A 3D microfluidic chip device for solution dilution and mixing in drug screening

Abstract

This invention discloses a 3D microfluidic chip device for solution dilution and mixing in drug screening, comprising: a cap, a solution loading and storage device fitted with the cap, a concentration gradient and mixing generator snapped into the solution loading and storage device, and a cell culture device connected to the concentration gradient and mixing generator. The solution loading and storage device, the concentration gradient and mixing generator, and the cell culture device are either connected or disconnected. The solution loading and storage device is used for loading and storing solutions; the concentration gradient and mixing generator is used for solution preparation; and the cell culture device is used for collecting the generated solution and creating a cell culture microenvironment for drug screening. According to this invention, the simplified and symmetrical structure avoids excessive intersections in the channel network, eliminates the need for auxiliary instruments, and allows for independent implementation of gradient dilution and mixing schemes for various drug solutions.

Description

Technical Field

[0001] This invention relates to the technical field of biological and pharmacological solution mixing, and particularly to a 3D microfluidic chip device for solution dilution and mixing for drug screening. Background Technology

[0002] Concentration gradients and solution mixing microenvironments play crucial roles in numerous fields, including mechanical engineering, chemical engineering, biology, and pharmacology. In anticancer drug development and disease treatment, achieving gradient concentrations and proper mixing of drug solutions is essential for determining optimal concentrations and combination therapy regimens. Microfluidic technology, through the creation of microchannel networks, enables complex solution processing and can construct microenvironments for drug screening in in vitro cell cultures. Compared to traditional titration plates, microfluidic chips offer more convenient and precise pretreatment processes, with unique advantages such as low reagent consumption and high throughput.

[0003] Constructing microenvironments for drug screening requires the preparation of drug solution dilution and mixing. Previously reported microfluidic chips can only be used for the dilution or mixing of single or two drug solutions, and require expensive equipment to operate, such as a pump for solution dispensing. Furthermore, previous microfluidic chips, limited by their spatial structure, could not simultaneously generate concentration gradients and perform mixing and combination of different solutions. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a 3D microfluidic chip device for solution dilution and mixing in drug screening. The simplified and symmetrical structure avoids excessive channel network intersections, eliminates the need for auxiliary instruments, and allows for independent implementation of gradient dilution and mixing schemes for various drug solutions. To achieve the above-mentioned objectives and other advantages of the present invention, a 3D microfluidic chip device for solution dilution and mixing in drug screening is provided, comprising:

[0005] A cap, a solution loading temporary storage device fitted with the cap, a concentration gradient and mixing generator snapped into the solution loading temporary storage device, and a cell culture device connected to the concentration gradient and mixing generator, wherein the solution loading temporary storage device, the concentration gradient and mixing generator, and the cell culture device are connected or disconnected.

[0006] The solution loading register is used for loading and temporarily storing solutions;

[0007] Concentration gradients and mixture generators are used for solution preparation;

[0008] Cell culture devices are used to collect generated solutions and create microenvironments for cell culture drug screening.

[0009] Preferably, the solution loading temporary storage device includes an outer ring loading chamber, a middle ring loading chamber, and an inner ring loading chamber from the outside to the inside. The middle ring loading chamber has a first channel of various specifications, and both the outer ring loading chamber and the inner ring loading chamber have a second channel of various specifications.

[0010] Preferably, the concentration gradient and mixing generator includes a concentration gradient generation section and a solution mixing section. The concentration gradient generation section is used for diluting the solution, and the solution mixing section is used for mixing and combining gradient solutions. The concentration gradient generation section and the solution mixing section are respectively provided with multiple third channels and multiple fourth channels. The multiple third channels are split and then converged to form a fourth channel. The multiple third channels and the multiple fourth channels form a channel network structure.

[0011] Preferably, the beginning of the third channel of the concentration gradient generation section corresponds one-to-one with the end of the first and second channels of the solution loading temporary storage device. The adjacent first and second channels in the solution loading temporary storage device converge to form the third channel of the concentration gradient generation section, and both the third and fourth channels are provided with 3D spiral channels.

[0012] Preferably, each third channel in the concentration gradient generation section splits into three smaller channels at its end. Each pair of adjacent third channels merges through a pair of smaller channels at their ends for solution mixing, while the other smaller channel on the outside flows straight downwards with the solution remaining constant. The end of the smaller channel connects to the beginning of the fourth channel in the solution mixing section.

[0013] Preferably, the cell culture device includes a cell culture chamber and a substrate disposed at the bottom of the cell culture chamber. The cell culture chamber is connected to the fifth channel. The cell culture chamber has multiple sub-chambers, and each sub-chamber has a dark channel on its side wall.

