Melanophore cultivation device and method

Abstract

The invention relates to the related technical field of cell culture, in particular to a melanocyte cultivation device and method.The melanocyte cultivation device comprises a cabinet body, a pushing mechanism is arranged on the inner side surface of the cabinet body, and a pushing-back mechanism is arranged on the surface of one side of the pushing mechanism. The pushing mechanism and the pushing-back mechanism are matched with each other, so that cells are observed more conveniently, the risks of environment instability and cell pollution caused by frequent opening of the incubator are reduced, the operation process is simplified, an operator only needs to set parameters and start corresponding programs on a display control panel, and the operation efficiency is improved. A stable and suitable growth environment can be provided for melanocytes, the cell growth and proliferation efficiency can be improved, the cell activity and quality are guaranteed, an operator can conveniently obtain culture dishes of different layers through the lifting function of the pushing mechanism, the requirements for observing and operating cells in different culture stages at the same time are met, and the culture efficiency is improved. And the flexibility and the efficiency of experimental operation are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field related to cell culture, and in particular to a melanocyte cultivation device and method. Background Art

[0002] Melanocytes are a type of neural crest-derived cells that exist in the basal layer of the skin epidermis, the hair bulb of the hair follicle, as well as the eyes and inner ear. Their core function is to give color to the skin, hair and eyes by synthesizing melanin, while protecting tissues from ultraviolet damage. Melanocytes are directly involved in the initiation and maintenance of the hair growth cycle through pigment transmission, signal regulation and microenvironment maintenance.

[0003] Enzymatic digestion refers to the use of enzymatic hydrolysis to decompose intercellular connecting substances and extracellular matrix components in tissues, separate cells from each other, and thus obtain single primary cells. Dispase II is a neutral protease that can hydrolyze the N-terminal peptide bonds of non-polar amino acid residues, hydrolyze fibronectin and type IV collagen, and has the specificity to cut leucine-phenylalanine bonds. Collagenase can cleave the Pro-X-Gly-Pro sequence, which is commonly found in the bond between glycine and neutral amino acids in collagen. Among proteases, only collagenase can degrade triple-helical natural collagen fibers commonly found in connective tissues such as skin, tendons, blood vessels and bones. Trypsin is widely used for cell dissociation, routine cell culture passage and primary tissue dissociation.

[0004] The tissue adhesion method refers to attaching tissue blocks directly to the surface of a culture dish, utilizing the cells' own migration ability to migrate out of the tissue block and grow attached to the wall. Under appropriate culture conditions, cells in the tissue block, such as fibroblasts and endothelial cells, will migrate from the cut edge to the surface of the culture dish, relying on their own motility to gradually detach from the tissue matrix. The migrated cells bind to the culture dish surface or pre-coated matrices such as collagen and fibronectin through adhesion molecules such as integrins, and then adhere to the wall and proliferate. The tissue block locally releases growth factors and nutrients, forming a microenvironment that is conducive to cell migration, while reducing damage to cells caused by enzymatic digestion.

[0005] Melanocytes are of great significance in skin physiology, hair coloration and disease treatment. For example, in the treatment of depigmentation diseases, the culture results of melanocytes can provide key support for disease improvement. Therefore, there is a special need for a melanocyte cultivation device and method.

[0006] However, existing melanocyte cultivation devices also have defects in the cell observation process. The traditional method requires frequently opening the incubator to remove the culture dishes, which will destroy the stable environment in the incubator, increase the risk of cell contamination, and is not conducive to the continuous cultivation of melanocytes. Summary of the Invention

[0007] The purpose of the present invention is to provide a melanocyte cultivation device and method to solve the problem that the existing melanocyte cultivation device proposed in the above background technology also has defects in the cell observation link. The traditional method requires frequent opening of the incubator to remove the culture dish, which will destroy the stable environment in the incubator, increase the risk of cell contamination, and is not conducive to the continuous cultivation of melanocytes.

[0008] To achieve the above-mentioned object, the present invention provides the following technical solutions: a melanocyte cultivation device, comprising a cabinet, a display control panel is installed on one side surface of the cabinet, a hinge is rotatably connected to one side surface of the cabinet, a cabinet door is rotatably connected to one side surface of the hinge, an observation bay window is installed on one side surface of the cabinet door, a stabilizing mechanism is provided on one side surface of the cabinet door, a gas sensor is installed on the inner side surface of the cabinet, a humidity sensor is installed on the inner side surface of the cabinet, a temperature sensor is installed on the inner side surface of the cabinet, a support frame is fixedly connected to the inner surface of the support frame, a placement plate is fixedly connected to the upper surface of the placement plate, a limiting block is fixedly connected to the inner side surface of the limiting block, a culture dish is slidably connected to the inner surface of the limiting block, a pushing mechanism is provided on the inner surface of the cabinet, and a push-back mechanism is provided on one side surface of the pushing mechanism; The pushing mechanism includes a motor, which is installed on the inner surface of the cabinet, a screw rod is fixedly connected to one end surface of the motor, a movable plate is threadedly connected to the outer surface of the screw rod, the inner surface of the movable plate is slidably connected to a limiting column, the upper surface of the movable plate is fixedly connected to a surrounding plate, a side surface of the surrounding plate is fixedly connected to a first multi-stage telescopic cylinder, one end surface of the first multi-stage telescopic cylinder is fixedly connected to a push plate, a side surface of the push plate is fixedly connected to two groups of stabilizing rubber columns, and a side surface of the surrounding plate is fixedly connected to an observation plate.

