CN111304079A - Method for thermally stimulating cultured cells in cells - Google Patents
Method for thermally stimulating cultured cells in cells Download PDFInfo
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- CN111304079A CN111304079A CN201910996034.3A CN201910996034A CN111304079A CN 111304079 A CN111304079 A CN 111304079A CN 201910996034 A CN201910996034 A CN 201910996034A CN 111304079 A CN111304079 A CN 111304079A
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- 210000004748 cultured cell Anatomy 0.000 title claims abstract description 27
- 210000004027 cell Anatomy 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 14
- 230000004936 stimulating effect Effects 0.000 title claims abstract description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 72
- 238000004113 cell culture Methods 0.000 claims abstract description 66
- 230000000638 stimulation Effects 0.000 claims abstract description 36
- 238000002474 experimental method Methods 0.000 claims abstract description 29
- 238000012544 monitoring process Methods 0.000 claims abstract description 26
- 238000010438 heat treatment Methods 0.000 claims abstract description 10
- 239000001963 growth medium Substances 0.000 claims description 12
- 239000000523 sample Substances 0.000 claims description 9
- 229920000915 polyvinyl chloride Polymers 0.000 claims description 6
- 239000004800 polyvinyl chloride Substances 0.000 claims description 6
- 230000004044 response Effects 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 238000013383 initial experiment Methods 0.000 claims description 3
- 230000001052 transient effect Effects 0.000 claims description 3
- 102000004310 Ion Channels Human genes 0.000 description 4
- 230000006399 behavior Effects 0.000 description 4
- 230000003915 cell function Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 241001528553 Malus asiatica Species 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000001514 detection method Methods 0.000 description 1
- 230000003760 hair shine Effects 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
- C12M41/18—Heat exchange systems, e.g. heat jackets or outer envelopes
- C12M41/22—Heat exchange systems, e.g. heat jackets or outer envelopes in contact with the bioreactor walls
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Abstract
The invention discloses a method for thermally stimulating cultured cells in cells. Firstly setting the temperature of a water bath as an experimental temperature, starting the water bath for heating, respectively placing a cell culture plate/bottle which is filled with cells to be cultured by thermal stimulation and a cell culture plate/bottle which is used as a blank control in a groove position of 2 underframe when the temperature of a water bath reaches a final experimental temperature, exposing the lower part of the cell culture plate/bottle above the water surface, monitoring the inner surface temperature of the blank control cell culture plate/bottle by an infrared temperature monitoring system, heating for a time reaching the temperature, then performing heating stimulation for a time required by the experiment, and rapidly taking out the cell culture plate/bottle containing the cultured cells after the completion of the experiment, thereby completing the accurate and instantaneous thermal stimulation experiment of the cultured cells. The invention can well realize the accurate and instantaneous thermal stimulation treatment on the cultured cells and can well meet the experimental requirements such as the accurate observation of the influence of the thermal stimulation on the cell behavior and the function.
Description
The technical field is as follows:
the invention belongs to the technical field of biology, and particularly relates to a method for thermally stimulating cultured cells in cells.
Background art:
in cell biology research work, cell culture is often required. Conventional cell culture conditions are: placing the cell culture dish or flask in an incubator at 37 deg.C and 5% CO2Culturing in the environment. In general, the temperature of the incubator is constant at 37 ℃. For example, in the experimental process, in order to achieve specific experimental purposes (e.g., observing the influence of thermal stimulation on cell behavior, researching the influence of a thermal sensitive ion channel or receptor on cell function after being heated, etc.), the cultured cells need to be subjected to thermal stimulation treatment. In this regard, conventional incubators currently available do not meet the experimental requirements (especially the need for transient or short-term accurate thermal stimulation experiments). Although temperature-adjustable incubators are available, the incubator has a slow temperature-increasing or temperature-decreasing speed, and cannot meet the requirement of instantaneous and accurate thermal stimulation on cultured cells.
