CN117448275A - CTCs release preparation and CTCs separation method - Google Patents

Abstract

The invention relates to a preparation for releasing CTCs and a method for separating CTCs, wherein functionalized homologous erythrocytes are utilized to capture CTCs, and plasma is used for assisting in releasing tumor cells captured by a functionalized erythrocyte bionic layer, the release efficiency is more than 70%, the purity of the released tumor cells is more than 95%, and the survival rate of the released cells is more than 95%.

Description

CTCs release preparation and CTCs separation method

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a release preparation for releasing CTCs captured by erythrocyte biomimetic materials and a separation method of the CTCs.

Background

Circulating Tumor Cells (CTCs) are cancer cells that circulate in the peripheral blood after shedding from primary or metastatic tumors. It can reach distant organs through blood, causing cancer metastasis leading to 90% of cancer-related deaths. The isolation and analysis of these CTCs in peripheral blood (liquid biopsies) has attracted attention because of its importance for early cancer diagnosis, treatment monitoring, prognostic assessment and metastasis diagnosis. However, the isolation of CTCs from billions of normal cells is a significant technical challenge. Various methods have been developed by researchers to specifically isolate CTCs from blood. These strategies typically exploit differences between CTC surface biomarkers or physical properties. Such as immunomagnetic beads based on surface modification to capture biomolecules, microfluidic devices to enhance cell surface contact, functionalized nanostructured surfaces based on cell-matrix affinity, and microfilter devices to separate CTCs based on size differences. Strategies that use physical property differences to sort CTCs can result in unnecessary false positive separation of normal blood cells due to overlapping white blood cells and CTCs physical properties, which can lead to low purity of CTCs, thereby interfering with the characterization of CTCs. While strategies that utilize surface biomarkers, such as the CellSearch system, are the only CTC detection devices on the market approved by the food and drug administration that use magnetic particles of surface modified antibodies to capture specific cells. Although high capture efficiencies of 80% can be achieved in this way, there is also a large number of non-specifically adsorbed leukocytes on the magnetic particles, which results in a purity of typically less than 0.5% of the captured CTCs. Clearly, non-specific binding of leukocytes to the surface of CTC capture devices by physical adsorption is a major factor leading to low purity. Thus, the relevant surface modifications should be focused on preventing nonspecific cell adhesion.

Based on this idea, the anti-fouling/anti-adhesion surface is preferred for the capture device surface, since leukocyte adhesion is a normal phenomenon of the body handling inflammatory reactions, and adhesion is enhanced when leukocytes in blood are activated by various stimuli in vitro, including the capture surface. For example, polyglycerol-based block polymers are used to prepare biospecific and bioinert interfaces, and erythrocyte membrane mimic surfaces, which consist of mussel-inspired self-adhesive polydopamine layers and phosphorylcholine zwitterionic polymers and polyethylene glycol (PEG) covalently anchored non-contaminating or cell-adhesion-resistant layers. And modifying tumor capture molecules such as folic acid, RGD, anti-EpCAM, human epithelial receptor 2 (Her 2) and Epithelial Growth Factor Receptor (EGFR) on the coating to enable the tumor capture molecules to capture tumor cells. Thanks to the anti-fouling capture surface, the resulting monolayer coating greatly inhibits the adhesion of non-specific cells, allowing the separation of CTCs with high selectivity and efficiency. However, the acquisition of such anti-cell adhesion polymer coatings inevitably involves complex organic synthesis and cumbersome chemical modification procedures, which are disadvantageous for clinical applications.

In recent years, studies have shown that cell membrane structure plays a key role in resisting bioadhesion and biofouling. In our previous studies, we also directly modified erythrocytes by functionalization to directly replace particles for CTC capture. Experimental results prove that the red blood cells have good leukocyte adhesion resistance. However, direct capture of CTCs with erythrocytes, whichever method is followed is still faced with a complex step of screening erythrocytes @ CTCs from background cells. To solve this problem, we contemplate constructing a biomimetic capture substrate comprised of red blood cells. However, in general, electrostatic repulsive force is generated on the surfaces of erythrocytes, which prevents them from approaching each other, resulting in failure to form a dense biomimetic structure. In the experimental process, we find that the red blood cells after being treated by the polybrene can be densely distributed on an adhesion glass slide to form a skin-like bionic structure, and meanwhile, the bionic layer still has excellent capability of resisting adhesion of white blood cells.

