Culture medium of lacrimal gland type organ and culture method of lacrimal gland type organ

Abstract

The invention discloses a culture medium of a lacrimal gland organ and a culture method of the lacrimal gland organ, and the culture medium of the lacrimal gland organ is composed of a basic culture medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, trehalose, vitamin C, glutathione, an adenylate cyclase activator, A8301, PGE2 and FGF10. The culture medium for lacrimal gland organ culture, provided by the invention, is prepared from various cell factor components according to an optimized proportion, and is simple and reasonable in components and rich in nutrition; when the culture medium is used for culturing the lacrimal gland organoid, the formation and growth of the organoid can be promoted, an in-vivo complex microenvironment is further simulated, the organoid is closer to an in-vivo physiological state, an organoid model is better constructed so as to evaluate the influence of drugs on lacrimal gland functions, and a new platform is provided for drug screening; and a new way is opened up for follow-up treatment of diseases such as xerophthalmia in the future.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a culture medium for lacrimal gland organoids and a culture method for lacrimal gland organoids. Background Art

[0002] The eye is one of the most important organs in the human body, and the lacrimal gland is the main exocrine gland of the eye. They are located in the eye socket, behind the upper eyelid, toward the temporal lobe of each eye, and secrete tears as the main component of the tear film. The aqueous layer of the tear film not only contains water to lubricate the ocular surface, but also contains a large number of antibacterial components that can protect the ocular surface from infection. When the lacrimal gland is damaged or inflamed, dry eye syndrome will occur, causing discomfort to the patient and may even eventually lead to vision loss. For many years, people knew very little about the (pathological) physiology of the lacrimal gland. Due to the lack of appropriate in vitro models, the model systems used to study the lacrimal gland (especially the human lacrimal gland) have been limited, which has led to a gap in knowledge about the function of the lacrimal gland under physiological and pathological conditions.

[0003] Currently, organoid technology can address existing deficiencies. Organoids are tissue analogs with a certain spatial structure formed by in vitro three-dimensional (3D) culture of adult or pluripotent stem cells. Although not true human organs, they can simulate real organs in structure and function, simulating the in vivo tissue structure and function to the greatest extent possible, and can be cultured stably and long-term. For example, a variety of functional organoid models have been developed for the liver, kidney, lung, and brain. In recent years, researchers have developed in vitro models to study the lacrimal gland in a dish (including lacrimal gland organoids derived from primary mouse and human tissue). These lacrimal gland organoids are three-dimensional structures derived from adult stem cells and cultured in an extracellular matrix supplemented with multiple growth factors to maintain their in vitro regenerative capacity. This shows that organoids represent a viable model to better understand the biology of the lacrimal gland and study the pathologies associated with tearing.

[0004] Organoids are formed by the differentiation of pluripotent stem cells or organ progenitor cells, self-assembling into structural and functional three-dimensional tissue structures. The formation of various organoids often requires components such as culture medium, matrix, growth factors, and small molecules, and the components and culture methods required by different organ types vary. However, existing technologies have little research on the specific culture conditions of lacrimal gland organoids, namely, the culture medium components and culture methods, and the culture results are poor, making it difficult to obtain a lacrimal gland organoid model that can be cultured stably and long-term in vitro. Summary of the Invention

[0005] In response to the problems in the above-mentioned background technology, the purpose of the present invention is to provide a culture medium for lacrimal gland organoids and a culture method for lacrimal gland organoids, so as to solve the shortcomings of conventional culture medium and the problem of small number and low viability of cells obtained from primary tissues. The lacrimal gland organoids cultured using the present invention can grow stably for a long time, continue to expand, and be repeatedly frozen.

[0006] In order to achieve the above object, the technical solution adopted by the present invention includes:

[0007] A lacrimal gland organoid culture medium comprising: a basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, an adenylate cyclase activator, and A8301;

[0008] Based on the basic culture medium, calculated by volume percentage:

[0009] R-spodin3 and B27 were 1% to 5%;

[0010] Noggin is 0.01% to 0.1%;

[0011] Acetyl-L-cysteine, adenylate cyclase activator and A8301 are 0.1% to 0.15% respectively.

[0012] Optionally, trehalose and vitamin C are further included, and the volume fractions of trehalose and vitamin C are 1% to 3% respectively.

[0013] Optionally, PGE2 and FGF10 are further included, with the volume fraction of PGE2 being 0.1% to 0.15% and the volume fraction of FGF10 being 0.01% to 0.1%.

[0014] Optionally, glutathione is also included, and the volume fraction of glutathione is 1% to 3%.

[0015] Optionally, the basal culture medium is DMEM / F12 culture medium for cell culture, containing 15 mM HEPES, 3151 mg / L D-glucose, 1200 mg / L sodium bicarbonate, and a pH value of 7.0-7.4.

