CN118165826A - Multi-channel three-dimensional co-culture microfluidic chip for bile duct cancer drug screening model - Google Patents
Multi-channel three-dimensional co-culture microfluidic chip for bile duct cancer drug screening model Download PDFInfo
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Abstract
The invention provides a multichannel three-dimensional co-culture microfluidic chip for a bile duct cancer drug screening model, and relates to the field of organ chip preparation, wherein a poly-polycarbonate membrane cell culture layer comprises a plurality of substructures; the straight flow channel layer comprises a multi-channel array layer and a first array cavity perforation film layer; the array type culture chamber comprises an array chamber basal layer and a second array chamber perforation membrane layer; the polycarbonate membrane cell culture layer is arranged between the first array chamber perforated membrane layer and the second array chamber perforated membrane layer; the multi-channel array layer and the first array cavity perforation film layer form a sealed flow channel and a cavity flow field through irreversible bonding; the array chamber substrate layer and the second array chamber perforated film layer form an array culture chamber by irreversible bonding. The invention simplifies the design of the conventional Christmas tree concentration gradient chip, adopts the parallel multi-channel array layer, can avoid concentration crosstalk caused by unstable diffusion time, and reduces the manufacturing difficulty.
Description
Technical Field
The invention relates to the field of organ chip preparation, in particular to a multichannel three-dimensional co-culture microfluidic chip for a bile duct cancer drug screening model.
Background
The organ chip is a bionic micro-fluidic chip which utilizes micro-processing technology, combines multi-disciplinary methods such as cell biology, biological tissue engineering and the like, reconstructs organ microstructure in vitro and simulates in vivo physiological functions and microenvironment. The method is widely applied to the aspects of human physiology research, disease simulation, toxicology research, drug research and the like at present, and provides a new research platform for exploring the occurrence and development mechanism of diseases, drug screening and drug effect evaluation.
Bile duct cancer, which is the second largest primary liver malignancy, is highly invasive and heterogeneous, has a poor prognosis for chemotherapy, and lacks an accurate treatment regimen. The release of paracrine signals by various cellular interactions in the tumor microenvironment drives the progression of cholangiocarcinoma, and is also an important factor affecting tumor therapy resistance, and the selection of an effective chemotherapy regimen is a treatment key. Traditional two-dimensional cell culture methods for researching cholangiocarcinoma cannot fully simulate multicellular interactions; the animal model brings inconvenience to experiments due to species difference and ethical problems, implantation of the ectopic xenogenic model is in a non-physiological environment and is rarely transferred, interaction between specific tumor cells and tumor microenvironment cannot be studied, however, in-situ xenogenic implantation model is excessively long in time, and application of real-time personalized medical treatment is limited; compared with a static mode of three-dimensional tumor microsphere and stent assisted culture, the microfluidic chip provides a strategy of dynamic perfusion culture, realizes nutrient exchange and drug stimulation, and performs drug effect evaluation in multiple dimensions.
Because the existing cholangiocarcinoma models can not be used for summarizing the physiological characteristics of the tumor microenvironment of patients, a pre-clinical cholangiocarcinoma drug screening model is needed.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a multichannel three-dimensional co-culture microfluidic chip for a bile duct cancer drug screening model.
In order to achieve the above object, the present invention provides the following solutions:
A multi-channel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model, comprising: a straight flow channel layer, an array type culture chamber and a poly-carbonate membrane cell culture layer; the poly-polycarbonate membrane cell culture layer comprises a plurality of substructures which are arranged in parallel and used for culturing substances to be cultured;
The straight flow channel layer comprises a multi-channel array layer and a first array cavity perforation film layer; the array culture chamber comprises an array chamber substrate layer and a second array chamber perforated membrane layer; the poly-polycarbonate membrane cell culture layer is disposed between the first array chamber perforated membrane layer and the second array chamber perforated membrane layer; the multi-channel array layer and the first array chamber perforated film layer form a sealed flow channel and a chamber flow field through irreversible bonding; the array chamber basal layer and the second array chamber perforated membrane layer form the array culture chamber through irreversible bonding, the distances between adjacent substructures are the same, and the length of each substructure is longer than that of the array culture chamber; the array type culture chamber is used for containing substances to be cultured and carrying out stimulation or detection treatment on the substances to be cultured.
