CN115920988A

CN115920988A - Reusable digital liquid drop generating device and vitrification refrigerating system

Info

Publication number: CN115920988A
Application number: CN202310078117.0A
Authority: CN
Inventors: 请求不公布姓名; 程奕昕; 潘挺睿
Original assignee: Suzhou Institute Of Higher Studies University Of Science And Technology Of China
Current assignee: Suzhou Institute Of Higher Studies University Of Science And Technology Of China
Priority date: 2023-02-08
Filing date: 2023-02-08
Publication date: 2023-04-07

Abstract

The invention provides a reusable digital liquid drop generating device, which comprises a base body and a digital liquid drop flow meter integrated in the base body and used for generating and removing liquid drops, wherein the digital liquid drop flow meter is provided with an air sealing cavity, a liquid drop generating part, a liquid drop removing part, a liquid circulation channel and a first gas circulation channel, the liquid drop generating part and the liquid drop 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 circulation channel is communicated with the liquid drop removing part, and the first gas circulation channel is communicated with a first air vent of the air sealing cavity. The invention not only can realize the accurate and adjustable generation of continuous liquid concentration gradient to improve the activity of cells in the liquid changing process to the maximum extent, but also can realize the repeated use of the digital flowmeter. Furthermore, the invention also provides a vitrification freezing system which combines the reusable digital liquid drop generating device and the microfluidic chip.

Description

Reusable digital liquid drop generating device and vitrification refrigerating system

Technical Field

The invention belongs to the technical field of biological sample micromanipulation, and particularly relates to a reusable digital liquid drop generating device and a vitrification freezing system adopting the device.

Background

Cryopreservation of biological samples generally refers to the preservation of the organism in liquid nitrogen at ultra-low temperatures (196 ℃ below zero) to maintain its activity after thawing recovery. Cryopreservation techniques are currently widely used for long-term storage of cells, tissues and organs, and have achieved breakthrough advances in a number of fields, such as in the field of assisted reproduction (freezing of ova, sperm, embryos, etc.) and stem cell freezing, among others. The vitrification freezing method is that high concentration freezing liquid is added to make the cell freeze fast in ultralow temperature environment to form irregular vitrification solid and avoid the formation of ice crystal during freezing. Because of its rapid freezing speed and low cell loss (no ice crystal generation), the vitrification rapid freezing technique is the most common low-temperature cryopreservation technique at present. However, one difficulty with the vitrification freezing technique is that the cells are exposed to a high concentration of freezing fluid, which is chemically toxic to the cells. In order to solve the problem, a common solution is to gradually change the buffer solution and the refrigerating fluid with concentration gradient for the cells, so that the cells gradually contact and adapt to the refrigerating fluid with the concentration from low to high to slowly reach the balance of the internal and external osmotic pressures, and reduce the chemical toxicity.

The current cell liquid changing modes are of two types, namely manual liquid changing and automatic liquid changing. However, both of these liquid changing methods are diluted by a conventional pipette-like dilution method, and usually only equilibrium liquid and freezing liquid with specific concentration gradients can be generated, so that it is difficult to generate a large-scale gradient across multiple concentrations. In order to minimize the influence of the liquid changing process on the activity of cells, an ideal liquid changing mode is to generate a plurality of precisely controllable concentration gradients continuously from low to high, and patent CN112430531B proposes a device capable of digital operation for biological sample microfluidic operation, so that precise quantification of liquid suction and removal is realized by a digital liquid drop flowmeter integrated in a microfluidic chip, and the liquid concentration gradient around the biological sample can be continuously adjusted by a digital liquid drop generation mode. However, the digital droplet flowmeter has high processing cost and a complex structure, and the design of integrating the digital droplet flowmeter with the microfluidic chip prevents the flowmeter from being reused, and limits the practicability to a certain extent.

Disclosure of Invention

The invention provides a novel reusable digital liquid drop generating device aiming at vitrification freeze thawing of a biological sample, which can not only realize accurate and adjustable generation of continuous liquid concentration gradient so as to improve the activity of cells in the liquid changing process to the maximum extent, but also realize the repeated use of a digital flow meter. Furthermore, the invention also provides a vitrification freezing system which combines the reusable digital liquid drop generating device with the microfluidic chip, wherein the microfluidic chip can be directly inserted into liquid nitrogen for freezing and thawing operation, the microfluidic chip is disposable, replaceable and disposable, and the digital liquid drop generating device can be reused and can be conveniently connected to the microfluidic chip.

