Cell temperature detection culture cavity and cell temperature measurement method

Abstract

The invention discloses a cell temperature detection culture chamber and a cell temperature measuring method.The cell temperature detection culture chamber comprises a carrier plate and a temperature sensor, the carrier plate is provided with a culture chamber, the temperature sensor is located on one side of the carrier plate, and the front end of the temperature sensor is inserted into the culture chamber; the temperature sensor is provided with at least two detection surfaces which are oppositely arranged, at least one detection surface faces the inlet of the culture chamber, and at least one detection surface faces the bottom of the culture chamber. Cells cannot be deposited on the detection surface facing the bottom of the culture cavity, and the solution temperature is obtained as a reference signal, so that two signals are accurately obtained.

Description

Technical Field

[0001] The present invention relates to a cell detection device, in particular to a cell temperature detection culture chamber, and also to a cell temperature measurement method. Background Art

[0002] There are many deficiencies in the detection of cell energy metabolism in cell culture chambers: existing technologies usually use destructive detection methods, such as destroying cells to extract components for detection. This is not only complicated to operate, but also cannot achieve real-time monitoring, making it difficult to accurately reflect the energy metabolism of cells under physiological conditions.

[0003] CN 116814403 A discloses a device and method for synchronously detecting the temperature and optical information of living cells. The device includes a cell temperature detection plate, which is mainly composed of a PCB adapter plate, a cell culture chamber, and a cable socket. The cell culture chamber and the cable socket are both located on the PCB adapter plate. A temperature sensor chip is provided inside the cell culture chamber. The temperature sensor chip includes an ambient temperature sensor and a cell temperature sensor. The cell temperature sensor is made of a transparent substrate material to facilitate the acquisition of cell optical information in conjunction with a microscope. During the cell culture process, the temperature variation is small, and two sensors are required to work simultaneously, one to provide an environmental reference signal and the other to provide a target signal (Zhang Fangzhou. Design of a multi-cell temperature sensor based on platinum resistance and development of microfluidic chips [D]. Southeast University, 2023.). In the solution of CN 116814403A, the ambient temperature sensor and the cell temperature sensor are arranged simultaneously in the cell culture chamber. Living cells cannot choose a growth site. Living cells may grow on the surfaces of both sensors at the same time. The measurement signals of the two sensors cannot be distinguished, and the measurement accuracy is low. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a cell temperature detection culture chamber to solve the problem that the existing cell culture chamber cannot accurately distinguish between the reference signal and the target signal. Another purpose of the present invention is to provide a method for measuring cell temperature.

[0005] Technical solution: The cell temperature detection culture chamber described in the present invention includes a carrier plate and a temperature sensor. A culture chamber is provided on the carrier plate. The temperature sensor is located on one side of the carrier plate, and its front end is inserted into the culture chamber. The temperature sensor has at least two oppositely arranged detection surfaces, at least one detection surface faces the entrance of the culture chamber, and at least one detection surface faces the bottom of the culture chamber.

[0006] Preferably, in order to facilitate obtaining optical information, the bottom of the culture chamber is transparent, and the temperature sensor has a transparent substrate.

[0007] Preferably, the gap between the carrier plate and the temperature sensor is filled with silicone sealant.

[0008] Preferably, the distance between the detection surface of the temperature sensor facing the bottom of the culture chamber and the bottom surface of the carrier plate is 2.4-2.6 mm.

[0009] Preferably, the carrier plate is provided with a through hole and annular blind holes surrounding the through hole, and the open end of the annular blind hole and the end of the through hole close to the opening of the annular blind hole are respectively sealed with transparent materials, so that the culture chamber formed by the through hole forms an air layer outside the culture chamber.

[0010] Preferably, the height of the transparent material connected to the through hole is greater than the height of the transparent material connected to the annular blind hole, and the transparent material is a cell climbing sheet.

