CN111826349B - Erythrocyte cluster based on size filtration method for enriching circulating tumor cells - Google Patents
Erythrocyte cluster based on size filtration method for enriching circulating tumor cells Download PDFInfo
- Publication number
- CN111826349B CN111826349B CN202010867920.9A CN202010867920A CN111826349B CN 111826349 B CN111826349 B CN 111826349B CN 202010867920 A CN202010867920 A CN 202010867920A CN 111826349 B CN111826349 B CN 111826349B
- Authority
- CN
- China
- Prior art keywords
- red blood
- ctcs
- antibody
- clusters
- erythrocyte
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0641—Erythrocytes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Abstract
The invention discloses a red blood cell cluster for enriching circulating tumor cells based on a size filtration method, and belongs to the technical field of biological medicines. Through designing a micron-scale targeted CTCs bionic material, namely combining erythrocytes with microspheres to obtain a erythrocyte cluster bionic material, the surfaces of the erythrocytes are modified with polybrene, so that electrostatic repulsion originally existing between the erythrocytes is eliminated, and further, the tight full-layer coverage of the erythrocytes on the surfaces of the microspheres can be realized. The encapsulation of the erythrocytes on the microspheres can avoid the nonspecific adsorption of the leukocytes on the surfaces of the microspheres, and Folic Acid (FA) modified on the surfaces of the erythrocyte clusters or antibodies capable of specifically recognizing and combining with the biomarkers on the surfaces of the CTCs can directly target the CTCs, so that the size of the CTCs is increased, and finally, the leukocytes in blood samples of clinical patients can be removed in a filtering mode, and the high-purity CTCs can be obtained efficiently.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and mainly relates to a red blood cell cluster for enriching circulating tumor cells based on a size filtration method.
Background
Recurrence and distant metastasis of local or regional tumor, and provides serious challenges for early diagnosis of tumor and cure of tumor. Recent years of molecular biology and clinical studies have shown that circulating tumor cells (circulating tumor cells, CTCs) lead to tumor invasion and the development of micrometastasis processes. CTCs are formed by the shedding of original tumor cells from primary tumors and entering the blood system of a human body, and finally transferred to other tissues and organs of the human body through the circulation and diffusion of peripheral blood, so that secondary tumors of the same type as the tumors of the primary parts are formed, namely, the metastasis and recurrence of the tumors and cancers are caused. By capturing the circulating tumor cells in the peripheral blood and analyzing the change of the number of the circulating tumor cells in the peripheral blood along with time, the tumor prognosis can be predicted earlier, and a simple and easy method and means are provided for the treatment response detection and the radiotherapy and chemotherapy curative effect analysis of the cancer drug.
Since the CTCs content in peripheral blood is very low, there are only several to tens of CTCs per milliliter of blood, and the content of the background blood cells reaches 10 9 In order of magnitude, there is a great deal of interference with CTCs separation and enrichment assays, so good CTCs separation and enrichment requires high sensitivity and selectivity, while still being able to maintain CTCs activity. Numerous methods of separation and enrichment of CTCs have been developed, one important class of which is based on the separation of CTCs from blood cells by different physical properties. There are obvious differences in sizes of red blood cells (4-8 μm), white blood cells (6-19 μm) and CTCs (13-22 μm), and CTCs can be obtained by filtration based on the size differences between the three. However, since there is overlap in size between leukocytes and CTCs, it is difficult to obtain CTCs with high efficiency by a simple filtration method while ensuring high purity of CTCs. In order to solve the contradiction, the targeting effect of the immune microsphere of the surface modified antibody on the CTCs is adopted to expand the size of the CTCs, so that the size range of the CTCs is staggered with the size range of the WBCs, and then the CTCs can be obtained by high-efficiency and high-purity filtration.
In practice, there is a degree of non-specific adsorption between the immune microspheres and the leukocytes, i.e., this can adversely affect the purity of the isolated CTCs. In order to reduce the nonspecific adsorption effect between the material and the leucocytes, the common thinking is to carry out bionic design on the surface of the material, for example, a monolayer cell membrane with the thickness of 9nm is wrapped on the surface of the nano material, or a DNA molecule is modified, so that the nonspecific adsorption effect of the nano material after the bionic design on the leucocytes is greatly reduced. However, these are bionic designs at nano-scale, and cannot enlarge the size difference between the white blood cells and the CTCs, so that the purity of the CTCs obtained by filtration needs to be improved.
