CN116410848B - Label-free high-invasiveness circulating tumor cell capturing and culturing chip - Google Patents
Label-free high-invasiveness circulating tumor cell capturing and culturing chip Download PDFInfo
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Abstract
The invention relates to the technical field of microfluidic chips, and discloses a label-free high-invasiveness circulating tumor cell capturing and culturing chip, which comprises an upper layer, a middle layer and a lower layer which are sequentially overlapped from top to bottom; the upper layer is provided with a cell suspension perfusion channel; the two ends of the cell suspension perfusion channel are respectively provided with a first liquid inlet and a first liquid outlet; the lower layer is provided with a leukocyte chemotactic separation area and a CTC chemotactic capture area corresponding to the cell suspension perfusion channel; the intermediate layer comprises a first PET film and a second PET film; the first PET film is arranged at a position corresponding to the leukocyte chemotactic separation area, and the second PET film is arranged at a position corresponding to the CTC chemotactic capture area; the invention can capture highly invasive CTC in peripheral blood without marking, can be used for clinical tumor drug screening, and can screen out therapeutic drugs with more pertinence to tumor metastasis.
Description
Technical Field
The invention relates to the technical field of microfluidic chips, in particular to a label-free high-invasiveness circulating tumor cell capturing and culturing chip.
Background
Circulating tumor cells CTCs are a collective term for the types of tumor cells present in the peripheral blood. Since CTCs undergo mostly apoptosis or phagocytosis after entering peripheral blood, CTCs in peripheral blood are currently acquired mainly for tumor detection. CTCs are generally recognized as nucleated cells expressing the epithelial marker cytokeratin CK, the epithelial cell adhesion molecule EpCAM, but not CD 45. However, the current technique of capturing CTCs by recognizing epithelial-specific markers has the disadvantage that CTCs that no longer overexpress EpCAM after epithelial-mesenchymal transition of EMT cannot be detected.
The existing methods for separating CTC mainly comprise the following steps: 1) Magnetic bead sorting of tumor markers (including EpCAM, CK8, CK18, CK19, etc.); 2) Sorting immune cell CD45 marker magnetic beads, screening and separating immune cells; 3) Density gradient centrifugation method: the required blood quantity is large and the impurities are more; 4) And (3) filtering: less tumor cells can be lost, different filter membranes are required to be formulated according to different tumors, and the production requirement is high; 5) Special instrument device: the threshold is high and the price is high. Currently, CTCs using existing screening methods are difficult to grow in vitro, one possible reason for this is that capturing CTCs using the cell surface marker (EpCAM) can hinder cell adhesion and proliferation in subsequent cell cultures. Physical methods such as centrifugation, filtration, etc. may also cause physical damage to the cells. Given that CTCs are captured without blocking cell surface markers, a reliable method to expand cells in vitro and maintain the captured CTC phenotype is possible. In recent years, CTC detection technology is continuously developed, and due to different enrichment and detection methods, the CTC detection method in clinical practice has not been standardized, and its main application is still limited to simple counting and determination of tumor metastasis capability.
CTCs in the peripheral blood have a small number of CTCs with higher invasiveness, and CTCs in this group are more easily attracted by a specific environment during the peripheral blood circulation, so that attachment and infiltration are generated, and distal metastasis is finally formed. If the CTCs of such a population can be effectively captured, cultured and used for drug sensitive testing of in vitro anticancer drugs, it is expected that therapeutic drugs more targeted to tumor metastasis can be screened out, which is of great significance for tumor patient treatment.
