CN107389420B - Cell enrichment and separation method - Google Patents
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Abstract
The invention provides a cell enrichment and separation method, which comprises the following steps: 1) combining a first type of magnetic bead with a biological sample, wherein the biological sample comprises a first type of cells and a second type of cells, the first type of magnetic bead is combined with a first type of antibody, and the first type of cells is combined with the first type of magnetic bead; 2) placing a capture magnetic head in a biological sample combined with a first type of magnetic beads, and adsorbing a first type of cells on a capture magnet device; 3) releasing the first type of cells from the capture head device to remove the first type of cells from the biological sample; 4) adding a second type of magnetic beads into the biological sample from which the first type of cells are removed, wherein the second type of cells are combined with the second type of magnetic beads; 5) placing the capture magnetic head in the biological sample combined with the second type of magnetic beads, and adsorbing the second type of cells on the capture magnet device; 6) releasing the second type of cells from the capture head assembly. The invention has high detection sensitivity and purity, and can be used for analyzing various gene markers of single cells or a small number of cells.
Description
Technical Field
The invention belongs to the technical field of cell separation, and particularly relates to a cell enrichment and separation method.
Background
Circulating Tumor Cells (CTCs) are important markers for studying tumor spreading mechanism and early detection, prevention and clinical treatment efficacy of tumors, because the number of CTCs existing in blood is small, the requirement on detection technology is high, and the technology on the market at present cannot meet the requirement on the market as a conventional detection item in terms of detection sensitivity and reasonable price. Most of the mature detection technologies in the market are the positive magnetic bead enrichment method or the negative magnetic bead enrichment method, but the detection sensitivity and the subsequent gene analysis of the cells are limited, for example, the detection sensitivity of the single positive magnetic bead enrichment method is limited and the purity of the captured target cells is not high, and the single negative magnetic bead enrichment method is also greatly limited in the purity of the target cells.
Disclosure of Invention
In view of the above-mentioned drawbacks in the background art, the main object of the present invention is to provide a cell enrichment and separation method, which combines a positive magnetic bead enrichment method and a negative magnetic bead enrichment method to improve the sensitivity, purity and stability of target cell detection.
In order to achieve the purpose, the invention adopts the following technical scheme: a cell enrichment isolation method, the method comprising:
1) combining a first type of magnetic beads with a biological sample, wherein the biological sample comprises a first type of cells and a second type of cells, a first type of antibody is combined with the first type of magnetic beads, and the first type of cells are combined with the first type of magnetic beads through the first type of antibody;
2) placing a capture magnetic head in the biological sample combined with the first type of magnetic beads, wherein the first type of cells are adsorbed on a capture magnet device;
3) releasing the first type of cells from the capture head assembly to remove the first type of cells from the biological sample;
4) combining a second type of magnetic beads with the biological sample from which the first type of cells are removed, wherein the second type of magnetic beads are combined with a second type of antibody, and combining the second type of cells with the second type of magnetic beads through the second type of antibody;
5) placing a capture magnetic head in the biological sample combined with the second type of magnetic beads, wherein the second type of cells are adsorbed on the capture magnet device;
6) releasing the second type of cells from the capture head assembly;
and when the first type of magnetic beads are negative magnetic beads, the second type of magnetic beads are positive magnetic beads, the first type of cells are non-target cells, and the second type of cells are target cells.
As a further preferred option, in the step 3), after releasing the first type of cells from the capture magnetic head, the capture magnet is placed in the biological sample bound to the first type of magnetic beads again, and the first type of cells are adsorbed again. This operation may be repeated multiple times until complete or near complete clearance of the first type of cells is achieved.
As a further preferred option, in the step 6), after releasing the second type of cells from the capture magnetic head, the capture magnet is placed in the second type of cells bound to the second magnetic beads again, and the second type of cells are adsorbed again. This operation can also be repeated multiple times until maximum capture of the second type of target cells is achieved.
Further preferably, in the step 3) or 6), a magnetic sleeve is disposed on the surface of the capture magnetic head, and the first or second type of cells are adsorbed on the magnetic sleeve of the capture magnetic head device.
As a further preference, the cells remaining after capture in step 5) and the target cells collected by positive enrichment are collected separately on containers or slides for cell identification, counting or gene hybridization analysis.
As a further preference, the tumor target cells are used for cell counting and multigenic marker analysis of single or small numbers of cells.
As a further preference, the biological sample is a blood sample or a biological fluid, the non-target cells include white blood cells and red blood cells, and the target cells include circulating tumor target cells.
Further preferably, the blood sample includes a raw blood sample, a blood sample from which red blood cells have been lysed, or a blood sample from which red blood cells have been removed by a density gradient cell separation process.
