Integrated in-situ drug screening microfluidic system and application thereof
Through the integrated in-situ drug screening microfluidic system, the microsphereization and variable concentration gradient generation module of acoustic drive cells are used, combined with the hydraulic gate regulation module, and the rapid generation of controllable cell clusters and multi-modal drug screening are achieved, solving the problem of insufficient efficiency and accuracy of drug screening in the prior art.
Patent Information
- Application Number
- CN202510444443.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-08-15
AI Technical Summary
It is difficult for the prior art to quickly and high throughput to obtain controllable cell clusters and perform drug screening in situ, and the existing concentration gradient generators are difficult to achieve time gradient regulation, resulting in insufficient drug screening efficiency and accuracy.
The integrated in-situ drug screening microfluidic system is adopted, combined with the acoustic cell microsphere stimulation module and the variable concentration gradient generation module, and the hydrogate regulation module realizes the spatiotemporal regulation of cell clusters, generates cell spheres of controllable size, and performs multi-mode high-throughput drug screening.
While achieving rapid and high-throughput acquisition of controllable-size cellular spheres, it improves the accuracy and efficiency of drug screening, and can perform drug screening in multiple concentrations and stimulation modes in situ, solving the problems in the prior art.
Smart Images
Figure CN120484954A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microfluidic chips, and in particular to an integrated in-situ drug screening microfluidic system and its application. Background Art
[0002] In the fields of tissue engineering and regenerative medicine, 3D cell spheroid culture technology has attracted considerable attention due to its significant biological advantages. Compared to traditional monodispersed cells or two-dimensional monolayer cultures, 3D cell spheroids can significantly enhance the therapeutic efficacy of stem cells by simulating the three-dimensional spatial structure of the in vivo microenvironment. This is manifested in enhanced cell survival, increased growth factor secretion, and long-term maintenance of multidirectional cell differentiation potential. In the field of tumor research, 3D cell spheroids have become an important model for drug screening due to their more physiologically relevant morphology and spatial arrangement. They can accurately replicate multiple key pathological features of real tumors, including phenotypic heterogeneity, growth dynamics, and cell-cell interactions, thereby obtaining more accurate results in drug screening.
[0003] However, when it comes to large-scale applications such as biomanufacturing and high-throughput drug screening, there are still multiple challenges. The current process for generating cell spheroids generally has problems such as long culture cycles and poor size uniformity. The long-term culture cycle places extremely high demands on the environment for maintaining cell culture. How to quickly and massively obtain size-controlled and uniform cell spheroids remains a challenge. At the same time, although culture strategies based on biological scaffolds or porous membranes can promote cell aggregation, they face problems with material biocompatibility and compatibility with sterilization processes. Existing materials that can withstand high temperature, high pressure or chemical sterilization often have cytotoxicity risks, while bio-friendly materials are difficult to sterilize.
[0004] Therefore, existing technologies make it difficult to quickly and conveniently perform in situ drug screening while simultaneously generating cell spheroids. Currently, common concentration gradient generators (such as Christmas tree-shaped and disc-shaped structures) are limited to constructing spatial concentration gradients. Most can only achieve spatial concentration gradient control, i.e., concentration differences between different outlets, but are unable to achieve temporal gradient control, i.e., concentration changes over time at the same outlet. This makes it difficult to perform in situ, real-time drug response monitoring after cell spheroid formation without destroying the three-dimensional structure, severely restricting the efficiency and data reliability of high-throughput screening.
[0005] Therefore, existing technologies lack a technology that can quickly and efficiently obtain cell clusters of controllable size and obtain the dynamic response of cell clusters in situ for drug screening. Summary of the Invention
[0006] In order to solve the problems existing in the prior art, the present invention provides an integrated in situ drug screening microfluidic system and its application, which combines the sound-driven cell microspherization stimulation module for quickly obtaining cell clusters with the variable concentration gradient generation module, and controls it through the hydraulic gate control module to achieve in situ spatiotemporal regulation of the chemical microenvironment outside the generated cell clusters, obtain the dynamic response of the cell clusters and then perform drug screening. It can achieve fast and convenient in situ drug screening operations while obtaining controllable size cell spheroids at a fast and high throughput, and significantly improve the accuracy of cell spheroid drug screening, the convenience of large-scale drug screening, and the efficiency of drug screening.
[0007] An integrated in situ drug screening microfluidic system includes a hydraulic gate control module, a variable concentration gradient generation module and an acoustic cell microspherization stimulation module located on both sides of the hydraulic gate control module; the variable concentration gradient generation module and the acoustic cell microspherization stimulation module are controlled and connected through the hydraulic gate control module.
