WO2024109896A1 - Rapid temperature control device for cell culture based on microfluidic chip - Google Patents

Abstract

The present invention provides a rapid temperature control device for cell culture based on a microfluidic chip, comprising a water bath incubator, a temperature control system, and a microfluidic chip and a temperature-sensing probe that are provided in the water bath incubator. The water bath incubator is arranged on a microscope stage or a motorized displacement stage of a time-lapse microscopy imaging system, a hollow window is provided at the bottom of an incubator body of the water bath incubator, a transparent window is provided at the top of the incubator body, and the centers of the transparent window and the hollow window are both coaxial with the optical axis of an objective lens of the time-lapse microscopy imaging system; a glass sheet substrate of the microfluidic chip is in close fit with the hollow window; a thermostat of the temperature control system is connected to the temperature-sensing probe so as to monitor the ambient temperature in the water bath incubator; the thermostat is used for heating the water in a water storage tank to a target temperature and maintaining a constant temperature, and the constant-temperature water in the water storage tank enters the water bath incubator through a water inlet pipe and then flows out of the water storage tank though a water outlet pipe, so that the constant-temperature water circulates inside the water bath incubator to achieve stable temperature control.

Description

A rapid temperature control device for cell culture based on microfluidic chip Technical Field

The invention relates to the field of biotechnology, and in particular to a rapid temperature control device based on microfluidic chip cell culture.

Background technique

Microfluidic chip technology combined with time-lapse microscopy technology can achieve long-term monitoring and cultivation of single cells. When microfluidic chips are used to culture and monitor the growth of cells or microorganisms in real time under microscopic imaging, temperature conditions suitable for biological growth are usually required. Different cells or microorganisms may not necessarily have the same growth conditions, including but not limited to temperature conditions. Controlling the rapid changes in temperature in the microfluidic chip and its surrounding environment is the key to the process.

At present, there are three main ways to control the temperature of the microfluidic chip culture environment. The first is a temperature control box that can be integrated on a microscope. It controls the temperature of the microfluidic chip on the microscope stage by heating the air to form convection, which can achieve a larger temperature control range; however, the temperature distribution uniformity is poor, and the air heating volume in a large space causes the temperature to rise and fall very slowly, and it is impossible to achieve a fast and stable temperature change effect. The second is a small constant temperature incubator based on metal heating that can be placed on the microscope stage, which directly heats or cools the microfluidic chip placed therein; the small constant temperature incubator can control the overall microfluidic chip to achieve a relatively fast heating process, but because it has no heat dissipation device, it takes a long time to wait during the cooling process. In addition, the microfluidic chip is in the air environment inside the incubator, and the heating rate is still limited by the large specific heat capacity of the air. The third method is to use semiconductor cooling chips placed directly under the microfluidic chip to perform heating or cooling operations through current control. Some research institutions also directly print thermoelectric semiconductor temperature control circuits on microfluidic chips to control the temperature on the chip. Semiconductor cooling chips usually need to be placed under the microfluidic chip, but the activity space of the microscope lens will be limited, so high-throughput experimental data acquisition cannot be achieved. For designs where the semiconductor cooling chip does not directly contact the microfluidic chip, uneven temperature conduction leads to poor temperature uniformity at different locations on the microfluidic chip. For direct processing of semiconductor materials onto microfluidic chips, it is required to be a special hard insulating sheet chip substrate, which has high requirements for thermoelectric materials. High-power microscopes usually use glass with a thickness of 0.17 mm as the chip substrate. This type of semiconductor temperature control method has a limited application range. In addition, microfluidic chips using semiconductor cooling methods are usually in contact with room temperature air, so microfluidic chips cannot stably maintain the expected temperature.

technical problem

Existing microfluidic chip rapid temperature control devices compatible with time-lapse microscopy imaging systems cannot achieve rapid temperature changes and stable temperature control.

