Cell thermal stimulation culture chamber

Abstract

The utility model discloses a cell heat stimulation culture cavity, cell heat stimulation culture cavity includes culture chamber and the chip of being located culture chamber bottom, is equipped with several orderly arranged stimulation unit on the chip inside culture chamber, and the material of stimulation unit is platinum, and line width d is equal to or more than 10um. In the utility model, stimulation unit can realize the control precision of 0.05 DEG C and submillimeter level stimulation area, and the difference between adjacent stimulation units is obvious, and the direction difference is obvious, forms accurate directive temperature stimulation, and the chip can be repeatedly used.

Description

Technical Field

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

[0002] In the field of biomedical research, thermal stimulation, as a non-invasive physical regulation method, plays a central role in the regulation of cell function. Its biological effects are mainly achieved through the heat shock transcription factor 1 (HSF1) pathway. Activation of this pathway can initiate the expression of various heat shock proteins (HSPs), thereby regulating processes such as cell metabolism, immune responses, and fate transformation. In the field of tumor therapy, photothermal therapy uses near-infrared light to excite nanomaterials to generate localized high temperatures (>42℃), inducing immunogenic cell death (ICD) in tumor cells, releasing danger signals such as HMGB1 and ATP, and activating anti-tumor immune responses.

[0003] Zhang Xiaojuan et al. used ITO glass as a heating source and substrate for cell culture chips to study the effect of temperature gradients on cells (Zhang Xiaojuan et al. "The effect of temperature distribution on myoblast growth and proliferation on ITO glass chips." Analytical Chemistry 41.8(2013):6.). Specifically, they used PMDS to fabricate a culture chamber with a diameter of 8 mm, and used conductive adhesive to fix copper electrodes to the conductive surface of ITO glass (5 cm side length, 1.1 mm thickness, 100 Ω resistance). After applying a certain voltage to the electrodes, the thin film resistance of the ITO glass conducted electricity and heated up, causing the glass surface temperature to rise. The temperature distribution varied greatly depending on the position and length of the electrodes. Placing electrodes parallel to both sides of the glass could obtain a uniformly changing, concentrically distributed temperature gradient. At the cell level, this scheme had low control precision and spatial resolution of thermal stimulation technology. Wolfrum et al. disclosed the effects of linear arrays of thermal stimulation elements on cells (10.1002 / adbi.201800138). Long-distance linear heating wires can cause temperature differences between the middle and the ends, and constant-temperature thermal stimulation has the problem of poor accuracy. Utility Model Content

[0004] Purpose of the utility model: The purpose of this utility model is to provide a cell thermal stimulation culture chamber that provides multi-angle and precise stimulation.

[0005] Technical solution: The present invention provides a cell thermal stimulation culture chamber, comprising a culture chamber and a chip located at the bottom of the culture chamber. The chip located inside the culture chamber is provided with a plurality of orderly arranged stimulation units. The stimulation units are made of platinum and have a linewidth d≥10μm.

[0006] Preferably, the structural parameters of the stimulation unit are: length L≥70μm, width W≥60μm.

[0007] Preferably, the stimulation units are arranged in a circular array.

[0008] Preferably, the distance between the center points of two oppositely positioned stimulation units is ≥1mm.

[0009] Preferably, the number of stimulation units is 4 to 12.

[0010] Preferably, the stimulation unit has two pins extending from its end, and the pin surfaces are plated with gold.

[0011] Preferably, the chip surface is covered with a silicon dioxide layer, the silicon dioxide layer having a thickness ≥0.2μm, and the silicon dioxide layer surface is covered with a polylysine layer.

[0012] Preferably, the chip is connected to a PCB adapter board, and the PCB adapter board is provided with a viewing window for observing the stimulation unit and the internal condition of the culture chamber.

[0013] Preferably, the PCB adapter board is provided with a ribbon cable connector for connecting the control circuit.

[0014] Preferably, the culture chamber is made of PDMS (polydimethylsiloxane), and the culture chamber and the chip are connected by plasma bonding.

