Pump-free perfusion micro-fluidic chip for multi-cell three-dimensional co-culture

Abstract

The utility model relates to the technical field of cell cultivators, in particular to a pump-free perfusion micro-fluidic chip for multi-cell three-dimensional co-culture, which comprises a liquid storage layer, a liquid inlet and outlet layer, an upper channel layer, a lower channel layer and a packaging layer, realizes a one-way circulation perfusion dynamic co-culture system without a pumping system, reduces the occupied space of equipment, and is convenient to operate. Operation is simple, culture medium consumption is reduced, and cost is reduced; the separation of free NK cells and tumor infiltration NK cells is realized, and conditions are provided for researching molecular biological characteristics of different types of NK cells. The migration and interaction of the tumor spheres and the NK cells are observed in real time at the same time, so that the problem that a microfluid pump integrated by an external system is needed for separating free immune cells and tumor infiltration immune cells by existing equipment is solved.

Description

Technical Field

[0001] The utility model relates to the technical field of cell culture devices, in particular to a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture. Background Art

[0002] Cancer seriously endangers human health, and its development is a long process, including various stages of tumor growth and tumor metastasis. The immune system plays a vital role in identifying and eradicating tumors, and tumor immunotherapy has therefore become a focus of attention. Cellular immunotherapy (Cellular Immunotherapy), also known as Adoptive Cell Therapy (ACT), is a treatment method that uses the body's own immune system, especially immune cells, to identify, locate and eliminate cancer cells. The core of this therapy is to utilize or enhance the patient's own immune cells so that they can fight tumors more effectively. Among them, tumor-infiltrating lymphocytes (TIL) are a type of immune cell that can penetrate the tumor matrix and enter the tumor to kill tumor cells. Therefore, the study of tumor-infiltrating lymphocytes plays an important role in cell immunotherapy.

[0003] Take natural killer cells (NKs) among immune cells as an example. NK cells are an important type of immune cell and are part of the innate immune system. NK cells have the ability to recognize and kill certain tumor cells and virus-infected cells without prior sensitization. The classification of NK cells is mainly based on their surface markers and functional characteristics. Among them, CD56 is an adhesion molecule on the surface of NK cells. About 90% of NK cells express CD56 receptors at a low density (CD56 dim / - ) and high density expression of CD16 (CD16 bright / + ), they have high cytotoxicity and antibody-mediated dependent cellular cytotoxicity (ADCC). The remaining 10% of NK cells express high density of CD56 (CD56 bright / + ) and low density expression of CD16 (CD56 bright / + CD16 dim or CD56 bright / + CD16 - ). Early studies have shown that CD56 bright / + Subtypes of NK cells mainly produce chemokines and cytokines, which play an anti-infection and anti-tumor role. Therefore, it is of great significance to study the subtypes of NKs and their role in the anti-tumor process.

[0004] Tumor-infiltrating natural killer (TINK) cells are a type of NK cell found in the tumor microenvironment. These cells infiltrate tumor tissues from the bloodstream and play a crucial role in immune surveillance and cancer cell elimination. Tumor microenvironmental conditions, such as intratumoral hypoxia, can reduce NK cell cytotoxicity. Furthermore, a limited supply of nutrients, such as glucose, can impair NK cell metabolism and function. Traditional methods for studying TINKs rely primarily on in vivo cell harvesting and in vitro cell enrichment. However, this approach is complex, time-consuming, and yields low cell numbers, hindering quantitative cell characterization and efficient molecular characterization. Therefore, researchers have developed ultra-low-adhesion 96-well plate culture and agar-coated plate culture methods to establish a three-dimensional co-culture model of tumor spheres and NK cells in vitro, enabling interaction between tumor cells and NK cells and improving experimental efficiency. Although this method can generate a large number of TINKs within 24 to 48 hours, the separation of free NKs from TINKs is difficult because tumor spheres, TINKs, and free NKs are all suspended in the culture medium. At the same time, traditional static in vitro three-dimensional culture cannot replicate the dynamic migration environment of NK cells, and requires microfluidic chip methods for biomimetic simulation.

