WO2019134405A1 - 培养装置及采用其的细胞培养方法 - Google Patents

培养装置及采用其的细胞培养方法 Download PDF

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Publication number
WO2019134405A1
WO2019134405A1 PCT/CN2018/107937 CN2018107937W WO2019134405A1 WO 2019134405 A1 WO2019134405 A1 WO 2019134405A1 CN 2018107937 W CN2018107937 W CN 2018107937W WO 2019134405 A1 WO2019134405 A1 WO 2019134405A1
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Prior art keywords
temperature sensitive
light emitting
temperature
sensitive layer
emitting structure
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PCT/CN2018/107937
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English (en)
French (fr)
Inventor
周适
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京东方科技集团股份有限公司
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Priority to US16/486,414 priority Critical patent/US20200032192A1/en
Publication of WO2019134405A1 publication Critical patent/WO2019134405A1/zh

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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
    • 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
    • C12M23/10—Petri dish
    • 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
    • C12M23/20—Material Coatings
    • 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
    • C12M23/46—Means for fastening
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • 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
    • C12M31/00—Means for providing, directing, scattering or concentrating light
    • C12M31/10—Means for providing, directing, scattering or concentrating light by light emitting elements located inside the reactor, e.g. LED or OLED
    • 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
    • C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates

Definitions

  • Embodiments of the present disclosure relate to a culture apparatus and a cell culture method using the same.
  • Cell membrane is a research hotspot in the field of tissue engineering in recent years, and has a wide range of applications in the treatment of diseases such as skin, cornea, heart and periodontitis.
  • the currently used cell membrane preparation device is a temperature-sensing culture dish device.
  • An embodiment of the present disclosure provides a culture apparatus, including:
  • a cold light emitting structure coupled to and overlapping the temperature sensitive layer, configured to emit cold light through the temperature sensitive layer.
  • the culture device further includes a heat insulating layer between the temperature sensitive layer and the luminescent light emitting structure, including a heat insulating material.
  • the luminescent light emitting structure includes scattering particles dispersed therein.
  • the luminescent light emitting structure includes a luminescent material that emits luminescent light after being excited or electrically excited, the luminescent material comprising at least one selected from the group consisting of a fluorescent material and a phosphorescent material.
  • the temperature sensitive layer is configured to be separated from the luminescent light emitting structure when the temperature exceeds a predetermined range.
  • the temperature sensitive layer comprises at least one selected from the group consisting of poly N-tetrahydrofurfuryl acrylamide, poly N-n-propyl acrylamide, and poly N-isopropyl acrylamide.
  • the culture device further includes a dish body, wherein the dish body includes a bottom wall and a side wall, the bottom wall and the side wall enclose an accommodation space, and the temperature sensitive layer is located in the Accommodate the space.
  • the luminescent light emitting structure comprises:
  • a light guide plate overlapping the temperature sensitive layer and located in the receiving space
  • the luminescent light emitting structure includes a plurality of luminescent LED light sources, located within the accommodating space, arranged in a matrix of rows and columns and overlapping the temperature sensitive layer.
  • the culture device further includes:
  • An electrode wiring layer is located inside the dish and on a side of the cold light emitting structure opposite to the temperature sensitive layer.
  • the culture device further includes a temperature adjustment layer located on the side wall of the dish.
  • the culture device further includes a fixation element located outside the dish and configured to secure the dish device.
  • the fixing element includes at least two positioning blocks, and each of the positioning blocks is provided with a positioning groove.
  • the fixation element is a locator and the dish is located within the locator.
  • the inner wall of the spacer is provided with a temperature regulating layer.
  • the culture device further includes an image recording device and a display unit electrically connected to each other,
  • the image recording device is configured to acquire an image of the temperature sensitive layer opposite to a surface of the cold light emitting structure
  • the display unit is configured to display the image acquired by the image recording device.
  • the culture device further includes: a temperature sensor and an alarm device electrically connected to each other,
  • the temperature sensor is configured to measure a temperature in the receiving space of the dish
  • the alarm device is configured to issue an alarm based on a temperature measured by the temperature sensor.
  • Another embodiment of the present disclosure provides a cell culture method using the culture device according to any one of the above, comprising:
  • the cold light emitting structure is used to emit cold light through the temperature sensitive layer to illuminate the cells to be cultured.
  • FIG. 1 is a schematic cross-sectional structural view of a petri dish device according to an embodiment of the present disclosure
  • FIG. 2 is a schematic cross-sectional structural view of another embodiment of a culture dish device according to an embodiment of the present disclosure
  • FIG. 3 is a schematic cross-sectional structural view of another embodiment of a petri dish device according to an embodiment of the present disclosure
  • FIG. 4 is a schematic diagram showing the external structure of a culture dish device according to an embodiment of the present disclosure
  • Figure 5 is a schematic view showing the external structure of the culture dish device according to an embodiment of the present disclosure.
  • FIG. 6 is a schematic perspective view showing the outer perspective of the culture dish device according to an embodiment of the present disclosure.
  • the temperature sensitive material is used as the base layer in the cell membrane culture process, so as to facilitate the harvesting and collection of the membrane at the later stage, and the temperature sensitive material is very sensitive to temperature changes, and the ordinary light source has a large heat dissipation amount, which may adversely affect the temperature sensitive material;
  • the actual culture liquid sampling often can only detect changes in the index of the culture fluid, such as pH, dissolved oxygen concentration, carbon dioxide concentration, lactic acid concentration, etc., but for the cell membrane area, thickness, uniformity, flatness and other cell membranes Important indicators, it is difficult to carry out effective detection; if the sensor is directly placed at the bottom of the culture vessel, it may be difficult to effectively measure the parameter change of the cell membrane due to the barrier of the temperature sensitive material. Therefore, the cell culture apparatus of the prior art is difficult to achieve real-time monitoring of the cell membrane culture process.
  • Embodiments of the present disclosure provide a culture dish apparatus and a culture method of a cell membrane capable of effectively controlling the temperature of a cell membrane culture process.
  • the culture dish device and the cell membrane culture method provided by the embodiments of the present disclosure can effectively control the temperature of the cell membrane culture process, facilitate the effective growth of the cell membrane, reduce the adverse effect of temperature on the cell membrane growth, and pass the present disclosure.
