A culture device for cell proliferation

Abstract

This invention discloses a cell proliferation culture device, belonging to the field of biomedical experimental equipment technology, comprising: a culture body, a microporous membrane, a microfluidic channel, a temperature control module, a sensor module, a sampler, and a control unit. The cell proliferation culture device of this invention can dynamically monitor the glucose consumption rate and lactate production rate during cell proliferation in real time, supports pollution-free dynamic sampling and rapid microenvironment switching, and is particularly suitable for studying the interaction between SIRT2 and HKII to regulate aerobic glycolysis and growth proliferation of colorectal cancer cells. It also has advantages such as real-time monitoring, high sensitivity, pollution-free sampling, and dynamic microenvironment control.

Description

Technical Field

[0001] This invention relates to the field of biomedical experimental equipment technology, and in particular to a culture device for cell proliferation, especially suitable for studying the interaction between SIRT2 and HKII in colorectal cancer cells to regulate aerobic glycolysis and growth proliferation. Background Technology

[0002] Colorectal cancer is a common malignant tumor, and its cells exhibit a significant Warburg effect, meaning they are highly active in aerobic glycolysis. Hexokinase II (HKII) is a key rate-limiting enzyme in glycolysis, and SIRT2 can regulate HKII activity through deacetylation modification, thereby affecting cell proliferation and metabolism.

[0003] Currently, conventional culture devices (such as culture flasks, culture plates, and ordinary cell culture incubators) used for studying tumor cell proliferation and glucose metabolism have the following technical shortcomings:

[0004] Real-time monitoring is not possible: detecting glucose consumption and lactate production requires interrupting culture, digesting cells, or collecting supernatant, making it impossible to obtain continuous dynamic data;

[0005] High risk of sampling contamination: Each sampling requires opening the culture container, which can easily introduce microbial contamination, and it is impossible to achieve multiple samplings without interference.

[0006] The inability to dynamically regulate the microenvironment limits the depth of mechanistic research because it prevents the rapid replacement of culture media containing inhibitors or drugs without disturbing the cells.

[0007] Therefore, in order to solve the problems existing in the prior art, a culture device for cell proliferation is needed. Summary of the Invention

[0008] The present invention aims to at least partially solve one of the technical problems in the above-mentioned technologies.

[0009] To achieve the above objectives, the first aspect of the present invention provides a culture device for cell proliferation, comprising a culture body having a cavity inside, wherein a microporous membrane is disposed at the bottom of the cavity; a microfluidic channel disposed at the bottom of the culture body; a temperature control module disposed at the bottom of the microfluidic channel; a sensor module disposed inside the cavity, wherein the sensor module is a three-in-one sensor array simultaneously including a glucose sensor, a lactate sensor, and a pH sensor; a sampler connected to the microfluidic channel; and a control unit electrically connected to the sensor module and the temperature control module, wherein the control unit has a built-in metabolic rate calculation module for calculating and displaying the glucose consumption rate and the lactate production rate in real time.

[0010] In addition, the cell proliferation culture device proposed above according to the present invention may also have the following additional technical features:

[0011] Furthermore, the flow rate adjustment range of the microfluidic channel is 0.1–5 μL / min.

[0012] Furthermore, the microporous membrane has a pore size of 0.4 μm and is made of polycarbonate.

[0013] Furthermore, the temperature control module uses PID control to maintain the temperature at 37±0.2℃.

[0014] Furthermore, the distance between the probe end of the sensor module and the microporous membrane is 0.5-1.5 mm.

[0015] Furthermore, the sampler is equipped with a sterile diaphragm.

[0016] A second aspect of this invention provides a method for studying SIRT2-regulated HKII activity using a cell proliferation culture apparatus, comprising the following steps:

[0017] S1: Inoculate SIRT2-overexpressing or knocked-down colorectal cancer cells into the culture medium; S2: Set the flow rate of the microfluidic channel to 0.5-2 μL / min and maintain a constant temperature of 37°C; S3: Continuously monitor the dynamic changes in glucose consumption rate and lactate production rate for 24-72 hours using the sensor module; S4: Take samples during the culture process using the sampler to detect HKII protein expression level and acetylation level; S5: Switch the culture medium containing HKII inhibitor through the microfluidic channel and continue to monitor cell metabolic response.

