Cell temperature detection culture cavity

Abstract

This invention discloses a cell temperature detection culture chamber, which includes a carrier plate and a temperature sensor. The culture chamber is disposed on the carrier plate, and the temperature sensor is located on one side of the carrier plate with its front end inserted into the culture chamber. The temperature sensor has at least two opposing detection surfaces, at least one of which faces the inlet of the culture chamber and at least one of which faces the bottom of the culture chamber. In this invention, cells are deposited on the detection surface facing the opening of the culture chamber by utilizing gravity to obtain the target signal, while cells cannot be deposited on the detection surface facing the bottom of the culture chamber, and the solution temperature is obtained as a reference signal. Thus, both signals are accurately obtained.

Description

Technical Field

[0001] This utility model relates to a cell detection device, and more particularly to a cell temperature detection culture chamber. Background Technology

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

[0003] CN 116814403 A discloses a device and method for simultaneously detecting the temperature and optical information of live cells. The device includes a cell temperature detection plate, which mainly consists of a PCB adapter board, a cell culture chamber, and a ribbon cable socket. The cell culture chamber and the ribbon cable socket are located on the PCB adapter board. A temperature sensing chip is installed inside the cell culture chamber. The temperature sensing 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 cell culture, the temperature change is relatively small, requiring two sensors to work simultaneously: one providing an environmental reference signal and the other providing a target signal (Zhang Fangzhou. Design of Multi-Cell Temperature Sensor Based on Platinum Resistance and Development of Microfluidic Chip [D]. Southeast University, 2023). The scheme in CN 116814403 A places the ambient temperature sensor and the cell temperature sensor simultaneously inside the cell culture chamber. Live cells cannot choose their growth sites, and they may grow on the surfaces of both sensors simultaneously. The measurement signals from the two sensors cannot be distinguished, resulting in low measurement accuracy. Utility Model Content

[0004] Purpose of the utility model: The purpose of this utility model is to provide a cell temperature detection culture chamber to solve the problem that existing cell culture chambers cannot accurately distinguish between reference signals and target signals.

[0005] Technical solution: The present invention provides a cell temperature detection culture chamber, comprising a carrier plate and a temperature sensor. The carrier plate is provided with a culture chamber, and the temperature sensor is located on one side of the carrier plate with its front end inserted into the culture chamber. The temperature sensor has at least two oppositely arranged detection surfaces, at least one detection surface facing the inlet of the culture chamber and at least one detection surface facing the bottom of the culture chamber.

[0006] Preferably, the bottom of the culture chamber is transparent to facilitate the acquisition of optical information, 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 2.4~2.6mm.

[0009] Preferably, the carrier plate has through holes and annular blind holes surrounding the through holes. The opening end of the annular blind holes and the end of the through holes near the opening of the annular blind holes are respectively sealed with transparent material, so that an air gap is formed outside the culture chamber formed by the through holes.

[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 spreader.

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

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

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

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

[0015] Beneficial Effects: Compared with the prior art, this utility model has the following significant advantages: 1. Accurate acquisition of reference and target signals: Utilizing gravity, cells are deposited on the detection surface facing the opening of the culture chamber to acquire the target signal, while cells cannot be deposited on the detection surface facing the bottom of the culture chamber, and the solution temperature is acquired as the reference signal, thus accurately obtaining both signals; 2. Improved detection accuracy: An air gap is formed outside the culture chamber to mitigate the impact of temperature fluctuations inside the incubator and the influence of ambient temperature fluctuations when entering and leaving the culture chamber, resulting in more stable reference and target signals, reduced noise, and improved resolution. The resolution of this device is 0.01℃. The small distance between the two detection surfaces reduces environmental influence, making temperature detection more accurate and stable, capable of detecting temperature differences as small as 0.1℃; 3. Greatly simplified sample introduction method, requiring no professional personnel to operate. Conventional cell culture methods can be used to detect cell temperature, making it easy to operate, avoiding cell damage, and ensuring the physiological state of the cells; 4. Simplified culture chamber structure, easy to manufacture; 5. Real-time monitoring: Real-time monitoring of cell energy metabolism is achieved through a MEMS temperature sensor, providing continuous and dynamic data. Attached Figure Description

[0016] Figure 1 This is a side view of the overall structure of the cell temperature detection culture chamber of this utility model;

[0017] Figure 2 This is a side cross-sectional view of the cell temperature detection culture chamber of this invention. Detailed Implementation

[0018] The technical solution of this utility model will be further described below with reference to the accompanying drawings.