At present, a microfluidic chip-based method is also used for researching the separation of CTC to some extent, and a common strategy is to adopt a physical mode; many studies emphasize the physical and biomechanical properties of CTCs, enabling them to be distinguished from other blood cells. Most CTCs are larger in size (17-52 μm) compared to erythrocytes (6-8 μm) and leukocytes (most 7-15 μm, monocytes 20 μm), with higher nuclear to cytoplasmic ratios and complex membrane folding. CTCs can be isolated from blood by using filters of different pore sizes or by providing arrays of micropillars of different spacing in the chip. However, this method has a problem of easy clogging when handling a large amount of cells. Meanwhile, the purity of the separated CTC is lower. Physical damage to cells can also occur when flow rates are controlled improperly. This method is only applicable to the sorting of CTCs and cannot be used to sort highly invasive CTCs. Another strategy for CTC separation by using a microfluidic chip is to adopt a biological manner, mostly adopt antigen and antibody affinities, and modify specific antibodies on the surface of a flow channel of the microfluidic chip aiming at specific markers (such as EpCAM and the like) on the surface of the CTC, so that CTCs can be specifically combined with the antibodies when cell suspension passes through the flow channel, and CTCs are captured. The other thinking is that a CTC specific antibody (such as EpCAM and the like) and a leukocyte specific antibody (such as CD45 and the like) are respectively combined with magnetic beads to prepare immune magnetic beads, the immune magnetic beads are respectively combined with CTC and leukocytes in a specific way, then the immune magnetic beads combined with the CTC are adsorbed by two stages of adsorption and rejection of a magnetic field, and the magnetic beads combined with the leukocytes are separated, so that the capture of the CTC can be realized. However, the total amount of the antibody modified by this method is constant, and when the amount of the treated cells exceeds the total amount of the antibody, some cells cannot bind to the antibody, resulting in loss. The throughput of the treated samples is therefore often low, whereas the immunomagnetic bead rules require pre-labelling of the treated samples with immunomagnetic beads. However, the captured CTCs all carry specific antibodies, so that label-free capture of CTCs cannot be achieved, and downstream analysis of CTCs is difficult. Meanwhile, the method can only capture CTC and cannot separate out highly invasive CTC.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a label-free high-invasiveness circulating tumor cell capturing and culturing chip.
The technical scheme adopted by the invention is as follows:
a label-free high invasive circulating tumor cell capturing and culturing chip comprises an upper layer, a middle layer and a lower layer which are sequentially overlapped from top to bottom; the upper layer is provided with a cell suspension perfusion channel; the two ends of the cell suspension perfusion channel are respectively provided with a first liquid inlet and a first liquid outlet; the lower layer is provided with a leukocyte chemotactic separation area and a CTC chemotactic capture area corresponding to the cell suspension perfusion channel; the intermediate layer comprises a first PET film and a second PET film; the first PET film is arranged at a position corresponding to the leukocyte chemotactic separation area, and the second PET film is arranged at a position corresponding to the CTC chemotactic capture area; the second liquid inlet and the second liquid outlet of the leukocyte chemotactic separation area are respectively provided with a through hole at the corresponding positions of the upper layer; and through holes are formed in the corresponding positions of the upper layer of the third liquid inlet and the third liquid outlet of the CTC chemotactic capture area.
Further, the cell suspension perfusion channel main body is of a rectangular structure, and two ends of the cell suspension perfusion channel main body are of a triangular structure; the triangular vertex positions correspond to the first liquid inlet and the first liquid outlet respectively.
Further, the leukocyte chemotactic separation area is communicated with the second liquid inlet through a first liquid inlet channel, and the second liquid outlet is communicated with the second liquid outlet through a first liquid outlet channel; the CTC chemotactic capture area is communicated with the third liquid inlet through the second liquid inlet channel, and the third liquid outlet is communicated with the third liquid outlet through the second liquid outlet channel.
Further, the main body of the leukocyte chemotactic separation zone is of a rectangular structure, two ends of the main body are of triangular structures, and the positions of the vertexes of the triangles correspond to one end of the first liquid inlet channel and one end of the first liquid outlet channel respectively; the main body of the CTC chemotactic capture area is of a rectangular structure, two ends of the main body are of triangular structures, and the positions of the vertexes of the triangles correspond to one ends of the second liquid inlet channel and the second liquid outlet channel respectively.
Further, the second liquid inlet is communicated with a first through hole arranged on the upper layer, and the second liquid outlet is communicated with a second through hole arranged on the upper layer; the third liquid inlet is communicated with a fourth through hole arranged on the upper layer, and the third liquid outlet is communicated with a third through hole arranged on the upper layer.
Furthermore, the vertex angles of the first liquid inlet and the first liquid outlet corresponding to the triangle are smaller than 45 degrees.
Furthermore, the vertex angles of the first liquid inlet channel and the first liquid outlet channel corresponding to the triangles are smaller than 45 degrees; the vertex angles of the second liquid inlet channel and the second liquid outlet channel corresponding to the triangles are smaller than 45 degrees.
Further, the first PET membrane covers a leukocyte chemotactic separation area, and the second PET membrane covers a CTC chemotactic capture area; the first PET film has a pore size of less than 8 μm and the second PET film has a pore size of greater than 8 μm.
Further, the flow rate of the cell suspension perfusion channel ranges from 0.1 ul/min to 4 ml/min.
Further, the upper layer and the lower layer are prepared from polydimethylsiloxane.
The beneficial effects of the invention are as follows:
(1) The invention can treat 95-97% of white blood cells through chemotaxis treatment, and can separate and capture CTC without labels.
