Separation method of yak sertoli cells

Abstract

The invention discloses a yak sertoli cell separation method which comprises the steps of yak testis disinfection, yak testis tissue separation, cell digestion, cell purification and the like, so that the whole process of the yak sertoli cell separation method is realized. The separation method of the yak sertoli cells is simple and convenient to operate, the separated yak sertoli cells are relatively high in purity, other purification measures are not needed, the subsequent culture stability is good, and the separation efficiency is high; meanwhile, the aspects of cell pollution, separation purification and identification, separation method cost and the like can be effectively controlled, and popularization and application are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and particularly relates to a method for isolating yak testicular Sertoli cells. Background Art

[0002] The testis is the most important reproductive organ of male animals, and Sertoli cells in the testis are an important component of seminiferous tubules. First of all, the number of Sertoli cells is one of the reasons determining the morphological size and normal function of the testis. Secondly, Sertoli cells are also the core functional cells for the formation of the blood-testis barrier (BTB). The tight junctions, adherens junctions and gap junctions established between adjacent Sertoli cells are the main structural basis for constituting the blood-testis barrier. During the normal spermatogenesis process of male mammals, Sertoli cells promote the disintegration of apoptotic germ cells through adhesion and phagocytosis to ensure the normal progress of spermatogenesis. Finally, Sertoli cells secrete many growth factors, including androgen-binding protein (ABP), transferrin (Tf), inhibin (INH), etc., to create a good microcirculation environment for the development of germ cells.

[0003] In recent years, many researchers have started to explore the application of Sertoli cells. Testicular Sertoli cells can promote cell growth. For example, various nutritional factors and growth factors secreted by them may affect the survival of dopaminergic neurons through different growth factor receptors expressed at different stages of dopaminergic neuron development. In addition, testicular Sertoli cells also have an immunosuppressive effect. Testicular Sertoli cells can kill activated lymphocytes in vitro. In addition, testicular Sertoli cells are also applied in the exploration of organ transplantation and the treatment of Parkinson's disease. All these indicate that the functions of testicular Sertoli cells are very extensive and have broad application prospects. Their specific action mechanisms and more extensive applications remain to be further developed.

[0004] In the prior art, the isolation of Sertoli cells often faces problems such as low purity, high contamination rate, and complex operation. In particular, there is a lack of effective solutions for plateau species such as yaks. Traditional methods using ordinary culture media (such as DMEM + 10% FBS) have defects such as slow cell growth and many cell debris, and the combination of digestive enzymes and purification steps have not been optimized. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for isolating yak testicular Sertoli cells to solve the problem that there is a lack of a suitable method for isolating yak testicular Sertoli cells in the prior art.

[0006] The above technical purpose of the present invention is achieved through the following technical solutions: A method for isolating yak testicular Sertoli cells, comprising the following steps:

[0007] Step S1 Tissue isolation:

[0008] A. Take the testicles of yaks aged 2 - 3 years from the slaughterhouse, cut off the excess fat, and wash and disinfect them.

[0009] B. Under the laminar flow hood, peel off the tunica vaginalis and tunica albuginea of the testicles, and then conduct the second washing and disinfection.

[0010] C. Cut a tissue block of about 2 cm³ from the parenchyma part into a centrifuge tube, cut the tissue block into minced pieces with an ophthalmic scissors, and transfer it to a 50 ml centrifuge tube, about 8 ml of tissue minced per tube.

[0011] Step S2 Cell digestion:

[0012] A. Add 5 times the volume of Digestive Enzyme I, digest in a 37 °C water bath for 40 min, shake and mix well every 3 min to disperse the seminiferous tubules in the tissue block.

[0013] B. Let the centrifuge tube stand, aspirate the upper digestive fluid, add 0.25% Trypsin - EDTA to 25 ml, digest in a 37 °C water bath for 10 min, shake and mix well every 3 min.

[0014] C. Add TM4 cell - specific medium to 50 ml to terminate the digestion. Filter the terminated digestive tissue fluid through a disposable cell strainer with a specification of 100 μm (100 μm) respectively, transfer the filtrate to a 1.5 ml centrifuge tube, and conduct a centrifugation operation at a centrifuge speed of 4000 revolutions per minute for 8 minutes, then discard the supernatant.