Technical Solutions

The present invention provides a rapid temperature control device based on microfluidic chip cell culture, the rapid temperature control device is compatible with a time-lapse microscopic imaging system, and comprises: a water bath incubator, which is arranged above a microscope stage or an electric displacement stage of the time-lapse microscopic imaging system; the water bath incubator comprises a box body and a water inlet and a water outlet arranged on the box body, a hollow window is arranged at the bottom of the box body, a transparent window is arranged at the top of the box body, and the centers of the hollow window and the transparent window are both coaxial with the objective lens optical axis of the time-lapse microscopic imaging system; a microfluidic chip is arranged on the hollow window of the water bath incubator, and a glass sheet substrate of the microfluidic chip is connected to the hollow window. The windows are tightly fitted; a temperature sensing probe is arranged in the water bath incubator and close to the microfluidic chip or on the microfluidic chip; a temperature control system includes a water reservoir, a thermostat, a water inlet pipe connected to the water inlet, and a water outlet pipe connected to the water outlet; wherein the thermostat is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to make the water in the water reservoir reach the target temperature and maintain a constant temperature, the constant temperature water in the water reservoir enters the water bath incubator through the water inlet pipe and flows out of the water reservoir through the water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control.

Optionally, the box body includes a base and a top cover arranged on the base, the hollow window is arranged on the base, and the transparent window is arranged on the top cover.

Optionally, the hollow window is designed to allow a high-power objective lens to image the maximum area of the microfluidic chip.

Optionally, the rapid temperature control device further comprises a chip pressing sheet disposed in the water bath incubator and detachably connected to the base, wherein the chip pressing sheet comprises a spring pin or a rubber ring for pressing the microfluidic chip.

Optionally, the chip pressing piece is connected to the base via screws.

Optionally, a waterproof rubber ring is provided on the peripheral side of the chip pressing sheet, a rubber ring groove is opened on the peripheral side of the upper surface of the base, and a waterproof rubber ring matching with the rubber ring groove is provided on the lower surface of the top cover.

Optionally, the base and the top cover are connected by screws.

Optionally, the box body is also provided with a chip pipeline inlet and a chip pipeline outlet.

Optionally, the chip pipeline inlet and the chip pipeline outlet are arranged on the top cover.

Optionally, the temperature control system further comprises a peristaltic pump, and the water inlet pipe is connected to the peristaltic pump so as to facilitate the transmission of the constant temperature water in the water reservoir to the interior of the water bath incubator.

Beneficial Effects

The beneficial effects of the present invention are:

The present invention provides a rapid temperature control device for cell culture based on a microfluidic chip, which is compatible with a time-lapse microscopy imaging system, and includes a water bath incubator, a temperature control system, and a microfluidic chip and a temperature sensing probe arranged in the water bath incubator. The water bath incubator is arranged above a microscope stage or an electric displacement stage of the time-lapse microscopy imaging system, a hollow window is arranged at the bottom of the water bath incubator, and a transparent window is arranged at the top of the water bath incubator, and the centers of the transparent window and the hollow window are coaxial with the optical axis of the objective lens of the time-lapse microscopy imaging system; the microfluidic chip is arranged on the hollow window, and its glass sheet base is closely matched with the hollow window; the thermostat of the temperature control system is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to heat the water in the water reservoir to reach the target temperature and maintain a constant temperature, and the constant temperature water in the water reservoir enters the water bath incubator through a water inlet pipe and flows out of the water reservoir through a water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control. The cell culture fluid enters the microfluidic chip through the chip pipeline inlet and flows out through the chip pipeline outlet to provide the nutritional environment required for continuous cell culture.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a schematic structural diagram of an embodiment of a rapid temperature control device for cell culture based on a microfluidic chip provided in the present application.

FIG. 2 is a schematic diagram of the exploded structure of the water bath incubator in the embodiment provided in FIG. 1 .

The following are the descriptions of the reference numerals:

10. Water bath incubator; 11. Base; 111. Hollow window; 112. Rubber ring groove; 113. Threaded hole; 12. Temperature sensor; 13. Chip pressing sheet; 131. Spring pin; 14. Top cover; 141. Transparent window; 142. Transparent plate; 143. Water-proof rubber ring; 144. Water inlet; 145. Water outlet; 146. Chip pipeline outlet; 147. Chip pipeline inlet; 20. Temperature control system; 21. Water reservoir; 22. Thermostat; 23. Peristaltic pump; 24. First pipeline; 25. Second pipeline; 26. Water inlet pipeline; 27. Water outlet pipeline; A. Microscope stage; B. Objective lens; C. Culture medium tube; D. Waste liquid tube.

