Sexual gland skin chip as well as preparation method and application thereof

Abstract

The invention discloses a gonad skin chip as well as a preparation method and application thereof, based on a micro-fluidic technology and a three-dimensional cell culture system, firstly, a culture unit containing skin tissue and gonad tissue is constructed in the chip, and nutrient substances and hormones are supplied through micro-fluidic channels respectively; the dynamic interaction of the skin and the gonad tissue is realized by accurately controlling the flow velocity of the culture medium and the hormone concentration gradient. The skin culture room of the chip supports growth and differentiation of skin cells; and the gonad culture room supports the culture of ovarian or testicular cells and the secretion of hormones. The obtained gonad skin chip can effectively simulate the skin-gonad interaction process, and provides an experimental platform for hormone research, dermatology and drug screening.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical engineering, and specifically relates to a gonad skin chip and a preparation method and application thereof. Background Art

[0002] Skin chip technology is an emerging microfluidic technology platform that can simulate the physiological environment of human skin and accurately reproduce the behavior of skin cells under different physiological conditions. With the deepening of skin research in the biomedical field, skin chips have gradually become an important tool for studying skin diseases, drug delivery, skin reactions and other issues. Traditional skin research mostly relies on animal models or two-dimensional cell culture systems. Although these methods have certain application value, they also have the problem of being unable to fully simulate the complex structure and physiological reactions of human skin. Skin chips provide a more realistic simulation for the study of skin-related diseases by integrating multiple cell types such as skin tissue structure, microvessels, nerves and hormones and their microenvironment.

[0003] In the application of skin chip technology, how to accurately control the interaction between skin cells and external stimuli (such as drugs and hormones) is one of its core challenges. Especially when simulating the effects of hormones on the skin, due to the large fluctuations in the types and concentrations of hormones, traditional chip models often cannot provide sufficient fine regulation. With the deepening of the understanding of the role of gonadal hormones (such as estrogen, testosterone, etc.) in skin physiology, the development of a skin chip that can simulate gonadal hormone responses has become an important topic in the current field of skin research.

[0004] Gonadal hormones play a vital role in the physiological functions of the skin, such as regulating skin growth, repair, and immune response. For the treatment of hormone-dependent skin diseases (such as acne, skin aging, etc.), an experimental platform that can accurately simulate the effects of hormones is needed. Therefore, researchers are increasingly interested in how to incorporate the dynamic changes of gonadal hormones into skin chips and test their role in drug absorption, metabolism, and response.

[0005] However, the current gonad skin chip technology still faces many challenges. For example, the selection and arrangement of cell types within the chip, the precise release and regulation of hormones, and the physiological changes of cells under different environments all limit the widespread application of this technology. Existing chips are often unable to simulate the interaction of multiple hormones and their effects on skin cells at the same time, and their application potential in personalized drug testing and disease research has not yet been fully realized. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide a skin chip that can more accurately simulate the effects of gonadal hormones on the skin in response to the deficiencies of the prior art.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows: A gonad skin chip comprises a gonad hormone releasing module and a skin culture module; the gonad hormone releasing modules are a group, each group of gonad hormone releasing modules culture a gonad cell globules and produce corresponding hormones; the skin culture module is provided with a group of inlets corresponding to the gonad hormone releasing modules for receiving the hormones produced by each gonad hormone releasing module; a group of skin culture chambers are provided in the skin culture module, and a bionic skin with a multi-layer structure of dermis, epidermal basal layer, epidermal granular layer and epidermal keratinized layer is formed in the skin culture chamber by cell culture; a graded flow channel is provided between the inlet of the skin culture module and the skin culture chamber.

[0008] Specifically, the gonadal hormone release module includes an inlet, a graded inlet channel, a pit array, a graded outflow channel and an outlet.

[0009] Specifically, the skin culture module includes a group of inlets, graded inlet channels, multi-level graded concentration gradient channels, a group of skin culture chambers and an outlet; the inlets of the skin culture module are respectively connected to the outlets of the gonadal hormone release module.

[0010] Specifically, the gonad skin chip of the present invention has a size of 2~3 cm × 1~3 cm, a microchannel width of 200-500 µm, and a microchannel depth of 100-300 µm, which ensures the growth and distribution of cells in the chip.