[0016] A method for culturing lacrimal gland organoids, obtained by culturing using any of the lacrimal gland organoid culture media described in the present invention, comprising the following steps:

[0017] (1) Wash the lacrimal gland tissue with PBS solution containing antibiotics and mince it with sterile ophthalmic scissors;

[0018] (2) Use tissue digestion solution to digest and terminate enzymatic hydrolysis;

[0019] (3) Filter the digested liquid to obtain a suspension of single cells and cell clusters, and collect the precipitate by centrifugation; add red blood cell lysis buffer, lyse the red blood cells at room temperature, and centrifuge at 4°C, 1500 rpm, and 5 min to obtain the precipitate;

[0020] (4) Resuspend the pellet in lacrimal gland organoid culture medium, count the pellet, mix it with Matrigel, and inoculate it into a culture plate. Invert it at 37°C until the gel drop solidifies.

[0021] (5) Add preheated lacrimal gland organoid culture medium and culture in a cell culture incubator at 37°C and 5% CO2. Replace the lacrimal gland organoid culture medium every 3 to 4 days. Organoids can be obtained after 7 to 14 days of culture.

[0022] Optional, specific steps include:

[0023] (1) The white lacrimal gland tissue around the mouse eye was completely peeled off to obtain the lacrimal gland tissue, which was then soaked in alcohol and sterile cleaning solution and then cut into pieces;

[0024] (2) Add 3-5 ml of tissue digestion solution to the minced lacrimal gland tissue and digest at 37°C with constant temperature and shaking for 15-20 min until the tissue is loose. Stop digestion when the suspension contains 50%-80% lacrimal gland single cells by volume. Add 2-3 times the volume of digestion solution to terminate digestion.

[0025] (3) Filter the collected filtrate through a 100 μm cell sieve into a centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet; add 2 to 3 times the volume of red blood cell lysis buffer to the harvested lacrimal gland cell pellet and resuspend it, place it at room temperature for 2 to 5 min, add 2 to 3 times the volume of sterile cleaning solution to dilute and terminate the lysis, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet;

[0026] (4) Resuspend the lacrimal gland cells in the lacrimal gland organoid culture medium, count them using a cell counter, calculate the preparation inoculum volume to be 180,000 to 240,000 per 30 μl, mix them according to the volume ratio of lacrimal gland organoid culture medium to matrix gel of 1:2, and add them dropwise to the culture plate; let them stand at 37°C and 5% CO2 for 2 minutes, solidify for 15 to 30 minutes, and add the lacrimal gland organoid culture medium; after culturing for 24 hours, lacrimal gland cell spheres are formed, and the lacrimal gland organoid culture medium is replaced every 3 to 4 days.

[0027] Optionally, in step (2), the tissue digestion fluid is used again for secondary digestion, and the digestion time is set to 10 to 15 minutes.

[0028] Optionally, the secondary digestion comprises: adding 3 to 5 ml of tissue digestion solution to the minced lacrimal gland tissue, digesting with constant temperature shaking at 37° C. for 10 to 15 minutes until the tissue is loose, and examining the number of single cells under a microscope; stopping the digestion when 30% to 50% of the lacrimal gland single cells are in the suspension, and adding 2 to 3 times the volume of the digestion solution to terminate the digestion;

[0029] The lacrimal gland single cells and cell clusters that have been digested were blown to make them uniformly suspended, and the tissue clusters were allowed to settle to the bottom of the tube. The upper suspension was aspirated and transferred to a new centrifuge tube. The tissue clusters were continued to add 2-3 mL of tissue digestion solution and digested at 37°C with constant temperature shaking for 10-15 minutes. The number of single cells was examined under a microscope. When the suspension contained 50%-80% lacrimal gland single cells, the digestion was stopped and 2-3 times the volume of the digestion solution was added to terminate the digestion.

[0030] Compared with the prior art, the present invention has the following effects:

[0031] 1) The culture medium for culturing lacrimal gland organoids provided by the present invention is composed of multiple cytokine components configured in optimized proportions. Its composition is simple and reasonable, and it is nutrient-rich. Cultivating lacrimal gland organoids using this culture medium can promote the formation and growth of organoids, further simulate the complex microenvironment in the body, make the organoids more similar to the physiological state in the body, and better construct organoid models to evaluate the effects of drugs on lacrimal gland function. This provides a new platform for drug screening and also opens up new avenues for the subsequent treatment of diseases such as dry eye in the future.

