CN102965339A - Kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and cell treatment method - Google Patents
Kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and cell treatment method Download PDFInfo
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- CN102965339A CN102965339A CN2011102567747A CN201110256774A CN102965339A CN 102965339 A CN102965339 A CN 102965339A CN 2011102567747 A CN2011102567747 A CN 2011102567747A CN 201110256774 A CN201110256774 A CN 201110256774A CN 102965339 A CN102965339 A CN 102965339A
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Abstract
The invention discloses a kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and provides a kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells and a cell treatment method which are strong in operationality, high in clinical safety, and convenient for clinical popularization. The kit comprises four reagents: No. A liquid is a diluent; No. B liquid is density fluid; No. C liquid is washing liquid; No. D liquid is erythrocyte-removing liquid. The invention fundamentally solves the problems of high cost, low cell activity, undefined human body influence for markers entering human body, pain for patients due to mobilization agent injection, cumbersome and inapplicable operations, and the like for current cell separation technology.
Description
Technical field
Present disclosure belongs to the biomedical technology Application Areas.Be specifically related to the method for stem cell in a kind of in-vitro separation human marrow, Cord blood and the peripheral blood, particularly a kind of human marrow, Cord blood, peripheral hematopoietic stem cells treatment kits and cell treatment process.
Background technology
Development along with present science and technology, the isolation technique that is used for the human blood cell constantly develops and optimizes, present stem cell method for separating and processing comprises immunomagnetic beads method, flow cytometer method, blood cell separator method, cultivates TRAP and density gradient centrifugation etc., but all has many drawbacks.(1) immunomagnetic beads method is combined with the cell response of the cell surface corresponding antigens positive by the antibody that is coated on the bead particulates, then by the magnetic absorption principle cell of non-absorption is got rid of, the cell that collection is adsorbed by magnetic bead antibody, this method cost is high, increased patient's burden, be unfavorable for popularizing of project, and cytoactive is had damage, affect the cell curative effect.(2) the flow cytometer method also is that utilization is carried out sorting, collection to the principle that target cell carries out mark identification by flow cytometer.The method existence screening cell scope is little, cell is labeled rear activity decreased and can't predicts the impact of marker in human body; And high (3) the blood cell separator method of the method cell contamination risk is only applicable to separate stem cells from peripheral blood, and application is restricted; The method need to increase the treatment burden to the agent of patient infusion stem cell mobilization, and the stem cell amount is little after separating simultaneously, liquid volume is large, and is dangerous to the patient; (4) the cultured and amplified in vitro rule need to be cultivated gathering the human blood cell, cleans and uses, and this method has increased Pollution risk, and stem cell is divided into unknown stem cell to unknown direction simultaneously, and incubation time is long.
Method for separating stem cell based on density gradient method obtains wider application clinically at present.But different methods and operation steps produce an effect are different, cut both ways, and in general, have following several problem:
(1) can not stdn.Be that 200610035900.5 " a kind of stem cell layering liquid and be used for the method that stem cell separates " needs operator oneself to prepare working fluid such as application number, and need to regulate proportion, this method has increased operator's artifical influence factor, is unfavorable for popularization and industrialization that stem cell separates.
(2) red corpuscle is removed unclean: be that 200610035900.5 " a kind of stem cell layering liquid and be used for the method that stem cell separates " is particularly for Cord blood such as application number, this patented method has greatly increased the patient in the time of final clinical application and has produced the risk of rejection except unclean red corpuscle; Application number is that 200910187212.4 " human marrow, Cord blood, peripheral blood cells treatment kits and cell treatment process " also exists identical shortcoming;
(3) the stem cell yield is not high: be 200610114475.9 " for separating of the test kit of BMNC " such as application number, its shortcoming is that the stem cell yield quantity of separating is not high, need to carry out cell cultures, but increased thus the cell contamination rate, the cell function that simultaneously cell in vitro amplification produces is unstable, is difficult to clinical treatment; Application number is the lymphocyte separation medium of the density 1.077 of 200610106875.5 " a kind of karyocyte in-vitro separation test kit and application method thereof " HISTOPAGUE077 of adopting in addition, the cell overwhelming majority after separating is lymphocyte, the stem cell that so directly causes can be used in treatment in the cell occupies the minority, and affects clinical efficacy;
(4) the separation and purification cost is too high: the patent that such as application number is 200710137781.9 " marrow umbilical cord blood stem cell in vitro separating kit and application method thereof ", added lin antibody in its reagent, cause cost greatly to improve, after dropping into clinical use, greatly increased patient's economical load, cause the patient cannot afford to consult a doctor, connect the cell therapy of daring not accept, and used lin antibody that cell is cleaned and caused larger burden, this kind material enters that the impact on human body also is a unknown number behind the human body, is still waiting further research.