(2) According to the invention, immunofluorescence staining characterization is performed on proteins related to invasiveness, so that CTC separated by a chip is proved to have stronger invasiveness, and the CTC can be maintained to be cultured for a long time in the chip;
(3) The invention captures and cultures the highly invasive CTC, breaks the limitation that the traditional CTC sorting is only used for counting, and widens the application of CTC in other aspects such as downstream analysis, drug test and the like.
Drawings
FIG. 1 is an exploded view of the structure of the present invention.
FIG. 2 is a schematic diagram showing the staining effect of the non-captured CTC with the chip, wherein a is the staining effect of DAPI, b is the staining effect of MMP-2, c is the staining effect of MMP-9, and d is the superposition effect of three staining effects of DAPI, MMP-2 and MMP-9.
FIG. 3 is a schematic diagram showing the staining effect of capturing CTC by using the chip, wherein a is the staining effect of DAPI, b is the staining effect of MMP-2, c is the staining effect of MMP-9, and d is the superposition effect of three staining effects of DAPI, MMP-2 and MMP-9.
In the figure: 1-upper layer, 101-cell suspension perfusion channel, 102-first inlet, 103-first outlet, 104-first through hole, 105-second through hole, 106-third through hole, 107-fourth through hole, 201-first PET film, 202-second PET film, 3-lower layer, 301-leukocyte chemotactic separation area, 302-CTC chemotactic capture area, 303-first inlet channel, 304-second outlet channel, 305-second inlet, 306-second outlet, 307-third inlet, 308-third outlet, 309-first outlet channel, 310-second inlet channel.
Detailed Description
The invention will be further described with reference to the drawings and specific examples.
As shown in figure 1, a label-free high invasive circulating tumor cell capturing and culturing chip comprises an upper layer 1, a middle layer and a lower layer 3 which are sequentially overlapped from top to bottom; the upper layer 1 is provided with a cell suspension perfusion channel 101; the two ends of the cell suspension perfusion channel are respectively provided with a first liquid inlet 102 and a first liquid outlet 103; the lower layer 3 is provided with a leukocyte chemotactic separation area 301 and a CTC chemotactic capture area 302 corresponding to the position of the cell suspension perfusion channel 101; the intermediate layer includes a first PET film 201 and a second PET film 202; the first PET film 201 is arranged at a position corresponding to the leukocyte chemotactic separation area 301, and the second PET film 202 is arranged at a position corresponding to the CTC chemotactic capture area 302; the second liquid inlet 305 and the second liquid outlet 306 of the leukocyte chemotactic separation zone 301 are respectively provided with a through hole at the corresponding position of the upper layer 1; and the third liquid inlet 307 and the third liquid outlet 308 of the CTC chemotactic capture area 302 are respectively provided with a through hole at the corresponding position of the upper layer 1. The first PET film 201 and the second PET film 202 have the same width and are slightly wider than the cell suspension perfusion channel 101. The first PET film 201 has a length greater than that of the second PET film 202, and the second PET film 202 has a length half of that of the first PET film 201. The first PET film 201 has a pore size of less than 8 μm, the second PET film 202 has a pore size of greater than 8 μm, and the first PET film has a pore size of 10 μm. This configuration allows blood cells to enter the leukocyte chemotactic separation area 301 through the first PET film 201 and tumor cells to enter the CTC chemotactic capture area 302 through the second PET film 202. Wherein the leukocyte chemotactic separation zone 301 is slightly smaller in size than the first PET film 201, can be completely covered therewith. The CTC chemotactic capture zone 302 is slightly smaller in size than the second PET film 202, and can be completely covered by it.
The cell suspension perfusion channel 101 is of a rectangular structure, and two ends of the cell suspension perfusion channel are of a triangular structure; the positions of the vertexes of the triangles correspond to the first liquid inlet 102 and the first liquid outlet 103 respectively. This structure makes the cells less likely to adhere to the side walls during injection. The flow rate of the cell suspension perfusion channel 101 ranges from 0.1 ul/min to 4 ml/min.
The leukocyte chemotactic separation zone 301 and the second liquid inlet 305 are communicated through a first liquid inlet channel 303, and the second liquid outlet 306 is communicated through a first liquid outlet channel 309; the CTC chemotactic capture zone 302 and the third fluid inlet 307 are in communication via a second fluid inlet channel 310 and the third fluid outlet 308 is in communication via a second fluid outlet channel 304. The culture medium or hydrogel material is injected through the first and second liquid inlet channels 303 and 310.
