A nociceptor skin organoid chip, its culture medium, construction method and application

Abstract

The present invention belongs to the technical field of biomedicine, and specifically relates to a nociceptor skin organoid chip and its culture medium, construction method and application. A culture medium for a nociceptor skin organoid chip, the culture medium is composed of fetal bovine serum and the following components added to the fetal bovine serum: epidermal growth factor, bovine pituitary extract, Normocin, B27, nerve growth factor, insulin, hydrocortisone, human brain-derived neurotrophic factor and capsaicin. Using this culture medium to co-culture a skin chip with nociceptors to obtain a nociceptor skin organoid chip, which can be used for the verification of pain perception effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a nociceptor skin organoid chip, a culture medium therefor, a construction method therefor, and an application thereof. Background Art

[0002] The skin is the largest organ of the human body and has abundant receptors, including nociceptors, tactile receptors, thermoreceptors, and baroreceptors, etc. Each receptor has different structures and functions. Nociceptors are one of the most widely distributed receptors in the skin. For the body to sense pain, it must rely on the normal functioning of nociceptors.

[0003] The skin organoid chip is a type of human organoid chip, which is a skin microphysiological system constructed in vitro through the cross-integration of cutting-edge technologies such as stem cells, biomaterials, and nanomanufacturing. There have been some remarkable progress in skin chips. For example, researchers have successfully generated skin cells from induced pluripotent stem cells (iPSCs) and used them to generate organized skin layers. These skin cells can remain viable in a 3-D system and differentiate into different types of skin cells, self-assembling to form skin layers that are very similar to human real skin. Researchers are also working on how to better understand the potential side effects of drugs on the skin through skin organoid chips, which is a challenging but very promising research field. The construction scheme of skin chips mainly includes designing and fabricating chips suitable for the growth and differentiation of skin cells and establishing corresponding pathway systems; developing biomaterials suitable for the growth of skin cells and constructing skin chips using human-derived skin cells and skin cells transformed from stem cells. On the basis of completing the corresponding biological evaluations, the current research on skin organoid chips has been continuously improved and advanced in terms of function, structure, cell types and viability, and model service life, providing a very important platform for various disease modeling and preclinical treatment evaluations. However, there are not many types of culture media for culturing skin organoids. Existing culture media include those containing Wnt3a, IGF, VEGF, and bFGF, but this culture medium is for culturing skin organoids and is not suitable for other organoids. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides a nociceptor skin organoid chip, a culture medium therefor, a construction method therefor, and an application thereof.

[0005] A culture medium for a nociceptor skin organoid chip, the culture medium consisting of fetal bovine serum and the following components added to the fetal bovine serum: epidermal growth factor at 80 ng / mL to 150 ng / mL, bovine pituitary extract at 10 μL / mL to 30 μL / mL, Normocin at 80 μg / mL to 150 μg / mL, B27 at 10 μL / mL to 30 μL / mL, nerve growth factor at 80 ng / mL to 150 ng / mL, insulin at 3 μg / mL to 5 μg / mL, hydrocortisone at 9 ng / mL to 11 ng / mL, human brain-derived neurotrophic factor at 9 ng / mL to 11 ng / mL, and capsaicin at 30 μM to 50 μM. Capsaicin is applied to activate the TRPV1 receptor. In the synaptic-like contact between keratinocytes and sensory neurons, when the TRPV1 receptor is activated, it may cause keratinocytes to release neurotransmitters, which bind to specific receptors on sensory neurons to complete the signal transduction process.

[0006] Preferably, the concentrations of the components in the culture medium are: epidermal growth factor 100 ng / mL, bovine pituitary extract 20 μL / mL, Normocin 100 μg / mL, B27 20 μL / mL, nerve growth factor 100 ng / mL, insulin 4 μg / mL, hydrocortisone 10 ng / mL, human brain-derived neurotrophic factor 10 ng / mL, and capsaicin 40 μM.

