CN115948326A - Method for synchronously separating porcine testicular supporting cells and testicular interstitial cells - Google Patents

Abstract

The invention discloses a method for synchronously separating porcine testicular supporting cells and testicular interstitial cells, which comprises the steps of S1 sterilizing pig testicles, S2 separating testicular tissues, S3 carrying out first enzyme digestion, S4 carrying out first physical separation, S5 carrying out second enzyme digestion, S6 carrying out second physical separation, S7 identifying cells and the like, thereby realizing the whole process of the method for synchronously separating the porcine testicular supporting cells and the testicular interstitial cells. The synchronous separation method of the porcine testicular sertoli cells and the testicular interstitial cells is simple and convenient to operate, the uniformity of the separated porcine testicular sertoli cells and testicular interstitial cells is high, the subsequent culture stability is good without other purification measures, and the separation efficiency is high; meanwhile, the method can effectively control the aspects of cell pollution, separation, purification and identification, the quantity and quantity of cytoplasm, the cost of the separation method and the like, and is favorable for popularization and application.

Description

A method for synchronous separation of Sertoli cells and Leydig cells of pig testis

technical field

The invention relates to the technical field of cell separation and culture, in particular to a method for synchronous separation of pig testicular Sertoli cells and testicular interstitial cells.

Background technique

Cells are the basic structural and functional units of animal organisms. Isolation of cells is an essential step for research in all related biological fields. Likewise, cells determine the fate of all research fields. With the advancement of technology and the development of all related fields, mastering diverse cell isolation techniques, reducing labor costs, labor time, reagent costs and increasing automation is of paramount importance.

Cells are separated from testicular tissue by a variety of separation methods (enzymatic digestion, mechanical method, combination of enzyme digestion and mechanical method, some by enzyme digestion and sieving, some by enzyme digestion and gradient solution). Enzymes, concentrations, sieve sizes, and digestion and processing times vary among the various separation methods. After years of research on the isolation and culture of Sertoli cells and stromal cells related to males, we have now been able to isolate Sertoli cells and stromal cells with a uniformity of more than 90%.

The culture of Sertoli cells and Leydig cells in vitro has become an important cell model for the study of testis function and reproductive toxicity. The technique of isolating two kinds of germ cells from pig testes at the same time has not been reported yet. Most of the research reports have to go through hypotonic treatment method Tris-HCL, differential attachment method, digestion method, repeated aspiration of germ cells, fluorescence activated cell sorting, density gradient centrifugation, starvation method of discontinuous gradient exchange of culture medium, etc. However, there are still problems in terms of cell contamination, separation and purification, identification, separation efficiency, cytoplasmic quantity and quantity, cost of separation method, and ease of operation.

Contents of the invention

The object of the present invention is to provide a method for synchronous separation of Sertoli cells and Leydig cells to solve the above defects.

In order to achieve the above object, the present invention provides the following technical solutions:

A method for synchronous separation of pig Sertoli cells and Leydig cells, comprising the following steps:

S1, disinfection of pig testis:

A. Put the testes of immature pigs taken from the farm into a 50mL centrifuge tube, and wash each testis with 30mL of povidone iodine upside down; then wash the testes with 30mL of 75% ethanol and shake for 1-2min; P/S PBS solution of penicillin and streptomycin was used to wash the testes and shake for 1-2min;

B. Repeat the above steps three times;

S2. Separation of testicular tissue:

Use large tweezers to fix the sterilized testis in a cell culture dish, and surgically remove the attached seminal vesicles, then repeat step A in S1 once for cleaning and disinfection; then use small tweezers to fix the testis in a 100mm culture dish, and place it horizontally on the buffy membrane Cut open, peel off the tunica albuginea with two tweezers, and then wet the testicular tissue with PBS containing 2% double antibody; put each 0.2-0.5g of testicular tissue in a 60mm Petri dish, and add a total of 1mL of tissue digestion solution a, use ophthalmic scissors to fully organize the testis into a homogenate;

S3, the first enzyme digestion:

Add 4mL of tissue digestion solution a to each 60mm Petri dish in step S2, mix the tissue homogenate and tissue digestion solution a thoroughly with a Pasteur pipette, and transfer to a 15mL centrifuge tube; at 34°C, Carry out the first digestion under the condition of 210g centrifugation, and time it for 45 minutes; transfer the centrifuged supernatant to centrifuge tube 1, and carry out the next step for the supernatant and the lower tissue respectively;

S4, the first physical separation:

