CN101469321A - Method for rapidly filtering and screening medulla ossium substrate stem cell - Google Patents
Method for rapidly filtering and screening medulla ossium substrate stem cell Download PDFInfo
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- CN101469321A CN101469321A CNA2007101734074A CN200710173407A CN101469321A CN 101469321 A CN101469321 A CN 101469321A CN A2007101734074 A CNA2007101734074 A CN A2007101734074A CN 200710173407 A CN200710173407 A CN 200710173407A CN 101469321 A CN101469321 A CN 101469321A
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Abstract
The invention relates to a filtering method, in particular to a method for quickly filtering and screening bone marrow stromal cells. The method adopts the technical proposal that the method for quickly filtering and screening the bone marrow stromal cells comprises the following steps: firstly, performing anticoagulant treatment on bone marrow blood to be used; secondly, filtering the bone marrow blood after anticoagulant treatment through a filtration recombiner; and thirdly, collecting stem cell-frame composite materials. The method has the advantages that the method quickly recombines the cells and a vector (namely a filter screen material) when filtering and screening the bone marrow stromal cells simultaneously, so as to integrate the two procedures of cell screening and cell-vector combination into a whole. The filtering method has short time consumption, does not need in vitro culture, can be simultaneously performed in an operation, and is more favorable for clinic use because the volume of the screened stromal cells is smaller than that of cell suspension finally obtained by density gradient centrifugation technology.
Description
Technical field
The present invention relates to a kind of filter method, specifically about a kind of method for rapidly filtering and screening medulla ossium substrate stem cell.
Background technology
The reparation that bone is damaged often needs bone grafting or bone alternate material to fill, and is the support carrier that is compounded with stem cell at the research focus of this field organizational project at present.Separate bone marrow stroma stem cell by adherent culture or density gradient centrifugation, carry out the means of amplification in vitro again, though can be compounded with the support carrier of cell in in-vitro separation bone marrow stroma stem cell, preparation, but still be in the laboratory study stage, and implant into body, still there are the problems in technical and the ethics, and culture cycle is long, easily pollute, the cost height influences Clinical Application and promotes.
The cell screening technology mainly contains at present: 1) flow cytometer partition method, 2) magnetic activated cell seperation 3) adherent sieve method, 4) density gradient centrifugation.1) the flow cytometer partition method is to use special fluorescence antibody to combine with the antigen sign of cell surface, uses flow cytometer then it is carried out sorting.2) but magnetic activated cell seperation is to utilize both conjugated proteins of little magnetic bead, again can be by the characteristics of attraction, through necessarily handling, with antibody sandwich on magnetic bead.By combining, form Ag-Ab magnetic bead immunocomplex with the cell surface specific antigens.When this mixture passes through separator column, under magneticaction, promptly separate, thereby make screening cell and other separating substances with mother liquor, reach the purpose of screening.3) adherent sieve method is the most frequently used bone marrow stroma stem cell screening method in present laboratory.By marrow blood is cultivated in culture dish, bone marrow stroma stem cell is adherent behind the 24-48h, bone marrow stroma stem cell can be separated with other not adherent medullary cell whereby.4) density gradient centrifugation is the method for the concentrating and separating cell used always, utilize the difference of different cellular constituent density gradients, concentrate in different levels by the centrifugal cell that can make, be used in combination the parting liquid of different specific weight, the cell of different specific weight is carried out layering separate.Above prior art has following shortcoming:
1) though the separation of flow cytometer partition method isolated cell is rapid, the purity height, the expense costliness is difficult to separate to the too low trace cell of content, can not handle a large amount of cell samples, and it is also very difficult to obtain aseptic cell suspension.2) activity influence of magnetic activated cell seperation pair cell is bigger, and the rate of recovery only has 60%-75%, may cause the cell complete deactivation, and same operation is complicated, costs an arm and a leg.Most critical be at present the screening antibody of bone marrow stroma stem cell surface specific still to be lacked common recognition, so use this method screening bone marrow stroma stem cell still to be in conceptual phase.3) though the adherent sieve method the most frequently used bone marrow stroma stem cell screening method that is present laboratory.But need the time longer, relate to cells in vitro and cultivate, clinical use acquires a certain degree of difficulty.4) density gradient centrifugation is the method for concentrating and separating cell commonly used, but often need to use separating medium (as Ficoll or Hypaque liquid etc.), the separating medium that uses for the separation of the stroma stem cell of large vol marrow blood is also bigger, and need the flush away separating medium at last, the special equipment of aseptic needs that keeps entire operation, the expense costliness.
