Pumpless perfusion microfluidic chip for multicellular three-dimensional co-culture and methods of use

Abstract

The present application relates to the technical field of cell culture device, in particular to a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture and a use method thereof, comprising a liquid storage layer, an inlet and outlet layer, an upper channel layer, a lower channel layer and a packaging layer, the present application realizes a one-way circulation perfusion dynamic co-culture system without a pumping system, the device occupies less space, is easy to operate, reduces the consumption of culture medium and reduces the cost; the separation of free NK cells and tumor infiltrating NK cells is realized, which provides conditions for the study of the molecular biological characteristics of different types of NK cells; the migration and interaction of tumor spheres and NK cells are simultaneously and real-time observed, thereby solving the problem of the external system integration of microfluidic pumps required for the separation of free immune cells and tumor infiltrating immune cells in the existing devices.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and in particular to a pumpless perfusion microfluidic chip for multi-cell three-dimensional co-culture and its usage method. Background Technology

[0002] Cancer seriously threatens human health, and its development is a long process, encompassing various stages of tumor growth and metastasis. The immune system plays a crucial role in recognizing and eradicating tumors, making tumor immunotherapy a focus of attention. Cellular immunotherapy, also known as adoptive cell therapy (ACT), is a treatment method that utilizes the body's own immune system, particularly immune cells, to recognize, locate, and eliminate cancer cells. The core of this therapy lies in utilizing or enhancing the patient's own immune cells, enabling them to more effectively fight tumors. Among these, tumor-infiltrating lymphocytes (TILs) are a type of immune cell capable of penetrating the tumor matrix and entering the tumor interior, playing a role in killing tumor cells. Therefore, research on tumor-infiltrating lymphocytes plays a vital role in cellular immunotherapy.

[0003] Taking natural killer (NK) cells as an example, NK cells are an important type of immune cell and 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. CD56 is an adhesion molecule on the surface of NK cells, and approximately 90% of NK cells express the CD56 receptor (CD56) at low density. dim / - ) and high-density expression of CD16 (CD16 bright / + These cells exhibit high cytotoxicity and antibody-dependent cytotoxicity (ADCC). The remaining 10% of NK cells highly express CD56 (CD56...). bright / + ) and low-density expression of CD16 (CD56) bright / + CD16 dim or CD56 bright / + CD16 - Early research indicated that CD56 bright / + Subtypes of NK cells mainly produce chemokines and cytokines, exerting anti-infection and anti-tumor effects. Therefore, studying NK cell subtypes and their roles in the anti-tumor process is of great significance.

[0004] Tumor-infiltrating natural killer (TINK) cells are NK cells that infiltrate the tumor microenvironment. These cells infiltrate tumor tissue from the bloodstream and play a crucial role in immune surveillance and elimination of cancer cells. The tumor microenvironment, such as intratumoral hypoxia, can reduce the cytotoxicity of NK cells. Furthermore, limited supply of nutrients (such as glucose) can impair the metabolism and function of NK cells. Traditional methods for studying TINK cells mainly involve in vivo cell acquisition and in vitro cell enrichment, but these methods are complex, time-consuming, and yield small cell numbers, hindering cell quantification and efficient molecular characterization. Therefore, researchers 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 TINK cells within 24–48 hours, separating free NK cells from tumor spheres and TINK cells is difficult because they are all suspended in the culture medium. Meanwhile, traditional static in vitro three-dimensional culture cannot replicate the dynamic migration environment of NK cells, requiring microfluidic chip methods for biomimetic simulation.

[0005] However, existing devices for separating free immune cells and tumor-infiltrating immune cells require microfluidic pumps integrated into external systems, and the devices occupy space and have high separation costs. Summary of the Invention

[0006] The purpose of this invention is to provide a pump-free perfusion microfluidic chip and its usage method for multi-cell three-dimensional co-culture, aiming to solve the problem that existing devices require externally integrated microfluidic pumps to separate free immune cells and tumor-infiltrating immune cells.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a pumpless perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a liquid storage layer, an inlet / outlet layer, an upper channel layer, a lower channel layer, and an encapsulation layer. The inlet / outlet layer is disposed between the liquid storage layer and the upper channel layer, the lower channel layer is disposed on the side of the upper channel layer away from the inlet / outlet layer, and the encapsulation layer is disposed on the 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 made of polydimethylsiloxane (PDMS) material, and the inlet / outlet layer and the encapsulation layer are made of polymethyl methacrylate (PMMA) material.

[0009] The liquid storage layer includes two liquid storage chambers, each of which is connected to the upper channel layer and the lower channel layer respectively; the area of ​​each liquid storage chamber is 143 mm². 2 The distance between the inlet and outlet is 7mm.