  • the culture dish device and the cell membrane culture method provided by the embodiments can also realize real-time detection of cell membrane growth parameters, and effectively monitor the growth process of the cell membrane.
  • this embodiment provides a petri dish apparatus including a dish body 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a temperature sensitive layer 106 and a luminescent light emitting structure 108 are disposed in the accommodating space M of the dish body 101.
  • the luminescent light emitting structure 108 is used to emit cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108.
  • the surface of the temperature sensitive layer 106 opposite the luminescent light emitting structure 108 is configured to be in direct contact with the cells to be cultured.
  • the luminescent light refers to visible light emitted from a non-incandescent light source at a low temperature, such as fluorescence, phosphorescence, bioluminescence or triboluminescence, etc., without a significant amount of infrared rays, so the heating effect is small.
  • the spacer layer 107 is, for example, a heat insulating layer including a heat insulating material having a thermal conductivity of less than 0.2 W/(m ⁇ K).
  • the temperature sensitive layer 106, the spacer layer 107, and the luminescent light emitting structure 108 overlap and are bonded to each other.
  • the spacer layer 107 may be omitted, and the temperature sensitive layer 106 and the cold light emitting structure 108 overlap and are bonded to each other.
  • the cell membrane 105 to be cultured is placed on the temperature sensitive layer 106, and the temperature sensitive layer 106 is used as a basal layer formed by the cell membrane to facilitate harvesting and collection of the cell membrane at a later stage.
  • the cold light emitting structure 108 of the present embodiment emits cold light to the cell membrane, which emits less heat, which can reduce the adverse effect of the light source on the growth of the cell membrane.
  • the arrangement of the spacer layer 107 effectively reduces the effect of the heat generated during the illumination of the luminescent emission structure on the internal ambient temperature of the culture dish apparatus. Therefore, the culture dish device provided in the embodiment can effectively control the temperature of the cell membrane culture process, and is beneficial to the effective growth of the cell membrane.
  • Embodiments of the present disclosure provide a petri dish apparatus, such as see FIG. 1, including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a temperature sensitive layer 106 and a luminescent light emitting structure 108 are disposed in the accommodating space M of the dish body 101.
  • the luminescent light emitting structure 108 is used to emit cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108.
  • the luminescent light emitting structure 108 includes scattering particles S that are uniformly dispersed in the luminescent light emitting structure 108 for uniform scattering of the luminescent light source emerging from the luminescent light emitting structure 108.
  • Embodiments of the present disclosure provide a petri dish apparatus, see FIG. 2, including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a temperature sensitive layer 106 and a luminescent light emitting structure 108 are disposed in the accommodating space M of the dish body 101.
  • the luminescent light emitting structure 108 is used to emit cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108. As shown in FIG.
  • the luminescent light emitting structure 108 includes a plurality of luminescent LED light sources 110 arranged in an array in the luminescent light emitting structure 108, using cold light LEDs that emit less heat relative to ordinary light sources.
  • the light source uniformly emits a cold light source to the cell membrane, which can reduce the adverse effect of the light source on the growth of the cell membrane.
  • scattering particles S are spaced apart in the cold light emitting structure 108.
  • the scattering particles are all located directly above the respective LED light sources 110, and there are no scattering particles directly above the gap of each of the LED light sources 110. In this way, it is advantageous for the cold light emitted from different regions of the cold light emitting structure 108 to be uniformly irradiated to the cell membrane 105 to be cultured.
  • Embodiments of the present disclosure provide a petri dish apparatus, see FIG. 3, including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a portion of the temperature sensitive layer 106 and the luminescent light emitting structure 108 is disposed in the accommodating space M of the dish 101, and the luminescent light emitting structure 108 is for emitting cold light between the temperature sensitive layer 106 and the portion of the luminescent light emitting structure 108.
  • a separation layer 107 is provided.
  • the luminescent light emitting structure 108 includes, for example, a luminescent LED light source 110 and a light guide plate 108'.
  • At least two luminescent LED light sources 110 are disposed outside the dish 101.
  • the light guide plate 108' is located inside the inner accommodation space M of the dish body 101.
  • the luminescent LED light source 110 is at the same horizontal position as the light guide plate 108' inside the dish.
  • the surface of the light guide plate 108' closest to the temperature sensitive layer 106 is flush with the surface of the luminescent LED light source 110 that is closest to the temperature sensitive layer 106.
  • the luminescent LED light source 110 emits luminescence, the luminescent light enters the light guide plate 108', and the cold light emitted from the cold light source is emitted to the cell membrane growth layer through the light guide plate 108'.
  • the density of the scattering particles dispersed around the cold light emitting structure 108 is greater than the density of the center, which is advantageous for better scattering of the cold LED light source around the cold light emitting structure to the cell membrane 105 to be cultured.
  • Embodiments of the present disclosure provide a culture dish apparatus including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a temperature sensitive layer 106 and a luminescent light emitting structure 108 are disposed in the accommodating space M of the dish body 101.
  • the luminescent light emitting structure 108 is used to emit cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108.
  • the luminescent light emitting structure 108 includes a luminescent material that can emit cold light upon exposure to light or electricity.
  • the cold light emitting material can emit light in a luminescent wavelength range such as fluorescence or phosphorescence after being electrically excited.
  • the cold light emitting material is an InGaN blue fluorescent material having an emission wavelength of about 460 nm, and a Ca3Sc2Si3O12:Ce green fluorescent material having an emission wavelength of about 500 nm.
  • the luminescent light emitted by the luminescent light emitting structure 108 is visible light.
  • luminescence has a wavelength in the range of 380 nm to 560 nm.
  • the light in the cold light wavelength range emits less heat, which can effectively reduce the influence of the heat generated by the light source on the growth of the cell membrane.
  • the luminescent material emitting the luminescent light can be selected from a luminescent material such as a fluorescent luminescent material or a phosphorescent luminescent material.
  • Embodiments of the present disclosure provide a culture dish apparatus including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • a temperature sensitive layer 106 and a luminescent light emitting structure 108 are disposed in the accommodating space M of the dish body 101.
  • the luminescent light emitting structure 108 is used to emit cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108.
  • the temperature sensitive layer 106 is, for example, a polymer temperature sensitive hydrogel or water to form a polymer temperature sensitive hydrogel, and the polymer temperature sensitive hydrogel can undergo a volume phase change with temperature.
