A reusable digital droplet generation device and vitrification system

Abstract

The application provides a reusable digital droplet generating device, which comprises a base body and a digital droplet flow meter integrated in the base body and used for droplet generation and removal, wherein the digital droplet flow meter is provided with an air sealing cavity, a droplet generating part, a droplet removing part, a liquid flow channel and a first gas flow channel; the droplet generating part and the droplet removing part are respectively arranged at an inlet and an outlet of the air sealing cavity and are communicated with the air sealing cavity; the liquid flow channel is communicated with the droplet removing part; and the first gas flow channel is communicated with a first air inlet of the air sealing cavity. The application can not only realize the generation of a continuous liquid concentration gradient with accurate adjustment to improve the activity of cells in the liquid exchange process to the maximum extent, but also realize the reuse of the digital flow meter. Furthermore, the application also provides a vitrification system which combines the reusable digital droplet generating device with a microfluidic chip.

Description

Technical Field

[0001] This invention belongs to the field of biological sample micromanipulation technology, specifically relating to a reusable digital droplet generation device and a vitrification freezing system using the above device. Background Technology

[0002] Cryopreservation of biological samples typically refers to preserving living organisms in liquid nitrogen at ultra-low temperatures (-196 degrees Celsius) to maintain their viability after thawing and rehydration. Cryopreservation technology is currently widely used for the long-term storage of cells, tissues, and organs, and has achieved breakthroughs in many fields, such as assisted reproduction (freezing of eggs, sperm, and embryos) and stem cell cryopreservation. Vitrification involves adding a high concentration of cryoprotectant to rapidly freeze cells at ultra-low temperatures (cooling rate approximately 10,000 degrees Celsius per minute), forming an irregular vitrified solid and avoiding the formation of ice crystals during freezing. Due to its rapid freezing speed and minimal cell loss (no ice crystal formation), vitrification is currently the most commonly used cryopreservation technique. However, a major challenge of vitrification is that cells are exposed to a high concentration of cryoprotectant, which is chemically toxic to cells. To address this issue, a common solution is to gradually replace the cells with buffer solutions and cryogenic solutions of varying concentrations, allowing the cells to gradually come into contact with and adapt to the increasing concentrations of the cryogenic solutions to slowly achieve osmotic pressure balance and reduce chemical toxicity.

[0003] Currently, there are two main types of cell media exchange methods: manual and automated. However, both of these methods use traditional pipette-like dilution techniques, typically producing only specific concentration gradients of equilibration and cryoprotectants, making it difficult to generate wide-ranging gradients across multiple concentrations. To minimize the impact of media exchange on cell viability, the ideal method is to generate multiple precise and controllable concentration gradients from low to high. Patent CN112430531B proposes a digitally operable device for microfluidic manipulation of biological samples. This device achieves precise quantification of liquid aspiration and removal through a digital droplet flowmeter integrated within a microfluidic chip, allowing for continuous adjustment of the liquid concentration gradient around the biological sample using digital droplet generation. However, the digital droplet flowmeter is costly to manufacture, structurally complex, and its integration with the microfluidic chip prevents reusability, limiting its practicality. Summary of the Invention

[0004] This invention addresses the vitrification and freeze-thaw processes of biological samples by proposing a novel, reusable digital droplet generation device. This device not only enables the precise and adjustable generation of a continuous liquid concentration gradient to maximize cell activity during fluid exchange but also allows for the reusability of a digital flow meter. Furthermore, this invention provides a vitrification freezing system that combines the aforementioned reusable digital droplet generation device with a microfluidic chip. The microfluidic chip allows for direct insertion into liquid nitrogen for freeze-thaw operations; it is disposable, replaceable, and reusable. The digital droplet generation device is reusable and can be easily connected to the microfluidic chip.

[0005] The first aspect of the present invention discloses a reusable digital droplet generating device, characterized in that it includes a substrate and a digital droplet flow meter integrated in the substrate for droplet generation and removal; the digital droplet flow meter is configured with an air-sealed cavity, a droplet generating section and a droplet removing section, the droplet generating section and the droplet removing section being respectively disposed at the inlet and outlet of the air-sealed cavity and communicating with the air-sealed cavity; the digital droplet flow meter is further configured with a liquid flow channel and a first gas flow channel, the liquid flow channel being connected to the droplet removing section, and the first gas flow channel being connected to a first vent of the air-sealed cavity.