[0032] 2) The lacrimal gland organoid culture method provided by the present invention adopts secondary digestion to shorten the digestion time, improve the viability of the lacrimal gland organoids, and increase the yield of the lacrimal gland organoids. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following detailed description, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

[0034] Figure 1 This is a picture of the lacrimal gland organoids cultured in EM-A medium for one week in Example 1 of the present invention, taken under a microscope at 20x magnification;

[0035] Figure 2 This is a picture of the lacrimal gland organoid cultured in EM-B medium for one week in Example 2 of the present invention, taken under a microscope at 20x magnification;

[0036] Figure 3 This is a picture taken at 20x magnification under a microscope of the lacrimal gland organoids cultured in EM-C medium for one week in Example 3 of the present invention;

[0037] Figure 4 This is a picture taken at 20x under a microscope of the lacrimal gland organoids cultured in EM-D medium for one week in Example 4 of the present invention;

[0038] Figure 5 Schematic diagram comparing the viability of lacrimal gland organoid spheres cultured in different EM culture media in Examples 1-4 of the present invention. DETAILED DESCRIPTION

[0039] In order to enable those skilled in the art to better understand the present invention, the present invention will be further described in detail below in conjunction with specific implementation methods.

[0040] R-spodin 3 (RSPO3): also known as cysteine-rich monothrombin domain-1 (CRISTIN1), protein with TSP1-type repeats (PWTSR), is a member of the R-Spondin protein family. RSPO-3 is widely expressed, with higher levels in the placenta, small intestine, fetal thymus, and lymph nodes. RSPO3 is an activator of the β-catenin signaling cascade, leading to TCF-dependent gene activation. RSPO3 plays a role in both the canonical Wnt / β-catenin-dependent pathway and the non-canonical Wnt signaling pathway, possibly by acting as an inhibitor of ZNRF3, an important regulator of the Wnt signaling pathway. RSPO3 also acts as a ligand for frizzled FZD8 and LRP6, and may negatively regulate the TGF-β pathway. These signaling pathways are involved in development, cell growth, and disease pathogenesis.

[0041] Recombinant human Noggin is a secreted homodimeric glycoprotein. Secreted Noggin is located on the cell surface due to its binding to heparin-containing proteoglycans and acts as an antagonist of bone morphogenetic proteins (BMPs). Noggin was originally identified as a BMP-4 antagonist, playing a crucial role in the proper formation of the head and other dorsal structures. As a result, Noggin has a high affinity for certain BMPs, such as BMP-4, but a lower affinity for BMP-7. It also has a high affinity for BMP-2, BMP-13, and BMP-14. During skeletal development, Noggin inhibits chondrocyte proliferation, thereby regulating normal joint formation. When human embryonic stem cells (hESCs) or neural stem cells are cultured under certain conditions in the adult central nervous system and peripheral tissues (such as the lung), the addition of Noggin to antagonize BMP activity can enable stem cells to proliferate while maintaining their undifferentiated state or to differentiate into dopaminergic neurons. In Noggin-deficient mice, enhanced BMP activity causes a series of developmental abnormalities, including failure of neural tube formation, delayed hair follicle development, axial skeletal deformities and joint lesions.

[0042] Human Leukocyte Antigen (B27): This is a serum-free supplement optimized to support the growth of embryonic, postnatal, and adult hippocampal and other central nervous system neurons, and is also used in the culture of organoids.

[0043] N-Acetyl-L-cysteine: It is a sulfhydryl-containing antioxidant that can increase the free radical scavenger pool in the cell and is also a mucolytic agent.

[0044] Trehalose (D-Trehalose anhydrous): is a typical stress metabolite that can form a unique protective film on the cell surface under harsh environmental conditions such as high temperature, extreme cold, high osmotic pressure, and dehydration, effectively protecting the biological molecular structure from destruction, thereby maintaining the life process and biological characteristics of living organisms.

[0045] Vitamin C: It participates in the body's complex metabolic processes, can promote growth and enhance resistance to disease, can be used as a nutritional supplement, antioxidant, and can also be used as a wheat flour improver.

[0046] Glutathione (GSH): It is a tripeptide composed of glutamic acid, cysteine ​​and glycine, containing a sulfhydryl group, which has antioxidant and integrated detoxification effects.

[0047] Adenylate cyclase activator (Forskolin): Acts on type I adenylate cyclase with an IC50 of 41 nM. It is a diterpenoid derived from the plant Coleus forskohlii that activates adenylate cyclase, which increases intracellular cAMP levels. Its anti-inflammatory and antioxidant effects are due to inhibition of macrophage activation, thereby reducing thromboxane B2 and superoxide levels. It is used to treat heart disease, hypertension, diabetes, and asthma.