Disclosure of Invention

The invention aims to overcome the defects and shortcomings of the prior art and provides a release preparation for releasing CTCs captured by erythrocyte biomimetic materials and a separation method of the CTCs.

The invention solves the technical problems by adopting the following scheme:

a CTCs release formulation for releasing circulating tumor cells captured by a red blood cell biomimetic material, the release formulation comprising plasma.

Further, the plasma is homologous to erythrocytes used in the erythrocyte biomimetic material.

The present invention also provides a method for isolating CTCs comprising the steps of:

(1) Capturing CTCs using a red blood cell biomimetic material;

(2) Soaking the erythrocyte bionic material with the captured CTCs in a release preparation to separate tumor cells from the erythrocyte bionic material; the release formulation according to any one of claims 1 to 2.

Further, the time for capturing CTCs by adopting the erythrocyte biomimetic material in the step (1) is 10-150 min, and more preferably 90-120 min.

Further, the erythrocyte biomimetic material comprises an erythrocyte layer, a linker which can be embedded into an erythrocyte membrane through hydrophilic and hydrophobic effects, and a capture molecule connected to the tail end of the linker.

Further, the linker comprises DSPE-PEG.

Further, the capture molecule is FA or an antibody that specifically recognizes and binds to CTCs surface biomarkers including, but not limited to, the epithelial markers cytokeratin, epithelial cell adhesion molecules, tumor embryo antigens, her receptor 2, venous endothelial cell molecules, epstein, sialylated lewis oligosaccharide-X, acetaldehyde dehydrogenase 1, vimentin, urokinase receptor, heparanase, prostate specific membrane antigen, CD44, CK18, CD133, CD90, CD45, or CD146; the CTCs targeted antibodies include, but are not limited to, any of the epithelial markers 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-CD 133, anti-CD90, anti-CD45, or anti-CD 146.

Further, the soaking condition is standing, shaking or cyclic leaching treatment, and the soaking time is more than 20 minutes.

Further, the preparation method of the plasma component in the release preparation comprises the following steps: centrifuging the whole blood, and collecting the pale yellow plasma at the upper layer.

Further, the method also comprises the following steps:

(3) Taking out the erythrocyte bionic material in the release preparation, flushing with buffer solution, and combining the flushing solution with the release preparation to obtain the isolated CTCs.

The invention utilizes plasma to assist in releasing tumor cells captured by the functionalized red blood cell bionic layer, the release efficiency is more than 70%, and the purity of the released tumor cells is more than 95%. The red blood cells are directly used as a material interface, redundant biochemical treatment is not needed to be carried out on the red blood cells, and the original biological characteristics of the red blood cells are maintained to the greatest extent. Meanwhile, almost all materials used in the capturing and releasing processes come from homologous blood, interference caused by foreign matters is avoided, and the final result also shows that the cell survival rate exceeds 95% and is comparable to that of a control group. The method has wide application prospect in the aspect of designing cell material biology/interfaces in future biomedical research based on cells.

Drawings

FIG. 1 is a schematic representation of the preparation of a biomimetic layer of erythrocytes for specific high purity capture of tumor cells according to the present invention;

FIG. 2 is a graph showing the capture time and capture efficiency of a functionalized red blood cell biomimetic layer for two tumor cells; b) Capturing SEM images of tumor cells on the functionalized red blood cell bionic layer, and performing false staining treatment in the figure; the scale bar in the figure is 4 μm;

FIG. 3 is a schematic representation of the release of CTC from a functionalized erythrocyte biomimetic layer of the present invention in plasma where DSPE ends are detached from erythrocyte membranes to release captured cells;

FIG. 4 shows the morphological change of functionalized erythrocytes during the release of plasma, wherein a) is a schematic representation of the release of DSPE-PEG-FA from erythrocytes in plasma; b) Changes in erythrocyte morphology and fluorescence before and after plasma addition;

FIG. 5 is a photograph of fluorescence of FITC-labeled functionalized red blood cell biomimetic layers before (left) and after (right) plasma immersion, scale 20 μm;