[0007] Preferably, it includes the following steps:

[0008] Constructing a nociceptor: Culturing human embryonic stem cells sequentially with a cell inhibitor, a first culture, and a second culture;

[0009] When culturing human embryonic stem cells, the cell inhibitor, the first culture, and the second culture are respectively added to the cell culture medium;

[0010] The cell inhibitor is a mixture of CHIR98014 and A83 - 01, wherein the final concentration of A83 - 01 is 1 μM to 3 μM and the final concentration of CHIR98014 is 0.1 μM to 0.3 μM;

[0011] The first culture is a mixture of A83 - 01, CHIR98014, DBZ, and PD173074, with the final concentration of A83 - 01 being 1 μM to 3 μM, the final concentration of CHIR98014 being 0.4 μM to 0.6 μM, the final concentration of DBZ being 0.9 μM to 1.1 μM, and the final concentration of PD173074 being 24 nM to 26 nM;

[0012] The second culture is a mixture of bradykinin, prostaglandin E2, histamine, 5-hydroxytryptamine, and ATP. The final concentration of bradykinin is 9 μM - 1.1 μM, the final concentration of prostaglandin E2 is 9 μM - 11 μM, the final concentration of histamine is 9 μM - 11 μM, the final concentration of 5-hydroxytryptamine is 9 μM - 11 μM, and the final concentration of ATP is 14 μM - 16 μM;

[0013] Construct a nociceptor skin organoid chip: Use the above-mentioned culture medium to co-culture the skin chip with the obtained nociceptors to obtain a nociceptor skin organoid chip.

[0014] Preferably, the cell inhibitor is added to E6 culture medium.

[0015] Preferably, the first culture is added to E6 culture medium.

[0016] Preferably, the second culture is added to Noci-3 culture medium;

[0017] Noci-3 culture medium is a DEME / F-12 culture medium containing N2 additive, B27 additive, 20 ng / mL - 30 ng / mL of BDNF / GDNF / NGF / NT-3, and 0.5 μM - 1 μM of PD0332991.

[0018] Preferably, after co-culturing for 20 h - 30 h, a reagent that promotes cells to enter the differentiation state is added to the culture medium.

[0019] Preferably, the reagent that promotes cells to enter the differentiation state is calcium chloride. After adding it to the culture medium, the final concentration is 1 mM - 2 mM.

[0020] The nociceptor skin organoid chip constructed by the above construction method.

[0021] The application of the above-mentioned nociceptor skin organoid chip in the preparation of a verification product for pain perception effects.

[0022] There is currently no complete skin chip with nociceptors added. Nociceptors are mainly distributed in the superficial layers of the epidermis and dermis. They are the peripheral processes of primary sensory neurons (DRGs), usually called peripheral processes. The fibers of these nerve endings contain abundant receptors that can sense tissue damage, chemical stimuli, etc. When the tissue is damaged or stimulated, nociceptors generate pain signals, which are transmitted to the brain through nerves, causing the sensation of pain. When a specific substance stimulates or activates nociceptors, the generated nerve impulses can be transmitted through afferent nerve fibers and activate neurons at all levels on the pain conduction pathway, ultimately causing the pain sensation. When combining the skin chip with nociceptors, it is necessary to form an ordered structure between the components in the original skin chip and the peripheral processes of nociceptors, and through self-organization, it has the function of sensing pain information. Among them, DRG neurons have a special "T" - shaped structure, and their peripheral processes are important components that make up nociceptors. Therefore, co - culturing DRGs or active peripheral nerve fibers with the skin chip is a feasible way to construct a skin chip containing nociceptors and having certain functions.

[0023] The guiding method for the formation of nociceptive sensory neurons is as follows: Cultivate human embryonic stem cells (hPSCs), apply CHIR98014 in a specific culture medium to transform hPSCs into SOX10 + neural crest cells, and then use A83 - 01, DBZ, and PD173074 in combination to further promote the maturation and differentiation of neural crest cells into specific neuron types, such as peptidergic or non - peptidergic dorsal root ganglion cells. In addition, to ensure the true conduction function of sensory nerve axons, we use a mixture of inflammatory factors (including histamine, prostaglandin, bradykinin, and adenosine) to enhance the transport and surface expression of the NaV1.7 channel, thereby enhancing the sensitivity of axons to nociceptive stimuli.