[0005] However, existing equipment for separating free immune cells and tumor-infiltrating immune cells requires a microfluidic pump integrated into an external system, and the equipment occupies a large space and has a high separation cost. Utility Model Content

[0006] The purpose of the utility model is to provide a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, aiming to solve the problem that existing equipment requires a microfluidic pump integrated in an external system to separate free immune cells and tumor-infiltrating immune cells.

[0007] To achieve the above-mentioned objectives, in a first aspect, the present invention provides a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a liquid reservoir layer, a liquid inlet and outlet layer, an upper channel layer, a lower channel layer and an encapsulation layer, wherein the liquid inlet and outlet layer is arranged between the liquid reservoir layer and the upper channel layer, the lower channel layer is arranged on a side of the upper channel layer away from the liquid inlet and outlet layer, and the encapsulation layer is arranged on a side of the lower channel layer away from the upper channel layer.

[0008] The liquid storage layer, the upper channel layer and the lower channel layer are supported by polydimethylsiloxane (PDMS) material, and the liquid inlet and outlet layer and the encapsulation layer are made of polymethyl methacrylate (PMMA) material.

[0009] Wherein, the area of ​​one of the liquid storage layers is 143mm 2 , the inlet and outlet spacing is 7mm.

[0010] Among them, the upper channel layer has a cell culture chamber, which is arranged on the upper channel layer, and the lower channel layer has a multi-layer paper-based composite fiber support, which is arranged on one side of the lower channel layer.

[0011] In a second aspect, a method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture is provided, which is used for the pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture described in the first aspect, comprising the following steps:

[0012] The liquid storage layer, the liquid inlet and outlet layer, the upper channel layer, the lower channel layer and the encapsulation layer are assembled to obtain a pump-free perfusion microfluidic chip;

[0013] Add cell culture medium or NK cell suspension to the pumpless perfusion microfluidic chip until the liquid fills the channel, and then add 200 μL of culture medium to each reservoir layer;

[0014] The pump-free perfusion microfluidic chip is placed in a culture container for culture.

[0015] The utility model is a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a liquid storage layer, a liquid inlet and outlet layer, an upper channel layer, a lower channel layer and an encapsulation layer, wherein the liquid inlet and outlet layer is arranged between the liquid storage layer and the upper channel layer, the lower channel layer is arranged on the side of the upper channel layer away from the liquid inlet and outlet layer, and the encapsulation layer is arranged on the side of the lower channel layer away from the upper channel layer. The utility model realizes a one-way circulation perfusion dynamic co-culture system without the need for a pumping system, reduces the space occupied by the equipment, is simple to operate, reduces culture medium consumption, and reduces costs; realizes the separation of free NK cells and tumor-infiltrating NK cells, and provides conditions for studying the molecular biological characteristics of different types of NK cells. It realizes the simultaneous real-time observation of the migration and interaction of tumor spheres and NK cells, thereby solving the problem that the existing equipment for separating free immune cells and tumor-infiltrating immune cells requires an external system-integrated microfluidic pump. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0017] Figure 1 This is a schematic structural diagram of a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture provided by the present invention.

[0018] Figure 2The present invention provides a flow chart of a method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture.

[0019] Figure 3 This is a schematic diagram of the one-way circulation flow of liquid in the chip.

[0020] Figure 4 This is a microscope observation picture of Example 1.

[0021] Figure 5 This is a microscope observation picture of Example 2.

[0022] Figure 6 This is the detection data diagram of Example 3.

[0023] Figure 7 This is a schematic diagram of Example 4 in which the upper channel layer of the chip is tilted 21° and the lower channel layer is tilted 15°.

[0024] Figure 8 and Figure 9 This is a schematic diagram of the pump-free single-cycle microfluidic array chip of Example 5.

[0025] Figure 10 、 Figure 11 、 Figure 12 and Figure 13 This is a schematic diagram of the pump-free dual-circulation microfluidic array chip of Example 5.