[0014] Compared with existing technologies, the advantages of this invention are: the three components can be flexibly disassembled and assembled, tightly connected, and the upper and lower channel network is equipped with an on / off switch, which is achieved by rotating the relative positions of the components to open and close the channels. The solution flows through the multi-layered 3D network of the chip under its own gravity, achieving predetermined dilution and mixing configurations, and collecting the solution in the cell culture chamber to create a cell perfusion culture microenvironment. This invention's 3D microfluidic chip adopts an inner, middle, and outer three-ring combination concept, which can simultaneously and efficiently achieve the dilution and mixing of multiple solution concentrations, and can implement various solution loading schemes, providing microenvironmental support for drug screening solution combinations. Attached Figure Description

[0015] Figure 1 A schematic diagram of the three-dimensional structure of a 3D microfluidic chip device for solution dilution and mixing for drug screening according to the present invention;

[0016] Figure 2This is a top view of the internal structure of the solution loading register of the 3D microfluidic chip device for drug screening, according to the present invention, for solution dilution and mixing of a drug screening solution.

[0017] Figure 3 A schematic diagram of a cell culture device for a 3D microfluidic chip device for drug screening solution dilution and mixing according to the present invention;

[0018] Figure 4 A top view of a cell culture apparatus for a 3D microfluidic chip device for drug screening solution dilution and mixing according to the present invention;

[0019] Figure 5 A schematic diagram of a solution combination scheme for a concentration gradient and a mixing generator under two solution mixing modes of a 3D microfluidic chip device for drug screening according to the present invention;

[0020] Figure 6 A bottom view of the concentration gradient and the internal channel structure of the mixing generator of the 3D microfluidic chip device for solution dilution and mixing in drug screening according to the present invention.

[0021] Figure 7 A schematic diagram of the concentration gradient and the internal channel structure of the mixing generator of the 3D microfluidic chip device for solution dilution and mixing in drug screening according to the present invention.

[0022] Figure 8 This is a three-dimensional structural diagram of the solution loading temporary storage device of the 3D microfluidic chip device for drug screening of the present invention, which is used for solution dilution and mixing. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Reference Figure 1-8A 3D microfluidic chip device for solution dilution and mixing in drug screening includes: a cap 1, a solution loading and storage device 2 fitted into the cap 1, a concentration gradient and mixing generator 3 snapped into the solution loading and storage device 2, and a cell culture device 4 connected to the concentration gradient and mixing generator 3. The solution loading and storage device 2, the concentration gradient and mixing generator 3, and the cell culture device 4 are interconnected or disconnected. The solution loading and storage device 2 is used for loading and storing solutions; the concentration gradient and mixing generator 3 is used for preparing solutions; and the cell culture device 4 is used for collecting the generated solutions and... The cell culture drug screening microenvironment is created through parameter matching at the connection points of each component, enabling easy assembly and separation. The concentration gradient and mixing generator 3 utilizes a 3D channel network, where the solution relies on its own gravity for multi-level, repeated diversion, convergence, and a 3D helical structure to achieve efficient solution configuration. The solution loading buffer 2 uses three large annular chambers, each with multiple downward diversion channels. The concentration gradient and mixing generator 3 performs solution dilution and mixing in two parts: multi-concentration gradient dilution, combining different volumes of mother liquor and diluent; and then diverting the solution, combining one portion with another diluent to form a mixed solution. The generated diluted and mixed solutions flow into the cell culture chamber, which contains 20 small chambers of the same depth and diameter to collect the generated solutions, thus creating the cell culture drug screening microenvironment.

[0025] The following describes in detail one drug loading scheme for this 3D microfluidic chip device: the inner ring chamber of the solution loading temporary storage 2 is loaded with diluent, and the outer and inner ring loading chambers are loaded with two drug solutions, in order to achieve gradient dilution of the two drug solutions and generate a combination of gradient solutions.

[0026] Furthermore, the solution loading temporary storage device 2 includes an outer ring loading chamber 21, a middle ring loading chamber 22, and an inner ring loading chamber 23 from the outside to the inside. The middle ring loading chamber 22 has first channels 25 of various specifications. The outer ring loading chamber 21 and the inner ring loading chamber 23 both have second channels 24 of various specifications. The total height of the solution loading temporary storage device 2 is 15.0 mm, and the maximum diameter of the outline is 25.2 mm. The lower end of the middle ring loading chamber 22 has 10 first channels 25 of 5 specifications each, and the lower ends of the outer ring loading chamber 21 and the inner ring loading chamber 23 each have five second channels 24 of various specifications, which prepare for the injection of solution into the channels of the components below.