[0009] Preferably, a carbon dioxide inlet valve is installed on the lower left surface of the cabinet, an oxygen inlet valve is installed on the lower left surface of the cabinet, and an exhaust valve is installed on the upper right surface of the cabinet. The carbon dioxide inlet valve, oxygen inlet valve, exhaust valve and gas sensor constitute a gas regulation module.

[0010] Preferably, a humidifier is installed on the inner lower surface of the cabinet, and a dehumidifier is installed on the inner upper surface of the cabinet. The humidifier, dehumidifier and humidity sensor constitute a humidity control module.

[0011] Preferably, a heating element is installed on the inner surface of the cabinet, and a cooling element is installed on the inner surface of the cabinet. The heating element and the cooling element are sheet structures. The heating element is installed on the inner bottom and left and right inner wall surfaces of the cabinet, and the cooling element is installed on the inner top and inner rear wall surface of the cabinet. The temperature sensor, heating element and cooling element constitute a temperature control module.

[0012] Preferably, the pushing mechanism also includes a stabilizing block, which is installed on one side surface of the observation plate, the inner surface of the stabilizing block is slidably connected to a limiting rod, the outer surface of the stabilizing block is slidably connected to a stabilizing plate, the inner surface of the stabilizing plate is provided with a stabilizing groove, and the upper surface of the observation plate is fixedly connected to the limiting plate.

[0013] Preferably, two groups of the stabilizing blocks are symmetrically arranged with respect to the central axis of the observation plate, the limiting rods are mounted on the inner surface of the stabilizing groove, and the inner dimensions of the stabilizing groove match the outer dimensions of the stabilizing blocks.

[0014] Preferably, the stabilizing mechanism includes a fixed block, which is installed on one side surface of the cabinet door, a sliding groove is provided on the inner surface of the fixed block, the inner surface of the sliding groove is slidably connected to the sliding block, the upper surface of the sliding block is fixedly connected to a clamping block, the lower surface of the sliding block is fixedly connected to a spring, the inner bottom surface of the sliding groove is fixedly connected to a stabilizing column, a fixed groove is provided on one side surface of the cabinet body, and a clamping groove is provided on the inner upper surface of the fixed groove.

[0015] Preferably, the sliding block and the clamping block are integrally formed, the sliding block and the clamping block are slidably connected to the stabilizing column, the outer size of the clamping block matches the inner size of the clamping slot, and the fixed block is slidably connected to the fixed slot.

[0016] Preferably, the push-back mechanism includes a placement block, which is installed on the lower surface of the observation plate, and the upper surface of the placement block is fixedly connected to a two-way multi-stage telescopic cylinder, and one end surface of the two-way multi-stage telescopic cylinder is fixedly connected to a connecting plate, and the upper surface of the connecting plate is fixedly connected to a pushing block, and two sides of the connecting plate are fixedly connected to two groups of limit blocks, and the inner surface of the observation plate is provided with a connecting plate groove, and the inner surface of the connecting plate groove is provided with two groups of limit block grooves.

[0017] A method for using a melanocyte cultivation device, comprising the following specific steps: Step 1. Preparation: Select the mouse pups required for the experiment. According to the experimental protocol for the isolation, extraction, and culture of mouse hair-derived melanocytes, rinse the pups' dorsal skin tissue with sterile saline to remove surface impurities. Cut the pieces into small pieces and place them in a digestion solution containing trypsin. Digest the pieces at 37°C for a period of time to promote cell dispersion. At the same time, thoroughly clean and disinfect the incubation cabinet. Set the temperature of the temperature control module to 37°C, the humidity of the humidity control module to 95%, the carbon dioxide concentration of the gas control module to 5%, and the oxygen concentration to 20% through the display control panel. Start each module to achieve a stable culture environment in the incubator. Step 2: Cell separation: The digested cell suspension is processed according to the experimental protocol. Utilizing the sterile environment and relevant operating space within the device, and using technologies such as microfluidic chips, melanocytes are separated from other cells based on cell size and charge differences, and the separated melanocyte suspension is collected. Step 3: Cell inoculation: The isolated melanocyte suspension is inoculated into a culture dish containing fresh culture medium using an automatic pipette. The culture dish is placed within the restriction block of the placement plate to ensure that it is firmly placed. The cabinet door is then closed and the culture is started. Step 4, culture process monitoring: During the culture process, the gas sensor, humidity sensor and temperature sensor monitor the environmental parameters in the incubator in real time and transmit the data to the display control panel. The display control panel automatically adjusts the gas adjustment module, humidity control module and temperature control module according to the preset parameters to ensure a stable culture environment. The operator can preliminarily observe the cell growth status through the observation bay window. When more detailed observation is needed, the motor of the pushing mechanism is started, and the screw drives the moving plate to move so that the observation plate is close to the culture dish. Then the first multi-stage telescopic cylinder is started, and the push plate pushes the culture dish to the observation plate. The limit plate is continued to be pushed to slide the observation plate into the observation bay window. Cell morphology is observed and photographed through the observation bay window and the observation plate. After the observation is completed, the two-way multi-stage telescopic cylinder is started to apply thrust to the observation bay window, so that the observation plate moves back to the initial position, and then the two-way multi-stage telescopic cylinder is started to connect one end of the connecting plate, and the pushing block is pushed to move the culture dish into the limit block above the placement plate; Step 5: Cell passaging: When the cultured melanocytes reach 80% to 90% confluence, cell passaging is performed. The push mechanism is activated to move the culture dish to a convenient position for operation. The digestion solution is aspirated by the automatic pipette to digest the cells in the culture dish. After the digestion is completed, fresh culture medium is aspirated and the digested cell suspension is dispensed into a new culture dish. The push mechanism is then activated to return the new culture dish to the culture position for continued cultivation. Step 6. Cell identification: Regularly remove a small amount of cells from the culture dish and identify the cells using morphological observation and immunocytochemical staining. Morphological observation mainly focuses on whether the cells have a dendritic morphology and whether there are brown-black granules in the cytoplasm. Immunocytochemical staining is performed on the target protein TYRP1 / Melan-A. Through fixation, permeabilization, blocking, primary antibody incubation, fluorescent secondary antibody incubation, nuclear staining and other steps, observe under a fluorescence microscope to confirm the cell type and activity.