Summary of the invention
The objective of the invention is to overcome the defective of prior art, a kind of method for rapidly filtering and screening medulla ossium substrate stem cell is provided.
The objective of the invention is to realize: in " the application of stem cells technology is carried out bone reparation and Study on Regeneration " by following technological method, find because the adhesion characteristics of human bone marrow stroma stem cell, (filter screen aperture 150-200 μ m when the marrow blood after the 250ml anti-freezing is filtered, thick about 300 μ m), there is 1/6～1/3 bone marrow stroma stem cell to be enriched on the filter screen approximately by selectivity.We cultivate after with the cell wash-out on the filter screen, can be in the cell colony (see figure 1)s of external formation alkali phosphorus enzyme (ALP) stained positive, and pointing out these cells is the bone marrow stroma stem cells that can be divided into the skeletonization precursor cell.With filter with after filter screen directly cut cultivation, can find that these cells can breed on filter screen, form cell colony (seeing Fig. 2,3).Filter screen is conventional blood transfusion filter, and the aperture is bigger, is about 150～200 μ m, and the thick about 300 μ m of filter screen are myeloid element and stroma stem cell diameter tens of times.Hematopoiesis such as red corpuscle a large amount of in the marrow are cell, are easy to deformation, are difficult for adhering to, and behind the adding antithrombotics, thrombocyte also is difficult for assembling or sticking, and filters unaffected.And bone marrow stroma stem cell is owing to exist stronger adhesion characteristics, and after filtering in short several minutes, some bone marrow stroma stem cells can stick on the filter screen.On porous tricalcium phosphate (implantable material), we also observe similar result equally, and bone marrow stroma stem cell can stick to the inwall (seeing Fig. 4,5) of material fast.Thus by experiment, but invented the rapid screening of carrying out bone marrow stroma stem cell with the material of Hui Zhi as screen material, invented parallel circulating type cell selective filter recombiner, and this filter recombiner of invention application filters at many levels or the means of circulating filtration farthest improve the screening efficiency of bone marrow stroma stem cell, thereby formed the method for original marrow blood being carried out rapidly filtering and screening medulla ossium substrate stem cell.
Technical scheme of the present invention is as follows:
A kind of method for rapidly filtering and screening medulla ossium substrate stem cell may further comprise the steps: (1) is carried out anti-freezing with stand-by marrow blood and is handled; (2) marrow blood after anti-freezing is handled filters by filter recombiner; (3) collect stem cell-prop composite; Wherein said filter recombiner is provided with filter screen, and this filter screen is made by the multiporous biological timbering material.Described filter recombiner comprises injection pump, first pipeline, second pipeline, the first right pipe connecting, the first left pipe connecting, the second right pipe connecting, the second left pipe connecting, the first unidirectional flap valve valve, the second unidirectional flap valve valve, opening-closing valve, venting hole, peristaltic pump wherein also comprises filter cartridge, and filter cartridge is provided with filter screen, removable cover, screen material is selected from bata-tricalcium phosphate, natural type inorganic bone NNB, decalcified bone matrix DBM, forging bone, poly(lactic acid)-polyglycolic acid (PLGA), collagen, freeze-drying spongy bone, filter screen mesh diameter are the 300-700 micron, and the filter screen mesh diameter is preferably 500 microns, first pipeline is provided with the second right pipe connecting, the second right pipe connecting is provided with internal thread, and second pipeline is provided with the second left pipe connecting, and the second left pipe connecting is provided with outside screw, filter cartridge one end is provided with internal thread, the filter cartridge the other end is provided with outside screw, and the filter cartridge internal thread is connected with the second left pipe connecting outside screw, and the filter cartridge outside screw is connected with the second right pipe connecting internal thread.It is identical with the pipe number that second pipeline is provided with the second left pipe connecting that first pipeline is provided with the second right pipe connecting.The pipe number of the second right pipe connecting and the second left pipe connecting is two, three, four, five, and filter cartridge is two boxes, three boxes, four boxes, five boxes.The pipe number of the second right pipe connecting and the second left pipe connecting is preferably two, and filter cartridge is preferably two boxes.First pipeline is provided with the first right pipe connecting, the first right pipe connecting is provided with internal thread, second pipeline is provided with the first left pipe connecting, the first left pipe connecting is provided with outside screw, peristaltic pump one end is provided with internal thread, the peristaltic pump the other end is provided with outside screw, and the peristaltic pump internal thread is connected with the first left pipe connecting outside screw, and the peristaltic pump outside screw is connected with the first right pipe connecting internal thread.