Embodiments of the present invention

Typical embodiments that embody the features and advantages of the present invention will be described in detail in the following description. It should be understood that the present invention can have various changes in different embodiments without departing from the scope of the present invention, and the descriptions and illustrations therein are essentially used for illustration purposes rather than for limiting the present invention.

In the description of the present application, it should be understood that the terms "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation on the present application. In addition, the terms "first" and "second" are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, the features defined as "first" and "second" may explicitly or implicitly include one or more of the features. In the description of the present application, the meaning of "multiple" is two or more, unless otherwise clearly and specifically defined.

In order to further illustrate the principle and structure of the present invention, preferred embodiments of the present invention are now described in detail with reference to the accompanying drawings.

Please refer to Figures 1 and 2. The present application provides a rapid temperature control device for cell culture based on a microfluidic chip. The rapid temperature control device is compatible with a time-lapse microscopy imaging system. It includes a water bath incubator 10, a temperature control system 20, and a microfluidic chip and a temperature sensing probe 12 arranged in the water bath incubator. The temperature sensing probe 12 is placed near or on the microfluidic chip for real-time temperature monitoring.

Please refer to Figure 2. The water bath incubator 10 is arranged above the microscope stage A or the electric translation stage of the time-lapse microscopy imaging system. It includes a box body and a water inlet 144 and a water outlet 145 arranged on the box body. A hollow window 111 is arranged at the bottom of the box body, and a transparent window 141 is arranged at the top of the box body. The centers of the transparent window 141 and the hollow window 111 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system. The lighting requirements are achieved by opening windows at the top and bottom of the box body.

Specifically, in this embodiment, the box body includes a base 11 and a top cover 14 disposed on the base 11 , and the base 11 and the top cover 14 form a containing chamber.

The base 11 is a groove structure and is compatible with the microscope stage A or the electric translation stage for time-lapse microscopic imaging experiments and applications. The base 11 with a groove structure can be filled with an aqueous solution to immerse the microfluidic chip disposed inside the water bath incubator 10. A hollow window 111 with a groove structure for supporting the microfluidic chip is provided in the middle of the base 11, and the glass sheet substrate of the microfluidic chip is tightly matched with the hollow window 111. Preferably, the design of the hollow window 111 satisfies the maximum area imaging of the microfluidic chip by a high-power objective lens. It can be understood that the size and shape of the base 11 can be adjusted and designed according to the application platform to match with different types and models of stages or electric translation stages.

Furthermore, in the present embodiment, a chip pressing plate 13 is also included, which is arranged in the water bath incubator 10 and is detachably connected to the base 11. The chip pressing plate 13 is connected to the base 11 by screws, and a spring pin 131 for pressing the glass substrate of the microfluidic chip is provided on the chip pressing plate 13. The glass substrate of the microfluidic chip is adapted to the hollow window 111. The spring pins 131 on the chip pressing plate 13 are evenly distributed directly above the hollow window 111, that is, directly above the glass substrate of the microfluidic chip. Through the squeezing of the spring pins 131, the glass substrate of the microfluidic chip is tightly fixed to the base 11. It can be understood that since the chip pressing plate 131 and the base 11 are detachably connected, microfluidic chips of different types and shapes can be easily replaced. On the premise of satisfying the glass substrate of the microfluidic chip, another structure that can replace the spring pin 131 is a rubber ring structure.

The top cover 14 is a convex groove structure corresponding to the base 11, and a transparent window 141 is provided in the middle thereof, and a transparent plate 142 is fixed in the transparent window 141. The centers of the transparent window 141 of the top cover 14 and the hollow window 111 of the base 11 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system. When the rapid temperature control device is integrated into the time-lapse microscopy imaging system, the transparent window 141 can project incident light onto the microfluidic chip in the water bath incubator 10. The transparent window 141 is large enough to avoid blocking the incident light. Since an oil immersion objective lens is required for high-magnification imaging, the distance between the objective lens B and the sample is only a few hundred microns, and the objective lens oil is filled in the middle. The objective lens B needs sufficient space to ensure a large range of sampling. The design of the hollow window 111 retains the imaging space of the microfluidic chip to the maximum extent.