[0018] Compared with the prior art, the present invention has the following beneficial effects: 1. The push mechanism and push-back mechanism of the present invention achieve precise movement and resetting of the culture dish and observation plate, facilitating cell observation while reducing the risk of environmental instability and cell contamination caused by frequent opening of the incubator; 2. The present invention simplifies the operation process of melanocyte culture. The operator only needs to set the parameters on the display control panel and start the corresponding program to provide a stable and suitable growth environment for melanocytes, which is conducive to improving cell growth and proliferation efficiency and ensuring cell activity and quality. 3. The lifting function of the pushing mechanism of the present invention enables operators to easily obtain culture dishes at different layers, meeting the needs of observing and operating cells at different culture stages at the same time, greatly improving the flexibility and efficiency of experimental operations. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is a side structural diagram of the present invention; Figure 2 This is a schematic diagram of the structure of the fixing block and the fixing groove cooperating with each other in the present invention; Figure 3 This is a schematic cross-sectional view of the stabilizing mechanism of the present invention; Figure 4 This is a schematic diagram of the structure of the dehumidifier and the cabinet body cooperating with each other in the present invention; Figure 5 This is a schematic diagram of the structure of the refrigeration element and the heating element cooperating with each other in the present invention; Figure 6 This is a schematic diagram of the structure of the propulsion mechanism of the present invention; Figure 7 This is a schematic diagram of the push-back mechanism structure of the present invention; Figure 8 This is a schematic diagram of the structure of the stabilizing block and the limiting rod cooperating with each other in the present invention; Figure 9 It is a schematic diagram of the structure of the cooperation between the limit block and the limit block groove of the present invention.

[0020] In the figure: 1. cabinet; 2. display control panel; 3. hinge; 4. cabinet door; 5. observation bay window; 6. stabilizing mechanism; 601. fixing block; 602. sliding slot; 603. sliding block; 604. clamping block; 605. spring; 606. stabilizing column; 607. fixing slot; 608. clamping slot; 7. gas sensor; 8. carbon dioxide inlet valve; 9. oxygen inlet valve; 10. exhaust valve; 11. humidity sensor; 12. humidifier; 13. dehumidifier; 14. temperature sensor; 15. heating element; 16. cooling element; 17. support frame; 18. placement plate; 19. limiting block; 20. culture dish; 21. , pushing mechanism; 2101, motor; 2102, screw rod; 2103, moving plate; 2104, limiting column; 2105, enclosure; 2106, first multi-stage telescopic cylinder; 2107, push plate; 2108, stabilizing rubber column; 2109, observation plate; 2110, stabilizing block; 2111, limiting rod; 2112, stabilizing plate; 2113, stabilizing groove; 2114, limiting plate; 22, push-back mechanism; 2201, placement block; 2202, bidirectional multi-stage telescopic cylinder; 2203, connecting plate; 2204, pushing block; 2205, limiting block; 2206, connecting plate groove; 2207, limiting block groove. DETAILED DESCRIPTION