The invention discloses in a first aspect a reusable digital droplet generator, comprising a substrate and a digital droplet flow meter integrated in the substrate for droplet generation and removal; the digital liquid drop flowmeter is provided with an air sealing cavity, a liquid drop generating part and a liquid drop removing part, wherein the liquid drop generating part and the liquid drop 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 digital droplet flow meter is further provided with a liquid circulation channel communicating with the droplet removing part and a first gas circulation channel communicating with the first vent of the air tight chamber.

As an alternative, the digital droplet flow meter is further provided with a second gas flow channel; the second gas circulation channel is communicated with the second vent of the air sealing cavity or communicated with the air sealing cavity through the first gas circulation channel.

As an alternative, the digital droplet generator further comprises a first reservoir; the liquid interface of the first liquid storage tank is connected with the liquid circulation channel of the digital liquid drop flow meter, and the gas interface of the first liquid storage tank is connected with the second gas circulation channel of the digital liquid drop flow meter; the gas interface of the first liquid storage tank is higher than the liquid interface.

In the first aspect of the invention, the most core digital liquid drop flow meter is reserved in the base body, and the liquid storage tank is externally connected, so that the use times of the digital liquid drop flow meter are not limited by the capacity of the liquid storage tank, the use flexibility and the use times are increased, and the use cost is reduced.

The second aspect of the invention discloses another reusable digital liquid drop generating device, which is characterized by comprising a base body, a digital liquid drop flowmeter and a second liquid storage tank, wherein the digital liquid drop flowmeter and the second liquid storage tank are integrated in the base body; the digital droplet flow meter is used for droplet generation and removal; the digital liquid drop flowmeter is provided with an air sealing cavity, a liquid drop generating part and a liquid drop removing part, wherein the liquid drop generating part and the liquid drop 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 digital droplet flow meter is further provided with a liquid flow channel, a first gas flow channel and a second gas flow channel; two ends of the liquid circulation channel are respectively communicated with the liquid drop removing part and the liquid interface of the first liquid storage tank, and the first gas circulation channel is communicated with the first air vent of the air sealing cavity; the gas interface of the second liquid storage tank is communicated with the first gas circulation channel through a second gas circulation channel, or is communicated with the second vent of the air sealing cavity through the second gas circulation channel; and the gas interface of the second liquid storage tank is higher than the liquid interface.

As an alternative, the second reservoir has a capacity of 1-5mL.

In the second aspect of the invention, the digital liquid drop flowmeter and the liquid storage tank are integrated in the matrix, so that the integration level is high, the volume is smaller, and the operation is simpler. And, through the reasonable capacity setting of liquid storage tank, can fully satisfy the demand of same user object.

The third aspect of the invention discloses a vitrification freezing system, which is characterized by comprising a digital liquid drop generating device and a micro-flow chip which are connected with each other; the digital droplet generator is the reusable digital droplet generator of the first aspect or the alternative aspect thereof, or the reusable digital droplet generator of the second aspect; the microfluidic chip comprises a microfluidic pipette and a cell sieve structure, and the microfluidic pipette is connected with a droplet generation part of a digital droplet flowmeter in the digital droplet generation device.

As an alternative, the microfluidic chip is connected with the liquid drop generating part of the digital liquid drop generating device through a hose; the hose is made of bio-inert material capable of being sterilized, and comprises any one of PE, PP, PEEK, PTFE, FEP and ETFE.

As an alternative, the vitrification refrigeration system further includes a gas pressure source for providing a positive pressure gas source or a negative pressure gas source; the air pressure source is connected with a first air flow channel of the digital liquid drop generating device through a communication pipeline.

As an alternative, the communication pipeline is made of a biological inert material capable of being sterilized, and the biological inert material comprises any one of PE, PP, PEEK, PTFE, FEP and ETFE.

As an alternative, a valve for controlling the passage of gas is provided in the communication line.

As an alternative, the valve is a millisecond response solenoid valve.

The invention has the following beneficial effects:

(1) The digital liquid drop flowmeter is independent of the design of the microfluidic chip, and the two are combined for use in an external connection mode, so that the structure is simple, the digital liquid drop type operation can be realized, and the repeatable use of the liquid drop digital flowmeter component with high processing cost can be realized.