[0015] D. Add 1 ml of TM4 cell - specific medium to each tube to resuspend the cells, then inoculate them into a T25 culture flask (4 ml culture system), and place them in a 37 °C constant temperature incubator.

[0016] Step S3 Cell culture:

[0017] After culturing for 7 h, perform the first medium change using the differential adhesion method, and then change the medium every 24 h, and take pictures to record the cell status. When the cells grow to more than 80%, conduct cell passage.

[0018] Preferably, in the tissue separation step, the operations of the two washings and disinfections are as follows: Wash the testicular tissue 2 - 3 times with pre - warmed (37 °C) PBS + 1% double antibody, and soak it in 75% alcohol for 10 min; Wash the testicular tissue 2 - 3 times with PBS + 1% double antibody, and soak it in 75% alcohol for 2 min.

[0019] Preferably, in the cell digestion step, the ratio of Digestive Enzyme I is 1 mg / ml Collagenase IV + 50 μg / ml DNAse I.

[0020] Preferably, in the cell digestion step, the formulation of the TM4 cell-specific medium is as follows: based on DMEM / F12 as the basal medium, supplemented with 5% horse serum, 2.5% fetal bovine serum, and 1% penicillin-streptomycin solution.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] (1) Selection of the medium: The use of the TM4 cell-specific medium is an innovative point of this method. Different media may have different effects on cell growth and maintenance. Cells obtained by separating and culturing using a common medium (DMEM medium, 10% FBS, 1% double antibody) in the past had a slower growth rate, probably because some growth factors suitable for supporting cell growth were added to the TM4 medium.

[0023] (2) Two-step enzyme digestion method: Although the two-step enzyme digestion method has also been mentioned in other literatures, the specific enzyme types and concentrations may be different, which may affect the cell separation efficiency. Adding DNase I to the tissue homogenate can reduce DNA suspension fragments and cell clumps, making it easier for cells to be released. In the past attempts, 50 μg / ml DNAseI was not used, and it was not easy to filter out the cell suspension during the cell filtration step, resulting in less cell suspension and fewer cells.

[0024] (3) Detailed operation steps: Detailed experimental steps are provided, including digestion time, temperature, and centrifugation conditions, which helps other researchers to replicate the experiment.

[0025] In summary, the innovation of this method lies in that it provides a detailed and possibly specially optimized experimental process and conditions for the isolation and culture of yak testicular Sertoli cells, which is more conducive to operation. Description of the Drawings

[0026] Figure 1 It is the morphological diagram of primary yak testicular Sertoli cells obtained by separating and culturing with TM4 medium and DNAseI. To better observe the cell morphological differences, attached Figure 1 Color drawings are used;

[0027] Figure 2 It is the morphological diagram of F1-generation yak testicular Sertoli cells obtained by culturing after cryopreservation. To better observe the cell resuscitation and culture effect after cryopreservation, attached Figure 2 Color drawings are used;

[0028] Figure 3 It is the diagram of identifying yak testicular Sertoli cells with specific antibodies WT1 and SOX9 for the Sertoli cells separated using TM4 medium and DNAseI. To better observe the cell identification effect, attached Figure 3 Color drawings are used;

[0029] Figure 4 The morphological diagram of primary yak testicular Sertoli cells obtained using a normal culture medium without adding DNAseI. For better observation of cell morphological differences, a Figure 4 color attached drawing is adopted;

[0030] Figure 5 The morphological diagram of primary yak testicular Sertoli cells obtained using a normal culture medium and DNAseI. For better observation of cell differences, a Figure 5 color attached drawing is adopted. Specific embodiments

[0031] The present invention will be specifically described below in combination with specific embodiments and examples, and the advantages and various effects of the present invention will be presented more clearly therefrom. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention rather than limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of conflict, this specification shall prevail.

[0033] Unless otherwise specifically stated, various raw materials, reagents, instruments, and equipment used in the present invention can be obtained through market purchase or can be prepared by existing methods.

[0034] The following is a further description of the present invention in combination with the attached Figures 1-5 drawings.