[0026] In the figure: 1-liquid storage layer, 2-liquid inlet and outlet layer, 3-upper channel layer, 4-lower channel layer, 5-encapsulation layer, 31-cell culture chamber, 41-multi-layer paper-based composite fiber scaffold. DETAILED DESCRIPTION

[0027] The following describes in detail embodiments of the present invention, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.

[0028] See also Figure 1 In the first aspect, the present invention provides a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a liquid reservoir layer 1, a liquid inlet and outlet layer 2, an upper channel layer 3, a lower channel layer 4 and an encapsulation layer 5, wherein the liquid inlet and outlet layer 2 is arranged between the liquid reservoir layer 1 and the upper channel layer 3, the lower channel layer 4 is arranged on the side of the upper channel layer 3 away from the liquid inlet and outlet layer 2, and the encapsulation layer 5 is arranged on the side of the lower channel layer 4 away from the upper channel layer 3.

[0029] The liquid storage layer 1 , the upper channel layer 3 and the lower channel layer 4 are supported by polydimethylsiloxane (PDMS) material, and the liquid inlet and outlet layer 2 and the encapsulation layer 5 are made of polymethyl methacrylate (PMMA) material.

[0030] The area of ​​one of the liquid storage layers 1 is 143 mm 2 , the inlet and outlet spacing is 7mm.

[0031] The upper channel layer 3 has a cell culture chamber 31 , which is arranged on the upper channel layer 3 . The lower channel layer 4 has a multi-layer paper-based composite fiber support 41 , which is arranged on one side of the lower channel layer 4 .

[0032] In this embodiment, the liquid storage layer 1 and the channel layer are made of PDMS material, and the remaining layers are cut from PMMA (acrylic sheet). Each of the five layers is provided with screw holes, and the five layers are assembled by screws to form an integrated microfluidic chip. The key to achieving one-way circulation of liquid lies in the setting of the inlet and outlet and the volume of liquid in the liquid storage layer 1. The area of ​​one liquid storage layer 1 is 143mm 2 The inlet and outlet spacing is 7 mm, and 200 μL of liquid is added to each liquid reservoir 1. When the shaker is tilted, the liquid forms a meniscus at the edge of the liquid reservoir 1 and covers the liquid inlet. The liquid then flows into the liquid inlet of one of the channels and flows out of the liquid outlet through the cell culture chamber. Conversely, when the shaker is tilted in the opposite direction, the liquid flows into the liquid inlet of the other channel and flows out of the liquid outlet, completing a unidirectional flow cycle; the culture container is divided into a culture dish and a culture dish cover. The culture container is designed according to the size of the pumpless microfluidic chip, can accommodate two chips, and is made of non-toxic materials with good biocompatibility.

[0033] The composite paper-based fiber scaffold for simulating the three-dimensional physical microenvironment of cell migration is the key to separating free NK cells and tumor-infiltrating NK cells, and is made of bio-friendly non-toxic materials. It can be stacked in space to form a multi-layer structure, and can also be split after stacking to facilitate microscopic observation of cells on each layer of paper-based fiber scaffold. In addition, cells other than free NK cells can adhere to the surface of the paper-based scaffold, and separation can be achieved by simply sucking away the liquid in the pump-free microfluidic chip.

[0034] See also Figures 2 to 13 In a second aspect, a method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture is provided, which is used for the pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture described in the first aspect, comprising the following steps:

[0035] S1 assembles the liquid storage layer 1, the liquid inlet and outlet layer 2, the upper channel layer 3, the lower channel layer 4 and the encapsulation layer 5 to obtain a pump-free perfusion microfluidic chip;

[0036] Specifically, the five-layer chip structure made of PDMS and PMMA acrylic plates was assembled layer by layer using screws in the order of encapsulation layer 5 - lower channel layer 4 - multi-layer paper-based composite fiber scaffold 41 containing tumor spheres (placed at the position of cell culture chamber 31) - upper channel layer 3 - liquid inlet and outlet layer 2 - liquid storage layer 1.