[0008] Furthermore, the variable concentration gradient generating module includes a drug solution inlet, a drug solution outlet, and a concentration gradient generating microfluidic channel connecting the drug solution inlet and the drug solution outlet; the concentration gradient generating microfluidic channel includes several channel layers, each channel layer is composed of at least one transverse flow channel and several Christmas tree-shaped micromixers; wherein, the channel layer is at least three layers, and the Christmas tree-shaped micromixers in the channel layer are equidistantly distributed on the transverse flow channel and are longitudinally connected to the transverse flow channel.
[0009] Furthermore, the drug solution inlet is connected to the lateral flow channel in the concentration gradient generating microfluidic channel, and the drug solution outlet is connected to the Christmas tree-shaped micromixer in the concentration gradient generating microfluidic channel; wherein, the number of the Christmas tree-shaped micromixers increases step by step with the increase in the number of channel layers.
[0010] Furthermore, the number of Christmas tree-shaped micromixers in the first channel layer of the concentration gradient generating microfluidic channel is greater than the number of the drug solution sampling ports, and the number of the drug solution sampling ports is equal to the number of Christmas tree-shaped micromixers in the last channel layer of the concentration gradient generating microfluidic channel; wherein the number of the drug solution sampling ports is at least three.
[0011] Furthermore, the acoustically driven cell microspherization stimulation module includes several cell generation units, each of which includes a first multifunctional inlet and outlet, a second multifunctional inlet and outlet, and a longitudinal liquid flow channel connecting the first and second multifunctional inlets and outlets, wherein a PDMS tip structure is embedded on the inner wall of one side of the longitudinal liquid flow channel; a piezoelectric transducer is provided below the second multifunctional inlet and outlet, and the piezoelectric transducer is a disc-shaped structure;
[0012] The number of cell generation units in the sound-driven cell microspherization stimulation module is equal to the number of drug solution outlets in the variable concentration gradient generation module.
[0013] Furthermore, the hydraulic gate control module includes a hydraulic gate cutoff flow channel, a plurality of fluid conduction interfaces arranged on the hydraulic gate cutoff flow channel, and a plurality of buffer injection ports; the plurality of buffer injection ports are longitudinally connected to the hydraulic gate cutoff flow channel through a buffer flow channel; the buffer flow channel is spaced between the plurality of conduction interfaces;
[0014] The spacing between the fluid conducting interfaces is equal, and the spacing between the buffer injection ports is equal.
[0015] Furthermore, a buffer inlet is set every two fluid conduction interfaces in the hydraulic gate control module, and the number of the buffer inlets is less than the number of the fluid conduction interfaces; the number of the fluid conduction interfaces in the hydraulic gate control module is consistent with the number of the drug solution outlets in the variable concentration gradient generating module.
[0016] Furthermore, the drug solution outlet of the variable concentration gradient generating module and the second multifunctional inlet and outlet of the sound-driven cell microspheroidization stimulation module are integrated and connected at the fluid conduction interface of the hydraulic gate control module.
[0017] The present invention also provides application of the above-mentioned integrated in-situ drug screening microfluidic system in in-situ drug screening.
[0018] Furthermore, the in situ drug screening microfluidic system for screening in situ drugs includes the following steps:
[0019] Step 1: First, the cell mixture is pumped into the acoustic-driven cell microspheroidization stimulation module. The signal generator switch is turned on, and a disc-shaped piezoelectric transducer generates an acoustic field, which excites the PDMS tip structure located in the flow channel of the acoustic-driven cell microspheroidization stimulation module to vibrate, generating acoustic vortices and aggregating the cells into compact spheres.
[0020] Step 2: After the cells have adhered to each other, adjust the output waveform of the signal generator and the distribution of the eddy current drag force and the acoustic radiation force so that the generated cell clusters are fixed at the PDMS tip structure;
[0021] Step 3: Pumping three different concentrations of drug solutions into the concentration gradient generator portion of the variable concentration gradient generation module through the inlet of the variable concentration gradient generation module; loading different concentrations of drug solutions into the cell clusters located in the acoustic-driven cell microspheroidization stimulation module through the first multifunctional port; and forming concentration gradients with different spatial distributions by adjusting the order and flow rate of the three different concentrations of drug solutions pumped in, thereby stimulating the cell clusters at the PDMS tip structure;
[0022] Step 4: Pumping buffer into the buffer inlet by opening and closing the hydraulic gate control module's air pump hydraulic gate control module. When the pumping pressure of the buffer inlet in the hydraulic gate control module is greater than the pumping pressure of the inlet of the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0023] Step 5: adjusting and exchanging the pumping pressure of the buffer solution inlet in the hydraulic gate control module and the pumping pressure of the drug solution inlet in the variable concentration gradient generation module, so that the drug solutions to be tested with different concentrations can stimulate the cell spheroids;
[0024] Step 6: Repeat steps 3-5 and simultaneously monitor the dynamic responses of cell clusters to chemical stimuli of different concentrations and frequencies in multiple channels.
