Method for obtaining sweat gland organoids from sweat gland cells and applications thereof

Abstract

The application discloses a method for culturing sweat gland cells to obtain a sweat gland organoid and application thereof, and belongs to the technical field of bioengineering, wherein the sweat gland cells can be cultured by using a three-dimensional culture medium provided by the application, and a sweat gland organoid with high cell activity and bidirectional differentiation potential is obtained; after being transplanted into a full-thickness injury mouse on the sole and back, the sweat gland organoid not only can promote the regeneration and function recovery of the sweat gland, but also can promote the healing of the skin wound, and can be used for preparing a cell product for repairing skin injury and recovering the function of an accessory organ, and has a wide application prospect.

Description

[0001] This application is a divisional application of application number 201910175914.4, filed on March 8, 2019, entitled "Method for obtaining sweat gland organoids by isolating and culturing sweat gland cells and its application". Technical Field

[0002] This invention belongs to the field of bioengineering technology, specifically relating to a method for culturing sweat gland cells, and particularly to a method for obtaining sweat gland organoids by culturing sweat gland cells and their application in skin repair of sweat gland damage. Background Technology

[0003] Sweat glands, as appendages of the skin—the largest organ in the human body—play a vital role in regulating body temperature, balancing body fluids, and promoting metabolism. Sweat glands develop during the embryonic period. After birth, when their structure is damaged, such as in a minor burn, the glands can repair themselves using the undamaged deeper tissues as a template. However, when the structure is completely destroyed, the glands cannot regenerate. For example, in severe deep burns, the wound healing site lacks sweat gland structure, and the patient cannot regain the sweating function at the site of the injury. Although advancements in medical technology have significantly improved the survival rate of patients with extensive burns, the lack of sweat gland function still significantly reduces their quality of life after recovery. Furthermore, several hereditary diseases exist that involve abnormal development and dysfunction of sweat glands, and currently, existing treatments cannot restore the normal structure and function of sweat glands. Therefore, promoting the reconstruction of sweat gland function in the affected area has become a crucial issue to be addressed in the healing process of skin wounds.

[0004] In recent years, with the deepening of research in stem cell biology and regenerative medicine, stem cell therapy has shown good tissue regeneration capabilities, and stem cell technology has brought new hope for sweat gland regeneration. Applying stem cell technology to promote sweat gland regeneration requires solving two key problems. First, the source of cells: the obtained cells must not only have strong proliferative capacity but also the potential to differentiate into sweat gland cells and perform sweat gland functions. Currently, studies have been reported on inducing differentiation of embryonic stem cells, epidermal stem cells, mesenchymal stem cells (bone marrow mesenchymal stem cells and umbilical cord mesenchymal stem cells), and hair follicle stem cells into sweat glands. However, these studies all suffer from difficulties in controlling the differentiation direction of stem cells and low differentiation efficiency. These problems limit the application of these stem cells as seed cells for sweat glands. It has been reported that although the number of sweat glands is determined before birth, a certain number of sweat gland stem cells still exist in adults. Therefore, if these sweat gland stem cells can be rapidly expanded, they will undoubtedly be the most ideal seed cells for promoting sweat gland regeneration. Second, the cell culture method: it is necessary not only to meet the number of cells to be transplanted but also to maintain the ability of cells to differentiate into functional sweat gland cells. In recent years, organoid research has developed rapidly. Organoids were named the 2017 Biosciences Technology of the Year by *Nature Methods*, specifically referring to three-dimensional cell cultures containing key characteristics representing organs. In vitro culture systems are based on self-renewing stem cell populations that differentiate into multiple organ-specific cell types, possessing similar spatial structures to their corresponding organs and reproducing some of their functions, thus providing highly physiological / pathologically relevant systems. To date, there have been reports of establishing organoid culture systems for most tissues and organs of the body, such as the brain, small intestine, liver, and pancreas. The successful establishment of organoid culture methods has accelerated research on tissue and organ development and diseases. Furthermore, under suitable conditions, organoids can undergo multiple passages, expanding the cell number and providing seed cells for cell therapy. However, there are currently no reports of in vitro organoid research on sweat glands. This may be because the developmental and regulatory mechanisms of sweat glands are not yet fully understood, making it difficult to establish a suitable culture system. Summary of the Invention

[0005] To address one or more problems existing in the prior art, one aspect of the present invention provides a method for culturing sweat gland organoids using sweat gland cells, comprising the following steps:

[0006] T1: Sweat gland cells were resuspended in matrix gel to obtain resuspended sweat gland cells; and

[0007] T2: Resuspended sweat gland cells are cultured using a three-dimensional culture medium, and the three-dimensional sweat gland-like structures collected after culture are cultured sweat gland organoids.

[0008] The formulation of the three-dimensional culture medium includes the following components: Advanced DMEM / F-12 medium, 0.01%-1% albumin, 0.2%-10% B-27 additive, 1-50mM HEPES, 0.1%-10% glutamine additive, 10-1000U / mL penicillin and 0.01-1mg / mL streptomycin.

