centrifugation time 15 min; After centrifugal end, remove supernatant, the DMEM substratum of the 10%FBS of 20 mL is joined in the centrifuge tube that removes supernatant liquor, slowly shake centrifuge tube, precipitation is is all blown and beaten, form solution.Then from wherein getting 20 ul to 1.5 mL centrifuge tubes, get two pipes, for cell counting and active detect (Trypan Blue); And then from wherein getting respectively 200 ul to the centrifuge tube of 1.5 mL, three pipes, the first pipe adds the physiological saline of 1 ul, the second pipe adds the homotype contrast PE of CD34, the 3rd pipe adds CD34 antibody, is placed on dark place and hatches 15 minutes after three pipes are mixed, and then flow cytometer detects.
The hemopoietic stem cell obtaining is carried out to cell counting, cytoactive detection, flow cytometer detection and colony culture experiment.By the enchylema of pipe 20 ul to cell counter, counting cells number 1.0 × 10
8individual mononuclearcell; The enchylema that another is managed to 20 ul, adds the trypan blue dye liquor of 20 ul concentration 0.08%, mixes, then get 4 ul to slide glass, examine under a microscope, do not observe cell and be colored, illustrate that cytoactive is better, and the form of cell fine (seeing Figure of description 3); Enchylema after cell detection by quantitative (CD34) is hatched three pipe 200 ul detects to flow cytometer, by detecting, the ratio of CD34 positive cell reaches 1.65%, this time detect CD34 positive cell and added negative control, data are more conservative, in normal experiment, obtain the high energy of ratio of CD34 positive cell reach 3.6%, this ratio that absolutely proves the hemopoietic stem cell that placenta lavation obtains is very high, a considerable number of, clinical using value very high (the results are shown in Figure of description 1); The hemopoietic stem cell obtaining is carried out to methylcellulose gum semisolid medium cultivation (CFU-GM), get hole of Tissue Culture Plate, in hole, add methylcellulose gum semisolid medium 3 mL, then get the enchylema of 1 ul, be added in methylcellulose gum semisolid medium, and separately get a hole and add 2 mL distilled water, after adding well, build culture plate lid, be placed in carbon dioxide cell incubator, 7 ℃ of the conditions of incubator, 5% CO
2, humidity 90% cultivates 14 days, observes complete cell colony, the hemopoietic stem cell that this explanation obtains there is multiplication capacity (the results are shown in Figure of description 2).
the method of embodiment bis-lavation hemopoietic stem cell from placenta
Placenta comes from local obstetrics and gynecology hospital.First, obtain pregnant woman or its family members agree to contribute after placenta, consult all audit reports of pregnant woman, confirm without the relevant virus infection of any virus, syphilis and blood, then inquire pregnant woman's family's medical history, infection and infection medical history.After determining that all are normally, gather female blood 5 mL, placenta splendid attire together with female blood is entered to placenta accumulating box, post label, bar code.In box in placenta storage and transport process, temperature remains on 4-10 ℃, in 24 hours, is transported to laboratory.
The method of reference example one is carried out the initial survey of placenta, pre-sterilization, placenta and female blood examination of placenta surveyed,
The lavaging method of placenta hemopoietic stem cell is changed into: the irrigating solution using while starting lavation is in every 500 mL DMEM substratum, to add 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions; Close constant flow pump, changing irrigating solution into 50 mL is the DMEM substratum of 100 mg/L containing the concentration of AMD3100, and with the speed lavation 5 minutes of drip irrigation washing lotion p.s.s 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(5 mg/ bottle) irrigating solution of 0.5 bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions, at receiving end, receive irrigating solution.
The method of reference example one is carried out concentrated and purified to hemopoietic stem cell, after processing sample, the hemopoietic stem cell obtaining is carried out to flow cytometer detection (the results are shown in accompanying drawing 4), the ratio of CD34 positive cell reaches 3.2%, the ratio that the hemopoietic stem cell that placenta lavation obtains is described is very high, a considerable number of.