(2) The digital liquid drop generating device is designed into two forms, namely a liquid storage tank and a liquid storage tank without the liquid storage tank, wherein the liquid storage tank with the liquid storage tank is designed to be thrown after the liquid storage tank is filled with liquid; the design without the liquid storage tank further separates the digital liquid drop flow meter from the liquid storage tank, so that the digital liquid drop flow meter can be repeatedly used, and the liquid storage tank is only required to be discarded after the liquid storage tank is filled with liquid.

(3) The invention adopts a digital liquid drop generation mode to realize continuous and accurate adjustment of digital concentration gradient, realize continuous digital liquid dilution and greatly improve the activity of cells in the liquid changing process.

(4) The digital liquid drop flowmeter has the advantages of simple control system, few parts, low manufacturing cost and convenience for user operation.

Drawings

Fig. 1 is a schematic structural view (front view) of a digital droplet generating apparatus according to embodiment 1.

Fig. 2 is a schematic structural diagram 2 (front view) of the digital droplet generating apparatus described in embodiment 1.

FIG. 3 is a schematic diagram of the connection of the vitrification freezing system described in example 2.

Fig. 4 is a flow diagram of the operation of a digital droplet generator, including both the application of negative and positive pressure.

Fig. 5 is a schematic structural view (front view) of another reusable digital droplet generator.

The attached drawings are marked as follows: 10-digital droplet generation device, 11-substrate, 12-digital droplet flowmeter, 121-air-sealed chamber, 122-droplet generation part, 123-droplet removal part, 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 pipette, 22-cell sieve structure; 30 air pressure source; 40-electromagnetic valve; 50-connecting lines; 60-liquid storage tank, 61-liquid interface and 62-gas interface.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following specific embodiments and accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the description of the present invention, if terms indicating orientation or positional relationship such as "upper", "lower", "inner", "outer", etc., appear on the drawings, they are merely for convenience of describing the present invention and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation, and thus, should not be construed as limiting the present invention. Moreover, the appearances of the phrases "first," "second," and the like herein are not necessarily all referring to the particular order or relative importance. To those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood in conjunction with specific situations. Furthermore, the terms "comprises" and "comprising," as well as any variations thereof, are intended to cover a non-exclusive inclusion, but may include other elements not expressly listed or inherent to such products or devices.

It is understood that cells of the present invention include oocytes, embryos, sperm, stem cells, blastocysts and like biological samples of humans or other organisms.

As shown in fig. 1, example 1 discloses a reusable digital droplet generator 10, which is mainly used for droplet generation and removal, and has an air-tight chamber to create an air-liquid interface, and further, the generated liquid can be temporarily stored after being removed. The digital droplet generator 10 mainly comprises a base body 11, and a digital droplet flow meter 12 and a liquid reservoir 13 which are integrated in the base body 11.

The digital droplet flowmeter 12 is also referred to as a "digital droplet generator 12" and has an air-tight chamber 121, and a droplet generating unit 122 and a droplet removing unit 123 respectively disposed at an inlet and an outlet of the air-tight chamber 121, wherein the droplet generating unit 122 is connected to an outlet of the microfluidic chip 20 through a hose, and the droplet removing unit 123 is connected to the liquid reservoir 13 through a liquid flow channel 124. The digital droplet flowmeter 12 is further provided with a gas flow channel 125, and one end of the gas flow channel 125 is connected to the vent of the air tight chamber 121, and the other end is connected to the air pressure source 30. The digital droplet flowmeter 12 is mainly used for realizing accurate quantification of liquid suction and removal, so that the liquid concentration gradient around the biological sample is continuously adjustable, and the specific structure and the working principle of the digital droplet flowmeter are not the key points of the present invention, and refer to US16538307 and chinese CN112430531B, which are not described herein again. The tube used in the present invention, for example, the tube connecting the droplet generating section 122 and the outlet of the microfluidic chip 20, may be made of any bio-inert material that can be sterilized, such as PE (polyethylene), PP (polypropylene), PEEK (polyether ether ketone), PTFE (polytetrafluoroethylene), FEP (fluorinated ethylene propylene), ETFE (ethylene tetrafluoroethylene), and the like.