Further preferably, the method further comprises: and when the first type of magnetic beads are positive magnetic beads, the second magnetic beads are negative magnetic beads, the first type of antibodies are target cell recognition antibodies, and the second antibodies are non-target cell recognition antibodies.
Further preferably, the first antibody is selected from the group consisting of CD45, CD14, and an erythrocyte antibody, and the second antibody is selected from the group consisting of EpCAM, Muc1, and a keratin antibody.
The invention has the beneficial effects that: according to the invention, a negative magnetic bead enrichment method and a positive magnetic bead enrichment method are combined, so that a biological sample can be enriched with negative magnetic beads and then enriched with positive magnetic beads, or enriched with positive magnetic beads and then enriched with negative magnetic beads; for example: the method comprises the steps of combining negative magnetic beads with a biological sample to combine a first class of antibodies on the negative magnetic beads with non-target cells in the biological sample, adsorbing the non-target cells by using a capture magnetic head, and fully separating the non-target cells from tumor target cells in the biological sample; and adding the positive magnetic beads into the biological sample containing the tumor target cells, culturing, combining the positive magnetic beads with the tumor target cells, and enriching the positive magnetic beads to obtain more pure positive tumor target cells. Therefore, the present invention can remove non-target cells, such as blood cells (white blood cells and red blood cells), from a large number of cells in a biological sample, and then isolate and extract tumor cells therein, wherein the background cell amount is greatly reduced, and the purity of the finally obtained tumor cells is greatly improved. The use of such products for subsequent cellular and genetic analysis would be advantageous for obtaining optimal analytical results. Therefore, the method improves the detection sensitivity, purity and detection stability of the target cells. For another example: the method can combine the positive magnetic beads with the biological sample to combine the antibodies on the positive magnetic beads with the tumor target cells in the biological sample, and then the capture magnetic head is utilized to adsorb the tumor target cells to separate the non-target cells from the tumor target cells in the biological sample; and adding negative magnetic beads into the non-target cells, combining the negative magnetic beads with the non-target cells, and enriching the negative magnetic beads to obtain the non-target cells. The target cells obtained by positive enrichment are collected, counted and subjected to cell and gene analysis, and the remaining cells of negative enrichment and positive enrichment can be used for cell and gene analysis of a glass slide and the like, so that the effect of non-selection analysis and detection of whole blood cells can be achieved.
Drawings
Fig. 1a-1b are schematic flow charts of a cell enrichment and separation method in which negative magnetic beads are enriched first and then positive magnetic beads are enriched in the embodiment 1 of the present invention.
FIG. 1c is a schematic analysis of multiple markers of tumor target cells.
FIG. 2 is a schematic flow chart of a cell enrichment and separation method in which positive magnetic beads are enriched first and negative magnetic beads are enriched second in embodiment 2 of the present invention.
FIGS. 3a-3d are schematic diagrams of multiple negative enrichments for removing magnetic material.
FIGS. 4a-4b are schematic diagrams of negative enrichment of single cells.
FIGS. 5a-5b are schematic diagrams of the negative enrichment process for the clearance of target cells of non-magnetic material.
Detailed Description
The invention provides a cell enrichment and separation method, which overcomes the defects of low detection sensitivity and low purity of target cells in the existing circulating tumor cell detection technology.
In order to solve the above problems, the main idea of the embodiment of the present invention is:
the cell enrichment and separation method provided by the embodiment of the invention comprises the following steps:
1) combining a first type of magnetic beads with a biological sample, wherein the biological sample comprises a first type of cells and a second type of cells, a first type of antibody is combined with the first type of magnetic beads, and the first type of cells are combined with the first type of magnetic beads through the first type of antibody;
2) placing a capture magnetic head in the biological sample combined with the first type of magnetic beads, wherein the first type of cells are adsorbed on a capture magnet device;
3) releasing the first type of cells from the capture head assembly to remove the first type of cells from the biological sample;
4) combining a second type of magnetic beads with the biological sample from which the first type of cells are removed, wherein the second type of magnetic beads are combined with a second type of antibody, and combining the second type of cells with the second type of magnetic beads through the second type of antibody;
5) placing a capture magnetic head in the biological sample combined with the second type of magnetic beads, wherein the second type of cells are adsorbed on the capture magnet device;
6) releasing the second type of cells from the capture head assembly;
and when the first type of magnetic beads are negative magnetic beads, the second type of magnetic beads are positive magnetic beads, the first type of cells are non-target cells, and the second type of cells are target cells.
