A microfluidic chip-based organoid droplet generation device and methods of use thereof

Abstract

This invention discloses an organoid droplet generation device based on a microfluidic chip and its usage method, belonging to the field of droplet microfluidics. It solves the technical problems of poor droplet consistency and chemical demulsification damaging cell viability in existing droplet microfluidic technologies. The device includes a microfluidic chip comprising a continuous phase injection channel and a dispersed phase injection channel connected by converging channels. A droplet collection tube is located at the bottom of the outlet of the droplet solidification zone. The droplet collection tube has a conical internal structure and a sieve at its bottom opening. This invention achieves oil and water phase separation through physical filtration and rinsing via the sieve at the bottom of the droplet collection tube, avoiding stress damage to organoid cells caused by chemical substances. It can prepare high-throughput organoid droplets with extremely low sample consumption, and the generated organoids have high consistency, which is beneficial for simultaneously screening multiple anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of droplet microfluidics, specifically to an organoid droplet generation device based on a microfluidic chip and its usage method. Background Technology

[0002] Primary tumor organoids are a technique that encapsulates patient-derived tumor cells in a three-dimensional matrix for in vitro culture. This technique preserves the genetic and phenotypic characteristics of tumors and has significant applications in precision medicine and drug screening. Traditional organoid culture methods often use manual dispensing to add a mixture of cell matrix gel to culture plates, which suffers from problems such as low throughput, inconsistent sizes, and poor reproducibility.

[0003] In recent years, droplet microfluidic technology has been introduced into organoid culture. Droplet microfluidics, an important branch of microfluidic platforms, is a novel technology for manipulating tiny volumes of liquid, namely droplets. Droplets are formed by one fluid within another immiscible carrier fluid, essentially an emulsification phenomenon. By rapidly emulsifying and generating uniform droplet structures in microfluidic chips, the standardization level of organoid preparation has been improved.

[0004] However, existing droplet generation systems generally rely on chemical demulsifiers to remove the oil phase, which may adversely affect the structural integrity of the droplets and subsequent drug sensitivity testing results, limiting their application in preclinical studies. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides an organoid droplet generation device based on a microfluidic chip and its usage method, solving the technical problems of poor droplet consistency and chemical demulsification damaging cell activity in existing droplet microfluidic technologies.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, an organoid droplet generation device based on a microfluidic chip is provided, including a microfluidic chip. The microfluidic chip includes a continuous phase injection channel and a dispersed phase injection channel connected by a confluence channel. The confluence channel is a T-shaped channel with both ends of its top connected to the continuous phase injection channel. The middle of the top of the confluence channel is connected to the dispersed phase injection channel. The bottom of the confluence channel is connected to a droplet solidification zone. A droplet guide tube is provided at the bottom of the outlet of the droplet solidification zone, and the droplets enter the droplet collection device below through the guide tube.

[0007] In this scheme, fluorinated oil and cell matrix gel suspension are used as the oil phase and dispersed phase, respectively. Droplets are generated by microfluidic chip using microfluidic focusing shearing. By adjusting the flow rates of the oil and dispersed phases in the continuous phase injection channel and the dispersed phase injection channel, the droplet diameter can be stabilized between 300 µm and 600 µm. Furthermore, the oil and water phases are separated by physical filtration and rinsing through a screen at the bottom of the droplet collection tube, avoiding stress damage to organoid cells caused by chemical substances. This scheme is particularly suitable for the construction of primary tumor organoids and drug sensitivity testing procedures, and can also be extended to three-dimensional culture scenarios such as stem cell spheres and embryoids.

[0008] Furthermore, the top-middle connection point of the confluence channel is the droplet generation region. Monodisperse droplets with diameters of 300 µm to 600 µm are generated in the confluence channel through interfacial shearing.

[0009] Furthermore, the dispersed phase injection channel is connected to the confluence channel via the sample storage area. The sample storage area provides a buffer space to inject the cell matrix gel suspension into the pre-cooled chip, preventing solidification during sample loading.

[0010] Furthermore, the droplet collection device includes a droplet collection tube and a sleeve located outside the droplet collection tube. The inside of the droplet collection tube has a conical structure, with a rounded corner buffer at the bottom opening and a screen installed on the bottom opening. The rounded corner buffer has a buffer arc at the bottom, which can reduce the impact shear force and prevent droplet breakage.

[0011] Furthermore, the sieve aperture is 50 µm to 150 µm. This aperture setting facilitates the interception of liquid droplets and the filtration of the oil phase.

[0012] Furthermore, the screen is made of nylon. Nylon is a stable material that does not easily affect the droplets.