  • the polymer temperature-sensitive hydrogel is a heat-increasing temperature-sensitive gel which exhibits a contracted state when it is lower than the phase transition temperature, and exhibits an expanded state when the temperature is higher than the phase transition temperature.
  • the polymeric temperature sensitive hydrogel may also be a heat shrinkable temperature sensitive gel that is in an expanded state below the phase transition temperature and in a contracted state when the temperature is above the phase transition temperature.
  • the temperature sensitive layer 106 is selected from one or more of a temperature sensitive material poly N-tetrahydrofurfuryl acrylamide, poly N-n-propyl acrylamide, poly N-isopropyl acrylamide, and these temperature sensitive materials are
  • the temperature change in the culture dish is sensitive.
  • the culture temperature in the culture dish device changes abnormally, that is, when the temperature in the culture dish deviates from the initial preset cell membrane growth temperature range, the temperature sensitive layer 106 is detached from the separation layer 107 to prevent temperature change from occurring on the cell membrane growth.
  • the adverse effects For example, the temperature sensitive layer 106 is detached from the separation layer 107 when the temperature deviates from the initial predetermined cell membrane growth temperature range of ⁇ 2 ° C to ⁇ 5 ° C.
  • the initial temperature of the culture dish is preset to a temperature range of 36 to 38 ° C, and when the temperature in the culture dish deviates from the initial preset cell membrane growth temperature range by ⁇ 3 ° C, the temperature in the culture dish device reaches At 40 ° C, the temperature sensitive layer 106 is detached from the separation layer 107 to prevent adverse effects of temperature changes on cell membrane growth.
  • the culture dish apparatus of this embodiment further includes a temperature sensor 112 and an alarm device 113.
  • the temperature sensor 112 is placed in the accommodating space M of the dish for monitoring the temperature inside the accommodating space M of the dish; the alarm device 113 is placed outside the dish 101 .
  • the temperature sensor 112 and the alarm device 113 are electrically connected.
  • the temperature sensor 112 transmits an alarm signal of an abnormal temperature change to the alarm device 113, and the alarm device 113 alarms.
  • the alarm device 113 may be a hummingbird alarm device or the like, which is not specifically limited herein.
  • the specific installation position and number of the alarm device and the temperature sensor are not specifically limited herein.
  • Embodiments of the present disclosure provide a culture dish apparatus including a dish 101.
  • the dish body includes a bottom wall W1 and a side wall W2.
  • the bottom wall W1 and the side wall W2 enclose a receiving space M.
  • the inside of the dish body 101 is provided with a temperature sensitive layer 106 and a luminescent light emitting structure 108 for emitting cold light, and a partition layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108.
  • an electrode wiring layer 109 is disposed in the housing space M of the dish of the petri dish apparatus, and the electrode wiring layer 109 is located on the other side of the cold light emitting structure 108 opposite to the temperature sensitive layer 106.
  • the electrode wiring layer 109 is disposed at the bottom of the dish for arranging the power supply lines required for the cold light source layer in the culture dish.
  • the embodiment provides a petri dish device, comprising a dish body 101.
  • the inside of the dish body 101 is provided with a temperature sensitive layer 106 and a cold light emitting structure 108, and the cold light emitting structure 108 is used for emitting cold light, the temperature sensitive layer 106 and A partition layer 107 is disposed between the cold light emitting structures 108.
  • a fixing member is disposed outside the culture dish, and the fixing member is a positioning block 103.
  • the positioning block 103 is provided with a positioning groove 104, and the positioning groove 104 is used for clamping with other devices to realize cultivation.
  • the dish device is fixed, and at least two positioning blocks are arranged outside the culture dish for fixing the culture dish device, so as to prevent the external force from affecting the environment inside the culture dish and affecting the growth of the cell membrane.
  • Embodiments of the present disclosure provide a petri dish apparatus including a dish body 101 having a temperature sensitive layer 106 and a cold light emitting structure 108 disposed therein, the cold light emitting structure 108 for emitting cold light, and the temperature sensitive layer 106 A separation layer 107 is disposed between the luminescent light emitting structure 108 and the luminescent light emitting structure 108.
  • the culture dish device is further provided with a temperature adjustment layer, and the temperature adjustment layer is a semiconductor temperature control element. As shown in FIG. 5, the temperature adjustment element 111 is closely arranged on the outer wall of the culture dish device for regulating the temperature inside the culture dish device. The heat generated by preventing the cold light emitting structure from emitting light for a long time causes a change in temperature in the petri dish device.
  • the temperature regulating layer may also be closely arranged on the inner wall of the culture dish device.
  • the temperature adjustment layer is, for example, a semiconductor refrigerating sheet, and the purpose of controlling the constant temperature is achieved by changing the magnitude of the current and the direction of the current.
  • the embodiment provides a petri dish device, comprising a dish body 101.
  • the inside of the dish body 101 is provided with a temperature sensitive layer 106 and a cold light emitting structure 108, and the cold light emitting structure 108 is used for emitting cold light, the temperature sensitive layer 106 and A partition layer 107 is disposed between the cold light emitting structures 108.
  • the outside of the dish body 101 is provided with a fixing member, the fixing member is a culture dish holder 103, and the culture dish device is further provided with a temperature adjusting layer and a temperature.
  • the adjustment layer is a semiconductor temperature control element, and the temperature adjustment element 111 is disposed on the inner wall of the culture dish holder 103, and the temperature adjustment elements 111 are closely arranged along the inner wall of the culture dish holder 103 to prevent the bottom light from being diverged.
  • the culture dish and the culture dish holder 103 are matched in shape, and the shape may be selected from any one of a circle, a rectangle, a square, a diamond, and a trapezoid.
  • the embodiment of the present disclosure provides a petri dish device, including a dish body 101 and a dish cover 102.
  • the inside of the dish body 101 is provided with a temperature sensitive layer 106 and a cold light emitting structure 108, and the cold light emitting structure 108 is used for emitting cold light.
  • a spacer layer 107 is disposed between the temperature sensitive layer 106 and the luminescent light emitting structure 108, and the temperature sensitive layer 106 and the spacer layer 107 are, for example, light transmitting layers.
  • the petri dish apparatus further includes an image recording device 112 and a display unit 113, and the image recording device 114 and the display unit 115 are electrically connected.
  • the image recording device 114 is configured to acquire an image of the temperature sensitive layer 106 opposite the surface of the luminescent light emitting structure 108.