[0048] 3-(6-Methyl-2-pyridyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-thiocarboxamide (A8301): A8301 is a potent inhibitor of TGF-β type I receptor ALK5 (IC50: 12 nm), ALK4 (IC50: 45 nm), and ALK7 (IC50: 7.5 nm) kinases. A8301 has a strong inhibitory effect on ALK5, inhibiting Smad2 / 3 phosphorylation and TGF-β-induced growth. A8301 has little or no effect on bone morphogenetic protein type I receptor, p38 mitogen-activated protein kinase, or extracellular-regulated kinase. Furthermore, A8301 inhibits TGF-β-induced epithelial-mesenchymal transition, suggesting that A8301 may be helpful in inhibiting the progression of advanced cancers.

[0049] Prostaglandin E2 (PGE2): is a hormone-like substance that is involved in a wide range of body functions, such as contraction and relaxation of smooth muscles, dilation and constriction of blood vessels, control of blood pressure and regulation of inflammation.

[0050] Fibroblast Growth Factor 10 (FGF10): The protein encoded by this gene is a member of the fibroblast growth factor (FGF) family. FGF family members have a wide range of mitogenic and cell survival activities and are involved in a variety of biological processes, including embryonic development, cell growth, morphogenesis, tissue repair, tumor growth and invasion. This protein exhibits mitogenic activity on keratinized epidermal cells but essentially no activity on fibroblasts, which is similar to the biological activity of FGF7. Studies on the mouse homolog have shown that this gene is required for embryonic epidermal morphogenesis, including brain development, lung morphogenesis and the initiation of lim bud formation. This gene is also considered a major factor in the wound healing process.

[0051] In a first aspect, the culture medium (EM) for lacrimal gland organoids provided by the present invention is composed of basal culture medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, trehalose, vitamin C, glutathione, adenylate cyclase activator, A8301, PGE2, and FGF10.

[0052] In the embodiments of the present disclosure, basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, adenylate cyclase activator, A8301;

[0053] In the embodiments of the present disclosure, basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, adenylate cyclase activator, A8301, trehalose, vitamin C;

[0054] In the embodiments of the present disclosure, basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, adenylate cyclase activator, A8301, trehalose, vitamin C, PGE2, FGF10;

[0055] In the embodiments of the present disclosure, basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, adenylate cyclase activator, A8301, trehalose, vitamin C, PGE2, FGF10, glutathione;

[0056] In the embodiments of the present disclosure, the basal culture medium is DMEM / F12 culture medium for cell culture, containing 15 mM HEPES, 3151 mg / L D-glucose, 1200 mg / L sodium bicarbonate, and a pH value of 7.0-7.4.

[0057] In the embodiments of the present disclosure, the content of each component is as follows: the volume fraction of R-spodin3 and B27 is 1% to 5%; the volume fraction of Noggin and FGF10 is 0.01% to 0.1%; the volume fraction of acetyl-L-cysteine, adenylate cyclase activator, A8301, and PGE2 is 0.1% to 0.15%; the volume fraction of trehalose, vitamin C, and glutathione is 1% to 3%.

[0058] In a second aspect, the present invention provides a method for culturing lacrimal gland organoids, the method comprising the following steps:

[0059] (1) Wash the lacrimal gland tissue with PBS solution containing antibiotics and mince it with sterile ophthalmic scissors;

[0060] (2) Use tissue digestion solution to digest and terminate enzymatic hydrolysis;

[0061] (3) Filter the digested liquid to obtain a suspension of single cells and cell clusters, and collect the precipitate by centrifugation;

[0062] (4) Add red blood cell lysis buffer, lyse the red blood cells at room temperature, and centrifuge at 4°C, 1500 rpm, for 5 min;

[0063] (5) Resuspend the pellet in the culture medium of the lacrimal gland organoids, count the pellet, mix it with Matrigel, and inoculate it into a culture plate. Invert it at 37°C until the gel drop solidifies.

[0064] (6) Add pre-warmed lacrimal gland organoid culture medium and culture in a cell culture incubator at 37°C and 5% CO2. Change the culture medium every 3-4 days. Organoids can be obtained after 7-14 days of culture.

[0065] In an embodiment of the present disclosure, the lacrimal gland organoid culture medium is the culture medium of the first aspect of the present invention.

[0066] In the embodiment of the present disclosure, the tissue digestion fluid is subjected to secondary digestion, and the digestion time is set to 10 to 15 minutes.

[0067] In this disclosure, the specific implementation steps include:

[0068] (1) Use sterile scissors and straight and curved forceps to dissect the mouse, completely peel off the white lacrimal gland tissue around its eyes to obtain the lacrimal gland tissue, soak and clean it with alcohol and sterile cleaning solution in turn, and then cut it into pieces;

[0069] (2) Add 3-5 ml of tissue digestion solution to the minced lacrimal gland tissue and digest at 37°C with constant temperature shaking for 15-20 min until the tissue is loose. When the suspension contains 50%-80% lacrimal gland single cells by volume, digestion is stopped and 2-3 times the volume of the digestion solution is added to terminate the digestion. The tissue digestion solution is obtained from Xiamen Model Biotechnology Co., Ltd.