The invention content is as follows:
the invention aims to solve the problem of short-time and accurate thermal stimulation on cultured cells in the process of cell biology experimental study, and provides a method for thermally stimulating the cultured cells in cells, which can meet the requirement of thermally stimulating the cultured cells (such as observing the influence of thermal stimulation on cell behaviors, researching the influence of a thermally sensitive ion channel or receptor on cell functions after being heated, and the like), particularly can well meet the experimental requirement of instantaneously and accurately thermally stimulating the adherent cultured cells, thereby realizing the accurate observation of the influence of thermal stimulation on the cell behaviors, and achieving the purposes of researching the influence of the thermally sensitive ion channel or receptor on the cell functions after being heated, and the like.
The method for stimulating the cultured cells with heat in the cells is characterized by comprising the following steps:
the thermal stimulation device comprises a water bath kettle, an infrared temperature monitoring system, a cell culture plate/bottle, a bottom frame, a fixed support and a stopwatch, wherein an infrared probe of the infrared temperature monitoring system is fixed on the fixed support, at least 2 bottom frames are arranged in the water bath kettle in parallel and horizontally, and a groove for placing the cell culture plate/bottle is arranged on each bottom frame;
a. pre-experiment: pure sterilized water is filled in a water bath kettle, a blank cell culture plate/bottle is placed in the groove position of 1 underframe, the lower part of the cell culture plate/bottle is submerged in the water, 4mm away from the water surface, the upper opening is exposed above the water surface, the infrared probe forms an angle of 45 degrees, aligning the inner wall of the blank cell culture plate/flask wetted with culture medium outside the distance of 5-6cm, setting the temperature of the water bath at least 10-12 ℃ higher than the desired stimulation temperature, then the water bath kettle is electrified to heat the water in the water bath kettle, simultaneously the infrared temperature monitoring system monitors the temperature change of the inner wall of the cell culture plate/bottle, stopping when the temperature of the inner wall can rise to the required stimulation temperature within 10 seconds, setting the temperature of the water bath as the initial experiment temperature, then repeatedly measuring for at least three times, and taking the average value as the final experiment temperature;
b. formal experiments: opening a water bath kettle, setting the temperature of the water bath kettle to the final experiment temperature obtained in the step a, then starting the water bath kettle to heat water in the water bath kettle, respectively placing a cell culture plate/bottle which needs to thermally stimulate cultured cells and is free of a culture medium and a cell culture plate/bottle which is wetted by the culture medium and is used as a blank control in groove positions of 2 underframe when the water bath temperature reaches the final experiment temperature value, immersing the lower part of the cell culture plate/bottle in water, keeping the lower part of the cell culture plate/bottle 4mm away from the water surface, exposing the upper part of the cell culture plate/bottle above the water surface, monitoring the temperature of the inner surface of the cell culture plate/bottle wetted by the culture medium and used as the blank control by an infrared temperature monitoring system at an angle of 45 degrees outside a distance of 5-6cm, firstly heating for a temperature reaching time, then starting a stopwatch, carrying out heating stimulation for the time required by the experiment, the cell culture plate/flask containing the cultured cells is removed, thus completing a precise and transient thermal stimulation experiment of the cultured cells.
The bottom frame is preferably a polyvinyl chloride bottom frame which is cuboid, a groove with a shape matched with the cell culture plate/bottle is arranged at the opening at the top, and the cell culture plate/bottle is clamped into the polyvinyl chloride bottom frame, so that the cell culture plate/bottle is fixed.
The water bath kettle is preferably a precise water bath kettle with the temperature resolution of +/-0.1 ℃.
The infrared temperature monitoring system is preferably an infrared temperature monitoring system with the response time of +/-100 ms and the temperature resolution of 0.1 ℃, and the infrared temperature monitoring system is an instrument known in the prior art, such as an IC-LI (integrated circuit-lithium) online infrared temperature monitoring system of the Huahong brand.
The current intercellular cell culture boxes and the related equipment cannot meet the requirement of short-time (within 1 minute) and accurate thermal stimulation on cultured cells in the research process of cell biology experiments (the most sensitive existing temperature-adjustable culture boxes require at least 3 minutes for increasing the temperature of 1 ℃). The invention can well solve the problem, can well realize the precise and instantaneous thermal stimulation treatment (the response time is +/-100 ms, the temperature resolution is 0.1 ℃) to the cultured cells, and can well meet the experimental requirements such as accurately observing the influence of the thermal stimulation on the cell behavior, researching the influence of a thermal sensitive ion channel or receptor on the cell function after being heated and the like, thereby improving the accuracy, the reliability and the repeatability of related scientific research experiments and improving the scientific research working quality.
