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CN108410798A - Cell separation microballon eluent and preparation method - Google Patents

Cell separation microballon eluent and preparation method Download PDF

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Publication number
CN108410798A
CN108410798A CN201810260416.5A CN201810260416A CN108410798A CN 108410798 A CN108410798 A CN 108410798A CN 201810260416 A CN201810260416 A CN 201810260416A CN 108410798 A CN108410798 A CN 108410798A
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China
Prior art keywords
cell
microballon
solution
eluent
6mmol
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CN201810260416.5A
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Chinese (zh)
Inventor
叶永清
黄能平
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Fujian Stemery Tech Co Ltd
Fujian Stemery Technology Co Ltd
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Fujian Stemery Tech Co Ltd
Fujian Stemery Technology Co Ltd
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Priority to CN201810260416.5A priority Critical patent/CN108410798A/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06Animal cells or tissues; Human cells or tissues
    • C12N5/0602Vertebrate cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00Methods for the dissociation of cells, e.g. specific use of enzymes

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  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
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  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention discloses cell separation microballon eluents and preparation method, relate generally to cell elution technique field.Include following components per 100ml:800~1200U of IdeS enzymes, 800~1200U of cysteine proteinase, bovine serum albumin BSA 8%~12% and trehalose 8%~12% based on mass-volume concentration, and 4~6mmol of glycine, 4~6mmol of sodium dihydrogen phosphate, 4~6mmol of potassium chloride, 4~6mmol of sodium chloride are 6.5 using 1MHcl adjustment eluent pH values.The present invention can make full use of the dissociation that Ides enzymes and glycine combine antigen or/and antibody, overcome respective disadvantage, microballon and intercellular antigen-antibody is set to combine closely disintegration, cell can be eluted completely from microballon and utmostly keep the existing characteristic of cell, to achieve the purpose that remove microballon under the premise of purifying cells, effect is good, and reaction is fast, is capable of the aim cell of easy economic acquisition high-purity noresidue.