[0024] The process of combining nociceptors with the existing skin chip requires forming an ordered structure between the components in the original skin chip and the peripheral processes of nociceptors, and through self - organization, it has the function of sensing pain information. Among them, the interaction between keratinocytes and neurons and the formation of synapse - like connections play a key role in information transmission. Since neurotransmitters are released through the vesicle release mechanism mediated by SNARE proteins only after TRPV1 on keratinocytes is activated, and then sensory neurons are activated to transmit pain signals, capsaicin Capsacine is used to activate keratinocytes during the fusion process of nociceptors and the original organ chip.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] 1. The present invention provides a culture medium for culturing a nociceptor skin organoid chip. By co-culturing a skin chip and nociceptors with this culture medium, a nociceptor skin organoid chip can be obtained, which can be used to verify the pain perception effect.

[0027] 2. The present invention has established a nociceptor skin organoid chip. By applying this platform, it greatly avoids the consumption of a large amount of experimental animal resources and testing costs caused by the screening of analgesic factors using animal experiments, laying a solid foundation for the future effects on the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram for creating a skin chip device. The customized microfluidic channels provide a good opportunity for good and uniform exposure to nanomaterials (NMs). Reconstructing the 3D skin microenvironment requires an air-liquid interface. Different cell types can be cultured on top of the porous membrane. Selecting the cell source is crucial for achieving biological functions. Currently, the introduction of cells of the immune system and vascular system has been carried out, and in the future, it is still necessary to include the skin microbiota, innervation, and skin appendages.

[0029] Figure 2 It is a fluorescence microscopic image of DRG neurite growth and cell migration in a three-dimensional double hydrogel structure, showing β-III tubulin-positive neurites and s100-positive ganglion cells. It can be seen that the neurites are restricted within the channels filled with the ECM. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0030] The following is a detailed description of the specific embodiments of the present invention, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the protection scope of the present invention. The experimental methods described in the embodiments of the present invention are all conventional methods unless otherwise specified.

[0031] Establishment of nociceptor components: A 3D hydrogel structure is fabricated using dynamic photolithography, with regions that permit and inhibit growth within the structure, thereby restricting the growth direction of nerve axons within the 3D structure. In previous studies, dorsal root ganglia from embryonic day 15 can be taken as grafts and transplanted into the above-mentioned 3D hydrogel structure for culture. Generally, mature neurites will grow after 7 days of culture (PMID: 25850799). However, for this type of culture, the resulting neuron types are not specific. In the present invention, neurons derived from stem cells rather than explants are used. By introducing specific small molecules into the culture medium, the differentiation of nociceptive neurons is promoted, and then they are directed to grow within the above-mentioned 3D hydrogel structure, so as to promote the development of free nerve endings of nociceptors composed of the peripheral processes of dorsal root ganglia. At this time, a preliminary nociceptor can be established.

[0032] It should be noted that the skin chip is a skin chip constructed by a method in the prior art. For example, Figure 1 As shown, the present invention only lists a skin chip constructed by one method, and the steps are as follows:

[0033] (1) Select different materials, such as PMMA (polymethyl methacrylate), PS (polystyrene), and PDMS (polydimethylsiloxane). Use CNC micro milling (Computer Numerical Control micro milling) technology to fabricate the chip.

[0034] (2) Cultivate different types of skin cells on the chip, including keratinocytes in the epidermis and fibroblasts in the dermis. Keratinocytes in the epidermis such as NHKs - normal human keratinocytes, and fibroblasts in the dermis such as HFFs - fibroblasts derived from human forehead skin. Adjust the seeding density according to the cell type: Generally, the NHKs in the epidermis are 10 6 cells / mL, and the HFFs in the dermis are 2.5×10 4 cells / cm 2 .

[0035] (3) During the culture process, use microfluidic channels to control the exposure and transcellular transport of nanomaterials, achieve perfusion flow, and control the flow rate at 1 μL / min to simulate the in vivo environment.

[0036] (4) Real-time monitoring of cell function and tissue integrity is achieved through an integrated sensor. At the same time, multiple parameters are automatically measured, such as pH, oxygen, glucose, or lactate, for multi-parameter evaluation. The integrated sensor is such as a TEER sensor.

[0037] The information on the culture media and reagents used in the present invention is as follows:

[0038] The model number of E8 medium is A1517001, and the manufacturer is thermofisher.

[0039] The model number of E6 medium is A1516401, and the manufacturer is thermofisher.