Description of the drawings:
FIG. 1 is a schematic structural view of a thermal stimulation apparatus of the present invention;
wherein 1, signal line; 2. a fixing clip; 3. fixing a bracket; 4. an infrared probe; 5. a water bath kettle; 6. a chassis; 7. a cell culture plate; 8. an infrared temperature monitoring system; 9. a power source;
FIG. 2 is a cell culture plate;
10, blank cell culture plate; 11. a cell culture plate containing cultured cells; 12. blank control culture wells; 13. a culture well containing cultured cells; 14. and detecting temperature infrared rays.
The specific implementation mode is as follows:
the following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1:
as shown in fig. 1 and 2, the thermal stimulation device comprises a water bath 5, an infrared temperature monitoring system 8, a cell culture plate 7, a bottom frame 6 and a fixing support 7, wherein an infrared probe 4 of the infrared temperature monitoring system (the infrared probe is connected with a host of the infrared temperature monitoring system through a signal line 1) is fixed on the fixing support 3 through a fixing clamp 2, at least 2 bottom frames 6 are arranged in the water bath 5, and a groove for placing the cell culture plate/bottle is arranged on each bottom frame;
a. pre-experiment: filling sterilizing purified water into a water bath kettle 5, wherein the liquid level is limited to be 0.4cm higher than the upper surface of a bottom frame 6, the liquid level is 2-3cm away from the upper edge of the water bath kettle, installing cell culture plates 7 on the groove positions of 1 bottom frame, the lower parts of the cell culture plates are submerged in water, the upper parts of the cell culture plates are exposed above the water level, ensuring that the bottoms of the cell culture plates are completely and horizontally immersed into the water, aligning the infrared probes 4 to the inner walls of blank cell culture plates wetted by culture media at an angle of 45 degrees and outside a distance of 5-6cm, setting the water bath temperature to be 10-12 ℃ higher than the required stimulation temperature (for example, the required stimulation temperature for an experiment is 52 ℃, and setting the water bath temperature to be 64 ℃, so that the heat conduction can be accelerated to pass through the bottom surface of the cell culture plates during the experiment, thereby accelerating the process of performing effective thermal stimulation on experimental cells), heating the water in the, an infrared temperature monitoring system (connected with a power supply 9) monitors the temperature change of the inner wall of a blank cell culture plate/bottle wetted by the culture medium, stops when the temperature of the inner wall can rise to the required stimulation temperature within 10 seconds, sets the water bath temperature as the initial experiment temperature, then repeatedly measures for three times, and takes the average value as the final experiment temperature;
b. formal experiments: setting the temperature of a water bath kettle as the final experiment temperature obtained in the step a, starting the water bath kettle to heat water in the water bath kettle, humidifying (namely immediately sucking away after adding a culture medium) a blank control culture plate 10 and sucking away the culture solution in a cell culture plate 11 needing to culture cells needing thermal stimulation on an ultra-clean workbench when the temperature of the water bath kettle rises to the final experiment temperature value, respectively placing the cell culture plate 11 containing the cells needing thermal stimulation and the cell culture plate 10 serving as a blank control on groove positions of 2 underframe, placing culture holes 13 containing adherent cells in the cell culture plate containing the cells needing thermal stimulation and culturing the cells in the blank control culture plate 10 containing control culture holes 12, wherein the lower part of the cell culture plate is 4mm below the water, the upper part of the cell culture plate is exposed above the water surface, and monitoring the culture medium humidified culture plate by an infrared temperature monitor, Blank control culture plate inner wall surface temperature, infrared probe are at 5-6cm departments apart from cell culture plate inner wall surface at 45 degrees angle, and temperature detection infrared ray 14 shines cell culture plate inner wall surface, presses stopwatch timing, and the heating (about 10 seconds) that reaches the warm time earlier, then carries out the heating stimulation of experiment required time, adds the lid rapidly after accomplishing, takes out the cell culture plate that contains the cultured cell, puts back in conventional incubator, accomplishes accurate and instantaneous heat stimulation experiment of cultured cell like this.