[0025] The advantages of the present invention are:
[0026] 1. The present invention utilizes acoustic-driven technology to rapidly generate and immobilize cell clusters in situ for drug screening. The size of the cell clusters can be controlled by adjusting the acoustic field parameters. A variable concentration gradient generation module combined with a hydraulic gate control module can exchange the pumping pressures of the drug solution inlet and the buffer outlet to achieve linear, exponential, or any desired concentration gradient, thereby enabling precise spatiotemporal control of the chemical microenvironment. The variable concentration gradient generation module, the hydraulic gate control module, and the acoustic-driven cell microspherization stimulation module are integrated into an integrated microfluidic chip for in situ drug screening, achieving the functions of cell microspherization, variable concentration gradient generation, and spatiotemporal control of the chemical microenvironment.
[0027] 2. The present invention integrates multiple channels. This integrated design significantly improves the efficiency and accuracy of drug screening. It can not only quickly obtain cell clusters of controllable size, but also perform in situ drug screening of multiple concentrations and stimulation modes. Compared with the existing technology, the present invention only needs to change the frequency of the piezoelectric transducer or the peak-to-peak value of the input voltage to prepare cell clusters of different sizes. The speed of preparing cell clusters is faster, and multiple cell clusters can be prepared at a time. After the cell clusters are generated, the frequency of the piezoelectric transducer or the peak-to-peak value of the input voltage can be used to achieve in situ fixation of the cell clusters. It can further achieve chemical stimulation of the cell clusters with adjustable amplitude and frequency, and realize multi-mode high-throughput multi-drug spatiotemporal adjustable drug concentration screening; it can solve the problem of the lack of rapid and high-throughput acquisition of controllable size cell clusters and in situ drug screening in the existing technology, and provide a powerful tool for the fields of tissue engineering, regenerative medicine and drug development. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 Schematic diagram of the structure of the integrated in situ drug screening microfluidic system of the present invention;
[0029] Figure 2 It is a schematic structural diagram of the variable concentration gradient generating module of the present invention;
[0030] Figure 3 This is a schematic structural diagram of the sound-driven cell microspheroidization stimulation module of the present invention;
[0031] Figure 4 It is a structural diagram of the hydraulic gate control module of the present invention;
[0032] Figure 5 This is a schematic diagram of the principle of acoustic-driven cell microspherization of the present invention;
[0033] Figure 6 is a linear concentration gradient distribution diagram in Example 1 of the present invention;
[0034] Figure 7 This is a graph representing the linear concentration gradient in Example 1 of the present invention;
[0035] Figure 8 This is the concave parabolic concentration gradient distribution diagram in Example 2 of the present invention;
[0036] Figure 9 This is a representation diagram of the concave parabolic concentration gradient in Example 2 of the present invention;
[0037] Figure 10 This is the upward convex parabolic concentration gradient distribution diagram in Example 3 of the present invention;
[0038] Figure 11 This is a characterization diagram of the upward convex parabolic concentration gradient in Example 3 of the present invention;
[0039] Figure 12 This is the exponential parabolic concentration gradient distribution diagram in Example 4 of the present invention;
[0040] Figure 13 This is a graph representing the exponential parabolic concentration gradient in Example 4 of the present invention;
[0041] Figure 14 This is a schematic diagram of the hydraulic gate control module of the present invention;
[0042] Figure numerals: 1. drug solution inlet; 11. first inlet; 12. second inlet; 13. third inlet; 2. concentration gradient generating microfluidic channel; 21. first transverse flow channel; 22. first Christmas tree-shaped micromixer; 23. second transverse flow channel; 24. second Christmas tree-shaped micromixer; 25. third transverse flow channel; 26. third Christmas tree-shaped micromixer; 3. drug solution outlet; 4. hydraulic gate shut-off flow channel; 5. first multifunctional inlet and outlet; 6. second multifunctional inlet and outlet; 41. fluid conduction interface; 7. buffer inlet; 71. buffer flow channel; 8. longitudinal liquid flow channel; 9. PDMS tip structure; 10. piezoelectric transducer. DETAILED DESCRIPTION
[0043] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.