[0040] The model number of DEME / F-12 medium is A4192001, and the manufacturer is thermofisher.

[0041] The catalog number of N2 supplement is 1750248, and the manufacturer is Gibco. In the use of the present invention, it is diluted 100 times.

[0042] The catalog number of B27 supplement is 17505044, and the manufacturer is Gibco. In the use of the present invention, it is diluted 50 times.

[0043] Noci-1 medium is E6 medium containing 2 μM A83-01 and 0.2 μM CHIR98014. Among them, A83-01 is a transforming growth factor-b inhibitor, and CHIR98014 is a GSK-3b inhibitor, which is used to activate the WNT signaling pathway.

[0044] Noci-2 medium is E6 medium containing 2 μM A83-01, 0.5 μM CHIR98014, 1 μM DBZ and 25 nM PD173074.

[0045] Noci-3 medium is DEME / F-12 medium containing N2 supplement, B27 supplement, 25 ng / mL of BDNF / GDNF / NGF / NT-3 and 1 μM PD0332991. Among them, the volume ratio of N2 supplement to DEME / F-12 medium is 1:1, and the volume ratio of B27 supplement to DEME / F-12 medium is 1:1.

[0046] The CEPT mixture is composed of 50 nM Chroman 1 (#HY-15392; MedChem Express), 5 mM Emricasan (#S7775; Selleckchem), polyamine supplement (#P8, diluted 1:1000; Sigma-Aldrich) and 0.7 mM Trans-ISRIB (product number #5284; Tocris) is used to improve cell viability and provide cell protection for cell passage.

[0047] Noci-2+CEPT medium is obtained by mixing Noci-2 medium and CEPT mixture at a volume ratio of 1:1.

[0048] The Noci-3+CEPT medium is obtained by mixing the Noci-3 medium and the CEPT mixture in a volume ratio of 1:1.

[0049] The IM cocktail+Noci-3 medium is obtained by adding an inflammatory mediator mixture (IM) to the Noci-3 medium. The components of the inflammatory mediator mixture are as follows: 1 μM bradykinin, 10 μM prostaglandin E2 (PGE-2), 10 μM histamine, 10 μM 5-hydroxytryptamine (5-HT), and 15 μM ATP.

[0050] Example 1

[0051] A method for constructing nociceptors, comprising the following steps:

[0052] (1) Human pluripotent stem cells (hPSCs) are routinely cultured in an E8 medium on a 6-well plate coated with vitronectin (VTN), washed with 0.5 mM EDTA for 5-6 minutes, and passaged every 3 days. The EDTA is prepared with PBS without magnesium and calcium.

[0053] (2) On day 1, for differentiation, the cells are counted using a Nexcelom cell counter and seeded in the E8 medium in a VTN-coated 6-well plate at a density of 1.5×10 5 cell / cm 2 .

[0054] (3) On day 2, the E8 medium in step (2) is replaced with the Noci-1 medium.

[0055] (4) The Noci-1 medium is replaced daily. On day 3, the cells are washed with PBS, 1 mL of Accutase is added to each well of the 6-well plate, incubated at 37 °C for 5 min, the cells are resuspended in 5 mL of the Noci-2 medium, gently pipetted up and down 10 times to disperse the cells into single cells, and then centrifuged at 300 g for 3 min at room temperature. The supernatant is removed, and the cells are suspended in the Noci-2+CEPT medium. According to the product instructions (STEMCELL Technologies), 5.5×10 6 cells / well are seeded into a 6-well AggreWell 800 culture plate for suspension culture and the formation of nociceptive neuron spheres.

[0056] (5) During days 4 to 14 (nocisphere stage), replace the Noci-2 + CEPT medium in step (4) with Noci-2 medium daily. The CEPT mixture is only used for 24 hours during cell aggregation and nocisphere formation and is no longer recommended for use during daily medium replacement. During the nocisphere stage, gently remove the Noci-2 medium (4 mL) and add fresh Noci-2 medium to the container wall to avoid disturbing the nocisphere balls.