Generally, after the blood sample is subjected to the negative magnetic bead binding treatment, most of the red blood cells and white blood cells are bound and removed, and the embodiment of the invention can use a repeatable automatic instrument to operate the capture magnetic head for multiple adsorptions, so that the red blood cells and the white blood cells in the blood sample can be completely and completely removed. At this time, the positive magnetic beads are added into the sample enriched with the negative magnetic beads, the positive magnetic beads are combined on the magnetic head, or are partially combined on the cells and then are combined on the magnetic head, the target cells in the sample solution are adsorbed on the magnetic head through the micro-speed scanning movement of the magnetic head in the sample solution, and are transferred to a specific collection container for counting the target cells and analyzing the multi-gene marker of single cells or a small number of cells. The embodiment of the invention has various advantages, firstly, the whole blood cells are used for detecting and analyzing the target cells through the operation, and the detection sensitivity is very high; on the other hand, since the number of non-specific cells is very small or none after the negative enrichment treatment, the purity of the cells for gene analysis is very high after the sample is re-enriched to extract the positive target cells. The residual cells after negative enrichment and positive enrichment are subjected to cell analysis on a glass slide, so that missing target cells of the positive enrichment marker are prevented from being lost. The cells extracted by positive enrichment are used for counting and multi-gene detection and analysis, the purity is high, and the analysis result is more reliable. The results of negative and positive enrichment are combined, the target cells are counted and the gene information is analyzed, and the complete effect equivalent to the whole blood cell analysis can be achieved. Meanwhile, the purity of the target cell is obviously improved, and more superiority is provided for the analysis of downstream genes and other multiple markers. In the sample after the negative processing, target cells which cannot be captured by the positive magnetic beads may exist in the residual cells after the positive magnetic bead enrichment processing, and at this time, the small amount of cells are all centrifuged on the glass slide to perform cell and gene level analysis, so that more comprehensive information of the target cells (such as blood circulation tumor cells and the like) can be obtained. Some free magnetic beads remain after negative enrichment or positive enrichment, and the free magnetic beads can be removed by adopting a filtration method.
The method provided by the embodiment of the invention can be used for enriching and separating cells in a sample, and can also be used for separating and extracting other biomolecule components (such as nucleic acid and protein).
In order to clearly understand the objects, technical solutions and advantages of the present invention, the present invention will be further described in detail with reference to the following embodiments. The specific examples described herein relate to specific data only to illustrate the present invention and are not intended to limit the invention.
Example 1
As shown in FIGS. 1a-1b, the cell enrichment and separation method of the embodiment 1 of the present invention comprises the following steps:
adding negative magnetic beads into a biological sample solution tank, wherein the negative magnetic beads are combined with antibodies, the biological sample comprises tumor target cells and non-target cells to be captured, and the antibodies are combined with the non-target cells; the biological sample is a raw blood sample, the antibody is CD45, and the non-target cells include red blood cells and white blood cells;
placing a capture magnetic head in the biological sample combined with the negative magnetic beads, adsorbing non-target cells in the sample solution to the magnetic head through the micro-speed scanning motion of the magnetic head in the sample solution, and transferring the non-target cells to a collection container; thus, the non-target cells in the biological sample are separated from the tumor target cells; in addition, in order to completely remove the red blood cells and white blood cells in the blood sample, the capture magnetic head can be placed in the biological sample for a plurality of times, and the adsorption of the non-target cells can be repeated for a plurality of times, for example, 3 times; the capture magnetic head can realize the automatic operation of the capture magnet through automatic instruments such as a magnetic capture instrument, silicon valley and the like, and can also adopt manual operation. The sleeve made of non-magnetic material can be used to cover the capture magnetic head, so as to prevent the magnetic beads and non-target cells from directly contacting the magnetic head when being adsorbed by the magnetic field of the capture magnetic head, thereby achieving the purposes of effectively collecting the non-target cells and not damaging the non-target cells.
Then, positive magnetic beads are added into the biological sample solution with the non-target cells removed, the positive magnetic beads are combined with an antibody molecule EpCAM, meanwhile, a capture magnetic head is placed in the biological sample solution, tumor target cells in the sample solution are adsorbed onto the magnetic head through the micro-speed scanning movement of the magnetic head in the sample solution, and the magnetic head is transferred into a collection container, and the magnetic head can be further used for counting target cells and analyzing the multi-gene markers of single cells or a small number of cells. The free magnetic beads in the magnetic substance enriched with the positive magnetic beads can be removed by using a filtering device.
The target cells enriched with the positive magnetic beads can be analyzed by gene amplification and analysis of multiple gene markers for DNA and RNA, or by direct analysis of protein markers of cells, analysis of cell structure and physical properties, analysis of foreign body markers, etc., as shown in FIG. 1 c.