[0019] Example: The cell temperature detection culture chamber mainly consists of an RTD (Resistive Temperature Detector, hereinafter referred to as the temperature sensor) and a carrier plate 1. The carrier plate 1 has a culture chamber with a transparent bottom, allowing light to enter. The temperature sensor is inserted through the side wall of the carrier plate 1, with its front end extending into the culture chamber. The temperature sensor has a transparent substrate and at least two opposing detection surfaces. One detection surface faces the inlet of the culture chamber, and the other faces the bottom. The detection surface facing the inlet is used to culture adherent cells and measure cell temperature. The detection surface facing the bottom is a certain distance from the bottom of the culture chamber and is used to detect the ambient temperature of the solution. Cell growth on this detection surface is prevented by gravity. The connection between the carrier plate 1 and the double-sided temperature sensor is filled with silicone sealant and allowed to dry completely for 24 hours before use. The tail of the double-sided temperature sensor is connected to the cell temperature detection system via a custom-sized flexible printed circuit (FPC).

[0020] The basic sensor used to form the temperature sensor is a platinum resistance thermometer (PTT). The fabrication method of the PTT is similar to the scheme disclosed in CN 115560873 A, specifically: using a 0.5mm thick glass substrate, a polyimide film is sequentially deposited on the surface as a thermal barrier layer, a Cr layer to improve adhesion, and a Pt resistance layer as a detection layer. The Pt resistance layer is then encapsulated with silicon nitride (Si3N4), which forms a transparent surface and ensures the rate at which heat radiates to the Pt resistance layer. Figure 2 The detection surface of base sensor 2 is further soaked in rat tail collagen solution to form a rat tail collagen surface layer, which facilitates cell adhesion. The detection surface of base sensor 3 does not require treatment. The glass substrates of base sensors 2 and 3 are bonded back-to-back (with the detection surfaces of the two base sensors facing outwards) to form the temperature sensor of this embodiment. The temperature sensor is inserted from the side wall of the carrier plate 1, with its front end extending into the culture chamber, so that the detection surface of base sensor 2 faces the entrance of the culture chamber and base sensor 3 faces the bottom of the culture chamber. Base sensor 2 detects cell temperature, and base sensor 3 detects ambient temperature. The difference between the two is used to obtain the cell temperature change signal. The substrate thickness can be further reduced to the nanometer level, as long as base sensors 2 and 3 are kept insulated.

[0021] The temperature sensor's detection surface near the bottom of the culture chamber is a certain distance from the bottom of the chamber to match the working distance of the microscope objective, ensuring the imaging quality of the inverted microscope and avoiding background interference when the distance limit is too close to the objective. Taking a 20x objective as an example, its working distance is 4.5mm, and the distance between the temperature sensor surface and the bottom surface of the carrier plate 1 is 2.4~2.6mm.

[0022] The culture chamber has an internal diameter of approximately 6 mm and a total volume of approximately 370 μL. The size of the culture chamber can be further enlarged or reduced proportionally by a certain factor, such as 0.9 to 1.5 times, to accommodate the temperature sensor and save on the amount of sample to be tested. Further reduction in the size of the culture chamber is not conducive to sensor assembly and cell culture operations. The inner ring diameter of the annular blind hole 4 is ≥8 mm, and the outer ring diameter is ≤18 mm.