  • the display unit 115 is configured to display the image acquired by the image recording device 114.
  • the image recording apparatus is configured to acquire and grow a growth process and growth parameters of the cell membrane 105 for displaying a growth process and a growth parameter of the cell membrane 105 to be cultured for acquisition by the image recording apparatus.
  • the image recording device 112 and the display unit 113 may be mounted on the dish cover 102, for example, see FIG. 4; or mounted on the outer wall of the dish, etc., and are not specifically limited herein.
  • the image recording device is a device that can be used to record images, such as a CCD image sensor, an infrared image sensor, or an electron microscope.
  • the display unit is an LCD, an OLED or other display device capable of displaying an image.
  • the prior art monitors the growth process of a cell membrane, usually by extracting the culture solution in the culture dish, and then detecting the change of the index in the culture solution, such as pH value, dissolved oxygen concentration, carbon dioxide concentration, lactic acid concentration and the like, and further analyzing The growth of the cell membrane.
  • the cell membrane formation process includes the incubation of the cell membrane in the latent period, the growth of the cell membrane during the growth phase, and the saturation of the cells in the stagnant phase. The cells stop proliferating.
  • direct monitoring of the above cell growth process is difficult to achieve by existing techniques.
  • the growth index of the cell membrane mainly refers to the growth of the cell membrane itself, such as the cell membrane area, thickness, uniformity and flatness.
  • the cell membrane 105 to be cultured is placed on the temperature sensitive layer 106.
  • the temperature sensitive layer 106 is used as a basal layer formed by the cell membrane to facilitate the harvest of the cell membrane in the later stage.
  • the cold light can reduce the disadvantage of the light source on the growth of the cell membrane.
  • the effect of the spacer layer 107 is further reduced by the influence of the heat generated during the illumination of the cold light emitting structure on the temperature of the cell membrane on the temperature sensitive layer 106; on the other hand, the cell membrane irradiated by the cold light is relatively cold and luminescent.
  • the topography of the cell structure can be observed on the other side of the emission structure 108, and the morphology of the cell membrane captured by the cold light illumination is recorded by the image recording device, and the captured cell membrane is passed through the analysis element in the image recording device.
  • the appearance of the film is calculated and calculated to include area, thickness, uniformity, flatness, etc.
  • the image recording device transmits the shape of the captured cell membrane and the parameters calculated by calculation, including area, thickness, uniformity, and flatness, to the display unit, and the display unit may include a cell membrane topography. Growth parameters such as area, thickness, uniformity, and flatness enable direct real-time detection of cell membrane growth.
  • the display unit can program to display a 3D simulation picture of the cell membrane growth process in the culture dish device, as well as other important parameter indicators.
  • the display unit is disposed on an outer wall of the dish, and the display unit is a transparent display unit.
  • the above embodiments are described by taking a temperature sensitive layer, a luminescent light emitting structure, and a separating layer parallel to the bottom wall W1 of the culture dish as an example, but between the temperature sensitive layer, the cold light emitting structure, and the separation layer. It is also possible to form a certain angle, for example: an angle of 0-90°.
  • the method is as follows: the temperature sensitive layer, the cold light emitting structure, and the partition layer form a three-dimensional structure, located at or near the center of the culture dish, and perpendicular to the bottom of the culture dish, wherein the temperature sensitive layer of the three-dimensional structure is located in the three-dimensional structure.
  • the cold light emitting structure is located inside the three-dimensional structure. That is, only the cell membrane to be cultured can be disposed on one side of the relative temperature sensing layer, and the cold light emitting structure is disposed on the other side of the temperature sensing layer, and the following methods are also similar.
  • Embodiments of the present disclosure provide a method for culturing a cell membrane, comprising:
  • Step 1 a temperature sensitive layer and a cold light emitting structure are disposed inside the culture dish body, and a separation layer is disposed between the temperature sensitive layer and the cold light emitting structure;
  • Step 2 adjusting a temperature range of the culture dish device, and placing a cell membrane to be cultured on a side of the temperature sensitive layer opposite to the cold light emitting structure;
  • Step 3 The cold light is emitted toward the cell membrane by the cold light emitting structure, and the cold light is emitted to the cell membrane at least partially through the temperature sensitive layer and the separation layer disposed in the culture dish device.
  • the temperature sensitive layer is detached from the separation layer when the temperature inside the culture dish device deviates from an initial predetermined cell membrane growth temperature range.
  • the temperature range of the culture dish device is adjusted. After the culture cell membrane 105 is attached to the temperature sensitive layer 106, the temperature sensitive layer 106 is used as a basal layer formed by the cell membrane to facilitate harvesting and collecting the cell membrane at a later stage.
  • the cold light emitting structure 108 of the present embodiment which emits less heat emits cold light to the cell membrane, which emits less heat, which can reduce the adverse effect of the light source on the growth of the cell membrane.
  • the setting of the partition layer 107 effectively reduces the cold light emission.
  • the morphology of the cell membrane can be observed from the other side of the relatively cold light emitting structure 108, and the morphology of the cell membrane captured by the cold light irradiation is recorded by the image recording device, and the image is passed through the image.
  • the analysis component in the recording device calculates parameters including area, thickness, uniformity, and flatness by analyzing the appearance of the captured cell membrane, and the image recording device calculates the morphology of the captured cell membrane and the analysis and calculation. Parameters such as area, thickness, uniformity, and flatness are transmitted to the display unit. At this time, the growth parameters including cell membrane morphology, area, thickness, uniformity, and flatness can be displayed in the display unit to realize the growth process of the cell membrane. Direct real-time detection.
  • the temperature sensitive layer 106 is selected from one or more of a temperature sensitive material poly N-tetrahydrofurfuryl acrylamide, poly N-n-propyl acrylamide, poly N-isopropyl acrylamide, and these temperature sensitive materials are
  • the temperature change in the culture dish is sensitive, and when the culture temperature in the culture dish device changes abnormally, that is, when the temperature in the culture dish deviates from the initial preset cell membrane growth temperature range, the temperature sensitive layer 106 is detached from the separation layer 107, Prevents adverse effects of temperature changes on cell membrane growth. For example, the temperature sensitive layer 106 is detached from the separation layer 107 when the temperature deviates from the initial predetermined cell membrane growth temperature range of ⁇ 2 ° C to ⁇ 5 ° C.