Preferably, the straight runner layers are distributed in an array, the straight runner layers are provided with a plurality of upper layer liquid inlets and upper layer liquid outlets, each upper layer liquid inlet is connected with a steel needle capillary tube through a polyethylene plastic hose, the upper layer liquid inlets are powered by a plurality of rows of injection pumps, the injection pumps are used for controlling a small amount of liquid to flow and change in the micro-channels so as to transport nutrient substances to the sealing runner, and the nutrient substances are fully acted on the surface through laminar diffusion and permeation of the upper layer runner to a lower layer cavity of the cell culture layer of the polycarbonate membrane; the liquid outlet is used for collecting substances to be detected.
Preferably, the method further comprises: a chip clamp;
The chip clamp is respectively arranged on an upper layer and a lower layer of an integrated structure formed by the straight flow channel layer and the array type culture layer; the chip clamp is used for fixing the straight flow channel layer and the array type culture chamber.
Preferably, an upper liquid through window is arranged on the upper chip clamp; the upper liquid inlet is communicated with the upper liquid inlet in an aligned manner; a lower liquid through window is arranged on the lower chip clamp; the lower liquid through window is communicated with the lower liquid inlet in an aligned mode.
According to the specific embodiment provided by the invention, the invention discloses the following technical effects:
The invention provides a multichannel three-dimensional co-culture microfluidic chip for a bile duct cancer drug screening model, which comprises the following components: a straight flow channel layer, an array type culture chamber and a poly-carbonate membrane cell culture layer; the poly-polycarbonate membrane cell culture layer comprises a plurality of substructures which are arranged in parallel and used for culturing substances to be cultured; the straight flow channel layer comprises a multi-channel array layer and a first array cavity perforation film layer; the array culture chamber comprises an array chamber substrate layer and a second array chamber perforated membrane layer; the poly-polycarbonate membrane cell culture layer is disposed between the first array chamber perforated membrane layer and the second array chamber perforated membrane layer; the multi-channel array layer and the first array chamber perforated film layer form a sealed flow channel and a chamber flow field through irreversible bonding; the array chamber basal layer and the second array chamber perforated membrane layer form the array culture chamber through irreversible bonding, the distances between adjacent substructures are the same, and the length of each substructure is longer than that of the array culture chamber; the array type culture chamber is used for containing substances to be cultured and carrying out stimulation or detection treatment on the substances to be cultured. The invention simplifies the design of the conventional Christmas tree concentration gradient chip, adopts the parallel multi-channel array layer, can avoid concentration crosstalk caused by unstable diffusion time, and reduces the manufacturing difficulty.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions of the prior art, the drawings that are needed in the embodiments will be briefly described below, it being obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is an exploded view of a chip assembly according to an embodiment of the present invention;
fig. 2 is a top view of a dc channel layer according to an embodiment of the present invention;
Fig. 3 is a schematic diagram of the overall structure of a dc channel layer according to an embodiment of the present invention;
FIG. 4 is a schematic view of a liquid inlet and a liquid outlet according to an embodiment of the present invention;
FIG. 5 is a top view of an array chamber perforated film layer provided in an embodiment of the invention;
FIG. 6 is a top view of a culture chamber provided in an embodiment of the invention;
FIG. 7 is a schematic view of a culture chamber according to an embodiment of the present invention;
FIG. 8 is a top view of a perforated film layer of a second array of chambers provided in accordance with an embodiment of the present invention;
FIG. 9 is a top view of a substrate layer of an array chamber provided in an embodiment of the invention;
fig. 10 is a schematic diagram of a chip fixture according to an embodiment of the present invention.