[0011] Preferably, the temperature sensor is a platinum thermal resistance sensor, the surface layer of the temperature sensor is silicon nitride, and the detection surface of the temperature sensor facing the entrance of the culture chamber is further coated with a rat tail collagen layer.

[0012] Preferably, the tail end of the temperature sensor is connected to a flexible flat cable for connecting to a cell temperature detection system.

[0013] Preferably, the inner diameter of the through hole is ≥6 mm, and the total volume is ≥370 μL.

[0014] Preferably, the inner ring diameter of the annular blind hole is ≥8 mm, and the outer ring diameter is ≤18 mm.

[0015] The method for measuring cell temperature using the aforementioned cell temperature detection culture chamber comprises the following steps:

[0016] (1) Add the cell suspension to the culture chamber, cover the detection surface of the temperature sensor facing the opening of the culture chamber, move the cell temperature detection culture chamber to the incubator, and culture;

[0017] (2) Remove the cell temperature detection culture chamber, remove the cells at the bottom of the culture chamber by enzymatic digestion, re-add the culture medium, and observe under a microscope. After the cells cover the detection surface of the temperature sensor facing the entrance of the culture chamber, obtain the signals from the two detection surfaces of the temperature sensor respectively, calculate the difference, and obtain the cell temperature.

[0018] Beneficial effects: Compared with the existing technology, the present invention has the following significant advantages: 1. Accurate acquisition of reference signal and target signal: Using gravity, cells are deposited on the detection surface facing the opening of the culture chamber to obtain the target signal. Cells cannot be deposited on the detection surface facing the bottom of the culture chamber, and the solution temperature is obtained as the reference signal, thereby accurately obtaining both signals; 2. Improved detection accuracy: An air layer is formed outside the culture chamber to reduce the impact of temperature fluctuations inside the incubator and the impact of ambient temperature fluctuations when the culture chamber enters and exits the incubator, thereby obtaining more stable reference signal and target signal, reducing noise and improving resolution. The resolution of this device is 0.01°C, the distance between the two detection surfaces is small, and the environmental impact is small, resulting in more accurate temperature detection and stable detection of temperature differences of 0.1°C; 3. Greatly simplified sampling method, no professional operation is required, conventional cell culture methods can be used to detect cell temperature, which is easy to operate, avoids damage to cells, and ensures the physiological state of cells; 4. Simplified culture chamber structure and easy to process; 5. Real-time monitoring: Real-time monitoring of cell energy metabolism is achieved through MEMS temperature sensors, providing continuous and dynamic data. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a side view of the overall structure of the cell temperature detection culture chamber of the present invention;

[0020] Figure 2 This is a side sectional view of the cell temperature detection culture chamber of the present invention. DETAILED DESCRIPTION

[0021] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0022] Example: The cell temperature detection culture chamber is mainly composed of an RTD (RTD, resistance temperature detector, hereinafter referred to as temperature sensor) and a carrier board 1. A culture chamber is provided on the carrier board 1. The bottom of the culture chamber is transparent so that light can enter the interior of the culture chamber. The temperature sensor is inserted from the side wall of the carrier board 1, and the front end extends into the culture chamber. The temperature sensor has a transparent base. The temperature sensor has at least two oppositely arranged detection surfaces, one detection surface facing the entrance of the culture chamber, and the other detection surface facing the bottom of the culture chamber. The detection surface facing the opening of the culture chamber is used to culture adherent cells and test cell temperature. The detection surface facing the bottom of the culture chamber is a certain distance away from the bottom of the culture chamber and is used to detect the ambient temperature of the solution. With the help of cell gravity, cells are prevented from growing on its detection surface. The connection between the carrier board 1 and the double-sided sensor is filled with silicone sealant and left to stand for 24 hours for the glue to dry thoroughly before use. The tail of the double-sided sensor is connected to the cell temperature detection system through a flexible circuit cable (FPC) of customized size.