A. First, use 100-mesh and 200-mesh stainless steel sieves to filter the cells in the supernatant of centrifuge tube 1 in step S3 in turn; secondly, use 10% FBS-containing DMEM/F-12 solution to collect 100-mesh and 200-mesh cells The cells on the mesh screen were placed in a 15mL centrifuge tube, and rinsed with DMEM/F-12 solution for 3 times; then, centrifuged at 34°C and 210g for 15min, and the collected cell population contained Leydig cells; Finally, use DMEM/F-12 solution containing 10% FBS to resuspend the above-mentioned cell population containing Leydig cells, insert it into a culture dish, and culture it after marking;

B. First, place 40 mesh, 100 mesh, 200, and 400 mesh stainless steel sieves in sequence, and sieve the lower tissue in step S3 in sequence; secondly, use 10% FBS-containing DMEM/F-12 solution for reaction To wash, collect the cells and tissues retained on the 200-mesh and 400-mesh sieves into 60mm culture dishes, and finally, collect them into 15mL centrifuge tube 2 and centrifuge to remove the supernatant;

S5, the second enzyme digestion:

Add tissue digestion solution b to each centrifuge tube 2 where the supernatant was removed in step B in S4, and digest while centrifuging at 34°C and 300g for 1 hour; Immediately add DMEM medium containing FBS to resuspend cells to terminate digestion;

S6, the second physical separation:

Collect the resuspended cells in step S5, pass through a 500-mesh sieve, and wash back with DMEM/F-12 solution containing 10% FBS. The collected cell population retained on the 500-mesh sieve contains Sertoli cells; Then rinse the cells with medium for 3 times, resuspend the above-mentioned cell population containing Sertoli cells with 10% FBS-containing DMEM/F-12 solution, put them into a petri dish, and culture after marking;

S7. Cell identification:

The morphology and structure of the cell population containing Leydig cells cultured in a petri dish in step A of S4 and the cell population containing Sertoli cells cultured in a petri dish in step S6 were both morphologically and structurally identified. The entire process of the method for the simultaneous isolation of Sertoli cells and Leydig cells.

Preferably, the proportion of the tissue digestion solution a is: 6 mg of P-type collagenase, 5.5 mg of Dispase collagenase, 5 mL of HBSS+phenol red, and 5 mL of PBS buffer.

Preferably, the proportioning of the tissue digestion liquid b is: 0.25% Trypsin, 45.00mL (5mL) and DNase I, 5.00mg (1.1mg/mL); DMEM, 9mL (90%); FBS, 1mL (10% ).

Preferably, the DMEM/F-12 solution of 10% FBS is the DMEM/F-12 medium containing 10% FBS, wherein by volume percentage, DMEM/F-12 medium accounts for 90%, FBS accounts for 10%.

Preferably, the DMEM/F-12 medium is a 1:1 liquid medium, the manufacturer of which is Cytiva, and the product number is SH30023.01.

Preferably, the methods for identification of cell morphology and structure in step S7 include microscopy identification, HE staining, Oil Red O staining, and immunofluorescence staining.

Preferably, the specific steps of the HE staining method are: first, take the cells to be stained, remove the culture medium and rinse with 1000 μL of PBS buffer; then use 95%, 85%, and 75% ethanol to treat each time, Second, soak in distilled water for 2 minutes, stain with hematoxylin staining solution for 15 minutes, and wash with distilled water to remove floating color; then, differentiate with differentiation solution for 3 minutes; rinse with tap water twice, each time for 2 minutes; finally, put eosin staining solution for 1 minute, distilled water Wash for 3 seconds and dehydrate quickly.

Preferably, the specific steps of the Oil Red O staining method are as follows: first, remove the cell culture medium, wash twice with PBS, stain the fixative with Oil Red O for 25 min; then discard the fixative, and wash twice with distilled water; Then, add 60% isopropanol to soak for 5 minutes, then discard 60% isopropanol, add the newly prepared Oil Red O staining solution and soak for 15 minutes, then discard the staining solution, wash with water 4 times until there is no excess dyeing solution ; Finally, add Mayer's hematoxylin staining solution, counterstain the cell nuclei for 2 minutes, then discard the staining solution and wash 4 times with water; then add Oil Red O buffer for 1 minute, discard, and add distilled water to cover the cells and observe under an inverted microscope.