FIG. 6 shows the effect of tumor cell release in a plasma environment, wherein a) is a schematic diagram of the process of red blood cell release targeting tumor cells in a plasma environment; b) For fluorescent photographs of erythrocytes targeting tumor cells, green is HeLa cells stained by FDA, red is functionalized erythrocytes stained by DiI; c) Bright field photographs of FA-RBCs isolated from tumor cells in plasma;

FIG. 7 is a morphology of a biomimetic layer of functionalized erythrocytes in the same region before and after plasma release;

FIG. 8 is a graph showing the relationship between tumor cell release efficiency in plasma and time;

FIG. 9 is a fluorescence micrograph of a functionalized red blood cell biomimetic layer before and after a plasma soak treatment;

FIG. 10 shows the capturing effect of the functionalized red blood cell biomimetic layer on tumor cells and white blood cells, wherein a) is a capturing effect bright field micrograph; b) A dark field Hoechst staining microscope photograph for capturing the effect; c) FDA staining photomicrographs of dark fields for capturing effects; d) Combining the photos for the bright field and dark field of the microscope;

FIG. 11 is data of purity of tumor cells captured by a functionalized red blood cell biomimetic layer in a mixed solution of tumor cells and white blood cells mixed in different proportions;

FIG. 12 shows purity data of tumor cells released by plasma immersion in a mixture of tumor cells and leukocytes mixed in different ratios;

FIG. 13 shows the survival of tumor cells released from plasma, wherein a) is a photograph of the released tumor cells; b) Plotting survival statistics;

FIG. 14 shows the released proliferation potency results of tumor cells.

Detailed Description

For a better understanding of the present invention, the following examples are further illustrative of the present invention, but the contents of the present invention are not limited to the following examples only.

Whole blood used in the examples and test examples of the present application was derived from healthy human blood, and both leukocytes and erythrocytes were isolated from homologous human blood.

Pre-staining and labelling of cells

Pre-staining the white blood cells by using FDA, preparing FDA solution with acetone, diluting 10 mu L to 1mL by using PBS, uniformly mixing 1mL of the previously extracted mononuclear cells with 1mL of FDA diluent, standing for 5 minutes under the dark condition, centrifugally cleaning for three times by using PBS (1000 rmp/min), and finally re-suspending by using 1mL of DMEM to obtain the FDA-stained white blood cells.

The Hoechest solution is prepared by using PBS, the concentration is 20 mug/mL, 1mL of Hoechest solution is uniformly mixed with 1mL of PBS-resuspended sea-tangle cells, the mixture is kept stand and dyed for 5 minutes under the dark condition, the mixture is centrifugally washed (1000 rmp/min) for three times by using PBS, and finally the mixture is resuspended by using 1mL of DMEM, so that the Hoechest-dyed sea-tangle cells are obtained.

HCT116 cells were labeled with Hoechest using the method described above.

Marking red blood cells by DiI, and centrifuging whole blood by using lymphocyte separating liquid to obtain pure red blood cells with other cells removed. And the washed red blood cell concentration (in/mL) was determined by a cell counting plate. Five million volumes of red blood cells were diluted to 200 μl with PBS for use. DiI solution with concentration of 5mM is prepared by absolute ethyl alcohol, 10 mu L is diluted to 1mL by PBS, and 20 mu L of diluted solution is fully mixed with 200 mu L of the solution to obtain five tens of millions of pure red blood cells. Then incubated in a shaker at 37℃for one hour. DiI was removed from the solution by centrifugation (400 g,1 min) three times in PBS to finally obtain DiI-labeled erythrocytes.

Example 1 preparation of functionalized erythrocyte biomimetic layer

A0.1 mg/mL concentration of DSPE-PEG-FA solution was prepared with PBS, 200. Mu.L was added to DiI-labeled erythrocytes, and the mixture was thoroughly blown by a pipette, followed by standing for 2 minutes. And (3) centrifugally cleaning for three times by using PBS, re-suspending by using a polybrene solution (10 mg/mL) prepared by using PBS for the third time, fully and uniformly blowing by using a pipette, standing for 1 minute, centrifugally cleaning for three times by using PBS, and re-suspending to 200 mu L by using PBS to obtain the functionalized red blood cells for later use.