Claims (4)
1. the method for a lavation hemopoietic stem cell from placenta, it is characterized in that, described method comprises the collection of placenta, the initial survey of placenta, pre-sterilization, placenta and female blood examination survey of placenta, the lavation of placenta hemopoietic stem cell, the concentrated and purified step of hemopoietic stem cell, and it is respectively:
(1) collection of placenta: collect placenta and female blood under Operation theatre gnotobasis, deposit in aseptic placenta accumulating box, post label, bar code, the temperature of placenta accumulating box remains on 4-10 ℃, sends to laboratory in 24 hours;
(2) initial survey of placenta: check in placenta accumulating box box, whether temperature, label, the date of service, accumulating box have seepage, whether have female blood blood sample;
(3) the pre-sterilization of placenta: the fetus face of placenta is launched to be positioned over to the bottom of placenta flushing box, with normal saline flushing placenta surface, open the water vent of placenta flushing box bottom surface, remove the physiological saline of flushing, check placenta Surface calcification situation;
(4) placenta and female blood examination are surveyed: got placenta and female blood sample are originally detected, and the project of detection comprises that hepatitis virus detects, virus of AIDS detects, venereal disease detects, tissue matching (HLA) is detected, hematopoietic stem/progenitor qualitative detection (CFU-GM);
(5) lavation of placenta hemopoietic stem cell: placenta is washed to blood clot and the hematocele on placenta by stroke-physiological saline solution under aseptic condition, adopt after sterilizing agent sterilization, perfusion liquid syringe needle is inserted in placenta Umbilical artery, placenta perfusion returnable bottle syringe needle inserts in umbilical vein, mosquito forceps clamps, slowly open constant flow pump, perfusion liquid, through sebific duct, switch, peristaltic pump, syringe needle, placenta artery, placenta, placenta vein, placenta perfusion returnable bottle syringe needle, finally receives irrigating solution in irrigating solution returnable bottle;
(6) hemopoietic stem cell is concentrated and purified: by lavation irrigating solution out, in whizzer with the centrifugal 15-20 minute of 1500-2000 rpm, remove supernatant liquor, the liquid of collecting precipitation and lower floor, the liquid of the precipitation of collecting and lower floor and physiological saline are mixed to obtain to mixed solution in the ratio of 2:1-1:2, then mixed solution and lymphocyte separation medium are joined respectively in centrifuge tube in the ratio of 2:1-1:2, the order of adding for first adding lymphocyte separation medium in centrifuge tube, slowly add again mixed solution, in adition process, note keeping the liquid level of lymphocyte separation medium smooth, after adding with the centrifugal 20-25 minute of 2200-2500 rpm, when centrifugal, slow acceleration slowed down slowly, after centrifugal end, in the middle of collecting, tunica albuginea layer is in new centrifuge tube, with the ratio of 2:1-1:2, adding physiological saline mixes, the centrifugal 10-15 minute of 1200-1500 rpm, after centrifugal end, remove supernatant liquor, add 10-20 mL physiological saline piping and druming precipitation, the centrifugal 10-15 minute of 1200-1500 rpm, after centrifugal end, removes supernatant liquor again, by the resuspended precipitation of DMEM substratum, collect hemopoietic stem cell group, resuspended cell mass is carried out to cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), active (Trypan Blue), the hemopoietic stem cell qualitative detection (CFU-GM) of detecting of hemopoietic stem cell.
According to claim 1 from placenta the method for lavation hemopoietic stem cell, it is characterized in that, the lavation step of described hemopoietic stem cell is:
The irrigating solution using while starting lavation is in every 500 mL DMEM substratum, to add 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions; Close constant flow pump, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(5 mg/ bottle) irrigating solution of 0.5 bottle, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions; Before preparation lavation DMEM substratum used, 10% FBS are used, be first positioned in the water-bath of 37 ℃, be preheating in advance 37 ℃.
According to claim 1 from placenta the method for lavation hemopoietic stem cell, it is characterized in that, the lavation step of described hemopoietic stem cell is:
The irrigating solution using while starting lavation is in every 500 mL DMEM substratum, to add 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2mL:12500 unit), mix the irrigating solution making, with the speed lavation 2 hours of drip irrigation washing lotion p.s.s 3, lavation 1000 mL irrigating solutions; Close constant flow pump, changing irrigating solution into 50 mL is the DMEM substratum of 100 mg/L containing the concentration of AMD3100, and with the speed lavation 5 minutes of drip irrigation washing lotion p.s.s 10, lavation, after lavation is complete; Close constant flow pump, after 30 minutes, irrigating solution is changed in every 500 mL DMEM substratum and adds 10% FBS 50 mL, 2 of nitroglycerin injections (1 mL:5 mg), 2 of heparin sodium injections (2 mL:12500 unit), AMD3100(5 mg/ bottle) irrigating solution of 0.5 bottle, open constant flow pump, with the speed lavation 2.5 hours of drip irrigation washing lotion p.s.s 3, lavation 1500 mL irrigating solutions, reclaim irrigating solution.
According to claim 1 from placenta the method for lavation hemopoietic stem cell, it is characterized in that, described hemopoietic stem cell is concentrated and purified can also be: by lavation irrigating solution out, centrifugal, centrifugal condition is: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 20 min, after centrifugal end, returnable bottle is transferred in aseptic operating platform, liquid-transfering gun siphons away supernatant liquid, stay hemocyte precipitation, toward the physiological saline that adds 10-20 mL in hemocyte precipitation, blown and beaten gently precipitation, get 50 mL centrifuge tubes, every pipe adds the lymphocyte separation medium of 20 mL, tilt containing the centrifuge tube of lymphocyte separation medium, with liquid-transfering gun absorption, blowing and beating the hemocyte getting up slowly joins on lymphocyte separation medium along centrifuge tube tube wall, the process adding is wanted slowly, keep lymphocyte separation medium liquid level smooth, every pipe adds hemocyte 25 mL, after moving liquid and completing, centrifugal, centrifugal condition: rotating speed 2200 rpm, pick-up period 600 s, deceleration times 600 s, centrifugation time 20 min, after centrifugal end, solution can divide four layers, with tunica albuginea layer in the middle of pipette, extract, every pipe 20 mL transfer in 50 new mL centrifuge tubes, and then every pipe adds the physiological saline of 20 mL, after mixing, centrifugal, centrifugal condition: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 15 min, after centrifugal end, transfer in aseptic operating platform, remove supernatant liquor, every pipe adds the physiological saline piping and druming precipitation of 20 mL, then centrifugal, centrifugal condition: rotating speed 1500 rpm, pick-up period 45 s, deceleration times 45 s, centrifugation time 15 min, after centrifugal end, remove supernatant liquor, with the DMEM substratum re-suspended cell of 10% FBS of 20 mL, collect hemopoietic stem cell group, cell mass is carried out to cell and mycotic culture, hemopoietic stem cell detection by quantitative (CD34), active (Trypan Blue), the hemopoietic stem cell qualitative detection (CFU-GM) of detecting of hemopoietic stem cell.
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