[0070] (3) Filter the collected filtrate through a 100 μm cell sieve into a centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet; add 2 to 3 times the volume of red blood cell lysis buffer to the harvested lacrimal gland cell pellet and resuspend it, place it at room temperature for 2 to 5 min, add 2 to 3 times the volume of sterile cleaning solution to dilute and terminate the lysis, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet;

[0071] (4) Resuspend the lacrimal gland cells in the lacrimal gland organoid culture medium, count them using a cell counter, and calculate the preparation inoculation volume to be 180,000 to 240,000 per 30 μl. Mix the lacrimal gland organoid culture medium and matrigel (low growth factor gold phenol red-free matrigel: a natural basement membrane matrix extracted from mouse tumors rich in extracellular matrix proteins, from Xiamen Model Biotechnology Co., Ltd.) in a volume ratio of 1:2, and add it dropwise to the culture plate; let it stand at 37°C and 5% CO2 for 2 minutes, solidify for 15 to 30 minutes, and add the lacrimal gland organoid culture medium; after culturing for 24 hours, lacrimal gland cell spheres are formed, and the lacrimal gland organoid culture medium is replaced every 3 to 4 days.

[0072] Specifically, in step (2), the tissue digestion solution is used again for secondary digestion, and the digestion time is set to 10 to 15 minutes.

[0073] Specifically, the secondary digestion process involves adding 3-5 ml of tissue digestion solution to the minced lacrimal gland tissue and incubating with shaking at 37°C for 10-15 minutes until the tissue is loose, followed by microscopic examination of the number of single cells. The digestion is stopped when 30%-50% of the lacrimal gland single cells are present in the suspension, and sterile washing solution (2-3 times the volume of the digestion solution) is added to terminate the digestion.

[0074] The lacrimal gland single cells and cell clusters that have been digested were blown to make them uniformly suspended. The large tissue clusters were allowed to settle to the bottom of the tube for 2 minutes. The upper suspension was aspirated and transferred to a new centrifuge tube. For large tissue clusters, 2-3 mL of tissue digestion solution was added. The cells were digested at 37°C with constant temperature and shaking for 10-15 minutes. The number of single cells was examined under a microscope. When the suspension contained 50%-80% lacrimal gland single cells, the digestion was stopped by adding 2-3 times the volume of the digestion solution and sterile washing solution was added to terminate the digestion.

[0075] The technical solution of the present invention is described in detail below with reference to specific embodiments and the accompanying drawings.

[0076] Example 1: Preparation of the lacrimal gland organoid culture medium EM-A of the present invention

[0077] 100 mL of DMEM / F12 (1:1) basal culture medium was added with the following components at the following concentrations: 3% of R-spodin3 and B27; 0.06% of Noggin; and 0.5% of N-acetyl-L-cysteine, adenylate cyclase activator, and A8301.

[0078] Example 2: Preparation of the lacrimal gland organoid culture medium EM-B of the present invention

[0079] 100 mL of DMEM / F12 (1:1) basal culture medium was added with the following components at the following concentrations: R-spodin3 and B27 were both 3%; Noggin was 0.06%; N-acetyl-L-cysteine, adenylate cyclase activator, and A8301 were both 0.5%; and trehalose and vitamin C were both 2%.

[0080] Example 3: Preparation of the lacrimal gland organoid culture medium EM-C of the present invention

[0081] 100 mL of DMEM / F12 (1:1) basal medium was added with the following components at the following concentrations: R-spodin3 and B27 were 3%; Noggin and FGF10 were 0.06%; N-acetyl-L-cysteine, adenylate cyclase activator, A8301, PGE2 were 0.5%; trehalose and vitamin C were 2%;

[0082] Example 4: Preparation of the Lacrimal Gland Organoid Culture Medium EM-D of the Present Invention

[0083] 100 mL of DMEM / F12 (1:1) basal culture medium was added with the following components at the following concentrations: R-spodin3 and B27 were both 3%; Noggin and FGF10 were both 0.06%; N-acetyl-L-cysteine, adenylate cyclase activator, A8301, PGE2 were all 0.5%; trehalose, vitamin C, and glutathione were all 2%.

[0084] Example 5: Using the lacrimal gland organoid culture medium of Example 1, Example 2, Example 3 and Example 4, lacrimal gland organoids were cultured as follows

[0085] The specific steps are as follows:

[0086] 4.1 Use sterile scissors and straight and curved forceps to dissect the mouse, completely peel off the white lacrimal gland tissue around its eyes to obtain the lacrimal gland tissue, transfer it to a 15ml centrifuge tube, add 10-12ml of alcohol and soak for 1min, pour out the alcohol, and then turn it upside down with sterile cleaning solution and wash it three times. Use sterile ophthalmic scissors or ophthalmic tissue scissors to cut the cleaned tissue into small pieces of approximately 1*1*1mm3. The sterile cleaning solution contains calcium and magnesium ions PBS and penicillin-streptomycin, and the concentration of penicillin-streptomycin is 10000U / mL.