[0027] Furthermore, the concentration gradient and mixing generator 3 includes a concentration gradient generation section and a solution mixing section. The concentration gradient generation section is used for solution dilution, and the solution mixing section is used for mixing and combining gradient solutions. The concentration gradient generation section and the solution mixing section are respectively provided with multiple third channels 31 and multiple fourth channels 32. The multiple third channels 31 are split and then converged to form the fourth channel 32. The multiple third channels 31 and the multiple fourth channels 32 form a channel network structure. Each second channel 24 at the lower end of the outer ring loading chamber 21 and the inner ring loading chamber 23 is connected to the nearest first channel 25 at the lower end of the middle ring loading chamber 22, which can mix solutions and diluents of different volumes to generate multiple concentration gradient solutions.

[0028] Furthermore, the beginning 33 of the third channel in the concentration gradient generation section corresponds one-to-one with the first channel 25 and the second channel 24 of the solution loading temporary storage unit 2. The beginning 33 of each third channel is connected to an adjacent first channel 25 and a second channel 24, and the channels converge. This is followed by a 3D spiral channel segment of the third channel 31, achieving gradient dilution of the solution. At the end of each third channel 31 in the concentration gradient generation section, three smaller channels 34 branch off. Every two adjacent smaller channels 34 originating from different third channels 31 are interconnected, and the channels converge. The solution in the other smaller channel remains unchanged. The end of this smaller channel connects to the beginning of the fourth channel 32 in the solution mixing section. This fourth channel is a 3D spiral channel, achieving solution mixing. Ultimately, both channels flow to the bottom. The flow from the top to the bottom of the channels is almost identical, ensuring the consistency and accuracy of the separation and mixing of the solution flowing through the channels.

[0029] The multi-channel solution convergence point between the concentration gradient generation section and the solution mixing section employs a multi-layer 3D spiral structure, facilitating uniform solution mixing. This structure is compact and has high space utilization. The mixing spiral structure in the concentration gradient generation section has a total of 6 rotations, a channel diameter of 1.9070 mm, and an outer diameter of 4.4070 mm; the mixing spiral structure in the solution mixing section has a total of 7 rotations, a channel diameter of 1.3484 mm, and an outer diameter of 3.1484 mm. A solution loading switch is provided between the concentration gradient and mixing generator 3 and the solution loading temporary storage unit 2. The liquid loading switch is used to control the connection and disconnection between the concentration gradient and mixing generator 3 and the solution loading temporary storage unit 2. The outer surface of the concentration gradient and mixing generator 3 is marked with "1" and "0", and the outer surface of the solution loading temporary storage unit 2 is marked with "V". When the "V" mark of the solution loading temporary storage unit 2 corresponds to the "1" mark of the concentration gradient generator 3, the channels of the two components are in a fully connected state; when the "V" mark of the solution loading temporary storage unit corresponds to the "0" mark of the concentration gradient and mixing generator, the channels of the two components are in a fully disconnected state.

[0030] Furthermore, the cell culture device 4 includes a cell culture chamber 41 and a substrate 5 disposed at the bottom of the cell culture chamber. The cell culture chamber 41 is connected to the end of the fourth channel. Multiple sub-chambers 42 are formed within the cell culture chamber 41. Each sub-chamber 42 has a dark channel 43 on its sidewall. The openings of the dark channels 43 are located at varying distances, some at the innermost part of the cell culture device 4 and others at the outermost part, employing different arched channel structures for different locations. Each small chamber can hold 12.6 μL of solution. Excess solution flows out of the microfluidic chip through the dark channel on the sidewall, providing a function of continuously replacing the solution within the chambers in real time. The substrate has no channels or textures and is tightly connected to the cell culture chamber, providing a flat surface to receive the final generated solution. Additionally, the top of the microfluidic chip is a cap to prevent debris from falling into the chip during operation, thus maintaining the integrity of the entire chip.

[0031] Working principle: After all components are assembled, the solution loading process begins. The cap is removed with tweezers, and the prepared mother liquor and diluent are slowly added to the corresponding chambers of the solution loading temporary container using a pipette. Then, the cap is placed back on top with tweezers to complete the solution loading process.