And (3) fully blowing the obtained functionalized red blood cells by using a liquid-transferring gun, dripping the functionalized red blood cells on an adhesion glass slide, standing the adhesion glass slide in a refrigerator at the temperature of 4 ℃ for 30 minutes, and flushing off the redundant red blood cells which cannot be adsorbed on the adhesion glass slide by using PBS (phosphate buffer solution), thereby obtaining the functionalized red blood cell bionic layer.

Example 2 capturing of tumor cells by functionalized erythrocyte biomimetic layer

The pre-labeled tumor cells were centrifuged to remove the complete medium and resuspended in basal medium, 200 μl of the resuspended tumor cell culture medium was mixed with the functionalized red cell biomimetic layer prepared in example 1, both cells were thoroughly and evenly blown by a pipette, and incubated in a 37 ℃ cell incubator for one hour to obtain the functionalized red cell biomimetic layer with tumor cells captured on the surface.

EXAMPLE 3 plasma Release

Whole blood was centrifuged directly (horizontal centrifuge, 200g,5 min) and the uppermost pale yellow plasma was collected for use. Taking a functionalized red blood cell bionic layer sample with tumor cells captured on the surface, removing the supernatant by centrifugation, adding plasma into the suspension, standing in a cell incubator at 37 ℃ for incubation for thirty minutes, and releasing the tumor cells.

Test example 1 test of time and efficiency of capturing tumor cells by functionalized erythrocyte biomimetic layer

To explore the optimal capture time of the functionalized erythrocyte biomimetic layer to tumor cells, heLa and HCT116 cells were selected as model cells, as they were tumor cells that highly expressed folate receptors on the cell membrane surface. Approximately 50,000 tumor cells (125,000 cells/mL PBS) were added dropwise to the functionalized erythrocyte biomimetic layer, and they were incubated in a cell incubator (37 ℃,5% CO 2). Taking out one group every 10 minutes, 30 minutes, 60 minutes, 90 minutes, 120 minutes and 150 minutes, lightly washing with PBS to obtain a functionalized red blood cell bionic layer for capturing the sea-pulling cells on the surface, and then imaging the captured cells by using a microscope to calculate the capturing efficiency. Cell capture efficiency was calculated according to the following formula:

where N is the number of cells on the obtained image, S is the surface area of the biomimetic layer, S is the surface area of the obtained image, and N is the total number of cells. 10 images were randomly taken. Three parallel experiments were performed in total. Two kinds of cells are subjected to an experiment to obtain the capturing efficiency of two kinds of tumor cells under different capturing time respectively.

The final statistics are shown in figure 2 a. It is known that the highest capture efficiency is stabilized at 85% or more when the incubation time is 90 to 120 minutes. However, beyond 120 minutes, the capture efficiency decreases, which may be related to the detachment of the functionalized erythrocytes from the adherent slide after aging at 37 ℃ for a long period of time, resulting in a decrease in capture efficiency.

Test example 2 Scanning Electron Microscope (SEM) observation of functionalized erythrocyte biomimetic layer capturing tumor cells on the surface

The functionalized erythrocyte biomimetic layer with captured tumor cells obtained in example 2 was immersed in glutaraldehyde (2.5%) electron microscope solution overnight, washed three times with DI water in a constant temperature shaker the next day, and then dehydrated stepwise with ethanol. The ethanol concentration during dehydration was 30%,50%,70%,80%,90%,95% from low to high, respectively. The dehydration time for each concentration is 10-15 minutes. The entire biomimetic layer was then freeze-dried and SEM imaged using a Zeiss Sigma scanning electron microscope, the results of which are shown in figure 2 b. The figure shows a false dyeing process, with a scale of 4. Mu.m.