Preferably, the specific steps of the immunofluorescence staining method are as follows: first, take the cells to be stained, remove the medium, and rinse once with 500 μL of DPBS+0.3% PVP buffer solution; add 4% PFA pre-cooled at 4°C 500 μL of fixative, after 15 minutes of fixation, discard the fixative; then, add 500 μL of DPBS+0.3% PVP buffer to rinse the cells three times, each time for 5 minutes; then add 500 μL of 0.5% Triton permeabilization solution, keep at room temperature for 30 minutes; then add 500 μL of DPBS + 0.3% PVP buffer to rinse the cells three times, each time for 5 minutes; then, add 500 μL 2% BSA, block at room temperature for 2 hours; remove the blocking solution, dilute the primary antibody with 2% BSA, and add the diluted primary antibody to the cell culture dish After incubation, absorb the antibody and add it again, incubate at 4°C for 12-14 hours; finally, aspirate the primary antibody, add 500 μL DPBS buffer to rinse for 3×15 min; add 200 μL secondary antibody working solution, and incubate at room temperature for 1 hour; remove the secondary antibody, the cells Rinse with DPBS buffer for 3×15min, add 200μL DAPI working solution, incubate at room temperature in the dark for 10min, observe and photograph the test results under an inverted fluorescent microscope

The beneficial effects of the present invention are:

The present invention is a synchronous separation method of pig testicular Sertoli cells and testicular interstitial cells, using two types of commercial enzyme preparations containing collagenase P and Collagenase/Dispase for the first enzyme digestion, and then physically separating to obtain testicular interstitial cells ; Then carry out the second enzymatic digestion with two types of commercial enzyme preparations containing trypsin and DNase I, and physically separate the Sertoli cells after digestion. The process of the invention is simple and efficient, and can realize synchronous separation of pig testicular Sertoli cells and testicular interstitial cells, and the separated cells are relatively uniform and stable in subsequent culture. Moreover, in the usual process of separating and culturing interstitial cells, many levels of germ cells will remain, so Tris-HCl solution hypotonic treatment is usually used to remove the remaining germ cells, and the method of the present invention is used to remove the remaining germ cells. When separating, a relatively pure mesenchymal cell population can be obtained without the need for hypotonic treatment with Tris-HCl solution. The invention is a synchronous separation method of pig testicular Sertoli cells and testicular interstitial cells, which is simple and convenient to operate, and the isolated pig testicular Sertoli cells and testicular interstitial cells have high uniformity, good follow-up culture stability, and high separation efficiency; at the same time It can be effectively controlled in terms of cell contamination, separation, purification and identification, cytoplasmic quantity and quantity, and cost of the separation method, which is conducive to industrial promotion and application.

Description of drawings

Fig. 1: the process flow chart of the inventive method among the embodiment 1;

Fig. 2: the process flow sheet of conventional method (control group) in embodiment 2;

Fig. 3: the conventional method (control group) in embodiment 2 and the pig testicular sertoli cell that the method of the present invention isolates in embodiment 1, the morphological observation contrast figure under the microscope of testicular interstitial cell;

Fig. 4: the comparison chart of the residual spermatogonia processed by the conventional method (control group) in Example 2 and the method of the present invention in Example 1;

Fig. 5: the morphological observation figure of the porcine Leydig cells separated by the method of the present invention in Example 1 after passage;

Fig. 6: the morphological observation figure in the primary culture of pig Sertoli cells isolated by the method of the present invention in Example 1;

Fig. 7: Diagram of the oil red O staining identification results of the pig testis primary Leydig cells and Sertoli cells isolated by the method of the present invention in Example 1;

Figure 8: HE staining identification results of Sertoli cells and Leydig cells in the primary culture stage isolated by the method of the present invention in Example 1;

Figure 9: a diagram of the results of immunofluorescence staining identification of specific markers of pig testicular Sertoli cells isolated by the method of the present invention in Example 1;

Fig. 10 is a diagram of identification results of immunofluorescent staining of specific markers of porcine Leydig cells isolated by the method of the present invention in Example 1.

Detailed ways

In order to facilitate the understanding of those skilled in the art, the present invention will be further described below in conjunction with specific embodiments.