FIG. 11 is a schematic diagram showing cell viability of an EA.hy926 cell porous membrane layer in chip culture for 12h, 24h, and 48h according to an embodiment of the present invention;
FIG. 12 is a schematic representation of apparent permeability analysis of EA.hy926 cell porous membranes using sodium fluorescein and dextran dyes of different molecular weights provided in an example of the present invention;
FIG. 13 is a schematic diagram showing the quantification of LX-2 and HUCCT-1 Cell activity during chip culture using Cell-TRACKER DIL/DID staining markers according to an example of the present invention;
FIG. 14 is a schematic diagram showing the expression of alpha-SMA and VEGF under co-culture compared with LX-2 alone;
FIG. 15 is a schematic diagram showing the detection of drug sensitivity of different drugs to human bile duct cancer cells HUCCT-1 by MTT method and chip liveness staining method according to an embodiment of the present invention;
FIG. 16 is a graph showing the effect of flow cytometry on HUCCT-1 apoptosis of drug treatments at different concentrations for 48h.
Reference numerals illustrate:
100-straight runner layers, 101-multichannel array layers, 102-chamber flow fields, 103-first array chamber perforated membrane layers, 104-sealed runners, 105-upper liquid inlets, 106-upper liquid outlets, 200-polycarbonate membrane cell culture layers, 202-channel holes, 300-array culture layers, 301-array chamber basal layers, 302-culture chambers, 303-second array chamber perforated membrane layers, 305-lower liquid inlets, 306-lower liquid outlets, 307-liquid inlets, 308-liquid outlets, 10-upper clamping plates, 11-upper liquid through windows, 13-lower liquid through windows and 13-intermediate observation windows.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The invention aims to provide a multichannel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model, which simplifies the design of a conventional Christmas tree concentration gradient chip, and can avoid concentration crosstalk caused by unstable diffusion time and reduce manufacturing difficulty by using a parallel multichannel array layer.
In order that the above-recited objects, features and advantages of the present invention will become more readily apparent, a more particular description of the invention will be rendered by reference to the appended drawings and appended detailed description.
As shown in fig. 1 to 10, the present invention provides a multi-channel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model, comprising: the cell culture device comprises a straight runner layer 100 (namely a straight runner layer PDMS), a poly-polycarbonate membrane cell culture layer 200, an array culture layer 300 (namely an array culture layer PDMS) and a chip clamp (namely an upper-layer and lower-layer acrylic clamp);
The straight flow channel layer 100 comprises a multi-channel array layer 101 and a first array chamber perforated membrane layer 103; the array culture layer 300 comprises an array chamber substrate layer 301 and a second array chamber perforated membrane layer 303; the multi-channel array layer 101 and the first array chamber perforated membrane layer 103 form a sealing flow channel 104 and a chamber flow field 102 through irreversible bonding; the array chamber substrate layer 301 and the second array chamber perforated membrane layer form the array culture chamber 302 by irreversible bonding, and the array culture layer 300 is used for accommodating substances to be cultured and performing stimulation or detection treatment on the substances to be cultured. The straight runner layers 100 are distributed in an array, the straight runner layers 100 are provided with a plurality of upper liquid inlets 105 and upper liquid outlets 106, the upper liquid inlets 105 are connected with a plurality of rows of injection pumps through polyethylene plastic hoses and steel needle capillaries, and the injection pumps are used for providing power and controlling a small amount of liquid to flow and change in the micro-channels so as to transport nutrient substances to the sealed runners 104; the liquid outlet is used for collecting substances to be detected and nutrient substances fully act on the surface. The chip fixture is respectively arranged on the upper layer and the lower layer of the integrated structure formed by the straight flow channel layer 100 and the array culture layer 300, and is fixed. An upper liquid through window 11 is arranged on the upper chip clamp (upper clamping plate 10); the upper liquid inlet window 11 is communicated with the upper liquid inlet 105 in an aligned manner; a lower liquid through window 12 is arranged on the lower chip clamp (lower clamping plate); the lower liquid inlet 12 is aligned with the lower liquid inlet 305.