[0021] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0022] Example 1: See also Figure 1-9 The present invention provides a technical solution: a melanocyte cultivation device, comprising a cabinet 1, a display control panel 2 is installed on one side surface of the cabinet 1, a hinge 3 is rotatably connected to one side surface of the cabinet 1, a cabinet door 4 is rotatably connected to one side surface of the hinge 3, an observation bay window 5 is installed on one side surface of the cabinet door 4, a stabilizing mechanism 6 is provided on one side surface of the cabinet door 4, a gas sensor 7 is installed on the inner surface of the cabinet 1, a humidity sensor 11 is installed on the inner surface of the cabinet 1, a temperature sensor 14 is installed on the inner surface of the cabinet 1, a support frame 17 is fixedly connected to the inner surface of the support frame 17, a placement plate 18 is fixedly connected to the upper surface of the placement plate 18, a limiting block 19 is fixedly connected to the inner surface of the limiting block 19, a culture dish 20 is slidably connected to the inner surface of the limiting block 19, a pushing mechanism 21 is provided on the inner surface of the cabinet 1, and a push-back mechanism 22 is provided on one side surface of the pushing mechanism 21; The pushing mechanism 21 includes a motor 2101, which is installed on the inner surface of the cabinet 1, and one end surface of the motor 2101 is fixedly connected to a screw rod 2102, and the outer surface of the screw rod 2102 is threadedly connected to a moving plate 2103, and the inner surface of the moving plate 2103 is slidably connected to a limiting column 2104, and the upper surface of the moving plate 2103 is fixedly connected to a panel 2105, and one side surface of the panel 2105 is fixedly connected to a first multi-stage telescopic cylinder 2106, and one end surface of the first multi-stage telescopic cylinder 2106 is fixedly connected to a push plate 2107, and one side surface of the push plate 2107 is fixedly connected to two groups of stable rubber columns 2108, and one side surface of the panel 2105 is fixedly connected to an observation plate 2109. The setting is that when in use, when it is necessary to observe cells, the motor 2101 is powered on, driving the screw rod 2102 to rotate, and the threaded connection between the screw rod 2102 and the movable plate 2103 enables the movable plate 2103 to move axially on the screw rod 2102. The limiting column 2104 plays a guiding and stabilizing role to prevent the movable plate 2103 from rotating with the screw rod 2102. When the movable plate 2103 moves to the placement plate 18 position below the culture dish 20 that needs to be observed, the first multi-stage telescopic cylinder 2106 is started, pushing the push plate 2107 and the stabilizing rubber column 2108 to push the culture dish 20 to move toward the observation plate 2109. When the culture dish 20 moves onto the observation plate 2109, the motor 2101 is started to move the observation plate 2109 to the vicinity of the observation bay window 5.

[0023] Furthermore, a carbon dioxide inlet valve 8 is installed on the lower left surface of the cabinet 1, an oxygen inlet valve 9 is installed on the lower left surface of the cabinet 1, and an exhaust valve 10 is installed on the upper right surface of the cabinet 1. The carbon dioxide inlet valve 8, the oxygen inlet valve 9, the exhaust valve 10 and the gas sensor 7 constitute a gas regulation module. Through the setting of the above structure, when in use, the gas sensor 7 monitors the concentrations of carbon dioxide and oxygen in the cabinet 1 in real time. When the carbon dioxide concentration is lower than the set value, the carbon dioxide inlet valve 8 opens and carbon dioxide gas enters the cabinet 1. When the carbon dioxide concentration is higher than the set value, the exhaust valve 10 opens to discharge excess gas. Similarly, the oxygen inlet valve 9 and the exhaust valve 10 work together to maintain a stable oxygen concentration. The gas regulation module can automatically and accurately control the gas composition in the incubator, provide melanocytes with a gas environment similar to the in vivo environment, meet the cell respiration and metabolic needs, be beneficial to cell growth and proliferation, and improve the quality of cell culture.

[0024] Furthermore, a humidifier 12 is installed on the lower inner surface of the cabinet 1, and a dehumidifier 13 is installed on the upper inner surface of the cabinet 1. The humidifier 12, the dehumidifier 13 and the humidity sensor 11 constitute a humidity control module. Through the setting of the above structure, when in use, the humidity sensor 11 detects the humidity in the cabinet 1 in real time. When the humidity is lower than the set range, the humidifier 12 starts to atomize the water and release it into the box to increase the humidity. When the humidity is higher than the set range, the dehumidifier 13 starts to remove excess water and reduce the humidity. The humidity control module ensures the stability of the humidity in the incubator, avoids dehydration of cells due to inappropriate humidity or metabolic abnormalities caused by excessive water, provides a suitable humidity environment for melanocytes, and ensures the normal physiological function and growth state of cells.

[0025] Furthermore, a heating element 15 is installed on the inner surface of the cabinet 1, and a cooling element 16 is installed on the inner surface of the cabinet 1. The heating element 15 and the cooling element 16 are sheet structures. The heating element 15 is installed on the inner bottom and left and right inner wall surfaces of the cabinet 1, and the cooling element 16 is installed on the inner top and inner rear wall surface of the cabinet 1. The temperature sensor 14, the heating element 15 and the cooling element 16 constitute a temperature control module. Through the setting of the above structure, when in use, the temperature sensor 14 monitors the temperature inside the cabinet 1 in real time. When the temperature is lower than the set suitable temperature, the heating element 15 is powered on to generate heat, thereby increasing the temperature inside the box. When the temperature is higher than the suitable temperature, the cooling element 16 starts cooling to reduce the temperature inside the box. The temperature control module creates a stable temperature environment for melanocytes, which meets the optimal temperature requirements for cell growth, reduces the adverse effects of temperature fluctuations on cells, promotes the healthy growth and proliferation of cells, and improves the success rate of cell culture.