  • the initial temperature of the culture dish is preset to a temperature range of 36 to 38 ° C, and when the temperature in the culture dish deviates from the initial preset cell membrane growth temperature range by ⁇ 3 ° C, the temperature in the culture dish device reaches At 40 ° C, the temperature sensitive layer 106 is detached from the separation layer 107 to prevent adverse effects of temperature changes on cell membrane growth.

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Abstract

提供一种培养装置及采用所述培养装置的细胞培养方法。所述培养装置包括：温度敏感层(106)，以及冷光发射结构(108)。所述冷光发射结构（108）与所述温度敏感层(106)连接且重叠，构造为发出冷光穿过所述温度敏感层(106)。这样，可有效降低光源散发的热量对于细胞膜片生长产生的不利影响。

Description

培养装置及采用其的细胞培养方法
本申请要求于2018年1月3日递交的中国专利申请第201810004126.4号的优先权，在此全文引用上述中国专利申请公开的内容以作为本申请的一部分。
技术领域
本公开的实施例涉及一种培养装置及采用其的细胞培养方法。
背景技术
细胞膜片是近年来组织工程领域的研究热点，在皮肤、角膜、心脏、牙周等相关疾病的治疗中具有广泛的应用。目前常用的细胞膜片制备装置是温度感应式培养皿装置。
发明内容
本公开实施例提供一种培养装置，包括：
温度敏感层，以及
冷光发射结构，与所述温度敏感层连接且重叠，构造为发出冷光穿过所述温度敏感层。
在一个示例中，所述培养装置还包括：所述温度敏感层和所述冷光发射结构之间的隔热层，包括热绝缘材料。
在一个示例中，所述冷光发射结构包括分散在其中的散射粒子。
在一个示例中，所述冷光发射结构包括受光或电激发后发射冷光的发光材料，所述发光材料包括选自荧光材料和磷光材料构成的组中的至少一种。
在一个示例中，所述温度敏感层构造为在温度超过预设范围时与所述冷光发射结构分离。
在一个示例中，所述温度敏感层包括选自聚N-四氢糠基丙烯酰胺、聚N-正丙基丙烯酰胺、聚N-异丙基丙烯酰胺构成的组中的至少一种。
在一个示例中，所述培养装置还包括皿体，其中，所述皿体包括底壁和 侧壁，所述底壁和所述侧壁围成一容纳空间，所述温度敏感层位于所述容纳空间内。
在一个示例中，所述冷光发射结构包括：
与所述温度敏感层重叠的且位于所述容纳空间内的导光板；以及
位于所述皿体之外的光源。
在一个示例中，所述冷光发射结构包括多个冷光LED光源，位于所述容纳空间内，排布为多行多列的矩阵且与所述温度敏感层重叠。
在一个示例中，所述的培养装置还包括：
电极布线层，位于所述皿体的内部，且位于所述冷光发射结构的相反于所述温度敏感层的一侧。
在一个示例中，所述培养装置还包括：温度调节层，位于所述皿体的所述侧壁。
在一个示例中，所述培养装置还包括：固定元件，位于所述皿体的外部，构造为固定所述培养皿装置。
在一个示例中，所述固定元件包括至少两个定位块，每个所述定位块中设置有定位槽。
在一个示例中，所述固定元件为定位架，所述皿体位于所述定位架内。
在一个示例中，所述定位架的内壁设置有温度调节层。
在一个示例中，所述的培养装置还包括彼此电连接的图像记录装置和显示单元，
其中，所述图像记录装置构造为获取所述温度敏感层的相反于冷光发射结构的表面的图像；
所述显示单元构造为显示所述图像记录装置获取的所述图像。
在一个示例中，所述培养装置还包括：彼此电连接的温度传感器和报警装置，
其中，所述温度传感器构造为测量所述皿体的所述容纳空间内的温度，
所述报警装置构造为根据所述温度传感器测得的温度发出警报。
本公开的另一实施例提供一种采用上述任一项所述培养装置的细胞培养方法，包括：
将待培养细胞置于所述温度敏感层相反于所述冷光发射结构的一侧；以 及
采用所述冷光发射结构发射冷光穿过所述温度敏感层而照射到所述待培养细胞。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例的附图作简单地介绍，显而易见地，下面描述中的附图仅仅涉及本公开的一些实施例，而非对本公开的限制。
图1为本公开的实施例提供的一实施例培养皿装置的剖面结构示意图；
图2为本公开的实施例提供的另一实施例培养皿装置的剖面结构示意图；
图3为本公开的实施例提供的另一实施例培养皿装置的剖面结构示意图；
图4为本公开的实施例提供的培养皿装置外部结构示意图；
图5为本公开的实施例提供的培养皿装置外部结构示意图；以及
图6为本公开的实施例提供的培养皿装置外部透视结构示意图。
具体实施方式
为使本公开实施例的目的、技术方案和优点更加清楚，下面将结合本公开实施例的附图，对本公开实施例的技术方案进行清楚、完整地描述。显然，所描述的实施例是本公开的一部分实施例，而不是全部的实施例。基于所描述的本公开的实施例，本领域普通技术人员在无需创造性劳动的前提下所获得的所有其他实施例，都属于本公开保护的范围。
细胞膜片培养过程中采用温敏材料作为基底层，以便于后期膜片的收获和采集，而温敏材料对于温度变化非常敏感，采用普通光源发散热量较大，可能对温敏材料产生不利影响；此外，实际培养液取样，往往只能检测培养液中的指标变化，如pH值、溶解氧浓度、二氧化碳浓度、乳酸浓度等，但是对于细胞膜片的面积、厚度、均一程度、平整程度等细胞膜片重要指标，难以进行有效检测；如果在培养容器底部直接设置传感器，由于存在温敏材料的阻隔，可能难以有效测量细胞膜片的参数变化。因此，现有技术中的细 胞培养装置难以实现对细胞膜片培养过程的实时监测。