[0087] 4.2 The minced lacrimal gland tissue was collected into a 50 ml centrifuge tube, 3-5 ml of tissue digestion solution was added, and the tissue was placed in a constant temperature shaker at 37°C for about 15-20 min to loosen the tissue. The number of single cells was examined microscopically. If the number was low (less than 30%), digestion was continued. When the suspension contained 50%-80% lacrimal gland single cells or when a 1 ml sterile pipette tip was not clogged, digestion was stopped by adding 2-3 times the volume of the digestion solution with sterile washing solution to terminate the digestion. The tissue digestion solution was obtained from Xiamen Model Biotechnology Co., Ltd.

[0088] 4.3 Filter the filtrate through a 100 μm cell sieve into a new centrifuge tube and centrifuge at 1500 rpm for 5 minutes. Discard the supernatant to harvest the lacrimal gland cell pellet. If a large number of red blood cells are clearly visible in the lacrimal gland cell pellet, add 2 to 3 volumes of red blood cell lysis buffer to the harvested lacrimal gland cell pellet and resuspend it. Pipet and let it stand at room temperature for 2 to 5 minutes. Then, add 2 to 3 volumes of sterile washing buffer to dilute and terminate lysis. Centrifuge at 1500 rpm for 5 minutes and discard the supernatant to harvest the lacrimal gland cell pellet.

[0089] 4.4 Resuspend the lacrimal gland cells in lacrimal gland organoid culture medium and count them using a cell counter. Calculate the inoculum size to be 180,000 to 240,000 cells / 30 μl. Keep the inoculum on ice and pre-chill the centrifuge tubes and pipette tips. Slowly and thoroughly mix the culture medium and Matrigel (Reduced Growth Factor Gold Phenol Red-Free Matrigel: a natural basement membrane matrix extracted from mouse tumors rich in extracellular matrix proteins, from Xiamen Model Biotechnology Co., Ltd.) in a 1:2 volume ratio, avoiding air bubbles. Add the mixture dropwise to the culture plate preheated in a CO2 incubator.

[0090] 4.5 Place the culture plate in an incubator (37°C, 5% CO2) and let it sit for 2 minutes. Gently shake the gel droplet until no noticeable flow occurs. Carefully invert the plate and allow it to fully solidify for approximately 15 to 30 minutes. After the gel droplet has fully solidified, add the culture medium of Example 1, Example 2, Example 3, and Example 4 to the culture dish and culture for 24 hours to form lacrimal gland cell spheres. Replace the culture medium every 3 to 4 days and culture in an incubator. Generally, lacrimal gland organoids can be obtained after 7 to 12 days of culture. Observe and photograph them under a microscope.

[0091] Different from the above steps, the two digestion operations in this embodiment include: 4.2 Collect the minced lacrimal gland tissue into a 50ml centrifuge tube, add 3-5ml of tissue digestion solution, place it in a constant temperature shaker at 37°C and oscillate and digest for about 10-15 minutes until the tissue is loose, and examine the number of single cells under a microscope. When the suspension contains 30% to 50% lacrimal gland single cells, the digestion is stopped and 2 to 3 times the volume of the digestion fluid is added to terminate the digestion. The digested lacrimal gland single cells and cell clusters are blown with a pipette to make them uniformly suspended. The large tissue clusters are allowed to settle to the bottom of the tube for 2 minutes. The upper lacrimal gland single cell and cell cluster suspension is aspirated into a new centrifuge tube. 2 to 3 mL of tissue digestion fluid is continued to be added to the large tissue clusters. The large tissue clusters are placed in a constant temperature shaker at 37°C and oscillated for about 10 to 15 minutes until the tissue is loose. The number of single cells is examined under a microscope. When the suspension contains 50% to 80% lacrimal gland single cells, the digestion is stopped and 2 to 3 times the volume of the digestion fluid is added to terminate the digestion. The tissue digestion fluid is obtained from Xiamen Model Biotechnology Co., Ltd.

[0092] The experimental results are shown in Table 1. The lacrimal gland organoid culture method in Example 5 was used. The lacrimal gland tissue sample was washed and sheared, and then digested twice using tissue digestion solution according to the steps. The cell suspension and trypan blue dye were mixed in a 1:1 ratio, and the cell concentration and viability after the two digestions were detected. As can be seen from the data in the table, the cell viability after two digestions was above 90%, and the viable cell concentration in the second digestion reached 1 / 2 times that of the first digestion, increasing the cell yield in a single digestion and avoiding the problem of low cell viability and small cell amount in the conventional single digestion time due to excessively long digestion time.