[0026] Furthermore, the pushing mechanism 21 also includes a stabilizing block 2110, which is installed on one side surface of the observation plate 2109. The inner surface of the stabilizing block 2110 is slidably connected to the limiting rod 2111, and the outer surface of the stabilizing block 2110 is slidably connected to the stabilizing plate 2112. The inner surface of the stabilizing plate 2112 is provided with a stabilizing groove 2113. The upper surface of the observation plate 2109 is fixedly connected to the limiting plate 2114. Through the setting of the above structure, when in use, the limiting plate 2114 prevents the culture dish 20 from moving excessively. In the process of pushing the culture dish 20 by the pushing mechanism 21, when the limiting plate 2114 above the observation plate 2109 is pushed by the pushing plate 2107, the observation plate 2109 starts to slide, and the stabilizing blocks 2110 on both sides of the observation plate 2109 slide along the limiting rod 2111 in the stabilizing groove 2113 of the stabilizing plate 2112, so that the observation plate 2109 is pushed into the observation bay window 5.

[0027] Furthermore, two groups of stabilizing blocks 2110 are symmetrically arranged around the central axis of the observation plate 2109, and the limiting rod 2111 is installed on the inner surface of the stabilizing groove 2113. The inner size of the stabilizing groove 2113 matches the outer size of the stabilizing block 2110. Through the arrangement of the above structure, when in use, the two symmetrically arranged groups of stabilizing blocks 2110 can provide stable support to the observation plate 2109 evenly from both sides. When the observation plate 2109 is subjected to force, the stabilizing block 2110 slides in the stabilizing groove 2113, and through cooperation with the limiting rod 2111, the force applied to the observation plate 2109 is balanced, so that the observation plate 2109 remains stable during movement.

[0028] Furthermore, the stabilizing mechanism 6 includes a fixed block 601, which is mounted on one side surface of the cabinet door 4. A sliding groove 602 is provided on the inner surface of the fixed block 601. A sliding block 603 is slidably connected to the inner surface of the sliding groove 602. A card block 604 is fixedly connected to the upper surface of the sliding block 603. A spring 605 is fixedly connected to the lower surface of the sliding block 603. A stabilizing column 606 is fixedly connected to the inner bottom surface of the sliding groove 602. There is a fixing groove 607, and a card slot 608 is opened on the inner upper surface of the fixing groove 607. Through the setting of the above structure, when in use, when the cabinet door 4 is closed, the fixing block 601 is inserted into the fixing groove 607, and the card block 604 moves upward under the elastic force of the spring 605 and is stuck in the card slot 608. The stabilizing column 606 guides the movement of the sliding block 603 and the card block 604 to prevent the sliding block 603 and the card block 604 from shaking. The stabilizing mechanism 6 enhances the stability of the cabinet door 4 when it is closed.

[0029] Furthermore, the sliding block 603 and the card block 604 are integrally formed, and the sliding block 603 and the card block 604 are slidably connected to the stabilizing column 606. The outer size of the card block 604 coincides with the inner size of the card slot 608, and the fixed block 601 is slidably connected to the fixed slot 607. Through the setting of the above structure, when in use, the integrally formed sliding block 603 and the card block 604 can accurately slide in the sliding slot 602 under the guidance of the stabilizing column 606. When the fixed block 601 is inserted into the fixed slot 607, the card block 604 can be accurately inserted into the card slot 608, thereby realizing stable fixation of the cabinet door 4 and improving the reliability and stability of the stabilizing mechanism 6.

[0030] Furthermore, the push-back mechanism 22 includes a placement block 2201, which is mounted on the lower surface of the observation plate 2109. The upper surface of the placement block 2201 is fixedly connected to a bidirectional multi-stage telescopic cylinder 2202. One end surface of the bidirectional multi-stage telescopic cylinder 2202 is fixedly connected to a connecting plate 2203. The upper surface of the connecting plate 2203 is fixedly connected to a pushing block 2204. Two sets of limit blocks 2205 are fixedly connected to the two side surfaces of the connecting plate 2203. The inner surface of the observation plate 2109 is provided with a connecting plate groove 220 6. Two groups of limit block grooves 2207 are provided on the inner surface of the connecting plate groove 2206. Through the arrangement of the above structure, when in use, after the observation is completed, the bidirectional multi-stage telescopic cylinder 2202 is started to apply thrust to the observation bay window 5, so that the observation plate 2109 moves back to the initial position, and then extends out to push the connecting plate 2203. The connecting plate 2203 drives the pushing block 2204 to push the culture dish 20 back to the limiting block 19 on the placement plate 18. The limiting block 2205 slides in the limiting block groove 2207 to ensure that the pushing process is smooth and accurate.