[0044] It should be noted that the various installation methods and technical terms mentioned in the present invention are technical terms that have long been clearly known in the relevant technical field and therefore will not be further explained. In addition, the same reference numerals are used for the same components, but this does not affect nor constitute an accurate understanding of the technical solution by those skilled in the art.
[0045] Example 1
[0046] This embodiment provides an integrated in situ drug screening microfluidic system. Figure 1 As shown, from top to bottom are a variable concentration gradient generation module, a hydraulic gate control module and an acoustic cell microspheroidization stimulation module; the variable concentration gradient generation module and the acoustic cell microspheroidization stimulation module are controlled and connected through the hydraulic gate control module.
[0047] like Figure 2As shown, the variable concentration gradient generating module comprises, from top to bottom, three equidistantly distributed drug solution inlets 1 (in this embodiment, the first inlet 11, the second inlet 12 and the third inlet 13, for pumping drugs of different concentrations), a concentration gradient generating microfluidic channel 2 and six drug solution outlets 3. The concentration gradient generating microfluidic channel 2 comprises three channel layers (from top to bottom, the first channel layer, the second channel layer and the third channel layer). The first channel layer consists of a first transverse flow channel 21 and four first Christmas tree-shaped micromixers 22 equidistantly distributed and vertically connected to the first transverse flow channel 21. The second channel layer consists of a second transverse flow channel 23 and five second Christmas tree-shaped micromixers 24 equidistantly distributed and vertically connected to the second transverse flow channel 23. The third channel layer consists of a third transverse flow channel 25 and six third Christmas tree-shaped micromixers 26 equidistantly distributed and vertically connected to the third transverse flow channel 25. Among them, the first channel layer and the second channel layer are connected through the first Christmas tree-shaped micromixer 22 and the second transverse flow channel 23, and the second channel layer and the third channel layer are connected through the second Christmas tree-shaped micromixer 24 and the third transverse flow channel 25; the three equidistantly distributed drug solution inlets 1 are vertically connected to the first transverse flow channel 21 in the concentration gradient generating microfluidic channel 2, and the six drug solution outlets 3 are longitudinally connected to the third Christmas tree-shaped micromixer 26 in the concentration gradient generating microfluidic channel 2.
[0048] like Figure 1 and Figure 3 As shown, the sound-driven cell microspherization stimulation module includes six cell generation units, each of which includes a first multi-functional inlet and outlet 5, a second multi-functional inlet and outlet 6, and a longitudinal liquid flow channel 8 connecting the first multi-functional inlet and outlet 5 and the second multi-functional inlet and outlet 6; a PDMS tip structure 9 is embedded on the inner wall of one side of the longitudinal liquid flow channel 8; and a disc-type piezoelectric transducer 10 is arranged below the second multi-functional inlet and outlet 6.
[0049] like Figure 4 As shown, the hydraulic gate control module includes a horizontally arranged hydraulic gate cut-off flow channel 4, six fluid conduction interfaces 41 are equidistantly arranged on the hydraulic gate cut-off flow channel 4, and two buffer flow channels 71 are spaced apart between the six fluid conduction interfaces 41 (the interval in this embodiment is set to set a buffer flow channel 71 between every two fluid conduction interfaces 41), one end of the buffer flow channel 71 is longitudinally connected to the hydraulic gate cut-off flow channel 4, and the other end of the buffer flow channel 71 is connected to the buffer injection port 7.
[0050] like Figure 1As shown, the integration of the integrated in situ drug screening microfluidic system is as follows: the drug solution outlet 3 of the variable concentration gradient generating module and the second multifunctional inlet and outlet 6 of the sound-driven cell microspheroidization stimulation module are connected at the fluid conduction interface 41 of the hydraulic gate control module, the first multifunctional inlet and outlet 5 is both the cell mixture inlet and the outlet of drug solutions and buffer solutions of different concentrations, and the second multifunctional inlet and outlet 6 is both the cell mixture outlet and the loading port of the buffer solution.