[0010] The upper channel layer has a cell culture chamber disposed on the upper channel layer, and the lower channel layer has a multi-layer paper-based composite fiber scaffold disposed on one side of the lower channel layer.

[0011] The second aspect is a method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, which is used in 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, inlet and outlet layers, upper channel layer, lower channel layer and 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 channel is full, and then add 200 μL of culture medium to each reservoir.

[0014] The pumpless perfusion microfluidic chip was placed in a culture container and cultured.

[0015] This invention discloses a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a reservoir layer, an inlet / outlet layer, an upper channel layer, a lower channel layer, and an encapsulation layer. The inlet / outlet layer is disposed between the reservoir layer and the upper channel layer. The lower channel layer is disposed on the side of the upper channel layer away from the inlet / outlet layer, and the encapsulation layer is disposed on the side of the lower channel layer away from the upper channel layer. This invention realizes a unidirectional circulating perfusion dynamic co-culture system without a pumping system, reducing equipment space, simplifying operation, reducing culture medium consumption, and lowering costs. It enables the separation of free NK cells and tumor-infiltrating NK cells, providing conditions for studying the molecular biological characteristics of different types of NK cells. It allows for simultaneous real-time observation of the migration and interaction of tumor spheroids and NK cells, thus solving the problem that existing devices require externally integrated microfluidic pumps to separate free immune cells and tumor-infiltrating immune cells. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0018] Figure 2This is a flowchart of the method for using the pumpless perfusion microfluidic chip for multi-cell three-dimensional co-culture provided by the present invention.

[0019] Figure 3 This is a schematic diagram of the unidirectional circulation of liquid within the chip.

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

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

[0022] Figure 6 This is the detection data graph for Example 3.

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

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

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

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

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

[0028] Please see Figure 1 In a first aspect, the present invention provides a pumpless perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising a liquid storage layer 1, an inlet / outlet layer 2, an upper channel layer 3, a lower channel layer 4, and an encapsulation layer 5. The inlet / outlet layer 2 is disposed between the liquid storage layer 1 and the upper channel layer 3, the lower channel layer 4 is disposed on the side of the upper channel layer 3 away from the inlet / outlet layer 2, and the encapsulation layer 5 is disposed 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 made of polydimethylsiloxane (PDMS) material, and the inlet / outlet layer 2 and the encapsulation layer 5 are made of polymethyl methacrylate (PMMA) material.

[0030] The liquid storage layer 1 includes two liquid storage chambers, each of which is connected to the upper channel layer 3 and the lower channel layer 4 respectively;

[0031] The area of ​​one of the liquid storage chambers is 143 mm². 2 The distance between the inlet and outlet is 7mm.

[0032] The upper channel layer 3 has a cell culture chamber 31, which is disposed on the upper channel layer 3. The lower channel layer 4 has a multilayer paper-based composite fiber scaffold 41, which is disposed on one side of the lower channel layer 4.

[0033] In this embodiment, the liquid storage layer 1 and the channel layer are both made of PDMS material, while the remaining layers are cut from PMMA (acrylic sheet). Each of the five layers has screw holes, and they are assembled into a single microfluidic chip using screws. The key to achieving unidirectional liquid circulation lies in the design of the inlet and outlet and the volume of liquid in the storage chamber. The area of ​​one storage chamber is 143 mm². 2 The inlet and outlet spacing is 7 mm, and 200 μL of liquid is added to each reservoir. When the shaker is tilted, the liquid forms a meniscus at the edge of the reservoir and covers the inlet. The liquid then flows in from the inlet of one channel, passes through the cell culture chamber, and flows out from the outlet. Conversely, when the shaker is tilted in the opposite direction, the liquid flows in from the inlet of the other channel and flows out from the outlet, completing a unidirectional flow cycle. The culture container consists of a culture dish and a culture dish lid. Designed to accommodate two chips based on the dimensions of a pumpless microfluidic chip, the culture container is made of a biocompatible, non-toxic material.

[0034] The composite paper-based fiber scaffold used to simulate the three-dimensional physical microenvironment for cell migration is key to achieving the separation of free NK cells and tumor-infiltrating NK cells. Both are made from bio-friendly, non-toxic materials. They can be spatially stacked to form multi-layered structures, and can also be disassembled after stacking, facilitating microscopic observation of cells on each layer of the paper-based fiber scaffold. Furthermore, cells other than free NK cells can adhere to the surface of the paper-based scaffold; separation can be achieved simply by aspirating the liquid from the pump-free microfluidic chip.