Disclosure of Invention
In order to solve the problems in the prior art, the invention aims to provide a red blood cell cluster for enriching circulating tumor cells based on a size filtration method. Through designing a micron-scale targeted CTCs bionic material, namely combining erythrocytes with microspheres to obtain a erythrocyte cluster bionic material, the surfaces of the erythrocytes are modified with polybrene, so that electrostatic repulsion originally existing between the erythrocytes is eliminated, and further, the tight full-layer coverage of the erythrocytes on the surfaces of the microspheres can be realized. The encapsulation of the erythrocytes on the microspheres can avoid the nonspecific adsorption of the leukocytes on the surfaces of the microspheres, and Folic Acid (FA) modified on the surfaces of the erythrocyte clusters or antibodies capable of specifically recognizing and combining with the biomarkers on the surfaces of the CTCs can directly target the CTCs, so that the size of the CTCs is increased, and finally, the leukocytes can be removed in a filtering mode, and the CTCs with high purity can be obtained with high efficiency.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
in a first aspect, a red blood cell cluster for enriching circulating tumor cells based on size filtration is provided, which is prepared by the following method:
(1) Folate or antibody targeted by CTC (cell-mediated) on surface of erythrocytes
First category: folic acid modification, namely uniformly mixing 10 mu L-50 mu L of healthy human fresh red blood cells with 100 mu L-1000 mu L of PBS solution of DSPE-PEG-FA with molecular weight 3400-10000 and concentration of 1mg/mL, standing and incubating for 30 min-2 h at 4-25 ℃, and centrifugally washing for three times by using PBS to obtain folic acid modified red blood cells; or alternatively, the process may be performed,
the second category: antibody modification capable of specifically recognizing and combining CTCs surface biomarkers, uniformly mixing 10 mu L-50 mu L of healthy human fresh red blood cells with 100 mu L-1000 mu L of PBS solution of DSPE-PEG-biotin with molecular weight 3400-10000 and concentration of 1mg/mL, standing and incubating for 30 min-2 h at 4-25 ℃, and centrifugally washing with PBS for three times to obtain red blood cells with surface modified biotin; uniformly mixing biological modified red blood cells with 100 mu L-1000 mu L of SA solution with the concentration of 100ug/mL, standing and incubating for 30 min-2 h at the temperature of 4-25 ℃, and centrifugally washing for three times by using PBS (phosphate buffer solution) to obtain the red blood cells with the surface modified SA; finally, uniformly mixing SA modified red blood cells with 100 mu L-1000 mu L of biotinylated CTCs targeting antibody solution with the concentration of 5ug/mL, standing and incubating for 30 min-2 h at the temperature of 4-25 ℃, and centrifugally washing for three times by using PBS to obtain the red blood cells of the surface modified CTCs targeting antibody;
(2) Modification of erythrocyte surface polybrene
Mixing 10 mu L-50 mu L of red blood cells with FA or CTC targeted antibodies modified on the surface with 100 mu L-500 mu L of polybrene PBS solution with the concentration of 1-100 mg/mL, standing and incubating for 30 min-2 h at 4-25 ℃, centrifugally washing with PBS for three times to obtain red blood cells with the surface modified polybrene and the FA or CTC targeted antibodies, and dispersing the red blood cells into the PBS solution for standby;
(3) Preparation of erythrocyte clusters
Washing the microsphere with the surface modification group with PBS for three times, adding more than 100 times of the number of the microsphere to the red blood cells in the step (2), uniformly mixing the two, centrifuging the mixture in a centrifuge at a centrifugal speed of 300 g-600 g for 1 min-30 min to ensure that the red blood cells and the microsphere are close to each other at the bottom of the centrifuge tube, standing and incubating the mixture at 4-25 ℃ for 20 min-2 h after centrifuging, and finally blowing off the mixture at the bottom of the centrifuge tube, and filtering the suspension by using a biocompatible microporous membrane with the aperture of 10 mu m; excess free red blood cells, microspheres that do not form red blood cell clusters, and defective red blood cell clusters of less than 10 μm can be removed by filtration; finally, clusters of red blood cells exceeding 10 μm in size were collected from the membrane.
Preferably, the surface biomarker of CTCs in step (1) of the above preparation method of erythrocyte clusters is any one of epithelial markers cytokeratin, epithelial cell adhesion molecule, tumor embryo antigen, human epidermal growth factor receptor 2, venous endothelial cell molecule, epstein, sialylated lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45 or CD146.
Preferably, the antibody specifically recognizing and binding to CTCs surface biomarker in step (1) of the above preparation method of erythrocyte clusters comprises any one of epithelial marker cytokeratin antibody, epithelial cell adhesion molecule antibody, tumor embryo antibody, HER 2 antibody, intravenous endothelial cell molecule antibody, epstein antibody, sialylated Lewis oligosaccharide-X antibody, acetaldehyde dehydrogenase 1 antibody, vimentin antibody, urokinase receptor antibody, heparanase antibody, prostate specific membrane antibody, anti-CD44, anti-CK18, anti-CD133, anti-CD90, anti-CD45 or anti-CD146.
Preferably, the molecular weight of DSPE-PEG-FA or DSPE-PEG-biotin in step (1) of the above method for preparing erythrocyte clusters is 5000.
Preferably, the modification reaction temperature in the steps (1) to (3) in the preparation method of the erythrocyte clusters is 4 ℃.
Preferably, in the preparation method of the erythrocyte cluster, the modification group on the microsphere surface in the step (3) is carboxyl or amino.
Preferably, the microsphere diameter of the surface modification group in the step (3) in the preparation method of the erythrocyte cluster is 5-6 μm.
In a second aspect, there is provided the use of the above-described red blood cell clusters in the preparation of a kit for identifying, capturing or enriching circulating tumor cells.