[0051] In this embodiment, the steps for obtaining cell microsphere clusters of controllable size based on the acoustic-driven cell microsphere stimulation module are as follows:
[0052] Step 1: First, inject the cell mixture into the acoustic-driven cell microspheroidization stimulation module through the first multifunctional inlet and outlet 5 by means of a syringe pump and fill the longitudinal liquid flow channel 8;
[0053] Step 2: Turn on the signal generator switch, so that the disc-shaped piezoelectric transducer 10 generates an acoustic field to excite the vibration of the PDMS tip structure 9 located in the longitudinal liquid flow channel 8 of the acoustic-driven cell microspheroidization stimulation module, generating acoustic eddy currents. Under the coupling effect of the eddy current drag force and the acoustic radiation force, the cells are aggregated into compact spheres (cell clusters);
[0054] Step 3: After the cells have completed adhesion, adjust the output waveform of the signal generator, the distribution of eddy current drag and acoustic radiation force, so that the generated cell clusters are fixed at the PDMS tip structure 9.
[0055] The steps of generating different concentration gradients of drugs in the integrated in situ drug screening microfluidic system in this embodiment are as follows:
[0056] Step 1: Pumping the drug solution with a concentration ratio of 0%, 50% and 100% into the first inlet 11, the second inlet 12 and the third inlet 13 (drug solution inlet 1) which are equally spaced in the variable concentration gradient generating module in sequence to generate a linear concentration gradient;
[0057] Step 2: Buffer is loaded into the six first multi-functional ports 5 through the two buffer inlets 7 of the hydraulic gate control module. When the pumping pressure of the buffer inlet 7 is greater than the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0058] Step 3: Regulate and exchange the pumping pressure of the buffer solution inlet 7 in the hydraulic gate control module and the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generation module, so that the drug solutions of different concentrations can stimulate the cell spheroids;
[0059] Step 4: Repeat steps 2-3 to monitor the dynamic responses of cell clusters in multiple channels to chemical stimuli of different concentrations and frequencies in the same field of view.
[0060] The dynamic responses of chemical stimuli of different concentrations and frequencies in this embodiment are as follows: Figure 6 、 Figure 7 and Figure 14 shown.
[0061] Example 2
[0062] The integrated in situ drug screening microfluidic system provided in this embodiment is the same as that in Example 1, except that the concentration gradients of the drug solutions are different. The steps for generating drug solutions with different concentration gradients in the integrated in situ drug screening microfluidic system in this embodiment are as follows:
[0063] Step 1: Pumping 50%, 0% and 100% concentration ratios of the drug solution to be tested into the first injection port 11, the second injection port 12 and the third injection port 13, which are equally spaced, in sequence into the variable concentration gradient generating module to generate a concave parabolic concentration gradient;
[0064] Step 2: Buffer is loaded into the six first multi-functional ports 5 through the two buffer inlets 7 of the hydraulic gate control module. When the pumping pressure of the buffer inlet 7 is greater than the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0065] Step 3: Regulate and exchange the pumping pressure of the buffer solution inlet 7 in the hydraulic gate control module and the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generation module, so that the drug solutions of different concentrations can stimulate the cell spheroids;
[0066] Step 4: Repeat steps 2-3 to monitor the dynamic responses of cell clusters in multiple channels to chemical stimuli of different concentrations and frequencies in the same field of view.
[0067] The dynamic responses of chemical stimuli of different concentrations and frequencies in this embodiment are as follows: Figure 8 、 Figure 9 and Figure 14 shown.
[0068] Example 3
[0069] The integrated in situ drug screening microfluidic system provided in this embodiment is the same as that in Example 1, except that the concentration gradients of the drug solutions are different. The steps for generating drug solutions with different concentration gradients in the integrated in situ drug screening microfluidic system in this embodiment are as follows:
[0070] Step 1: Pumping the drug solution with a concentration ratio of 0%, 100% and 50% into the first injection port 11, the second injection port 12 and the third injection port 13, which are equally spaced, in sequence into the variable concentration gradient generating module to generate an upward convex parabolic concentration gradient;
[0071] Step 2: Buffer is loaded into the six first multi-functional ports 5 through the two buffer inlets 7 of the hydraulic gate control module. When the pumping pressure of the buffer inlet 7 is greater than the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0072] Step 3: Regulate and exchange the pumping pressure of the buffer solution inlet 7 in the hydraulic gate control module and the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generation module, so that the drug solutions of different concentrations can stimulate the cell spheroids;
[0073] Step 4: Repeat steps 2-3 to monitor the dynamic responses of cell clusters in multiple channels to chemical stimuli of different concentrations and frequencies in the same field of view.
[0074] The dynamic responses of chemical stimuli of different concentrations and frequencies in this embodiment are as follows: Figure 10 、 Figure 11 and Figure 14 shown.
