Separation and culture method for different cell types of lindel gland

Abstract

The invention relates to the technical field of cell separation and culture, and discloses a method for separating and culturing different cell types of musk glands of forest musk deer, which comprises the following steps of: 1, taking off the musk glands from the forest musk deer just dead of illness, simply separating glands, using one part of the musk glands for separating primary cells, and using the other part of the musk glands for separating secondary cells; one part is treated with formalin and then sent to be subjected to HE staining slicing, and deep glands, superficial glands and sebaceous glands are distinguished; according to the method, the musk gland cells of the musk deer can serve as primary cell strains to be stored for subsequent musk secretion research, 0-generation cells are digested when growing to 40%-50%, cryopreservation and passage are conducted, so that the amplification purpose is achieved, sufficient cell strains are provided for subsequent research, and the cryopreserved 0-generation, 1-generation and 2-generation musk gland cells are stored in a liquid nitrogen tank, so that the musk gland cells of the musk deer can be obtained. And according to the three gland cells, mass amplification and cryopreservation are carried out after successful separation, and the method is used for subsequent fragrance secretion research of the fragrant gland cells.

Description

Technical Field

[0001] The invention relates to the technical field of cell separation and culture, in particular to a method for separating and culturing different cell types of musk gland. Background Art

[0002] Musk deer are small mammals living in Asia. They belong to the family Muskidae and usually live in forests or mountainous areas. They feed on grass, leaves, fruits and insects. Studies have found that the scent gland epithelial cells in male Musk deer can secrete scent gland hormone (musk). Musk has the effects of awakening the mind, promoting blood circulation and menstruation, and reducing swelling and relieving pain. Therefore, it is often used to make spices and medicines. However, the yield of natural musk is extremely low. Each male Musk deer can only produce about 15 grams per year, so musk is extremely precious.

[0003] In order to meet the market demand for musk, researchers are actively seeking methods to obtain musk through in vitro cell biological culture. However, when using the culture medium prepared by existing technology to isolate and culture forest musk glandular epithelial cells, the cell growth state is poor, the cells are prone to non-directional differentiation during the subculture process, and the purpose of amplification cannot be achieved well.

[0004] Therefore, we provide a method for isolating and culturing different cell types of the forest musk gland. The present invention has conducted in-depth research on the optimization of the culture medium and the cell passage and amplification method. By adding appropriate amounts of growth factors and hormones, the growth rate and purity of the forest musk gland epithelial cells are significantly improved. In addition, the use of enzyme digestion combined with mechanical scraping in the subculture effectively reduces the risk of non-directional cell differentiation and ensures the stability of the cells and the ability to secrete musk. This innovation provides a scientific basis for the sustainable use of musk and is of great significance to the protection and development of musk resources. In subsequent experiments, we conducted a detailed analysis of the growth characteristics of the forest musk gland epithelial cells and found that under optimized culture conditions, the growth cycle of the cells was significantly shortened and the ability to secrete musk was significantly improved. Summary of the invention

[0005] The object of the present invention is to provide a method for separating and culturing different cell types of the musk gland to solve the problems raised in the above-mentioned background technology.

[0006] To achieve the above object, the present invention provides the following technical solution: a method for separating and culturing different cell types of musk gland, comprising the following steps:

[0007] Step 1: Use a newly dead forest musk deer, remove the scent glands, and simply separate the glands. One part is used to separate primary cells, and the other part is treated with formalin and sent for HE staining and sectioning to distinguish deep glands, superficial glands and sebaceous glands;

[0008] Step 2, reagent preparation: 0.25% Trypsin-EDTA Phenol Red, DMEM / F-12 culture medium, 1×D-PBS buffer, medical 75% alcohol, FBS, stop solution (DMEM / F12+FBS), complete culture medium, serum-free non-programmed cell freezing solution (without phenol red);

[0009] Step 3. Equipment preparation: fluorescence inverted microscope and microscopic image analysis system, cell culture box, clean bench, pipette, centrifuge, -4℃ refrigerator, -80℃ refrigerator, liquid nitrogen tank and pH meter;

[0010] Step 4: Preparation of experimenters: lay a water-proof pad on the operating table, and the experimenters and veterinarians wear masks, sterile surgical gowns and rubber gloves. Sterilize the surgical instruments with high temperature and high pressure and keep them ready for use. And disinfect the laboratory with ultraviolet light.