Specifically, the straight flow channel layer 100 includes a multi-channel array layer 101 and a first array chamber perforated membrane layer 103; the array culture layer 300 further comprises an array chamber substrate layer 301, an array culture chamber 302, and a second array chamber perforated membrane layer 303; forming a sealing runner 104 and a cavity flow field 102 by irreversibly bonding the multichannel array layer 101 and the first array cavity perforated membrane layer 103; the array chamber substrate layer 301 and the second array chamber perforated film layer 303 form an array culture chamber 302 through irreversible bonding, and are used for accommodating substances to be cultured, assisting the assembly of the customized acrylic clamp, and further performing stimulation or detection treatment on the substances to be cultured in the chip. The straight runner layers 100 are distributed in an array, and are distributed in an array mode in a 3×7 mode, and comprise upper liquid inlets 105, are connected to a plurality of rows of injection pumps through polyethylene plastic hoses and steel needle capillaries, are powered by the injection pumps, can accurately control a small amount of liquid to flow in micro channels for changing, and transport nutrient substances to the sealing runners 104. A poly-Polycarbonate (PC) membrane culture layer (poly-polycarbonate membrane cell culture layer 200), the PC membrane containing a regular ordered porous structure with a diameter of 0.4 μm, the ultra-high tensile strength maintaining pore size gaps, preventing cell migration and allowing molecular transport; smooth, thin, glass-like surfaces, very low absorption maximizes critical solution recovery; the structural arrangement can also increase the spatial arrangement, in order to overcome the limitation of 2D cell culture, physical hydrogel such as a rat tail collagen coating surface is used for promoting cell adhesion and growth, and PC membranes full of cells respectively correspond to substance exchange areas of PDMS, and relatively closed upper and lower culture chambers 302 are formed by assembling upper and lower layers of PDMS.
Optionally, the chip holder in this embodiment includes: the upper acrylic clamp, the lower acrylic clamp and the eight upper and lower connecting holes penetrate through the upper and lower connecting holes through screws to assemble and fix the microfluidic chip, the positions of the upper liquid through window 11 and the lower liquid through window 12 corresponding to the upper clamping plate 10 are respectively communicated with the liquid inlet and the liquid outlet in an aligned mode, the middle observation window 13 corresponds to the position of the detection channel, and the physiological state of cells is conveniently monitored by combining the related biological sensors.
The multilayer superposition design in this embodiment can be used to construct a three-dimensional co-cultured cholangiocarcinoma in vitro model, and the main module comprises an upper multichannel perfusion layer (straight flow channel layer 100), a middle poly-polycarbonate membrane cell culture layer 200 and a lower co-cultured cell chamber layer (array culture chamber 302), so that the micro-physiological structure and functions of vascular endothelial cells, cholangiocytic epithelial cells and hepatic stellate cells are highly simulated, the relative closed environment formed inside the chip is close to the spatial characteristics of cells in a physiological state, and a porous membrane interlayer is arranged in the chip to realize the barrier function of endothelial cells and realize the tumor microenvironment of cell-cell interaction.
The Polydimethylsiloxane (PDMS) material adopted by the chip has the advantages of good biocompatibility, high elasticity and air permeability, ensures the gas exchange of O 2 and CO 2 in the cell culture process, and allows the cells to maintain the cell viability for a long time through the diffusion of the gas in the chamber.