One, embodiment 1:

As shown in Figure 1, a method for the synchronous isolation of pig Sertoli cells and Leydig cells, comprising the following steps:

S1, disinfection of pig testis:

A. Put the testes of immature pigs taken from the farm into a 50mL centrifuge tube, and wash each testis with 30mL of povidone iodine upside down; then wash the testes with 30mL of 75% ethanol and shake for 1-2min; P/S PBS solution of penicillin and streptomycin was used to wash the testes and shake for 1-2min;

B. Repeat the above steps three times;

S2. Separation of testicular tissue:

Use large tweezers to fix the sterilized testis in a cell culture dish, and surgically remove the attached seminal vesicles, then repeat step A in S1 once for cleaning and disinfection; then use small tweezers to fix the testis in a 100mm culture dish, and place it horizontally on the buffy membrane Cut open, peel off the tunica albuginea with two tweezers, and then wet the testicular tissue with PBS containing 2% double antibody; put each 0.2-0.5g of testicular tissue in a 60mm Petri dish, and add a total of 1mL of tissue digestion solution a, use ophthalmic scissors to fully organize the testis into a homogenate;

S3, the first enzyme digestion:

Add 4mL of tissue digestion solution a to each 60mm Petri dish in step S2, mix the tissue homogenate and tissue digestion solution a thoroughly with a Pasteur pipette, and transfer to a 15mL centrifuge tube; at 34°C, Carry out the first digestion under the condition of 210g centrifugation, and time it for 45 minutes; transfer the centrifuged supernatant to centrifuge tube 1, and carry out the next step for the supernatant and the lower tissue respectively;

S4, the first physical separation:

A. First, use 100-mesh and 200-mesh stainless steel sieves to filter the cells in the supernatant of centrifuge tube 1 in step S3 in turn; secondly, use 10% FBS-containing DMEM/F-12 solution to collect 100-mesh and 200-mesh cells The cells on the mesh screen were placed in a 15mL centrifuge tube, and rinsed with DMEM/F-12 solution for 3 times; then, centrifuged at 34°C and 210g for 15min, and the collected cell population contained Leydig cells; Finally, use DMEM/F-12 solution containing 10% FBS to resuspend the above-mentioned cell population containing Leydig cells, insert it into a culture dish, and culture it after marking;

B. First, place 40 mesh, 100 mesh, 200, and 400 mesh stainless steel sieves in sequence, and sieve the lower tissue in step S3 in sequence; secondly, use 10% FBS-containing DMEM/F-12 solution for reaction To wash, collect the cells and tissues retained on the 200-mesh and 400-mesh sieves into 60mm culture dishes, and finally, collect them into 15mL centrifuge tube 2 and centrifuge to remove the supernatant;

S5, the second enzyme digestion:

Add tissue digestion solution b to each centrifuge tube 2 where the supernatant was removed in step B in S4, and digest while centrifuging at 34°C and 300g for 1 hour; Immediately add DMEM medium containing FBS to resuspend cells to terminate digestion;

S6, the second physical separation:

Collect the suspended cells after resuspension in step S5, pass through a 500-mesh sieve, and wash back with DMEM/F-12 solution containing 10% FBS. The collected cell population on the 500-mesh sieve contains the testis Sertoli cells; then rinse the cells with culture medium for 3 times, resuspend the above-mentioned cell population containing Sertoli cells in DMEM/F-12 solution containing 10% FBS, insert them into a culture dish, and culture after marking;

S7. Cell identification:

The morphology and structure of the cell population containing Leydig cells cultured in a petri dish in step A of S4 and the cell population containing Sertoli cells cultured in a petri dish in step S6 were both morphologically and structurally identified. The entire process of the method for the simultaneous isolation of Sertoli cells and Leydig cells.

Among them, the proportion of tissue digestion solution a is: P-type collagenase 6 mg, Dispase collagenase 5.5 mg, HBSS+phenol red 5 mL, PBS buffer 5 mL. The ratio of tissue digestion liquid b is: 0.25% Trypsin, (5mL) and DNase I, 5.00mg (1.1mg/mL); DMEM, 9mL (90%); FBS, 1mL (10%).

Wherein, the DMEM/F-12 solution of 10% FBS is the DMEM/F-12 medium containing 10% FBS, wherein by volume percentage, DMEM/F-12 medium accounts for 90%, and FBS accounts for 10%. DMEM/F-12 medium is 1:1 liquid medium=.

Wherein, the methods for identification of cell morphology and structure in step S7 include microscopy identification, HE staining, Oil Red O staining, and immunofluorescence staining.

Two, embodiment 2:

For the conventional method control group.