The disclosed a kind of method for rapidly filtering and screening medulla ossium substrate stem cell of the present invention, its advantage shows: 1) method for rapidly filtering and screening medulla ossium substrate stem cell, it is the adhesion characteristics that utilizes stroma stem cell itself, material gives over to carrying out the screening of cell the selectivity storage of stem cell during by filtration, do not add other material, the interference of pair cell is little.2) when bone marrow stroma stem cell carries out filtering screening, carried out the compound fast of cell and carrier (being screen material), cell screening and cell-carrier compound two procedures is united two into one.3) filtration method weak point consuming time need not vitro culture, can art in the same period carry out, and the stroma stem cell volume ratio of screening is much smaller in the last cell suspension volume that obtains of density gradient centrifugation, is more conducive to clinical use.
Description of drawings
3 holes are the filter screen cell elutriant behind the filtration marrow blood on Fig. 1,100 μ l/ wares; 3 holes are original marrow blood 100 μ l/ wares down, through external evoked cultivation 10 days, show alkali phosphorus enzyme (ALP) stained positive cell colony.
The filter screen that Fig. 2 will filter after using is directly cut, and cultivates 8 days, and a large amount of alkali phosphorus enzymes (ALP) positive cell colony growth (40x) is arranged.
On Fig. 3 filter screen the positive bone marrow stroma stem cell of alkali phosphorus enzyme (ALP) adhere to, the wire drawing growth (amplification of Fig. 2,200x).
Fig. 4 marrow blood is after porous tricalcium phosphate filters, and the part bone marrow stroma stem cell sticks to the surface and the inwall (scanning electron microscope 500x) of material.
Fig. 5 marrow blood is after porous tricalcium phosphate filters, and bone marrow stroma stem cell sticks to (scanning electron microscope 5000x) on the material walls.
Fig. 6 is a filter recombiner structural representation of the present invention.
Fig. 7 is another structural representation of filter recombiner of the present invention.
The marrow blood of Fig. 8 after with the anti-freezing filter screen that the multiporous biological timbering material is made of flowing through filters.
Fig. 9 utilizes the distinctive adhesion characteristics of bone marrow stroma stem cell, makes bone marrow stroma stem cell selectivity storage on filter screen stay enrichment, and remaining medullary cell is then filtered.
Figure 10 bed filtration helps the height screening of bone marrow stroma stem cell, and has also carried out the compound of cell-scaffold cell screening the time, and two procedures unites two into one, preparation stem cell-prop composite.
Embodiment
Below in conjunction with embodiment the present invention is further described.
See also Fig. 6.
The first unidirectional flap valve valve 1 is arranged, opening-closing valve 2, the second unidirectional flap valve valves 3 among Fig. 6, venting hole 4, peristaltic pump 5, filter cartridge 6, injection pump 8, the first right pipe connecting 11, the first left pipe connectings 12, the second right pipe connecting 13, second left pipe connecting 14, the first pipelines 31, the second pipelines 32, filter screen 61, removable cover 62.
Method of work of the present invention is as follows: open opening-closing valve 2,250ml marrow blood after the anti-freezing processing is entered first pipeline 31 by injection pump 8, close opening-closing valve 2, open peristaltic pump 5 switches, marrow blood flows into the second right pipe connecting 13 respectively, entering filter cartridge 6 filters, flow to second pipeline 32 then, under peristaltic pump 5 wrigglings and filter screen 61 effects, enrichment is stayed in the selectivity storage on filter screen of the bone marrow stroma stem cell in the 250ml marrow blood, filters remaining medullary cell, close peristaltic pump 5 switches, open removable cover 62, take out enrichment bone marrow stroma stem cell filter screen 61, it is standby that collection obtains stem cell-prop composite.