[0023] An air jacket surrounds the culture chamber. Specifically, an opening is made at the bottom of a rectangular or cylindrical carrier plate 1 to form a through hole penetrating the carrier plate 1 and an annular blind hole 4 near the top of the carrier plate 1. The end of the through hole near the opening of the annular blind hole 4 and the opening end of the annular blind hole 4 are sealed with transparent materials 5 and 6, respectively. 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 jacket around the culture chamber.

[0024] During cell temperature measurement, the operator may open the incubator door to add culture medium or stimulate drugs, causing temperature fluctuations inside the incubator (in culture chambers without an air gap, the volume remains constant, and the temperature change during incubator opening and closing is approximately 0.5K). The linearity of basic sensors 2 and 3 differs slightly, and temperature fluctuations affect measurement accuracy. The air gap is used to reduce temperature fluctuations in the culture chamber (with an air gap, simultaneous detection in the culture chamber results in a maximum temperature change of 0.3K) and reduce background noise. The height of transparent material 5 is greater than that of transparent material 6; both transparent materials 5 and 6 are cell spreaders.

[0025] The method for measuring cell temperature in a cell culture chamber using cell temperature detection is as follows:

[0026] The cell temperature detection culture chamber was sterilized and prepared for use. Adherent cells (A549) were removed from the incubator, the culture medium was removed, and the cells were washed once with PBS. An appropriate amount of 0.25% trypsin-EDTA was added, and the cells were digested in the incubator for 2 min. The cells were centrifuged, and the cell pellet was collected and resuspended in fresh culture medium. 200 μL of a cell suspension with a concentration of 10000 cells / mL was added to the culture chamber, ensuring the liquid completely covered the surface of the temperature sensor. The cell temperature detection culture chamber was then transferred to the incubator, and cultured for another 24 h.

[0027] The cell temperature detection culture chamber was removed, washed once with PBS, and a small amount of trypsin was added to cover the cells at the bottom of the culture chamber for digestion. After washing with PBS again to remove the bottom cells, 300 μL of fresh culture medium was added for cell culture. Under a microscope, the cells were observed until they just covered the detection surface of the temperature sensor facing the entrance of the culture chamber. Temperature was then measured, and the detection surface signals from basic sensor 2 and basic sensor 3 were acquired separately. The difference between the two signals was calculated as the cell temperature. The temperature difference between the cells and the ambient temperature measured using this method was 0.1℃.

[0028] The temperature of the culture chamber can be obtained using the cell temperature detection method for adherent cells, including but not limited to A549, HMEC-1, C3H10T, 1 / 2 clone8, and HeLa cells.

Claims

1. A cell temperature detection culture cavity, comprising a carrier plate (1) and a temperature sensor, characterized in that, The carrier plate is provided with a culture chamber. 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, with at least one detection surface facing the inlet of the culture chamber and at least one detection surface facing the bottom of the culture chamber.

2. The cell temperature detection incubation chamber of claim 1, wherein, The bottom of the culture chamber is transparent, and the temperature sensor has a transparent substrate.

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

4. The cell temperature detection culture cavity according to claim 1, wherein, 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 2.4~2.6mm.

5. The cell temperature detection culture cavity according to claim 1, wherein, The carrier plate (1) is provided with through holes and annular blind holes (4) surrounding the through holes. The opening end of the annular blind holes (4) and the end of the through hole near the opening of the annular blind holes (4) are respectively sealed with transparent material, so that an air interlayer is formed outside the culture chamber formed by the through holes.

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 cavity according to any one of claims 1 to 6, wherein, The temperature sensor is a platinum resistance thermometer, the surface of the temperature sensor is silicon nitride, and the detection surface of the temperature sensor facing the inlet of the culture chamber is further coated with a rat tail collagen layer.

8. The cell temperature detection culture cavity according to any one of claims 1 to 5, wherein, 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 5, characterized in that, The inner diameter of the through hole is ≥6mm and the total volume is ≥370 μL. The inner ring diameter of the annular blind hole (4) is ≥8mm and the outer ring diameter is ≤18mm.

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