A conventional synchronous separation method of Sertoli cells and Leydig cells of pig testis is shown in Figure 2. The separation method can be briefly described as follows: the testicular tissue is normally cut into pieces, fully digested with type IV collagenase and then placed in a centrifuge. Centrifuge in the tube, and the Sertoli cells sink to the bottom of the tube due to their larger specific gravity. Therefore, it is necessary to repeatedly rinse and suspend the upper layer of cells by slight shaking of PBS, and collect the upper layer of cell suspension in a new centrifuge tube for the separation of interstitial cells, and the lower layer of sediment after repeated rinsing is Sertoli cells. After the lower layer was fully digested with 0.25% trypsin, the only sieve (400 mesh) was carried out, and the cells were collected by centrifugation in the filtrate as Sertoli cells.

Three, embodiment 1 and embodiment 2 different methods separate the porcine testis Sertoli cell, the morphological observation of testicular stromal cell under the microscope

Using 2-day-old Anqing Liubai pig testis tissue as raw material, and using the conventional method in Example 2 (control group) and the method of the present invention in Example 1 to isolate Sertoli cells and Leydig cells.

Fig. 3 is the comparison chart of microscopic morphological observation of pig Sertoli cells and Leydig cells separated by the conventional method (control group) in Example 2 and the method of the present invention in Example 1. Wherein, Fig. 3-A is the micromorphological figure (Scale bar=50 μm) of the Leydig cells isolated by the conventional method (control group) in Example 2 after culturing for 12 hours; Fig. 3-B is the micromorphological figure of the present invention in Example 1 The micromorphological figure (Scale bar=100 μ m) of the Leydig cells isolated by the method after culturing for 12 hours; Micromorphology (Scalebar=50 μm); FIG. 3-D is the micromorphological diagram (Scale bar=100 μm) of the pig Sertoli cells isolated by the method of the present invention in Example 1 after 14 hours of culture.

As shown in Figure 3, the results show that through 12h of adherent culture, most of the Leydig cells separated by the method of the present invention in Example 1 have just completed attachment, showing a circular outline (see Figure 3-B), while in Example 1 The Leydig cells in the 2 routine methods (control group) showed various shapes (see Figure 3-A). Similarly, after 14 hours of adherent culture, the Sertoli cells isolated by the method of the present invention in Example 1 also showed a relatively uniform colony-like and fibroblast-like cell population (see Figure 3-D), while in Example 2 Sertoli cell populations isolated by the conventional method (control group) were more mixed (see Figure 3-C). This shows that the homogeneity of the porcine Sertoli cells and Leydig cells isolated by the method of the invention is higher. 4. Comparison of residual spermatogonia in primary Leydig cells separated by different methods in Example 1 and Example 2.

During the isolation of primary mesenchymal cells, the residual spermatogonia is very common, so a short-term immersion in Tris-HCl buffer is required to inactivate and remove the residual spermatogonia in the culture. In this regard, we cultured the cells obtained by the method of the present invention in Example 1 and the conventional method (control group) in Example 2 respectively through in vitro culture. When the cells reached a nearly continuous monolayer, they were observed under a microscope and photographed.

Fig. 4 is a comparison chart of residual spermatogonia treated by the conventional method (control group) in Example 2 and the method of the present invention in Example 1 (Scale bar=100 μm). As shown in the figure, the results show that when the primary pig Leydig cells isolated by the conventional method (control group) in Example 2 grow to a continuous monolayer, they present a fibroblast-like morphology and arrangement, and the cell surface is mixed with adherent cells. Insufficient cells are considered to be residual spermatogonia (see Figure 4-A); after soaking in Tris-HCl for 7 minutes, the cell contamination suspected to be spermatogonia can be removed, and a relatively uniform cell population can be obtained (see Figure 4 -B). Correspondingly, after culturing the cells obtained by the method of the present invention in Example 1, under a microscope, it can be seen that the cell populations are relatively uniform and there are no obvious residual spermatogonia (see Figure 4.C). Therefore, Tris-HCl soaking treatment is not required.

The above results show that the established method of the present invention in Example 1 may have more advantages in isolating and obtaining porcine Leydig cells.

Five, the morphological observation of the porcine Leydig cells separated by the method of the present invention in embodiment 1 after passage.

In order to further identify the cell types isolated by the method of the present invention in Example 1, the primary isolated cells need to be further expanded and cultured. During the expansion and culture process, the morphological changes of the mesenchymal cells during growth were observed day by day under a phase-contrast microscope.

