Microfluidic platform for screening tumor individualized treatment drugs

Abstract

The utility model relates to the field of organ chips, in particular to a micro-fluidic platform for screening tumor individualized treatment drugs. The micro-fluidic platform comprises a chip which comprises an upper-layer chip and a lower-layer chip, a first channel, a first-stage mixing structure, a second channel, a second-stage mixing structure, a first cell culture area and a third channel which are communicated in sequence are arranged on the upper-layer chip, a medicine inlet is formed in the first channel, and a discharge port is formed in the third channel; the lower-layer chip is provided with a second cell culture area, and the second cell culture area is arranged corresponding to the first cell culture area; a temperature control device is arranged on the lower surface of the lower-layer chip and comprises a heating film, an isolation layer and a power source, and the heating film is arranged on the isolation layer and electrically connected with the power source. According to the utility model, the temperature control device is arranged, so that the chip can be continuously heated when cell observation is needed, the temperature of the chip is always maintained at 37 DEG C, and the accuracy of an experiment can be ensured.

Description

Technical Field

[0001] The utility model relates to the field of organ chips, and in particular to a microfluidic platform for screening individualized tumor therapeutic drugs. Background Art

[0002] Microfluidic chip technology is a technology that integrates basic operating units such as sample preparation, reaction, separation, and detection in biological, chemical, and medical analysis processes onto micrometer-scale chips to automatically complete the entire analysis process. Its basic characteristics are the flexible combination and large-scale integration of multiple unit technologies on a tiny platform. It has low reagent consumption, high detection throughput, short operating time, low pollution, and low cost. It has a trend of replacing traditional testing laboratories in the future. In the biomedical field, it can be used for drug synthesis analysis, medical in vitro diagnosis, bionic skin tissue and organs, single cell analysis, nucleic acid analysis, drug screening and delivery, and other scenarios.

[0003] Drug screening microfluidic chips can be divided into four levels in the field of drug screening: molecules, cells, organs and whole animals. Cell culture technology based on microfluidic platforms is an important technical platform for animal cell culture and microenvironment manipulation today. Among them, microfluidic organ chips are expected to partially replace animal experiments and are widely used in drug development and screening, as well as drug toxicology and pharmacology research.

[0004] CN116836790A discloses an organoid chip device for high-throughput drug testing, including a chip assembly and a fixed assembly, the chip assembly is fixedly arranged around the fixed assembly; the chip assembly includes an upper chip, a sealing film and a lower chip arranged in sequence from top to bottom, the upper chip is provided with a first main channel, a Tesla micromixer, a collection channel and a first outlet channel, the channel inlets of multiple Tesla micromixers are all connected to the first main channel, the first inlet channel is connected to the first main channel, the channel outlet of the Tesla micromixer is connected to the collection channel, and the first outlet channel is connected to the collection channel; a hollow area is provided on the sealing film, and an organoid culture chamber is provided on the lower chip, and the hollow area corresponds to the collection channel and the organoid culture chamber respectively. This organoid chip device has the following disadvantages: when observing tumor cells, the chip needs to be taken out of the constant temperature device, so the chip is at room temperature for a long period of time, which makes it difficult to ensure the accuracy of the experiment. Utility Model Content

[0005] In view of the technical problems existing in the prior art, the purpose of the utility model is to provide a microfluidic platform for screening drugs for personalized tumor treatment. The microfluidic platform is applied to drug screening of tumor cells, provides the required growth environment for tumor cell culture, and can observe the survival of tumor cells under the action of drugs in real time.

[0006] In order to achieve the above purpose, the utility model adopts the following technical solutions:

[0007] A microfluidic platform for screening individualized tumor therapeutic drugs comprises a chip, wherein the chip comprises an upper chip and a lower chip; the upper chip is provided with a first channel, a first-level mixing structure, a second channel, a second-level mixing structure, a first cell culture area and a third channel which are connected in sequence, the first channel is provided with a drug inlet, and the third channel is provided with a discharge outlet; the lower chip is provided with a second cell culture area, which is provided corresponding to the first cell culture area; a temperature control device is provided on the lower surface of the lower chip, the temperature control device comprises a heating film, an insulating layer and a power supply, and the heating film is provided on the insulating layer and is electrically connected to the power supply.

[0008] As a preferred embodiment, the insulating layer is glass, the heating film is a nano heating film, and the nano heating film is coated on the glass.

[0009] As a preference, the temperature control device further comprises a temperature sensor and a control circuit board, the temperature sensor is arranged on the heating film and is electrically connected to the control circuit board, and the control circuit board is electrically connected to the power supply.

