CN113088494B - Method for releasing tumor cells captured by erythrocyte biomimetic material - Google Patents
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Abstract
The invention discloses a method for releasing tumor cells captured by a red blood cell bionic material, and belongs to the technical field of biological medicines. The invention finds that DSPE-PEG-X molecules can fall off from erythrocyte membranes under the action of a complete culture medium, the method of the invention incubates the erythrocyte bionic material for capturing tumor cells with the culture medium, and the tumor cells are released from the erythrocyte bionic material after incubation; the red blood cell bionic material comprises pure red blood cells modified with DSPE-PEG-X (X represents FA, RGD or biotin), red blood cells modified by nano materials, red blood cell clusters, a plane red blood cell bionic layer and the like. The tumor cells released by the method of the invention can be directly cultured under the condition of complete culture medium without the need of liquid changing treatment.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a method for releasing tumor cells captured by a red blood cell bionic material.
Background
Local or regional tumor recurrence and distant metastasis present serious challenges for early diagnosis and cure of tumors. Molecular biological and clinical studies in recent years have shown that Circulating Tumor Cells (CTCs) contribute to the invasion and micrometastasis process of tumors. The CTCs are formed by the shedding of original tumor cells from a primary tumor and the entering of blood systems of human bodies, and are finally transferred to other tissues and organs of the human bodies through the circulation and the diffusion of peripheral blood, so that secondary tumors of the same type as the primary tumors are formed, namely, the metastasis and the 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, convenient and easy method and means are provided for the treatment response detection of cancer medication and the treatment effect analysis of radiotherapy and chemotherapy.
Since the content of CTCs in peripheral blood is very low, there are only a few to several tens of CTCs per ml of blood,the content of background blood cells reaches 10 9 The magnitude of the interference is greatly interfered for the separation and enrichment detection of the CTCs, so that the good separation and enrichment of the CTCs requires high sensitivity and high selectivity, and the activity of the CTCs can be maintained. Numerous methods for separation and enrichment of CTCs have been developed, including separation methods based on differences in physical properties (size, density, dielectrophoresis, etc.) and immunoaffinity methods based on surface-modified antibodies. Since there is a certain overlap in the physical properties of CTCs and leukocytes, it is difficult to efficiently remove leukocytes from isolated CTCs, which makes it difficult to achieve both high efficiency and high purity by a separation method based on only one physical property. The immunoaffinity method based on the surface modified antibody can specifically target antigens on the CTCs, and although the antigen-antibody combination has high specificity, the purity of the CTCs which are actually separated can be adversely affected due to the fact that the conventional materials have a certain non-specific adsorption on unrelated cells.
In order to reduce the nonspecific adsorption of the material for modifying the antibody to irrelevant cells as much as possible, one method in the prior art is to design bionic materials based on red blood cells, including pure red blood cells, red blood cells modified by nano materials, red blood cell clusters, planar red blood cell bionic layers and the like. The materials have the common point that red blood cells are used as a capture interface of circulating tumor cells, the interface is of a biological membrane structure of the red blood cells, DSPE-PEG-X (X represents FA or biotin) type molecules are inserted into a phospholipid bilayer of a cell membrane by utilizing hydrophilic and hydrophobic acting forces, and antibody molecules capable of specifically targeting the surface antigen of the circulating tumor cells are modified by utilizing a biotin-SA specific binding mode (FA can directly target the circulating tumor cells). The biomembrane structure has excellent non-specific adsorption resistance to other irrelevant cells, so that the high-purity enrichment of the circulating tumor cells can be realized.
For subsequent culture analysis, it is often necessary to release the enriched high purity circulating tumor cells. For such biomimetic materials based on erythrocytes, a known method for releasing circulating tumor cells captured on their surface is the erythrocyte lysate release method. The red blood cell lysate in the market comprises two types, one is an enzyme lysate, and the enzyme lysate contains an enzyme which can attack a specific red blood cell surface antigen, can cause red blood cell deformation, biological channel expansion and swelling lysis, or cause red blood cell degeneration; the other type is ammonium chloride type lysate, ammonium ions cannot permeate cell membranes, and other ions can pass through the cell membranes, so that the ion concentration difference between the inside and the outside of cells, namely osmotic pressure difference, is caused, and finally, external water is diffused into the cells to burst red blood cells. The two types of erythrocyte lysates are difficult to completely break up a cell membrane phospholipid molecular layer, so that DSPE-PEG-X molecules modified based on hydrophilic and hydrophobic acting forces are difficult to release by cell membranes, and the bonding force between denatured erythrocytes acted by the erythrocyte lysates and circulating tumor cells still exists, so that the high-efficiency release of the circulating tumor cells is difficult to realize. In addition, the erythrocyte lysate cannot achieve 100% accurate lysis of erythrocytes, which always leads to lysis of more or less other cell types. In view of the above, there is a need to search for an efficient and mild method for releasing circulating tumor cells from a capture material of the erythroid series.