[0011] Step 5, separation of scent gland tissue: Make the musk deer lie on the operating table in a supine position, expose the location of the scent sac, shave the hair around the scent sac, use tweezers to grab the sterilized alcohol cotton and dip it in alcohol to wipe the scent sac for disinfection, then take the alcohol cotton and dip it in iodine to wipe it, deiodine it with alcohol, use high-temperature sterilized surgical instruments to separate the entire musk deer scent sac along the tissue around the scent sac, place it in a sterile transfer box, and quickly transfer it to a Class III laboratory;

[0012] Step 6: Digestion and culture of scent gland tissue: transfer the centrifuge tube containing the tissue to the clean bench, take out each scent gland tissue from the centrifuge tube, cut it into minced meat with ophthalmic scissors in a 1.5 ml centrifuge tube containing 0.1 ml 1×D-PBS for later use, and add 0.5 ml of type I collagenase with a concentration of 1 mg / ml to the minced meat;

[0013] Step 7. Primary cell culture and storage: After culturing in a cell culture incubator at 37° C., 5% CO 2 and 95% humid air for 48 hours, transfer the culture dish to an ultra-clean workbench.

[0014] Preferably, in step five, under a stereoscope, according to the distribution of various tissues in the cross-section of the scent sac reported in relevant literature, ophthalmic scissors are used to separate the muscle connective tissue and other tissues on the surface of the scent gland to expose the surface of the scent gland, and multiple portions of musk deer gland tissue of about 2cm*3cm in size are separated and taken out, and temporarily placed in a 15ml centrifuge tube containing 1% penicillin-streptomycin saline.

[0015] Preferably, in step six, the mixture is mixed by pipetting and then placed in a cell culture incubator for digestion for 8 to 15 minutes, then taken out and transferred to a 15 ml centrifuge tube with a pipette, and immediately stopped by adding 5 ml of DMEM / F12 containing 10% serum. The mixture is mixed by pipetting and then centrifuged at 1500r for 3 minutes, washed once with the stop solution, the supernatant is removed, 2 ml of complete culture medium is added, the mixture is mixed by pipetting, and the mixture is inoculated into a 35 mm culture dish, and the dish is transferred to a cell culture incubator with culture conditions of 37°C, 5% CO2 and 95% humid air for culture.

[0016] Preferably, in step seven, the original culture medium is aspirated with a pipette, 2 ml of complete culture medium is added, and the culture is continued, and observation is made every 48 hours until the primary cells grow by more than 60%, and cell subculture and expansion are performed.

[0017] The present invention provides a method for separating and culturing different cell types of forest musk glands. It has the following beneficial effects:

[0018] (1) The present invention has conducted in-depth research on the optimization of culture medium and cell subculture and amplification methods, and significantly improved the growth rate and purity of forest musk glandular epithelial cells by adding appropriate amounts of growth factors and hormones. In addition, the use of enzyme digestion combined with mechanical scraping in subculture effectively reduces the risk of non-directional cell differentiation and ensures the stability of the cells and the ability to secrete musk.

[0019] (2) The present invention preserves the scent gland cells of musk deer as primary cell lines for subsequent scent secretion research. When the 0th generation cells grow to 40%-50%, they are digested, frozen and passaged to achieve the purpose of expansion, providing sufficient cell lines for subsequent research. The frozen 0th, 1st and 2nd generation musk deer scent gland cells are stored in a liquid nitrogen tank, and their information is recorded for subsequent use. According to the three types of glandular cells, they are amplified and frozen in large quantities after successful separation for subsequent scent secretion research of scent gland cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram of the separation process of the musk gland of the present invention;

[0021] Figure 2 This is a HE-stained section view of the whole gland of the musk deer of the present invention;

[0022] Figure 3 This is a superficial view of the forest musk gland of the present invention;

[0023] Figure 4 This is a deep view of the musk gland of the present invention.