[0035] Please see Figures 2 to 13 Secondly, a method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, using the pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture described in the first aspect, includes the following steps:

[0036] S1 assembles the liquid storage layer 1, the inlet / 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;

[0037] Specifically, the five-layer chip structure made of PDMS and PMMA acrylic sheets is assembled layer by layer using screws in the following order: encapsulation layer 5 - lower channel layer 4 - multi-layer paper-based composite fiber scaffold 41 containing tumor spheres (placed at the cell culture chamber 31) - upper channel layer 3 - inlet / outlet layer 2 - storage chamber layer 1.

[0038] S2 adds cell culture medium or NK cell suspension to the pumpless perfusion microfluidic chip until the channel is full of liquid, and then adds 200 μL of culture medium to each reservoir.

[0039] Specifically, use a 1mL sterile syringe to add cell culture medium or NK cell suspension to the upper and lower channel inlets respectively until the channel is full of liquid. Finally, add 200 μL of culture medium or NK cell suspension to each reservoir.

[0040] S3 places the pumpless perfusion microfluidic chip in a culture container for cultivation.

[0041] Specifically, the chip is placed in a specific culture container, which is then placed on a shaker with the upper channel tilted at 21° and the lower channel tilted at 15°. The shaker is set to swing at 60-second intervals and cultured at 37°C with 5% CO2 for 24 hours.

[0042] Example 1

[0043] The migration behavior of tumor spheres in a flowing state was studied using a pump-free microfluidic chip.

[0044] First, an agar@paper composite fiber scaffold was placed in a 96-well plate, with an initial inoculation density of 1.5 × 10⁶ cells / well. 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0045] A layer of agar@paper-based composite fiber scaffold containing tumor spheres and two layers of matrix gel@paper-based composite fiber scaffold (simulating extracellular matrix) are stacked in a bottom-middle-top order to form a multilayer paper-based composite fiber scaffold.

[0046] The chip was assembled according to the implementation plan, culture medium was injected into the channels and reservoirs, and the tumor spheres were dynamically cultured in three dimensions. After 24 hours, the chip and multiple layers of paper substrate were separated, and each layer of paper substrate was placed in a 96-well plate for observation under an inverted microscope.

[0047] Example 2

[0048] Pump-free microfluidic chips were used for dynamic co-culture of tumor spheres and NK cells, as well as semi-quantitative analysis of tumor-infiltrating NK cells.

[0049] Agar@paper composite fiber scaffolds were placed in 96-well plates, with an initial inoculation density of 1.5 × 10⁶ cells / well. 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0050] A layer of agar@paper containing tumor spheres and two layers of blank matrigel@paper composite fiber scaffolds (simulating extracellular matrix) are stacked in a bottom-middle-top order to form a multilayer paper-based composite fiber scaffold.

[0051] The chip was assembled according to the implementation plan. NK cell suspension labeled with a green fluorescent probe of the DiO cell membrane was injected into the channels and reservoirs for dynamic three-dimensional co-culture. After 24 hours, the chip and multilayer paper base were separated. Each layer of paper base was placed in a 96-well plate for observation under an inverted microscope, with static culture as a control.

[0052] The distribution area (green fluorescent area) of NK cells in each layer of paper-based scaffold was measured using ImageJ software to characterize the migration of NK cells.

[0053] Example 3

[0054] Pump-free microfluidic chips are used for dynamic co-culture of tumor spheres and NK cells, as well as for NK cell subtype detection.

[0055] Agar@paper composite fiber scaffolds were placed in 96-well plates, with an initial inoculation density of 1.5 × 10⁶ cells / well. 5 Human prostate cancer cells were cultured at 37°C and 5% CO2 for 24 hours to form tumor spheres.

[0056] A layer of agar@paper containing tumor spheres and two layers of matrix glue@paper-based composite fiber scaffold (simulating extracellular matrix) are stacked in a bottom-middle-top order to form a multilayer paper-based composite fiber scaffold.

[0057] Assemble the chip according to the implementation plan, inject NK cell suspension labeled with DiO cell membrane green fluorescent probe into the channel and reservoir, perform dynamic three-dimensional co-culture, collect the old culture medium after 24 hours, and obtain free NK cells by centrifugation at 1000 rpm for 3 min.

[0058] The chip and multiple layers of paper base were separated, and the cells of each layer of paper base were digested with 0.25% trypsin to prepare a single-cell suspension.

[0059] Free NK cells and cells obtained from each digestion layer were stained with CD56 and CD16 antibody staining reagents and detected by flow cytometry.