本公开的实施例提供一种能够有效控制细胞膜片培养过程温度的培养皿装置以及细胞膜片的培养方法。本公开的实施例提供的培养皿装置及细胞膜片培养方法，可实现细胞膜片培养过程的温度的有效控制，有利于细胞膜片的有效生长，降低温度对于细胞膜片生长的不利影响，并且通过本公开的实施例提供的培养皿装置及细胞膜片培养方法还能够实现细胞膜片生长参数的实时检测，有效监控细胞膜片的生长过程。
如图1所示，本实施例提供一种培养皿装置，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。
例如，温度敏感层106的相反于冷光发射结构108的表面构造为与待培养细胞直接接触。
这里，冷光是指来低温下从非白炽光源发出的可见光，如荧光，磷光，生物发光或摩擦发光等，没有明显的红外线量，因此加热效果很小。
分隔层107例如为隔热层，包括导热率小于0.2W/(m·K)的热绝缘材料。温度敏感层106、分隔层107和冷光发射结构108彼此重叠且结合在一起。在另一示例中，分隔层107可以省略，温度敏感层106和冷光发射结构108彼此重叠且结合在一起。
如图1所示，待培养细胞膜片105放置于温度敏感层106之上，以温度敏感层106作为细胞膜片生成的基底层，以便于后期对细胞膜片的收获和采集。相对于普通热光源，本实施例的冷光发射结构108向细胞膜片出射冷光，其散发热量少，可降低光源对于细胞膜片生长产生的不利影响。分隔层107的设置有效降低冷光发射结构发光过程中所产生的热量对于培养皿装置内部环境温度的影响。因此，本实施例提供的培养皿装置，可实现细胞膜片培养过程的温度的有效控制，有利于细胞膜片的有效生长。
本公开实施例提供一种培养皿装置，例如参见图1，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108，所 述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。所述冷光发射结构108包括散射粒子S，所述散射粒子均匀分散于冷光发射结构108中，用于冷光发射结构108内出射的冷光光源均匀散射出去。
本公开实施例提供一种培养皿装置，参见图2，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。如图2所示，所述冷光发射结构108包括多个冷光LED光源110，所述冷光LED光源110阵列式排布于所述冷光发射结构108内，利用相对于普通光源散发热量少的冷光LED光源，均匀向细胞膜片出射冷光光源，可降低光源对于细胞膜片生长产生的不利影响。
可选的，在冷光发射结构108中间隔式分散有散射粒子S。例如，散射粒子均位于各个LED光源110的正上方，各个LED光源110的间隙的正上方没有散射粒子。这样，有利于冷光发射结构108不同区域出射的冷光之间的也能够均匀的向待培养细胞膜片105照射。
本公开实施例提供一种培养皿装置，参见图3，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108的一部分，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108的所述部分之间设置有分隔层107。如图3所示，冷光发射结构108例如包括冷光LED光源110和导光板108’。至少两个冷光LED光源110设置于皿体101外部。导光板108’位于皿体101的内部容纳空间M内。所述冷光LED光源110与所述皿体内部的导光板108’处于同一水平位置。例如，导光板108’的最靠近温度敏感层106的表面与冷光LED光源110的最靠近温度敏感层106的表面齐平。当冷光LED光源110发射冷光，冷光照射进入导光板108’内，通过导光板108’将冷光源发出的冷光向细胞膜片生长层出射。将冷光LED光源110置于皿体101的外部，可进一步降低冷光LED光源110在皿体内产生热量对于细胞膜片生长环境的影响。
可选的，所述散射粒子在冷光发射结构108周边分散的密度大于中心的 分散密度，有利于将冷光发射结构周边的冷光LED光源较好的散射向待培养细胞膜片105照射。
本公开实施例提供一种培养皿装置，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。所述冷光发射结构108包括受光或电激发后可以发射冷光的发光材料。冷光发光材料在受到电激发后能够发射荧光或磷光等冷光波长范围的光，例如，冷光发光材料为发射波长在460nm左右的InGaN蓝光荧光材料、发射波长在500nm左右的Ca3Sc2Si3O12:Ce绿色荧光材料中的一种或两种。例如，冷光发射结构108发出的冷光为可见光。例如，冷光的波长范围在380nm-560nm。
冷光波长范围内的光散发的热量较低，可有效降低光源产生热量对于细胞膜片生长的影响，发射冷光的发光材料可选自荧光发光材料或磷光发光材料等冷光材料。
本公开实施例提供一种培养皿装置，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的容纳空间M内设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。温度敏感层106例如本身为高分子温敏水凝胶或者遇水能够形成高分子温敏水凝胶，且该高分子温敏水凝胶能够随温度变化而发生体积相变。例如，该高分子温敏水凝胶为热涨温敏凝胶，其在低于相转变温度时呈收缩状态，当温度高于相转变温度时则呈现出膨胀状态。该高分子温敏水凝胶也可以为热缩温敏凝胶，其在低于相转变温度时呈膨胀状态，而当在温度高于相转变温度时处于收缩状态。
所述温度敏感层106选用温敏材料聚N-四氢糠基丙烯酰胺、聚N-正丙基丙烯酰胺、聚N-异丙基丙烯酰胺中的一种或几种，这些温敏材料对于培养皿内温度的变化反应灵敏。当培养皿装置内培养温度发生异常改变时，即当培养皿内温度偏离初始预设的细胞膜片生长温度范围时，温度敏感层106从分隔层107脱离，以防止温度的改变对细胞膜片生长产生的不利影响。例如： 温度偏离初始预设的细胞膜片生长温度范围±2℃-±5℃时温度敏感层106从分隔层107脱离。例如其中一种方式：培养皿内温度初始预设的培养温度范围是36～38℃，当培养皿内温度偏离初始预设的细胞膜片生长温度范围±3℃时，即培养皿装置内温度达到40℃时，温度敏感层106从分隔层107脱离，以防止温度的改变对细胞膜片生长产生的不利影响。