[0057] (6) Collect the nocisphere balls contained in a 6-well AggreWell 800 culture plate into a 50 mL test tube, let it stand for 5 minutes, remove the Noci-2 medium, wash with 10 mL PBS, and let it stand again. After removing the PBS, dissociate the spheres using the MACS EB isolation kit numbered 130-096-348 according to the product instructions of Miltenyi Biotec. Resuspend the cell pellet in Noci-3 + CEPT medium, and then pass the cells through a 70-micron cell strainer. Next, count the cells at 2×10 6 cells / well and seed them into a 6-well plate coated with Geltrex, and culture using Noci-3 medium + CEPT.

[0058] (7) Days 15 to 28: After 24 hours, replace the Noci-3 + CEPT medium in step (6) with fresh Noci-3 medium. The nociceptive neurons can be cultured up to day 28 or longer for further maturation, and the Noci-3 medium is replaced every 2 - 3 days.

[0059] (8) Cryopreservation and recovery: If cryopreservation is required, on day 14, cryopreserve the isolated neurosphere cells (5 million cells per milliliter) in Noci-3 + CEPT medium + 10% dimethyl sulfoxide for use as needed. The thawing and patching of sensory nerve cells should be subjected to 24-hour CEPT treatment, where Noci-3 + CEPT medium + 10% dimethyl sulfoxide means adding 10% (volume fraction) of dimethyl sulfoxide to the Noci-3 + CEPT medium.

[0060] (9) Inducing increased Nav1.7 channel expression: On days 15 - 28 of culture, first mix the stock solution of IM with a portion of the medium in the culture dish, dilute it, and then add it back to the original well to prevent the cells from being exposed to high concentrations of the compound. After incubating with IM cocktail + Noci-3 medium for 4 hours every day, replace the IM cocktail + Noci-3 medium with Noci-3 medium to obtain nociceptors, as Figure 2 shown.

[0061] Example 2

[0062] A method for constructing a nociceptor skin organoid chip, comprising the following steps:

[0063] In a humidity environment of 37 °C and 5% CO2, the skin chip cultured by microfluidic technology is co-cultured with the nociceptors obtained in Example 1 using a co-culture medium. The specific process is as follows: Select a standard 6-well plate, and coat the surface of the culture plate with Geltrex matrix protein - SNARE protein to promote cell adhesion. Place the skin chip into the wells of the culture plate, and inoculate the nociceptor cell tissue onto or near the skin chip to interact with the cells in the chip. After culturing for 24 hours, add 1.5 mM of CaCl2 to the co-culture medium, and maintain the culture for 4 to 6 days. Update 50% of the co-culture medium every 2 to 3 days to obtain a nociceptor skin organoid chip.

[0064] Among them, the co-culture medium is composed of fetal bovine serum and the following substances added to fetal bovine serum: epidermal growth factor at 100 ng / mL and bovine pituitary extract at 20 μL / mL, and contains Normocin at a concentration of 100 g / mL, B27 at a concentration of 20 μL / mL, nerve growth factor (NGF) at a concentration of 100 ng / mL, insulin at a concentration of 4 μg / mL, hydrocortisone at a concentration of 10 ng / mL, human brain-derived neurotrophic factor (BDNF) at a concentration of 10 ng / mL, and capsaicin at 40 μM.

[0065] When constructing the nociceptor skin organoid chip, since differentiated keratinocytes are more likely to form synapses with nociceptive cells, the added CaCl2 causes more keratinocytes to enter the differentiated state. Capsaicin at a concentration of 40 μM is applied in the co-culture medium to activate the TRPV1 receptor. In the synapse-like contact between keratinocytes and sensory neurons, when the TRPV1 receptor is activated, it may cause keratinocytes to release neurotransmitters, which bind to specific receptors on sensory neurons to complete the signal transduction process. Therefore, capsaicin is applied to activate the TRPV1 receptor.

[0066] Example 3

[0067] A comparison was made between the organizational structure changes and the changes in afferent signals of a nociceptor skin organ chip and a mouse after being irradiated with the same laser. The specific experimental process is as follows:

[0068] Take the prepared nociceptor skin organoid chip and divide it into experimental group 1 and the control group. Additionally, take 6-week-old C57 mice as experimental group 2 for laser plantar irradiation. Both experimental group 1 and experimental group 2 received laser irradiation with a specific 808 nm laser at a power output of 2.5 W for a duration of 10 s, and the irradiation area was 2 mm 2 . Except for not receiving irradiation, the control group was treated the same as experimental group 1.