Disclosure of Invention
The present invention aims at overcoming the demerits and demerits of available technology, and provides one kind of method for releasing tumor cell captured by red blood cell bionic material.
The core technology discovery of the invention is derived from the summary of the results of multiple failure experiments for capturing circulating tumor cells by erythrocytes. The core technology for preparing the bionic material of the red blood cells for enriching the circulating tumor cells is that the surface of the red blood cells is modified with molecules of specific target circulating tumor cells, the modification process needs to modify DSPE-PEG-X (X is FA, RGD or biotin) type molecules on the surface of the red blood cell membrane, the appearances of the red blood cells before and after modification can be obviously changed, and the preparation method can be used as a proof for successful modification of the DSPE-PEG-X type molecules. First, taking the modification of DSPE-PEG-FA as an example, FIG. 1 shows a bright field image and an SEM image of an erythrocyte optical microscope comparing the unmodified and two DSPE-PEG-FA surface modification amounts. As is apparent from FIG. 1, the morphology of the red blood cells modified with DSPE-PEG-FA is changed from the original cake shape to the spinous red blood cells with sharp protrusions on the surface, and the larger the modification amount, the more circular the shape of the spinous red blood cells, which shows that the morphology of the red blood cells can be changed by the different modification amount of DSPE-PEG-FA. Similarly, erythrocytes were also modified with DSPE-PEG-FITC, and FIG. 2 is a fluorescence micrograph of modified echinoid erythrocytes, thereby also illustrating that morphological changes of erythrocytes are related to the modification of DSPE-PEG-FITC.
After modification of the erythrocyte surface with a molecule specifically targeting circulating tumor cells (folic acid for example), it was mixed with freshly passaged Hela cells in DMEM complete medium and incubated at 37 ℃ for 2 h. After that, the mixed cells were blown to observe the trapping effect, but no HeLa cells were observed which were trapped by folic acid-modified erythrocytes (originally, echiniform erythrocytes), whereas folic acid-modified acupunctate erythrocytes almost completely changed back to folic acid-modified cake erythrocytes, as shown in FIG. 3 (a).
As can be seen from the results shown in FIGS. 1 and 2, the appearance of the red blood cells is changed from cake shape to spiky sphere shape due to the modification of DSPE-PEG-X molecules on the surface of the red blood cells, and the phenomenon that the red blood cells are changed from spiky sphere shape to cake shape in the experimental process shown in FIG. 3(a) indicates the shedding of the modified DSPE-PEG-FA molecules, and it is preliminarily hypothesized that the shedding phenomenon may be related to the existence of DMEM complete medium. To confirm this hypothesis, the Hela cells without DMEM complete medium after washing with PBS were subjected to the same incubation operation as the folic acid-modified spiked globular erythrocytes, and as a result, no phenomenon was observed in which the folic acid-modified spiked globular erythrocytes changed back to cake-like erythrocytes before folic acid modification, and the spiked globular erythrocytes successfully targeted the Hela cells, as shown in fig. 3(b), thereby preliminarily demonstrating that DMEM complete medium causes DSPE-PEG-FA to be detached from the surface of the spiked globular erythrocytes. For more evidence, DMEM complete medium is directly added into the spine spherical erythrocytes modified with DSPE-PEG-FA, and the spine spherical erythrocytes are incubated at 37 ℃ for 2 hours, so that the morphology of the spine erythrocytes is finally changed back to a cake shape, as shown in FIG. 3(c), and the phenomenon that the DSPE-PEG-FA falls off from the surfaces of the spine spherical erythrocytes is again proved by the DMEM complete medium.