[0006] As an optional feature, the digital droplet flow meter is also equipped with a second gas flow channel; the second gas flow channel is connected to the second vent of the air-sealed cavity, or is connected to the air-sealed cavity through the first gas flow channel.

[0007] As an optional solution, the digital droplet generating device further includes a first liquid storage tank; the liquid interface of the first liquid storage tank is connected to the liquid flow channel of the digital droplet flow meter, and the gas interface of the first liquid storage tank is connected to the second gas flow channel of the digital droplet flow meter; the gas interface of the first liquid storage tank is set higher than the liquid interface.

[0008] The first aspect of this invention retains the core digital droplet flow meter in the substrate and externally connects the liquid storage tank, so that the number of times the digital droplet flow meter can be used is not limited by the capacity of the liquid storage tank, which increases the flexibility of use and the number of times it can be used, and reduces the cost of use.

[0009] A second aspect of the present invention discloses another reusable digital droplet generating device, characterized in that it includes a substrate and a digital droplet flow meter and a second liquid storage tank integrated in the substrate; the digital droplet flow meter is used for droplet generation and removal; the digital droplet flow meter is configured with an air-sealed cavity, a droplet generating section and a droplet removing section, the droplet generating section and the droplet removing section being respectively disposed at the inlet and outlet of the air-sealed cavity and communicating with the air-sealed cavity; the digital droplet flow meter is further configured with a liquid flow channel, a first gas flow channel and a second gas flow channel; the two ends of the liquid flow channel are respectively connected to the liquid interfaces of the droplet removing section and the first liquid storage tank, and the first gas flow channel is connected to the first vent of the air-sealed cavity; the gas interface of the second liquid storage tank is connected to the first gas flow channel through the second gas flow channel, or connected to the second vent of the air-sealed cavity through the second gas flow channel; the gas interface of the second liquid storage tank is positioned higher than the liquid interface.

[0010] As an alternative, the capacity of the second storage tank is 1-5 mL.

[0011] A second aspect of this invention integrates the digital droplet flow meter and the liquid storage tank into a single substrate, resulting in high integration, smaller size, and simpler operation. Furthermore, the reasonable capacity setting of the liquid storage tank can fully meet the needs of the same user.

[0012] A third aspect of the present invention discloses a vitrification freezing system, characterized in that it includes a digital droplet generating device and a microfluidic chip interconnected; the digital droplet generating device adopts the reusable digital droplet generating device described in the first aspect or its alternative, or adopts the reusable digital droplet generating device described in the second aspect; the microfluidic chip includes a microfluidic pipette and a cell sieve structure, the microfluidic pipette being connected to the droplet generating section of the digital droplet flowmeter in the digital droplet generating device.

[0013] As an alternative, the microfluidic chip is connected to the droplet generation unit of the digital droplet generation device via a flexible tube; the flexible tube is made of a sterilizable bio-inert material, including any one of PE, PP, PEEK, PTFE, FEP, and ETFE.

[0014] As an alternative, the vitrification refrigeration system also includes a pressure source for providing a positive or negative pressure gas source; the pressure source is connected to the first gas flow channel of the digital droplet generating device via a connecting pipe.

[0015] As an optional solution, the connecting pipeline is made of a sterilizable bio-inert material, including any one of PE, PP, PEEK, PTFE, FEP, and ETFE.

[0016] As an alternative, the connecting pipeline is equipped with a valve for controlling the flow of gas.

[0017] As an alternative, the valve is a solenoid valve with a millisecond-level response.

[0018] The present invention has the following beneficial effects:

[0019] (1) This invention combines the digital droplet flow meter with the microfluidic chip design by using an external connection. The structure is simple and can realize both digital droplet operation and the reusability of the high-cost droplet digital flow meter component.