Filter recombiner of the present invention, comprise injection pump 8, first pipeline 31, second pipeline 32, the first right pipe connecting 11, the first left pipe connecting 12, the second right pipe connecting 13, the second left pipe connecting 14, the first unidirectional flap valve valve 1, the second unidirectional flap valve valve 3, opening-closing valve 2, venting hole 4, peristaltic pump 5, also comprise filter cartridge 6, filter cartridge 6 is provided with filter screen 61, removable cover 62, described injection pump 8 connections first pipeline 31, the first pipelines 31 are provided with the first right pipe connecting 11 and second right pipe connecting 13, the first right pipe connectings 11 and the second right pipe connecting 13 and are respectively equipped with internal thread, second pipeline 32 is provided with the first left pipe connecting 12, second left pipe connecting 14, the first left pipe connectings 12 and the second left pipe connecting 14 are respectively equipped with outside screw, and peristaltic pump 5 one ends are provided with internal thread, peristaltic pump 5 the other ends are provided with outside screw, peristaltic pump 5 internal threads are connected with first left pipe connecting 12 outside screws, and peristaltic pump 5 outside screws are connected with first right pipe connecting 11 internal threads, and filter cartridge 6 one ends are provided with internal thread, filter cartridge 6 the other ends are provided with outside screw, filter cartridge 6 internal threads are connected with second left pipe connecting 14 outside screws, and filter cartridge 6 outside screws are connected with second right pipe connecting 13 internal threads, and second pipeline 32 is provided with opening-closing valve 2, venting hole 4, the first right pipe connecting 11 is provided with first unidirectional flap valve valve 1, the first pipeline 31 and is provided with the second unidirectional flap valve valve 3.
It should be noted that the pipe number of second right pipe connecting 13, the second left pipe connectings 14 is two in the present embodiment, filter cartridge 6 is two boxes, and filter screen 61 materials are selected from bata-tricalcium phosphate, and filter screen 61 mesh diameters are 500 microns.
See also Fig. 7.
The first unidirectional flap valve valve 1 is arranged, opening-closing valve 2, the second unidirectional flap valve valves 3 among Fig. 7, venting hole 4, peristaltic pump 5, filter cartridge 6, injection pump 8, the first right pipe connecting 11, the first left pipe connectings 12, the second right pipe connecting 13, second left pipe connecting 14, the first pipelines 31, the second pipelines 32, filter screen 61, removable cover 62.
Method of work of the present invention is as follows: open opening-closing valve 2,300ml marrow blood after the anti-freezing processing is entered first pipeline 31 by injection pump 8, close opening-closing valve 2, open peristaltic pump 5 switches, marrow blood flows into the second right pipe connecting 13 respectively, entering filter cartridge 6 filters, flow to second pipeline 32 then, under peristaltic pump 5 wrigglings and filter screen 61 effects, enrichment is stayed in the selectivity storage on filter screen of the bone marrow stroma stem cell in the 300ml marrow blood, filters remaining medullary cell, close peristaltic pump 5 switches, open removable cover 62, take out enrichment bone marrow stroma stem cell filter screen 61, it is standby that collection obtains stem cell-prop composite.
Filter recombiner of the present invention, comprise injection pump 8, first pipeline 31, second pipeline 32, the first right pipe connecting 11, the first left pipe connecting 12, the second right pipe connecting 13, the second left pipe connecting 14, the first unidirectional flap valve valve 1, the second unidirectional flap valve valve 3, opening-closing valve 2, venting hole 4, peristaltic pump 5, also comprise filter cartridge 6, filter cartridge 6 is provided with filter screen 61, removable cover 62, described injection pump 8 connections first pipeline 31, the first pipelines 31 are provided with the first right pipe connecting 11 and second right pipe connecting 13, the first right pipe connectings 11 and the second right pipe connecting 13 and are respectively equipped with internal thread, second pipeline 32 is provided with the first left pipe connecting 12, second left pipe connecting 14, the first left pipe connectings 12 and the second left pipe connecting 14 are respectively equipped with outside screw, and peristaltic pump 5 one ends are provided with internal thread, peristaltic pump 5 the other ends are provided with outside screw, peristaltic pump 5 internal threads are connected with first left pipe connecting 12 outside screws, and peristaltic pump 5 outside screws are connected with first right pipe connecting 11 internal threads, and filter cartridge 6 one ends are provided with internal thread, filter cartridge 6 the other ends are provided with outside screw, filter cartridge 6 internal threads are connected with second left pipe connecting 14 outside screws, and filter cartridge 6 outside screws are connected with second right pipe connecting 13 internal threads, and second pipeline 32 is provided with opening-closing valve 2, venting hole 4, the first right pipe connecting 11 is provided with first unidirectional flap valve valve 1, the first pipeline 31 and is provided with the second unidirectional flap valve valve 3.