Wherein the digital droplet flow meter 12 is further provided with a gas flow channel 126. The liquid reservoir 13 mainly stores liquid removed by the droplet removing section 123 after being sucked from the micro flow pipette 21 in the micro flow control chip 20. On one hand, the liquid interface 131 of the liquid storage tank 13 is connected with the liquid drop removing part 123 at the outlet of the digital liquid drop generator 12 through the liquid flowing channel 124; on the other hand, the gas port 132 of the reservoir 13 is connected to the gas flow channel 125 of the digital droplet flowmeter 12 via the gas flow channel 126. It is noted that the gas port 132 of the reservoir 13 is typically disposed above the liquid port 131, i.e., the gas port 132 of the reservoir 13 is disposed higher than the liquid port 131. When the liquid level in the liquid reservoir 13 is about to be level with 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 the requirement, the capacity of the liquid storage tank can be generally designed to be 1-5mL, the liquid storage tank can be used for about 10-20 times under the capacity, and the use requirement of the same user can be fully met. Optionally, the air-tight chamber 121 is provided with two air vents, one of which is connected through a gas communication channel 125 to the outside, and the other of which is connected through a gas communication channel 126 to a gas interface 132 of the reservoir 13, as shown in fig. 2.

It is understood that the specific processing and material selection of the digital droplet generation apparatus 10 are not the main point of the present invention, and can be referred to in US16538307 and chinese patent CN112430531B, and the processing and material selection of the microfluidic chip in the following description can also be referred to in the above patent, which is not described herein again.

Referring to fig. 3, embodiment 2 discloses a vitrification freezing system, which mainly includes a digital droplet generation device and a microfluidic chip, wherein the digital droplet generation device can use the reusable digital droplet generation device 10 described in embodiment 1, and 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 generation unit 122 of the digital droplet generation apparatus 10 through a hose. The air pressure source 30 external to the digital droplet generator 10 may provide negative or positive pressure to draw in or squeeze out liquid.

Further, the system may further include an air pressure source 30, the air pressure source 30 is connected to the air passage 125 in the droplet generator 12 through the connecting pipe 50 to communicate with the air-tight chamber 121, and the air-tight chamber 121 may be communicated with the atmosphere. The air pressure source 30 may be connected to a positive pressure air source or a negative pressure air source, and the connection line 50 may have a solenoid valve 40 therein, and the positive pressure or the negative pressure may be output to be applied to the connection line 50 by controlling the opening or the disconnection of the solenoid valve 40. The connecting line 50 can also be made of a biologically inert material that can be sterilized, 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 connection pipe 50 can be ensured by the interference fit at the time of connection. The solenoid valve 40 may be a high-precision millisecond-level response solenoid valve, but may be a manual valve, a solenoid valve, or other types of valves as long as the function of opening and closing the gas inlet passage is achieved.

In connection with the working state of the vitrification freezing system shown in fig. 4: the air pressure source 30 intermittently applies negative pressure through the electromagnetic valve 40, and the liquid in the microfluidic chip 20 connected to the digital droplet generation apparatus 10 can be gradually and gradually sucked into the digital droplet flow meter 12 and then into the liquid storage tank 13. After the liquid storage tank 13 is filled, the digital droplet generation device 10 is disconnected from the microfluidic chip 20 and the connecting pipeline 50, the substrate 11 is discarded, then the brand-new digital droplet generation device 10 is replaced, and the digital droplet generation device 10 is connected with the microfluidic chip 20 and the connecting pipeline 50 again through the hose. The pressure source 30 intermittently applies positive pressure through the electromagnetic valve 40, and can push the liquid in the droplet generating portion 122 into the microfluidic chip 20, so as to extrude the cells in the microfluidic chip 20, and the liquid level in the liquid storage tank 13 is kept substantially unchanged in the process.

In other embodiments, as shown in fig. 5, the reservoir may not be integrated in the substrate 11, but may be a detachable part of the digital droplet generator 10. The liquid reservoir 60 is provided with a liquid port 61 and a gas port 62, and when in use, the liquid port 61 of the liquid reservoir 60 is connected with the liquid flow channel 124 on the digital droplet generator 12 through a hose, and the gas port 62 of the liquid reservoir 60 is connected with the gas flow channel 126. With this embodiment, when the liquid in the liquid storage tank 60 is full, the liquid storage tank 60 can be directly detached and discarded, and the base 11 can be reused, that is, the liquid interface 61 of the new liquid storage tank 60 is connected to the liquid flow channel 124 by the hose, and the gas interface 62 of the new liquid storage tank 60 is connected to the gas flow channel 126. Accordingly, the vitrification freezing system disclosed in example 2 should include a liquid storage tank 60 in addition to the digital droplet generating device, the microfluidic chip and the pressure source, and the other configurations are substantially unchanged.