[0032] Then comes the solution dilution and mixing stage, such as... Figure 1 Hold the concentration gradient and mixing generator 3 with your left hand and apply slight downward pressure to stabilize the connection position. Rotate the solution loading temporary storage unit 2 counterclockwise with your right hand until the "V" mark on its surface is collinear with the "1" mark of the concentration gradient and mixing generator. At this point, all channels between the components of the microfluidic chip are fully connected. Under the influence of gravity, the solution flows from the solution loading temporary storage unit 2 along the layer-by-layer channel network. After being split, converged, and fully mixed by the 3D spiral channel network, it flows to the cell culture vessel. When there is too much solution in the chamber, the excess solution flows out of the device through the dark channels on the side wall of the chamber. When the solution loading temporary storage unit is rotated clockwise until the "V" mark is collinear with the "0" mark of the concentration gradient and mixing generator, the upper end stops supplying the solution, and the solution mixing ends.

[0033] After the experiment, the mixed solution was collected and disposed of. First, the components of the microfluidic chip needed to be separated. Following a top-to-bottom order, the cap, solution loading buffer, upper sealing ring, concentration gradient and mixing generator, and bottom sealing ring were separated and immersed in a beaker containing deionized water. Using a pipette, the solutions from each chamber of the cell culture device were sequentially removed according to their numbers and dropped into corresponding numbered EP tubes for subsequent application and analysis experiments. Finally, the microfluidic chip was cleaned. Each component was repeatedly rinsed and soaked with deionized water, dried, rinsed with anhydrous ethanol, dried again, and then placed separately in a storage box. Before operation, cell culture was completed in the cell culture device, and the chip was loaded with solution. The operation process provides a perfusion culture microenvironment for cell culture drug screening experiments.

[0034] The implementation of solution dilution and mixing involves two solution mixing modes. For the sake of description and explanation, drug solutions A and B represent two stock solutions, and Am and Bn (where m and n are integers from 1 to 5) represent solutions of different concentrations obtained from diluting the stock solutions. The values ​​of m and n indicate the concentration magnitude, and AmBn represents the mixed solution obtained by combining solutions Am and Bn. The solution preparation effect is divided into two parts. The first part dilutes the solutions at concentration gradients, and the second part mixes and combines the solutions. The two parts need to be closely connected. The first part provides the base solution for the solution mixing in the second part. At the same time, some solutions generated in the first part do not participate in the mixing process in the second part and are directly output as multiple concentration gradient solutions. The second part mixes the solutions and finally outputs them as a combined solution. Due to the need for solution dilution and mixing, and the large number of combinations, a large number of complex channel networks are required to implement solution collection and separation. Setting up a channel network in 3D space is a feasible strategy. Considering minimizing the complexity of the spatial structure, symmetry of the channel network structure, and maximizing space utilization, the ring design concept is applied, and the channel network extends from top to bottom. Nearby channel networks are set up with structures that have separation and convergence functions.

[0035] For the first part, the two solutions are mixed with diluents to generate solutions with five concentration gradients. Both mother solution A and diluent can be divided into five portions of unequal volume. Mother solution A and diluent are mixed one-to-one to generate five concentration gradient Am solutions; similarly, five concentration gradient Bn solutions are generated. Considering symmetry and simplification of solution concentration analysis, the concentration gradients of the five Am and five Bn solutions are the same. For the second part, the concentration gradient solutions are mainly mixed and combined. The five concentration gradient Am and five concentration gradient Bn solutions generated in the first part are each divided into two portions. One portion is directly output as the concentration gradient solution, and the other portion serves as the base solution for the second part. Solutions Am and Bn are mixed and combined to generate AmBn solutions, which are then output. For the latter, as... Figure 5 As shown, selecting the shortest path between Am and Bn solutions and combining them generates 10 AmBn solutions. Due to limitations in spatial structure and combination distribution, other Am and Bn combinations require the channel network to bend and connect, resulting in inconsistencies with the channel network shown in the figure. This leads to longer and more complex paths, significantly increasing the difficulty of subsequent design, manufacturing, and analysis, and introducing unnecessary challenges. Mixing five concentrations of Am and five concentrations of Bn yields 25 AmBn combinations. Furthermore, considering changing the positions of A and B, 20 more AmBn combinations can be obtained. The five AmBn combinations A1B1, A2B2, A3B3, A4B4, and A5B5 are not feasible, but this is understandable. The 20 feasible AmBn combinations are sufficient to meet the concentration gradient and mixed solution analysis requirements for drug screening. It is important to note that the distribution of Am and Bn is not arbitrary. It is necessary to avoid the situation where A and B are mixed in the same concentration gradient, that is, to obtain five AmBn combinations such as A1B1, A2B2, A3B3, A4B4 and A5B5. When the positions of A and B are changed, these five AmBn combination solutions will be repeated. In the end, only 15 AmBn combinations can be obtained in total. This phenomenon should be avoided.