Preferably, the method for enriching CTCs by using the erythrocyte clusters in the application mainly comprises the following steps: 1) Treating the blood sample with the erythrocyte lysate to remove erythrocytes in the blood sample, and avoiding interference of erythrocytes in the sample on capturing CTCs by the functionalized erythrocyte clusters; 2) Adding the functionalized red cell clusters in a blood sample without red cells, and carrying out incubation at 4 ℃ for a period of time to enable the red cell clusters to be specifically combined with CTCs; 3) Separating the sample in the step 2) by a 20 mu m porous filter membrane filtration mode, namely realizing the high-purity enrichment of the circulating tumor cells; 4) And adding a sodium citrate solution with the concentration of 4% prepared by the erythrocyte lysate into the erythrocyte clusters captured with the CTCs and obtained by filtering in the step 3), and realizing the release process of the CTCs on the basis of most erythrocyte shedding on the surfaces of the clusters.
Preferably, the method for enriching CTCs by using the erythrocyte clusters in the application mainly comprises the following steps: 1) Obtaining a blood sample of a tumor patient after operation or chemotherapy, and obtaining a lymphocyte layer through a percoll cell separation liquid; 2) Adding the functionalized red cell clusters of the invention into a lymphocyte layer, and incubating at 4 ℃ to perform capturing; 3) Separating the sample in the step 2) by a 20 mu m porous filter membrane filtration mode, namely realizing the high-purity enrichment of the circulating tumor cells; 4) Adding 4% sodium citrate solution prepared from erythrocyte lysate into the erythrocyte clusters captured with CTCs obtained by filtering in the step 3), and realizing the release process of CTCs on the basis of most erythrocyte shedding on the surfaces of the clusters; 5) After the captured cells were subjected to three-fluorescence staining, the number of CTCs captured was quantitatively analyzed by flow cytometry, and the capturing was analyzed by confocal laser.
The principle of the technical scheme for enriching the red blood cell clusters of the circulating tumor cells based on the size filtration method is as follows (see figure 1):
1. principle of erythrocyte modification:
in order to enable the targeting of erythrocyte clusters to CTCs, folic acid or antibodies targeting to erythrocyte surface modification CTCs are required, and the principle is divided into the following two categories:
the first is a folate molecule (FA) that specifically recognizes and binds to the CTCs surface folate receptor (FA receptor) on the surface modification of erythrocytes. The modification of folic acid is realized by embedding a functional molecule into erythrocyte membrane, one end of which is hydrophobic distearoyl phosphatidylethanolamine (DSPE), the DSPE end is embedded into the hydrophobic erythrocyte membrane through hydrophobic interaction, the other end is FA, and the DSPE and the FA are connected through hydrophilic polyethylene glycol (PEG) to form the functional molecule DSPE-PEG-FA.
The second type is an antibody which can specifically recognize and bind to a CTCs surface biomarker on the surface of erythrocytes, and the specific method is as follows: firstly, embedding a functional molecule on the surface of an erythrocyte membrane, wherein one end of the functional molecule is hydrophobic distearoyl phosphatidylethanolamine (DSPE), the DSPE end is embedded into the hydrophobic erythrocyte membrane through hydrophobic interaction, the other end of the functional molecule is biotin (biotin) capable of being specifically combined with streptavidin (SA for short), and the DSPE and the biotin are connected through hydrophilic polyethylene glycol (PEG) to form the functional molecule DSPE-PEG-biotin; then, through the specific combination mode of SA and biotin, SA is continuously modified on the basis of a functional molecule DSPE-PEG-biotin to be used as a bridging molecule for modifying antibodies targeted by CTCs; finally, biotinylated CTCs-targeted antibodies that specifically recognize and bind to biomarkers on the surface of CTCs are modified on the SA basis by specific binding of SA to biotin. The CTCs targeted antibodies described include, but are not limited to, epithelial marker cytokeratin antibodies, epithelial cell adhesion molecule antibodies, tumor embryo antibodies, human epidermal growth factor receptor 2 antibodies, venous endothelial cell molecule antibodies, annexin antibodies, sialylated lewis oligosaccharide-X antibodies, acetaldehyde dehydrogenase 1 antibodies, vimentin antibodies, urokinase receptor antibodies, heparanase antibodies, prostate specific membrane antibodies, anti-CD44, anti-CK18, anti-CD133, anti-CD90, anti-CD45, or anti-CD146. The CTCs surface biomarker specifically recognized by the above antibody is CTCs surface biomarker cytokeratin, epithelial cell adhesion molecule, tumor embryo antigen, human epidermal growth factor receptor 2, venous endothelial cell molecule, epstein, sialylated lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45 or CD146.
Whether the DSPE-PEG-FA used for modifying the FA or the DSPE-PEG-biotin used in the first step of modifying the antibody is modified by a molecule with a molecular weight of 5000. Because the molecular weight corresponds to the length of the PEG chain segments, after the PEG chain segments with larger molecular weight are modified on the surface of the red blood cells, the electrostatic repulsive force between the PEG chain segments is larger, so that the appearance of the red blood cells is amplified into a sphere from the original biconvex central concave cake shape. The rigidity of the deformed red blood cells is enhanced, so that the formed red blood cell cluster surface is difficult to deform, and the adsorption effect of the repellet white blood cells can be well removed.