In conclusion, the digital liquid drop flowmeter is independent of the design of the microfluidic chip, and the digital liquid drop flowmeter and the microfluidic chip are combined in an external connection mode, so that the digital liquid drop operation can be realized, and the repeatable use of a liquid drop digital flowmeter component with high processing cost can be realized.

Finally, it should be noted that while the above describes exemplifying embodiments of the invention with reference to the accompanying drawings, the invention is not limited to the embodiments and applications described above, which are intended to be illustrative and instructive only, and not limiting. Those skilled in the art, having the benefit of this disclosure, may effect numerous modifications thereto without departing from the scope of the invention as defined by the appended claims.

Claims

1. A reusable digital droplet generator comprising a substrate and a digital droplet flow meter integrated in the substrate for droplet generation and removal; the digital liquid drop flowmeter is provided with an air-tight cavity, a liquid drop generating part and a liquid drop removing part, wherein the liquid drop generating part and the liquid drop removing part are respectively arranged at an inlet and an outlet of the air-tight cavity and are communicated with the air-tight cavity; the digital droplet flow meter is further provided with a liquid circulation channel communicating with the droplet removing part and a first gas circulation channel communicating with the first vent of the air tight chamber.

2. The digital drop generator of claim 1, wherein the digital drop flow meter is further configured with a second gas flow channel; the second gas circulation channel is communicated with the second vent of the air sealing cavity or communicated with the air sealing cavity through the first gas circulation channel.

3. The digital droplet generator of claim 2 further comprising a first reservoir; the liquid interface of the first liquid storage tank is connected with the liquid circulation channel of the digital liquid drop flow meter, and the gas interface of the first liquid storage tank is connected with the second gas circulation channel of the digital liquid drop flow meter; the gas interface of the first liquid storage tank is higher than the liquid interface.

4. A reusable digital liquid drop generating device is characterized by comprising a base body, a digital liquid drop flowmeter and a second liquid storage tank, wherein the digital liquid drop flowmeter and the second liquid storage tank are integrated in the base body; the digital droplet flow meter is used for droplet generation and removal; the digital liquid drop flowmeter is provided with an air sealing cavity, a liquid drop generating part and a liquid drop removing part, wherein the liquid drop generating part and the liquid drop 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 digital droplet flow meter is also provided with a liquid flow channel, a first gas flow channel and a second gas flow channel; two ends of the liquid circulation channel are respectively communicated with the liquid drop removing part and the liquid interface of the first liquid storage tank, and the first gas circulation channel is communicated with the first air vent of the air sealing cavity; the gas interface of the second liquid storage tank is communicated with the first gas circulation channel through a second gas circulation channel, or is communicated with the second vent of the air sealing cavity through the second gas circulation channel; and the gas interface of the second liquid storage tank is higher than the liquid interface.

5. The digital droplet generator of claim 2, wherein the second reservoir has a capacity of 1-5mL.

6. A vitrification freezing system is characterized by comprising a digital liquid drop generating device and a micro-flow chip which are connected with each other; the digital droplet generation device adopts the reusable digital droplet generation device of any one of claims 1 to 3 or the reusable digital droplet generation device of claim 4 or 5; the microfluidic chip comprises a microfluidic pipette and a cell sieve structure, and the microfluidic pipette is connected with a droplet generation part of a digital droplet flowmeter in the digital droplet generation device.

7. The vitrification freezing system of claim 6 wherein the microfluidic chip is connected to a droplet generation section of the digital droplet generation device by a hose; the hose is made of bio-inert material capable of being sterilized, and comprises any one of PE, PP, PEEK, PTFE, FEP and ETFE.

8. The vitrification freezing system of claim 6 or 7 further comprising an air pressure source for providing a positive pressure air source or a negative pressure air source; the air pressure source is connected with a first gas circulation channel of the digital liquid drop generating device through a communication pipeline; and a valve for controlling the gas circulation is arranged in the communication pipeline.

9. The vitrification freezing system of claim 8 wherein the connecting channel is made of a bio-inert material capable of being sterilized, including any one of PE, PP, PEEK, PTFE, FEP, ETFE.

10. The vitrification freezing system of claim 8 wherein the valve is a millisecond response solenoid valve.