The chip is manufactured by using a standard soft lithography method, and the process mainly comprises mask design, template manufacture and chip processing, and specifically comprises the following steps: the structure was designed using Auto CAD software and printed as a film mask with a full chip size of 33X 37mm. The standard soft lithography method is adopted to prepare the silicon wafer template, and the specification is as follows: the upper layer flow chip contains 7 channels, each channel having a diameter of 0.4mm and a distance between channels of 4mm. The diameters of the liquid inlet and the liquid outlet are 1mm, the liquid inlet channel 307 is 5mm long, the liquid outlet channel 308 is 6mm long, and the interval channel between the liquid inlet and the liquid outlet is 3mm long; the four layers of chips are provided with 21 rounded rectangles with the size of 4.6X2 mm as substance exchange areas. The middle layer chip is provided with a round hole with the diameter of 2mm as a channel for communicating the lower layer chip; the liquid outlet channel 308 of the lower array chamber chip is 3mm long, and a liquid inlet and a liquid outlet corresponding to the upper layer are respectively arranged on the left side of the channel. The height of the template for manufacturing the silicon wafer is 200 mu m. . And weighing the PDMS prepolymer RTV615 type adhesive A and the PDMS prepolymer B according to a ratio of 10:1, and cutting a structural part PDMS after curing, wherein the thickness of each layer of PDMS chip is respectively 3mm, 1mm and 2mm from top to bottom. And (3) punching holes at the inlet end and the outlet end of the channel by using a 0.5mm puncher, wherein the pore size is proper to the size of a steel needle inserted into the connecting pipe.
Sterilization of microfluidic chip devices: before performing a cell experiment, placing the PDMS chip in absolute ethyl alcohol, performing ultrasonic treatment for 5min, washing with deionized water for 5min, and drying at 80 ℃ for later use; the device is sterilized by ultraviolet rays by a steel needle connected with an inlet and an outlet and a polyethylene elastic plastic tube connected with a syringe pump.
Preparation and application methods of the porous membrane: a polycarbonate porous membrane with a pore diameter of 0.4 μm was cut into a rectangle with a specification of 30X 0.5mm, stored in 75% ethanol, washed three times with DPBS, placed in 60mm dishes in order, and air-dried in an ultra clean bench. Coating two sides of a porous membrane by using 0.36mg/ml of rat tail collagen, carefully attaching the rat tail collagen to the bottom of a dish, incubating for 2 hours at 37 ℃ and then inoculating cells, wherein the cell density is more than 1X 10 7 cells/ml, incubating in a cell incubator, changing the liquid after 24 hours, and then culturing for 48 hours again, and observing under a microscope until the cells are completely full of the porous membrane for experiments.
Cell co-culture in PDMS chip: the epithelial cells HUCCT-1 of the human intrahepatic bile duct cancer and the human hepatic stellate cells LX-2 are mixed into a cell suspension according to a ratio of 1:2, the cell density is larger than 1X 10 6/ml, and the cell density of each cell layer is inoculated according to the total number of the simulated human intrahepatic cells. The cell mixture was mixed with Matrigel at 1:1 and injected into the cell culture chamber 302 and placed in a 5% CO 2 incubator at 37℃for 4 hours. Matrigel polymerizes at room temperature to form a three-dimensional matrix with bioactivity, which is helpful for simulating the 3D structure of cells in vivo and providing a favorable physiological microenvironment for co-culture of the cells.
And after the multi-layer module is assembled from bottom to top, fixing and sealing the multi-layer module by using an acrylic clamp and screws.
The channel inlet is connected with the injection pump through the steel needle and the polyethylene elastic plastic pipe, cell culture liquid perfusion is carried out at a stable flow rate, the flow rate is 1 mu l/min in the experiment, and the influence degree of the control fluid shear force on cells is low. Co-culture medium EA.hy926/HUCCT-1/LX-2 medium was prepared at a 1:1:1 volume ratio and 10ng/mL vascular endothelial growth factor VEGF was added.