[0075] Example 4
[0076] The integrated in situ drug screening microfluidic system provided in this embodiment is the same as that in Example 1, except that the concentration gradients of the drug solutions are different. The steps for generating drug solutions with different concentration gradients in the integrated in situ drug screening microfluidic system in this embodiment are as follows:
[0077] Step 1: Pumping the drug solutions with concentrations of 0%, 50%, and 100% into the first, second, and third injection ports 11, 12, and 100% at equal intervals into the variable concentration gradient generating module, and at the same time, appropriately reducing the air pump pressure at the second and third injection ports 12, 13 where the drug solutions with concentrations of 50% and 100% are pumped, thereby generating an exponential parabolic concentration gradient;
[0078] Step 2: Buffer is loaded into the six first multi-functional ports 5 through the two buffer inlets 7 of the hydraulic gate control module. When the pumping pressure of the buffer inlet 7 is greater than the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0079] Step 3: Regulate and exchange the pumping pressure of the buffer solution inlet 7 in the hydraulic gate control module and the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generation module, so that the drug solutions of different concentrations can stimulate the cell spheroids;
[0080] Step 4: Repeat steps 2-3 to monitor the dynamic responses of cell clusters in multiple channels to chemical stimuli of different concentrations and frequencies in the same field of view.
[0081] The dynamic responses of chemical stimuli of different concentrations and frequencies in this embodiment are as follows: Figure 12 、 Figure 13 and Figure 14 shown.
[0082] The steps for using the integrated in situ drug screening microfluidic system of the present invention are as follows:
[0083] Step 1: First, inject the cell mixture into the acoustic-driven cell microspheroidization stimulation module through the first multifunctional inlet and outlet 5 by means of a syringe pump and fill the longitudinal liquid flow channel 8;
[0084] Step 2: Turn on the signal generator switch, so that the disc-shaped piezoelectric transducer 10 generates an acoustic field to excite the vibration of the PDMS tip structure 9 located in the longitudinal liquid flow channel 8 of the acoustic-driven cell microspheroidization stimulation module, generating acoustic eddy currents. Under the coupling effect of the eddy current drag force and the acoustic radiation force, the cells are aggregated into compact spheres (cell clusters);
[0085] Step 3: After the cells have completed adhesion, the output waveform of the signal generator, the distribution of the eddy current drag force and the acoustic radiation force are adjusted so that the generated cell clusters are fixed at the PDMS tip structure 9;
[0086] Step 4: Pump different concentrations of drug solutions into the multiple drug solution inlets 1 of the variable concentration gradient generation module in sequence, and set different pumping modes according to the different spatial distributions of the required concentration gradients (when a linear concentration gradient is required, the three inlets 1 are pumped into the drug solutions with concentrations of 0, 50%, and 100% in sequence; when an upward convex parabolic concentration gradient is required, the three inlets 1 are pumped into the drug solutions with concentrations of 0, 100%, and 50% in sequence; when a downward convex parabolic concentration gradient is required, the three inlets 1 are pumped into the drug solutions with concentrations of 50%, 0, and 100% in sequence; when an exponential concentration gradient is required, the three inlets 1 are pumped into the drug solutions with concentrations of 0, 100%, and 50% in sequence; At the same time, the three sampling ports 1 are sequentially pumped with drug solutions of 0%, 50%, and 100% concentrations, and the air pump pressure at the sampling port 1 for pumping drugs of 50% and 100% concentrations is appropriately reduced; at the same time, more drug solution sampling ports 1 and more concentration gradient generating microfluidic channels 2 can be provided to obtain more concentration gradient distributions); different concentrations of drug solutions of the target drug are loaded into the longitudinal liquid flow channel 8 of the acoustically driven cell microspheroidization stimulation module (cell clusters fixed in place) through the second multifunctional inlet and outlet 6 (connected to the fluid conduction interface 41 of the hydraulic gate control module) to stimulate the cell clusters;
[0087] Step 5: Loading buffer solution into the first multifunctional inlet and outlet 5 connected to the fluid communication interface 41 of the hydraulic gate control module through the buffer solution inlet 7 of the hydraulic gate control module. When the pumping pressure of the buffer solution inlet 7 is greater than the pumping pressure of the drug solution inlet 1 in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, causing the cell spheroids to enter a buffer state.
[0088] Step 6: Adjust the pumping pressure of the buffer solution inlet 7, and simultaneously exchange the pumping pressures of the buffer solution inlet 7 and the drug solution inlet 1, so that different concentrations of the drug solution to be tested stimulate the cell spheroids;
[0089] Step 7: Repeat steps 5-6 to monitor the dynamic responses of cell clusters to chemical stimuli of different concentrations and frequencies in multiple channels in the same field of view.