[0069] Observation indicators include: 1. Observe the morphological changes of the skin in experimental group 1, experimental group 2, and the control group through histological HE staining, and evaluate the degree of tissue damage. 2. Through electrophysiological recordings, such as single-fiber recordings of peripheral nerve trunks or using microelectrode arrays to record the nerve signals of the peripheral processes of nociceptors in the three groups.

[0070] Data collection and analysis: Collect morphological, pathological, and electrophysiological data, and use statistical methods to compare the differences between the two experimental groups and the control group.

[0071] Conclusion: By comparing, the data of experimental group 1 and experimental group 2 are close, and there are potential changes in the peripheral processes in both. The control group does not show manifestations such as inflammatory cell infiltration, changes in skin stratification, and disordered cell arrangement; no afferent nerve impulses, that is, changes in action potentials, can be recorded on the peripheral processes of nociceptors.

[0072] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that any value between the two endpoints of each numerical range and the two endpoints themselves can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0073] Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of the present invention.

[0074] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims

1. A method for constructing a nociceptor skin organoid chip, characterized in that, Comprising the following steps: Constructing nociceptors: culturing human embryonic stem cells successively with a cell inhibitor, a first culture medium, and a second culture medium; When culturing human embryonic stem cells, adding the cell inhibitor, the first culture medium, and the second culture medium into the culture medium respectively; The cell inhibitor is a mixture of CHIR98014 and A83-01, the final concentration of A83-01 is 1 μM - 3 μM, and the final concentration of CHIR98014 is 0.1 μM - 0.3 μM; The first culture medium is a mixture of A83-01, CHIR98014, DBZ, and PD173074, the final concentration of A83-01 is 1 μM - 3 μM, the final concentration of CHIR98014 is 0.4 μM - 0.6 μM, the final concentration of DBZ is 0.9 μM - 1.1 μM, and the final concentration of PD173074 is 24 nM - 26 nM; The second culture medium is a mixture of bradykinin, prostaglandin E2, histamine, 5-hydroxytryptamine, and ATP, the final concentration of bradykinin is 0.9 μM - 1.1 μM, the final concentration of prostaglandin E2 is 9 μM - 11 μM, the final concentration of histamine is 9 μM - 11 μM, the final concentration of 5-hydroxytryptamine is 9 μM - 11 μM, and the final concentration of ATP is 14 μM - 16 μM; Constructing a nociceptor skin organoid chip: co-culturing the skin chip with the obtained nociceptors using a co-culture medium to obtain a nociceptor skin organoid chip; The co-culture medium consists of fetal bovine serum and the following substances added to fetal bovine serum: epidermal growth factor at 100 ng / mL and bovine pituitary extract at 20 μL / mL, and contains Normocin at a concentration of 100 mg / mL, B27 at a concentration of 20 μL / mL, nerve growth factor at a concentration of 100 ng / mL, insulin at a concentration of 4 μg / mL, hydrocortisone at a concentration of 10 ng / mL, human brain-derived neurotrophic factor at a concentration of 10 ng / mL, and capsaicin at 40 μM.

2. The construction method according to claim 1, wherein The cell medium to which the cell inhibitor is added is E6 medium.

3. The construction method according to claim 1, wherein The cell medium to which the first culture medium is added is E6 medium.

4. The construction method according to claim 1, characterized in that The cell medium to which the second culture medium is added is Noci-3 medium; Noci-3 medium is a DEME / F-12 medium containing N2 additive, B27 additive, 20 ng / mL - 30 ng / mL of BDNF / GDNF / NGF / NT-3, and 0.5 μM - 1 μM of PD0332991.

5. The construction method according to claim 1, characterized in that, Adding a reagent that promotes cells to enter a differentiation state to the culture medium after co-culturing for 20 h - 30 h.

6. The construction method according to claim 5, characterized in that, The reagent that promotes cells to enter a differentiation state is calcium chloride, and after adding it to the culture medium, the final concentration is 1 mM - 2 mM.

7. A nociceptor skin organoid chip constructed by the construction method according to any one of claims 1 - 6.

8. Use of the nociceptor skin organoid chip according to claim 7 in the preparation of a product for verifying pain perception effects.

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