(5) do not consider that albumen precipitation, cell condensation factor are to the later stage risk used of stem cell: adopt centrifugal mode separation and purification stem cell, in operating process often because bleeding of the umbilicus, peripheral blood sample quality are different, stem cell produces cohesion and precipitation in purge process, if but this precipitation is not in time removed, in the application process in later stage, cause easily patient's Microembolization, affect result for the treatment of, severe patient can jeopardize patient's life security.
Summary of the invention
Technical problem to be solved by this invention is for human marrow, Cord blood, peripheral blood cells treatment kits and the cell treatment process that a kind of strong operability, clinical safety are high, be convenient to clinical expansion is provided, it has fundamentally solved, and the cost that exists in the existing cell separation technology is high, cytoactive is low, marker is failed to understand the human influence after entering human body, need the injection mobilization agent cause the patient suffering, the loaded down with trivial details inapplicable problem such as clinical.But this test kit is easy to storage transportation industrialization, convenient to use simultaneously.
Technical scheme of the present invention is: human marrow, Cord blood, peripheral blood cells treatment kits, its technical essential are to comprise following four kinds of reagent in the described test kit:
A liquid is diluent: PBS liquid;
B liquid is density fluids: Sodium Diatrizoate-ficoll 400# or HISTOPAQUE1077 or Ficoll; The density that Sodium Diatrizoate-ficoll 400# is mixed with by ficoll and urografic acid methylglucamine salt is 1.075 ± 0.001 density fluids;
C liquid is washings: 0.9% sodium chloride injection;
D liquid is for removing red corpuscle liquid: 0.8% ammonium chloride solution;
A kind of application human marrow as claimed in claim 1, Cord blood, peripheral blood cells treatment kits and cell treatment process, its technical essential is: will contain the marrow of sodium citrate anticoagulant or Cord blood or peripheral blood and add A liquid, mixing in the ratio more than or equal to 1: 1; The sample volume good according to dilution, get the 15ml centrifuge tube of proper amt, respectively fill first the B liquid of 4ml, respectively get again 10ml and dilute good sample and be taped against on the B liquid, then with 500-850g centrifugal 25 minutes, tell the stem cell layer, collect the stem cell layer and clean cell 1 time with C liquid afterwards, with the C liquid re-suspended cell of 5-10ml, add the approximately long-pending D liquid of triploid, mixing, reacted 6 minutes, with 350-550g centrifugal 10 minutes again, remove supernatant, clean cell 1-2 time with C liquid after collecting stem cell, filter with 70 micron filters more as required, clean cell 1-2 time with C liquid after collecting the stem cell after filtering, be diluted to the 5-50 milliliter with C liquid behind the centrifugal concentrating, stand-by.
Advantage of the present invention and positive technique effect:
(1) the present invention can separate the stem cell in human marrow or Cord blood or the peripheral blood, be about to 4 kinds of agent combination application in the test kit, by B reagent density gradient centrifugation, according to various cell density differences they are scattered in top-down reagent level, remove the blood plasma in human marrow or human Cord blood or the peripheral blood, thrombocyte, oxyphorase, granulocyte and most of red corpuscle etc., the stem cell rate of recovery of separating through the present invention can reach more than 85%;
(2) the present invention reacts by the long-pending D liquid of the adding of the stem cell after will concentrating triploid, make erythroclasis, the stem cell by centrifugal collection purifying again, reach and remove erythrocytic effect, risk and the red corpuscle of having avoided stem cell patient in follow-up application process to produce rejection disturb;
(3) stem cell that the present invention is good with purifying is filtered by strainer, and the precipitated impurities that removal may exist by the stem cell of present method preparation, has been avoided the Microembolization danger that causes owing to precipitation in the follow-up stem cell application process.
(4) this test kit comprises all solution of separation and purification test kit, need not the other prep solution of user, and easy user's use preparation work also avoids the artificial pollution of user or third party to pollute introducing.
(5) the present invention is applicable to the stem cell separation of human marrow, Cord blood, peripheral blood, and for different samples, reagent forms identical, has avoided domestic more existing patents can only separate human marrow and Cord blood, can not separate the problem of peripheral blood.
(6) reagent raw material of the present invention is cheap, and layoutprocedure is simple, quality controllable, unmanageable biological products of functional quality not, and it is low to have cost, without any marker, keeps original cytoactive, and cell quantity can satisfy the advantages such as needs of clinical treatment.
(7) the present invention's needs of volume per sample, the reagent solution that adds in proportion different volumes operates and gets final product, be applicable to operation or the operation in enormous quantities of clinical short run sample, avoided the waste of sample, be clinically in the urgent need to a kind of test kit and method of isolated cell.