In the embodiment, the sample and the negative magnetic beads are mixed, most of negative cells can be removed after enrichment, on the basis, the sensitivity of the detection target cells can be improved by positively enriching the target cells, meanwhile, the purity of the target cells obtained by positively enriching can also be greatly improved, and in addition, the residual cells after enrichment are counted and subjected to gene analysis, so that the limitation of positive magnetic bead antibodies is overcome, the positive magnetic bead enrichment result and the analysis result of the residual cells are integrated, and the counting, cell analysis and gene analysis results are more comprehensive and approximate to whole blood cell analysis.
Example 2
As shown in fig. 2, the cell enrichment and separation method of embodiment 2 of the present invention includes the following steps:
adding positive magnetic beads into a biological sample solution tank, wherein the positive magnetic beads are combined with antibodies, the biological sample comprises tumor target cells and non-target cells to be captured, and the antibodies are combined with the tumor target cells; the biological sample is a raw blood sample, the antibody is Muc1, and the non-target cells include red blood cells and white blood cells;
placing a capture magnetic head in a biological sample combined with the positive magnetic beads, adsorbing tumor target cells in a sample solution to the magnetic head through the micro-speed scanning motion of the magnetic head in the sample solution, and transferring the tumor target cells to a collection container; the obtained target cells are collected, counted and subjected to cellular and genetic analysis. Thus, the non-target cells in the biological sample are separated from the tumor target cells; the capture magnetic head can realize the automatic operation of the capture magnet through automatic instruments such as a magnetic capture instrument, silicon valley and the like, and can also adopt manual operation.
Then adding negative magnetic beads to the biological sample solution with the tumor target cells removed, wherein the negative magnetic beads are combined with the antibody CD14, simultaneously placing a capture magnetic head in the biological sample solution, adsorbing non-target cells in the sample solution to the magnetic head through the micro-speed scanning movement of the magnetic head in the sample solution, transferring the non-target cells to a collection container, removing most of the non-target cells, transferring the rest cells to a glass slide for cell and gene analysis, and also performing the whole blood cell analysis and detection.
Example 3
This example is similar to example 1, except that: the biological sample is a blood sample with red blood cells cracked; the antibody combined by the negative magnetic beads is a red blood cell antibody, and the antibody combined by the positive magnetic beads is a keratin antibody.
Example 4
This example is similar to example 2, except that: the biological sample is a blood sample obtained by cracking red blood cells or a blood sample obtained by removing red blood cells through density gradient cell separation. The antibody combined by the negative magnetic beads is a red blood cell antibody, and the antibody combined by the positive magnetic beads is a keratin antibody.
Comparative example 1
Negative magnetic beads are placed in a biological sample tank, and are subjected to multiple times of negative enrichment treatment through multiple times of cyclic adsorption of a capture magnetic head, so that most of magnetic substances can be eliminated, for example, as shown in fig. 3a-3d, more than 99.99% of the magnetic substances are removed after 3 times of negative enrichment treatment (shown in fig. 3b-3 d), and in addition, after the negative enrichment treatment, the magnetic substances in the sample basically cannot cause any influence on later positive enrichment. The above described multiple adsorbtions of the capture head can be operated using repeatable automated instrumentation in this embodiment.
Comparative example 2
FIGS. 4a-4b are schematic diagrams of negative enrichment of single cells. The single cells marked by the negative magnetic beads are put into different sample tanks, and the single cells can be removed by a negative magnetic bead enrichment method, which shows that the embodiment can remove even single non-specific cells and ensure that the cells are not brought to subsequent processing steps such as: and the enrichment of the positive magnetic beads ensures the thoroughness of the enrichment of the negative magnetic beads.
Comparative example 3
FIGS. 5a-5b are schematic diagrams of the negative enrichment method for the clarity of target cells for nonmagnetic material. The negative magnetic beads and the positive cells are mixed, and in principle, the magnetic beads cannot be combined with the positive cells. By negative bead enrichment, most of the beads can be removed, but cells that are not free of this material are not cleared. Thus, negative bead enrichment only removes cells and material bound by negative beads, but not positive cells, and negative enrichment does not remove target cells that are not magnetically labeled, as shown in FIGS. 5a-5 b.