Similarly, the spiked spherical erythrocytes obtained by modifying DSPE-PEG-FITC were also incubated with DMEM complete medium, and the morphology of the spiked erythrocytes was changed back to the cake erythrocytes, as shown in FIGS. 4(a) and 4 (b). Meanwhile, the green fluorescent red blood cells with the surface modified by the DSPE-PEG-FITC also fall off along with the DSPE-PEG-FITC to become red blood cells with no fluorescence on the surface, and meanwhile, the dispersion liquid is changed from no fluorescence to uniform green fluorescence, as shown in FIG. 4 (c). The red blood cells were washed three more times with PBS, the DMEM complete medium containing exfoliated DSPE-PEG-FITC was removed, and the green fluorescence that turned back to the surface of the cake-like red blood cells was substantially not observed by fluorescence microscopy, as shown in FIG. 4 (d). Thus, the DMEM complete medium can realize the effect of shedding the DSPE-PEG-FITC from the erythrocyte membrane.
Similarly, when the echinocyte modified by DSPE-PEG-biotin was incubated with DMEM complete medium, it was also observed that the morphology of the echinocyte was changed back to a pie-shaped erythrocyte, as shown in FIGS. 5(a) and 5 (b). Thus, the DMEM complete medium can realize the effect of shedding the DSPE-PEG-biotin from the erythrocyte membrane.
By combining the experimental phenomena, the invention proves that the DMEM complete medium can realize the effect of shedding DSPE-PEG-X (X is FA, FITC or biotin) from an erythrocyte membrane, so that other DSPE-PEG-X analogs (such as DSPE-PEG-RGD) can be released under the action of the DMEM complete medium.
Biomimetic materials designed based on red blood cells are generally required to capture circulating tumor cells by modifying the red blood cell membrane at the capture interface with molecules of the DSPE-PEG-X (X stands for FA, RGD or biotin) type. If the modified product is DSPE-PEG-biotin, the specific binding mode of the biotin-SA is also needed, SA is taken as a bridge molecule, and finally, an antibody molecule specifically targeting the circulating tumor cells is modified. When the bionic material based on the red blood cells captures the circulating tumor cells, the capture of the circulating tumor cells by the interface red blood cells depends on a large number of molecular bridges connected between the two, one end of each molecular bridge is embedded into the red blood cell membrane by virtue of hydrophobic interaction force between the DSPE end and the red blood cell membrane, and the other end of each molecular bridge forms specific combination with antigens on the surface of the tumor cells by virtue of specific molecules or antibodies. This was inspired since the DMEM complete medium could achieve the shedding of DSPE-PEG-X type molecules from the erythrocyte membrane, which was the result of the release of circulating tumor cells by the DSPE ends on the interface erythrocytes that captured circulating tumor cells, and was finally confirmed by the experiments in the examples.
Meanwhile, the invention also discovers that the complete culture medium commonly used for cell culture can achieve similar effects, such as DMEM high-sugar complete culture medium, RPMI-1640 complete culture medium, McCoy's 5A complete culture medium and MEM complete culture medium, and corresponding experimental results are shown in the embodiment of the invention.
Based on the above, the purpose of the invention is realized by the following technical scheme:
a method for releasing tumor cells captured by a red blood cell biomimetic material, comprising the steps of: and incubating the erythrocyte biomimetic material for capturing the tumor cells by using a culture medium, and releasing the tumor cells from the erythrocyte biomimetic material after incubation. The red blood cell bionic material is pure red blood cells modified with DSPE-PEG-X (X represents FA, RGD or biotin), red blood cells modified with nano materials, red blood cell clusters, a plane red blood cell bionic layer and the like, if the modified DSPE-PEG-biotin is DSPE-PEG-biotin, the specific combination mode of the biotin-SA is further required to be utilized, SA is taken as a bridge molecule, and finally, antibody molecules specifically targeting tumor cells are modified.
Further, the tumor cells include but are not limited to Hela cells, MCF-7 cells, SKOV3 cells, HT29 cells, and the like.
Further, the medium is a complete medium, including but not limited to DMEM complete medium, DMEM high-sugar complete medium, RPMI-1640 complete medium, McCoy's 5A complete medium, MEM complete medium, and the like.
Further, the incubation condition is incubation for 0.1 min-2 h at 37 ℃.
Still further, the method for releasing tumor cells captured by the erythrocyte biomimetic material comprises the following steps:
(1) and removing the buffer solution in the system after the erythrocyte biomimetic material captures the tumor cells. The buffer may be PBS buffer.
(2) The medium was added to the trapping system and the trapping system was completely submerged.