Embodiment
Below in conjunction with implementation two examples, further set forth the present invention.Present embodiment only is used for explanation the present invention and is not used in restriction the scope of protection of present invention.
Embodiment 1:
The present invention has developed a kind of marrow, Cord blood, peripheral blood cells treatment kits, and wherein test kit comprises following four kinds of reagent:
A liquid is diluent: PBS solution;
B liquid is density fluids: Sodium Diatrizoate-ficoll 400# or HISTOPAQUE1077 or Ficoll; The density that Sodium Diatrizoate-ficoll 400# is mixed with by ficoll and Sodium Diatrizoate is 1.075 ± 0.001 density fluids;
C liquid is washings: 0.9% sodium chloride injection;
D liquid is for removing red corpuscle liquid: 0.8% ammonium chloride solution;
The present invention also discloses a kind of cell treatment process of using the mentioned reagent box, and its concrete steps are: will contain the marrow of sodium citrate anticoagulant or Cord blood or peripheral blood and add A liquid, mixing in the ratio more than or equal to 1: 1; The sample volume good according to dilution, get the 15ml centrifuge tube of proper amt, respectively fill first the B liquid of 4ml, respectively get again 10ml and dilute good sample and be taped against on the B liquid, then with 500-850g centrifugal 25 minutes, tell the stem cell layer, collect the stem cell layer and clean cell 1 time with C liquid afterwards, with the C liquid re-suspended cell of 5-10ml, add the approximately long-pending D liquid of triploid, mixing, reacted 6 minutes, with 350-550g centrifugal 10 minutes again, remove supernatant, clean cell 1-2 time with C liquid after collecting stem cell, filter with 70 micron filters more as required, clean cell 1-2 time with C liquid after collecting the stem cell after filtering, be diluted to the 5-50 milliliter with C liquid behind the centrifugal concentrating, stand-by.
Understand and explanation the present invention for better, the below utilizes embodiment to elaborate.
Embodiment 2:
Use test kit of the present invention and separate the cord blood stem cell method steps:
Test kit is composed as follows:
A liquid is diluent: PBS solution;
B liquid is density fluids: Ficoll solution;
C liquid is washings: 0.9% chloride injection liquor;
D liquid is for removing red corpuscle liquid: 0.8% ammonium chloride solution;
Above-mentioned A liquid through sterilization in 10 pounds, 10 minutes, is detected cellulose content≤0.5EU/ml in it, bottling, 4 ℃ of C preserve.B liquid detects its endotoxin content≤0.5EU/ml through sterilization in 10 pounds, 10 minutes, bottling.
It is as follows to utilize the mentioned reagent box to separate 100ml cord blood stem cell method steps:
(1) gets A liquid 100ml, by 1: 1 dilution proportion bleeding of the umbilicus, mixing, can save backup by 4 degree;
(2) get the centrifuge tube of 20 15ml, 4ml adds in each pipe for subsequent use with 10ml pipette, extract B liquid;
(3) with each 10ml of pipette, extract bleeding of the umbilicus of 10ml, slowly be superimposed on the B liquid liquid level along tube wall, note keeping clearly interface;
(4) horizontal centrifugal, rotating speed 2114rpm, centrifugation time 25 minutes, temperature 21 degree;
(5) get 4 50ml centrifuge tubes, each adds 35ml C liquid, and is for subsequent use;
(6) step 4 centrifugal after, sample solution is divided into four layers in the centrifuge tube, draws in 4 centrifuge tubes of the 3rd layer of (the narrow band of white cloud and mist layer) average mark in step 5 mixing with pasteur pipet; Horizontal centrifugal 1674rpm * 20 minute, temperature 21 degree;
(7) suck supernatant with the 25ml transfer pipet, use the C liquid of 10ml pipette, extract 5ml in a centrifuge tube, re-suspended cell, and successively 4 tube cells are pooled in the 50ml centrifuge tube, softly blow and beat mixing;
(8) if hypercythemia in the step 7 then slowly adds the D liquid of 15ml in centrifuge tube, mixing reacted 6 minutes, wherein every 2 minutes mixings once; Horizontal centrifugal 1674rpm * 10 minute, temperature 21 degree;
(9) suck supernatant with the 10ml transfer pipet, add the C liquid of 20ml, mixing; Horizontal centrifugal, rotating speed 1674rpm, centrifugation time 20 minutes, temperature 21 degree;
(10) if obviously visible macrobead precipitation appears in resuspended cell, can adopt strainer to filter; Carry out filtered liquid centrifugal again; Horizontal centrifugal, rotating speed 1674rpm, centrifugation time 20 minutes, temperature 21 degree;
(11) suck supernatant with the 10ml transfer pipet, add the C liquid of 20ml, mixing; Horizontal centrifugal, rotating speed 1674rpm, centrifugation time 20 minutes, temperature 21 degree;
(12) suck supernatant with the 10ml transfer pipet, add as required the C liquid re-suspended cell of 5ml or 20ml, save backup.