[0090] The working principle of the integrated in situ drug screening microfluidic system of the present invention is as follows:
[0091] (1) First, cell clusters are formed and fixed in the flow channel using acoustic driving technology; then, the required drug concentration gradient is generated through a variable concentration gradient generation module; finally, chemical stimuli of different concentrations and frequencies are applied to the cell clusters using hydraulic gating technology, and the dynamic response of the cell clusters is observed.
[0092] (2) Working principle of the present invention for preparing cell spheres:
[0093] like Figure 5As shown, first, the longitudinal liquid flow channel 8 is filled with the cell mixture through the first multi-functional inlet and outlet 5, and the signal generator switch of the disc-type piezoelectric transducer is turned on (the sound field is on). The disc-type piezoelectric transducer is used to generate a sound field that excites the PDMS tip structure, and excites the PDMS tip structure to vibrate through body acoustic waves, thereby generating acoustic vortexes near the PDMS tip structure, thereby capturing cells on both sides of the PDMS tip structure, and can generate two cell balls of similar size through a PDMS tip structure. By adjusting the length and angle of the PDMS tip structure, cell balls (cell clusters) of various sizes can also be generated at the same time. At the same time, by adjusting the sound field frequency, the closer to the PDMS resonance frequency, the stronger the acoustic vortex can be generated, thereby generating larger cell balls (cell balls of various sizes can be generated using the same PDMS tip structure). After the cell balls meet the requirements, the signal generator switch of the disc-type piezoelectric transducer can be turned off (the sound field is off).
[0094] (3) The working principle of the present invention to generate concentration gradient:
[0095] Drug solutions of different concentrations are pumped in through different drug solution inlets that are equally spaced, and mixed with drug solutions of different concentrations in another flow channel at the branch of the flow channel (mixed layer by layer along the multiple channel layers of the microfluidic channel that generates the concentration gradient, and mixed through the transverse flow channels and longitudinal Christmas tree-shaped micromixers in the channel layer) to obtain a new drug solution concentration. After multiple mixing, a drug solution with a concentration gradient is generated at the drug solution outlet. As the number of concentration gradient generator layers increases, the number of drug solution concentrations obtained gradually increases.
[0096] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention may be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the present invention is defined by the appended claims rather than the foregoing description. It is intended that all variations that fall within the meaning and range of equivalents of the claims be embraced within the present invention. Any reference sign in a claim should not be construed as limiting the claim to which it relates.
[0097] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any minor modifications, equivalent replacements, and improvements made to the above embodiments based on the technical essence of the present invention shall be included in the scope of protection of the technical solution of the present invention.
Claims
1. An integrated in situ drug screening microfluidic system, characterized in that: It includes a hydraulic gate control module, a variable concentration gradient generation module and an acoustic cell microspheroidization stimulation module located on both sides of the hydraulic gate control module; the variable concentration gradient generation module and the acoustic cell microspheroidization stimulation module are controlled and connected through the hydraulic gate control module.
2. The integrated in situ drug screening microfluidic system according to claim 1, characterized in that: The variable concentration gradient generating module comprises a drug solution inlet (1), a drug solution outlet (3), and a concentration gradient generating microfluidic channel (2) communicating with the drug solution inlet (1) and the drug solution outlet (3); the concentration gradient generating microfluidic channel (2) comprises a plurality of channel layers, each channel layer being composed of at least one transverse flow channel and a plurality of Christmas tree-shaped micromixers; wherein the channel layers are at least three layers, and the Christmas tree-shaped micromixers in the channel layers are equidistantly distributed on the transverse flow channel and are longitudinally connected to the transverse flow channel.
3. The integrated in situ drug screening microfluidic system according to claim 2, characterized in that: The drug solution inlet (1) is connected to the lateral flow channel in the concentration gradient generating microfluidic channel (2), and the drug solution outlet (3) is connected to the Christmas tree-shaped micromixer in the concentration gradient generating microfluidic channel (2); wherein the number of the Christmas tree-shaped micromixers increases step by step with the increase in the number of channel layers.
4. The integrated in situ drug screening microfluidic system according to claim 3, characterized in that: The number of Christmas tree-shaped micromixers in the first channel layer of the concentration gradient generating microfluidic channel (2) is greater than the number of the drug solution sampling ports (1), and the number of the drug solution sampling ports (3) is equal to the number of Christmas tree-shaped micromixers in the last channel layer of the concentration gradient generating microfluidic channel (2); wherein the number of the drug solution sampling ports (1) is at least three.