[0011] Furthermore, the present invention also provides a method for preparing the gonad skin chip, comprising the following steps: (1) Preparation of chip substrate Mixing polydimethylsiloxane and a curing agent and pouring them into a mold, and cutting them after curing to obtain a PDMS substrate; preparing a PDMS sheet of the same size as the PDMS substrate, and bonding the two to prepare a chip substrate with a gonadal hormone release module and a chip substrate with a skin culture module respectively; (2) Preparation of bionic skin The skin cells are cultured in their respective culture media, and after the cells reach a certain seeding density, they are uniformly introduced into the skin culture chamber of the chip substrate having the skin culture module in step (1), and the growth factors in the culture media are adjusted to promote the growth and differentiation of various cells and form a multi-layer structure of the skin; (3) Embedding of biomimetic gonad cell spheroids The gonadal cells are suspended in a culture medium and then added to a chip substrate having a gonadal hormone release module to form gonadal cell aggregates; gonadal hormones with a stable concentration are obtained through cell culture; (4) The chip substrate with the gonad hormone release module and the chip substrate with the skin culture module are connected through microfluidic channels to enable the exchange of nutrients and hormones between the two to simulate the physiological interaction between skin and gonad tissue.

[0012] Specifically, in step (1), the mold is a positive mold of a photocurable resin having a microchannel structure constructed by 3D printing, and is subjected to silanization and hydrophobic treatment.

[0013] Specifically, in step (1), the support material of the gonad skin chip is polydimethylsiloxane (PDMS), which has good physical and chemical properties and biocompatibility and is suitable for long-term use. After the PDMS substrate and the PDMS sheet are cleaned with isopropyl alcohol, a plasma cleaning agent is used to activate the Si-CH3 functional groups on the surface of the PDMS substrate and the PDMS sheet, and the PDMS is bonded by Si-O-Si linking to achieve the bonding of the two.

[0014] Specifically, in step (2), the skin cells include epidermal cells, fibroblasts and skin immune cells.

[0015] Specifically, in step (2), the culture temperature is maintained at 35-38°C and the carbon dioxide concentration is 4-6 vt.% to simulate the physiological environment in vivo.

[0016] Specifically, in step (3), the gonadal cells include at least testicular cells and / or ovarian cells; they are added to the corresponding pit array of the gonadal hormone release module through a microchannel; during cell culture, the sedimentation, aggregation and formation of cell polymers of the gonadal cells are ensured to obtain androgens and / or estrogens with stable concentrations.

[0017] Furthermore, the present invention also claims to protect the use of the above-mentioned gonad skin chip in drug screening, skin care or hormone research, and utilizes the gonad skin chip of the present invention to simulate the physiological interaction between skin and gonad tissue, thereby playing the role of an experimental platform. Beneficial Effects

[0018] (1) The present invention uses microfluidic technology to construct a culture unit including skin tissue and gonad tissue on a chip. The skin unit is composed of cells such as skin fibroblasts and keratinocytes to simulate the epidermis and dermis of the skin; the gonad unit is composed of ovarian or testicular cells to simulate the function of the gonads. The two units are connected by a microfluidic channel so that nutrients and hormones can be exchanged between the two layers, simulating the physiological interaction between the skin and gonadal tissues. The use of specially designed microfluidic channels makes the signal transmission and hormone delivery between the skin layer and the gonadal layer more precise, and the dynamic interaction between the skin and gonads is achieved by adjusting the flow rate and hormone concentration gradient. The resulting gonad skin chip can effectively simulate the skin-gonad interaction process and provide an experimental platform for hormone research, dermatology and drug screening.

[0019] (2) The present invention can accurately simulate the multi-layered structure and physiological functions of the skin and is suitable for fields such as drug screening, skin care and hormone research. Through cell co-culture and drug-induced reactions, the interaction between gonads and skin cells can be truly reproduced, with high physiological relevance. The chip can simulate the long-term gonadal response to the skin in vitro, saving animal experiments, reducing costs, and has high application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0021] Figure 1 Schematic diagram of the structure of the gonadal hormone circulation channel formed with a concentration gradient in the embodiment.

[0022] Figure 2 is a schematic diagram of embedding gonadal cells in the gonadal hormone releasing system of the embodiment.