[0093] Table 1 Organoid cell data after two digestions

[0094]

[0095]

[0096] The experimental results are as follows Figure 1-4As shown, the images of the lacrimal gland organoids cultured in the four culture media in the specific examples for one week were taken under a microscope at 20x and imported into Image J to measure the size of the lacrimal gland organoids according to the corresponding 200 μm scale bar. The culture media of Examples 1-4 can all effectively culture liver cancer organoids. Judging from the data size in the picture, the diameter of the lacrimal gland organoid cell spheres cultured in the EM-A medium in Example 1 for one week is small, approximately between 35 and 45 μm, and the growth is slow; the diameter of the lacrimal gland organoid cell spheres cultured in the EM-B medium in Example 2 for one week is between 55 and 125 μm, and the growth is slightly faster than that of the EM-A medium in Example 1; the diameter of the lacrimal gland organoid cell spheres cultured in the EM-C medium in Example 3 for one week is between 115 and 220 μm, and the growth rate is faster; the diameter of the lacrimal gland organoid cell spheres cultured in the EM-D medium in Example 4 for one week is between 195 and 310 μm, and the growth rate is the fastest. In addition, the lacrimal gland organoids cultured in the EM-C medium in Example 3 and the EM-D medium in Example 4 are in good condition compared with the organoids cultured in the EM-A medium in Example 1 and the EM-B medium in Example 2, with clear boundaries, no disintegration, strong cell aggregation, and good refractive index. This shows that the growth trends of lacrimal gland organoid spheres cultured in the four different culture media for one week are quite different. In comprehensive comparison, the EM-D culture medium in Example 4 has an outstanding culture effect.

[0097] Lacrimal gland organoid cell sphere viability detection: Lacrimal gland organoid cell spheres cultured to the 3rd, 5th, 7th, and 10th days were taken respectively, and the organoid viability analysis kit Organoid Vitality Assay Kit (purchased from MCE) was used for cell viability detection. The specific steps are as follows: 1) Lacrimal gland organoid cell inoculation: The lacrimal gland organoid cells were inoculated into a 96-well plate in advance, and four groups were set up to take 100 μl of the EM culture medium in Examples 1-4 for culture, and the culture time was 1-10 days; 2) 96-well plate preparation: The 96-well cell culture plate on the 3rd day of culture was taken out, and the original complete culture medium in the well to be tested was aspirated and discarded; 2) Add detection working solution: 100 μl of organoid viability detection working solution (1x) was added to each well along the wall of the well, and the culture wells without organoids and dye were set as dyes Place the negative control wells in the incubator and incubate for 30-120 minutes; 3) Detect fluorescence: Use a fluorescence microplate reader (Ex / Em = 560 / 590nm) to detect fluorescence and record the fluorescence intensity; 4) Replace the culture medium: After the inspection is completed, discard the working solution in the 96-well plate, re-add the corresponding fresh complete culture medium, and continue culturing; 5) Repeat the test: Repeat the above organoid viability test steps (2-4) on the 5th, 7th, and 10th days respectively; 6) Analyze the results: Analyze the relative viability of the organoids based on the total fluorescence intensity (minus the dye negative control).

[0098] The experimental results are as follows Figure 5 As shown, on the 3rd day, there was little difference in the viability test of the lacrimal gland organoids cultured in the EM culture media of different components in Examples 1-4. On the 5th, 7th and 10th days, more obvious changes appeared. Among them, the lacrimal gland organoids cultured in the EM culture media in Examples 3 and 4 had higher activity than the lacrimal gland organoids cultured in the EM culture media in Examples 1 and 2. From the overall data, it can be seen that the activity of the lacrimal gland organoid spheres cultured in the four different composition culture media is ranked from high to low: EM-D culture medium in Example 4 > EM-C culture medium in Example 3 > EM-B culture medium in Example 2 > EM-A culture medium in Example 1.

[0099] The above are only specific embodiments of the present invention, but the present invention is not limited to the above embodiments. Without departing from the design concept and principle of the present invention, various modifications, improvements and changes of the present invention should be included in the scope of protection of the present invention.

Claims

1. A lacrimal gland organoid culture medium, characterized in that include: basal medium, R-spodin3, Noggin, B27, N-acetyl-L-cysteine, adenylate cyclase activator, and A8301; Based on the basic culture medium, calculated by volume percentage: R-spodin3 and B27 were 1% to 5%; Noggin is 0.01% to 0.1%; Acetyl-L-cysteine, adenylate cyclase activator and A8301 are 0.1% to 0.15% respectively.

2. The lacrimal gland organoid culture medium according to claim 1, wherein The invention also comprises trehalose and vitamin C, and the volume fractions of trehalose and vitamin C are 1% to 3% respectively.