[0018] According to the present invention, a culture device for cell proliferation can dynamically monitor the glucose consumption rate and lactate production rate during cell proliferation in real time, support pollution-free dynamic sampling and rapid switching of the microenvironment, and is particularly suitable for the study of the interaction between SIRT2 and HKII to regulate aerobic glycolysis and growth and proliferation of colorectal cancer cells. It has the advantages of real-time monitoring, high sensitivity, pollution-free sampling and dynamic control of the microenvironment. Attached Figure Description

[0019] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments taken in conjunction with the accompanying drawings, wherein:

[0020] Fig. 1 This is a schematic diagram of the overall structure of a cell proliferation culture device according to an embodiment of the present invention;

[0021] Fig. 2 This is a flowchart illustrating the workflow of a cell proliferation culture apparatus according to an embodiment of the present invention.

[0022] Fig. 3 This is a flowchart illustrating the sensor data acquisition and processing process of a cell proliferation culture device according to an embodiment of the present invention.

[0023] Fig. 4 This is a time-series diagram of cell culture and metabolic monitoring of a cell proliferation culture device according to an embodiment of the present invention;

[0024] As shown in the figure:

[0025] 1. Culture medium; 2. Microporous membrane; 3. Microfluidic channel; 4. Temperature control module; 5. Sensor module; 6. Sampler; 7. Control unit. Detailed Implementation

[0026] Embodiments of the present invention are described in detail below, with examples of the embodiments illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0027] The following description, in conjunction with the accompanying drawings, describes a culture apparatus for cell proliferation according to an embodiment of the present invention.

[0028] like Figs. 1 to 4 As shown, an embodiment of the present invention provides a cell proliferation culture device, including a culture body 1 with a cavity inside. A microporous membrane 2 is disposed at the bottom of the cavity. A microfluidic channel 3 is disposed at the bottom of the culture body 1. A temperature control module 4 is disposed at the bottom of the microfluidic channel 3. A sensor module 5 is disposed inside the cavity. The sensor module 5 is a three-in-one sensor array that simultaneously includes a glucose sensor, a lactate sensor, and a pH sensor. A sampler 6 is connected to the microfluidic channel 3. A control unit 7 is electrically connected to the sensor module 5 and the temperature control module 4. The control unit 7 has a built-in metabolic rate calculation module for calculating and displaying the glucose consumption rate and lactate production rate in real time.

[0029] Preferably, the culture medium 1 is made of a transparent biocompatible material, which facilitates in-situ observation of cell growth status and is non-toxic and free of leachates, ensuring the authenticity of experimental data.

[0030] It should be noted that the flow rate of microfluidic channel 3 is adjustable from 0.1 to 5 μL / min and is connected to the culture medium storage tank and waste liquid tank, which can realize low shear force culture, automatic medium change and rapid drug switching, and is suitable for dynamic intervention experiments.

[0031] It should be noted that the microporous membrane 2 has a pore size of 0.4 μm and is made of polycarbonate, and is used for cell adhesion growth and the diffusion of metabolites.

[0032] It should be noted that the temperature control module 4 uses PID control to maintain the temperature at 37±0.2℃, which is more accurate than traditional incubators, ensuring the stable operation of enzyme activity and metabolic pathways.

[0033] It should be noted that the distance between the probe end of the sensor module 5 and the microporous membrane 2 is 0.5-1.5 mm, which can detect the immediate concentration of metabolites on the cell membrane surface. The detection response time is shortened from 24 hours to 4-6 hours, significantly improving the sensitivity.

[0034] It should be noted that the sampler 6 is equipped with a sterile diaphragm for collecting culture medium samples without contamination.

[0035] It should be noted that the control unit 7 can automatically draw dynamic curves, generate metabolic parameter change graphs in real time, and support comparative analysis of multiple sets of data, which is convenient for studying the interaction mechanism between SIRT2 and HKII.

[0036] The present invention discloses a method for studying SIRT2-regulated HKII activity using a cell proliferation culture device, comprising the following steps: S1: Inoculating SIRT2-overexpressing or knocked-down colorectal cancer cells into a culture medium 1; S2: Setting the flow rate of the microfluidic channel 3 to 0.5-2 μL / min and maintaining a constant temperature of 37°C; S3: Continuously monitoring for 24-72 hours using a sensor module 5 and recording the dynamic changes in glucose consumption rate and lactate production rate; S4: Taking samples during the culture process using a sampler 6 to detect the expression level and acetylation level of HKII protein; S5: Switching the culture medium containing HKII inhibitors through the microfluidic channel 3 and continuing to monitor the cell metabolic response.