[0060] Example 4

[0061] Multi-layered pumpless microfluidic chips are integrated into a single pumpless microfluidic device.

[0062] After assembling each layer of the structure into a single unit, the chip is printed as a whole using a 3D printer and non-toxic materials.

[0063] A lid is installed in the cell culture chamber to create a certain space inside the chamber, preventing a large amount of liquid from remaining in the chamber during the shaking process and thus preventing successful perfusion. The lid is also printed using a 3D printer and non-toxic materials.

[0064] Finally, a multilayer paper-based composite fiber scaffold is placed in the cell culture chamber of the chip, the lid is closed, and the lid is secured with rubber bands or tape to prevent the lid from being impacted by the liquid during perfusion.

[0065] Liquid is injected into both the upper and lower channels from the inlet until it overflows from the outlet. 400 μL of liquid is added to each of the storage chambers. The device is then placed on a shaker with the upper channel tilted at 21° and the lower channel at 15°, with a 5-second swing interval, to achieve liquid perfusion.

[0066] Example 5

[0067] The multi-layered pumpless microfluidic chip was fabricated into an integrated pumpless single-cycle / dual-cycle microfluidic array chip.

[0068] Pump-free single-cycle microfluidic array chip: A micropore array for cell culture is set in the lower channel of a multi-layered pump-free microfluidic chip, while the upper channel does not have a cell culture chamber, integrating each layer into a single unit. A cap is placed at the micropore array, and the chip and cap are printed using a 3D printer and non-toxic materials. After printing, the cap is placed on the chamber and secured to prevent it from being impacted by liquid during perfusion.

[0069] Liquid is injected into both the upper and lower channels from the inlet until it overflows from the outlet. 400 μL of liquid is added to each of the storage chambers. The device is then placed on a shaker with the upper channel tilted at 21° and the lower channel at 15°, with a 5-second swing interval, to achieve liquid perfusion.

[0070] Pump-free dual-circulation microfluidic array chip: Two sets of upper and lower channels are set on a multi-layered pump-free microfluidic chip. The upper channels do not contain cell culture chambers. A micropore array is placed between the two sets of lower channels, connecting the lower channels to the micropore array. Each layer of the structure is integrated into a single unit. A cap is provided to prevent liquid evaporation from the reservoir and array chambers. The chip and cap are printed using a 3D printer and non-toxic materials.

[0071] Inject liquid A into the upper and lower channels through one set of inlets until the liquid overflows from the outlet. Add 100 μL of liquid A to each of the corresponding storage chambers. Then, inject liquid B into the upper and lower channels through the other set of inlets until the liquid overflows from the outlet. Add 100 μL of liquid B to each of the corresponding storage chambers and cover the chambers. Place the device on a shaker with the upper channel tilted at 21° and the lower channel tilted at 15°, with a shaking interval of 5 seconds, to achieve liquid perfusion.

[0072] The above description is merely a preferred embodiment of the pumpless perfusion microfluidic chip and its usage method for multi-cell three-dimensional co-culture of the present invention. Of course, it should not be construed as limiting the scope of the present invention. Those skilled in the art can understand that implementing all or part of the above embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the 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, an inlet / outlet layer, an upper channel layer, a lower channel layer, and a sealing layer. The inlet / outlet layer is disposed between the liquid storage layer and the upper channel layer. The lower channel layer is disposed on the side of the upper channel layer away from the inlet / outlet layer. The sealing layer is disposed on the side of the lower channel layer away from the upper channel layer. The upper channel layer has a cell culture chamber, which is disposed on the upper channel layer; the lower channel layer has a multi-layer paper-based composite fiber scaffold, which is disposed on one side of the lower channel layer. The liquid storage layer, the upper channel layer, and the lower channel layer are made of polydimethylsiloxane material, and the liquid inlet / outlet layer and the encapsulation layer are made of polymethyl methacrylate material. The liquid storage layer includes two liquid storage chambers, each of which is connected to the upper channel layer and the lower channel layer respectively; The area of ​​one of the liquid storage chambers is 143 mm². 2 The distance between the inlet and outlet is 7mm.

2. A method for using a pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture, comprising the pump-free perfusion microfluidic chip for multi-cell three-dimensional co-culture as described in claim 1, characterized in that, Includes the following steps: The liquid storage layer, inlet and outlet layers, upper channel layer, lower channel layer and encapsulation layer are assembled to obtain a pump-free perfusion microfluidic chip; Add cell culture medium or NK cell suspension to the pumpless perfusion microfluidic chip until the channel is full, and then add 200 μL of culture medium to each reservoir. The pumpless perfusion microfluidic chip was placed in a culture container and cultured.

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