(3) And (3) incubating the capture system at the temperature of 37 ℃ for 0.1 min-2 h, releasing the tumor cells from the erythrocyte bionic material, and transferring the whole system to an incubator to re-culture the released tumor cells.
The invention has the beneficial effects that: since the complete medium can be used for culturing a variety of tumor cells, the tumor cells released by the action of the complete medium can be directly cultured under the condition of the complete medium without performing a liquid change treatment on the tumor cells. In the traditional method for releasing the erythrocyte lysate, the erythrocyte lysate needs to be removed firstly after release, and then the culture medium is added to realize re-culture of the released tumor cells, so that the method has obvious advantages compared with the traditional method.
Drawings
FIG. 1 is a bright field and SEM images of unmodified erythrocytes and erythrocytes modified with varying amounts of DSPE-PEG-FA. Wherein (a) is a bright field pattern with a scale bar of 20 microns; (b) is an SEM image, scale bar 5 microns.
FIG. 2 is a bright field (a) and fluorescence (b) plot of DSPE-PEG-FITC-modified Burr spherical erythrocytes, on a scale: 20 microns.
FIG. 3 is a bright field diagram of folate-modified red blood cells under different conditions. Wherein (a) is a bright field image of folic acid modified red blood cells after the capture of Hela cells in a DMEM complete medium environment; (b) is a bright field picture of the red blood cells with modified folic acid after capturing Hela cells in a PBS environment; (c) is a bright field diagram of folic acid modified red blood cells after incubation in a DMEM complete medium environment, and a scale bar is as follows: 20 microns.
FIG. 4 is a diagram showing the state of DSPE-PEG-FITC-modified erythrocytes in different cases. Wherein (a) is a fluorescence map of DSPE-PEG-FITC-modified erythrocytes; (b) is a bright field picture after DSPE-PEG-FITC modified red blood cells are treated by a DMEM complete culture medium; (c) is a fluorescence image after DSPE-PEG-FITC modified red blood cells are treated by a DMEM complete culture medium; (d) is a fluorescence image obtained after DSPE-PEG-FITC modified red blood cells are treated by DMEM complete medium and then washed by PBS, and the proportion is as follows: 20 microns.
FIG. 5 is a bright field diagram of DSPE-PEG-biotin modified erythrocytes in different cases. Wherein (a) is a bright field pattern of DSPE-PEG-biotin-modified erythrocytes; (b) is a bright field diagram of DSPE-PEG-biotin modified red blood cells treated by DMEM complete medium, and is scaled as follows: 20 microns.
FIG. 6 is a graph showing the results of releasing of red blood cell-captured HeLa cells modified with DSPE-PEG-FA using DMEM complete medium. Wherein (a) is a bright field pattern prior to release; (b) is the bright field pattern after release, scale: 20 microns.
FIG. 7 is a graph showing the results of the release of red blood cell-captured MCF-7 cells modified with DSPE-PEG-RGD on the surface thereof using DMEM high-glucose complete medium. Wherein (a) is a bright field pattern prior to release; (b) is the bright field pattern after release, scale: 20 microns.
FIG. 8 is a graph showing the results of magnetic erythrocyte-captured MCF-7 cells surface-modified with EpCAM antibody released in RPMI-1640 complete medium. Wherein (a) is a bright field pattern prior to release; (b) is the bright field pattern after release, scale: 20 microns.
FIG. 9 is a graph showing the results of releasing magnetic red blood cell clusters-captured SKOV3 cells with McCoy's 5A complete medium, which were surface-modified with polybrene and DSPE-PEG-FA. Wherein (a) is a bright field pattern prior to release; (b) is the bright field pattern after release, scale: 10 microns.
FIG. 10 is a graph showing the results of the release of HeLa cells trapped in a planar biomimetic layer of red blood cells surface-modified with polybrene and DSPE-PEG-RGD in MEM complete medium. Wherein (a) is a bright field pattern prior to release; (b) is the bright field pattern after release, scale: 20 microns.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
The erythrocytes modified with different amounts of DSPE-PEG-FA in fig. 1 were prepared: to red blood cell dispersion (whole separation by Percoll separation liquid)Obtaining pure red blood cells from blood, washing the red blood cells for three times by using a PBS solution, dispersing the red blood cells by using the PBS solution to obtain a red blood cell dispersion solution), adding DSPE-PEG-FA into the red blood cells, incubating, centrifuging and washing to obtain the red blood cells with the surfaces modified with folic acid. Wherein, when preparing red blood cells with less surface modification, the proportion of the original red blood cells to the DSPE-PEG-FA is 5 × 10 7 Adding 0.02mg DSPE-PEG-FA into each erythrocyte; when preparing red blood cells with larger surface modification amount, the proportion of the used amount of the primary red blood cells to the used amount of the DSPE-PEG-FA is 5 x 10 7 0.04mg of DSPE-PEG-FA was added to each erythrocyte.