The bottom frame is a polyvinyl chloride bottom frame which is cuboid, a groove with a shape matched with the cell culture plate is arranged at the opening at the top, and the cell culture plate is clamped into the polyvinyl chloride bottom frame, so that the cell culture plate is fixed.
The water bath kettle is a precise water bath kettle with the temperature resolution of +/-0.1 ℃.
The infrared temperature monitoring system is an infrared temperature monitoring system with response time of +/-100 ms and temperature resolution of 0.1 ℃, and particularly is an IC-LI online infrared temperature monitoring system of the Huahong brand.
Claims (4)
1. A method for thermally stimulating cultured cells in cells, comprising the steps of:
the thermal stimulation device comprises a water bath kettle, an infrared temperature monitoring system, a cell culture plate/bottle, a bottom frame, a fixed support and a stopwatch, wherein an infrared probe of the infrared temperature monitoring system is fixed on the fixed support, at least 2 bottom frames are arranged in the water bath kettle in parallel and horizontally, and a groove for placing the cell culture plate/bottle is arranged on each bottom frame;
a. pre-experiment: pure sterilized water is filled in a water bath kettle, a blank cell culture plate/bottle is placed in the groove position of 1 underframe, the lower part of the cell culture plate/bottle is submerged in the water, 4mm away from the water surface, the upper opening is exposed above the water surface, the infrared probe forms an angle of 45 degrees, aligning the inner wall of the blank cell culture plate/flask wetted with culture medium outside the distance of 5-6cm, setting the temperature of the water bath at least 10-12 ℃ higher than the desired stimulation temperature, then the water bath kettle is electrified to heat the water in the water bath kettle, and simultaneously, the temperature change of the inner wall of the cell culture plate/bottle is monitored by an infrared temperature monitoring system, stopping when the temperature of the inner wall can rise to the required stimulation temperature within 10 seconds, setting the temperature of the water bath as the initial experiment temperature, then repeatedly measuring for at least three times, and taking the average value as the final experiment temperature;
b. formal experiments: opening a water bath kettle, setting the temperature of the water bath kettle to the final experiment temperature obtained in the step a, then starting the water bath kettle to heat water in the water bath kettle, respectively placing a cell culture plate/bottle which needs to thermally stimulate cultured cells and is free of a culture medium and a cell culture plate/bottle which is wetted by the culture medium and is used as a blank control in groove positions of 2 underframe when the water bath temperature reaches the final experiment temperature value, immersing the lower part of the cell culture plate/bottle in water, keeping the lower part of the cell culture plate/bottle 4mm away from the water surface, exposing the upper part of the cell culture plate/bottle above the water surface, monitoring the temperature of the inner surface of the cell culture plate/bottle wetted by the culture medium and used as the blank control by an infrared temperature monitoring system at an angle of 45 degrees outside a distance of 5-6cm, firstly heating for a temperature reaching time, then starting a stopwatch, carrying out heating stimulation for the time required by the experiment, the cell culture plate/flask containing the cultured cells is removed, thus completing a precise and transient thermal stimulation experiment of the cultured cells.
2. The method of claim 1, wherein the base frame is a polyvinyl chloride base frame, and the top opening is provided with a groove with a shape corresponding to the cell culture plate/bottle, so that the cell culture plate/bottle can be clamped into the polyvinyl chloride base frame, thereby fixing the cell culture plate/bottle.
3. The method of claim 1, wherein the water bath is a precision water bath with a temperature resolution of ± 0.1 ℃.
4. The method of claim 1, wherein the infrared temperature monitoring system is one with a response time of ± 100ms and a temperature resolution of 0.1 ℃.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910996034.3A CN111304079A (en) | 2019-10-18 | 2019-10-18 | Method for thermally stimulating cultured cells in cells |
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| CN201910996034.3A CN111304079A (en) | 2019-10-18 | 2019-10-18 | Method for thermally stimulating cultured cells in cells |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112825818A (en) * | 2020-12-31 | 2021-05-25 | 杭州电子科技大学 | Novel fruit fly thermal stimulation experiment device |
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2019
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Application publication date: 20200619 |