Description

Cell separation microballon eluent and preparation method
Technical field
The present invention relates to cell elution technique fields, specifically cell separation microballon eluent and preparation method.
Background technology
Cell is the important base of scientific research institution and drug research mechanism as the basic constitutional unit of human body and animal Plinth raw material.Almost all of Basic of Biology research and medicament research and development will use cell, and the product quality of cell is direct It is related to the quality of entire scientific experiment and drug research.Cell is present in tissue or blood, accurately studies certain spy Determine cell just to need to isolate and purify out from different types of tissue and blood by specific cells, currently used cell separation Purification process is that the principle combined using antigen-antibody is combined cell with the microballon of labelled antibody, then utilizes different physics Method separates microballon, achievees the purpose that cell purification.After this step, some does not divide microballon and cell From directly use.Some is using complicated oligomeric amino acid competitive binding bead surface antibody, to detach cell.
However, existing cell isolation method, including method of magnetic etc., there is microballon and remove that of high cost, effect is poor asks Topic.It is mainly manifested in:(1) prior art is bad to the microballon removal effect of cell surface, and part microballon is caused not remove, with It cell and enters subsequent experiment or human body, harmful effect is caused to experiment link and result, or even caused to human body irreversible Injury.(2) prior art be used for remove microballon surface bound antibody cost it is higher, such as synthesis amino acid price of living alone as a widow It is very expensive, substantially increase the cost of this link.It above problem generally existing and is not solved always so that the separation of microballon The problem of being detached as cell link, it would be highly desirable to the follow-up of the relevant technologies.
Invention content
The purpose of the present invention is to provide cell separation microballon eluents and preparation method, it can make microballon and cell Between antibody disintegrate, to achieve the purpose that remove microballon under the premise of purifying cells, effect is good, and reaction is fast, can be easy The aim cell of economic acquisition high-purity noresidue.
The present invention to achieve the above object, is achieved through the following technical solutions:
Cell separation microballon eluent, includes following components per 100ml:IdeS 800~1200U of enzyme, cysteine 00~1200U of proteinase 8, bovine serum albumin BSA 8%~12% and trehalose 8%~12% based on mass-volume concentration, and 4~6mmol of glycine, 4~6mmol of sodium dihydrogen phosphate, 4~6mmol of potassium chloride, 4~6mmol of sodium chloride.
As a kind of optimal proportion, it includes following components that the above-mentioned cell per 100ml, which is detached with microballon eluent,:IdeS Enzyme 1000U, cysteine proteinase 1000U, bovine serum albumin BSA 10% and trehalose 10% based on mass-volume concentration, with And glycine 5mmol, sodium dihydrogen phosphate 5mmol, potassium chloride 5mmol, sodium chloride 5mmol.
And preferably match by the following method:
(1) sodium dihydrogen phosphate of 6g, the sodium chloride and 3.75g glycine of the potassium chloride 2.92g of 3.73g, with 1L's are weighed Deionized water dissolving, it is 6.5 that sodium hydroxide, which adjusts pH value, obtains elution buffer;
(2) trehalose for weighing 10g is uniformly dissolved with the elution buffer of 100mL, obtains the first solution;
(3) BSA for weighing 10g, the first solution described in 100mL are uniformly dissolved, and obtain the second solution;
(4) the Ides enzymes of 1000U are weighed, the cysteine proteinase of 1000U, the second solution dissolving described in 100mL is It is even, obtain third solution;
(5) the third solution adjusts pH value to 6.5 using 1M Hcl, is filtered to get institute using 0.22um sterile filters Solution is obtained to preserve at -20 °.Above-mentioned cell separation is with the application method of microballon eluent:
(1) the microballon purifies and separates cell for being connected with antibody is utilized so that bead surface is connected with aim cell;
(2) elution buffer is added, and the cell eluent of respective volume, mixing microballon is added;
(3) it is incubated 20min;
(4) it centrifuges, it is aim cell to remove supernatant, precipitation, is used for subsequent experimental.
Preferably, the water used in reaction is deionized water.
The prior art is compared, the beneficial effects of the present invention are:
This product is a kind of colourless transparent solution, and microballon can be made to be combined complete disintegration with intercellular antigen-antibody, from And achieve the purpose that remove microballon under the premise of purifying cells.It, merely can not be complete using Ides enzymes according to experimental result So that cell is detached from from the microballon of combination, may between cell microballon more close space in conjunction with and Ides enzyme molecular weights are existing Completely antibody can not cut to larger related.But the knot of antigen-antibody can be destroyed with the glycine of 0.1M-1M Close, but need to adjust pH value to 2-3 or so, and low PH solution has damaging influence for the cell of separation, cannot keep thin Born of the same parents' primary characteristic, it is that the eluent that composition configures is used for microballon and cell to use using ides enzymes and glycine and other reagents Separation elution, cell can be eluted quickly and keep cell primary characteristic, from pearl to reach in purifying cells completely Under the premise of remove the purpose of microballon, effect is good, and reaction is fast, of low cost, is capable of easy economic acquisition high-purity noresidue Aim cell.