[0023] Figure 3 The figure is a picture of the culture effect of testicular cells and ovarian cells in the chip substrate. Among them, a is the cell aggregation structure of testicular cells after 5 minutes and 48 hours of culture; b is the cell aggregation structure of ovarian cells after 5 minutes and 48 hours of culture; c is the ovarian cell aggregation structure verified by cytoskeleton staining; d is the testicular cell aggregation structure verified by cytoskeleton staining; e is the concentration change of testosterone and estradiol in the chip outflow within 24 hours.

[0024] Figure 4 It is a working schematic diagram of the gonad skin chip of the present invention. DETAILED DESCRIPTION

[0025] The present invention can be better understood with reference to the following examples. Example 1

[0026] Preparation of gonadal skin chip In this embodiment, the gonad skin chip includes a gonad hormone release module and a skin culture module. The gonad hormone release module includes an inlet, a graded inlet channel, a pit array, a graded outflow channel, and an outlet; the skin culture module (such as Figure 1 The chip was fabricated by 3D printing (as shown in Figure 1) and consisted of two inlets, a graded inlet channel, a 10-level graded concentration gradient channel, ten skin culture chambers, and one outlet. The channel width was set to 200 µm and the depth to 150 µm to accommodate cell growth and fluid flow. A photocurable resin positive mold with a designed microchannel structure was constructed using 3D printing, and silanization and hydrophobic treatment were completed. Polydimethylsiloxane (PDMS) was selected as the support material for the chip. PDMS and its curing agent were mixed in a mass ratio of 10:1, and vacuum degassing was performed after stirring evenly to ensure that bubbles were removed. Then, the mixture was poured into the positive mold, baked at 80°C for 2h, and demolded after cooling. After demolding, the PDMS was cut 1 cm away from the boundary of the channel and chamber area to complete the preparation of the chip substrate. A PDMS sheet with a thickness of 3 mm and the same size as the PDMS substrate was prepared. Isopropyl alcohol (IPA) was used to clean the chip substrate and PDMS sheet, and then a plasma cleaner was used to activate the Si-CH3 functional groups on the chip substrate and PDMS sheet surface, and PDMS was bonded through Si-O-Si links. Finally, holes were punched at the designed positions and the microfluidic pipelines were connected to complete the preparation of the gonad skin chip. Example 2

[0027] Preparation of bionic skin Human primary keratinocytes and human primary fibroblasts were cultured in complete skin cell culture medium. When the cells reached a seeding density of 75%, they were made into a culture medium with a density of 1×10 6 Cell suspensions of 10 cells / mL are uniformly introduced into the skin culture chamber of the chip substrate with the skin culture module at a flow rate of 15ul / min. During the cell culture process, the growth factors in the culture medium are adjusted to promote cell growth and differentiation and form a multilayer structure with dermis, epidermal basal layer, epidermal granular layer and epidermal keratinized layer. During the epidermal culture process, the culture medium in the skin culture chamber needs to be reduced after 3 days of culture so that the liquid level of the culture medium is lower than the upper layer of the epidermis to form a gas-liquid interface required for keratin differentiation. The bionic skin culture temperature is maintained at 37°C and the carbon dioxide concentration is 5% to simulate the physiological environment in the body. Example 3

[0028] Embedding of biomimetic gonadal cell spheroids Testicular cells and ovarian cells were suspended in complete culture medium and added to two well arrays in the chip substrate with gonadal hormone release modules through microchannels. During cell culture, the sedimentation, aggregation and formation of cell aggregates of gonadal cells were ensured (e.g. Figure 2 As shown in Figure 3), the cell aggregates of testicular cells and ovarian cells can continuously secrete sex hormones within 6, 12 and 24 hours of culture. Within 24 hours, the concentration of testosterone in the chip effluent can be maintained at 1.1 ng / mL, and the concentration of estradiol can be maintained at 1.4 ng / mL (Figure 3e). Example 4

[0029] Combination Figure 4 The working schematic diagram of the gonad skin chip shown in the figure, the simulation operation of the gonad skin chip of the present invention is as follows: The outlet of the gonadal hormone release module was connected to the two inlets of the skin culture module, and androgens and estrogens flowed into the skin culture chamber through a 10-level graded concentration gradient channel. After testing, the concentrations of estradiol and testosterone in the 1st to 10th concentration gradients were 0.15 / 0.95, 0.30 / 0.73, 0.46 / 0.57, 0.62 / 0.46, 0.74 / 0.40, 0.9 / 0.34, 1.06 / 0.29, 1.22 / 0.22, 1.36 / 0.14, and 1.52 / 0.07, respectively, and all concentration units were ng / mL. After 24 hours of sex hormone stimulation, the bionic skin was collected for subsequent histopathological tests.