This example enables assessment of cell viability and structural function of each cell layer: because PDMS has good optical transparency, the physiological state of the cells can be monitored in real time by using a fluorescence microscope. To verify the structure and function of cells in the three-dimensional culture mode of the chip, after 48 hours of normal operation, the structure of each cell layer was visualized by fluorescent staining, and the chip was disassembled for biological characterization of each cell layer.
First, a porous membrane layer of 2D cells was constructed using ea.hy926 cells to mimic the tissue barrier function of the capillary wall. TRITC Phallodin fluorescent staining shows the network fiber structure distribution of the cellular microfilament skeleton on the porous membrane, while live and dead staining of cells using AM and PI staining. The results show that cells are spread on the surface of the porous membrane to form a complete coverage, and the state structure is good. The cultured EA.hy926 cell porous membrane layer and the chip are combined and cultured for 48 hours, and the cell viability is more than 90% as shown in FIG. 11. Expression of ZO-1 is a biomarker that indicates tight junctions between endothelial cells. Immunofluorescence analysis shows that after 72h of endothelial cell layer culture, a tightly connected monolayer can be formed, meeting the requirements of drug permeability modeling based on porous membranes.
Porous membrane endothelial cell layer barrier function analysis: a chip containing only single-layer porous membrane endothelial cells is designed, and prepared DMEM complete culture medium is simultaneously applied to the upper layer and the lower layer of the chip, wherein the upper layer culture medium is respectively prepared into DMEM complete culture solution containing 1 mu M of 70kDa, 40kDa and 10kDa fluorescein isothiocyanate FITC-dextran solution and 2mg/ml fluorescein sodium. After the system is stable, collecting the underflow liquid every half hour, detecting the fluorescence intensity by using an enzyme-labeled instrument, and comparing the fluorescence intensity with a standard curve to obtain the apparent permeability according to a formula. From the view of figure 12 (n=4,) The result shows that the endothelial cell layer has a barrier function, allows selective permeation of substances with different molecular weights, and has strong interception capability on macromolecular substances; in immunofluorescence analysis of its ZO-1 protein, it was shown that the endothelial cell layer can form a tightly linked monolayer, indicating that drug permeability studies can be performed based on porous membranes, and that analytes can diffuse through dynamic fluids to the lower layer of the endothelial cell monolayer.
Verifying the functional characteristics of the co-culture system of the chip: this example uses Matrigel to help mimic the extracellular matrix of cells in vivo, providing a favorable physiological microenvironment for the cells. The quantitative ratios of the two cells (HUCCT-1 and LX-2) stained with Cell-TRACKERDID/DIL were counted 24h and 48h after 3D culture of the chip, and the results of fluorescence quantification, as shown in fig. 13 (where (n=4, ns P >0.05, P < 0.01)), showed good Cell activity. Alpha-smooth muscle actin (alpha-SMA) is one of the markers of hepatic stellate cell activation, with alpha-SMA being expressed in increased amounts with increased liver fibrosis. Cytokeratin 19 (CK 19) is an epithelial cell structural protein, and specifically stains bile duct epithelial cells and intrahepatic bile duct cancer cells. Compared with the single culture of LX-2, the immunofluorescence detection quantification result is shown in fig. 14 (n=5, ns P >0.05, P < 0.0001), and CK19 and α -SMA are positive in specific expression, indicating that HUCCT-1 and LX-2 have good structure and function in the chip co-culture mode. As shown in the multi-index immunoassay result of the multi-channel chip in FIG. 14, the expression of the secretory factor Vascular Endothelial Growth Factor (VEGF) is increased, so that LX-2 cells are activated and have fibrosis proliferation. The data of this example demonstrate that the microenvironment of the chip is more conducive to the biomimetic growth of tumor cells.
In addition, the microporous unit of multicellular co-culture in the embodiment has high correlation to tumor microenvironment research, can provide analysis data of cell polarization, differentiation and cytotoxicity, can carry out high-efficiency drug screening under the background of the platform, and can carry out real-time monitoring on cytotoxicity analysis of the estimated drugs.