The preparation of the DSPE-PEG-FITC modified spiculated globular erythrocytes of fig. 2 was: adding DSPE-PEG-FITC into the erythrocyte dispersion liquid, incubating, centrifuging, and washing to obtain the surface modified FITC burr spherical erythrocyte. Wherein the dosage ratio of primary erythrocyte to DSPE-PEG-FITC is 5 × 10 7 0.04mg DSPE-PEG-FITC was added to each erythrocyte.
The folate-modified red blood cells of FIG. 3 were prepared as follows: adding DSPE-PEG-FA into the erythrocyte dispersion liquid, incubating, centrifuging, and washing to obtain the burr spherical erythrocyte with surface modified folic acid. Wherein the dosage ratio of primary red blood cells to DSPE-PEG-FA is 5 × 10 7 0.02mg of DSPE-PEG-FA was added to each erythrocyte.
The DSPE-PEG-FITC-modified erythrocytes of FIG. 4 were prepared as the DSPE-PEG-FITC-modified Burr-spherical erythrocytes of FIG. 2.
The DSPE-PEG-biotin modified erythrocytes in FIG. 5 were prepared: adding DSPE-PEG-biotin into the erythrocyte dispersion liquid, incubating, centrifuging and washing to obtain the echinoid erythrocyte with surface modified biotin. Wherein the dosage proportion of the primary red blood cells to the DSPE-PEG-biotin is 5 × 10 7 0.04mg of DSPE-PEG-biotin was added to each erythrocyte.
Example 1 Release of Red blood cells having DSPE-PEG-FA modified on the surface thereof captured Hela cells in DMEM complete Medium
(1) Capture
The system uses red blood cells with the surface modified with DSPE-PEG-FA to capture Hela cells, and the total volume of the capture system is 0.2 mL.
The preparation process of the erythrocyte modified with DSPE-PEG-FA on the surface comprises the following steps: adding DSPE-PEG-FA into the erythrocyte dispersion liquid, incubating, centrifuging, and washing to obtain the burr spherical erythrocyte with surface modified folic acid. Wherein the dosage ratio of primary red blood cells to DSPE-PEG-FA is 5 × 10 7 0.02mg of DSPE-PEG-FA was added to each erythrocyte.
And (3) capturing Hela cells: quantitative measuring 10 5 Hela cells were washed with PBS and dispersed, and 10 was quantitatively added thereto 7 Mixing the red blood cells with the surfaces modified with the DSPE-PEG-FA uniformly, centrifuging the mixture in a centrifuge at 1500rpm for 3min, and then incubating the mixture at 37 ℃ for 1h to realize the capture of the pure red blood cells modified with the DSPE-PEG-FA on the Hela cells.
(2) Releasing
First, the cells of the whole system were centrifuged at a centrifugation speed of 1500rpm, and the supernatant was removed. Then 1mL of DMEM complete medium was added and the cells were gently blown into suspension. Finally, the whole system is placed in an environment at 37 ℃ for incubation for 2 h. The settled cells were intermittently subjected to a suspension procedure during incubation to ensure that the DMEM complete medium was in sufficient contact with the capture system.
The picture before release is shown in fig. 6(a), and the picture after release is shown in fig. 6(b), and the release efficiency is about 95%.
[ example 2 ] MCF-7 cells captured by erythrocytes modified with DSPE-PEG-RGD on their surfaces were released in DMEM high-sugar complete Medium
(1) Capture
The system uses red blood cells with the surface modified with DSPE-PEG-RGD to capture MCF-7 cells, and the total volume of the capture system is 0.4 mL.