[0023] The basic sensor of the temperature sensor is a platinum thermal resistor sensor. The manufacturing method is similar to the solution disclosed in CN 115560873 A. Specifically, a 0.5mm thick glass substrate is used, and a polyimide film is sequentially provided on the surface as a thermal barrier layer, a Cr layer to improve adhesion performance, and a Pt resistor layer as a detection layer. The Pt resistor layer is encapsulated with silicon nitride (Si3N4). The silicon nitride forms a transparent surface and ensures the heat radiation rate to the Pt resistor layer. Figure 2 Taking the most basic temperature sensor as an example, the sensing surface of base sensor 2 is further soaked in a rat tail collagen solution to form a rat tail collagen surface layer to facilitate cell attachment. The sensing surface of base sensor 3 remains untreated. The glass substrates of base sensors 2 and 3 are bonded back-to-back (with their sensing surfaces facing outward) to form the temperature sensor of this embodiment. The sensing surface of base sensor 2 faces the culture chamber entrance, while that of base sensor 3 faces the bottom of the culture chamber. Base sensor 2 detects cell temperature, while base sensor 3 detects ambient temperature. The difference between the two is used to obtain a cell temperature change signal. The substrate thickness can be further reduced, down to the nanometer level, to ensure insulation between base sensors 2 and 3.

[0024] The temperature sensor's detection surface, located near the bottom of the culture chamber, is positioned a certain distance from the chamber bottom to match the working distance of the microscope objective lens. This ensures imaging quality when using an inverted microscope and prevents background interference when the objective lens is too close. For example, a 20x objective lens has a working distance of 4.5 mm, and the distance between the temperature sensor and the bottom of carrier plate 1 is 2.4 to 2.6 mm.

[0025] The culture chamber has an internal diameter of approximately 6 mm and a total volume of approximately 370 μL. The chamber size can be further proportionally enlarged or reduced by a certain factor, for example, 0.9 to 1.5 times, to accommodate the temperature sensor and conserve the sample volume. Further reduction in the chamber size would be detrimental to sensor assembly and cell culture operations. The inner diameter of the annular blind hole 4 is ≥8 mm, and the outer diameter is ≤18 mm.

[0026] An air layer surrounding the culture chamber is provided outside the culture chamber. The specific operation is as follows: an opening is formed at the bottom of a rectangular or cylindrical carrier plate 1 to form a through hole passing through the carrier plate 1 and an annular blind hole 4 near the top of the carrier plate 1. One end of the through hole near the opening of the annular blind hole 4 and the open end of the annular blind hole 4 are respectively sealed with transparent materials 5 and 6. The through hole with one end closed serves as the culture chamber, and the annular blind hole 4 with the opening closed forms an air layer around the culture chamber.

[0027] During the cell temperature measurement process, the operator will open the incubator door to add culture medium or stimulate drugs, etc. The opening of the door will cause the temperature fluctuation inside the incubator (the culture chamber without air sandwich structure, the volume remains the same, and the temperature change of the culture chamber is about 0.5K during the opening and closing of the incubator). The linearity of the base sensors 2, 3 is slightly different, and the temperature fluctuation has an impact on the measurement accuracy. The air sandwich is used to reduce the temperature fluctuation of the culture chamber (the air sandwich structure is set, the culture chamber is detected synchronously, and the maximum temperature change is 0.3K), and the background noise is reduced. The height of the transparent material 5 is greater than the height of the transparent material 6, and the transparent materials 5, 6 are cell climbing sheets.

[0028] The method for measuring the cell temperature of the cell temperature detection culture chamber by using the cell temperature is as follows:

[0029] The cell temperature detection culture chamber is sterilized and ready for use. The adherent cultured cells (A549) are taken out of the incubator, the culture medium is removed, and the cells are washed once with PBS. An appropriate amount of 0.25% trypsin-EDTA is added to the culture chamber to digest the cells for 2min. The cells are centrifuged, and the cell pellet is resuspended in fresh culture medium. 200μL of cell suspension with a concentration of 10000 cells / mL is added to the culture chamber, and the liquid completely covers the surface of the temperature sensor. The cell temperature detection culture chamber is transferred to the incubator for continuous culture for 24h.