[0009] In some embodiments, the formulation of the three-dimensional culture medium includes the following components: Advanced DMEM / F-12 medium, 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 100U / mL penicillin, and 0.1mg / mL streptomycin.

[0010] In some embodiments, in step T1, the matrix gel is Matrigel or BME; the concentration of the matrix gel is 6-9 mg / mL; and the concentration of sweat gland cells in the resuspended sweat gland cells is 1 × 10⁻⁶. 2 pcs / mL - 2×10 3 per mL.

[0011] In some embodiments, in step T2, the culture conditions are: cultured at 37°C in a 5% CO2 incubator for 5-12 days, wherein the three-dimensional culture medium is replaced every 3 days; preferably, the culture time is 5-7 days.

[0012] In some embodiments, the formulation of the three-dimensional culture medium further includes one or more of the following components: antioxidants, epidermal growth factor, basic fibroblast growth factor, EDA signaling pathway agonists, Sirt1 protein inhibitors, Wnt signaling pathway agonists, TGFβ signaling pathway inhibitors, adenylate cyclase agonists, and BMP4 signaling pathway agonists.

[0013] In some embodiments, the antioxidant is N-acetyl-L-cysteine ​​at a concentration of 0.1-10 mM; the epidermal growth factor is EGF at a concentration of 5-200 ng / mL; the basic fibroblast growth factor is bFGF at a concentration of 2-200 ng / mL; the EDA signaling pathway agonist is EDA at a concentration of 2-200 ng / mL; the Sirt1 protein inhibitor is nicotinamide at a concentration of 1-100 mM; the Wnt signaling pathway agonist is Wnt3a at a concentration of 10-1000 ng / mL; the TGFβ signaling pathway inhibitor is A83-01 at a concentration of 0.1-10 μM; the adenylate cyclase agonist is forsocrine FSK at a concentration of 1-100 μM; and the BMP4 signaling pathway agonist is BMP4 at a concentration of 2-200 ng / mL.

[0014] Another aspect of the present invention provides a three-dimensional culture medium for the above-described method, the formulation of which includes the following components: Advanced DMEM / F-12 culture medium, 0.01%-1% albumin, 0.2%-10% B-27 additive, 1-50 mM HEPES, 0.1%-10% glutamine additive, 10-1000 U / mL penicillin and 0.01-1 mg / mL streptomycin.

[0015] In some embodiments, the culture medium is formulated with the following components: Advanced DMEM / F-12 medium, 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 100U / mL penicillin, and 0.1mg / mL streptomycin.

[0016] In some embodiments, the formulation of the three-dimensional culture medium further includes one or more of the following components: antioxidants, epidermal growth factor, basic fibroblast growth factor, EDA signaling pathway agonists, Sirt1 protein inhibitors, Wnt signaling pathway agonists, TGFβ signaling pathway inhibitors, adenylate cyclase agonists, and BMP4 signaling pathway agonists.

[0017] In some embodiments, the antioxidant is N-acetyl-L-cysteine ​​at a concentration of 0.1-10 mM; the epidermal growth factor is EGF at a concentration of 5-200 ng / mL; the basic fibroblast growth factor is bFGF at a concentration of 2-200 ng / mL; the EDA signaling pathway agonist is EDA at a concentration of 2-200 ng / mL; the Sirt1 protein inhibitor is nicotinamide at a concentration of 1-100 mM; the Wnt signaling pathway agonist is Wnt3a at a concentration of 10-1000 ng / mL; the TGFβ signaling pathway inhibitor is A83-01 at a concentration of 0.1-10 μM; the adenylate cyclase agonist is forsocrine FSK at a concentration of 1-100 μM; and the BMP4 signaling pathway agonist is BMP4 at a concentration of 2-200 ng / mL.

[0018] In some embodiments, the formulation of the three-dimensional culture medium is selected from any of the following formulations:

[0019] Formula 1: Add 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 100U / mL penicillin and 0.1mg / mL streptomycin to Advanced DMEM / F-12 medium;

[0020] Formula 2: Add 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 1mM N-acetyl-L-cysteine, 50ng / mL EGF, 20ng / mL bFGF, 20ng / mL EDA, 100U / mL penicillin and 0.1mg / mL streptomycin to Advanced DMEM / F-12 medium;

[0021] Formula 3: Add 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 1mM N-acetyl-L-cysteine, 10mM nicotinamide, 20ng / mL EDA, 50ng / mL EGF, 20ng / mL bFGF, 100ng / mL Wnt3a, 1μM A83-01, 10μM FSK, 100U / mL penicillin and 0.1mg / mL streptomycin to Advanced DMEM / F-12 medium.

[0022] Formula 4: Add the following to Advanced DMEM / F-12 medium: 0.1% albumin, 2% B-27 additive, 10mM MEPES, 1% glutamine additive, 1mM N-acetyl-L-cysteine, 10mM nicotinamide, 20ng / mL EDA, 50ng / mL EGF, 20ng / mL bFGF, 100ng / mL Wnt3a, 1μM A83-01, 10μM FSK, 20ng / mL BMP4, 100U / mL penicillin, and 0.1mg / mL streptomycin.