It should be noted that the pipe number of second right pipe connecting 13, the second left pipe connectings 14 is five in the present embodiment, filter cartridge 6 is five boxes, and filter screen 61 materials are selected from collagen, and filter screen 61 mesh diameters are 400 microns.
See also Fig. 8,9,10.
Filter recombiner 60 is arranged among Fig. 8, filter screen 61, bone marrow stroma stem cell 71, other medullary cell 72,
Filter recombiner 60 is arranged among Fig. 9, filter screen 61, bone marrow stroma stem cell 71, other medullary cell 72,
Filter recombiner 60 is arranged, filter screen 61, bone marrow stroma stem cell 71, other medullary cell 72, stem cell-prop composite 7 among Figure 10.
Extract the 250ml marrow blood in the operation, carry out anti-freezing and handle.Select for use tricalcium phosphate as filter screen 61 materials, the filter opening aperture can make medullary cell pass through smoothly.Marrow blood after the anti-freezing is injected filter recombiner 60 filter (Fig. 8).Utilize the distinctive adhesion characteristics of bone marrow stroma stem cell, make bone marrow stroma stem cell 71 selectivity storage on filter screen stay enrichment, filter remaining medullary cell 72 (Fig. 9).The height screening that helps bone marrow stroma stem cell is filtered on many planes.Compound this two procedures with cell screening and cell-scaffold unites two into one simultaneously, preparation stem cell-prop composite 7 (Figure 10).
Claims (10)
1, a kind of method for rapidly filtering and screening medulla ossium substrate stem cell may further comprise the steps:
(1) stand-by marrow blood being carried out anti-freezing handles;
(2) marrow blood after anti-freezing is handled filters by filter recombiner;
(3) collect stem cell-prop composite;
Wherein said filter recombiner is provided with filter screen (61), and this filter screen (61) is made by the multiporous biological timbering material.
2, method according to claim 1, described filter recombiner comprises injection pump (8), first pipeline (31), second pipeline (32), the first right pipe connecting (11), the first left pipe connecting (12), the second right pipe connecting (13), the second left pipe connecting (14), the first unidirectional flap valve valve (1), the second unidirectional flap valve valve (3), opening-closing valve (2), venting hole (4), peristaltic pump (5), it is characterized in that: also comprise filter cartridge (6), filter cartridge (6) is provided with filter screen (61), removable cover (62).
3, method according to claim 1 is characterized in that: filter screen (61) biologic bracket material is selected from bata-tricalcium phosphate, natural type inorganic bone NNB, decalcified bone matrix DBM, forging bone, poly(lactic acid)-polyglycolic acid (PLGA), collagen, freeze-drying spongy bone.
4, method according to claim 1 is characterized in that: filter screen (61) mesh diameter is the 300-700 micron.
5, method according to claim 4 is characterized in that: filter screen (61) mesh diameter is 500 microns.
6, method according to claim 2, it is characterized in that: first pipeline (31) is provided with the second right pipe connecting (13), the second right pipe connecting (13) is provided with internal thread, second pipeline (32) is provided with the second left pipe connecting (14), the second left pipe connecting (14) is provided with outside screw, filter cartridge (6) one ends are provided with internal thread, filter cartridge (6) the other end is provided with outside screw, filter cartridge (6) internal thread is connected with second left pipe connecting (14) outside screw, and filter cartridge (6) outside screw is connected with second right pipe connecting (13) internal thread.