[0020] (2) The digital droplet generating device of the present invention is designed in two forms, namely with a liquid storage tank and without a liquid storage tank. The design with a liquid storage tank can be discarded after the liquid storage tank is filled with liquid. The design without a liquid storage tank further separates the digital droplet flow meter and the liquid storage tank, which can realize the repeated use of the digital droplet flow meter. The liquid storage tank can be discarded after it is filled with liquid.

[0021] (3) The present invention uses digital droplet generation to achieve continuous and precise adjustment of digital concentration gradient, realize continuous digital liquid dilution, and greatly improve cell activity during liquid exchange.

[0022] (4) The digital droplet flowmeter control system used in this invention is simple, has few components, low manufacturing cost, and is easy for users to operate. Attached Figure Description

[0023] Figure 1 This is a schematic diagram (front view) of the digital droplet generation device described in Example 1.

[0024] Figure 2 This is a schematic diagram of the digital droplet generation device described in Example 1. Figure 2 (Front view)

[0025] Figure 3 This is a connection diagram of the vitrification freezing system described in Example 2.

[0026] Figure 4 This is a flowchart of the digital droplet generation device, including both negative and positive pressure scenarios.

[0027] Figure 5 This is a schematic diagram (front view) of another reusable digital droplet generation device.

[0028] Figure captions: 10-Digital droplet generating device; 11-Substrate; 12-Digital droplet flow meter; 121-Air-sealed cavity; 122-Droplet generating section; 123-Droplet removal section; 124-Liquid flow channel; 125-Gas flow channel; 126-Gas flow channel; 13-Reservoir; 131-Liquid interface; 132-Gas interface; 20-Microfluidic chip; 21-Microfluidic pipe; 22-Cell sieve structure; 30-Pressure source; 40-Solenoid valve; 50-Connecting pipeline; 60-Reservoir; 61-Liquid interface; 62-Gas interface. Detailed Implementation

[0029] The technical solution of the present invention will now be clearly and completely described with reference to specific embodiments and accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] In the description of this invention, the use of terms such as "upper," "lower," "inner," and "outer" to indicate orientation or positional relationship is based on the orientation or positional relationship shown in the accompanying drawings. These terms are used solely for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the use of terms such as "first" and "second" is used to distinguish similar objects and is not necessarily used to describe a specific order or relative importance. Those skilled in the art can understand the specific meaning of the above terms in this invention in conjunction with the specific circumstances. In addition, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion, but rather to include other units not explicitly listed or inherent to these products or devices.

[0031] It is understood that the cells in this invention include biological samples such as oocytes, embryos, sperm, stem cells, and blastocysts from humans or other organisms.

[0032] like Figure 1 As shown, Embodiment 1 discloses a reusable digital droplet generating device 10, mainly used for droplet generation and removal. It has an air-sealed cavity to generate a gas-liquid interface, and furthermore, the generated liquid can be temporarily stored after removal. The digital droplet generating device 10 mainly includes a substrate 11 and a digital droplet flow meter 12 and a liquid storage tank integrated in the substrate 11.

[0033] The digital droplet flow meter 12, also known as the "digital droplet generator 12," has an air-sealed chamber 121 and droplet generating and removing parts 122 and 123 respectively located at the inlet and outlet of the air-sealed chamber 121. The droplet generating part 122 is connected to the outlet of the microfluidic chip 20 via a flexible tube, and the droplet removing part 123 is connected to the storage tank 13 via a liquid flow channel 124. The digital droplet flow meter 12 also has a gas flow channel 125, one end of which is connected to the vent of the air-sealed chamber 121, and the other end is connected to the pressure source 30. The digital droplet flow meter 12 is mainly used to achieve precise quantitative measurement of liquid aspiration and removal, making the liquid concentration gradient around the biological sample continuously adjustable. Its specific structure and working principle are not the focus of this invention. For details, please refer to US Patent US16538307 and Chinese Patent CN112430531B, which will not be elaborated here. It should be noted that the tubing used in this invention, such as the tubing connecting the droplet generation unit 122 and the outlet of the microfluidic chip 20, can all be made of sterilizable bio-inert materials, such as PE (polyethylene), PP (polypropylene), PEEK (polyether ether ketone), PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), ETFE (ethylene tetrafluoroethylene), etc.