The technical scheme in the embodiment of the application at least has the following technical effects or advantages:
according to the invention, a negative magnetic bead enrichment method and a positive magnetic bead enrichment method are combined, so that a biological sample can be enriched with negative magnetic beads and then enriched with positive magnetic beads, or enriched with positive magnetic beads and then enriched with negative magnetic beads; for example: combining negative magnetic beads with a biological sample to combine a first class of antibodies on the negative magnetic beads with non-target cells in the biological sample, adsorbing the non-target cells by using a capture magnetic head, and separating the non-target cells from tumor target cells in the biological sample; and adding the positive magnetic beads into the tumor target cells, culturing, combining the positive magnetic beads with the tumor target cells, and enriching the positive magnetic beads to obtain purer positive tumor target cells. Therefore, the present invention can remove non-target cells, such as blood cells (white blood cells and red blood cells), from a large number of cells in a biological sample, and then isolate and extract tumor cells therein, wherein the background cell amount is greatly reduced, and the purity of the finally obtained tumor cells is greatly improved. The use of such products for subsequent cellular and genetic analysis would be advantageous for obtaining optimal analytical results. Therefore, the method improves the detection sensitivity, purity and detection stability of the target cells. For another example: the method can combine the positive magnetic beads with the biological sample to combine the antibodies on the positive magnetic beads with the tumor target cells in the biological sample, and then the capture magnetic head is utilized to adsorb the tumor target cells to separate the non-target cells from the tumor target cells in the biological sample; and adding negative magnetic beads into the non-target cells, combining the negative magnetic beads with the non-target cells, and enriching the negative magnetic beads to obtain the non-target cells. The target cells obtained by positive enrichment are collected, counted and subjected to cell and gene analysis, and the cells obtained by negative enrichment can be used for cell and gene analysis of glass slides and the like, and can also play a role in whole blood cell analysis and detection.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention. It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (5)
1. A cell enrichment and separation method is characterized in that: the method comprises the following steps:
1) combining a first type of magnetic beads with a biological sample, wherein the biological sample comprises a first type of cells and a second type of cells, a first type of antibody is combined with the first type of magnetic beads, and the first type of cells are combined with the first type of magnetic beads through the first type of antibody;
2) placing a capture magnetic head in the biological sample combined with the first type of magnetic beads, wherein the first type of cells are adsorbed on a capture magnet device;
3) releasing the first type of cells from the capture head assembly to remove the first type of cells from the biological sample;
4) combining a second type of magnetic beads with the biological sample from which the first type of cells are removed, wherein the second type of magnetic beads are combined with a second type of antibody, and combining the second type of cells with the second type of magnetic beads through the second type of antibody;
5) placing a capture magnetic head in the biological sample combined with the second type of magnetic beads, wherein the second type of cells are adsorbed on the capture magnet device;
6) releasing the second type of cells from the capture head assembly;
when the first type of magnetic beads are positive magnetic beads, the second type of magnetic beads are negative magnetic beads, the first type of antibodies are target cell recognition antibodies, and the second type of antibodies are non-target cell recognition antibodies;
the biological sample is a blood sample or a biological fluid, the non-target cells comprise white blood cells and red blood cells, and the target cells comprise tumor target cells;
the blood sample comprises a raw blood sample, a blood sample subjected to red blood cell lysis or a blood sample subjected to density gradient cell separation treatment and red blood cell removal;
respectively collecting the residual cells after the capture in the step 5) and the target cells collected by positive enrichment on a container or a glass slide for cell identification, counting or gene hybridization analysis;
the target cells are used for cell counting and multigene marker analysis of single or small numbers of cells.
2. The cell enrichment and separation method according to claim 1, wherein: in the step 3), after the first type of cells are released from the capture magnetic head, the capture magnet is placed in the biological sample combined with the first type of magnetic beads again, and the first type of cells are adsorbed again.
3. The cell enrichment and separation method according to claim 1, wherein: in the step 6), after the second type of cells are released from the capture magnetic head, the capture magnet is placed in the second type of cells combined with the second magnetic beads again, and the second type of cells are adsorbed again.
4. The cell enrichment and separation method according to claim 1, wherein: in the step 3) or 6), a magnetic sleeve is arranged on the surface of the capture magnetic head, and the first or second type of cells are adsorbed on the magnetic sleeve of the capture magnetic head device.
5. The cell enrichment and separation method according to claim 1, wherein: the first type of antibody is selected from CD45, CD14 and erythrocyte antibody, and the second type of antibody is selected from EpCAM, Muc1 and keratin antibody.
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| WO2020037684A1 (en) * | 2018-08-24 | 2020-02-27 | 深圳华大生命科学研究院 | Separation and extraction device for cells and biomolecules, and test system |
| CN109752530A (en) * | 2019-01-31 | 2019-05-14 | 宜昌美光硅谷生命科技股份有限公司 | A kind of liquid phase cellular immunity identification method and its application |
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| CN107389420A (en) | 2017-11-24 |
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