Claims (4)
1. a human marrow, Cord blood, peripheral blood cells treatment kits and cell treatment process is characterized in that comprising in the described test kit following four kinds of reagent:
A liquid is diluent: PBS liquid;
B liquid is density fluids: Sodium Diatrizoate-ficoll 400# or HISTOPAQUE1077 or Ficoll; The density that Sodium Diatrizoate-ficoll 400# is mixed with by ficoll and urografic acid methylglucamine salt is 1.077 ± 0.001 density fluids;
C liquid is washings: 0.9% sodium chloride solution;
D liquid is erythrocyte cracked liquid: 0.8% ammonium chloride solution.
2. use human marrow as claimed in claim 1, Cord blood, peripheral blood cells treatment kits and cell treatment process for one kind, it is characterized in that the method comprises the following steps:
The marrow of sodium citrate anticoagulant or Cord blood or peripheral blood be will contain and A liquid, mixing added in the ratio more than or equal to 1: 1; The sample volume good according to dilution, get the 15ml centrifuge tube of proper amt, respectively fill first the B liquid of 4ml, respectively get again 10ml and dilute good sample and be taped against on the B liquid, then with 500-850g centrifugal 25 minutes, tell the stem cell layer, collect the stem cell layer and clean cell 1 time with C liquid afterwards, with the C liquid re-suspended cell of 5-10ml, add the approximately long-pending D liquid of triploid, mixing, reacted 6 minutes, with 350-550g centrifugal 10 minutes again, remove supernatant, clean cell 1-2 time with C liquid after collecting stem cell, filter with strainer more as required, clean cell 1-2 time with C liquid after collecting the stem cell after filtering, be diluted to the 5-50 milliliter with C liquid behind the centrifugal concentrating, stand-by.
3. an application such as claim 1 and 2 described human marrows, Cord blood, peripheral blood cells treatment kits and cell treatment process is characterized in that using 0.8% ammonium chloride solution to process residual red corpuscle in the stem cell.
4. an application such as claim 1 and 2 described human marrows, Cord blood, peripheral blood cells treatment kits and cell treatment process, it is characterized in that using the stem cell after 70 micron pore size strainers filter purifying, to remove cell or the precipitated impurities of cohesion.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN2011102567747A CN102965339A (en) | 2011-09-01 | 2011-09-01 | Kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and cell treatment method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2011102567747A CN102965339A (en) | 2011-09-01 | 2011-09-01 | Kit for treating human bone marrow, umbilical cord blood, and peripheral blood cells, and cell treatment method |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567631A (en) * | 2016-01-19 | 2016-05-11 | 齐湘杰 | Kit for culturing chain type activated immune cells |
| CN106434545A (en) * | 2016-11-11 | 2017-02-22 | 北正赛欧（北京）生物科技有限公司 | Method and kit for separating high-purity umbilical cord blood stem cells |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN107821383A (en) * | 2016-10-27 | 2018-03-23 | 杭州华得森生物技术有限公司 | Circulating tumor cell Sample preservation liquid and its application |
| CN113082053A (en) * | 2021-04-06 | 2021-07-09 | 武汉济源高科技有限公司 | Kit for treating habitual abortion caused by reduction or deletion of blocking antibody |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567631A (en) * | 2016-01-19 | 2016-05-11 | 齐湘杰 | Kit for culturing chain type activated immune cells |
| CN107821383A (en) * | 2016-10-27 | 2018-03-23 | 杭州华得森生物技术有限公司 | Circulating tumor cell Sample preservation liquid and its application |
| CN106434545A (en) * | 2016-11-11 | 2017-02-22 | 北正赛欧（北京）生物科技有限公司 | Method and kit for separating high-purity umbilical cord blood stem cells |
| CN106434545B (en) * | 2016-11-11 | 2019-12-03 | 北正赛欧（北京）生物科技有限公司 | The separation method and kit of high-purity cord blood stem cell |
| CN106969964A (en) * | 2017-02-23 | 2017-07-21 | 宁波美晶医疗技术有限公司 | The negative enrichment method and kit of rare cell in a kind of blood based on micro-fluidic and immune Magneto separate |
| CN113082053A (en) * | 2021-04-06 | 2021-07-09 | 武汉济源高科技有限公司 | Kit for treating habitual abortion caused by reduction or deletion of blocking antibody |
| CN113082053B (en) * | 2021-04-06 | 2022-05-24 | 武汉济源高科技有限公司 | Kit for treating habitual abortion caused by reduction or deletion of blocking antibody |
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