[0031] Working principle: After turning on the device, the operator sets the temperature, humidity, gas concentration and other parameters required for culture through the display control panel 2. The device enters the working preparation state. The gas sensor 7, humidity sensor 11 and humidifier 12 start to monitor the environmental parameters in the cabinet 1 in real time and feed the data back to the display control panel 2. The culture dish 20 containing melanocytes is placed on the placement plate 18. The limiting block 19 limits and fixes the culture dish 20. The cabinet door 4 is closed. The fixing block 601 is inserted into the fixing groove 607. The card block 604 moves upward under the elastic force of the spring 605 and is locked. The card slot 608 and the stabilizing column 606 guide the movement of the sliding block 603 and the card block 604 to prevent the sliding block 603 and the card block 604 from shaking. The stabilizing mechanism 6 enhances the stability of the cabinet door 4 when it is closed. When it is necessary to observe the cells, the motor 2101 is powered on to drive the screw rod 2102 to rotate. The screw rod 2102 is threadedly connected to the movable plate 2103 so that the movable plate 2103 moves axially on the screw rod 2102. The limiting column 2104 plays a guiding and stabilizing role, preventing the movable plate 2103 from rotating with the screw rod 2102. When the movable plate 2103 moves to the cell to be observed After the placement plate 18 below the culture dish 20 is in position, the first multi-stage telescopic cylinder 2106 is started, pushing the push plate 2107 and the stabilizing rubber column 2108 to push the culture dish 20 to move toward the observation plate 2109. When the culture dish 20 moves onto the observation plate 2109, the motor 2101 is started to move the observation plate 2109 to the vicinity of the observation bay window 5. The limit plate 2114 prevents the culture dish 20 from moving excessively. During the process of the pushing mechanism 21 pushing the culture dish 20 to move, when the limit plate 2114 above the observation plate 2109 is pushed by the push plate 2107, the observation plate 2109 begins to slide. The stabilizing blocks 2110 on both sides of 2109 slide along the limiting rod 2111 in the stabilizing groove 2113 of the stabilizing plate 2112, so that the observation plate 2109 is pushed into the observation bay window 5. After the observation is completed, the bidirectional multi-stage telescopic cylinder 2202 is started to apply thrust to the observation bay window 5, so that the observation plate 2109 moves back to the initial position, and then extends to push the connecting plate 2203. The connecting plate 2203 drives the pushing block 2204 to push the culture dish 20 back to the limiting block 19 on the placement plate 18. The limiting block 2205 slides in the limiting block groove 2207 to ensure that the pushing process is smooth and accurate.

[0032] Example 2: A method for using a melanocyte cultivation device, comprising the following specific steps: Step 1. Preparation: Select the mouse pups required for the experiment. According to the experimental protocol for the isolation, extraction, and culture of mouse hair-derived melanocytes, rinse the pups' back skin tissue with sterile saline to remove surface impurities, cut into small pieces, and place in a digestion solution containing trypsin. Digest at 37°C for a period of time to promote cell dispersion. At the same time, thoroughly clean and disinfect the cabinet 1 of the incubation device. Set the temperature of the temperature control module to 37°C, the humidity of the humidity control module to 95%, the carbon dioxide concentration of the gas control module to 5%, and the oxygen concentration to 20% through the display control panel 2. Start each module to achieve a stable culture environment in the incubator. Step 2: Cell separation: The digested cell suspension is processed according to the experimental protocol. Utilizing the sterile environment and relevant operating space within the device, and using technologies such as microfluidic chips, melanocytes are separated from other cells based on cell size and charge differences, and the separated melanocyte suspension is collected. Step 3: Cell inoculation: The separated melanocyte suspension is inoculated into a culture dish 20 containing fresh culture medium using an automatic sampler. The culture dish 20 is placed in the limiting block 19 of the placement plate 18 to ensure that it is firmly placed. The cabinet door 4 is then closed to start the culture. Step 4: Monitoring the culture process: During the culture process, the gas sensor 7, humidity sensor 11 and temperature sensor 14 monitor the environmental parameters in the incubator in real time and transmit the data to the display control panel 2. The display control panel 2 automatically adjusts the gas adjustment module, humidity control module and temperature control module according to the preset parameters to ensure a stable culture environment. The operator can observe the cell growth status through the observation bay window 5. When a more detailed observation is required, the motor 2101 of the pushing mechanism 21 is started, and the screw rod 2102 drives the moving plate 2103 to move, so that the observation plate 2109 is close to the culture dish 20, and then the step 4 is started. A multi-stage telescopic cylinder 2106 and a push plate 2107 push the culture dish 20 to the observation plate 2109, and continue to push the limit plate 2114 to slide the observation plate 2109 into the observation bay window 5. Cell morphology is observed and photographed through the observation bay window 5 and the observation plate 2109. After the observation is completed, the bidirectional multi-stage telescopic cylinder 2202 is started to apply a thrust to the observation bay window 5, so that the observation plate 2109 moves back to the initial position. Then, the bidirectional multi-stage telescopic cylinder 2202 is started to connect one end of the connecting plate 2203, pushing the push block 2204 to move the culture dish 20 into the limit block 19 above the placement plate 18; Step 5, cell passaging: When the cultured melanocytes reach 80% to 90% confluence, cell passaging is performed. The pushing mechanism 21 is activated to move the culture dish 20 to a convenient position for operation. The digestion liquid is aspirated by the automatic sampler to digest the cells in the culture dish 20. After the digestion is completed, fresh culture medium is aspirated and the digested cell suspension is dispensed into a new culture dish 20. The pushing mechanism 22 is then activated to return the new culture dish 20 to the culture position for continued cultivation. Step 6. Cell identification: A small amount of cells are periodically removed from the culture dish 20 and identified by morphological observation and immunocytochemical staining. Morphological observation mainly focuses on whether the cells have a dendritic morphology and whether there are brown-black granules in the cytoplasm. Immunocytochemical staining is performed for the target protein TYRP1 / Melan-A. Through fixation, permeabilization, blocking, primary antibody incubation, fluorescent secondary antibody incubation, nuclear staining and other steps, the cells are observed under a fluorescence microscope to confirm the cell type and activity.