The preparation process of the red blood cells with the surface modified with DSPE-PEG-RGD comprises the following steps: adding DSPE-PEG-RGD into the erythrocyte dispersion liquid, incubating, centrifuging, and washing to obtain the burr spherical erythrocyte with the surface modified with DSPE-PEG-RGD. Wherein the proportion of the usage of the primary red blood cells and the usage of the DSPE-PEG-RGD is 5 × 10 7 0.02mg of DSPE-PEG-RGD was added to each red blood cell.
And (3) capturing Hela cells: quantitative measuring 10 5 An MCF-7 cell and PBSWashing and dispersing, and quantitatively adding 10 7 The red blood cells of which the surfaces are modified with DSPE-PEG-RGD are evenly mixed and then are centrifuged for 3min at 1500rpm in a centrifuge, and then the mixture is incubated for 1h at 37 ℃, so that the capture of the pure red blood cells modified with DSPE-PEG-RGD to MCF-7 cells can be realized.
(2) Releasing
First, the cells of the whole system were centrifuged at a centrifugation speed of 1500rpm, and the supernatant was removed. Then 1.2mL of DMEM high-sugar complete medium was added and the cells were gently resuspended by air-suspension. Finally, the whole system is placed in an environment at 37 ℃ for incubation for 2 h. The settled cells were intermittently subjected to a suspension procedure during incubation to ensure that the DMEM high-glucose complete medium was in sufficient contact with the capture system.
The picture before release is shown in fig. 7(a), and the picture after release is shown in fig. 7(b), and the release efficiency is about 93%.
Example 3 Release of MCF-7 cells captured by magnetic erythrocytes having EpCAM antibody modified on their surface in RPMI-1640 complete Medium
(1) Capture
The magnetic erythrocyte with the surface modified with the EpCAM antibody is prepared by loading magnetic nanoparticles into the erythrocyte by an electroporation method, the surface of the erythrocyte is also modified with DSPE-PEG-biotin/SA/biotin-anti EpCAM, and the total volume of the capture system is 0.3 mL.
The preparation process of the magnetic red blood cell with the EpCAM antibody modified on the surface comprises the following steps: in the first step, red blood cells are loaded with magnetic nanoparticles. To contain 10 9 Adding 100 mu L of 5mg/mL 20nm magnetic ferroferric oxide nanoparticle suspension into 500 mu L of suspension of fresh red blood cells, uniformly mixing the suspension and the suspension, transferring the mixture into a 2cm electric transfer cup in two times, and loading the magnetic nanoparticles into the red blood cells in an electroporation mode. Removing non-magnetic red blood cells by magnetic separation, and removing free magnetic nanoparticles by means of low-speed centrifugation at 1000rpm, thereby obtaining pure magnetic red blood cells. In the second step, magnetic erythrocytes modify the EpCAM antibody. Quantitatively take out 1.5X 10 8 Adding quantitative red blood cell, adding PBS solution containing 0.06mg DSPE-PEG-biotin with molecular weight of 2000, and incubating at 4 deg.CCentrifuging and washing for three times by PBS (phosphate buffer solution) for 30min to obtain surface-modified biotin red blood cells; uniformly mixing biotin-modified red blood cells with 200 mu L of SA solution with the concentration of 100 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by PBS to obtain red blood cells with surface modified SA; and finally, uniformly mixing the SA modified erythrocyte with 500 mu L of biotinylated EpCAM antibody solution with the concentration of 5 mu g/mL, standing and incubating for 30min at 37 ℃, and centrifugally washing for three times by PBS to obtain the magnetic erythrocyte with the surface modified EpCAM antibody.
The process of capturing MCF-7 cells: quantitative measuring 10 5 MCF-7 cells were washed with PBS and dispersed, and 10 was quantitatively added thereto 7 And uniformly mixing the magnetic red blood cells with the surface modified with the EpCAM antibody, centrifuging the mixture in a centrifuge at 1500rpm for 3min, and incubating the mixture at 37 ℃ for 1h to capture the MCF-7 cells by the magnetic red blood cells with the surface modified with the EpCAM antibody.
(2) Releasing
Firstly, the MCF-7 cells captured by the magnetic red blood cells are separated and washed by a magnetic separation mode on a magnetic frame. Then, the supernatant was removed and 1mL of RPMI-1640 complete medium was added, and the cells were gently resuspended by blowing. Finally, the whole system is placed in an environment at 37 ℃ for incubation for 2 h. The settled cells were intermittently subjected to a suspension operation during incubation to ensure that the complete medium of RPMI-1640 was in sufficient contact with the capture system.