[0034] The digital droplet flow meter 12 also includes a gas flow channel 126. The liquid reservoir 13 primarily stores the liquid drawn from the microfluidic pipette 21 in the microfluidic chip 20 and removed by the droplet removal section 123. On one hand, the liquid interface 131 of the reservoir 13 is connected to the droplet removal section 123 at the outlet of the digital droplet generator 12 via the liquid flow channel 124; on the other hand, the gas interface 132 of the reservoir 13 is also connected to the gas flow channel 125 of the digital droplet flow meter 12 via the gas flow channel 126. It should be noted that the gas interface 132 of the reservoir 13 is typically positioned above the liquid interface 131, meaning the gas interface 132 of the reservoir 13 is higher than the liquid interface 131. When the liquid level in the reservoir 13 is about to reach the gas flow channel 126, the use of the digital droplet generator 10 is terminated, and the entire substrate 11 is discarded. The size of the liquid storage tank 13 can be designed according to requirements, generally with a capacity of 1-5 mL. This capacity typically allows for 10-20 uses, fully meeting the needs of the same user. Optionally, the air-sealed chamber 121 has two vents. One vent connects to the outside via a gas flow channel 125, and the other vent connects to the gas interface 132 of the liquid storage tank 13 via a gas flow channel 126. Figure 2 As shown.

[0035] It is understood that the specific processing technology and material selection of the digital droplet generation device 10 are not the focus of this invention. Please refer to US Patent US16538307 and Chinese Patent CN112430531B. The processing and material selection of the microfluidic chip mentioned below can also be found in the above patents, and will not be repeated here.

[0036] Combination Figure 3 As shown, Embodiment 2 discloses a vitrification freezing system, which mainly includes a digital droplet generating device and a microfluidic chip. The digital droplet generating device can be the reusable digital droplet generating device 10 described in Embodiment 1. The microfluidic chip 20 mainly includes a microfluidic pipette 21 and a cell sieve structure 22. The microfluidic chip 20 is connected to the droplet generating section 122 of the digital droplet generating device 10 via a flexible tube. An external pressure source 30 for the digital droplet generating device 10 can provide negative or positive pressure to draw in or expel liquid.

[0037] Furthermore, the system may also include a pressure source 30, which is connected to the gas flow channel 125 in the droplet generator 12 via a connecting pipe 50, and then communicates with the air-sealed cavity 121, allowing the air-sealed cavity 121 to be connected to the atmosphere. The pressure source 30 can be connected to a positive or negative pressure gas source. The connecting pipe 50 contains a solenoid valve 40, which can be controlled to open or close to output positive or negative pressure to be applied to the connecting pipe 50. The connecting pipe 50 can also be made of a sterilizable bio-inert material, such as PE (polyethylene), PP (polypropylene), PEEK (polyetheretherketone), PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), ETFE (ethylene tetrafluoroethylene), etc. The airtightness of both ends of the connecting pipe 50 can be ensured by interference fit during connection. The solenoid valve 40 can be a high-precision millisecond-level response solenoid valve, but manual valves, solenoid valves, or other types of valves can also be selected, as long as they can realize the function of controlling the opening and closing of the gas entry channel.

[0038] Combination Figure 4The operating state of the vitrification freezing system is shown as follows: The pressure source 30 intermittently applies negative pressure through the solenoid valve 40, drawing liquid dropwise from the microfluidic chip 20 connected to the digital droplet generating device 10 into the digital droplet flow meter 12, and then into the storage tank 13. After the storage tank 13 is full, the connection between the digital droplet generating device 10 and the microfluidic chip 20, as well as the connecting pipe 50, is disconnected, and the substrate 11 is discarded. A brand new digital droplet generating device 10 is then installed, and the digital droplet generating device 10 is reconnected to the microfluidic chip 20 and the connecting pipe 50 using a flexible tube. Alternatively, the pressure source 30 intermittently applies positive pressure through the solenoid valve 40, pushing the liquid in the droplet generating section 122 into the microfluidic chip 20, thereby squeezing out the cells in the microfluidic chip 20. During this process, the liquid level in the storage tank 13 remains essentially constant.