[0024] Figure 5 It is a view of the sebaceous gland of the musk deer of the present invention. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0026] Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0027] The preferred embodiment of the method for separating and culturing different cell types of the musk gland provided by the present invention is as follows: Figure 1-4 As shown: The method for isolating and culturing different cell types of the musk gland includes the following steps:

[0028] Step 1: Use a newly dead forest musk deer, remove the scent glands, and simply separate the glands. One part is used to separate primary cells, and the other part is treated with formalin and sent for HE staining and sectioning to distinguish deep glands, superficial glands and sebaceous glands;

[0029] Step 2, reagent preparation: 0.25% Trypsin-EDTA Phenol Red, DMEM / F-12 culture medium, 1×D-PBS buffer, medical 75% alcohol, FBS, stop solution (DMEM / F12+FBS), complete culture medium, serum-free non-programmed cell freezing solution (without phenol red);

[0030] Step 3. Equipment preparation: fluorescence inverted microscope and microscopic image analysis system, cell culture box, clean bench, pipette, centrifuge, -4℃ refrigerator, -80℃ refrigerator, liquid nitrogen tank and pH meter;

[0031] Step 4: Preparation of experimenters: lay a water-proof pad on the operating table, and the experimenters and veterinarians wear masks, sterile surgical gowns and rubber gloves. Sterilize the surgical instruments with high temperature and high pressure and keep them ready for use. And disinfect the laboratory with ultraviolet light.

[0032] Step 5, separation of scent gland tissue: make the musk deer lie on the operating table in a supine position, expose the location of the scent sac, shave the hair around the scent sac, use tweezers to clamp the sterilized alcohol cotton and dip it in alcohol to wipe the scent sac part for disinfection, then take the alcohol cotton and dip it in iodine to wipe it, deiodinate it with alcohol, use high-temperature sterilized surgical instruments to separate the entire musk deer scent sac along the tissue around the scent sac, place it in a sterile transfer box, and quickly transfer it to a third-level laboratory; under a stereoscope, according to the distribution of various tissues in the cross section of the scent sac reported in relevant literature, use ophthalmic scissors to separate the muscle connective tissue and other tissues on the surface of the scent gland, expose the surface of the scent gland, separate and take out multiple pieces of musk deer scent gland tissue of about 2cm*3cm in size, and put them into a 15ml centrifuge tube containing 1% penicillin-streptomycin physiological saline for temporary storage;

[0033] Step 6, digestion and culture of scent gland tissue: transfer the centrifuge tube containing the tissue to the clean bench, take out each scent gland tissue from the centrifuge tube, cut it into minced meat with ophthalmic scissors in a 1.5ml centrifuge tube containing 0.1ml 1×D-PBS for later use, add 0.5ml of type I collagenase with a concentration of 1mg / ml to the minced meat, mix it by pipetting, put it in a cell culture incubator for digestion for 8-15min, take it out, transfer it to a 15ml centrifuge tube with a pipette, immediately add 5ml of DMEM / F12 containing 10% serum to stop, mix it by pipetting, centrifuge it at 1500r for 3min, wash it with stop solution once, remove the supernatant, add 2ml of complete culture medium, mix it by pipetting, inoculate it into a 35mm culture dish, and transfer it to a cell culture incubator with culture conditions of 37°C, 5% CO2 and 95% humid air for culture;

[0034] Step 7. Primary cell culture and preservation: After culturing in a cell culture incubator at 37°C, 5% CO2 and 95% humid air for 48 hours, transfer the culture dish to an ultra-clean workbench, aspirate the original culture medium with a pipette, add 2 ml of complete culture medium, continue culturing, and observe every 48 hours until the primary cells grow by more than 60% for cell subculture and amplification.

[0035] Experimental evaluation:

[0036] 1. The obtained primary cells can grow adherently in the culture medium.

[0037] 2. Identify the primary cells obtained in subsequent primary cell isolation experiments.

[0038] Results and analysis:

[0039] The extraction and cultivation of primary cells is a slow process. After 6 days of inoculation, it was observed that the cells migrated well, the cells grew well, and the cell proliferation rate was normal, indicating that the scent gland cells of this musk deer can be preserved as primary cell lines for subsequent scent secretion research. When the 0th generation cells grow to 40%-50%, they are digested, frozen and passaged to achieve the purpose of amplification, providing sufficient cell lines for subsequent research. The frozen 0th, 1st, and 2nd generation forest musk gland cells are stored in a liquid nitrogen tank, and their information is recorded for subsequent use. According to the three types of glandular cells, they are amplified and frozen in large quantities after successful separation for subsequent scent secretion research of scent gland cells.

