CN1793340A - Method for separating cell and special separating liquid for cell - Google Patents
Method for separating cell and special separating liquid for cell Download PDFInfo
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- CN1793340A CN1793340A CNA2005101303267A CN200510130326A CN1793340A CN 1793340 A CN1793340 A CN 1793340A CN A2005101303267 A CNA2005101303267 A CN A2005101303267A CN 200510130326 A CN200510130326 A CN 200510130326A CN 1793340 A CN1793340 A CN 1793340A
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Abstract
The invention discloses a cell separating method and special cell separating medium. The special cell separating medium gains is solvent gained by adding surface active agent into general cell separating medium. The cell separating method includes the following steps: mixing the surface active agent and current cell separating medium in given volumetric proportion; centrifuging to gain special cell separating medium; adding cell separating sample into the special cell separating medium; and centrifuging; colleting cloud layer cell and washing to gain purified cell. The invention greatly shortens sample adding and cell separating time, simplifies operation. And it is fit for large-scale separating. Its effect is stable and reliable. And high purity goal cell can be gained. The separating method is simple, and has strong operability. The invention has high practical application value and broad market prospect in cell biology, functional genome, and cell engineering fields.
Description
Technical field
The present invention relates to a kind of method and special-purpose cellular segregation liquid of isolated cell.
Background technology
Appearance and the development of stem cell (myeloid-lymphoid stem cell, multipotential stem cell, special energy stem cell) have greatly promoted the progress of cell engineering.Modern biotechnologies such as the cytodiagnosis of cell therapy, disease, cell vaccine, cell chip, RNAi progressively have been used widely at cell, body and population level, become the important component part in modern biotechnology field.Thereby stem cell all has important effect and huge commercial value at aspects such as fundamental research and production popularizations.
Density gradient centrifugation is separated the cell of different volumes size, different mass, make dissimilar cells be separated and purifying, by being applied to fields such as cell transgenosis, medical diagnosis on disease, functional genomics research and cell therapy after amplification in vitro, the engineering operation.Popular cellular segregation liquid composition is parting liquids such as sucrose, Ficoll-Hypaque (Ficoll) and Percoll at present, be mainly used in and separate XY sperm, lymphocyte, granulocyte etc., also the someone utilizes its protein that separates the differing molecular quality simultaneously, utilizes cellular segregation liquid can also obtain karyocyte in a large number in addition and is used to extract genome and carries out the genetically engineered operation.The Application Areas of cellular segregation liquid constantly enlarges, and separating effect also improves constantly, and provides effective means for cell engineering and functional genomics research.
But existing cellular segregation liquid all exist complicated operating process, application of sample speed slowly, be unfavorable for the mass-producing cellular segregation, particularly the tyro be difficult to shortcoming such as grasps, greatly influenced follow-up study and applied work.Tu Chu performance is the most: when isolated cell, need slowly to add sample separation, to prevent the destruction of cellular segregation liquid liquid level.If the parting liquid liquid level is destroyed, can not carry out cellular segregation (because of cell suspension mixes with parting liquid).In order to guarantee the intact of liquid level, the application of sample person need be through the training of certain hour, and needs patient operation, could obtain good effect, has restricted seriously that mass cell is isolating to carry out.
Summary of the invention
It is higher to the objective of the invention is a kind of separation efficiency, can be used for mass cell isolated cells parting liquid.
Cellular segregation liquid provided by the present invention is to add the reagent that obtains behind the tensio-active agent in conventional cellular segregation liquid.
Described conventional cellular segregation liquid can be any one cellular segregation liquid commercially available or that prepare according to a conventional method, as sucrose solution, lymphocyte separation medium, Ficoll, Percoll or Metrizamide etc.
The selection of the tensio-active agent that is added is diversified, gets final product so long as have the related reagent of following physicochemical property: density is less than 1, and water insoluble and pair cell is without any detrimentally affect.Dynamical cure effect by itself and cellular segregation liquid guarantees the stability of parting liquid liquid level between the two, makes the cell can be effectively separated.Specifically, the available tensio-active agent is one or more or performance other composition close or similar with character and they in paraffin oil, triglyceride level, phosphatide, the sheath ester.