2. Principle of red blood cell cluster design:
unmodified erythrocytes (4-8 μm) and carboxyl polystyrene microspheres (5-6 μm) can be mutually adsorbed through the hydrogen bond action between membrane proteins and carboxyl or amino groups on the surfaces of the microspheres, and because electrostatic repulsive force exists between the unmodified erythrocytes, the surface adsorption state of the microspheres is difficult to form fully filled surface layers. Through electrostatic adsorption of cationic polymer polybrene molecules on the surface of unmodified erythrocytes, electrostatic repulsive force between erythrocytes can be greatly weakened, so that adsorption of the surface erythrocytes of the microsphere on the full layer is realized. The obtained red blood cell clusters (13-22 μm) with complete shapes can avoid the non-specific adsorption of core microspheres and white blood cells. When preparing the red cell clusters, excessive red cells should be used to ensure that the carboxyl polystyrene microspheres are dispersed as far as possible, so that a large number of red cell clusters with single microspheres as cores can be prepared. Thus, quantitative preparation of the red blood cell clusters is achieved by controlling the amount of microspheres added.
The modification of the polybrene can enable the surface of the polystyrene microsphere to adsorb a large number of red blood cells, and then the addition of the deflocculant sodium citrate can neutralize the charge of the polybrene, so that the polybrene is invalid, and a large number of red blood cells are dropped from the red blood cell clusters. In addition, after the erythrocyte lysate is added at the same time, the erythrocyte can be further separated from the surface of the polystyrene microsphere through the lysis action on the erythrocyte. Therefore, the erythrocyte clusters can be removed from the surface of the clusters by adding the erythrocyte clusters into the sodium citrate solution prepared by the erythrocyte lysate.
3. Principle of enrichment and isolation of CTCs by erythrocyte clusters:
since the surface of the red blood cell cluster (13-22 μm) is modified with the FA or CTCs targeting antibody, the specific binding can be carried out with CTCs (13-22 μm) in a blood sample containing CTCs, the combined overall size is larger than 20 μm (as shown in figure 3), and the size range is larger than the size range of white blood cells (6-19 μm).
In the red cell clusters, a few clusters with surface defects exist, the defects of individual red cells on the surface of the clusters can lead the core microsphere to be partially exposed to white cells, the rigidity of the surface of the red cells is higher, the deformability is poorer, the site size of the defects is smaller, so that the white cells with larger size are difficult to nonspecifically adsorb on the surface of the exposed microsphere, and after the white cells with smaller size are nonspecifically adsorbed, the influence on the whole size of the defective red cell clusters is small and is less than 20 mu m (as shown in figure 4).
Thus, by filtration through a 20 μm porous filter membrane, excessive red blood cell clusters, free leukocytes, and defective red blood cell clusters of nonspecifically adsorbed leukocytes in the mixed sample after targeting CTCs can be removed by filtration together, as shown in fig. 5. And adding a sodium citrate solution with the concentration of 4% prepared by the erythrocyte lysate into the CTCs captured by the clusters obtained by filtering, so that the release process of the CTCs can be realized on the basis of most erythrocyte shedding on the surfaces of the clusters. Erythrocyte lysate was purchased from the company solarbio.
The invention has the beneficial effects that: (1) The invention provides a functional red blood cell cluster for efficiently and highly specifically identifying and capturing circulating tumor cells, which can be used for specifically identifying and capturing micro CTCs in simulated blood samples and clinical patient blood samples in vitro, thereby being expected to play an important role in the early warning and prevention fields of cancer metastasis; (2) The invention provides a functionalized red blood cell cluster for efficiently and specifically identifying and capturing circulating tumor cells, which can realize more firm and efficient capturing of the circulating tumor cells by virtue of a three-dimensional surface structure, and has the capturing efficiency up to more than 90 percent, and the surface of the red blood cell cluster is composed of red blood cells, so that the red blood cell cluster can avoid nonspecific adsorption of white blood cells, is not phagocytized by macrophages, and can realize the CTCs capturing purity of more than 80 percent in a simulated blood sample containing a large number of white blood cells or a clinical patient blood sample by combining an experimental method of size filtration.
Drawings
FIG. 1 is a schematic diagram of a size filtration method;
FIG. 2 (a) is a bright field photograph of red blood cell clusters formed by FA-and polybrene-modified red blood cells and 5-6 μm carboxyl polystyrene microspheres;
FIG. 2 (b) is a photograph of red blood cell clusters stained with DiI;
FIG. 3 (a) is a bright field photograph of a red blood cell cluster after targeting MCF-7 in PBS;
FIG. 3 (b) is a fluorescence photograph of fluorescent red blood cell clusters after targeting MCF-7 in PBS;
FIG. 4 is a schematic size diagram of defective red blood cell clusters after nonspecific adsorption of leukocytes;
FIG. 5 is a schematic diagram of the removal of nonspecifically adsorbed leukocytes by filtration;
FIG. 6 (a) is a graph showing the efficiency statistics of capturing MCF-7 by red blood cell clusters in PBS;
FIG. 6 (b) is a purity statistic for capture of MCF-7 by red blood cell clusters in buffy coat cells;
FIG. 7 is a photograph showing the result of defective red blood cell clusters prepared by folic acid red blood cells of comparative example 2 using 5 to 6 μm polystyrene microspheres with surface modified carboxyl groups and unmodified polybrene;
FIG. 8 (a) is a graph showing the efficiency statistics of capturing MCF-7 in PBS for the red blood cell clusters of comparative example 2;
FIG. 8 (b) is a purity statistic for capturing MCF-7 in a simulated blood sample;
FIG. 9 is a photograph showing the result of preparing a defective red blood cell cluster in comparative example 3, wherein the incubation temperature is 37℃during the preparation of the red blood cell cluster;
FIG. 10 (a) is a graph showing the statistics of the efficiency of capturing MCF-7 in PBS by the red blood cell clusters of comparative example 3;
FIG. 10 (b) captures purity statistics of MCF-7 in a simulated blood sample.