Test example 3 Effect verification of linker shedding in plasma Environment

FIG. 3 is a schematic representation of the release of CTC in plasma by the functionalized erythrocyte biomimetic layer of the present application. Functionalized erythrocytes were prepared by replacing DSPE-PEG-FA with DSPE-PEG-FITC in PBS environment, centrifuging to remove PBS, and resuspending them with plasma. The change in morphology and fluorescence of the erythrocytes before and after plasma addition was observed by fluorescence microscopy, and as shown in FIG. 4b, it was found that the erythrocytes recovered from the original sea urchin shape to the original double concave round dish shape after plasma immersion treatment, and the disappearance of green fluorescence representing DSPE-PEG-FITC on the erythrocytes was also seen from the fluorescence photograph. Erythrocyte morphology recovery was due to the shedding of PEG on the membrane, and the disappearance of FITC fluorescence on erythrocytes also verifies that DSPE-PEG-FA, which was also modified by hydrophilic-hydrophobic forces in the plasma environment, also shed from the erythrocyte membrane. This is because lipoproteins in plasma can transfer modified DSPE on the erythrocyte membrane, so that DSPE-PEG-FA is detached from the erythrocyte membrane and adsorbed on lipoproteins in plasma. This experiment demonstrates that plasma does have the ability to release DSPE-PEG molecules on modified erythrocyte membranes.

And preparing the functionalized red blood cells prepared by replacing DSPE-PEG-FITC with DSPE-PEG-FA in PBS environment into a FITC marked functionalized red blood cell bionic layer. After plasma immersion rinsing, FITC fluorescence on the functionalized red blood cell biomimetic layer was substantially lost, as shown in fig. 5. Experimental results demonstrate that plasma-assisted DSPE shedding is equally applicable to functionalized erythrocyte biomimetic layers.

Test example 4 efficacy validation of tumor cell release in plasma environment.

FIG. 6a is a schematic of the erythrocyte release process for targeting tumor cells in a plasma environment. DiI is a lipophilic fluorescent substance, can enter between phospholipid bilayer of cell membrane by hydrophobic force, and can emit red fluorescence under excitation of 550nm light. Tumor cells were additionally labeled with FDA. The DiI labeled functionalized erythrocytes were then used to capture tumor cells in basal medium, and as shown in FIG. 6b, it was observed that green FDA labeled tumor cells were encapsulated by a layer of red DiI labeled functionalized erythrocytes under fluorescence. The ability of functionalized erythrocytes to capture tumor cells was demonstrated.

All cells were centrifuged to remove basal medium from the dispersion and re-dispersed with plasma, and after 20 minutes, detachment of erythrocytes from tumor cells was observed, as shown in fig. 6 c. Furthermore, it can be seen that most of the erythrocytes are restored from sea urchin shapes to the double concave circular disk shape of normal erythrocytes, which suggests that plasma can indeed release tumor cells captured by functionalized erythrocytes.

Test example 5 effect of tumor cell release captured by functionalized erythrocyte biomimetic layer in plasma environment was verified.

The functionalized biomimetic layer, which captured the tumor cells, was completely immersed in plasma, gently shaken in a thermostatted shaker at 37 ℃ for a period of time, and gently swirled three times with PBS. The functionalized red cell biomimetic layer of the same region before and after release was then observed under a microscope and photographed as shown in fig. 7. It can be found that tumor cells are shed after plasma soaking and washing, and the bionic layer remains intact. Experimental results demonstrate the ability of plasma release to specifically capture tumor cells.

In plasma, the release rate of tumor cells was very fast, and by counting the data, it was found that more than 80% of tumor cells were released within about 10 minutes. The relationship between release efficiency and time is shown in fig. 8.

Test example 6 Effect of plasma treatment on leukocytes non-specifically adhered to functionalized erythrocyte biomimetic layer

The plasma treatment does not affect the white blood cells which are non-specifically adhered to the functionalized red blood cell bionic layer. Sea Law cells (Hoechst labeled) labeled with different dyes and white blood cells (FDA labeled) extracted from whole blood were labeled with 1:40, and dripping the mixture on the prepared functionalized red cell bionic layer, incubating for 90 minutes, and then soaking the mixture in plasma for 20 minutes. Fluorescence micrographs of the functionalized red blood cell biomimetic layers before and after plasma soaking treatment were taken. As a result, as shown in fig. 9, after plasma treatment, the blue fluorescence of Hoechst-labeled hela cells captured by the functionalized red blood cell biomimetic layer was almost completely lost, but the green fluorescence of FDA-labeled white blood cells was still observed. It is demonstrated that plasma can specifically release tumor cells captured by folic acid. Whereas leukocytes which adhere non-specifically to the biomimetic layer are not affected by this.