Described tensio-active agent is 0.1-10 with conventional cellular segregation liquid blended volume ratio: 1.
In addition, described tensio-active agent and relevant composition also can be used for the separation of animal and plant cell dispersion, the many aspects such as preparation of density gradient centrifugation liquid.
Above-mentioned special-purpose cellular segregation liquid also can be used for preparing gradient separations liquid, and method is: add the less relatively conventional cellular segregation liquid of density in above-mentioned dedicated separation liquid, of short duration centrifugal or static parking greater than after 1 minute just can be carried out cellular segregation; Can also add the littler conventional cellular segregation liquid of density on this basis again, till reaching requirement, to be used for various objectives cell, DNA or proteic separation.
Above-mentioned cellular segregation liquid need be kept under 4 ℃ of conditions, preserve half a year after, still can obtain separating effect preferably, the cell rate of recovery can reach 75-85%, cell motility rate＞95%, separation purity＞95%.In long period transportation or preservation process, can form the running balance interface, therefore need before use parting liquid is mixed, so that rebuild stable interface after centrifugal.
Second purpose of the present invention provides a kind of method of isolated cell.
The method of isolated cell provided by the present invention may further comprise the steps:
1) getting volume ratio is cell sample 0.1-10 to be separated: 1 special-purpose cellular segregation liquid, and centrifugal;
2) cell sample to be separated is directly added step 1) in the special-purpose cellular segregation liquid of centrifugal treating, centrifugal:
3) individual cells in the collection cloud and mist layer, washing obtains pure cell.
In above-mentioned cell isolation method, the centrifugal condition in the step 1) is: 100-4000g 10 seconds-10 minutes, is preferably: 1000g, 3 minutes; Step 2) centrifugal condition in is: 200-10000g 10-30 minute, is preferably: 1000g, 20 minutes.
The invention provides a kind of method and special-purpose cellular segregation liquid of isolated cell.In 3 day time, finished nearly 400 monocytic lock out operation of individuality with cellular segregation liquid of the present invention, obtained satisfied experiment effect, prove that this cellular segregation liquid can shorten application of sample and cellular segregation time greatly, easy working specification, be suitable for large-scale cellular segregation, be specially adapted to not have the empirical scientific worker of relating operation to use, effect stability, reliable can obtain highly purified purpose cell; In addition, can utilize multichannel pipettor to carry out application of sample for a plurality of samples, and the vigor of isolated cell is not affected.Researchs such as separated cell can be directly used in genome extraction, karyotyping, cell cultures, the cultivation of going down to posterity, cell transgenosis, cell induction differentiation, cell therapy and breeding for disease resistance also can be used for the purposes such as separation of the preparation of density gradient separation liquid, different mass molecule or compound simultaneously.This cell isolation method is simple, and is workable.The present invention has higher actual application value in cytobiology, functional genomics and cell engineering field, and market outlook are wide.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is for to carry out isolating synoptic diagram with cellular segregation liquid of the present invention to mononuclearcell
Fig. 2 is by isolating chicken mononuclearcell being carried out Jim Sa (Giemsa) dyeing and CD14 fluorescent-labeled antibody detected result
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The preparation of embodiment 1, cellular segregation liquid
The interpolation concentration of volume percent is 50% paraffin oil in Ficoll, obtains cellular segregation liquid.
The preparation of embodiment 2, cellular segregation liquid
Earlier making Percoll reach physiological osmotic pressure with ordinary method, is 80% triglyceride level to wherein adding concentration of volume percent then, obtains cellular segregation liquid.If needed, centrifugal after, the Percoll working fluid of other concentration can be successively adds by normal speed.
The preparation of embodiment 3, cellular segregation liquid
Adding concentration of volume percent in Metrizamide is 500% phosphatide, obtains cellular segregation liquid.