Detailed Description
A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever.
EXAMPLE 1 preparation of Normal erythrocyte clusters Using surface-modified carboxyl 5-6 μm polystyrene microspheres
Modification process of erythrocytes: mixing 30 μL of healthy human fresh red blood cells with 100 μL of PBS solution of DSPE-PEG-FA (1 mg/mL) with molecular weight of 5000, standing at 4deg.C for incubation for 30min, and centrifuging and washing with PBS for three times to obtain folic acid modified red blood cells. And mixing the red blood cells with the surface modified folic acid with a polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at the temperature of 4 ℃, centrifugally washing for three times by using PBS to obtain the red blood cells with the surface modified polybrene and folic acid, and dispersing the red blood cells into the PBS solution for standby.
Preparation of red cell clusters: washing 5-6 mu m polystyrene microsphere with carboxyl on the surface with PBS for three times, adding more than 100 times of microsphere count number of surface modified polybrene and folic acid red blood cells, uniformly mixing the two, centrifuging at a centrifugal speed of 400g for 3min in a centrifuge to ensure that the red blood cells and the polystyrene microsphere are mutually close to the bottom of the centrifuge tube, standing and incubating the mixture at the bottom of the centrifuge tube for 10min at 4 ℃ after centrifuging, blowing off the mixture at the bottom of the centrifuge tube, filtering by a PDMS porous filter membrane with the aperture of 10 mu m, filtering and removing excessive red blood cells and severely incomplete red blood cell clusters to obtain flower-type red blood cell clusters, and taking a bright field photo under an optical microscope as shown in fig. 2 (a).
The preparation process of the fluorescent red blood cell clusters comprises the following steps: firstly, 30 mu L of healthy human fresh red blood cells are uniformly mixed with 1000 mu L of PBS solution of DiI (10 mu g/mL), standing and incubating is carried out for 60min at 37 ℃, the red blood cells dyed by DiI are obtained by centrifugal washing with PBS for three times, and then the red blood cells are prepared according to the steps described above to obtain a red blood cell cluster dyed by DiI, and a fluorescent photograph taken under a fluorescent microscope is shown in fig. 2 (b).
Example 2 capturing MCF-7 cells by erythrocyte clusters or fluorogenic erythrocyte clusters
1. Erythrocyte clusters captured MCF-7 cells in PBS: mu.L of a suspension of red blood cell clusters (density 1.2X10) 7 mu.L of MCF-7 cell suspension (density 5X 10) was added 5 And (3) per mL), placing the mixture at the bottom of the centrifuge tube after uniform mixing, standing and incubating for 120min at 4 ℃, blowing off the mixture at the bottom of the centrifuge tube after incubation, filtering by a PDMS porous filter membrane with the aperture of 20 mu m, and filtering and removing excessive erythrocyte clusters to obtain MCF-7 cells captured by the erythrocyte clusters, wherein a bright field photo taken under an optical microscope is shown in fig. 3 (a).
2. Fluorescent red cell clusters captured MCF-7 cells in PBS: mu.L of the fluorescent red cell cluster suspension prepared in example 1 (density 1.2X10) 7 mu.L of MCF-7 cell suspension pre-stained with FDA (density 5X 10) 5 And (3) per mL), placing the mixture at the bottom of the centrifuge tube after uniform mixing, standing and incubating for 120min at 4 ℃, blowing off the mixture at the bottom of the centrifuge tube after incubation, filtering by a porous filter membrane with the aperture of 20 mu m, filtering and removing excessive erythrocyte clusters, and obtaining fluorescent MCF-7 cells captured by fluorescent erythrocyte clusters, wherein a fluorescent photograph taken under a fluorescent microscope is shown in fig. 3 (b).
3. Evaluation of efficiency of erythrocyte clusters to capture MCF-7 cells in PBS: a total of three experiments were performed, each containing five samples, each with 50 μl of red blood cell cluster suspension (density 1.2x10 7 Five samples per group were added with 10uL, 20uL, 30uL, 40uL, 50uL of MCF-7 cell suspension previously stained with FDA (density 5X 10) 5 And (3) each mL), namely 5000, 10000, 15000, 20000 and 25000 MCF-7 cells are added, and after the samples of each group are uniformly mixed, the samples are placed at 4 ℃ for standing and incubation for 120min. After incubation, 200 mu L of PBS solution is added into each sample and uniformly blown, the sample is dropped on a glass slide, and the quantitative ratio of the MCF-7 cells targeted by the erythrocyte clusters to the free MCF-7 cells is directly counted under a fluorescence microscope, so that the efficiency of capturing the MCF-7 cells in the PBS by the erythrocyte clusters can be converted to be approximately 90%, and the result is shown in a figure 6 (a).