[0010] As a preferred embodiment, the temperature control device is detachably connected or adhesively connected to the lower chip.

[0011] As a preference, it further comprises a porous membrane, which is arranged between the upper chip and the lower chip, and is arranged corresponding to the first cell culture area.

[0012] As a preference, the porous membrane is made of PC, PE or PVDF.

[0013] As a preference, the first-stage mixing structure includes an S-shaped bend and a fishbone structure, the first channel is connected to the S-shaped bend, the S-shaped bend is connected to the fishbone structure, and the fishbone structure is connected to the second channel.

[0014] As a preference, a plurality of first-stage mixing structures are arranged on the upper chip, and the plurality of first-stage mixing structures are arranged in parallel.

[0015] As a preferred embodiment, the first cell culture area includes a plurality of first cell culture chambers, which are arranged in parallel; a first sample inlet is arranged at one end of the first cell culture chamber, and a first sample outlet is arranged at the other end;

[0016] The second cell culture area includes a plurality of second cell culture chambers, which are arranged in parallel; a second sample inlet is arranged at one end of the second cell culture chamber, and a second sample outlet is arranged at the other end.

[0017] As a preference, the first cell culture chamber comprises a plurality of first cell culture chambers, and the plurality of first cell culture chambers are arranged in series and are connected.

[0018] As a preferred embodiment, a plurality of second-stage mixing structures are arranged on the upper chip, and each of the second-stage mixing structures is arranged in a ring on the outside of the first cell culture chamber; the second-stage mixing structure includes an upper arc segment, a lower arc segment and a direct current segment, and the upper arc segment and the lower arc segment are respectively arranged at two ends of the first cell culture chamber, and the upper arc segment and the lower arc segment are connected by the direct current segment, and each first cell culture chamber is connected to the direct current segment.

[0019] In general, the utility model has the following advantages:

[0020] 1. The utility model is provided with a temperature control device, which can continuously heat the chip when cell observation is required, so that the chip is always maintained at 37°C, which can ensure the accuracy of the experiment.

[0021] 2. The utility model encapsulates the heating film at the bottom of the chip through glass. The glass can prevent the heat of the heating film from being lost to the outside and also facilitates observation of the cells.

[0022] 3. The utility model realizes precise temperature control of the chip by setting a temperature sensor and a control circuit board.

[0023] 4. The temperature control device of the utility model is detachably connected to the lower chip, thereby reducing the maintenance cost between the heating film and the lower chip; the temperature control device is adhesively connected to the lower chip, thereby reducing the difficulty of assembling the heating film and the lower chip.

[0024] 5. The utility model separates vascular endothelial cells from tumor cells through a porous membrane, so that the entire chip forms a bionic model with a multi-dimensional space, which evaluates the sensitivity and resistance of tumor cells to a certain concentration of drugs, so as to obtain a more accurate individualized treatment plan, avoid affecting the efficacy due to too low drug concentration, and avoid unnecessary damage to normal human cells due to too high drug concentration.

[0025] 6. The first-stage mixing structure and the second-stage mixing structure of the utility model constitute a drug concentration gradient generator, and the second-stage mixing structure surrounds the first cell culture chamber, so that the two fluids are evenly mixed, thereby achieving the effect of theoretical concentration of the drug being consistent with the actual concentration, preventing the problem of uneven mixing or laminar flow of the two drugs when the drugs are mixed, thereby avoiding the impact of the inability to accurately obtain the theoretical concentration on tumor cells or normal cells.

[0026] 7. The utility model realizes a three-layer structure combining two layers of chips and one layer of porous membrane by using the concentration difference penetration principle, without the need for glue of other materials and clamps and screws for clamping, and the chip bonding effect is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1It is a schematic diagram of the structure of the microfluidic platform for screening individualized tumor therapeutic drugs of the utility model;

[0028] Figure 2 It is a schematic diagram of the structure of the upper chip of the utility model;

[0029] Figure 3 It is a schematic diagram of the structure of the lower chip of the utility model;

[0030] Figure 4 It is a preparation flow chart of the chip of the utility model;

[0031] Wherein: 1: upper chip, 11: first channel, 12: first level mixing structure, 121: S-shaped bend, 122: fishbone structure, 13: second channel, 14: second level mixing structure, 15: first cell culture area, 151: first cell culture chamber, 152: first sample inlet, 153: first sample outlet, 16: third channel, 17: drug inlet, 18: discharge port, 2: lower chip, 21: second cell culture area, 211: second cell culture chamber, 212: second sample inlet, 213: second sample outlet, 3: temperature control device, 31: isolation layer, 32: power supply, 4: porous membrane. DETAILED DESCRIPTION