[0035] Example: A method for isolating yak testicular Sertoli cells, comprising the following steps:

[0036] S1. Tissue separation:

[0037] A. Briefly cut off the excess fat from the testicles of 2 - 3-year-old yaks taken from the slaughterhouse, and place them in preheated (37°C) PBS + 1% double antibody to wash the testicular tissue 3 times, then bring them back to the laboratory;

[0038] B. Wash the testicular tissue with preheated (37°C) PBS + 1% double antibody 2 - 3 times, and soak it in 75% alcohol for 10 min;

[0039] C. After peeling off the tunica vaginalis and tunica albuginea of the testicles in a laminar flow hood, wash the testicular tissue with PBS + 1% double antibody 2 - 3 times, and soak it in 75% alcohol for 2 min;

[0040] D. Cut a tissue block of about 2 cm³ of the substantial part into a centrifuge tube, use an ophthalmic scissors to cut the tissue block into minced pieces, and transfer it to a 50 ml centrifuge tube, about 8 ml of tissue minced per tube.

[0041] S2. Cell digestion:

[0042] A. Add 5 times the volume of digestive enzyme Ⅰ (1 mg / ml collagenase Ⅳ + 50 μg / ml DNAse Ⅰ), digest in a 37°C water bath for 40 min, shake and mix well every 3 min to disperse the seminiferous tubules in the tissue block;

[0043] B. Let the centrifuge tube stand, aspirate the upper digestive fluid (try to aspirate it as clean as possible), add 0.25% trypsin-EDTA to 25 ml, digest in a 37°C water bath for 10 min, shake and mix well every 3 min;

[0044] C. Add TM4 cell-specific medium to 50 ml to terminate digestion. Filter the terminated digestion tissue fluid through a disposable cell strainer (100 μm) respectively, transfer the filtrate to a 1.5 ml centrifuge tube, centrifuge at 4000 rpm for 8 min, and discard the supernatant;

[0045] D. Add 1 ml of TM4 cell-specific medium to each tube to resuspend the cells, then transfer them to a T25 culture flask (4 ml culture system), and place them in a 37°C constant temperature incubator.

[0046] S3. Cell culture:

[0047] After culturing for 7 h, perform the first medium change using the differential adhesion method, then change the medium every 24 h, and take pictures to record the cell status. When the cells grow to more than 80%, perform cell passage.

[0048] S4. Cell identification:

[0049] A. Inoculate yak testicular Sertoli cells into a 12-well plate. When the cell confluence rate reaches 70%, discard the cell culture medium, wash with PBS 1 - 2 times, 5 min each time;

[0050] B. Add 4% paraformaldehyde fixative to the cell culture plate, fix at room temperature for 30 min, wash with PBS 3 times, 5 min each time;

[0051] C. Add 500 μL of 0.1% Triton X-100 (prepared with PBS) to each well, incubate at room temperature for 20 min, wash with PBS 3 times, 5 min each time;

[0052] D. Prepare 5% bovine serum albumin (BSA) with PBS, block at room temperature for 1.5 h, and discard;

[0053] E. Dilute Sox9 and WT1 primary antibodies with 5% BSA at a ratio of 1:200 and incubate overnight at 4°C; recover the primary antibodies, wash with PBS 3 times, 5 minutes each time;

[0054] F. Dilute the secondary antibody at a ratio of 1:500, incubate in the dark at room temperature for 1.5 h, wash with PBS 3 times, 5 minutes each time;

[0055] G. Add an appropriate amount of DAPI for nuclear staining for 30 min, wash with PBS 3 times, 5 minutes each time, and then observe and photograph under an inverted fluorescence microscope.

[0056] Figure 1 Figure of the morphology of primary yak testicular Sertoli cells obtained by separating and culturing with TM4 medium and DNAseI; Figure 2 Figure of the morphology of F1 generation yak testicular Sertoli cells obtained by culturing after cryopreservation; Figure 3 Figure for identifying yak testicular Sertoli cells by specific antibodies WT1 and SOX9 for the Sertoli cells separated using TM4 medium and DNAseⅠ; Figure 4 Figure of the morphology of primary yak testicular Sertoli cells obtained using ordinary medium without adding DNAseI; Figure 5 Figure of the morphology of primary yak testicular Sertoli cells obtained using ordinary medium and DNAseI.

[0057] As Figure 1 shown, for the cells separated using TM4 and DNAseI, the background is clean, the cell quantity is large, and the growth rate is fast.