4. Purity assessment of red blood cell clusters to capture MCF-7 cells in the buffy coat: a total of three experiments were performed, each containing five samples, each with 1000 μl of red blood cell cluster suspension (density 1.2x10 7 mu.L of FDA-stained buffy coat cell suspension (density 2X 10) 6 Each mL) of five samples were added to 10uL, 20uL, 30uL, 40uL, 50uL of MCF-7 cell suspension (density 5X 10) previously stained with Hoechst 5 Each mL), namely, 5000, 10000, 15000, 20000 and 25000 MCF-7 cells are added, and the samples of each group are uniformly mixed and then are placed at 4 ℃ for standingIncubate for 120min. After incubation, the mixture at the bottom of the centrifuge tube is blown off, the mixture is filtered by a porous filter membrane with the aperture of 20 mu m, excessive red blood cell clusters, free white blood cells and defective red blood cell clusters with nonspecifically adsorbed white blood cells are filtered and removed, the filter membrane is directly placed under a fluorescence microscope for observation, the ratio between the number of the Hoechst-dyed MCF-7 cells and the number of the FDA-dyed white blood cells on the surface of the filter membrane is directly counted, and the purity of the red blood cell clusters for capturing the MCF-7 cells in a white membrane layer can be obtained through conversion, and the overall result is close to more than 80 percent, as shown in fig. 6 (b).
Comparative example 1 preparation of erythrocyte clusters Using polystyrene microspheres having 5 to 6 μm without surface modification groups
Except that the polystyrene microsphere used in the preparation process is provided with no surface modification group, other experimental conditions are completely the same as those in example 1, and finally, the erythrocyte cluster is not obtained, and modified erythrocytes cannot be adsorbed on the surface of the polystyrene microsphere without the modification group, so that the erythrocyte cluster cannot be obtained after filtration through a 10 mu m membrane.
Comparative example 2 incomplete erythrocyte clusters prepared with folic acid erythrocytes having 5-6 μm polystyrene microspheres with surface modified carboxyl groups and unmodified polybrene
Except that folic acid red blood cells are not modified on the surface in the preparation process of the incomplete red blood cell cluster, other experimental conditions are completely the same as those of the embodiment 1, and the incomplete red blood cell cluster is finally obtained, namely, the surface of the core microsphere only adsorbs a small amount of red blood cells, and the exposed microsphere surface is obviously present. In addition to the incomplete clusters of erythrocytes, there are fully naked polystyrene microspheres to which erythrocytes are not adsorbed. As particularly shown in fig. 7.
Evaluation of efficiency of the incomplete erythrocyte clusters to capture MCF-7 cells in PBS: the experimental procedure was exactly the same as in example 2, and the efficiency of capturing MCF-7 cells in PBS was nearly 80% for the final defective red blood cell clusters, and the results are shown in FIG. 8 (a).
Purity assessment of the capture of the incomplete erythrocyte clusters in the buffy coat of MCF-7 cells: the experimental procedure was exactly the same as in example 2, and the purity of the finally obtained defective red blood cell clusters for capturing MCF-7 cells in the buffy coat was less than 10%, and the results are shown in FIG. 8 (b).
[ comparative example 3 ] incomplete erythrocyte clusters prepared at 37℃during the preparation of erythrocyte clusters
Except that the incubation temperature used was 37℃in the preparation of the red cell clusters, the experimental conditions were exactly the same as in example 1, and the resulting red cell clusters were essentially defective. I.e. only a small amount of erythrocytes are adsorbed on the surface of the core microsphere, and the naked microsphere surface is obviously present. In addition to the incomplete clusters of erythrocytes, there are fully naked polystyrene microspheres to which erythrocytes are not adsorbed. As shown in particular in fig. 9.
Evaluation of efficiency of the defective erythrocyte clusters in PBS to capture MCF-7 cells. The experimental procedure was exactly the same as in example 2, and the efficiency of capturing MCF-7 cells in PBS was nearly 70% for the final defective red blood cell clusters, and the results are shown in FIG. 10 (a).
The defective red blood cell clusters captured the purity assessment of MCF-7 cells in the buffy coat. The experimental procedure was exactly the same as in example 2, and the purity of the finally obtained defective red blood cell clusters for capturing MCF-7 cells in the buffy coat was less than 10%, and the results are shown in FIG. 10 (b).