A water inlet 144, a water outlet 145, a chip pipeline inlet 147 and a chip pipeline outlet 146 are provided on one side of the top cover 14. The first pipeline 24 connected to the culture liquid tube C enters the water bath incubator 10 from the chip pipeline inlet 147 of the top cover 14 and is connected to the microfluidic chip. The microfluidic core is connected to the second pipeline 25. The second pipeline 25 passes through the water bath incubator 10 from the chip pipeline outlet 146 of the top cover 14 and is connected to the waste liquid pipe D, so as to provide the nutritional environment required for continuous cell culture. It can be understood that the water inlet 144, the water outlet 145, the chip pipeline inlet 146 and the chip pipeline outlet 147 can also be set at other positions of the top cover 14 according to actual needs.

Preferably, the first pipeline 24 and the second pipeline 25 can be placed in a non-window space position in the water bath incubator 10 as required, and the outer sides of the chip pipeline inlet 147 and the chip pipeline outlet 146 are sealed and fixed to the top cover 14 with pipeline blade rings and split joints.

The top cover 14 and the base 11 are provided with corresponding threaded holes 113 around them, so that the two can be connected by screws. Furthermore, a rubber ring groove 112 is provided on the upper surface of the base 11, and a waterproof rubber ring 143 that cooperates with the rubber ring groove 112 is provided on the lower surface of the top cover 14. The waterproof rubber ring 143 is squeezed by screwing the base 11 and the top cover 14 to waterproof the connection between the two. The base 11 and the top cover 14 are combined into a water bath incubator 10 with a water storage function, which can quickly control the temperature of the microfluidic chip immersed therein.

Please refer to FIG. 1 again. The temperature control system 20 includes a water reservoir 21, a thermostat 22, a peristaltic pump 23, a water inlet pipe 26 connected to the water inlet 144, and a water outlet pipe 27 connected to the water outlet 145. The water inlet pipe 26 and the water outlet pipe 27 are placed together in the water reservoir 21. The water inlet pipe 26 is connected to the peristaltic pump 23 so that the constant temperature water in the water reservoir 21 is transferred to the water bath incubator 10 so that the water fills the inside of the water bath incubator 10. The thermostat 22 is used to make the water in the water reservoir 21 reach the target temperature and maintain the constant temperature. The constant temperature water enters the water bath incubator 10 through the water inlet pipe 26 and flows out of the water reservoir 21 through the water outlet pipe 27, thereby realizing the circulation of the constant temperature water in the water bath incubator 10 to stabilize the temperature control; the thermostat 22 is connected to the temperature sensing probe 12 to monitor the ambient temperature in the water bath incubator 10. If the ambient temperature of the microfluidic chip needs to be changed quickly, the target temperature of the thermostat 22 can be adjusted and set to quickly replace the water in the water reservoir 21 with water of the target temperature for circulation, so that the monitoring temperature of the microfluidic chip reaches the target temperature. The temperature disturbance of the microfluidic chip in the water bath environment is extremely small, the temperature switching is accurate and stable, and rapid temperature change can be achieved. The temperature switching is completed within 3 minutes and maintained stable, and the temperature range can reach 50°C.

It is understandable that the number and position of the chip pipeline inlet 147, the chip pipeline outlet 146, the water inlet 144 and the water outlet 145 of the water bath incubator 10 can be arbitrarily designed and changed under the premise of satisfying rapid temperature change.

The present application provides a rapid temperature control device for cell culture based on a microfluidic chip. The rapid temperature control device is compatible with a time-lapse microscopy imaging system and includes a water bath incubator 10, a temperature control system 20, and a microfluidic chip and a temperature sensing probe 12 arranged in the water bath incubator 10. Among them, the water bath incubator 10 is arranged above the microscope stage A or the electric translation stage of the time-lapse microscopy imaging system, a hollow window 111 is arranged at the bottom of the water bath incubator 10, and a transparent window 141 is arranged at the top of the water bath incubator 10, and the centers of the transparent window 141 and the hollow window 111 are coaxial with the optical axis of the objective lens B of the time-lapse microscopy imaging system; the microfluidic chip is arranged on the hollow window 111, and its glass sheet base is tightly matched with the hollow window 111; the thermostat 22 of the temperature control system 20 is connected to the temperature sensing probe 12 to monitor the ambient temperature in the water bath incubator 10; the thermostat 22 is used to make the water in the water reservoir 21 reach the target temperature and maintain a constant temperature, the constant temperature water in the water reservoir 21 enters the water bath incubator 10 through the water inlet pipe 26 and flows out of the water reservoir 21 through the water outlet pipe 27, thereby realizing the circulation of constant temperature water in the water bath incubator 10 to stabilize the temperature control. This rapid temperature control device has been realized and its feasibility and effectiveness have been verified by long-term experiments. The rapid temperature control device was used to culture Escherichia coli cells at 37°C for 6 hours and then switched to 10°C within 3 minutes to obtain time-lapse microscopy experimental results. The experimental data obtained can be analyzed and counted using image analysis software.