[0058] As Figure 4 shown, without adding DNAseI, the cell quantity obtained after 7 h is small. The reason is that during filtration, the cells were not fully released, resulting in a small cell suspension; when cultured to 90 h, there are many cell debris and the background is dirty.

[0059] As Figure 5 shown, when using ordinary medium plus DNAseI, the cell quantity obtained after 7 h is significantly more than Figure 4 that, but the cell growth is relatively slow, probably because some growth factors suitable for the growth of Sertoli cells are added in TM4 medium.

[0060] As Figure 3 shown, for the cells separated using TM4 medium and DNAseI, the cell purity is high.

[0061] Finally, it should also be noted that the terms "comprising", "including" or any other variant are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus comprising a series of elements not only includes those elements but also other elements not expressly listed, or elements inherent to such process, method, article or apparatus. Although the preferred embodiments of the present invention have been described, those skilled in the art can make additional changes and modifications to these embodiments once they learn the basic creative concept. Therefore, the appended claims are intended to be construed as including the preferred embodiments as well as all changes and modifications falling within the scope of the present invention.

[0062] The above-described embodiments merely represent the specific implementation manners of the present application. The description is relatively specific and detailed, but it should not be construed as a limitation to the protection scope of the present application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the technical solution of the present application, several variations and improvements can still be made, and these all fall within the protection scope of the present application.

Claims

1. A method for isolating yak testicular Sertoli cells, characterized in that, It includes the following steps: S1. Tissue separation: A. Take the testicles of yaks aged 2 - 3 years old from the slaughterhouse, cut off the excess fat, and perform cleaning and disinfection; B. Peel off the tunica vaginalis and tunica albuginea of the testicles on the ultra-clean workbench, and then perform a second cleaning and disinfection; C. Cut off a tissue block of about 2 cm in substantial part and transfer it to a centrifuge tube. Use an ophthalmic scissors to cut the tissue block into minced pieces and transfer them to a 50-ml centrifuge tube, with about 8 ml of tissue mince in each tube. 3 Transfer the tissue block to a centrifuge tube. Use an ophthalmic scissors to cut the tissue block into minced pieces and transfer them to a 50-ml centrifuge tube, with about 8 ml of tissue mince in each tube. S2. Cell digestion: A. Add 5 times the volume of digestive enzyme I, digest in a 37°C water bath for 40 min, shake and mix well every 3 min to disperse the seminiferous tubules in the tissue mass; B. Let the centrifuge tube stand, suck away the upper digestive fluid, add 0.25% trypsin - EDTA to 25 ml, digest in a 37°C water bath for 10 min, shake and mix well every 3 min; C. Add TM4 cell - specific medium to 50 ml to terminate digestion. Filter the terminated digestive tissue fluid through a disposable cell strainer with a specification of 100 μm (100 μm) respectively, transfer the filtrate to a 1.5 ml centrifuge tube, and perform centrifugation at a centrifuge speed of 4000 revolutions per minute for 8 minutes, and discard the supernatant; D. Add 1 ml of TM4 cell - specific medium to each tube to resuspend the cells, then inoculate them into a T25 culture flask (4 ml culture system), and place them in a 37°C constant - temperature incubator. S3. Cell culture: Perform the first medium change using the differential adhesion method 7 h after culturing, then perform medium changes every 24 h, and take pictures to record the cell status. When the cells grow to more than 80%, perform cell passage.

2. The isolation method of yak testicular Sertoli cells according to claim 1, characterized in that, In the tissue separation step, the operations of the two cleanings and disinfections are as follows: Wash the testicular tissue 2 - 3 times with pre - warmed PBS + 1% double antibody at 37°C, and soak it in 75% alcohol for 10 min; Wash the testicular tissue 2 - 3 times with PBS + 1% double antibody, and soak it in 75% alcohol for 2 min.

3. The isolation method of yak testicular Sertoli cells according to claim 1, characterized in that, In the cell digestion step, the ratio of digestive enzyme I is 1 mg / ml collagenase IV and 50 μg / ml DNAseI.

4. The method for isolating yak testicular Sertoli cells according to claim 1, wherein In the cell digestion step, the ratio of the TM4 cell - specific medium is: Based on DMEM / F12 as the basal medium, add 5% horse serum, 2.5% fetal bovine serum, and 1% penicillin - streptomycin solution.

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