[0033] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A melanocyte cultivation device, comprising a cabinet (1), characterized in that: A display control panel (2) is mounted on one side surface of the cabinet (1), a hinge (3) is rotatably connected to one side surface of the cabinet (1), a cabinet door (4) is rotatably connected to one side surface of the hinge (3), an observation bay window (5) is mounted on one side surface of the cabinet door (4), a stabilizing mechanism (6) is provided on one side surface of the cabinet door (4), a gas sensor (7) is mounted on the inner side surface of the cabinet (1), a humidity sensor (11) is mounted on the inner side surface of the cabinet (1), and the cabinet (1) is provided with a plurality of air conditioners. A temperature sensor (14) is installed on the inner surface, a support frame (17) is fixedly connected to the inner surface of the cabinet (1), a placement plate (18) is fixedly connected to the upper surface of the support frame (17), a limiting block (19) is fixedly connected to the upper surface of the placement plate (18), a culture dish (20) is slidably connected to the inner surface of the limiting block (19), a pushing mechanism (21) is provided on the inner surface of the cabinet (1), and a push-back mechanism (22) is provided on one side surface of the pushing mechanism (21); The pushing mechanism (21) comprises a motor (2101), the motor (2101) being mounted on the inner surface of the cabinet (1), one end surface of the motor (2101) being fixedly connected to a screw rod (2102), the outer surface of the screw rod (2102) being threadedly connected to a movable plate (2103), the inner surface of the movable plate (2103) being slidably connected to a limiting column (2104), the upper surface of the movable plate (2103) being fixedly connected to a surrounding plate (2105), one side surface of the surrounding plate (2105) being fixedly connected to a first multi-stage telescopic cylinder (2106), one end surface of the first multi-stage telescopic cylinder (2106) being fixedly connected to a push plate (2107), one side surface of the push plate (2107) being fixedly connected to two groups of stabilizing rubber columns (2108), and one side surface of the surrounding plate (2105) being fixedly connected to an observation plate (2109).

2. The melanocyte cultivation device according to claim 1, characterized in that: A carbon dioxide inlet valve (8) is installed on the lower left surface of the cabinet (1), an oxygen inlet valve (9) is installed on the lower left surface of the cabinet (1), and an exhaust valve (10) is installed on the upper right surface of the cabinet (1). The carbon dioxide inlet valve (8), the oxygen inlet valve (9), the exhaust valve (10) and the gas sensor (7) constitute a gas regulation module.

3. The melanocyte cultivation device according to claim 1, characterized in that: A humidifier (12) is installed on the inner lower surface of the cabinet (1), and a dehumidifier (13) is installed on the inner upper surface of the cabinet (1). The humidifier (12), the dehumidifier (13) and the humidity sensor (11) constitute a humidity control module.

4. The melanocyte cultivation device according to claim 1, characterized in that: A heating element (15) is installed on the inner surface of the cabinet (1), and a cooling element (16) is installed on the inner surface of the cabinet (1). The heating element (15) and the cooling element (16) are sheet-like structures. The heating element (15) is installed on the inner bottom and left and right inner wall surfaces of the cabinet (1), and the cooling element (16) is installed on the inner top and inner rear wall surface of the cabinet (1). The temperature sensor (14), the heating element (15) and the cooling element (16) constitute a temperature control module.

5. The melanocyte cultivation device according to claim 1, characterized in that: The pushing mechanism (21) further comprises a stabilizing block (2110), the stabilizing block (2110) being mounted on a side surface of the observation plate (2109), the inner surface of the stabilizing block (2110) being slidably connected to a limiting rod (2111), the outer surface of the stabilizing block (2110) being slidably connected to a stabilizing plate (2112), the inner surface of the stabilizing plate (2112) being provided with a stabilizing groove (2113), and the upper surface of the observation plate (2109) being fixedly connected to a limiting plate (2114).

6. The melanocyte cultivation device according to claim 5, characterized in that: The stabilizing blocks (2110) are symmetrically arranged in two groups about the central axis of the observation plate (2109), and the limiting rods (2111) are installed on the inner surface of the stabilizing groove (2113). The inner dimensions of the stabilizing groove (2113) match the outer dimensions of the stabilizing blocks (2110).

7. The melanocyte cultivation device according to claim 1, characterized in that: The stabilizing mechanism (6) comprises a fixed block (601), the fixed block (601) being mounted on a side surface of the cabinet door (4), a sliding groove (602) being provided on the inner surface of the fixed block (601), a sliding block (603) being slidably connected to the inner surface of the sliding groove (602), a clamping block (604) being fixedly connected to the upper surface of the sliding block (603), a spring (605) being fixedly connected to the lower surface of the sliding block (603), a stabilizing column (606) being fixedly connected to the inner bottom surface of the sliding groove (602), a fixed groove (607) being provided on a side surface of the cabinet body (1), and a clamping groove (608) being provided on the inner upper surface of the fixed groove (607).