[0013] On the other hand, a method for using an organoid droplet generation device based on a microfluidic chip is provided, comprising the following steps: S1. Place the microfluidic chip on a temperature control platform and pre-cool it to 4°C; S2. Fluorinated oil and cell matrix suspension are injected into the continuous phase injection channel and the dispersed phase injection channel, respectively. S3. After the droplets are generated, raise the temperature of the microfluidic chip to 37°C and incubate for 30-60 minutes until the droplets are completely solidified. S4. After the droplet collection tube collects the droplet oil phase mixture and filters out the oil phase in the droplet oil phase mixture, add 0.5 mL of culture medium to the droplet collection tube to rinse the residual oil phase on the surface of the droplets, replace the new collection tube sleeve, add 1.5 mL of culture medium and blow to form a droplet suspension. S5. Take the droplet suspension and evenly distribute it into a 96-well plate or a 384-well plate, with multiple organoid droplets in each well. Place the plate in an incubator and incubate until the organoid droplets mature. Perform drug sensitivity testing after the droplets mature.

[0014] Furthermore, the flow rate ratio of fluorinated oil to cell matrix gel suspension in the continuous phase injection channel and the dispersed phase injection channel is 2 to 6, so that the droplet diameter is stabilized between 300 µm and 600 µm.

[0015] This invention discloses an organoid droplet generation device based on a microfluidic chip and its usage method, the beneficial effects of which are: This invention achieves separation of the oil and water phases through physical filtration and rinsing using a screen at the bottom of the droplet collection tube, avoiding stress damage to organoid cells caused by chemical substances. It can prepare high-throughput organoid droplets with extremely low sample consumption, and the generated organoids have high consistency, which is beneficial for screening multiple anti-tumor drugs simultaneously. Attached Figure Description

[0016] Figure 1 A three-dimensional view of an organoid droplet generation device; Figure 2 A top view of an organoid droplet generation device; Figure 3 This is a side view of an organoid droplet generation device; Figure 4 An exploded view of the droplet collection tube; The components are: 1. Continuous phase injection channel; 2. Dispersed phase injection channel; 3. Sample storage area; 4. Droplet generation area; 5. Droplet solidification area; 6. Guide tube; 7. Droplet collection device; 71. Droplet collection tube; 72. Rounded corner buffer zone; 73. Screen; 74. Sleeve. Detailed Implementation

[0017] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0018] This embodiment provides an organoid droplet generation device based on a microfluidic chip, which solves the technical problems of poor droplet consistency and chemical demulsification damaging cell structures in existing droplet microfluidic technologies. It is shown in detail below.

[0019] refer to Figures 1-2An organoid droplet generation device based on a microfluidic chip is disclosed, comprising a microfluidic chip including a continuous phase injection channel 1 and a dispersed phase injection channel 2 connected by a confluence channel. The confluence channel is a T-shaped channel with its top two ends connected to the continuous phase injection channel 1, its top middle section connected to the dispersed phase injection channel 2, and its bottom section connected to a droplet solidification zone 5. The top middle section of the confluence channel forms the droplet generation zone 4. Monodisperse droplets with a diameter of 300 µm to 600 µm are generated in the confluence channel through interfacial shearing.

[0020] refer to Figures 3-4 A droplet guide tube 6 is installed at the bottom of the outlet of the droplet solidification zone 5. The droplets enter the droplet collection tube 71 of the droplet collection device 7 through the guide tube 6. After the droplets are intercepted and the oil phase is filtered by the screen 73 at the bottom of the droplet collection tube 71, 0.5 mL of culture medium is added to the droplet collection tube 71 to rinse off the residual oil phase on the surface of the droplets. The droplet collection tube 71 has a conical structure inside and a rounded corner buffer zone 72 is provided at the bottom opening. The rounded corner buffer zone 72 has a buffer arc, which can reduce the impact shear force generated by blowing the culture medium when collecting droplets and prevent droplet breakage.

[0021] The mesh size of sieve 73 is 50 µm to 150 µm and the material is nylon.

[0022] As a further embodiment, the dispersed phase injection channel 2 is connected to the confluence channel via the sample storage area 3. The confluence channel includes multiple sections of meandering bends. The sample storage area provides a buffer space, allowing the cell matrix gel suspension to be injected into the pre-cooled chip, preventing solidification during sample loading.

[0023] In this embodiment, fluorinated oil and cell matrix gel suspension are used as the oil phase and dispersed phase, respectively. Droplets are generated by microfluidic chip using microfluidic focusing shearing. By adjusting the flow rates in the oil and dispersed phases in continuous phase injection channel 1 and dispersed phase injection channel 2, the droplet diameter can be stabilized between 300 µm and 600 µm. Furthermore, the oil and water phases are separated by physical filtration and rinsing through the screen 73 at the bottom of the droplet collection tube 71, avoiding stress damage to organoid cells caused by chemical substances. This method is particularly suitable for the construction of primary tumor organoids and drug sensitivity testing procedures, and can also be extended to three-dimensional culture scenarios such as stem cell spheres and embryoids.