Test example 7 anti-adhesion Effect of functionalized erythrocyte biomimetic layer on leukocytes

Preparing mixed solutions (Hale: WBC 1:1;1:5;1:10;1:20; 1:40) of tumor cells and white blood cells mixed in different proportions, and performing a capturing experiment through the prepared functionalized red blood cell bionic layer. Tumor cells and leukocytes were pre-stained by host and FDA, respectively, and the captured results are shown in fig. 10. It was found that only a small number of green spots representing white blood cells adhere non-specifically to the biomimetic layer. The functionalized erythrocyte bionic layer has extremely strong anti-leukocyte adhesion capability and simultaneously has the function of specifically capturing tumor cells.

The counted pre-stained leucocytes and sea-drawn cells are added into a monocyte suspension extracted from two milliliters of blood to be uniformly mixed, DMEM is added to fix the volume to 1 milliliter, the mixture is respectively dripped on the FA-RBCs functionalized erythrocyte bionic layer prepared before, after incubation is carried out for 90 minutes at 37 ℃, DPBS is used for gently flushing for 3 times, and observation is carried out under a fluorescence microscope. The capture purity was calculated by taking 20 photographs at random for each group, and the cell capture purity was calculated according to the following formula:

where x is the total number of sea-tangled cells (blue) in the 20 pictures and y is the total number of white blood cells (green) in the 20 pictures.

The data statistics were performed on the cells captured by the functionalized erythrocyte biomimetic layer according to the method described above, and the capture purity was calculated as shown in fig. 11. It was found that the ratio of the sea-tangle cells to the white blood cells reached 1: after 40, the capture purity of the functionalized erythrocyte biomimetic layer to the sea-Law cells still exceeds 70%.

Test example 8 purity statistics of plasma released tumor cells

The plasma can specifically release tumor cells captured by the functionalized red blood cell bionic layer, and meanwhile, the white blood cells which are non-specifically adhered in gaps of the functionalized red blood cell bionic layer are not affected. The purity of the tumor cells collected after plasma immersion is thus theoretically further improved.

The plasma was added dropwise to the biomimetic layer with captured tumor cells and placed in a shaker at 37℃for 20min (40 r/min). All liquids were collected, the collected liquids were observed under a fluorescence microscope, 20 photographs were randomly taken for each group, purity after release was calculated, and purity of cells released was calculated according to the following formula:

where a is the total number of sea-tangled cells (blue) in 20 photographs and b is the total number of white blood cells (green) in 20 photographs. According to the above method for counting released tumor cells, the final statistics result is shown in fig. 12, and it can be found that the purity of the released tumor cells after plasma immersion exceeds 95%, and the purity after release is improved by approximately 20% compared with the purity when captured.

Test example 9 identification of the viability and proliferation potency of plasma released tumor cells

The activity of tumor cells released from the plasma after capture was determined by FDA and Propidium Iodide (PI) double staining. FDA/PI solution composed of 2. Mu.M/mL FDA and 4. Mu.M/mL PI was prepared for use. Firstly, adding DMEM to sea-drawn cells counted in the previous step to fix the total volume to 1 milliliter, dripping the sea-drawn cells on the functionalized red blood cell bionic layer prepared in the previous step, incubating the sea-drawn cells at 37 ℃ for 90 minutes, lightly flushing the sea-drawn cells with PBS for 3 times, and observing the capturing condition under a microscope. Then, the plasma extracted from the blood was dropped onto the biomimetic layer in which the cells were trapped, and the mixture was placed in a shaker at 37℃for 20min (40 r/min). All cell suspensions were then collected.

Centrifuging at 1500rpm/min for 3 min, removing supernatant, adding the FDA/PI solution prepared before, blowing uniformly by a pipette, standing at room temperature for 30 min, centrifuging with PBS solution, washing for 3 times, and observing with a fluorescence microscope (M-shot MF43, ming and Mey, guangzhou). 10 photographs were taken at random and the viability of the cells was calculated. The survival rate was calculated by the following formula:

where a is the total number of surviving cells (green) in the 20 photographs and b is the total number of dead cells (red) in the 20 photographs.