[0015] Beneficial Effects: Compared with existing technologies, this invention has the following significant advantages: 1. Multi-angle, precise stimulation: It has multiple independently working stimulation units, which can achieve temperature control accuracy of ±0.05℃ (uncertainty) and sub-millimeter-level stimulation area control. The differences between adjacent stimulation units are obvious, and the directional differences are obvious, forming precise directional temperature stimulation; 2. The stimulation units are close to the cell size. The parameters of each stimulation unit can be set separately to study cell behavior. It is suitable for stimulating a small number of cells under the same culture conditions, reducing the impact of differences caused by changes in culture conditions on the final experimental results; 3. Simultaneous stimulation at different temperatures: It can construct temperature gradient changes higher than 0.5K within a 0.5mm range, filling the gap in the single mode of conventional temperature stimulation methods; 4. Real-time in-situ monitoring of temperature changes of stimulation units. The temperature is precisely controllable, and it is possible to accurately study changes in cell behavior under different temperature stimulation conditions. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the control circuit structure of the cell thermal stimulation device of this utility model;

[0017] Figure 2 This is a schematic diagram of the cell thermal stimulation device of this utility model;

[0018] Figure 3 This is a schematic diagram of the chip structure of the cell thermal stimulation device of this utility model;

[0019] Figure 4 for Figure 3 Enlarged view of the middle section structure;

[0020] Figure 5 This is a dimensioned diagram of the stimulation unit of the cell thermal stimulation device of this utility model;

[0021] Figure 6 The diagram shows the test results of applying the same current to all stimulation units of the cell thermal stimulation device of this invention at the same time.

[0022] Figure 7 The graph shows the test results of applying different currents to all stimulation units of the cell thermal stimulation device of this invention. Detailed Implementation

[0023] The technical solution of this utility model will be further described below with reference to the accompanying drawings.

[0024] Example: Figure 2 As shown, the cell thermal stimulation device mainly consists of a chip 13 and a culture chamber 14. The chip 13 is located at the bottom of the culture chamber 14. The culture chamber 14 is made of a cube with a side length of 3 mm. A through hole with a diameter of 2 mm is opened on the cube, and the inside of the through hole is the culture chamber 14. The material of the culture chamber 14 is PDMS, which is connected to the chip 13 by plasma bonding and has good biocompatibility.

[0025] like Figure 3 , Figure 4 The chip 13 uses a transparent material as a substrate and is located inside the culture chamber 14. Several orderly arranged stimulation units 22 are disposed on the chip 13. The arrangement of the stimulation units 22 can be a circular array or a rectangular array, with a circular array being preferred. The circular array arrangement can be consistent with a Cartesian coordinate system or a clock direction, facilitating the description of direction during directional thermal stimulation experiments. A highly transparent glass sheet is preferred as the substrate.

[0026] Stimulation unit 22 is made of platinum, such as Figure 5 In the structural parameters of the stimulation unit 22, the linewidth d ≥ 10 μm, which takes into account both thermal stimulation and temperature detection functions and can be used repeatedly.

[0027] When arranging the stimulation units 22 in a circular array, to ensure a significant temperature gradient between the stimulation units 22 and the central region of the culture chamber 14, and to guarantee a temperature gradient between adjacent thermal stimulation units 22 for precise directional thermal stimulation, the minimum length L of the stimulation unit 22 is 70 μm, and the minimum width W is 60 μm. The distance between the center points of two oppositely positioned stimulation units 22 is at least 1 mm (i.e., the distance between the center of each stimulation unit 22 and the center point of the culture chamber 14 is at least 0.5 mm). Four to twelve stimulation units 22 are preferred. Reducing the structural parameters of the stimulation units 22 helps to achieve more precise regional temperature stimulation, but further reduction will decrease the yield rate and make them more susceptible to damage during use, and will also hinder the arrangement of the pins 21.

[0028] Each stimulation unit 22 is led out by two pins 21. The pins 21 are 3mm long and 0.3mm wide. The surface of the pins 21 is gold-plated to facilitate connection to the PCB. The adjacent pins 21 are spaced 0.2mm apart.

[0029] The surface of chip 13 is covered with 0.2μm thick silicon dioxide to isolate the liquid from contact with the stimulation unit 22. The thickness of the cover layer ensures the reliability of the cover layer and facilitates the reuse of chip 13. The thickness of the silicon dioxide layer can be increased to 0.5μm. The surface of silicon dioxide is treated with materials such as polylysine to make it suitable for cell growth.