[0039] Combination Figure 5 As shown, in other embodiments, the liquid storage tank may not be integrated into the substrate 11, but may serve as a detachable component of the digital droplet generating device 10. The liquid storage tank 60 is provided with a liquid interface 61 and a gas interface 62. In use, the liquid interface 61 of the liquid storage tank 60 is connected to the liquid flow channel 124 on the digital droplet generator 12 via a flexible hose, and the gas interface 62 of the liquid storage tank 60 is connected to the gas flow channel 126. In this embodiment, when the liquid in the liquid storage tank 60 is full, the liquid storage tank 60 can be directly disassembled and discarded. The substrate 11 can be reused; that is, the liquid interface 61 of a new liquid storage tank 60 can be reconnected to the liquid flow channel 124 via a flexible hose, and the gas interface 62 of the new liquid storage tank 60 can be connected to the gas flow channel 126. Accordingly, the vitrification freezing system disclosed in Embodiment 2, in addition to the digital droplet generating device, microfluidic chip, and gas pressure source, should also include the liquid storage tank 60, with other configurations remaining essentially unchanged.

[0040] In summary, this invention combines the digital droplet flow meter with the microfluidic chip design by using an external connection, thereby achieving both digital droplet operation and the reusability of the high-cost droplet digital flow meter component.

[0041] Finally, it should be noted that although the embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art, guided by this specification, can make many other forms without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

Claims

1. A reusable digital droplet generation device, characterized in that, The device includes a substrate and a digital droplet flow meter integrated within the substrate for droplet generation and removal. The digital droplet flow meter is configured with an air-sealed cavity, a droplet generation section, and a droplet removal section. The droplet generation section and droplet removal section are respectively located at the inlet and outlet of the air-sealed cavity and communicate with it. The digital droplet flow meter also includes a liquid flow channel, a first gas flow channel, and a second gas flow channel. The liquid flow channel communicates with the droplet removal section, the first gas flow channel communicates with a first vent of the air-sealed cavity, and the second gas flow channel communicates with a second vent of the air-sealed cavity, or the second gas flow channel communicates with the air-sealed cavity through the first gas flow channel. The droplet generation section is configured to be externally connected to a microfluidic chip, which is independent of the digital droplet generation device.

2. The digital droplet generation device as described in claim 1, characterized in that, It also includes a first liquid storage tank; the liquid interface of the first liquid storage tank is connected to the liquid flow channel of the digital droplet flow meter, and the gas interface of the first liquid storage tank is connected to the second gas flow channel of the digital droplet flow meter; the gas interface of the first liquid storage tank is set higher than the liquid interface.

3. The digital droplet generation device as described in claim 2, characterized in that, The capacity of the first storage tank is 1-5 mL.

4. A vitrification freezing system, characterized in that, The device includes an interconnected digital droplet generating device and a microfluidic chip; the digital droplet generating device is a reusable digital droplet generating device as described in any one of claims 1 to 3; the microfluidic chip is designed independently of the digital droplet generating device and is connected to the droplet generating section of the digital droplet flowmeter in the digital droplet generating device via an external connection; the microfluidic chip includes a microfluidic pipette and a cell sieve structure, and the microfluidic pipette is connected to the droplet generating section of the digital droplet flowmeter in the digital droplet generating device.

5. The vitrification freezing system as described in claim 4, characterized in that, The microfluidic chip is connected to the droplet generation part of the digital droplet generation device via a flexible tube; the flexible tube is made of a sterilizable bio-inert material, including any one of PE, PP, PEEK, PTFE, FEP, and ETFE.

6. The vitrification freezing system as described in claim 4 or 5, characterized in that, It also includes a pressure source for providing a positive or negative pressure gas source; the pressure source is connected to the first gas flow channel of the digital droplet generating device through a connecting pipe; the connecting pipe is equipped with a valve for controlling the gas flow.

7. The vitrification freezing system as described in claim 6, characterized in that, The connecting pipeline is made of a sterilizable bio-inert material, including any one of PE, PP, PEEK, PTFE, FEP, and ETFE.

8. The vitrification freezing system as described in claim 6, characterized in that, The valve is a solenoid valve with a millisecond-level response.

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