The picture before release is shown in fig. 8(a), and the picture after release is shown in fig. 8(b), and the release efficiency is about 88%.
Example 4 release of magnetic Red Cluster captured SKOV3 cells with polybrene and DSPE-PEG-FA modified surfaces in McCoy's 5A complete Medium
(1) Capture
The magnetic erythrocyte cluster with the surface modified with polybrene and DSPE-PEG-FA used by the system is prepared by adsorbing full-layer erythrocytes on the surface of a magnetic microsphere with surface modified carboxyl, the surface of the adsorbed erythrocytes is modified with polybrene and DSPE-PEG-FA, and the total volume of the capture system is 0.5 mL.
Magnetic red with surface modified by polybrene and DSPE-PEG-FAPreparation of cell clusters: in the first step, DSPE-PEG-FA is modified. Quantitatively taking out 2X 10 based on the content of red blood cells in the red blood cell dispersion 8 Quantitatively adding 0.08mg of PBS solution containing DSPE-PEG-FA with molecular weight of 2000 into erythrocytes, standing and incubating for 30min at 4 ℃, and centrifuging and washing with PBS for three times to obtain DSPE-PEG-FA modified erythrocytes. And secondly, polybrene modification. Mixing the red blood cells modified with the DSPE-PEG-FA with 300 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifuging and washing for three times by PBS to obtain the red blood cells modified with the polybrene and the DSPE-PEG-FA, and dispersing the red blood cells into the PBS solution for later use. And thirdly, preparing red cell clusters. Washing the magnetic microspheres with the surface modified carboxyl groups for three times by PBS, adding modified red blood cells with the number being 100 times of the counted number of the microspheres, uniformly mixing the magnetic microspheres and the modified red blood cells, centrifuging the mixture in a centrifuge at a centrifugation speed of 400g for 1min to ensure that the red blood cells and the microspheres are centrifuged to the bottom of a centrifuge tube to ensure that the red blood cells and the microspheres are close to each other, standing and incubating the mixture at 4 ℃ for 30min after centrifugation, finally blowing and suspending the mixture at the bottom of the centrifuge tube, carrying out magnetic separation and washing on the mixture by a magnetic frame, washing and removing excessive free red blood cells, and finally obtaining the magnetic red blood cell cluster with the surface modified by polybrene and DSPE-PEG-FA.
Capturing SKOV3 cell process: quantitative measuring 10 5 SKOV3 cells were washed with PBS and dispersed, and 10 was quantitatively added thereto 7 The magnetic red blood cell clusters with the surfaces modified with polybrene and DSPE-PEG-FA are evenly mixed and then centrifuged for 3min at 1500rpm in a centrifuge, and then incubated for 1h at 37 ℃, so that the SKOV3 cells can be captured by the magnetic red blood cell clusters with the surfaces modified with polybrene and DSPE-PEG-FA.
(2) Releasing
Firstly, SKOV3 cells captured by magnetic red blood cell clusters were separated and washed by magnetic separation on a magnetic rack. Then, the supernatant was removed and 1.4mL of McCoy's 5A complete medium was added and the cells were gently blown into suspension. Finally, the whole system is placed in an environment at 37 ℃ for incubation for 2 h. The settled cells were intermittently subjected to a suspension procedure during incubation to ensure that the McCoy's 5A complete medium was in sufficient contact with the capture system.
The picture before release is shown in fig. 9(a), and the picture after release is shown in fig. 9(b), and the release efficiency is about 85%.
Example 5 HeLa cells captured by erythrocyte plane biomimetic layer with polyamine and DSPE-PEG-RGD modified on surface were released in MEM complete medium
(1) Capture
The red blood cell plane bionic layer used in the system and modified with polybrene and DSPE-PEG-RGD on the surface is prepared by a mode of tightly adsorbing a layer of red blood cells on the surface of an amino glass slide (with the size of 1cm multiplied by 3cm), and the surface of the adsorbed red blood cells is modified with polybrene and DSPE-PEG-RGD.