本实施例所述培养皿装置还包括温度传感器112和报警装置113。例如参见图3，所述温度传感器112置于所述皿体的容纳空间M内，用于监测皿体的容纳空间M内的温度情况；所述报警装置113置于所述皿体101的外部。所述温度传感器112和所述报警装置113电连接。在培养皿内温度发生改变温度敏感层106从隔离层分隔层107脱离时，同时温度传感器112将温度异常变化的报警信号传递给报警装置113，报警装置113报警。所述报警装置113可以是蜂鸟报警装置等，在此不做具体限定。所述报警装置与所述温度传感器的具体安装位置和数量在此不做具体限定。
本公开实施例提供一种培养皿装置，包括皿体101。所述皿体包括底壁W1和侧壁W2。底壁W1和侧壁W2围成一容纳空间M。所述皿体101的内部设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。如图2所示，所述培养皿装置的皿体的容纳空间M内还设置有电极布线层109，电极布线层109位于冷光发射结构108相反于温度敏感层106的另一侧。电极布线层109设置在皿体底部，用于布置培养皿内冷光光源层所需电源线路。
本实施例提供一种培养皿装置，包括皿体101，所述皿体101的内部设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。如图2和图4所示，培养皿外部设置有固定元件，所述固定元件为定位块103，定位块103内设置有定位槽104，定位槽104用于与其他装置的卡合而实现培养皿装置的固定，培养皿外部设置有至少两个定位块，用于固定培养皿装置，避免外界作用力影响培养皿内环境进而影响细胞膜片的生长。
本公开实施例提供一种培养皿装置，包括皿体101，所述皿体101的内部设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107。所 述培养皿装置还设置有温度调节层，温度调节层选用半导体温控元件，如图5所示，温度调节元件111紧密排列于培养皿装置的外壁，用于调控培养皿装置内的温度，防止冷光发射结构发射光时间过长而产生的热量引起培养皿装置内温度的改变。
可选的，温度调节层也可紧密排列于培养皿装置的内壁。
例如，温度调节层例如为半导体制冷片，通过改变电流大小，电流方向来达到控制恒定温度的目的。
本实施例提供一种培养皿装置，包括皿体101，所述皿体101的内部设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107，如图6所示，皿体101外部设置有固定元件，所述固定元件为培养皿架103，所述培养皿装置还设置有温度调节层，温度调节层选用半导体温控元件，所述温度调节元件111设置于培养皿架103内壁，温度调节元件111沿培养皿架103内壁紧密排列，防止底部光线发散。
所述的培养皿和培养皿架103的形状相配合设计，形状可选自圆形、矩形、正方形、菱形、梯形中的任意一种。
本公开实施例提供一种培养皿装置，包括皿体101和皿盖102，所述皿体101的内部设置有温度敏感层106和冷光发射结构108，所述冷光发射结构108用于出射冷光，温度敏感层106和冷光发射结构108之间设置有分隔层107，所述温度敏感层106和所述分隔层107例如均为光透过层。例如，参见图4，所述培养皿装置还包括图像记录装置112和显示单元113，所述图像记录装114和显示单元115电连接。所述图像记录装置114构造为获取所述温度敏感层106的相反于冷光发射结构108的表面的图像。所述显示单元115构造为显示所述图像记录装置114获取的所述图像。例如，所述图像记录装置用于获取并待培养细胞膜片105的生长过程及生长参数，所述显示单元用于显示图像记录装置用于获取的待培养细胞膜片105的生长过程及生长参数。
可选的，所述图像记录装置112和显示单元113可以安装在皿盖102上，例如参见图4；或者安装于皿体外壁等，在此不做具体限定。
可选的，所述图像记录装置为如CCD图像传感器、红外图像传感器、 或电子显微镜等可用于记录影像的装置。
可选的，所述显示单元为LCD、OLED或其他可显示图像的显示器件。
现有技术监测细胞膜片的生长过程，通常情况下是提取培养皿中的培养液，然后检测培养液中的指标变化，如pH值、溶解氧浓度、二氧化碳浓度、乳酸浓度等参数值，进而分析细胞膜片的生长情况。细胞膜片的生成过程包括潜伏期细胞膜片贴壁、增长期细胞膜片生长过程和停滞期细胞数量达到饱和度后，细胞停止增殖，然而通过现有的技术难以实现对上述细胞生长过程的直接监测。
细胞膜片的生长指标主要是指其细胞膜片面积、厚度、均一度、平整度等细胞膜片自身参数的增长。
将待培养细胞膜片105放置于温度敏感层106之上，待培养细胞膜片105贴壁于温度敏感层106后，以温度敏感层106作为细胞膜片生成的基底层，以便于后期对细胞膜片的收获和采集，利用相对于普通光源散发热量少的冷光发射结构108出射冷光，冷光至少部分经过温度敏感层106和分隔层107向细胞膜片方向出射，一方面冷光可降低光源对于细胞膜片生长产生的不利影响，所设置的分隔层107的可进一步降低冷光发射结构发光过程中所产生的热量对于温度敏感层106上细胞膜片的温度影响；另一方面，透过冷光照射的细胞膜片，从相对与冷光发射结构108的另一侧可观测到细胞膜片的形貌，通过所述图像记录装置记录透过冷光照射所捕捉的细胞膜片的形貌，通过图像记录装置中的分析元件通过所捕捉到的细胞膜片外貌分析计算得出包括面积、厚度、均一度、平整度等参数，图像记录装置将所捕捉的细胞膜片的形貌、以及经过分析计算得出包括面积、厚度、均一度、平整度等参数传送给显示单元，此时显示单元中可呈现包括细胞膜片形貌、面积、厚度、均一度、平整度等生长参数，实现对细胞膜片生长过程的直接实时检测。
进一步，显示单元可通过程序设定显示所述培养皿装置中细胞膜片生长过程的3D模拟图片，以及其他重要参数指标。
可选的，所述显示单元设置于所述皿体的外壁，所述显示单元为透明显示单元。
可以理解的是，上述实施例均是以温度敏感层、冷光发射结构、分隔层相互平行于培养皿底壁W1为例进行介绍的，但温度敏感层、冷光发射结构、 分隔层三者之间形成一定角度也是可以的，例如：角度为0-90°。例如：方式为：温度敏感层、冷光发射结构、分隔层形成立体结构，位于培养皿的约中心位置或者靠近培养皿内壁位置，且垂直于培养皿底部，其中，立体结构的温度敏感层位于立体结构外侧，靠近培养液，冷光发射结构位于立体结构内侧。即只需待培养的细胞膜片能设置在相对温度感应层的一侧，冷光发射结构设置在相对温度感应层的另一侧即可，下述方法也类同。