FDA is a fluorescent reagent capable of carrying out fluorescent staining on living cells, which can not emit fluorescence, but can easily pass through cell membranes, nonspecific lipase is arranged in the living cells, and the living cells can be hydrolyzed to obtain polar substances with green fluorescence, wherein the excitation wavelength is 470nm, and the wavelength of emitted light is 530nm. In contrast, the dead cells do not have nonspecific lipase, and they cannot be hydrolyzed and therefore do not fluoresce. While Propidium Iodide (PI) does not have the ability to penetrate the cell membrane of living cells, but it can penetrate the cell membrane of dead cells and combine with genetic material in the nuclei of dead cells to produce red fluorescence, wherein the excitation light wavelength is 535nm and the emission light wavelength is 617nm, thereby realizing staining of dead cells.

The released tumor cells are shown in FIG. 13 a. Tumor cells exhibiting green fluorescence FDA were then counted as viable cells, and cells exhibiting red fluorescence PI were counted as dead cells. At the same time, the same staining treatment was performed on tumor cells that were not subjected to any treatment to prepare a control group, and the final statistical result was plotted as shown in fig. 13b. The survival rate of tumor cells released by plasma after capturing the functionalized erythrocyte biomimetic layer is almost the same as that of a control group without any operation, which also proves that the whole capturing and releasing method is very mild to cells and the cell activity is maintained to the greatest extent.

In order to evaluate the proliferation capacity of tumor cells after release by capture, post-release re-culture experiments were performed. Specifically, the released tumor cells were collected from the washed PBS by centrifugation, transplanted into a 6-well plate, and DMEM medium containing 10% FBS was added to a 96-well plate to incubate with a 37℃cell incubator. Every 1 hour, 12 hours, 24 hours, the proliferation of cells was observed under a microscope.

The released tumor cells were able to proliferate and after one hour, the tumor cells were seen to successfully adhere to the wall and start growing, as shown in FIG. 14. After 12 hours incubation, the tumor cells were completely adherent in a spindle shape. After 24 hours of culture, the tumor cells have shown a clear tendency to confluence and a good morphological distribution. All cells showed good growth behaviour. The experimental result shows that the tumor cells which are released by assistance of plasma after being captured by the functionalized erythrocyte bionic layer have good cell activity, and the proliferation capability of the tumor cells is not affected.

While the invention has been described with respect to the preferred embodiments, it will be understood that the invention is not limited thereto, but is capable of modification and variation without departing from the spirit of the invention, as will be apparent to those skilled in the art.

Claims (10)

1. A CTCs release formulation for releasing circulating tumor cells captured by a red blood cell biomimetic material, said release formulation comprising plasma.

2. The CTCs release formulation of claim 1, wherein the plasma is homologous to erythrocytes used in the erythrocyte biomimetic material.

3. A method for isolating CTCs comprising the steps of:

(1) Capturing CTCs using a red blood cell biomimetic material;

(2) Soaking the erythrocyte bionic material with the captured CTCs in a release preparation to separate tumor cells from the erythrocyte bionic material; the release formulation according to any one of claims 1 to 2.

4. A method of isolating CTCs according to claim 3, wherein step (1) captures CTCs using erythrocyte biomimetic material for a period of 10 to 150 minutes.

5. A method of isolating CTCs according to claim 3, wherein the erythrocyte biomimetic material comprises a layer of erythrocytes, a linker embedded within the erythrocyte membrane by hydrophilic-hydrophobic interactions, and a capture molecule attached to the terminal end of the linker.

6. The method of isolating CTCs of claim 4, wherein said linker comprises DSPE-PEG.

7. The method of isolating CTCs according to claim 4, wherein the capture molecule is FA or an antibody that specifically recognizes and binds to a biomarker on the surface of CTCs.

8. The method of isolating CTCs according to claim 3, wherein the soaking conditions are standing, shaking or cyclic rinsing for a period of 20min or more.

9. A method of isolating CTCs according to claim 3, wherein the preparation of the plasma component in the release formulation comprises the steps of: centrifuging the whole blood, and collecting the pale yellow plasma at the upper layer.

10. The method of isolating CTCs of claim 3, further comprising the steps of:

(3) Taking out the erythrocyte bionic material in the release preparation, flushing with buffer solution, and combining the flushing solution with the release preparation to obtain the isolated CTCs.