[0037] S2: adding cell culture medium or NK cell suspension to the pumpless perfusion microfluidic chip until the liquid fills the channel, and then adding 200 μL of culture medium to each liquid reservoir;

[0038] Specifically, a 1 mL sterile syringe was used to add cell culture medium or NK cell suspension to the upper and lower channel inlets respectively until the liquid filled the channel, and finally 200 μL of culture medium or NK cell suspension was added to each reservoir cavity.

[0039] S3: placing the pump-free perfusion microfluidic chip in a culture container for culture.

[0040] Specifically, the chip was placed in a specific culture container, and the culture container was placed on a shaker with the upper channel tilted at 21° and the lower channel tilted at 15°. The shaker was set to swing at an interval of 60 seconds and cultured at 37°C and 5% CO2 for 24 hours.

[0041] Example 1

[0042] A pump-free microfluidic chip was used to study the migration behavior of tumor spheroids under flow conditions.

[0043] First, agar@paper composite fiber scaffolds were placed in a 96-well plate, and the initial inoculation number was 1.5×10 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0044] A layer of agar@paper-based composite fiber scaffold containing tumor spheres and two layers of matrigel@paper-based composite fiber scaffold (simulating extracellular matrix) were stacked in the order of bottom-middle-top to form a multi-layer paper-based composite fiber scaffold.

[0045] The chip was assembled according to the implementation plan, and culture medium was injected into the channels and liquid reservoirs for dynamic three-dimensional culture of tumor spheres. After 24 hours, the chip and multi-layer paper base were separated, and each layer of paper base was placed in a 96-well plate for observation under an inverted microscope.

[0046] Example 2

[0047] A pump-free microfluidic chip was used for dynamic co-culture of tumor spheres and NK cells and semi-quantification of tumor-infiltrating NK cells.

[0048] The agar@paper composite fiber scaffold was placed in a 96-well plate, and the initial inoculation number was 1.5×10 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0049] A layer of agar@paper containing tumor spheres and two layers of blank matrigel@paper paper-based composite fiber scaffolds (simulating extracellular matrix) were stacked in the order of bottom-middle-top to form a multi-layer paper-based composite fiber scaffold.

[0050] The chip was assembled according to the implementation plan, and a NK cell suspension labeled with a DiO cell membrane green fluorescent probe was injected into the channel and reservoir for dynamic three-dimensional co-culture. After 24 hours, the chip and multi-layer paper substrate were separated, and each layer of paper substrate was placed in a 96-well plate for observation under an inverted microscope. Static culture was used as a control.

[0051] ImageJ software was used to measure the distribution area (green fluorescence area) of NK cells in each layer of paper-based scaffold to characterize the migration amount of NK cells.

[0052] Example 3

[0053] A pump-free microfluidic chip was used for dynamic co-culture of tumor spheres and NK cells and detection of NK cell subtypes.

[0054] The agar@paper composite fiber scaffold was placed in a 96-well plate, and the initial inoculation number was 1.5×10 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0055] A layer of agar@paper containing tumor spheres and two layers of matrigel@paper-based composite fiber scaffold (simulating extracellular matrix) were stacked in the order of bottom-middle-top to form a multi-layer paper-based composite fiber scaffold.

[0056] The chip was assembled according to the implementation plan, and a NK cell suspension labeled with a DiO cell membrane green fluorescent probe was injected into the channel and the reservoir for dynamic three-dimensional co-culture. After 24 hours, the old culture medium was collected and centrifuged at 1000 rpm for 3 minutes to obtain free NK cells.

[0057] The chip and the multiple layers of paper substrate were separated, and the cells in each layer of paper substrate were digested with 0.25% trypsin to prepare a single-cell suspension.

[0058] Free NK cells and cells obtained from digestion of each layer were stained with CD56 and CD16 antibody staining reagents and detected by flow cytometry.

[0059] Example 4

[0060] Merge multi-layer pump-free microfluidic chips into an integrated pump-free microfluidic device

[0061] After combining each layer of the structure into one, the entire chip is printed using a 3D printer and non-toxic materials.