[0030] The cell temperature detection culture chamber is taken out, washed once with PBS, and a small amount of trypsin is added to cover the cells at the bottom of the culture chamber for digestion and PBS washing. The bottom cells are removed, and 300μL of fresh culture medium is added for cell culture. Through microscopic observation, the temperature measurement is performed when the cells just cover the detection surface of the temperature sensor facing the entrance of the culture chamber. The detection surface signals of the base sensor 2 and the base sensor 3 are obtained respectively, and the difference between the two is calculated, which is the cell temperature. Through this method, the difference between the cell temperature and the environmental temperature is 0.1℃.

[0031] The cell temperature detection culture chamber can be used to obtain the temperature of adherent cells, including but not limited to A549, HMEC-1, C3H10T, 1 / 2 clone8, and Hela cells.

Claims

1. A cell temperature detection culture chamber, comprising a carrier plate (1) and a temperature sensor, characterized in that: A culture chamber is provided on the carrier plate, and the temperature sensor is located on one side of the carrier plate, with its front end extending into the culture chamber and a certain distance from the bottom of the culture chamber. The temperature sensor has at least two oppositely arranged detection surfaces, at least one detection surface facing the entrance of the culture chamber, and at least one detection surface facing the bottom of the culture chamber.

2. The cell temperature detection culture chamber according to claim 1, characterized in that: The bottom of the culture chamber is transparent, and the temperature sensor has a transparent base.

3. The cell temperature detection culture chamber according to claim 1, characterized in that: The gap between the carrier plate (1) and the temperature sensor is filled with organic silicon sealant.

4. The cell temperature detection culture chamber according to claim 1, characterized in that: The distance between the detection surface of the temperature sensor facing the bottom of the culture chamber and the bottom surface of the carrier plate (1) is 2.4 to 2.6 mm.

5. The cell temperature detection culture chamber according to claim 1, characterized in that: The carrier plate (1) is provided with a through hole and an annular blind hole (4) surrounding the through hole (1). The open end of the annular blind hole (4) and the end of the through hole close to the opening of the annular blind hole are respectively sealed with transparent materials, so that an air layer is formed outside the culture chamber formed by the through hole.

6. The cell temperature detection culture chamber according to claim 5, characterized in that: The height of the transparent material connected to the through hole is greater than the height of the transparent material connected to the annular blind hole (4), and the transparent material is a cell climbing sheet.

7. The cell temperature detection culture chamber according to any one of claims 1 to 6, characterized in that: The temperature sensor is a platinum thermal resistance sensor, the surface layer of the temperature sensor is silicon nitride, and the detection surface of the temperature sensor facing the entrance of the culture chamber is further coated with a rat tail collagen layer.

8. The cell temperature detection culture chamber according to any one of claims 1 to 5, characterized in that: The tail end of the temperature sensor is connected to a flexible flat cable for connecting to a cell temperature detection system.

9. The cell temperature detection culture chamber according to claim 2, characterized in that: The inner diameter of the through hole is ≥6 mm, the total volume is ≥370 μL, the inner ring diameter of the annular blind hole (4) is ≥8 mm, and the outer ring diameter is ≤18 mm.

10. A method for measuring cell temperature using the cell temperature detection culture chamber according to claim 1, characterized in that: The following steps are involved: (1) Add the cell suspension to the culture chamber, cover the detection surface of the temperature sensor facing the opening of the culture chamber, move the cell temperature detection culture chamber to the incubator, and culture; (2) Remove the cell temperature detection culture chamber, remove the cells at the bottom of the culture chamber by enzymatic digestion, re-add the culture medium, and observe under a microscope. After the cells cover the detection surface of the temperature sensor facing the entrance of the culture chamber, obtain the signals from the two detection surfaces of the temperature sensor respectively, calculate the difference, and obtain the cell temperature.

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