[0023] In another aspect, this invention provides a sweat gland organoid, which is obtained by culturing according to the above-described method. The application of this sweat gland organoid in the preparation of products for repairing full-thickness skin injuries and / or sweat gland defects also falls within the scope of this invention.

[0024] Based on the above technical solution, sweat gland cells can be cultured under the three-dimensional culture conditions provided by this invention to obtain sweat gland organoids. These organoids have a morphology similar to sweat glands and retain strong stem cell characteristics, enabling rapid expansion to obtain a large number of sweat gland organoids. These organoids possess bidirectional differentiation potential, capable of differentiating into epidermal cells and sweat gland cells. They can be used to prepare drugs or products that promote skin wound healing in mice with full-thickness injuries and to regenerate and restore sweat gland function in mice with damaged sweat glands, showing broad application prospects. Compared with existing technologies, this invention has the following beneficial effects:

[0025] 1) This invention utilizes sweat gland organs obtained through three-dimensional culture of sweat gland cells. These cells exhibit high viability, with most remaining viable after 30 days of continuous culture and passage, and only a small number dying. The resulting sweat gland organs, after a period of culture, express CK14, Ki67 (a cell proliferation marker indicating strong proliferative capacity), αSMA, and SOX9 proteins, demonstrating that the cultured sweat gland organs retain stem cell characteristics. Furthermore, compared to sweat gland cells, the expression of stem components is increased, while the expression of differentiated components is decreased, indicating that three-dimensional culture enriches sweat gland stem / progenitor cells and allows for the rapid expansion of a large number of sweat gland seed cells. This establishes an effective method for culturing and obtaining sweat gland organs.

[0026] 2) The sweat gland organoids obtained by the present invention through three-dimensional culture contain both sweat gland stem / progenitor cells and some mature sweat gland cells. They have bidirectional differentiation potential and can differentiate into sweat gland cells and epidermal cells. After transplantation into mice with full-thickness injuries to the soles and backs, they can not only promote the regeneration and functional recovery of sweat glands, but also promote the healing of skin wounds. They can be used to prepare cell products that repair skin damage and restore the function of accessory organs, and have broad application prospects.

[0027] 3) This invention provides, for the first time, a three-dimensional culture medium for culturing sweat gland cells to obtain sweat gland organoids. It employs a mixed culture method of matrix gel and cells, adding other components to the Advanced DMEM / F-12 medium. The resulting sweat gland organoid cells exhibit high viability and high stem cell content. Traditional two-dimensional culture of sweat gland cells causes them to lose their in vivo biological characteristics. In contrast, the sweat gland organoids generated by this invention through three-dimensional culture are organoid tissues composed of sweat gland stem cells and mature cells, resembling in vivo sweat gland tissue in morphology and structure, thus maintaining and preserving the biological characteristics of in vivo sweat glands. Furthermore, the three-dimensional sweat gland culture system screened using this invention can yield a large number of sweat gland organoids in a short period, thus providing an ideal seed resource of sweat gland cells. Attached Figure Description

[0028] Figure 1 These are bright-field images of the sweat gland cell separation process at different times, observed under a phase-contrast microscope.

[0029] Figure 2 These are immunofluorescence staining images of sweat gland tissue;

[0030] Figure 3 These are the results of immunofluorescence staining of the isolated sweat gland cells;

[0031] Figure 4 These are morphological images of sweat gland organoids formed by culturing in different three-dimensional culture medium formulations;

[0032] Figure 5 These are morphological images of sweat gland organoids at different times during the three-dimensional culture process;

[0033] Figure 6 This is the result of immunofluorescence staining of sweat gland organoids;

[0034] Figure 7 This is the result of relative gene expression in sweat gland organoids;

[0035] Figure 8 This is a diagram showing the changes in wound repair in mice with full-thickness skin damage, illustrating the effect of sweat gland organoids on skin repair.

[0036] Figure 9 This is a diagram showing the changes in skin thickness during the repair of full-thickness injury in mice with sweat gland organoids.

[0037] Figure 10 This is a diagram showing the effect of sweat gland organoids on the recovery of sweat gland function in mice with sweat gland damage. Detailed Implementation

[0038] To achieve efficient isolation of sweat gland cells for the cultivation of sweat gland organoids with high cell activity, strong stemness, and bidirectional differentiation potential, this invention provides a method for isolating sweat gland cells based on a combination of digestive enzymes. The isolated sweat gland cells are cultured using a specialized three-dimensional culture medium based on a mixture of matrix gel and cells provided by this invention, resulting in sweat gland organoids with high cell activity, strong stemness, and bidirectional differentiation potential. This fills the gap in modern medicine for culturing sweat gland stem cells to form sweat gland organoids.

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0040] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0041] Unless otherwise specified, the methods used in the following examples are conventional methods. For specific steps, please refer to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold SpringHarbor).

[0042] Unless otherwise specified, the percentage concentrations mentioned are mass / mass (W / W, g / 100g) percentage concentrations, mass / volume (W / V, g / 100mL) percentage concentrations, or volume / volume (V / V, mL / 100mL) percentage concentrations.

[0043] The following materials used in this invention are all commercially available: Matrigel and BME are available from Corning; Advanced DMEM / F-12 culture medium, albumin (trade name Albumin), B-27 supplement (trade name B-27 supplement), 10 mM HEPES ((4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid)), and glutamine supplement (trade name GlutaMAX) The supplements are available from Gibco; N-acetyl-L-cysteine ​​and nicotinamide (a Sirt1 protein inhibitor) are available from Sigma; EGF (epidermal growth factor), bFGF (basic fibroblast growth factor), Wnt3a (Wnt signaling pathway agonist), EDA (EDA signaling pathway agonist), and BMP4 (BMP4 signaling pathway agonist) are available from R&D; A83-01 (TGFβ signaling pathway inhibitor) and forskolin (adenylate cyclase agonist, FSK) are available from SELLECK; penicillin and streptomycin are available from North China Pharmaceutical Group. Those skilled in the art should understand that the choice of material suppliers should not be a limitation of this invention.

[0044] Example 1: Isolation and acquisition of sweat gland cells

[0045] 1.1 The method for obtaining sweat gland cells according to the present invention includes the following steps:

[0046] 1) After washing the mouse paw skin tissue with PBS, subcutaneous tissues such as subcutaneous fascia, fat and muscle were removed to obtain pretreated skin tissue;

[0047] 2) The pretreated skin tissue was digested with the tissue separation enzyme Dispase at a concentration of 2 mg / mL, a digestion temperature of 4℃, and a digestion time of 12 hours. After centrifugation at 200g for 5 minutes, the supernatant was discarded and the precipitate was retained to obtain the dermal tissue of the skin tissue.

[0048] 3) The dermal tissue obtained in step 2) is further digested using a combination of digestive enzymes: 2 U / mL collagenase A, 0.5 U / mL hyaluronidase, and 6 U / mL elastase. The digestion temperature is 37°C, and the digestion time is 15-20 minutes. Centrifuge at 200g for 5 minutes, discard the supernatant, and retain the precipitate. To more effectively separate sweat gland cells from other cell types (mainly fibroblasts) in the dermal tissue, the digestion, centrifugation, and discarding of supernatant and retention of precipitate can be repeated multiple times.

[0049] 4) Add the above digestive enzyme combination to the precipitate after centrifugation in step 3) and continue digestion under the same conditions as in step 3). Under a microscope, sweat gland cell clusters are observed, indicating that other cells in the dermis have been removed and all sweat gland cells have been separated from the skin tissue.

[0050] 5) After aspirating the sweat gland cell clusters under a microscope, digest them with Accutase enzyme at a digestion temperature of 37°C for 5 minutes. After passing through a 70-micron sieve, centrifuge at 200g for 5 minutes to collect the precipitate. The cells obtained are the sweat gland cells.

[0051] Figure 1 Bright-field images at different time points during the sweat gland cell separation process, observed under a phase-contrast microscope, are shown. Image A shows the mouse paw pad; image B shows the excised skin of the foot containing the paw pad; image C shows the dermal tissue after Dispase digestion to remove the epidermis; image D shows the sweat gland precipitate collected after digestion of the dermis with a combination of digestive enzymes and low-speed centrifugation, as observed under a microscope; image E shows the selected sweat gland cell clusters; and image F shows sweat gland cells inoculated on day 0. Figure 1 It is evident that the sweat gland cell separation method provided by this invention can effectively achieve the separation of sweat gland cells.

[0052] 1.2 Immunofluorescence staining detection of sweat gland tissue

[0053] Immunofluorescence staining was used to detect cell type markers in sweat gland tissue, including sweat gland stem / progenitor cell markers CK14 (keratin 14), CK5 (keratin 5), and αSMA (myofibroblast), pluripotent stem cell marker SOX9 (transcription factor SOX9), differentiation marker CK10 (keratin 10), functional markers AQP5 (aqueous channel protein 5) and αATP (sodium-potassium pump), as well as sweat gland-specific markers CK18 (keratin 18) and CEA (ectoderm dysplasia antigen), and sweat gland stemness marker CK19 (keratin 19).

[0054] Detection Procedure: Mouse paw skin was collected, fixed in 4% paraformaldehyde for 12 hours, dehydrated, and then embedded in paraffin for sectioning, with a section thickness of 4 micrometers. After dewaxing, antigen retrieval was performed on the paraffin sections, followed by blocking with 10% goat serum for 1 hour. Primary antibodies were then added: CK14, CK10, CK5, CK19, CK18, αSMA, SOX9, AQP5, αATP, and CEA. The sections were incubated at 4°C for 10 hours, washed, and then inoculated with the fluorescent secondary antibody Alexa. 488, Alexa Incubate at 568 for 1 hour, then incubate with DAPI at room temperature for 10 minutes. After mounting, observe the immunofluorescence staining images of the sweat gland tissue under a fluorescence microscope. The results are as follows: Figure 2 As shown.

[0055] according to Figure 2 As can be seen, CK14 staining results show that CK14 is strongly expressed in the basal layer of the epidermis, while in the sweat gland tissue, it is expressed in the ductal portion of the sweat gland ( Figure 2 The stained area represented by D in the middle layer highly expresses CK14, similar to the basal layer of the epidermis, while it is highly expressed in the glandular part of the sweat gland ( Figure 2 The staining results (S indicates the stained area) show that CK14 is weakly expressed only in the outer cells; CK10 staining results show that CK10 is highly expressed in all layers of the epidermis except the basal layer and in the ductal part of the sweat gland, while no CK10 expression is observed in the glandular part of the sweat gland; CK5 staining results show that CK5 is highly expressed in all parts of the epidermis and in the ductal part of the sweat gland, while the expression in the glandular part is similar to that of CK14, with weak CK5 expression detected only in the outer cells; αSMA, SOX9, CK19, CK18 and CEA staining results show that these sweat gland markers are only expressed in the glandular part of the sweat gland, and not in the ductal part or the epidermis; while the functional markers of sweat glands, αATP and AQP5, are only expressed in the inner cells of the glandular part of the sweat gland. Figure 2 The results showed that sweat gland tissue contains both stem cells and mature cells, and sweat gland cells containing both stem cells and mature cells can be separated from sweat gland tissue using appropriate separation methods.

[0056] 1.3 Immunofluorescence staining detection of isolated sweat gland cells

[0057] The cell type markers of the sweat gland cells isolated in this example were detected by immunofluorescence staining, including sweat gland stem / progenitor cell markers CK14, CK5, and αSMA, pluripotent stem cell marker SOX9, differentiation marker CK10, functional markers AQP5 and αATP, epidermal stem cell marker P63 (transcription factor), sweat gland-specific markers CK18 and CEA, and sweat gland stemness marker CK19.

[0058] Detection procedure: The isolated sweat gland cells were fixed with 4% paraformaldehyde for 10 minutes, dehydrated overnight with 20% sucrose solution, embedded in OCT, and frozen sectioned to a thickness of 8 μm. The membrane was perforated with 0.2% Triton X-100, blocked with goat serum for 1 hour, and then incubated with primary antibodies: AQP5, P63, CK14, CK5, CK10, SOX9, αSMA, CK19, and CK18 at 4°C for 10 hours. After washing, the secondary fluorescent antibody Alexa was added. 488, Alexa Incubate at 568 for 1 hour, then incubate with DAPI at room temperature for 10 minutes. After mounting, observe under a fluorescence microscope. The results are as follows: Figure 3 As shown.

[0059] according to Figure 3 It can be seen that the isolated sweat gland cells can express sweat gland functional markers AQP5 (green fluorescence), CK18 (green fluorescence), stem cell markers CK19 (green fluorescence), αSMA (red fluorescence), CK14 (green fluorescence), and P63 (red fluorescence), indicating that the isolated sweat gland cells are a mixture of mature functional cells (AQP5 positive, CK18 positive) and stem cells (αSMA positive, CK19 positive, CK14 positive, and P63 positive), and contain multiple subtypes of stem cells.

[0060] Based on the above results, the sweat gland cell separation method of the present invention uses collagenase A combined with hyaluronidase and elastase to digest dermal tissue, which can accelerate the digestion speed and remove dermal tissue and other types of cells in a short time, thereby achieving efficient separation of sweat gland cells. The separated sweat gland cells can express CK5, CK14, CK19, CK18 and αSMA, indicating that the separated sweat gland cells contain stem cells. Among them, cells expressing CK19, CK18 and αSMA can differentiate into sweat gland cells, while cells expressing CK5 and CK14 can serve as skin stem / progenitor cells and differentiate into epidermal cells or sweat gland cells.

[0061] Example 2: Culture of sweat gland organoids

[0062] 2.1 This embodiment involves three-dimensional culture of the sweat gland cells isolated in Example 1 to obtain sweat gland organoids, which includes the following steps:

[0063] 1): The sweat gland cells isolated in Example 1 were resuspended in a matrix gel to obtain resuspended sweat gland cells; specifically, the sweat gland cells isolated in Example 1 were resuspended in a diluted matrix gel ( BME Type 2 (concentration 7.2 mg / mL) was resuspended, and the concentration of sweat gland cells after resuspending was 1 × 10⁻⁶. 3 cells / mL;

[0064] 2): The resuspended sweat gland cells were cultured using a three-dimensional culture medium. The three-dimensional sweat gland-like structures collected after culture were the cultured sweat gland organoids. The culture conditions were 37℃ and 5% CO2 incubator, and the culture time was 5-12 days.

[0065] The formulation of the three-dimensional culture medium used for culturing sweat gland cells can be any one of the following formulations 1-4 (the concentration of each component is the final concentration in the culture medium, where % represents the volume concentration):

[0066] Formula 1: Add 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 100U / mL penicillin, and 0.1mg / mL streptomycin to Advanced DMEM / F-12 medium. Change the medium every 3 days.

[0067] Formula 2: Add 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 1mM N-acetyl-L-cysteine, 50ng / mL EGF, 20ng / mL bFGF, 20ng / mL EDA, 100U / mL penicillin, and 0.1mg / mL streptomycin to Advanced DMEM / F-12 medium. Change the medium every 3 days.

[0068] Formula 3: Add the following to Advanced DMEM / F-12 medium: 0.1% albumin, 2% B-27 additive, 10 mM HEPES, 1% glutamine additive, 1 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 20 ng / mL EDA, 50 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Wnt3a, 1 μM A83-01, 10 μM FSK, 100 U / mL penicillin, and 0.1 mg / mL streptomycin. Change the medium every 3 days.

[0069] Formula 4: Add the following to Advanced DMEM / F-12 medium: 0.1% albumin, 2% B-27 additive, 10 mM HEPES, 1% glutamine additive, 1 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 20 ng / mL EDA, 50 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Wnt3a, 1 μM A83-01, 10 μM FSK, 20 ng / mL BMP4, 100 U / mL penicillin, and 0.1 mg / mL streptomycin. Change the medium every 3 days.

[0070] The aforementioned three-dimensional (3D) culture for the large-scale expansion and culture of sweat gland organoids refers to the co-culturing of carriers with three-dimensional structures (three-dimensional refers to spatial dimensions, generally length, width, and height; specifically, in cell culture, this means the formation of small, three-dimensional mound-like clumps of matrix gel dropped onto the bottom wall of a culture dish) with cells in vitro, as opposed to two-dimensional (2D) adherent culture (two-dimensional refers to a plane; specifically, in cell culture, this means cells are flat and attached to the bottom wall of a culture dish). The three-dimensional culture medium provided by this invention specifically employs a matrix gel-cell co-culture method, adding albumin (commercially available, used to provide nutrients and maintain cell growth), B-27 supplement (commercially available, used to provide nutrients and maintain cell growth), HEPES buffer (a zwitterionic chemical buffer), and glutamine supplement (commercially available, GlutaMAX) to the Advanced DMEM / F-12 medium. Supplements include: amino acid additives to promote growth; N-acetyl-L-cysteine ​​(antioxidant); epidermal growth factor (promoting cell proliferation and differentiation, such as EGF); basic fibroblast growth factor (promoting cell growth, such as bFGF); Wnt signaling pathway agonists (maintaining and regulating stem cell characteristics, such as Wnt3a); EDA signaling pathway agonists (promoting cell differentiation and maintaining cell stemness, such as EDA); Sirt1 protein inhibitors (promoting cell proliferation and differentiation, such as nicotinamide); BMP4 signaling pathway agonists (promoting cell differentiation and maintaining cell stemness, such as BMP4); TGFβ signaling pathway inhibitors (maintaining stem cell characteristics, such as A83-01); adenylate cyclase agonists (maintaining pluripotent stem cell characteristics, such as forsocrine-FSK); antibiotics (preventing bacterial contamination, such as penicillin and streptomycin); and combinations thereof. Using the three-dimensional culture medium provided in this embodiment, the sweat gland cells obtained in Example 1 can be cultured to obtain a three-dimensional sweat gland-like structure, which is a sweat gland organoid.

[0071] 2.2 Morphology of sweat gland organoids cultured with different three-dimensional culture medium formulations

[0072] At the end of the culture, three-dimensional sweat gland-like structures were selected as cultured sweat gland organoids for testing. The morphology of the 3D cultured sweat gland organoids for 5 days was observed under a microscope (Leica DM1, 100× magnification). Figure 4 As shown, where Figure 4 Image A shows the morphology of sweat gland organoids obtained using three-dimensional culture medium formulation 1. Figure 4 Image B shows the morphology of sweat gland organoids obtained using three-dimensional culture medium formulation 2. Figure 4Image C represents the morphology of sweat gland organoids obtained using three-dimensional culture medium formulation 3. Figure 4 Image D shows the morphology of sweat gland organoids obtained using three-dimensional culture medium formulation 4. From Figure 4 As observed in the AD diagram, sweat gland organoids cultured using different three-dimensional culture medium formulations exhibited a variety of cell morphologies (i.e., cell populations composed of multiple cell types), such as clonal, ductal, and glandular structures. This also confirms that the sweat gland cells isolated from Example 1 contain different sweat gland stem / progenitor cells, which proliferate and differentiate into sweat gland organoids with diverse morphologies under three-dimensional culture conditions. These organoids contain both sweat gland stem / progenitor cells and some mature sweat gland cells. During the culture and proliferation process, the isolated sweat gland cells were observed to have high cell viability and vigorous proliferation, generally reaching a diameter of 200 μm in 5-7 days. The passage cycle was short, and the growth was rapid. Therefore, this indicates that the three-dimensional culture medium used for culturing sweat gland cells is suitable for the formation of sweat gland organoids.

[0073] Figure 5 The morphological images of sweat gland organoids at different time points during three-dimensional culture are also shown. Figure 5 As can be seen, after 3 days of 3D culture, sweat gland cells proliferated to form multiple clone-like or duct-like cell spheroids with strong refractive index, indicating vigorous cell vitality. By day 5 of culture, sweat gland organoids grew to tubular or glandular structures with a diameter of about 200 μm. By day 30 of culture, sweat gland organoids grew to vesicle-like structures, indicating that the 3D culture system established in this example can promote the rapid proliferation of sweat gland cells, form a large number of sweat gland organoids in a short period of time, and maintain the survival of organoids until day 30.

[0074] 2.3 Immunofluorescence staining detection of sweat gland organoids

[0075] Immunofluorescence staining was used to detect cell type markers of sweat gland organoids cultured for 5 days in this example, including sweat gland stem / progenitor cell markers CK14, CK5, and αSMA, pluripotent stem cell marker SOX9, differentiation marker CK10, functional markers AQP5 and αATP, as well as sweat gland-specific markers CK18 and sweat gland stem marker CK19.

[0076] Detection Procedure: The sweat gland organoids cultured in this example were fixed with 4% paraformaldehyde for 10 minutes, dehydrated overnight with 20% sucrose solution, embedded in OCT, and frozen sectioned to a thickness of 8 μm. The sections were perforated with 0.2% Triton X-100, blocked with 10% goat serum for 1 hour, and then incubated with primary antibodies: AQP5, αATP, CK14, CK5, CK10, SOX9, αSMA, CK19, and CK18 at 4°C for 10 hours. After washing, the secondary fluorescent antibody Alexa was added. 488, Alexa Incubate at 568 for 1 hour, then incubate with DAPI at room temperature for 10 minutes. After mounting, observe under a fluorescence microscope. The results are as follows: Figure 6 As shown.

[0077] according to Figure 6 Immunofluorescence staining results of sweat gland organoids showed that sweat gland organoids expressed sweat gland-specific marker CK18 and stemness marker CK19; at the same time, functional markers AQP5 and αATP were also expressed in small amounts, while the expression of sweat gland stem / progenitor cell marker αSMA and pluripotent stem cell marker SOX9 was strong, indicating that sweat gland organoids generated by sweat gland cells have strong stemness; similarly, the high expression of CK14 and CK5 and the absence of CK10 proves the stemness potential of sweat gland organoids and that they have not differentiated into epidermis.

[0078] 2.4 Relative gene expression of sweat gland organoids

[0079] The relative gene expression of sweat gland organoids cultured for 5 days in this embodiment was analyzed, and the results are as follows: Figure 7 As shown, EPC represents the epidermal cells isolated from the primary culture; D0 represents the isolated sweat gland cells; P0, P1, and P2 represent the first, second, and third generation sweat gland organoids generated from the primary culture, respectively.

[0080] Figure 7 The results showed that the gene expression of CK14, CK5, and CK19 enriched the stem cells of sweat glands through three-dimensional culture, and the resulting sweat gland organoids maintained strong stem characteristics across different generations, while the functional marker of maturity (AQP5) was significantly weakened. Furthermore, the high expression of the sweat gland-specific marker CK18 and the absence of the epidermal cell maturation marker CK10 confirmed that the generated sweat gland organoids maintained sweat gland characteristics and did not differentiate into the epidermis.

[0081] Example 3: Effect of sweat gland organoids on skin repair in mice with full-thickness skin damage

[0082] Full-thickness skin excision was performed on SCID mice with combined T and B cell immunodeficiency to create a model. The wound defect area was covered with three-dimensional (3D) cultured sweat gland organoids as described in Example 2. After 3, 7, 14, and 21 days of repair, the wound and surrounding skin tissue were excised, and the repair status was detected by H&E staining.

[0083] Test results as follows Figure 8 and Figure 9 As shown, where Figure 8The diagram illustrates the wound repair changes in mice with full-thickness skin injuries, showing the effect of sweat gland organoids on skin repair. Results showed that the wound healing area in the control group (control mice with simple surgical excision and wounds covered only with 3M dressings) was significantly smaller than that in the transplantation group (wounds inoculated with sweat gland organoids cultured in Example 2, followed by 3M dressing coverage). Furthermore, the control group showed a large number of inflammatory cells in the wound area, with no significant dermal collagen regeneration. The transplantation group using the 3D cultured sweat gland organoids from Example 2 showed better epidermal regeneration, faster and longer migration of epidermal cells in the wound area, clearer inflammation, and more ideal dermal collagen regeneration.

[0084] Figure 9 The diagram shows the changes in skin thickness during the repair of full-thickness injury in mice by sweat gland organoids. Figure 9 The results showed that on day 14, the wounds of mice in the cell group had basically healed, while the wounds of mice in the control group had not yet healed. From this point (day 14), the skin thickness and full-thickness skin thickness of the mice were compared. The results showed that from day 14 to day 21, the skin thickness and full-thickness skin thickness of the control group were much lower than those of the cell group.

[0085] The results of this example show that, compared with the control group, the sweat gland organoids cultured in three dimensions (3D) in Example 2 can significantly promote the repair of skin damage in mice with full-thickness skin injury.

[0086] Example 4: Effect of sweat gland organoids on the recovery of sweat gland function in mice with sweat gland damage

[0087] A sweat gland damage model was created by placing the skin of the soles of C57 mice on a 65°C metal plate for 15 seconds. The next day, the three-dimensionally cultured sweat gland organoids from Example 2 were transplanted into the damaged mouse sole skin, and iodine-starch function and staining tests were performed at 3, 7, 14, and 21 days after cell transplantation.

[0088] The results are as follows Figure 10 The results showed that, compared with the control group (where the skin of the soles of C57 mice was placed on a 65°C metal plate for 15 seconds to cause sweat gland damage, and then inoculated with physiological saline), the cell group (where the skin of the soles of C57 mice was placed on a 65°C metal plate for 15 seconds to cause sweat gland damage, and then inoculated with the three-dimensionally cultured sweat gland organoids of Example 2) mice began to show sweating on the 7th day after surgery, and the number of detected sweat spots increased significantly over time. In contrast, no sweating was detected in the control group until three weeks after the injury. H&E staining results also confirmed this phenomenon, showing that black spots were detected in the cell group from the 14th day, and the number of black spots increased significantly on the 21st day.

[0089] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be considered part of the present invention.

Claims

1. A method of culturing sweat gland organoids using sweat gland cells, characterized by, Includes the following steps: T1: Sweat gland cells were resuspended in matrix gel to obtain resuspended sweat gland cells; and T2: Resuspended sweat gland cells are cultured using a three-dimensional culture medium, and the three-dimensional sweat gland-like structures collected after culture are cultured sweat gland organoids. The formulation of the three-dimensional culture medium is as follows: 0.1% albumin, 2% B-27 additive, 10mM HEPES, 1% glutamine additive, 1mM N-acetyl-L-cysteine, 10mM nicotinamide, 20ng / mL EDA, 50ng / mL EGF, 20ng / mL bFGF, 100ng / mL Wnt3a, 1μM A83-01, 10μM FSK, 20ng / mL BMP4, 100U / mL penicillin, and 0.1mg / mL streptomycin are added to Advanced DMEM / F-12 medium. The sweat gland cells mentioned above are obtained by a method comprising the following steps: S1: After washing the mouse paw skin tissue with PBS, the subcutaneous tissue was removed to obtain pretreated skin tissue; S2: The pretreated skin tissue is digested with the first digestive enzyme, centrifuged, the supernatant is discarded and the precipitate is retained to obtain the dermal tissue of the skin tissue; the first digestive enzyme is 2 mg / mL tissue dissociation enzyme Dispase; the digestion conditions are: digestion temperature of 4℃, digestion time of 12 hours; centrifugation conditions are: centrifugation at 200g for 5 minutes; S3: Digest the dermal tissue described in S2 with a second digestive enzyme, centrifuge, discard the supernatant and retain the precipitate, repeat the second digestive enzyme digestion on the precipitate, centrifuge and discard the supernatant and retain the precipitate N times to obtain tissue precipitate, where N is a natural number; The second digestive enzyme is a combination of 2 U / mL collagenase A, 0.5 U / mL hyaluronidase, and 6 U / mL elastase; the digestion conditions are: digestion temperature of 37℃, digestion time of 15-20 minutes; and centrifugation conditions are: centrifugation at 200g for 5 minutes. S4: The tissue precipitate obtained in S3 was digested again with the second digestive enzyme, and sweat gland cell clusters were observed under a microscope; the digestion conditions were: digestion temperature of 37℃ and digestion time of 15-20 minutes. S5: Remove the sweat gland cell clusters described in S4, digest them with a third digestive enzyme, pass them through a sieve, centrifuge, discard the supernatant and retain the precipitate to obtain sweat gland cells; the third digestive enzyme is Accutase; the digestion conditions are: digestion temperature of 37℃, digestion time of 5 minutes; the size of the sieve is 70 micrometers; the centrifugation conditions are: centrifugation at 200g for 5 minutes.

2. The method of claim 1, wherein, In step T1, the matrix gel is Matrigel or BME; the concentration of the matrix gel is 6-9 mg / mL; and the concentration of sweat gland cells in the resuspended sweat gland cells is 1 × 10⁻⁶. 2 pcs / mL - 2×10 3 pcs / mL; and / or In step T2, the culture conditions are: cultured at 37°C in a 5% CO2 incubator for 5-12 days, with the three-dimensional culture medium being replaced every 3 days.

3. The method of claim 2, wherein, In step T2, the culture time is 5-7 days.

4. A three-dimensional culture medium for use in the method according to any one of claims 1-3, characterized in that, The culture medium was formulated as follows: 0.1% albumin, 2% B-27 additive, 10 mM HEPES, 1% glutamine additive, 1 mM N-acetyl-L-cysteine, 10 mM nicotinamide, 20 ng / mL EDA, 50 ng / mL EGF, 20 ng / mL bFGF, 100 ng / mL Wnt3a, 1 μM A83-01, 10 μM FSK, 20 ng / mL BMP4, 100 U / mL penicillin, and 0.1 mg / mL streptomycin were added to Advanced DMEM / F-12 medium.

5. A sweat gland organoid, characterized in that, The sweat gland organoids are obtained by culturing according to any one of claims 1-3.

6. The use of the sweat gland organoids of claim 5 in the preparation of products for repairing full-thickness skin damage and / or sweat gland defects.

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