Description of the drawings
Attached drawing 1 is the floating bead microscopy figure before the elution of the embodiment of the present invention 4, and there are many cell, (volume is big for connection in floating bead It is floating bead, is cell with floating bead and small size together).
Attached drawing 2 is the floating bead microscopy figure (be hardly visible in floating bead and be combined with cell) after the elution of the embodiment of the present invention 4.
Attached drawing 3 is the content of cd4 cell in 4 aim cell of the embodiment of the present invention.
Attached drawing 4 is the enlarged drawing of attached drawing 1 of the present invention.
Specific implementation mode
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, people in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Range.
Involved instrument, reagent, material etc., are existing in the prior art unless otherwise noted in following embodiments Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Involved experimental method in following embodiments, inspection Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
Embodiment 1:Cell separation microballon eluent, includes following components per 100ml:IdeS enzymes 1000U, half Guang ammonia Pepsin 1000U, the BSA10% based on mass-volume concentration and trehalose 10% and glycine 5mmol, sodium dihydrogen phosphate 5mmol, potassium chloride 5mmol, sodium chloride 5mmol.
Concrete configuration method is:
(1) sodium dihydrogen phosphate of 6g, the sodium chloride and 3.75g glycine of the potassium chloride 2.92g of 3.73g, with 1L's are weighed Deionized water dissolving, it is 6.5 that sodium hydroxide, which adjusts pH value, obtains elution buffer;
(2) trehalose for weighing 10g is uniformly dissolved with the elution buffer of 100mL, obtains the first solution;
(3) BSA for weighing 10g, the first solution described in 100mL are uniformly dissolved, and obtain the second solution;
(4) the Ides enzymes of 1000U are weighed, the cysteine proteinase of 1000U, the second solution dissolving described in 100mL is It is even, obtain third solution;
(5) the third solution adjusts pH value to 6.5 using 1M Hcl, is filtered to get institute using 0.22um sterile filters Solution is obtained to preserve at -20 °.
Embodiment 2:Cell separation microballon eluent, includes following components per 100ml:IdeS enzymes 800U, half Guang ammonia Pepsin 900U, the BSA8% based on mass-volume concentration and trehalose 9% and glycine 4mmol, sodium dihydrogen phosphate 4.5mmol, potassium chloride 4.2mmol, sodium chloride 5.5mmol.
Embodiment 3:Cell separation microballon eluent, includes following components per 100ml:IdeS enzymes 1100U, half Guang ammonia Pepsin 1200U, the BSA12% based on mass-volume concentration and trehalose 11% and glycine 6mmol, sodium dihydrogen phosphate 5.5mmol, potassium chloride 5.8mmol, sodium chloride 4.5mmol.
Embodiment 4:Using the cell separation microballon eluent of embodiment 1 to being connected with the cd4 cell of cd4 cell microballon It is eluted.
1) the microballon 1mL purifies and separates 1x10 of CD4 antibody is connected with using the surfaces 0.1mg/ml7(single core is thin by a PBMC Born of the same parents) in cd4 cell so that bead surface is connected with cd4 cell;
2) 20mL elution buffers are added, and the cell eluent of 1mL, mixing microballon is added;
3) it is incubated 20min;
4) it centrifuges, it is aim cell to remove supernatant, precipitation;
5) aim cell and same sample PBMC carry out cell count;
6) 1x10 is taken5The aim cell of acquisition and same sample PBMC are added the anti-CD4-FITC antibody of 5 μ g and are protected from light incubation 30min;
7) PBS is washed 2 times, flow cytometer detection.
Testing result is as follows:
1) in terms of microscopic examination result:Floating bead microscopy figure before elution, that there are many cells is (bulky for drift for connection in floating bead Pearl is cell with floating bead and small size together).Microscopy image is shown in attached drawing 1.
Floating bead microscopy figure (be hardly visible in floating bead and be combined with cell) after elution:Microscopy image is shown in attached drawing 2.
2) it is counted by flow cytometer detection combination cell, the elution efficiency of the cell eluent is about 98% (referring to attached drawing 3):
In sample 1,1x1073.35x10 containing cd4 cell in a PBMC6It is a, it is captured by floating bead, after cell elution action, Gained cell quantity is 3.3x106A, wherein the quantity of cd4 cell is 3.17x106It is a.
In sample 2,1x1073.5x10 containing cd4 cell in a PBMC6It is a, it is captured by floating bead, after cell elution action, institute It is 3.43x10 to obtain cell quantity6A, wherein the quantity of cd4 cell is 3.3x106It is a.
In sample 3,1x1073.65x10 containing cd4 cell in a PBMC6It is a, it is captured by floating bead, after cell elution action, Gained cell quantity is 3.53x106A, wherein the quantity of cd4 cell is 3.39x106It is a.
Table 1:Sample PBMC and cd4 cell quantity contained by elution gained aim cell