[0036] The solution loading and generation process preferably involves solution dilution and mixing in a two-solution mixing mode. Two drug solutions are loaded into the outer and inner ring chambers at the top of the solution loading buffer, and a diluent is loaded into the middle ring chamber. After the chip operation is complete, the solutions are collected in a cell culture device. Five concentration gradient solutions are generated for each of the two drug solutions, as well as a mixture of the two drug solutions. Other feasible loading schemes are as follows:

[0037] (a) The same drug solution is loaded in the outer and inner ring chambers at the top of the solution loading temporary container, and a diluent is loaded in the middle ring drug solution. A single drug solution produces at least 15 drug solutions with different concentration gradients.

[0038] (ii) The outer and inner ring chambers at the top of the solution loading temporary container are loaded with dilution solutions, and the middle ring chamber is loaded with drug solutions, so as to generate drug solutions with 15 concentration gradients for a single drug.

[0039] (III) Each of the outer ring chamber, inner ring chamber and middle ring chamber at the upper end of the solution loading temporary container is loaded with a drug solution to achieve mixing between multiple concentration gradients of the three drug solutions.

[0040] In summary, this 3D microfluidic chip can perform solution dilution and mixing configurations using 1 to 3 solutions with different loading site combinations. For the verification and analysis of the microfluidic chip, experiments with only one solution loading scheme are sufficient to deduce the volume ratio of each solution involved in the mixing, thereby establishing the chip's mechanism for solution dilution and mixing. Using this quantitative basis of the mechanism, and with only the concentration of the loading mother liquor known, the solution generated by the chip under various solution loading schemes can be calculated, revealing the solution conditions for the chip to create a drug screening microenvironment.

[0041] The number of devices and processing scales described herein are intended to simplify the description of the present invention, and applications, modifications, and variations of the present invention will be apparent to those skilled in the art.

[0042] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A 3D microfluidic chip device for solution dilution and mixing in drug screening, characterized in that, include: The cap (1), the solution loading temporary storage device (2) fitted with the cap (1), the concentration gradient and mixing generator (3) snapped into the solution loading temporary storage device (2), and the cell culture device (4) connected to the concentration gradient and mixing generator (3) are connected or disconnected. The solution loading temporary storage device (2) is used for loading and temporarily storing the solution; the solution loading temporary storage device (2) includes an outer ring loading chamber (21), a middle ring loading chamber (22) and an inner ring loading chamber (23) from the outside to the inside. The middle ring loading chamber (22) is provided with a first channel (25) of various specifications. The outer ring loading chamber (21) and the inner ring loading chamber (23) are both provided with a second channel (24) of various specifications. Concentration gradient and mixing generator (3) are used for solution preparation; Cell culture apparatus (4) is used to collect generated solutions and create a microenvironment for cell culture drug screening; The concentration gradient and mixing generator (3) includes a concentration gradient generation section and a solution mixing section. The concentration gradient generation section is used for diluting the solution, and the solution mixing section is used for mixing and combining gradient solutions. The concentration gradient generation section and the solution mixing section are respectively provided with multiple third channels (31) and multiple fourth channels (32). The multiple third channels (31) are diverted and then converged to form a fourth channel (32). The multiple third channels (31) and the multiple fourth channels (32) form a channel network structure. The beginning (33) of the third channel of the concentration gradient generation section corresponds one-to-one with the end of the first channel (25) and the second channel (24) of the solution loading temporary storage (2). The adjacent first channel (25) and second channel (24) in the solution loading temporary storage (2) converge to form the third channel (31) of the concentration gradient generation section, and a 3D spiral channel is provided in the fourth channel (32). Each third channel (31) of the concentration gradient generation section splits into three small channels (34) at its end. Each pair of adjacent third channels (31) is combined through a pair of small channels (34) at its end to mix the solution. Another small channel (34) on the outside flows straight down to keep the solution unchanged. The end of the small channel (34) is connected to the beginning of the fourth channel (32) of the solution mixing section. A 3D spiral channel is provided in the fourth channel (32).

2. The 3D microfluidic chip device for solution dilution and mixing for drug screening as described in claim 1, characterized in that, The cell culture device (4) includes a cell culture chamber (41) and a substrate (5) disposed at the bottom of the cell culture chamber. The cell culture chamber (41) is connected to the end of the fourth channel (32). Multiple sub-chambers (42) are opened in the cell culture chamber (41), and a dark channel (43) is opened on the side wall of each sub-chamber (42).

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