[0040] By sending the tissues to solarbio for HE sections, we can see the distribution of sebaceous glands in the deep and superficial layers.

[0041] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0042] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for separating and culturing different cell types of musk glands, characterized in that: The following steps are included: Step 1: Use a newly dead forest musk deer, remove the scent glands, and simply separate the glands. One part is used to separate primary cells, and the other part is treated with formalin and sent for HE staining and sectioning to distinguish deep glands, superficial glands and sebaceous glands; Step 2, reagent preparation: 0.25% Trypsin-EDTA PhenolRed, DMEM / F-12 culture medium, 1×D-PBS buffer, medical 75% alcohol, FBS, stop solution (DMEM / F12+FBS), complete culture medium, serum-free non-programmed cell freezing solution (without phenol red); Step 3. Equipment preparation: fluorescence inverted microscope and microscopic image analysis system, cell culture box, clean bench, pipette, centrifuge, -4℃ refrigerator, -80℃ refrigerator, liquid nitrogen tank and pH meter; Step 4: Preparation of experimenters: lay a water-proof pad on the operating table, and the experimenters and veterinarians wear masks, sterile surgical gowns and rubber gloves. Sterilize the surgical instruments with high temperature and high pressure and keep them ready for use. And disinfect the laboratory with ultraviolet light. Step 5, separation of scent gland tissue: Make the musk deer lie on the operating table in a supine position, expose the location of the scent sac, shave the hair around the scent sac, use tweezers to grab the sterilized alcohol cotton and dip it in alcohol to wipe the scent sac for disinfection, then take the alcohol cotton and dip it in iodine to wipe it, deiodine it with alcohol, use high-temperature sterilized surgical instruments to separate the entire musk deer scent sac along the tissue around the scent sac, place it in a sterile transfer box, and quickly transfer it to a Class III laboratory; Step 6: Digestion and culture of scent gland tissue: transfer the centrifuge tube containing the tissue to the clean bench, take out each scent gland tissue from the centrifuge tube, cut it into minced meat with ophthalmic scissors in a 1.5 ml centrifuge tube containing 0.1 ml 1×D-PBS for later use, and add 0.5 ml of type I collagenase with a concentration of 1 mg / ml to the minced meat; Step 7. Primary cell culture and storage: After culturing in a cell culture incubator at 37°C, 5% CO2 and 95% humid air for 48 hours, transfer the culture dish to an ultra-clean workbench.

2. The method for separating and culturing different cell types of the forest musk gland according to claim 1, characterized in that: In the step five, under a stereoscope, according to the distribution of various tissues in the cross section of the scent sac reported in relevant literature, ophthalmic scissors are used to separate the muscle connective tissue and other tissues on the surface of the scent gland to expose the surface of the scent gland, and multiple portions of musk deer gland tissue of about 2 cm*3 cm in size are separated and taken out, and temporarily placed in a 15 ml centrifuge tube containing 1% penicillin-streptomycin saline.

3. The method for separating and culturing different cell types of the musk gland according to claim 1, characterized in that: In the step six, the mixture is mixed by pipetting and then placed in a cell culture incubator for digestion for 8 to 15 minutes, then taken out and transferred to a 15 ml centrifuge tube by pipetting, and immediately stopped by adding 5 ml of DMEM / F12 containing 10% serum. The mixture is mixed by pipetting and then centrifuged at 1500r for 3 minutes, washed once with the stop solution, the supernatant is removed, 2 ml of complete culture medium is added, the mixture is mixed by pipetting, and the mixture is inoculated into a 35 mm culture dish, and the dish is transferred to a cell culture incubator with culture conditions of 37°C, 5% CO2 and 95% humid air for culture.

4. The method for separating and culturing different cell types of the forest musk gland according to claim 1, characterized in that: In step seven, the original culture medium is sucked out with a pipette, 2 ml of complete culture medium is added, and the culture is continued, and observation is made every 48 hours until the primary cells grow by more than 60%, and then the cells are passaged and expanded.

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