As an optional implementation mode, aiming at the problem of lack of a drug sensitivity detection model for simulating the bile duct cancer tumor microenvironment, the chip model is utilized to perform single drug administration and combined drug administration to induce tumor cell apoptosis, and under the injection pump perfusion strategy, the processes of drug delivery, chip cell culture, stimulation, marking, multiple cell response detection and the like are integrated into a whole by utilizing the laminar flow characteristics in the chip microchannel, so that in-situ drug sensitivity detection is realized. The method can simultaneously generate 7 drug action conditions and 21 response effects in one operation, and can simultaneously analyze the apoptosis induction degree of different drugs.
In order to verify the feasibility of in-vitro drug sensitivity detection of the microfluidic chip platform, resveratrol (RV) is used for induction treatment for 48 hours in the embodiment, and the anti-tumor cell activity of the sample is detected. The use of single drug RV in 96-well plate and chip model respectively in 48 hours, detection results for such as figure 15 shows A, C, in chip RV drug sensitivity is dose dependent, calculated IC50 is 116.7 u M, than 96-well plate RVIC50, shows in chip system drug response sensitivity higher, and further using the chip system for cell co-culture drug effect evaluation. Based on MTT assay results, CIS 16. Mu.M was selected for administration in combination with RV (10, 25, 50, 100, 200. Mu.M), respectively. Under the same mode of the chip, drug sensitivity detection tests of HUCCT-1 single culture group, HUCCT-1 co-culture group and LX-2 co-culture group are respectively carried out, and the apoptosis rates of HUCTT-1 cells of the two groups are compared. As shown in the B of FIG. 15, compared with the single culture group, the apoptosis rate of HUCCT-1 cells in the co-culture group is reduced by about two times after the induction of 10-100 mu M of the chip drug RV for 48 hours, which suggests that the existence of hepatic stellate cells reduces the drug sensitivity of bile duct cancer cells and possibly participates in drug resistance generation of bile duct cancer cells.
To confirm whether the drug inhibited tumor cell growth by inducing HUCCT-1 apoptosis, this example used flow cytometry to examine the apoptosis rate of different concentrations of RV, CIS and their combination for treatment of HUCCT-1 cells for 48 h. The results show that both RV and CIS can effectively induce HUCCT-1 cells to apoptosis as shown in figure 16A; compared with a single drug group, RV combined with CIS can obviously promote HUCCT-1 cells to apoptosis.
To further investigate whether hepatic stellate cells (LX-2) affect drug sensitivity of tumor cells by apoptosis. The flow cytometry detection result shows that as shown in FIG. 16B, the combined drug (RV+CIS16. Mu.M) has more remarkable effect of promoting HUCCT-1 cells to apoptosis than the single RV drug; under the combined action, the apoptosis rate of HUCCT-1 cells of the co-culture group is obviously reduced by about two times compared with that of the single culture group (shown in table 1). These data again demonstrate that hepatic stellate cells (LX-2) are likely to inhibit bile duct cancer cell HUCCT-1 sensitivity to drugs.
TABLE 1 ratio of apoptosis rates of combination drugs on single cells and co-cultured groups HUCCT-1
The invention can realize the complex mechanical force microenvironment of three-dimensional multicellular co-culture, reappear the local tumor microenvironment of bile duct cancer, simulate the physiological microenvironment of liver sinus endothelial cells-bile duct epithelial cells-hepatic stellate cells, realize the barrier function of endothelial cells and have the tumor microenvironment of cell-cell and cell-cell interstitial interactions. The multicellular co-culture in vitro model constructed based on the platform can be used for investigating the sensitivity of anti-tumor drugs and the preliminary exploration of anti-tumor cell apoptosis cells by combined medication. Provides a platform with small sample quantity, low cost and high flux for exploring the evaluation of the anti-cholangiocarcinoma drug effect, and is helpful for improving the drug curative effect and the accurate treatment of tumors
The organ chip is based on micro-fluidic technology, micro-processing technology and bionic principle, and can simulate the complex microstructure, micro-environment and physiological functions of specific organ tissues of human body. A plurality of perfusion channels and a specially designed micro culture chamber can be integrated in the chip, the fluid flow speed is accurately controlled, and the physical and chemical gradient change is maintained in the chip by virtue of the laminar flow state; the medium is prepared by micromachining porous membranes with nanoscale apertures and the like, so that the mass transfer efficiency of nutrient substances is improved, the cell survival rate and the function are improved, and a favorable technical support is provided for in-vitro cell research, disease modeling, drug screening and individuation treatment.