[0037] As one possible scenario, according to another embodiment of the present invention, the step of culturing the colorectal cancer cell line HCT116 using a culture device for cell proliferation is as follows: S1: culturing HCT116 cells at a rate of 1×10⁻⁶... 5 Cells / cm² were seeded in culture medium 1 and adhered to the microporous membrane 2. S2: The flow rate of microfluidic channel 3 was set to 1 μL / min, and the temperature control module 4 maintained 37℃. S3: Sensor module 5 monitored glucose consumption and lactate production in real time, and the data was uploaded to control unit 7. S4: After 48 hours of culture, the glucose consumption rate was 12.5 mmol / L / day, and the lactate production rate was 22.3 mmol / L / day. S5: Samples were taken through sampler 6, and Western blot analysis showed that HKII protein expression was upregulated.

[0038] As a possible scenario, according to another embodiment of the present invention, the steps for studying the effect of SIRT2-regulated HKII activity on cell proliferation using a cell proliferation culture device are as follows: S1: SIRT2-siRNA is transfected into SW480 cells and seeded into culture medium 1; S2: After continuous monitoring for 48 hours, control unit 7 shows that after SIRT2 knockdown, glucose consumption rate decreases by about 40% and lactate production rate decreases by about 35%; S3: At the 24th hour of culture, samples are taken through sampler 6, and the detection shows that HKII acetylation level increases and activity decreases; S4: The culture medium containing the HKII inhibitor 3-bromopyruvate is switched through microfluidic channel 3, and monitoring continues for 24 hours, and the cell proliferation rate further decreases.

[0039] The results show that the cell proliferation culture device of the present invention can be effectively used for the study of the interaction between SIRT2 and HKII to regulate glucose metabolism and growth and proliferation of colorectal cancer cells.

[0040] In summary, the cell proliferation culture device according to the embodiments of the present invention can monitor the glucose consumption rate and lactate production rate during the cell proliferation process in real time, support pollution-free dynamic sampling and rapid switching of the microenvironment, and is particularly suitable for the study of the interaction between SIRT2 and HKII to regulate aerobic glycolysis and growth and proliferation of colorectal cancer cells. It also has the advantages of real-time monitoring, high sensitivity, pollution-free sampling, and dynamic control of the microenvironment.

[0041] In the description of this specification, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0042] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0043] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A culture device for cell proliferation, characterized in that, include: The culture body (1) has a cavity inside, and a microporous membrane (2) is provided at the bottom of the cavity. A microfluidic channel (3) is disposed at the bottom of the culture body (1); Temperature control module (4) is located at the bottom of the microfluidic channel (3); The sensor module (5) is located inside the cavity. The sensor module (5) is a three-in-one sensor array that simultaneously includes a glucose sensor, a lactic acid sensor and a pH sensor. The sampler (6) is connected to the microfluidic channel (3); The control unit (7) is electrically connected to the sensor module (5) and the temperature control module (4). The control unit (7) has a built-in metabolic rate calculation module for calculating and displaying the glucose consumption rate and lactic acid production rate in real time.

2. The culture device for cell proliferation according to claim 1, characterized in that, The flow rate adjustment range of the microfluidic channel (3) is 0.1–5 μL / min.

3. The culture device for cell proliferation according to claim 1, characterized in that, The microporous membrane (2) has a pore size of 0.4 μm and is made of polycarbonate.

4. The culture device for cell proliferation according to claim 1, characterized in that, The temperature control module (4) is a PID controller, which maintains the temperature at 37±0.2℃.

5. A culture device for cell proliferation according to claim 1, characterized in that, The distance between the probe end of the sensor module (5) and the microporous membrane (2) is 0.5-1.5 mm.

6. A culture device for cell proliferation according to claim 1, characterized in that, The sampler (6) is equipped with a sterile diaphragm.

7. A method for studying SIRT2-regulated HKII activity using a cell proliferation culture apparatus according to any one of claims 1 to 6, characterized in that, Includes the following steps: S1: Inoculate SIRT2-overexpressing or knocked-down colorectal cancer cells into the culture medium (1); S2: Set the flow rate of the microfluidic channel (3) to 0.5-2 μL / min and maintain a constant temperature of 37℃; S3: Continuously monitor for 24-72 hours using the sensor module (5) and record the dynamic change curves of the glucose consumption rate and the lactic acid production rate; S4: During the culture process, samples are taken using the sampler (6) to detect the expression level and acetylation level of HKII protein; S5: Switch the culture medium containing HKII inhibitor through the microfluidic channel (3) and continue to monitor the cell metabolic response.

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