Claims (9)
1. A red blood cell cluster based on size filtration for enriching circulating tumor cells, which is prepared by the following method:
(1) Folate or antibody targeted by CTC (cell-mediated) on surface of erythrocytes
First category: folic acid modification, namely uniformly mixing 10 mu L-50 mu L of healthy human fresh red blood cells with 100 mu L-1000 mu L of PBS solution of DSPE-PEG-FA with molecular weight 3400-10000 and concentration of 1mg/mL, standing and incubating for 30 min-2 h at 4 ℃, and centrifugally washing for three times by using PBS to obtain folic acid modified red blood cells; or alternatively, the process may be performed,
the second category: antibody modification capable of specifically recognizing and combining CTCs surface biomarkers, uniformly mixing 10 mu L-50 mu L of healthy human fresh red blood cells with 100 mu L-1000 mu L of PBS solution of DSPE-PEG-biotin with molecular weight 3400-10000 and concentration of 1mg/mL, standing at 4 ℃ for incubation for 30 min-2 h, and centrifugally washing with PBS for three times to obtain red blood cells of the surface modified biotin; uniformly mixing biological modified red blood cells with 100 mu L-1000 mu L of SA solution with the concentration of 100ug/mL, standing and incubating for 30 min-2 h at the temperature of 4 ℃, and centrifugally washing for three times by using PBS (phosphate buffer solution) to obtain the red blood cells with the surface modified SA; finally, uniformly mixing SA modified red blood cells with 100-1000 mu L of biotinylated CTCs targeting antibody solution with the concentration of 5ug/mL, standing at 4 ℃ for incubation for 30 min-2 h, and centrifugally washing with PBS for three times to obtain the red blood cells of the surface modified CTCs targeting antibody;
(2) Modification of erythrocyte surface polybrene
Mixing 10 mu L-50 mu L of red blood cells of the antibody with the FA or CTC targeting on the surface and 100 mu L-500 mu L of polybrene PBS solution with the concentration of 1-100 mg/mL, standing and incubating for 30 min-2 h at the temperature of 4 ℃, centrifugally washing for three times by using PBS to obtain red blood cells of the antibody with the FA or CTC targeting on the surface, and dispersing the red blood cells into the PBS solution for standby;
(3) Preparation of erythrocyte clusters
Washing the microsphere with the surface modification group with PBS for three times, adding more than 100 times of the number of the microsphere to the red blood cells in the step (2), uniformly mixing the two, centrifuging the mixture in a centrifuge at a centrifugal speed of 300 g-600 g for 1 min-30 min to ensure that the red blood cells and the microsphere are close to each other at the bottom of the centrifuge tube, standing and incubating the mixture at the bottom of the centrifuge tube at 4 ℃ for 20 min-2 h after centrifuging, and finally blowing off the mixture at the bottom of the centrifuge tube, and filtering the suspension by using a biocompatible microporous membrane with the aperture of 10 mu m; excess free red blood cells, microspheres that do not form red blood cell clusters, and defective red blood cell clusters of less than 10 μm can be removed by filtration; finally, clusters of red blood cells exceeding 10 μm in size were collected from the membrane.
2. The erythrocyte pellet of claim 1, wherein the surface biomarker of CTCs in step (1) of the process for preparing the erythrocyte pellet is any one of the epithelial markers cytokeratin, epithelial cell adhesion molecule, tumor embryo antigen, her 2, venous endothelial cell molecule, epstein, sialylated lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45 or CD146.
3. The red blood cell cluster of claim 1, wherein the antibody that specifically recognizes and binds to a CTCs surface biomarker in step (1) of the preparation method of the red blood cell cluster comprises any one of an epithelial marker cytokeratin antibody, an epithelial cell adhesion molecule antibody, a tumor embryo antibody, a her 2 antibody, a vein endothelial cell molecule antibody, an annexin antibody, a sialylated lewis oligosaccharide-X antibody, an acetaldehyde dehydrogenase 1 antibody, a vimentin antibody, a urokinase receptor antibody, a heparanase antibody, a prostate-specific membrane antibody, an anti-CD44, an anti-CK18, an anti-CD133, an anti-CD90, an anti-CD45, or an anti-CD146.
4. The red blood cell cluster according to claim 1, wherein the molecular weight of DSPE-PEG-FA or DSPE-PEG-biotin in step (1) of the method of preparing the red blood cell cluster is 5000.
5. The red blood cell cluster according to claim 1, wherein the modification group on the microsphere surface in the step (3) is a carboxyl group or an amino group.
6. The red blood cell cluster according to claim 1, wherein the microspheres of the surface modifying group of step (3) have a diameter of 5 to 6 μm.
7. Use of the red blood cell cluster of any one of claims 1-6 for the preparation of a kit for identifying, capturing or enriching circulating tumor cells.
8. The use according to claim 7, wherein the method for enriching circulating tumor cells with red blood cell clusters in said use essentially comprises the steps of: 1) Treating the blood sample with the erythrocyte lysate to remove erythrocytes in the blood sample, so as to avoid interference of erythrocytes in the sample on capturing CTCs by erythrocyte clusters; 2) Adding the red blood cell clusters of any one of claims 1-6 to a red blood cell-free blood sample, and allowing the red blood cell clusters to specifically bind to CTCs by incubation at 4 ℃ for a period of time; 3) Separating the sample in the step 2) by a 20 mu m porous filter membrane filtration mode, namely realizing the high-purity enrichment of the circulating tumor cells; 4) And adding a sodium citrate solution with the concentration of 4% prepared by the erythrocyte lysate into the erythrocyte clusters captured with the CTCs and obtained by filtering in the step 3), and realizing the release process of the CTCs on the basis of most erythrocyte shedding on the surfaces of the clusters.