[0032] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0033] like Figures 1 to 3 As shown, a microfluidic platform for screening personalized tumor therapeutic drugs includes a chip, and the chip includes an upper chip and a lower chip; the upper chip is provided with a first channel, a first-level mixing structure, a second channel, a second-level mixing structure, a first cell culture area and a third channel which are connected in sequence, the first channel is provided with a drug inlet, and the third channel is provided with a discharge outlet; the lower chip is provided with a second cell culture area, and the second cell culture area is provided corresponding to the first cell culture area; a temperature control device is provided on the lower surface of the lower chip, the temperature control device includes a heating film, an insulating layer and a power supply, and the heating film is provided on the insulating layer and is electrically connected to the power supply.

[0034] The bottom of the lower chip in this embodiment is connected to the heating film. The power supply provides electric energy to the heating film for heat generation. The heating film provides a surface heat source for the chip. The surface heat source has the advantages of rapid temperature rise, small thermal hysteresis and wide thermal radiation. The insulating layer blocks the heat from being transferred to the outside of the chip, thereby ensuring that the temperature of the chip is maintained at 37°C.

[0035] As a preferred embodiment, the insulating layer is glass, the heating film is a nano heating film, and the nano heating film is coated on the glass. Specifically, the bottom of the lower chip of this embodiment is connected to the glass coated with the nano heating film. Since the glass coated with the nano heating film has a high transparency, almost close to glass, the observation effect of the cell is good. The power supply is a lithium ion battery or other lithium battery, and the heating film is electrically connected to the power supply through a power line.

[0036] Preferably, the temperature control device further includes a temperature sensor and a control circuit board. The temperature sensor is arranged on the heating film and is electrically connected to the control circuit board. The control circuit board is electrically connected to the power supply. The temperature sensor can detect the temperature of the heating film. When the temperature sensor senses that the temperature on the heating film is lower than 37°C, the temperature sensor transmits the information that heating is required to the control circuit board, and the control circuit board controls the power supply to supply electric energy to the heating film; when the temperature sensor senses that the temperature is maintained at 37°C, the temperature sensor transmits the information of stopping heating to the control circuit board, and the control circuit board controls the power supply to stop supplying electric energy to the heating film. In this way, the temperature of the upper chip and the lower chip is controlled, and the upper chip and the lower chip are precisely controlled.

[0037] As a preferred implementation manner, the temperature control device is bonded and connected to the lower chip.

[0038] In this embodiment, the edge of the insulating layer is sealed with the edge of the lower chip by glue. The area of ​​the insulating layer is larger than the area of ​​the heating film. The insulating layer can completely cover the heating film to prevent the heat of the heating film from being lost quickly, and has good safety performance.

[0039] As another preferred embodiment, the temperature control device is detachably connected to the lower chip, which reduces the maintenance cost between the temperature control device and the chip.

[0040] Preferably, the microfluidic platform for screening personalized tumor therapeutic drugs further comprises a porous membrane, which is arranged between the upper chip and the lower chip, and is arranged corresponding to the first cell culture area. The porous membrane is made of PC, PE or PVDF.

[0041] In this embodiment, the microfluidic platform for screening personalized tumor therapeutic drugs is set as a three-layer structure, the upper layer is the first cell culture area, the middle layer is a permeable porous membrane, and the lower layer is the second cell culture area. Taking the lung organ chip as an example, the first cell culture area is covered with vascular endothelial cells, and the second cell culture area is covered with tumor cells. The tumor cells are wrapped by extracellular matrix molecules. The microfluidic platform for screening personalized tumor therapeutic drugs forms a bionic model with multi-dimensional space.

[0042] As a preferred embodiment, the first-stage mixing structure includes an S-shaped bend and a fishbone structure, the first channel is connected to the S-shaped bend, the S-shaped bend is connected to the fishbone structure, and the fishbone structure is connected to the second channel. A plurality of first-stage mixing structures are arranged on the upper chip, and the plurality of first-stage mixing structures are arranged in parallel. The first cell culture area includes a plurality of first cell culture chambers, and the plurality of first cell culture chambers are arranged in parallel; a first sample inlet is arranged at one end of the first cell culture chamber, and a first sample outlet is arranged at the other end; the second cell culture area includes a plurality of second cell culture chambers, and the plurality of second cell culture chambers are arranged in parallel; a second sample inlet is arranged at one end of the second cell culture chamber, and a second sample outlet is arranged at the other end. The first cell culture chamber includes a plurality of first cell culture chambers, and the plurality of first cell culture chambers are arranged in series and connected. A plurality of second-stage mixing structures are arranged on the upper chip, and each of the second-stage mixing structures is arranged in a ring on the outside of the first cell culture chamber; the second-stage mixing structure comprises an upper arc segment, a lower arc segment and a direct current segment, and the upper arc segment and the lower arc segment are respectively arranged at the two ends of the first cell culture chamber, and the upper arc segment and the lower arc segment are connected by the direct current segment, and each first cell culture chamber is connected to the direct current segment.