本公开实施例提供一种细胞膜片的培养方法，包括：
Step1：培养皿体内部设置温度敏感层和冷光发射结构，在所述温度敏感层和所述冷光发射结构之间设置分隔层；
Step2：调节培养皿装置温度范围，将待培养细胞膜片置于所述温度敏感层相反所述冷光发射结构的一侧；
Step3：通过冷光发射结构向所述细胞膜片方向出射冷光，冷光至少部分经过所述温度敏感层和培养皿装置内设置的分隔层向细胞膜片方向出射。
进一步，当所述培养皿装置内温度偏离初始预设的细胞膜片生长温度范围时时，所述温度敏感层从所述分隔层脱离。
调节培养皿装置温度范围，待培养细胞膜片105贴壁于温度敏感层106后，以温度敏感层106作为细胞膜片生成的基底层，以便于后期对细胞膜片的收获和采集。
相对于普通热光源，散发热量少的本实施例的冷光发射结构108向细胞膜片出射冷光，其散发热量少，可降低光源对于细胞膜片生长产生的不利影响，分隔层107的设置有效降低冷光发射结构发光过程中所产生的热量对于培养皿装置内部环境温度的影响。透过冷光照射的细胞膜片，从相对与冷光发射结构108的另一侧可观测到细胞膜片的形貌，通过所述图像记录装置记录透过冷光照射所捕捉的细胞膜片的形貌，通过图像记录装置中的分析元件通过所捕捉到的细胞膜片外貌分析计算得出包括面积、厚度、均一度、平整度等参数，图像记录装置将所捕捉的细胞膜片的形貌、以及经过分析计算得出包括面积、厚度、均一度、平整度等参数传送给显示单元，此时显示单元中可呈现包括细胞膜片形貌、面积、厚度、均一度、平整度等生长参数，实现对细胞膜片生长过程的直接实时检测。
所述温度敏感层106选用温敏材料聚N-四氢糠基丙烯酰胺、聚N-正丙 基丙烯酰胺、聚N-异丙基丙烯酰胺中的一种或几种，这些温敏材料对于培养皿内温度的变化反应灵敏，当培养皿装置内培养温度发生异常改变时，即当培养皿内温度偏离初始预设的细胞膜片生长温度范围时，温度敏感层106从分隔层107脱离，以防止温度的改变对细胞膜片生长产生的不利影响。例如：温度偏离初始预设的细胞膜片生长温度范围±2℃-±5℃时温度敏感层106从分隔层107脱离。例如其中一种方式：培养皿内温度初始预设的培养温度范围是36～38℃，当培养皿内温度偏离初始预设的细胞膜片生长温度范围±3℃时，即培养皿装置内温度达到40℃时，温度敏感层106从分隔层107脱离，以防止温度的改变对细胞膜片生长产生的不利影响。
有以下几点需要说明：
(1)本实发明公开的实施例附图中，只涉及到与本公开的实施例公开实施例涉及到的结构，其他结构可参考通常设计。
(2)在不冲突的情况下，本公开的实施例公开同一实施例及不同实施例中的特征可以相互组合。
以上实施方式仅被配置为说明本公开的实施例，而并非对本公开的实施例的限制，有关技术领域的普通技术人员，在不脱离本公开的实施例的精神和范围的情况下，还可以做出各种变化和变型，因此所有等同的技术方案也属于本公开的实施例的范畴，本公开的保护范围应由权利要求限定。

Claims (18)

  1. 一种培养装置，包括：
    温度敏感层，以及
    冷光发射结构，与所述温度敏感层连接且重叠，构造为发出冷光穿过所述温度敏感层。
  2. 根据权利要求1所述的培养装置，还包括：所述温度敏感层和所述冷光发射结构之间的隔热层，包括热绝缘材料。
  3. 根据权利要求1或2所述的培养装置，其中，所述冷光发射结构包括分散在其中的散射粒子。
  4. 根据权利要求1至3中任一项所述的培养装置，其中，所述冷光发射结构包括受光或电激发后发射冷光的发光材料，所述发光材料包括选自荧光材料和磷光材料构成的组中的至少一种。
  5. 根据权利要求1至4中任一项所述的培养装置，其中，所述温度敏感层构造为在温度超过预设范围时与所述冷光发射结构分离。
  6. 根据权利要求5所述的培养装置，其中，所述温度敏感层包括选自聚N-四氢糠基丙烯酰胺、聚N-正丙基丙烯酰胺、聚N-异丙基丙烯酰胺构成的组中的至少一种。
  7. 根据权利要求1至6中任一项所述的培养装置，还包括皿体，其中，所述皿体包括底壁和侧壁，所述底壁和所述侧壁围成一容纳空间，所述温度敏感层位于所述容纳空间内。
  8. 根据权利要求7所述的培养装置，其中，所述冷光发射结构包括：
    与所述温度敏感层重叠的且位于所述容纳空间内的导光板；以及
    位于所述皿体之外的光源。
  9. 根据权利要求7所述的培养装置，其中，所述冷光发射结构包括多个冷光LED光源，位于所述容纳空间内，排布为多行多列的矩阵且与所述温度敏感层重叠。
  10. 根据权利要求9所述的培养装置，还包括：
    电极布线层，位于所述皿体的内部，且位于所述冷光发射结构的相反于所述温度敏感层的一侧。
  11. 根据权利要求7所述的培养装置，还包括：温度调节层，位于所述皿体的所述侧壁。
  12. 根据权利要求7所述的培养装置，还包括：固定元件，位于所述皿体的外部，构造为固定所述培养皿装置。
  13. 根据权利要求12所述的培养装置，其中，所述固定元件包括至少两个定位块，每个所述定位块中设置有定位槽。
  14. 根据权利要求12所述的培养装置，其中，所述固定元件为定位架，所述皿体位于所述定位架内。
  15. 根据权利要求14所述的培养装置，其中，所述定位架的内壁设置有温度调节层。
  16. 根据权利要求7至15中任一项所述的培养装置，还包括：彼此电连接的图像记录装置和显示单元，
    其中，所述图像记录装置构造为获取所述温度敏感层的相反于冷光发射结构的表面的图像；
    所述显示单元构造为显示所述图像记录装置获取的所述图像。
  17. 根据权利要求7至16中任一项所述的培养装置，还包括：彼此电连接的温度传感器和报警装置，
    其中，所述温度传感器构造为测量所述皿体的所述容纳空间内的温度，
    所述报警装置构造为根据所述温度传感器测得的温度发出警报。
  18. 一种采用上述任一项所述培养装置的细胞培养方法，包括：
    将待培养细胞置于所述温度敏感层相反于所述冷光发射结构的一侧；以及
    采用所述冷光发射结构发射冷光穿过所述温度敏感层而照射到所述待培养细胞。
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