The main body of the leukocyte chemotactic separation zone 301 is of a rectangular structure, two ends of the main body are of triangular structures, and the positions of the vertexes of the triangles correspond to one ends of the first liquid inlet channel 303 and the first liquid outlet channel 309 respectively; the main body of the CTC chemotactic capture area 302 has a rectangular structure, two ends of the main body have triangular structures, and the vertex positions of the triangles correspond to one ends of the second liquid inlet channel 310 and the second liquid outlet channel 304 respectively. This structure allows the leukocyte chemotactic separation area 301 and the CTC chemotactic capture area 302 to form a hexagonal structure.
The second liquid inlet 305 is communicated with the first through hole 104 arranged on the upper layer 1, and the second liquid outlet 306 is communicated with the second through hole 105 arranged on the upper layer 1; the third liquid inlet 307 is communicated with the fourth through hole 107 arranged on the upper layer 1, and the third liquid outlet 308 is communicated with the third through hole 106 arranged on the upper layer 1.
The vertex angles of the first liquid inlet 102 and the first liquid outlet 103 corresponding to the triangles are smaller than 45 degrees. The vertex angles of the first liquid inlet channel 303 and the first liquid outlet channel 309 corresponding to the triangles are smaller than 45 degrees; the vertex angles of the second liquid inlet channel 310 and the second liquid outlet channel 304 corresponding to the triangles are smaller than 45 degrees.
The upper layer 1 and the lower layer 3 are made of polydimethylsiloxane. The three-layer structure is combined into a whole by a vacuum plasma bonding method.
When in use, the method is carried out according to the following steps:
step 1: taking 10 ml of whole blood sample, preprocessing the sample by adopting erythrocyte lysate, lysing erythrocytes to obtain mixed cells of leucocytes and CTC, and re-suspending the mixed cells in 10 mL serum-free RPMI-1640 medium for later use.
Step 2: leukocyte chemotactic factors (C5 a, IL-2, etc., but not limited to the two) are mixed with a serum-free RPMI-1640 medium to prepare the leukocyte chemotactic medium.
Step 3: CTC chemokines (TGF- β, FGF, IGF, EGF, etc., not limited to the above four) were mixed with 10-15% serum-containing methacryloylated gelatin to prepare CTC capture matrices.
Step 4: the leukocyte chemotactic medium and the CTC trapping matrix were injected into the leukocyte chemotactic separation area 301 and the CTC chemotactic trapping area 302 of the chip, respectively, the liquid injection port was closed with a plug, and the methacryloylated gelatin was irradiated with an ultraviolet lamp for 20 seconds, so that the matrix in a liquid state was solidified into a hydrogel state.
Step 5: two hoses for the storage tube filled with the mixed cell suspension are connected with a cell suspension liquid inlet and a cell suspension liquid outlet (namely a first liquid inlet 102 and a first liquid outlet 103) on the top layer of the chip to form a closed liquid circulation perfusion system, and the peristaltic pump is used as power to drive the cell suspension in the storage tube to flow through the chip at a stable flow rate and then return to the storage tube. The flow rate of the liquid can be set with reference to the blood flow rate of peripheral veins of the capillary network of the human body so as to simulate the environment of peripheral blood circulation inside and outside the human body. The liquid circulation time is controlled between 24 and 48 and h, and during the period, the chip and the cell suspension storage tube are placed in a 5% carbon dioxide incubator to be suitable for the environment of cell culture.
Step 6: 24-48 h, the leukocytes in the mixed cell suspension have been largely chemotactically migrated into the underlying leukocyte chemotactic separation zone 301, and the highly invasive CTCs will also undergo chemotactic migration from the upper layer 1 of the chip into the hydrogel material of the CTC capturing zone. At this point, the cell suspension reservoir tube may be replaced with a nutrient medium reservoir tube, continuing to maintain liquid perfusion, enabling the captured highly invasive CTCs to continue culture growth in the hydrogel material for downstream analysis.
The invention can treat 95-97% of white blood cells through chemotaxis treatment, can label-free separation and capture CTC, and performs immunofluorescence staining characterization on invasiveness related proteins MMP-2 and MMP-9, wherein the characterization results are shown in figures 2 and 3. Fig. 2 is a schematic view of the staining effect of CTCs not captured by the present chip, and fig. 3 is a schematic view of the staining effect of CTCs captured by the present chip. As can be seen from the figure, CTCs isolated by the chip secreted a greater amount of invasiveness-related protein, confirming that CTCs isolated by the chip were more invasive. And which can be maintained in culture in the chip for a long period of time. The method provides a brand-new separation idea and method for the label-free separation of CTC, breaks the limitation that the traditional CTC separation is only used for counting through the capture and culture of highly invasive CTC, widens the application of CTC in other aspects such as downstream analysis, drug testing and the like, and has a stronger application value.