[0024] This embodiment also provides a method for using an organoid droplet generation device based on a microfluidic chip, including the following steps: S1. Place the microfluidic chip on a temperature control platform and pre-cool it to 4°C; S2. Fluorinated oil and cell matrix gel suspension are injected into continuous phase injection channel 1 and dispersed phase injection channel 2 respectively; wherein, the flow rate ratio of fluorinated oil and cell matrix gel suspension in continuous phase injection channel 1 and dispersed phase injection channel 2 is 2~6, so that the droplet diameter is stabilized between 300 µm and 600 µm.

[0025] S3. After the droplets are generated, raise the temperature of the microfluidic chip to 37°C and incubate for 30-60 minutes until the droplets are completely solidified. S4. After the droplet collection tube 71 collects the droplet oil phase mixture and filters out the oil phase in the droplet oil phase mixture, slowly inject culture medium from the top of the tube to wash it, repeat 3 times; then add 0.5 mL of culture medium to the droplet collection tube 71 to rinse the residual oil phase on the surface of the droplets, replace the new collection tube sleeve, add 1.5 mL of culture medium and gently blow to form a droplet suspension. S5. Take the droplet suspension and evenly distribute it into a 96-well plate or a 384-well plate, with multiple organoid droplets in each well. Place the plate in an incubator and incubate until the organoid droplets mature. Perform drug sensitivity testing after the droplets mature.

[0026] Although specific embodiments of the invention have been described in detail with reference to the accompanying drawings, this should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by a person skilled in the art without inventive effort within the scope described in the claims still fall within the scope of protection of this patent.

Claims

1. A microfluidic chip-based organoid droplet generation device, characterized in that, The microfluidic chip includes a continuous phase injection channel (1) and a dispersed phase injection channel (2) connected by a confluence channel. The confluence channel is a T-shaped channel with both ends of its top connected to the continuous phase injection channel (1). The middle of the top of the confluence channel is connected to the dispersed phase injection channel (2). The bottom of the confluence channel is connected to the droplet solidification zone (5). A guide tube (6) is provided at the bottom of the outlet of the droplet solidification zone (5). The droplets enter the droplet collection device (7) below through the guide tube (6).

2. The organoid droplet generation device based on a microfluidic chip according to claim 1, characterized in that, The top and middle connection point of the confluence channel is the droplet generation area (4).

3. The organoid droplet generation device based on a microfluidic chip according to claim 1, characterized in that, The dispersed phase injection channel (2) is connected to the confluence channel through the sample storage area (3).

4. The organoid droplet generating device according to claim 1, characterized in that, The droplet collecting device (7) includes a droplet collecting tube (71) and a sleeve (74) located outside the droplet collecting tube (71). The droplet collecting tube (71) has a conical structure inside, a rounded corner buffer zone (72) is provided at the bottom opening, and a screen (73) is provided on the bottom opening.

5. The organoid droplet generating device according to claim 1, characterized in that, The sieve (73) has an aperture of 50µm to 150µm.

6. The organoid droplet generating device according to claim 1, characterized in that, The screen (73) is made of nylon.

7. The method of using the organoid droplet generation device based on a microfluidic chip according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1. Place the microfluidic chip on a temperature control platform and pre-cool it to 4°C; S2. Fluorinated oil and cell matrix suspension are injected into the continuous phase injection channel (1) and the dispersed phase injection channel (2), respectively. S3. After the droplets are generated, raise the temperature of the microfluidic chip to 37°C and incubate for 30-60 minutes until the droplets are completely solidified. S4. After the droplets enter the droplet collection device (7) through the guide tube (6), collect the droplet oil phase mixture and filter out the oil phase in the droplet oil phase mixture, add 0.5 mL of culture medium to the droplet collection tube (71), rinse the residual oil phase on the surface of the droplets, replace the new collection tube sleeve (74), add 1.5 mL of culture medium and blow to form a droplet suspension. S5. Distribute the droplet suspension evenly into 96-well or 384-well plates, with each well containing multiple organoid droplets. Incubate the plates in an incubator and perform drug sensitivity testing once the organoid droplets have matured.

8. The method of use according to claim 7, characterized in that, The flow rate ratio of fluorinated oil and cell matrix suspension in the continuous phase injection channel (1) and the dispersed phase injection channel (2) is 2 to 6.

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