Although the present invention has been described with reference to several typical embodiments, it should be understood that the terms used are illustrative and exemplary, rather than restrictive. Since the present invention can be embodied in a variety of forms without departing from the spirit or essence of the invention, it should be understood that the above embodiments are not limited to any of the foregoing details, but should be interpreted broadly within the spirit and scope defined by the appended claims, so all changes and modifications falling within the scope of the claims or their equivalents should be covered by the appended claims.

Claims (10)

1. A rapid temperature control device for cell culture based on a microfluidic chip, wherein the rapid temperature control device is compatible with a time-lapse microscopic imaging system, and is characterized in that it comprises:

   A water bath incubator, which is arranged above a microscope stage or an electric translation stage of a time-lapse microscopy imaging system; the water bath incubator comprises a box body and a water inlet and a water outlet arranged on the box body, a hollow window is arranged at the bottom of the box body, and a transparent window is arranged at the top of the box body, and the centers of the hollow window and the transparent window are both coaxial with the objective lens optical axis of the time-lapse microscopy imaging system;

   A microfluidic chip, which is arranged on the hollow window of the water bath incubator, and the glass substrate of the microfluidic chip is closely matched with the hollow window;

   A temperature sensing probe is arranged in the water bath incubator and close to the microfluidic chip or arranged on the microfluidic chip;

   A temperature control system, comprising a water reservoir, a thermostat, a water inlet pipe connected to the water inlet, and a water outlet pipe connected to the water outlet;

   Among them, the thermostat is connected to the temperature sensing probe to monitor the ambient temperature in the water bath incubator; the thermostat is used to make the water in the water reservoir reach the target temperature and maintain a constant temperature, and the constant temperature water in the water reservoir enters the water bath incubator through the water inlet pipe and flows out of the water reservoir through the water outlet pipe, thereby realizing the circulation of constant temperature water in the water bath incubator to stabilize the temperature control.
2. The rapid temperature control device according to claim 1 is characterized in that the box body includes a base and a top cover arranged on the base, the hollow window is arranged on the base, and the transparent window is arranged on the top cover.
3. The rapid temperature control device according to claim 2 is characterized in that the design of the hollow window satisfies the maximum area imaging of the microfluidic chip by a high-power objective lens.
4. The rapid temperature control device according to claim 2 is characterized in that it also includes a chip pressing sheet disposed in the water bath incubator and detachably connected to the base, the chip pressing sheet including a spring pin or a rubber ring for pressing the microfluidic chip.
5. The rapid temperature control device according to claim 4 is characterized in that the chip pressing sheet is connected to the base by screws.
6. The rapid temperature control device according to claim 2 is characterized in that a waterproof rubber ring is provided on the peripheral side of the chip pressing sheet, a rubber ring groove is opened on the peripheral side of the upper surface of the base, and a waterproof rubber ring matching the rubber ring groove is provided on the lower surface of the top cover.
7. The rapid temperature control device according to claim 6 is characterized in that the base and the top cover are connected by screws.
8. The rapid temperature control device according to claim 1 is characterized in that a chip pipeline inlet and a chip pipeline outlet are also provided on the box body.
9. The rapid temperature control device according to claim 8 is characterized in that the chip pipeline inlet and the chip pipeline outlet are arranged on the top cover.
10. The rapid temperature control device according to claim 1 is characterized in that the temperature control system also includes a peristaltic pump, and the water inlet pipe is connected to the peristaltic pump to facilitate the transmission of the constant temperature water in the water reservoir to the interior of the water bath incubator.