Claims (6)

1. cell separation microballon eluent, it is characterised in that:Include following components per 100ml:IdeS 800~1200U of enzyme, 800~1200U of cysteine proteinase, based on mass-volume concentration bovine serum albumin BSA 8%~12% and trehalose 8%~ 12% and 4~6mmol of glycine, 4~6mmol of sodium dihydrogen phosphate, 4~6mmol of potassium chloride, 4~6mmol of sodium chloride.
2. cell separation microballon eluent according to claim 1, it is characterised in that:Include following components per 100ml: IdeS enzyme 1000U, cysteine proteinase 1000U, bovine serum albumin BSA 10% and trehalose based on mass-volume concentration 10% and glycine 5mmol, sodium dihydrogen phosphate 5mmol, potassium chloride 5mmol, sodium chloride 5mmol.
3. cell separation microballon eluent according to claim 1, it is characterised in that:It is to match by the following method:
(1) weigh the sodium dihydrogen phosphate of 6g, the sodium chloride and 3.75g glycine of the potassium chloride 2.92g of 3.73g, with 1L go from Sub- water dissolution, it is 6.5 that sodium hydroxide, which adjusts pH value, obtains elution buffer;
(2) trehalose for weighing 10g is uniformly dissolved with the elution buffer of 100mL, obtains the first solution;
(3) BSA for weighing 10g, the first solution described in 100mL are uniformly dissolved, and obtain the second solution;
(4) the Ides enzymes of 1000U, the cysteine proteinase of 1000U are weighed, the second solution described in 100mL is uniformly dissolved, obtains Third solution;
(5) the third solution adjusts pH value to 6.5 using 1M Hcl, is filtered using 0.22um sterile filters to get gained is molten Liquid is preserved at -20 °.
4. cell separation microballon eluent according to claim 1, it is characterised in that:It is the nothing that pH value is 5.5~6.5 Color clear solution.
5. cell separation microballon eluent according to claim 1, it is characterised in that:Its application method is:
(1) the microballon purifies and separates cell for being connected with antibody is utilized so that bead surface is connected with aim cell;
(2) elution buffer is added, and the cell eluent of respective volume, mixing microballon is added;
(3) it is incubated 20min;
(4) it centrifuges, it is aim cell to remove supernatant, precipitation, is used for subsequent experimental.
6. the preparation method of cell separation microballon eluent, it is characterised in that:Include the following steps:
(1) weigh the sodium dihydrogen phosphate of 6g, the sodium chloride and 3.75g glycine of the potassium chloride 2.92g of 3.73g, with 1L go from Sub- water dissolution, it is 6.5 that sodium hydroxide, which adjusts pH value, obtains elution buffer;
(2) trehalose for weighing 10g is uniformly dissolved with the elution buffer of 100mL, obtains the first solution;
(3) BSA for weighing 10g, the first solution described in 100mL are uniformly dissolved, and obtain the second solution;
(4) the Ides enzymes of 1000U, the cysteine proteinase of 1000U are weighed, the second solution described in 100mL is uniformly dissolved, obtains Third solution;
(5) the third solution adjusts pH value to 6.5 using 1M hydrochloric acid solutions, is filtered to get institute using 0.22um sterile filters Solution is obtained to preserve at -20 °.
CN201810260416.5A 2018-03-27 2018-03-27 Cell separation microballon eluent and preparation method Pending CN108410798A (en)

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CN112522194A (en) * 2020-11-26 2021-03-19 北京贝康医学检验所有限公司 Method for capturing fetal nucleated red blood cells

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Publication number Priority date Publication date Assignee Title
CN112522194A (en) * 2020-11-26 2021-03-19 北京贝康医学检验所有限公司 Method for capturing fetal nucleated red blood cells

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