[0043] Specifically, the upper chip of the present embodiment is provided with two drug inlets, three S-shaped bends, three fishbone structures, four upper arc segments, eight direct current segments, four lower arc segments, four first cell culture chambers and one discharge port. The two drug inlets are both connected with the first channel. The two drug inlets are connected with the inlets of the three S-shaped bends through the first channel. The outlets of the three S-shaped bends are respectively connected with the inlets of the three fishbone structures. The outlets of the three fishbone structures are connected with the inlets of the four upper arc segments through the second channel. The outlet of each upper arc segment is connected with the inlet of a lower arc segment through two direct current segments. Each first cell culture chamber includes three first cell culture chambers connected in series. Both sides of each first cell culture chamber are connected with the direct current segment of the second-stage mixing structure. The outlets of the four lower arc segments are connected with the discharge port through the third channel. An S-shaped flow channel is provided between the third channel and the discharge port.

[0044] The drug inlet at the head end of the upper chip is used to connect the drug delivery pipeline and the injection pump. The two drug inlets are through holes with a diameter of 100-200μm, which can respectively inject two drugs to be verified into the upper chip at the same pressure and flow rate; three first-stage mixing structures and four second-stage mixing structures constitute a drug concentration gradient generator. After the drug flows into the first-stage mixing structure from the first channel, the first-stage mixing begins. An S-shaped bend and a fishbone structure constitute a first-stage mixing structure, wherein the width of the S-shaped bend is 150-300μm and the depth is 100μm, and the width of the fishbone structure is about 200-300μm and the depth is 100μm, which can better make the drug The first stage of mixing is performed; after the drug flows from the fishbone structure through the second channel into the second stage of mixing structure, the second stage of mixing begins, an upper arc segment, two direct current segments and a lower arc segment constitute a second stage of mixing structure, and enter the first cell culture chamber in a circumferential manner, so that the drug can be better mixed in the second stage; four first cell culture chambers and four second cell culture chambers constitute a cell culture chamber, and the middle of the cell culture chamber is separated by a porous membrane, wherein the thickness of the porous membrane is 8-20 μm, and the pore size is 0.1-10 μm, and the thickness and pore size of the porous membrane can vary according to the cell type; the exhaust port at the end of the upper chip is used to discharge excess drugs and gases in the drug pushing process.

[0045] like Figure 4 As shown, the manufacturing method of the chip of the utility model:

[0046] S1. Material preparation: prepare the upper chip, the lower chip and a porous membrane of appropriate size;

[0047] S2. Preparation of PDMS mixed glue: PDMS glue and curing agent are mixed in a mass ratio of 5:1 and stirred thoroughly, and vacuum degassing is performed to prepare PDMS mixed glue;

[0048] S3, PDMS mixed glue coating: Pour the PDMS mixed glue on the polished clean silicon wafer, and place it on a glue spreader for spin coating. Set the speed of the glue spreader to 500 rpm for 20 seconds, and then set the speed of the glue spreader to 3000 rpm for 60 seconds.

[0049] S4, chip assembly: dip the surface to be bonded of the upper chip prepared in step S1 into the PDMS mixed glue on the silicon wafer, then lay the porous membrane flat on the second cell culture area of ​​the lower chip, and finally gently cover the upper chip on the porous membrane;

[0050] S5. Chip forming: The combined chip is placed horizontally at room temperature for 6-8 hours to complete chip preparation.

[0051] The utility model combines two layers of chips and a layer of porous membrane through the concentration difference penetration principle. The concentration difference penetration principle refers to the use of the different mass ratios of PDMS glue and curing agent in the PDMS mixed glue. For example, the mass ratio of PDMS glue and curing agent is usually 10:1, and the mass ratio of PDMS glue and curing agent used for bonding is usually 5:1 to 7:1. Therefore, the curing agent will be redistributed between the interfaces due to the concentration difference, and finally penetrate the porous membrane to reach equilibrium, thereby achieving bonding.