8. The melanocyte cultivation device according to claim 7, characterized in that: The sliding block (603) and the clamping block (604) are integrally formed. The sliding block (603) and the clamping block (604) are slidably connected to the stabilizing column (606). The outer dimensions of the clamping block (604) match the inner dimensions of the clamping slot (608). The fixed block (601) is slidably connected to the fixed slot (607).

9. The melanocyte cultivation device according to claim 1, characterized in that: The push-back mechanism (22) comprises a placement block (2201), the placement block (2201) being mounted on the lower surface of the observation plate (2109), the upper surface of the placement block (2201) being fixedly connected to a bidirectional multi-stage telescopic cylinder (2202), one end surface of the bidirectional multi-stage telescopic cylinder (2202) being fixedly connected to a connecting plate (2203), the upper surface of the connecting plate (2203) being fixedly connected to a push block (2204), the two side surfaces of the connecting plate (2203) being fixedly connected to two groups of limit blocks (2205), the inner surface of the observation plate (2109) being provided with a connecting plate groove (2206), and the inner surface of the connecting plate groove (2206) being provided with two groups of limit block grooves (2207).

10. A method for using a melanocyte cultivation device, characterized in that: The melanocyte cultivation device according to any one of claims 1 to 9, wherein the specific steps are as follows: Step 1. Preparation: Select the mouse pups required for the experiment. According to the experimental protocol for the separation, extraction and culture of mouse hair-derived melanocytes, rinse the skin tissue on the back of the pups with sterile saline to remove surface impurities, cut into small pieces and place in a digestion solution containing trypsin. Digest at 37°C for a period of time to promote cell dispersion. At the same time, thoroughly clean and disinfect the cabinet (1) of the culture device. Set the temperature of the temperature control module to 37°C, the humidity of the humidity control module to 95%, the carbon dioxide concentration of the gas regulation module to 5%, and the oxygen concentration to 20% through the display control panel (2). Start each module to achieve a stable culture environment in the incubator. Step 2: Cell separation: The digested cell suspension is processed according to the experimental protocol. Utilizing the sterile environment and relevant operating space within the device, and using technologies such as microfluidic chips, melanocytes are separated from other cells based on cell size and charge differences, and the separated melanocyte suspension is collected. Step 3, cell inoculation: The separated melanocyte suspension is inoculated into a culture dish (20) containing fresh culture medium by means of an automatic sampler, the culture dish (20) is placed in the limiting block (19) of the placement plate (18), ensuring that it is placed firmly, and then the cabinet door (4) is closed to start the culture; Step 4: Monitoring the culture process: During the culture process, the gas sensor (7), humidity sensor (11) and temperature sensor (14) monitor the environmental parameters in the incubator in real time and transmit the data to the display control panel (2). The display control panel (2) automatically adjusts the gas adjustment module, humidity control module and temperature control module according to the preset parameters to ensure a stable culture environment. The operator can observe the cell growth status through the observation bay window (5). When a more detailed observation is required, the motor (2101) of the driving mechanism (21) is started, and the screw rod (2102) drives the moving plate (2103) to move, so that the observation plate (2109) is close to the culture dish (20), and then the first multi-stage telescopic gas is started. Cylinder (2106), push plate (2107) pushes the culture dish (20) to the observation plate (2109), continues to push the limit plate (2114) to make the observation plate (2109) slide into the observation bay window (5), and observes the cell morphology and takes photos through the observation bay window (5) and the observation plate (2109). After the observation is completed, start the two-way multi-stage telescopic cylinder (2202), apply a thrust to the observation bay window (5), so that the observation plate (2109) moves back to the initial position, and then start the two-way multi-stage telescopic cylinder (2202) to connect one end of the connecting plate (2203), push the push block (2204) so ​​that the culture dish (20) moves into the limiting block (19) above the placement plate (18); Step 5, cell passage: When the cultured melanocytes grow to 80% to 90% confluence, cell passage is performed, the pushing mechanism (21) is activated to move the culture dish (20) to a position convenient for operation, and the digestion liquid is sucked out by the automatic sampler to digest the cells in the culture dish (20). After the digestion is completed, fresh culture medium is sucked out and the digested cell suspension is dispensed into a new culture dish (20), and the pushing mechanism (22) is activated to put the new culture dish (20) back to the culture position for continued cultivation; Step 6. Cell identification: Regularly remove a small amount of cells from the culture dish (20) and identify the cells using morphological observation and immunocytochemical staining. Morphological observation mainly focuses on whether the cells have a dendritic morphology and whether there are brown-black particles in the cytoplasm. Immunocytochemical staining is performed on the target protein TYRP1 / Melan-A. Through fixation, permeabilization, blocking, primary antibody incubation, fluorescent secondary antibody incubation, nuclear staining and other steps, the cells are observed under a fluorescence microscope to confirm the cell type and activity.

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