[0062] A lid is set on the cell culture chamber to form a certain space in the chamber to prevent a large amount of liquid from being retained in the chamber during the swinging of the shaker, resulting in unsuccessful perfusion. The lid is also printed using a 3D printer and non-toxic materials.

[0063] Finally, place a multilayer paper-based composite fiber scaffold in the cell culture chamber of the chip, cover it with a lid, and secure it with a rubber band or tape to prevent the lid from being impacted by the liquid during perfusion.

[0064] Inject liquid into the upper and lower channels from the inlet until it overflows from the outlet. Add 400 μL of liquid to each reservoir chamber and place the device on a rocker with the upper channel tilted at 21° and the lower channel tilted at 15°, with a rocking interval of 5 seconds, to achieve liquid perfusion.

[0065] Example 5

[0066] The multi-layer pump-free microfluidic chip is made into an integrated pump-free single-cycle / dual-cycle microfluidic array chip.

[0067] Pumpless, single-loop microfluidic array chip: A multilayer, pumpless microfluidic chip features a microwell array for spheroid culture in the lower channel. The upper channel lacks a cell culture chamber, integrating each layer into a single structure. A cover is placed over the microwell array. The chip and cover are printed using a 3D printer and non-toxic materials. After printing, the cover is placed over the chamber and secured to prevent impaction during perfusion.

[0068] Inject liquid into the upper and lower channels from the inlet until it overflows from the outlet. Add 400 μL of liquid to each reservoir chamber and place the device on a rocker with the upper channel tilted at 21° and the lower channel tilted at 15°, with a rocking interval of 5 seconds, to achieve liquid perfusion.

[0069] Pumpless dual-circulation microfluidic array chip: A multilayer pumpless microfluidic chip features two sets of upper and lower channels. The upper channels lack cell culture chambers, and a microwell array is placed between the two sets of lower channels, connecting them. Each layer is integrated into a single structure. A lid is installed to prevent evaporation of liquid from the reservoir and array chambers. The chip and lid are printed using a 3D printer and non-toxic materials.

[0070] Inject liquid A into the upper and lower channels through the inlet of one set until the liquid overflows from the outlet. Add 100 μL of liquid A to each corresponding reservoir. Then, inject liquid B into the upper and lower channels through the inlet of the other set until the liquid overflows from the outlet. Add 100 μL of liquid B to each corresponding reservoir and close the lid. Place the device on a rocker with the upper channel tilted at 21° and the lower channel tilted at 15°, with a rocking interval of 5 seconds, to achieve liquid perfusion.

[0071] The above disclosure is merely a preferred embodiment of the pumpless perfusion microfluidic chip for multi-cell three-dimensional co-culture of the present invention and is certainly not intended to limit the scope of the present invention. Persons skilled in the art will appreciate that any equivalent variations made by implementing all or part of the processes of the above embodiment in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, characterized in that: It includes a liquid storage layer, a liquid inlet and outlet layer, an upper channel layer, a lower channel layer and an encapsulation layer. The liquid inlet and outlet layer is arranged between the liquid storage layer and the upper channel layer, the lower channel layer is arranged on the side of the upper channel layer away from the liquid inlet and outlet layer, and the encapsulation layer is arranged on the side of the lower channel layer away from the upper channel layer.

2. The pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture according to claim 1, characterized in that: The liquid storage layer, the upper channel layer and the lower channel layer are supported by polydimethylsiloxane material, and the liquid inlet and outlet layer and the packaging layer are made of polymethyl methacrylate material.

3. The pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture according to claim 2, characterized in that: The area of ​​one of the liquid storage layers is 143 mm 2 , the inlet and outlet spacing is 7mm.

4. The pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture according to claim 3, characterized in that: The upper channel layer has a cell culture chamber, which is arranged on the upper channel layer; the lower channel layer has a multi-layer paper-based composite fiber scaffold, which is arranged on one side of the lower channel layer.

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