[0030] Chip 13 is connected to PCB adapter board 11, corresponding to the position of stimulation unit 22 on chip 13. A 3mm diameter viewing window on PCB adapter board 11 allows observation and recording of cell ecology using a microscope. PCB adapter board 11 has a ribbon cable connector 12 for connecting to control circuit 2, which in turn connects to computer 1. The computer records temperature data and live cell workstation image data in real time for later analysis.

[0031] Using control circuit 2, different currents can be applied to each stimulation unit 22 to achieve thermal stimulation at different temperature gradients. While heating, the voltage information of each stimulation unit is collected to calculate the resistance value, monitoring the real-time temperature change of the stimulation unit surface. A pre-compensation algorithm is used to eliminate thermal inertia errors. Considering temperature detection applications, the maximum operating current of each thermal stimulation unit 22 is set at 5mA. During thermal stimulation, control circuit 2 outputs current for heating, pausing heating every 2 seconds to detect the temperature. The temperature detection time is no less than 20ms, ensuring that the stimulation unit 22 has reached a stable state and guaranteeing detection accuracy. Control circuit 2 can calculate the temperature change trend and adjust the output current or time accordingly.

[0032] Cellular thermal stimulation device test: A 1mA current was simultaneously applied to 12 stimulation units 22 and heated continuously for 5 minutes. The surface temperature distribution of chip 13 was as follows. Figure 6As shown, a significant temperature difference of 0.7K can be formed between the stimulation unit 22 and the center of the chip 13.

[0033] Currents of 1, 0.5, 0.25, 0.1, 0.05, 0.025, and 0 were applied to 12 stimulation units (symmetrically positioned on both sides of the vertical midline) for 5 minutes. The surface temperature distribution of chip 13 is as follows. Figure 7 As shown, a significant temperature difference can be formed between the center of the stimulation unit 22 and the chip 13, and a temperature difference can also be formed between two adjacent stimulation units.

[0034] Cell culture: An ethylene oxide-sterilized cell heat stimulation device was used for standby. After adherent cell culture, trypsin digestion was performed to prepare a cell suspension. 10 μL of the cell suspension with a concentration of 10,000 cells / mL was added to culture chamber 14 and cultured in an incubator for 12 hours. The heat cable connector 12 was connected to control circuit 2, the target temperature of stimulation unit 22 was input into computer 1, the program was started, and the live cell workstation observed and recorded image information.

[0035] Comparative Example: The chip 13 in this comparative example is similar to that in the embodiment, except that the size of the stimulation unit 22 is 150μm long, 120μm wide, and 0.3μm linewidth. There are 4 stimulation units 22 arranged in a circular array. The distance between the centers of two opposite stimulation units 22 is 1mm. This comparative example scheme is similar to that in the embodiment, and a temperature gradient can be formed on the surface of the chip 13. However, it requires a larger operating current (greater than 5mA) for the stimulation unit 22, which causes the lifespan of the stimulation unit 22 to decrease rapidly and make it unusable.

Claims

1. A cell thermal stimulation culture chamber, comprising a culture chamber (14) and a chip (13) located at the bottom of the culture chamber, characterized in that, The chip (13) located inside the culture chamber (14) is provided with several orderly arranged stimulation units (22). The stimulation unit (22) is made of platinum, with a line width d ≥ 10 μm, a length L ≥ 70 μm, and a width ≥ 60 μm. There are 4 to 12 stimulation units (22). The stimulation units (22) are arranged in a circular array, and the distance between the center points of two opposite stimulation units (22) is ≥ 1 mm.

2. The cell heat stimulation culture chamber according to claim 1, characterized in that, The stimulation unit (22) is led out by two pins (21) at its end, and the surface of the pins (21) is plated with gold.

3. The cell heat stimulation culture chamber according to claim 1, characterized in that, The chip (13) is covered with a silicon dioxide layer with a thickness ≥0.2μm and a polylysine layer.

4. The cell heat stimulation culture chamber according to claim 1, characterized in that, The chip (13) is connected to the PCB adapter board (11). The PCB adapter board (11) is provided with a window for observing the stimulation unit (22) and the internal condition of the culture chamber. The PCB adapter board (11) is provided with a ribbon cable plug (12) for connecting the control circuit (2).

5. The cell thermostimulation culture chamber according to claim 1, characterized in that, The culture chamber (14) is made of PDMS, and the culture chamber (14) and the chip (13) are connected by plasma bonding.

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