The design of the invention simplifies the design of the conventional Christmas tree concentration gradient chip, avoids concentration crosstalk caused by unstable diffusion time, and reduces the manufacturing difficulty. The method can be suitable for drug sensitivity evaluation of single drug administration and combined drug administration, has the characteristics of high flux and low cost, and has wide conversion application prospect.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The principles and embodiments of the present invention have been described herein with reference to specific examples, the description of which is intended only to assist in understanding the methods of the present invention and the core ideas thereof; also, it is within the scope of the present invention to be modified by those of ordinary skill in the art in light of the present teachings. In view of the foregoing, this description should not be construed as limiting the invention.
Claims (4)
1. A multichannel three-dimensional co-culture micro-fluidic chip for bile duct cancer drug screening model is characterized by comprising: a straight flow channel layer, an array type culture chamber and a poly-carbonate membrane cell culture layer; the poly-polycarbonate membrane cell culture layer comprises a plurality of substructures which are arranged in parallel and used for culturing substances to be cultured;
The straight flow channel layer comprises a multi-channel array layer and a first array cavity perforation film layer; the array culture chamber comprises an array chamber substrate layer and a second array chamber perforated membrane layer; the polycarbonate membrane cell culture layer is arranged between the first array chamber perforated membrane layer and the second array chamber perforated membrane layer to form an upper chamber and a lower chamber; the multi-channel array layer and the first array chamber perforated film layer form a sealed flow channel and a chamber flow field through irreversible bonding; the array chamber basal layer and the second array chamber perforated membrane layer form the array culture chamber through irreversible bonding, the distances between adjacent substructures are the same, and the length of each substructure is longer than that of the array culture chamber; the array type culture chamber is used for containing substances to be cultured and carrying out stimulation or detection treatment on the substances to be cultured.
2. The multi-channel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model according to claim 1, wherein the straight flow channel layers are distributed in an array, the straight flow channel layers are provided with a plurality of upper liquid inlets and upper liquid outlets, each upper liquid inlet is connected with a steel needle capillary tube through a polyethylene plastic hose, the upper liquid inlets are powered by a plurality of rows of injection pumps, the injection pumps are used for controlling a small amount of liquid to flow and change in the micro-channels so as to transport nutrient substances to the sealing flow channels, and laminar diffusion through the upper flow channels permeates into a lower cavity of the polycarbonate membrane cell culture layer and fully acts on the surface; the liquid outlet is used for collecting the substance to be detected.
3. The multi-channel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model according to claim 2, further comprising: a chip clamp;
The chip clamp is respectively arranged on an upper layer and a lower layer of an integrated structure formed by the straight flow channel layer and the array type culture chamber; the chip clamp is used for fixing the straight flow channel layer and the array type culture chamber.
4. The multi-channel three-dimensional co-culture microfluidic chip for a cholangiocarcinoma drug screening model according to claim 3, wherein an upper liquid-passing window is arranged on the upper chip fixture; the upper liquid inlet is communicated with the upper liquid inlet in an aligned manner; a lower liquid through window is arranged on the lower chip clamp; the lower liquid through window is communicated with the lower liquid inlet in an aligned mode.
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