The vitro culture and the observation of embodiment 4, the monocytic separation of chicken and isolated cell
One, the monocytic separation of chicken
Get 2 times of cellular segregation liquid to sample to be separated (chicken peripheral blood) volume, the embodiment that mixes 1 preparation in centrifuge tube, behind centrifugal 3 minutes of the 1000g, directly sample is added, need not consider liquid level destruction (because of stabilizing layer between two-phase), 1000g is centrifugal 20 minutes then, sepn process separates mononuclearcell as shown in Figure 1 in the cloud and mist layer, through both obtaining pure cell after the washing.In the sepn process, the interface is clear, and recovery speed is fast, and the rate of recovery of mononuclearcell can reach 75-85%, proves with cellular segregation liquid of the present invention to obtain cellular segregation effect preferably.
Two, the vitro culture of isolated cell and observation
The isolating chicken mononuclearcell of step 1 is cultivated with the conventional adherent method of differential, carried out Jim Sa (Giemsa) dyeing and CD14 fluorescent-labeled antibody (U.S. NeoMarkers company) detected at the 7th day that cultivates, ((a) is the result of cultivation monocyte Giemsa staining after 7 days to detected result, and wherein a-2 is the amplification of picture a-1 square frame part as shown in Figure 2; (b) be to cultivate the indirect immunostaining result of monocytic CD14 after 7 days; (c) be the indirect immunostaining of CD14 positive control.B-1, c-1 are being seen cells under the microscope bright field, and b-2, c-2 are the result that corresponding cell is observed under fluorescent microscope.Scale is 30 μ m), detected result shows that the monocytic purity that is obtained can reach 100%.
Embodiment 5, person monocytic cell's separation
Get 0.1 times of special-purpose cellular segregation liquid to sample to be separated (human peripheral) volume, the embodiment that mixes 2 preparations in centrifuge tube, behind centrifugal 6 minutes of the 200g, slowly sample is added, need not consider liquid level destruction (because of stabilizing layer between two-phase), 2000g is centrifugal 20 minutes then, sepn process separates mononuclearcell as shown in Figure 1 in the cloud and mist layer, through both obtaining pure cell after the washing.In the sepn process, the interface is clear, and recovery speed is fast, and the rate of recovery of mononuclearcell can reach 80-90%, proves with special-purpose cellular segregation liquid of the present invention to obtain cellular segregation effect preferably.
Embodiment 6, the monocytic separation of ox
Get 10 times of special-purpose cellular segregation liquid to sample to be separated (ox peripheral blood) volume, the embodiment that mixes 3 preparations in centrifuge tube, behind centrifugal 3 minutes of the 1000g, fast sample is added, need not consider liquid level destruction (because of stabilizing layer between two-phase), 5000g is centrifugal 10 minutes then, sepn process separates mononuclearcell as shown in Figure 1 in the cloud and mist layer, through both obtaining pure cell after the washing.In the sepn process, the interface is clear, and recovery speed is fast, and the rate of recovery of mononuclearcell can reach 75-85%, proves with special-purpose cellular segregation liquid of the present invention to obtain cellular segregation effect preferably.
Claims (9)
1, a kind of special-purpose cellular segregation liquid is to add the reagent that obtains behind the tensio-active agent in conventional cellular segregation liquid.
2, special-purpose cellular segregation liquid according to claim 1 is characterized in that: described conventional cellular segregation liquid is any one commercially available cellular segregation liquid.
3, special-purpose cellular segregation liquid according to claim 2 is characterized in that: described conventional cellular segregation liquid is sucrose solution, lymphocyte separation medium, Ficoll, Percoll or Metrizamide.
4, according to the described special-purpose cellular segregation liquid of claim 1, it is characterized in that: the density of described tensio-active agent is less than 1, and is water insoluble.
5, special-purpose cellular segregation liquid according to claim 4 is characterized in that: described tensio-active agent is one or more in paraffin oil, triglyceride level, phosphatide or the sheath ester.