7, method according to claim 6 is characterized in that: it is identical with the pipe number that second pipeline (32) is provided with the second left pipe connecting (14) that first pipeline (31) is provided with the second right pipe connecting (13).
8, method according to claim 7 is characterized in that: the second right pipe connecting (13), the pipe number of the second left pipe connecting (14) are two, three, four, five, and filter cartridge (6) is two boxes, three boxes, four boxes, five boxes.
9, method according to claim 7 is characterized in that: the second right pipe connecting (13), and the pipe number of the second left pipe connecting (14) is two, filter cartridge (6) is two boxes.
10, method according to claim 2, it is characterized in that: first pipeline (31) is provided with the first right pipe connecting (11), the first right pipe connecting (11) is provided with internal thread, second pipeline (32) is provided with the first left pipe connecting (12), the first left pipe connecting (12) is provided with outside screw, peristaltic pump (5) one ends are provided with internal thread, peristaltic pump (5) the other end is provided with outside screw, peristaltic pump (5) internal thread is connected with first left pipe connecting (12) outside screw, and peristaltic pump (5) outside screw is connected with first right pipe connecting (11) internal thread.
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Cited By (6)
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| CN101851606A (en) * | 2010-04-07 | 2010-10-06 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
| CN102260628A (en) * | 2010-05-31 | 2011-11-30 | 上海康德莱企业发展集团医疗器械有限公司 | Device for filtering, screening, enriching and quick-compositing stem cells |
| CN104587523A (en) * | 2014-06-10 | 2015-05-06 | 上海交通大学医学院附属第九人民医院 | Enriched bone marrow mesenchymal stem cell loaded self-curing calcium phosphate cement, and preparation method thereof |
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| CN107312707A (en) * | 2017-08-17 | 2017-11-03 | 卢建熙 | The harvester and acquisition method of mescenchymal stem cell |
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| GB0404656D0 (en) * | 2004-03-02 | 2004-04-07 | Univ Manchester | Treatment of spinal conditions |
| CN100438928C (en) * | 2005-09-02 | 2008-12-03 | 南方医院 | Injection type tissue engineering bone renovation material and construct method thereof |
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| CN101851606A (en) * | 2010-04-07 | 2010-10-06 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
| CN101851606B (en) * | 2010-04-07 | 2012-05-30 | 魏红江 | Cumulus oophorus-oocyte compound cutting filter method |
| CN102260628A (en) * | 2010-05-31 | 2011-11-30 | 上海康德莱企业发展集团医疗器械有限公司 | Device for filtering, screening, enriching and quick-compositing stem cells |
| CN104587523A (en) * | 2014-06-10 | 2015-05-06 | 上海交通大学医学院附属第九人民医院 | Enriched bone marrow mesenchymal stem cell loaded self-curing calcium phosphate cement, and preparation method thereof |
| CN106635769A (en) * | 2016-11-07 | 2017-05-10 | 深圳市达科为生物工程有限公司 | Device and method for separating cells |
| CN106635769B (en) * | 2016-11-07 | 2019-06-28 | 深圳市达科为生物工程有限公司 | Cell separation apparatus and cell isolation method |
| CN107312707A (en) * | 2017-08-17 | 2017-11-03 | 卢建熙 | The harvester and acquisition method of mescenchymal stem cell |
| CN107312707B (en) * | 2017-08-17 | 2020-05-19 | 卢建熙 | Mesenchymal stem cell collecting device and method |
| CN108785779A (en) * | 2018-09-12 | 2018-11-13 | 上海交通大学医学院附属第九人民医院 | A kind of stem cell in vitro enrichment isolation system and method |
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| CN101469321B (en) | 2012-07-25 |
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Application publication date: 20090701 Assignee: SHANGHAI KINDLY MEDICAL INSTRUMENTS Co.,Ltd. Assignor: SHANGHAI NINTH PEOPLE'S HOSPITAL SHANGHAI JIAOTONG University SCHOOL OF MEDICINE Contract record no.: X2022310000049 Denomination of invention: A Rapid Filtering Method for Screening Bone Marrow Stromal Stem Cells Granted publication date: 20120725 License type: Exclusive License Record date: 20230510 |