[0030] The present invention provides a gonad skin chip and its preparation method and application ideas and methods. There are many methods and ways to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented by existing technologies.

Claims

1. A gonad skin chip, characterized in that: It comprises a gonadal hormone releasing module and a skin culture module; the gonadal hormone releasing module is a group, each group of gonadal hormone releasing modules culture a gonadal cell globules and produce corresponding hormones; the skin culture module is provided with a group of inlets corresponding to the gonadal hormone releasing modules for receiving the hormones produced by each gonadal hormone releasing module; a group of skin culture chambers are provided in the skin culture module, and a bionic skin with a multi-layer structure of dermis, epidermal basal layer, epidermal granular layer and epidermal keratinized layer is formed in the skin culture chamber by cell culture; a graded flow channel is provided between the inlet of the skin culture module and the skin culture chamber.

2. The gonad skin chip according to claim 1, characterized in that: The gonadal hormone release module comprises an inlet, a graded inlet channel, a pit array, a graded outflow channel and an outlet.

3. The gonad skin chip according to claim 1, characterized in that: The skin culture module comprises a group of inlets, graded inlet channels, multi-level graded concentration gradient channels, a group of skin culture chambers and an outlet; the inlets of the skin culture module are respectively connected to the outlets of the gonad hormone release module.

4. The method for preparing the gonad skin chip according to claim 1, characterized in that: The steps include: (1) Preparation of chip substrate Mixing polydimethylsiloxane and a curing agent and pouring them into a mold, and cutting them after curing to obtain a PDMS substrate; preparing a PDMS sheet of the same size as the PDMS substrate, and bonding the two to prepare a chip substrate with a gonadal hormone release module and a chip substrate with a skin culture module respectively; (2) Preparation of bionic skin The skin cells are cultured in their respective culture media, and after the cells reach a certain seeding density, they are uniformly introduced into the skin culture chamber of the chip substrate having the skin culture module in step (1), and the growth factors in the culture media are adjusted to promote the growth and differentiation of various cells and form a multi-layer structure of the skin; (3) Embedding of biomimetic gonad cell spheroids The gonadal cells are suspended in a culture medium and then added to a chip substrate having a gonadal hormone release module to form gonadal cell aggregates; gonadal hormones with a stable concentration are obtained through cell culture; (4) The chip substrate with the gonad hormone release module and the chip substrate with the skin culture module are connected through microfluidic channels to enable the exchange of nutrients and hormones between the two to simulate the physiological interaction between skin and gonad tissue.

5. The method for preparing the glandular skin chip according to claim 4, characterized in that: In step (1), the mold is a positive mold of a photocurable resin having a microchannel structure constructed by 3D printing, and is subjected to silanization and hydrophobic treatment.

6. The method for preparing the glandular skin chip according to claim 4, characterized in that: In step (1), the PDMS substrate and the PDMS sheet are cleaned with isopropyl alcohol, and then a plasma cleaning agent is used to activate the Si-CH3 functional groups on the surface of the PDMS substrate and the PDMS sheet, and the PDMS is bonded via Si-O-Si links to achieve bonding between the two.

7. The method for preparing the glandular skin chip according to claim 4, characterized in that: In step (2), the skin cells include epidermal cells, fibroblasts and skin immune cells.

8. The method for preparing the glandular skin chip according to claim 4, characterized in that: In step (2), the culture temperature is maintained at 35-38°C and the carbon dioxide concentration is 4-6 vt.% to simulate the physiological environment in vivo.

9. The method for preparing the glandular skin chip according to claim 4, characterized in that: In step (3), the gonadal cells include at least testicular cells and / or ovarian cells; they are added to the corresponding pit array of the gonadal hormone release module through the microchannel; during cell culture, the sedimentation, aggregation and formation of cell polymers of the gonadal cells are ensured to obtain androgens and / or estrogens with stable concentrations.

10. Use of the gonad skin chip according to claim 1 in drug screening, skin care or hormone research.

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