[0052] The use of the utility model:

[0053] In order to evaluate the sensitivity of tumor cells of cancer patients to single or multiple chemotherapy drugs administered by intravenous injection, after the chip is prepared,

[0054] S1: Tumor cells encapsulated by basement membrane extract (BME hydrogel, a substitute for extracellular matrix) were injected into the second cell culture area.

[0055] In step S1, since BEM hydrogel is in liquid state at 4°C, tumor cells need to be centrifuged, mixed, suspended and injected into the chip with BEM at 4°C, and then placed at 37°C for 30 minutes to allow BEM to solidify into a gel.

[0056] S2: Inject vascular endothelial cells into the first cell culture area.

[0057] In step S2, the endothelial cells are suspended in the culture medium by centrifugation and injected into the upper channel of the chip for culture at 37°C and about 5% carbon dioxide. The endothelial cells are finally attached to the porous membrane to verify the actual effect of the drug on the tumor cells in the 3D gel matrix by passing through the endothelial cell barrier.

[0058] S3: Inject drugs into the drug inlet to verify the effect of the drugs on tumor cells.

[0059] In step S3, if the effect of a single drug on tumor cells is to be verified, a drug of a certain concentration is injected into one of the drug inlets, and a culture medium is injected into the other drug inlet;

[0060] If you want to verify the combined effect of two drugs, inject drug A into one of the drug inlets and inject drug B into the other drug inlet. The concentration can be selected according to the actual situation.

[0061] S4: Evaluate the survival of tumor cells under the action of drugs, and then derive the sensitivity and resistance of the patient's tumor cells to a certain concentration of drugs.

[0062] The above-mentioned embodiments only express several implementation methods of the utility model, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the utility model. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the utility model, and these all belong to the protection scope of the utility model. Therefore, the protection scope of the utility model patent shall be based on the attached claims.

Claims

1. A microfluidic platform for screening personalized tumor therapeutic drugs, characterized by: The invention comprises a chip, which comprises an upper chip and a lower chip; the upper chip is provided with a first channel, a first-level mixing structure, a second channel, a second-level mixing structure, a first cell culture area and a third channel which are connected in sequence, the first channel is provided with a drug inlet, and the third channel is provided with a discharge outlet; the lower chip is provided with a second cell culture area, which is provided corresponding to the first cell culture area; a temperature control device is provided on the lower surface of the lower chip, the temperature control device comprises a heating film, an insulating layer and a power supply, the heating film is provided on the insulating layer and is electrically connected to the power supply.

2. A microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: The insulating layer is glass, and the heating film is a nano heating film, which is coated on the glass.

3. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: The temperature control device also includes a temperature sensor and a control circuit board. The temperature sensor is arranged on the heating film and is electrically connected to the control circuit board. The control circuit board is electrically connected to the power supply.

4. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: The temperature control device is detachably connected or adhesively connected to the lower chip.

5. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: It also includes a porous membrane, which is arranged between the upper chip and the lower chip, and the porous membrane is arranged corresponding to the first cell culture area.

6. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: The first-stage mixing structure includes an S-shaped bend and a fishbone structure, the first channel is connected to the S-shaped bend, the S-shaped bend is connected to the fishbone structure, and the fishbone structure is connected to the second channel.

7. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: A plurality of first-stage mixing structures are arranged on the upper chip, and the plurality of first-stage mixing structures are arranged in parallel.

8. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 1, characterized in that: The first cell culture area includes a plurality of first cell culture chambers, which are arranged in parallel; a first sample inlet is arranged at one end of the first cell culture chamber, and a first sample outlet is arranged at the other end; The second cell culture area includes a plurality of second cell culture chambers, which are arranged in parallel; a second sample inlet is arranged at one end of the second cell culture chamber, and a second sample outlet is arranged at the other end.

9. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 8, characterized in that: The first cell culture chamber includes a plurality of first cell culture chambers, and the plurality of first cell culture chambers are arranged in series and are connected.

10. The microfluidic platform for screening personalized tumor therapeutic drugs according to claim 8, characterized in that: A plurality of second-stage mixing structures are arranged on the upper chip, and each of the second-stage mixing structures is arranged in a ring on the outside of the first cell culture chamber; the second-stage mixing structure comprises an upper arc segment, a lower arc segment and a direct current segment, and the upper arc segment and the lower arc segment are respectively arranged at the two ends of the first cell culture chamber, and the upper arc segment and the lower arc segment are connected by the direct current segment, and each first cell culture chamber is connected to the direct current segment.

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