5. The integrated in situ drug screening microfluidic system according to claim 1, characterized in that: The acoustic-driven cell microspherization stimulation module comprises a plurality of cell generation units, each of which comprises a first multifunctional inlet and outlet (5), a second multifunctional inlet and outlet (6), and a longitudinal liquid flow channel (8) connecting the first multifunctional inlet and outlet (5) and the second multifunctional inlet and outlet (6), wherein a PDMS tip structure (9) is embedded on the inner wall of one side of the longitudinal liquid flow channel (8); a piezoelectric transducer (10) is provided below the second multifunctional inlet and outlet (6), and the piezoelectric transducer (10) is a disc-shaped structure; The number of cell generation units in the acoustic-driven cell microsphere stimulation module is equal to the number of drug solution sample outlets (3) in the variable concentration gradient generation module.
6. The integrated in situ drug screening microfluidic system according to claim 5, characterized in that: The hydraulic gate control module comprises a hydraulic gate cut-off flow channel (4), a plurality of fluid conducting interfaces (41) arranged on the hydraulic gate cut-off flow channel (4), and a plurality of buffer injection ports (7); the plurality of buffer injection ports (7) are longitudinally connected to the hydraulic gate cut-off flow channel (4) via a buffer flow channel (71); the buffer flow channel (71) is arranged at intervals between the plurality of conducting interfaces (41); The spacing between the fluid conducting interfaces (6) is equal, and the spacing between the buffer injection ports (7) is equal.
7. The integrated in situ drug screening microfluidic system according to claim 5, characterized in that: A buffer injection port (7) is provided every two fluid conduction interfaces (41) in the hydraulic gate control module, and the number of the buffer injection ports (7) is less than the number of the fluid conduction interfaces (41); the number of the fluid conduction interfaces (41) in the hydraulic gate control module is consistent with the number of the drug solution sample outlets (3) in the variable concentration gradient generating module.
8. The integrated in situ drug screening microfluidic system according to claim 5, characterized in that: The drug solution sample outlet (3) of the variable concentration gradient generating module and the second multifunctional inlet and outlet (6) of the sound-driven cell microsphere stimulation module are integrated and connected at the fluid conduction interface (41) of the hydraulic gate control module.
9. Application of the integrated in situ drug screening microfluidic system according to claim 1 in in situ drug screening.
10. The use according to claim 9, characterized in that The in situ drug screening microfluidic system for screening in situ drugs comprises the following steps: Step 1: first pump the cell mixture into the acoustic-driven cell microspherization stimulation module; Step 2: Turn on the signal generator switch again, generate an acoustic field through the disc-shaped piezoelectric transducer (10), excite the PDMS tip structure (9) located in the flow channel of the acoustic-driven cell microspheroidization stimulation module to vibrate, generate acoustic vortexes and aggregate the cells into compact spheres; Step 3: After the cells have completed adhesion, adjust the output waveform of the signal generator and the distribution of the eddy current drag force and the acoustic radiation force so that the generated cell clusters are fixed at the PDMS tip structure (9); Step 4: three different concentrations of drug solutions to be tested are pumped into the concentration gradient generator portion of the variable concentration gradient generation module through the inlet; different concentrations of drug solutions to be tested are loaded into the cell clusters located in the acoustic-driven cell microspheroidization stimulation module through the first multifunctional port; and by adjusting the order and flow rate of the three different concentrations of drug solutions to be tested being pumped in, concentration gradients with different spatial distributions are formed to stimulate the cell clusters at the PDMS tip structure; Step 5, by switching the hydraulic gate of the air pump of the buffer inlet (7) in the hydraulic gate control module, pumping buffer into the buffer inlet (7), when the pumping pressure of the buffer inlet (7) in the hydraulic gate control module is greater than the pumping pressure of the drug solution inlet (1) in the variable concentration gradient generating module, the drug solution to be tested is instantaneously cut off, so that the cell spheroid enters a buffer state; Step 5: Regulating and exchanging the pumping pressure of the buffer solution inlet (7) in the hydraulic gate control module and the pumping pressure of the drug solution inlet (1) in the variable concentration gradient generation module, so that the drug solutions to be tested with different concentrations can stimulate the cell spheroids; Step 6: Repeat steps 3-5 and simultaneously monitor the dynamic responses of cell clusters in multiple channels to chemical stimuli of different concentrations and frequencies.