3. The lacrimal gland organoid culture medium according to claim 1 or 2, characterized in that It also includes PGE2 and FGF10, with the volume fraction of PGE2 being 0.1% to 0.15% and the volume fraction of FGF10 being 0.01% to 0.1%.

4. The lacrimal gland organoid culture medium according to claim 3, characterized in that It also includes glutathione, and the volume fraction of glutathione is 1% to 3%.

5. The lacrimal gland organoid culture medium according to claim 1 or 2, characterized in that The basic culture medium is DMEM / F12 culture medium, which is used for cell culture and contains 15 mM HEPES, 3151 mg / L D-glucose, 1200 mg / L sodium bicarbonate, and a pH value of 7.0-7.

4.

6. A method for culturing lacrimal gland organoids, characterized in that: The method comprises culturing the lacrimal gland organoid culture medium according to any one of claims 1 to 5, comprising the following steps: (1) Wash the lacrimal gland tissue with PBS solution containing antibiotics and mince it with sterile ophthalmic scissors; (2) Use tissue digestion solution to digest and terminate enzymatic hydrolysis; (3) Filter the digested liquid to obtain a suspension of single cells and cell clusters, and collect the precipitate by centrifugation; add red blood cell lysis buffer, lyse the red blood cells at room temperature, and centrifuge at 4°C, 1500 rpm, and 5 min to obtain the precipitate; (4) Resuspend the pellet in lacrimal gland organoid culture medium, count the pellet, mix it with Matrigel, and inoculate it into a culture plate. Invert it at 37°C until the gel drop solidifies. (5) Add preheated lacrimal gland organoid culture medium and culture in a cell culture incubator at 37°C and 5% CO2. Replace the lacrimal gland organoid culture medium every 3 to 4 days. Organoids can be obtained after 7 to 14 days of culture.

7. The method for culturing lacrimal gland organoids according to claim 6, wherein: The specific steps include: (1) The white lacrimal gland tissue around the mouse eye was completely peeled off to obtain the lacrimal gland tissue, which was then soaked in alcohol and sterile cleaning solution and then cut into pieces; (2) Add 3-5 ml of tissue digestion solution to the minced lacrimal gland tissue and digest at 37°C with constant temperature and shaking for 15-20 min until the tissue is loose. Stop digestion when the suspension contains 50%-80% lacrimal gland single cells by volume. Add 2-3 times the volume of digestion solution to terminate digestion. (3) Filter the collected filtrate through a 100 μm cell sieve into a centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet; add 2 to 3 times the volume of red blood cell lysis buffer to the harvested lacrimal gland cell pellet and resuspend it, place it at room temperature for 2 to 5 min, add 2 to 3 times the volume of sterile cleaning solution to dilute and terminate the lysis, centrifuge at 1500 rpm for 5 min, discard the supernatant to harvest the lacrimal gland cell pellet; (4) Resuspend the lacrimal gland cells in the lacrimal gland organoid culture medium, count them using a cell counter, calculate the preparation inoculum volume to be 180,000 to 240,000 per 30 μl, mix them according to the volume ratio of lacrimal gland organoid culture medium to matrix gel of 1:2, and add them dropwise to the culture plate; let them stand at 37°C and 5% CO2 for 2 minutes, solidify for 15 to 30 minutes, and add the lacrimal gland organoid culture medium; after culturing for 24 hours, lacrimal gland cell spheres are formed, and the lacrimal gland organoid culture medium is replaced every 3 to 4 days.

8. The method for culturing lacrimal gland organoids according to claim 6 or 7, wherein: In the step (2), the tissue digestion fluid is used again for secondary digestion, and the digestion time is set to 10 to 15 minutes.

9. The method for culturing lacrimal gland organoids according to claim 8, wherein: The secondary digestion comprises: adding 3 to 5 ml of tissue digestion solution to the minced lacrimal gland tissue, digesting at 37° C. with constant temperature and shaking for 10 to 15 minutes until the tissue is loose, and examining the number of single cells under a microscope; stopping the digestion when 30% to 50% of the lacrimal gland single cells are in the suspension, and adding 2 to 3 times the volume of the digestion solution to terminate the digestion; The lacrimal gland single cells and cell clusters that have been digested were blown to make them uniformly suspended, and the tissue clusters were allowed to settle to the bottom of the tube. The upper suspension was aspirated and transferred to a new centrifuge tube. The tissue clusters were continued to add 2-3 mL of tissue digestion solution and digested at 37°C with constant temperature shaking for 10-15 minutes. The number of single cells was examined under a microscope. When the suspension contained 50%-80% lacrimal gland single cells, the digestion was stopped and 2-3 times the volume of the digestion solution was added to terminate the digestion.

Images