The preparation process of the red blood cell plane bionic layer with the surface modified with polybrene and DSPE-PEG-RGD comprises the following steps: firstly, modifying the surface of red blood cells with DSPE-PEG-RGD. Adding DSPE-PEG-RGD into the erythrocyte dispersion liquid, incubating, centrifuging, and washing to obtain the burr spherical erythrocyte with the surface modified with DSPE-PEG-RGD. Wherein the proportion of the usage of the primary red blood cells and the usage of the DSPE-PEG-RGD is 5 × 10 7 0.02mg of DSPE-PEG-RGD was added to each red blood cell. And secondly, polybrene modification. Mixing the red blood cells modified with the DSPE-PEG-RGD with 300 mu L polybrene PBS solution with the concentration of 10mg/mL, standing and incubating for 30min at 4 ℃, centrifugally washing for three times by PBS to obtain the red blood cells modified with the polybrene and the DSPE-PEG-RGD, and dispersing the red blood cells into the PBS solution for later use. And thirdly, preparing a red blood cell plane bionic layer. And dripping the red blood cell suspension with the surface modified with polybrene and DSPE-PEG-RGD on the surface of an amino glass slide, standing for 2h at 4 ℃, leaching the amino glass slide with PBS, and removing unadsorbed red blood cells to obtain the red blood cell plane bionic layer with the surface modified with polybrene and DSPE-PEG-RGD.
The process of capturing Hela cells: quantitative measuring 10 5 And washing and dispersing the Hela cells by PBS, dripping the Hela cells onto the red blood cell plane bionic layer of which the surface is modified with polybrene and DSPE-PEG-RGD, and standing for 2h at 37 ℃, so that the capture of the Hela cells by the red blood cell plane bionic layer of which the surface is modified with polybrene and DSPE-PEG-RGD can be realized.
(2) Releasing
First, the whole erythrocyte planar biomimetic layer with captured Hela cells was rinsed with PBS to remove a small amount of non-captured Hela cells. Then placing the red blood cell plane bionic layer in a culture dish with the upward plane bionic layer, adding 3mL of MEM complete culture medium to completely infiltrate the red blood cell plane bionic layer, and completely sealing the culture dish by using a sealing film to prevent the culture medium from leaking. And finally, putting the whole culture dish into a constant-temperature shaking table at 37 ℃ for incubation for 2h, and keeping the speed of 20rpm in the incubation process to shake the culture dish so as to ensure that the MEM complete culture medium can sufficiently release the captured Hela cells.
The picture before release is shown in fig. 10(a), and the picture after release is shown in fig. 10(b), and the release efficiency is about 88%.
The examples provided above are merely illustrative of the method of the present invention and are not intended to limit the remainder of the disclosure in any way. Other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (6)
1. A method for releasing tumor cells captured by a red blood cell biomimetic material, characterized in that: the method comprises the following steps: incubating the erythrocyte biomimetic material for capturing the tumor cells with a complete culture medium, and releasing the tumor cells from the erythrocyte biomimetic material after incubation;
the red blood cell bionic material comprises pure red blood cells modified by DSPE-PEG-X, red blood cells modified by nano materials, red blood cell clusters or a plane red blood cell bionic layer; wherein X in the DSPE-PEG-X represents FA, RGD or biotin.
2. The method for releasing tumor cells captured by a biomimetic material of red blood cells according to claim 1, characterized in that: the tumor cells comprise Hela cells, MCF-7 cells, SKOV3 cells or HT29 cells.
3. The method for releasing tumor cells captured by a biomimetic material of red blood cells according to claim 1, characterized in that: the complete medium comprises DMEM complete medium, DMEM high-sugar complete medium, RPMI-1640 complete medium, McCoy's 5A complete medium or MEM complete medium.
4. The method for releasing tumor cells captured by a biomimetic material of red blood cells according to claim 1, characterized in that: the incubation condition is incubation for 0.1 min-2 h at 37 ℃.
5. The method for releasing tumor cells captured by a biomimetic material of red blood cells according to claim 1, characterized in that: the method comprises the following steps:
(1) removing the buffer solution in the system after the erythrocyte biomimetic material captures the tumor cells;
(2) adding a complete culture medium into the capture system, and completely immersing the capture system;
(3) and (3) incubating the capture system at the temperature of 37 ℃ for 0.1 min-2 h, and releasing the tumor cells from the erythrocyte bionic material.
6. Method for releasing tumour cells trapped by a biomimetic material of erythrocytes according to claim 5, characterized in that: the buffer solution in the step (1) is PBS buffer solution.
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| US10787499B2 (en) * | 2017-02-13 | 2020-09-29 | Regents Of The University Of Minnesota | EpCAM targeted polypeptides, conjugates thereof, and methods of use thereof |
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