Claims (9)
1. The label-free high invasive circulating tumor cell capturing and culturing chip is characterized by comprising an upper layer (1), a middle layer and a lower layer (3) which are sequentially overlapped from top to bottom; a cell suspension perfusion channel (101) is arranged on the upper layer (1); a first liquid inlet (102) and a first liquid outlet (103) are respectively arranged at two ends of the cell suspension perfusion channel; a leukocyte chemotactic separation area (301) and a CTC chemotactic capture area (302) are arranged at the position of the lower layer (3) corresponding to the cell suspension perfusion channel (101); the intermediate layer comprises a first PET film (201) and a second PET film (202); the first PET film (201) is arranged at a position corresponding to the leukocyte chemotactic separation area (301), and the second PET film (202) is arranged at a position corresponding to the CTC chemotactic capture area (302); the second liquid inlet (305) and the second liquid outlet (306) of the leukocyte chemotactic separation area (301) are respectively provided with a through hole at the corresponding position of the upper layer (1); the third liquid inlet (307) and the third liquid outlet (308) of the CTC chemotactic capture area (302) are respectively provided with a through hole at the corresponding position of the upper layer (1); the first PET film (201) has a pore size of 5 μm, and the second PET film (202) has a pore size of 10 μm; the flow rate range of the cell suspension perfusion channel (101) is 0.1 mu l/min-4 ml/min;
the leukocyte chemotactic factor is mixed with serum-free RPMI-1640 culture medium to prepare leukocyte chemotactic culture medium, and the CTC chemotactic factor is mixed with methacryloylated gelatin containing 10-15% of serum to prepare CTC capturing matrix; the leukocyte chemotactic medium and the CTC capture matrix are respectively injected into a leukocyte chemotactic separation area (301) and a CTC chemotactic capture area (302) of the chip.
2. The label-free high invasive circulating tumor cell capturing and culturing chip according to claim 1, wherein the main body of the cell suspension perfusion channel (101) is of a rectangular structure, and two ends of the cell suspension perfusion channel are of a triangular structure; the positions of the vertexes of the triangles are respectively corresponding to the first liquid inlet (102) and the first liquid outlet (103).
3. The label-free highly invasive circulating tumor cell capturing and culturing chip according to claim 1, characterized in that the leukocyte chemotactic separation area (301) and the second liquid inlet (305) are communicated through a first liquid inlet channel (303), and the second liquid outlet (306) is communicated through a first liquid outlet channel (309); the CTC chemotactic capture area (302) is communicated with the third liquid inlet (307) through a second liquid inlet channel (310), and the third liquid outlet (308) is communicated with the third liquid outlet channel (304).
4. A label-free highly invasive circulating tumor cell capturing and culturing chip according to claim 3, wherein the main body of the leukocyte chemotactic separation area (301) is in a rectangular structure, two ends are in a triangular structure, and the vertex positions of the triangles correspond to one end of the first liquid inlet channel (303) and one end of the first liquid outlet channel (309) respectively; the main body of the CTC chemotactic capture area (302) is of a rectangular structure, two ends of the main body are of triangular structures, and the vertex positions of the triangles correspond to one end of the second liquid inlet channel (310) and one end of the second liquid outlet channel (304) respectively.
5. A label-free highly invasive circulating tumor cell capturing and culturing chip according to claim 3, wherein the second liquid inlet (305) is communicated with the first through hole (104) arranged on the upper layer (1), and the second liquid outlet (306) is communicated with the second through hole (105) arranged on the upper layer (1); the third liquid inlet (307) is communicated with a fourth through hole (107) arranged on the upper layer (1), and the third liquid outlet (308) is communicated with a third through hole (106) arranged on the upper layer (1).
6. The label-free high invasive circulating tumor cell capturing and culturing chip according to claim 2, wherein the vertex angles of the corresponding triangles of the first liquid inlet (102) and the first liquid outlet (103) are smaller than 45 °.
7. The label-free highly invasive circulating tumor cell capturing and culturing chip according to claim 4, wherein the vertex angles of the corresponding triangles of the first liquid inlet channel (303) and the first liquid outlet channel (309) are smaller than 45 °; the vertex angles of the second liquid inlet channel (310) and the second liquid outlet channel (304) corresponding to the triangles are smaller than 45 degrees.
8. The label-free highly invasive circulating tumor cell capturing and culturing chip of claim 1, wherein the first PET film (201) covers a leukocyte chemotactic separation area (301) and the second PET film covers a CTC chemotactic capturing area (302).
9. The label-free highly invasive circulating tumor cell capturing and culturing chip according to claim 1, wherein the upper layer (1) and the lower layer (3) are made of polydimethylsiloxane.
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