9. The use according to claim 7, characterized in that the method for enriching CTCs with red blood cell clusters in said use essentially comprises the following steps: 1) Obtaining a blood sample of a tumor patient after operation or chemotherapy, and obtaining a lymphocyte layer through a percoll cell separation liquid; 2) Adding the red blood cell clusters of any one of claims 1-6 to a lymphocyte layer for co-incubation at 4 ℃ to perform capture; 3) Separating the sample in the step 2) by a 20 mu m porous filter membrane filtration mode, namely realizing the high-purity enrichment of the circulating tumor cells; 4) Adding 4% sodium citrate solution prepared from erythrocyte lysate into the erythrocyte clusters captured with CTCs obtained by filtering in the step 3), and realizing the release process of CTCs on the basis of most erythrocyte shedding on the surfaces of the clusters; 5) After the captured cells were subjected to three-fluorescence staining, the number of CTCs captured was quantitatively analyzed by flow cytometry, and the capturing was analyzed by confocal laser.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010175489 | 2020-03-13 | ||
| CN2020101754891 | 2020-03-13 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111826349A CN111826349A (en) | 2020-10-27 |
| CN111826349B true CN111826349B (en) | 2023-08-15 |
Family
ID=72917923
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010867920.9A Active CN111826349B (en) | 2020-03-13 | 2020-08-26 | Erythrocyte cluster based on size filtration method for enriching circulating tumor cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111826349B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112011434B (en) * | 2020-08-26 | 2022-01-04 | 武汉大学 | Red blood cell bionic coating for enriching circulating tumor cells |
| CN113201493B (en) * | 2021-04-13 | 2022-07-05 | 武汉大学 | Preparation method of erythrocyte clusters with ideal morphology |
| CN113151176B (en) * | 2021-04-13 | 2022-07-05 | 武汉大学 | Circulating tumor cell enrichment method based on diethylaminoethyl agarose microspheres and spherical erythrocytes |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2406654A1 (en) * | 2000-04-20 | 2001-11-01 | The University Of British Columbia | Methods of enhancing splp-mediated transfection using endosomal membrane destabilizers |
| CN101469323A (en) * | 2007-12-28 | 2009-07-01 | 英科新创(厦门)科技有限公司 | Method for separating red corpuscle from whole blood |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN108588018A (en) * | 2018-01-09 | 2018-09-28 | 段莉 | A kind of function red blood cell of targeting circulating tumor cell CTCs |
-
2020
- 2020-08-26 CN CN202010867920.9A patent/CN111826349B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2406654A1 (en) * | 2000-04-20 | 2001-11-01 | The University Of British Columbia | Methods of enhancing splp-mediated transfection using endosomal membrane destabilizers |
| CN101469323A (en) * | 2007-12-28 | 2009-07-01 | 英科新创(厦门)科技有限公司 | Method for separating red corpuscle from whole blood |
| CN104178454A (en) * | 2013-05-24 | 2014-12-03 | 益善生物技术股份有限公司 | Enrichment and analysis method for circulating tumor cells |
| CN108588018A (en) * | 2018-01-09 | 2018-09-28 | 段莉 | A kind of function red blood cell of targeting circulating tumor cell CTCs |
Non-Patent Citations (1)
| Title |
|---|
| Taoye Zhang 等."Ultradense Erythrocyte Bionic Layer Used to Capture Circulating Tumor Cells and Plasma-Assisted High-Purity Release".《ACS Appl. Mater. Interfaces》.2021,第13卷第24543−24552页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111826349A (en) | 2020-10-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111826351B (en) | Magnetic red blood cell cluster for enriching circulating tumor cells based on magnetic separation method | |
| CN111826349B (en) | Erythrocyte cluster based on size filtration method for enriching circulating tumor cells | |
| WO2022041644A1 (en) | Erythrocyte biomimetic coating for enriching circulating tumor cells | |
| Franquesa et al. | Update on controls for isolation and quantification methodology of extracellular vesicles derived from adipose tissue mesenchymal stem cells | |
| CN109507418B (en) | Magnetic nanoparticle with cell-like structure, immunomagnetic nanoparticle, and preparation method and application thereof | |
| JP6661368B2 (en) | Multi-sort cell separation method | |
| US20140315295A1 (en) | Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof | |
| US20200009563A1 (en) | Methods and Systems for Circulating Tumor Cell Capture | |
| Cui et al. | ZnO nanowire-integrated bio-microchips for specific capture and non-destructive release of circulating tumor cells | |
| WO2009140876A1 (en) | Integration methods of enriching and detecting rare cells from biological body fluids samples | |
| CN105087493B (en) | Three kinds of monoclonal antibody coupling immunomagnetic beads of combination are applied in tumour cell sorting | |
| CN110036111A (en) | The probe based on lipid for extracellularly separating | |
| CN107287107A (en) | A kind of circulating tumor cell separation equipment, system and method | |
| CN106701684A (en) | High-efficiency circulating tumor cell enrichment method | |
| CN108588018A (en) | A kind of function red blood cell of targeting circulating tumor cell CTCs | |
| CN109161530B (en) | Separation method of circulating tumor cells based on phenylboronic acid | |
| Jørgensen et al. | A melt-electrowritten filter for capture and culture of circulating colon cancer cells | |
| CN106635769B (en) | Cell separation apparatus and cell isolation method | |
| CN114958743A (en) | Separation detection method and application of neutrophil exosome | |
| CN110054696B (en) | Mussel bionic polypeptide composite magnetic bead and preparation method and application thereof | |
| Wu et al. | Positively charged magnetic nanoparticles for capture of circulating tumor cells from clinical blood samples | |
| US11860157B2 (en) | Polymer microfilters, devices comprising the same, methods of manufacturing the same, and uses thereof | |
| CN112718027B (en) | Microfluidic chip and temperature-sensitive material composite system and preparation method and application thereof | |
| CN116064225A (en) | Microfluidic chip for detecting circulating tumor cells and preparation method and application thereof | |
| CN108548920A (en) | A kind of detection method for the kit detecting circulating tumor cell using immunomagnetic beads negative sense absorption joint flow cytometry |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |