Epidermal organoid culture medium and culture method

Abstract

The present invention discloses an epidermal organoid culture medium and a culture method, belonging to the field of organoid technology. The epidermal organoid culture medium includes: a basal culture medium, a specific additive factor, CHIR99021 and Forskolin; wherein the specific additive factor includes the following components at the final concentration: Glutamax, 0.5-2×; HEPES, 5-20mM; N-Acetylcysteine, 1-5mM; Nicotinamide, 1-20mM; Penicillin streptomycin, 0.5-5×; B27, 0.5-2×, etc. The culture medium of the present invention can increase the proliferation rate of epidermal organoids, shorten the construction time of epidermal organoids, increase the construction success rate of epidermal organoids, and the sample is easy to obtain, the performance of the epidermis can be guaranteed, and it is easy to industrialize production.

Description

Technical Field

[0001] The present invention relates to the technical field of organoids, and in particular to an epidermal organoid culture medium and a culture method. Background Art

[0002] Skin is the largest organ in the human body and is mainly divided into three layers, namely the epidermis, dermis and subcutaneous tissue. As a barrier for the human body, it is directly exposed to a variety of stressors in the external environment, such as solar or electromagnetic radiation, temperature and humidity, microorganisms, cosmetics, topical drugs and mechanical stress. In recent years, with the rapid development of the global pharmaceutical and cosmetics industry, various new technologies and methods have emerged, and pharmaceutical and cosmetic raw materials and dosage forms have been continuously updated, which has brought greater challenges to the testing and supervision of such products. At the same time, in order to adapt to the development trend of the global pharmaceutical and cosmetics industry, the development, verification and promotion of more effective pharmaceutical and cosmetic raw material evaluation research and regulatory tools are imminent.

[0003] Classical two-dimensional (2D) culture methods have been used as valuable tools to provide primary epidermal cells for the study of skin stem cell biology and stem cell therapy for extensive and severe skin injuries. However, these 2D culture techniques rely on the basal layer of murine fibroblasts (3T3 cells) and culture media containing fetal bovine serum or bovine pituitary extracts. The introduction of exogenous substances by these factors can adversely affect clinical safety and interfere with mechanistic studies of skin diseases. Therefore, in recent years, people have been studying safer and more effective clinical culture systems. Until recently, chemically defined xeno-free culture methods for long-term culture of human epidermal cells have been successfully developed using small molecules or laminin-based matrices. However, these epidermal cells expanded in 2D form do not replicate the cellular composition and physiological structure of the human epidermis in vivo. In addition, a variety of methods have been developed to efficiently generate physiologically meaningful three-dimensional skin equivalents of human skin, especially multilayered epidermis reconstructed under air-liquid interaction model conditions, which provide exquisite three-dimensional models of skin for skin research and regenerative medicine. However, the epidermis reconstructed using the air-liquid interaction model has a limited lifespan and ability to be propagated, which may limit the large-scale application of this model system in industrial or clinical projects. Therefore, the development of more complex in vitro models that are closer to the real human epidermis and can evaluate different types of drugs and cosmetics is the trend and focus of future research and development.

[0004] Currently, there are few reports on the research of epidermal organoids, and the culture conditions are unclear. Epidermal organoids are usually not easy to culture successfully and grow very slowly during the culture process. Summary of the invention

[0005] In order to solve the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide an epidermal organoid culture medium and a culture method to solve the problems of low culture success rate, long culture cycle, poor survival ability and subculture ability of existing epidermal organoids during the culture process.

[0006] The technical solution of the present invention to solve the above technical problems is as follows: provide an epidermal organoid culture medium, comprising: a basal culture medium, a specific additive factor, CHIR99021 and Forskolin; wherein the specific additive factor comprises the following components at final concentrations: Glutamax, 0.5-2×; HEPES, 5-20mM; N-Acetylcysteine, 1-5mM; Nicotinamide, 1-20mM; Penicillin streptomycin, 0.5-5×; B27, 0.5-2×; A83-01, 200-1000nM; Y-27632, 1-20μM; FGF-10, 5-50ng / mL; R-spondin1, 200-1000ng / mL; Noggin, 50-200ng / mL and EGF, 1-100ng / mL.

[0007] The beneficial effects of the present invention are as follows: One of the methods for obtaining epidermal organoids in the prior art is to induce pluripotent stem cells to differentiate and regenerate into three-dimensional spherical organoids under in vitro conditions, which has a low culture success rate, a long culture cycle, and a complex culture process. Another method for obtaining epidermal organoids in the prior art is to reconstruct a multilayer skin model under gas-liquid interaction model conditions, and the epidermis reconstructed by this method has a short-term survival ability and passaging ability. In response to these problems in the prior art, the present invention provides a new epidermal organoid culture medium, which can effectively improve the success rate of epidermal organoids and the number of epidermal organoids formed by culturing epidermal organoids, and can shorten the culture cycle.

[0008] The present invention helps to promote the proliferation of epidermal cells by adding Wnt pathway activator CHIR99021 and adenylate cyclase activator Forskolin to conventional culture medium, and activates Wnt and cyclic adenosine monophosphate (cAMP) pathways, which helps to improve the success rate and growth rate of epidermal organoid culture. In addition, specific added factors are also added to the culture medium of the present invention, and these factors cooperate with CHIR99021 and Forskolin to control cell processes by regulating signal transduction pathways, gene expression or metabolism, further improving the success rate of culture and the growth rate of epidermal organoids.

[0009] Further, the specific added factors include the following components at final concentrations: Glutamax, 1×; HEPES, 10 mM; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1×; B27, 1×; A83-01, 500 nM; Y-27632, 10 μM; FGF-10, 10 ng / mL; R-spondin1, 500 ng / mL; Noggin, 100 ng / mL and EGF, 50 ng / mL.

[0010] Furthermore, the final concentration of CHIR99021 in the epidermal organoid culture medium is 0.5-5 μM; preferably, the final concentration of CHIR99021 in the epidermal organoid culture medium is 3 μM.

[0011] Furthermore, the final concentration of Forskolin in the culture medium of epidermal organoids is 5-50 μM; preferably, the final concentration of Forskolin in the culture medium of epidermal organoids is 10 μM.

[0012] Furthermore, the basic culture medium is Advanced DMEM / F-12 culture medium.

[0013] The method for culturing epidermal organoids using the above-mentioned epidermal organoid culture medium comprises the following steps:

[0014] The epidermal tissue is processed to obtain epidermal cells, the epidermal cells are resuspended and mixed with matrix gel, and then inoculated, and after solidification, the above-mentioned epidermal organoid culture medium is added for culture to obtain epidermal organoids.

[0015] Furthermore, the epidermal tissue is processed specifically by taking the epidermal tissue, digesting it with a digestive enzyme, then terminating the digestion, centrifuging it, discarding the supernatant, and obtaining epidermal cells.

[0016] Furthermore, the epidermal tissue is mammalian epidermal tissue, such as human epidermal tissue, mouse epidermal tissue, etc.

[0017] Furthermore, the digestive enzyme is trypsin.

[0018] Further, after adding digestive enzymes, digestion is carried out at 35-40°C for 20-50 minutes.

[0019] Furthermore, after adding epidermal organoid culture medium, the cells were incubated at 37°C and 5% CO 2 Cultivated under conditions.

[0020] Furthermore, the culture medium is replaced every 2-3 days during the culture process, and the culture is carried out for 4-10 days to obtain epidermal organoids.

[0021] The present invention has the following beneficial effects:

[0022] The culture medium of the present invention can increase the proliferation rate of epidermal organoids, shorten the construction time of epidermal organoids, and improve the success rate of epidermal organoid construction. The samples are easy to obtain, the performance of the epidermis can be guaranteed, and industrial production is easy. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram showing the culture results of epidermal organoids in Example 1.

[0024] Figure 2 This is a diagram showing the culture results of epidermal organoids in Example 2.

[0025] Figure 3 This is a diagram showing the culture results of epidermal organoids in Example 3.

[0026] Figure 4 This is the HE staining identification result of epidermal organoids in Example 1.

[0027] Figure 5 This is the qPCR test result of epidermal organoids in Example 1.

[0028] Figure 6 This is a diagram showing the culture results of epidermal organoids in Comparative Example 1.

[0029] Figure 7 This is a diagram showing the culture results of epidermal organoids in Comparative Example 2.

[0030] Figure 8 This is a diagram showing the culture results of epidermal organoids in Comparative Example 3.

[0031] Fig. 9 This is a diagram showing the culture results of epidermal organoids in Comparative Example 4.

[0032] Fig.10 This is a diagram showing the culture results of epidermal organoids in Comparative Example 5.

[0033] Fig.11 This is a diagram showing the culture results of epidermal organoids in Comparative Example 6.

[0034] Fig.12 This is a diagram of the culture results of epidermal organoids in Comparative Example 7. DETAILED DESCRIPTION

[0035] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0036] Embodiment 1:

[0037] An epidermal organoid culture medium, comprising: a basal culture medium, a specific added factor, CHIR99021 and Forskolin;

[0038] Among them, the basic culture medium is Advanced DMEM / F-12 medium;

[0039] Specific added factors include the following components at final concentrations: Glutamax, 1×; HEPES, 10 mM; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillin streptomycin, 1×; B27, 1×; A83-01, 500 nM; Y-27632, 10 μM; FGF-10, 10 ng / mL; R-spondin1, 500 ng / mL; Noggin, 100 ng / mL and EGF, 50 ng / mL.

[0040] The final concentration of CHIR99021 in the epidermal organoid culture medium was 3 μM, and the final concentration of Forskolin was 10 μM.

[0041] The method for culturing epidermal organoids using epidermal organoid culture medium comprises the following steps:

[0042] (1) Soak mice within 3 days of birth in 75% alcohol to kill them. After the mice show no signs of life, transfer them to DPBS (phosphate buffered saline) and submerge them for 5 minutes. Carefully peel off the skin on the back of the mice with straight scissors, and cut off excess fat tissue and fascia with curved scissors to minimize adhesion of subcutaneous tissue. Soak them completely in DPBS and wash them several times until the blood is removed.

[0043] (2) Place the mouse back skin with the dermis side facing up and the epidermis side facing down in a 100 mm culture dish and flatten it. Add 10 mL of 1 mg / mL Thermolysin (MCE, HY-P1748) and digest it in a refrigerator at 4°C for 16 hours.

[0044] (3) Carefully peel off the mouse epidermis with a toothless nickel and transfer it to a 1.5 mL EP tube. Add 500 μL of 0.05% trypsin-EDTA (Gibco, 25300054) and cut it into pieces. Collect the cut epidermal tissue in a 15 mL centrifuge tube and add 4.5 mL of 0.05% trypsin-EDTA. Digest at 37°C for 30 min.

[0045] (4) Add 10 mL of DPBS to terminate digestion. Pipette up and down 15 times with a Pasteur pipette, filter through a 70 μm filter, collect the filtrate, and centrifuge at 2000 rpm and 4°C for 8 min. After centrifugation, first use a 1 mL pipette tip to aspirate the supernatant and collect the cell pellet at the bottom of the centrifuge tube.

[0046] (5) Resuspend the collected cell pellet by adding 1.5-2 times the volume of undiluted Matrigel (Corning, 356231) and mixing. Carefully pipette up and down 10 times to mix thoroughly, avoiding the generation of bubbles.

[0047] (6) Pipette 30 μL of cell suspension and add it to the center of each well of a preheated 48-well plate so that the sample forms a dome structure in the center of each well.

[0048] (7) Place the inoculated culture dish in a CO 2 Place the gel drop in an incubator at 37°C for 5 minutes. After gently shaking the gel drop and no obvious flow, carefully turn it upside down and wait for it to fully solidify for 25 minutes. Add 500 μL of epidermal organoid culture medium, take pictures under a microscope, and place it in an incubator at 37°C and 5% CO. 2 Cultivated under conditions.

[0049] (8) Observe daily, replace the culture medium every 2 days, and take photos under a microscope after 7 days of culture.

[0050] Embodiment 2:

[0051] An epidermal organoid culture medium, comprising: a basal culture medium, a specific added factor, CHIR99021 and Forskolin;

[0052] Among them, the basic culture medium is Advanced DMEM / F-12 medium;

[0053] Specific added factors include the following components at final concentrations: Glutamax, 0.5×; HEPES, 5mM; N-Acetylcysteine, 1mM; Nicotinamide, 1mM; Penicillin streptomycin, 0.5×; B27, 0.5×; A83-01, 200nM; Y-27632, 1μM; FGF-10, 5ng / mL; R-spondin1, 200ng / mL; Noggin, 50ng / mL and EGF, 1ng / mL.

[0054] The final concentration of CHIR99021 in the epidermal organoid culture medium was 3 μM, and the final concentration of Forskolin was 10 μM.

[0055] The method for culturing epidermal organoids is the same as in Example 1.

[0056] Embodiment 3:

[0057] An epidermal organoid culture medium, comprising: a basal culture medium, a specific added factor, CHIR99021 and Forskolin;

[0058] Among them, the basic culture medium is Advanced DMEM / F-12 medium;

[0059] Specific added factors include the following components at final concentrations: Glutamax, 2×; HEPES, 20 mM; N-Acetylcysteine, 1 mM; Nicotinamide, 1 mM; Penicillin streptomycin, 2×; B27, 2×; A83-01, 1000 nM; Y-27632, 20 μM; FGF-10, 50 ng / mL; R-spondin1, 1000 ng / mL; Noggin, 100 ng / mL and EGF, 100 ng / mL.

[0060] The final concentration of CHIR99021 in the epidermal organoid culture medium was 3 μM, and the final concentration of Forskolin was 10 μM.

[0061] The method for culturing epidermal organoids is the same as in Example 1.

[0062] Comparative Example 1:

[0063] The difference between Comparative Example 1 and Example 1 is that CHIR99021 is removed from the culture medium, and the rest is the same as Example 1.

[0064] Comparative Example 2:

[0065] The difference between Comparative Example 2 and Example 1 is that Forskolin is removed from the culture medium, and the rest is the same as Example 1.

[0066] Comparative Example 3:

[0067] The difference between Comparative Example 3 and Example 1 is that FGF-10 is removed from the culture medium, and the rest is the same as Example 1.

[0068] Comparative Example 4:

[0069] The difference between Comparative Example 4 and Example 1 is that EGF is removed from the culture medium, and the rest is the same as Example 1.

[0070] Comparative Example 5:

[0071] The difference between Comparative Example 5 and Example 1 is that N-Acetylcysteine ​​is removed from the culture medium, and the rest is the same as Example 1.

[0072] Comparative Example 6:

[0073] The difference between Comparative Example 6 and Example 1 is that CHIR99021, Forskolin, FGF-10, EGF and N-Acetylcysteine ​​are removed from the culture medium, and the rest is the same as Example 1.

[0074] Comparative Example 7:

[0075] The difference between Comparative Example 7 and Example 1 is that CHIR99021 in the culture medium is replaced by Wnt3A recombinant protein.

[0076] Test example:

[0077] 1. The mouse epidermal organoids obtained after 7 days of culture in Example 1-3 were observed. The results are shown in Figure 1-3 ;in, Figure 1 This is a diagram of the culture results of epidermal organoids in Example 1; Figure 2 This is a diagram of the culture results of epidermal organoids in Example 2; Figure 3 This is a diagram showing the culture results of epidermal organoids in Example 3.

[0078] Depend on Figure 1-3 It can be seen that the epidermal organoids cultivated by the method of the present invention are large in number and have good activity.

[0079] 2. The mouse epidermal organoids obtained in Example 1 were identified by hematoxylin-eosin staining (HE), and the mouse epidermal organoids after culture were embedded and sectioned for staining. The results are shown in Figure 4 .

[0080] Depend on Figure 4 It can be seen that there are large and flat basal cells and hardened keratinized substances in the center of the epidermal organoid structure. It can be seen that the HE histological characteristics of the mouse epidermal organoid constructed in the present invention are consistent with the characteristics of the epidermis.

[0081] 3. Detection of epidermal specific expression markers

[0082] Mouse skin tissue (positive control), mouse liver tissue (negative control), and mouse epidermal organoids cultured in Example 1 were taken, and RNA was extracted according to the standard RNA extraction procedure, and then reverse transcribed into cDNA for qPCR detection to detect the expression levels of epidermal specific markers keratin 10 (KRT10) and keratin 14 (KRT14). The results are shown in Figure 5 .

[0083] Depend on Figure 5 It can be seen that epidermal-specific markers are highly expressed in mouse epidermal organoids.

[0084] 4. Detection of the number and viability of epidermal organoids

[0085] The mouse epidermal organoids were cultured in the culture medium of Example 1-3 and Comparative Example 1-7 for 7 days. The morphology of the mouse epidermal organoids obtained in Comparative Example 1-7 was as follows: Figure 6-12 As shown. Among them, Figure 6-Figure 12 The morphologies of mouse epidermal organoids obtained in Comparative Example 1, Comparative Example 2, Comparative Example 3, Comparative Example 4, Comparative Example 5, Comparative Example 6 and Comparative Example 7 respectively.

[0086] The number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7 are shown in Table 1:

[0087] Table 1 Number and viability of organoids formed in Examples 1-3 and Comparative Examples 1-7

[0088]

[0089] From Table 1 and Figure 6-Figure 12 It can be seen that when CHIR99021 is removed from the culture medium, the organoids grow slowly, the number of organoids is less than that in Examples 1-3, and compared with Examples 1-3, there are more hardened keratinocytes in the middle of the organoids in Comparative Example 1.

[0090] When Forskolin was removed from the culture medium, the growth rate of the organoids slowed down, the organoids had a small diameter, were less in number than in Examples 1-3, and had poor activity.

[0091] When FGF10 was removed from the culture medium, compared with Examples 1-3, the organoids grew slowly during the culture process, the number of organoids decreased, and the activity was poor.

[0092] When EGF was removed from the culture medium, the number of organoids formed decreased and the viability was lower compared with Examples 1-3.

[0093] When N-Acetylcysteine ​​was removed from the culture medium, the organoids grew slowly and had a low viability during the culture process compared with the culture medium of Examples 1-3.

[0094] When N-Acetylcysteine, EGF, FGF10, CHIR99021 and Forskolin were removed from the culture medium, the number of organoids formed was significantly reduced compared to Examples 1-3, and the viability was lower, proving that N-Acetylcysteine, EGF, FGF10, CHIR99021 and Forskolin are key components in the mouse epidermal organoid culture medium.

[0095] When CHIR99021 in the culture medium was replaced with Wnt3A recombinant protein, the number of organoids formed and the viability of the organoids were significantly reduced compared with Examples 1-3. After replacing the above components, the cells grew slowly and were morphologically non-uniform in size.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An application of an epidermal organoid culture medium in improving the viability of epidermal organoids, characterized in that: The epidermal organoid culture medium comprises: a basal culture medium, specific additive factors, CHIR99021 and Forskolin; wherein the specific additive factors comprise the following components at final concentrations: Glutamax, 0.5-2×; HEPES, 5-20 mM; N-Acetylcysteine, 1-5 mM; Nicotinamide, 1-20 mM; Penicillin streptomycin, 0.5-5×; B27, 0.5-2×; A83-01, 200-1000 nM; Y-27632, 1-20 μM; FGF-10, 5-50 ng / mL; R-spondin1, 200-1000 ng / mL; Noggin, 50-200 ng / mL and EGF, 1-100 ng / mL.

2. The use according to claim 1, characterized in that: Specific added factors include the following components at final concentrations: Glutamax, 1×; HEPES, 10 mM; N-Acetylcysteine, 1 mM; Nicotinamide, 10 mM; Penicillinstreptomycin, 1×; B27, 1×; A83-01, 500 nM; Y-27632, 10 μM; FGF-10, 10 ng / mL; R-spondin1, 500 ng / mL; Noggin, 100 ng / mL and EGF, 50 ng / mL.

3. The use according to claim 1, characterized in that: The final concentration of CHIR99021 in the epidermal organoid culture medium is 0.5-5 μM, and the final concentration of Forskolin is 5-50 μM.

4. The use according to claim 3, characterized in that: The final concentration of CHIR99021 in the epidermal organoid culture medium was 3 μM, and the final concentration of Forskolin was 10 μM.

5. The use according to claim 1, characterized in that: The basic culture medium was Advanced DMEM / F-12 medium.

6. The use according to claim 1, characterized in that: The following steps are involved: The epidermal tissue is processed to obtain epidermal cells, the epidermal cells are resuspended and mixed with matrix gel, and then inoculated, and after solidification, epidermal organoid culture medium is added for culture to obtain epidermal organoids.

7. The use according to claim 6, characterized in that: The epidermal tissue is processed specifically as follows: the epidermal tissue is taken, digested with a digestive enzyme, then digestion is terminated, centrifuged, and the supernatant is discarded to obtain epidermal cells.

8. The use according to claim 6 or 7, characterized in that: The epidermal tissue is mammalian epidermal tissue.

9. The use according to claim 7, characterized in that: After adding the digestive enzyme, digest at 35-40°C for 20-50 minutes; the digestive enzyme is trypsin.

10. The use according to claim 6, characterized in that: After adding epidermal organoid culture medium, culture was carried out at 37°C and 5% CO2.

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