6, according to each described special-purpose cellular segregation liquid of claim 1-5, it is characterized in that: described tensio-active agent is 0.1-10 with conventional cellular segregation liquid blended volume ratio: 1.
7, a kind of method of isolated cell may further comprise the steps:
1) get volume and be the cell sample 0.1-10 to be separated described special-purpose cellular segregation liquid of claim 1 doubly, centrifugal;
2) cell sample to be separated is directly added step 1) in the special-purpose cellular segregation liquid of centrifugal treating, centrifugal;
3) individual cells in the collection cloud and mist layer, washing obtains pure cell.
8, the method for isolated cell according to claim 7 is characterized in that: the centrifugal condition is in the described step 1): 100-4000g, 10 seconds-10 minutes; Step 2) centrifugal condition in is: 200-10000g, 10-30 minute.
9, the method for isolated cell according to claim 8 is characterized in that: the centrifugal condition is in the described step 1): 1000g, 3 minutes; Step 2) centrifugal condition in is: 1000g, 20 minutes.
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| CNB2005101303267A CN100393871C (en) | 2005-12-09 | 2005-12-09 | Method for separating cell and special separating liquid for cell |
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| CN100393871C CN100393871C (en) | 2008-06-11 |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103992984A (en) * | 2014-05-21 | 2014-08-20 | 中国水产科学研究院淡水渔业研究中心 | Method for treating whole blood used for fish chromosome technique based on whole blood culture method |
| US9241959B2 (en) | 2013-03-13 | 2016-01-26 | Mingqi TANG | Kits and methods for processing stem cells from bone marrow or umbilical cord blood |
| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| CN109321524A (en) * | 2018-11-05 | 2019-02-12 | 中国医学科学院血液病医院（血液学研究所） | The cellifugal method of one kind point |
| CN110923189A (en) * | 2019-12-07 | 2020-03-27 | 顾霆 | Preparation method of liquid-state separated phases of various solvents |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5254342A (en) * | 1991-09-30 | 1993-10-19 | University Of Southern California | Compositions and methods for enhanced transepithelial and transendothelial transport or active agents |
| CN1294254C (en) * | 2005-01-21 | 2007-01-10 | 周建业 | Method for removing cells for heterogeneous cardiovascular transplant |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9241959B2 (en) | 2013-03-13 | 2016-01-26 | Mingqi TANG | Kits and methods for processing stem cells from bone marrow or umbilical cord blood |
| US10842820B2 (en) | 2013-03-13 | 2020-11-24 | Mingqi Tang | Kits and methods for processing stem cells from bone marrow or umbilical cord blood |
| CN103992984A (en) * | 2014-05-21 | 2014-08-20 | 中国水产科学研究院淡水渔业研究中心 | Method for treating whole blood used for fish chromosome technique based on whole blood culture method |
| CN106047795A (en) * | 2016-06-22 | 2016-10-26 | 杭州海世嘉生物科技有限公司 | Sample density separation liquid and preparation method thereof |
| CN106047795B (en) * | 2016-06-22 | 2019-06-14 | 杭州海世嘉生物科技有限公司 | A kind of sample rate separating liquid and preparation method thereof |
| CN106754714A (en) * | 2016-11-11 | 2017-05-31 | 北正赛欧（北京）生物科技有限公司 | The method that cord blood sample dilution, kit and treatment Cord blood obtain stem cell |
| CN106754714B (en) * | 2016-11-11 | 2020-05-19 | 北正赛欧（北京）生物科技有限公司 | Umbilical cord blood sample diluent, kit and method for processing umbilical cord blood to obtain stem cells |
| CN109321524A (en) * | 2018-11-05 | 2019-02-12 | 中国医学科学院血液病医院（血液学研究所） | The cellifugal method of one kind point |
| CN110923189A (en) * | 2019-12-07 | 2020-03-27 | 顾霆 | Preparation method of liquid-state separated phases of various solvents |
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| CN100393871C (en) | 2008-06-11 |
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