Fig. 5 is the morphological observation diagram (Scale bar=200 μm) of the porcine Leydig cells separated by the method of the present invention in Example 1 after passage. As shown in FIG. 5 , the results showed that, similar to when the primary cells were isolated, the cells appeared in a state of just adherent at 12 hours after inoculation, with a round outline (see FIG. 5-A ). After 24 hours after inoculation, the cells appeared as long fibers and completed relatively firm attachment (see Figure 5-B). After subculture, the cells grew vigorously. When the culture time reached 2 days (see Figure 5-C) and 3 days (see Figure 5-D), the cell outlines were clear, the three-dimensional texture was strong, and the cytoplasm was more prominent near the nucleus. Visible when approaching a continuous monolayer after 3 days in culture, the cells have a high degree of homogeneity (see Figure 5-D).

6. Morphological observation in the primary culture of the pig Sertoli cells separated by the method of the present invention in Example 1.

Because of the Sertoli cells obtained by the conventional method (control group) in Example 2 and the method of the present invention in Example 1, there is no difference in morphology, and there is a relatively consistent change process in the growth process, so it is mainly for the Sertoli cells in Example 1. The cells isolated by the method of the invention were observed.

The changes in cell morphology and growth were observed day by day with a phase contrast microscope, and photographed and recorded. Fig. 6 is a morphological observation diagram of the primary culture of porcine Sertoli cells isolated by the method of the present invention in Example 1, wherein A: Scale bar = 100 μm, B: 200 μm, C: 50 μm, D: 200 μm. As shown in Figure 6, the results showed that Sertoli cells of pig testis adhered to the bottom of the dish in the form of colonies after 14 hours of attachment, the boundaries between cells were not clear, and the structure of the nucleus was not easily observed, but the cytoplasm contained a large number of highly refractive vacuoles , presumably it is a typical lipid droplet structure in Sertoli cells. After 2 days of in vitro culture, the cytoplasm of the colony-like cells gradually stretched outward, the intercellular space became clear, and the structure of the nucleus was easily observed. After 3 days of in vitro culture, the cells gradually separated and diffused, the lipid droplet structure became less, and the refractive properties became weaker.

Seven, the oil red O staining identification of pig Leydig cells and Sertoli cells separated by the method of the present invention in embodiment 1.

In order to further identify the cells separated by the method of the present invention in Example 1, we performed Oil Red O staining on the primary mesenchymal cells and supporting cells.

Oil red O staining method, the specific steps are: first, remove the cell culture medium, wash twice with PBS, add oil red O staining fixative for 25min; then discard the fixative, wash twice with distilled water; then, add 60% Immerse in isopropanol for 5 minutes, then discard 60% isopropanol, add the newly prepared Oil Red O staining solution and soak for 15 minutes, then discard the staining solution, wash with water 4 times until there is no excess dyeing solution; finally, add Mayer Hematoxylin staining solution, counterstain cell nuclei for 2 minutes, then discard the staining solution and wash 4 times with water; then add Oil Red O buffer for 1 minute, discard, and add distilled water to cover the cells and observe under an inverted microscope.

Fig. 7 is a picture of the oil red O staining identification results of the primary Leydig cells and Sertoli cells isolated by the method of the present invention in Example 1 (Scale bar = 50 μm). As shown in Figure 7, the results show that: the cytoplasm of the primary Leydig cells of the pig testis is relatively uniformly distributed with fine and tiny lipid droplet particles (see Figure 7-A, Figure 7-B), while the Sertoli cells are near the nucleus or Lipid droplets appeared in the cytoplasmic poles (see Figure 7-C, Figure 7-D). In contrast, the lipid droplets in primary Leydig cells of porcine testis are smaller and dense (see Figure 7-A, Figure 7-B); while the lipid droplets in Sertoli cells are relatively large and scattered, and there are more The diameter of the lipid droplets can be as small as the nucleus (see Figure 7-C, Figure 7-D). In addition, lipid droplets observed under a phase-contrast microscope appear brighter orange-red (see Figure 7-A, Figure 7-C); while under a non-phase-contrast microscope, lipid droplets appear dark red to blue ( See Figure 7-B, Figure 7-D).

8. HE staining identification of Sertoli cells and Leydig cells in the primary culture stage isolated by the method of the present invention in Example 1.

In order to further identify the cell types isolated by the method of the present invention in Example 1, we performed HE staining on Sertoli cells and Leydig cells in the primary culture stage.

HE staining method, the specific steps are as follows: first, take the cells to be stained, fully remove the medium and rinse with 1000 μL of PBS buffer; then use 95%, 85%, and 75% ethanol to treat each time, each time for 2 minutes; , soaked in distilled water for 2 minutes, stained with hematoxylin staining solution for 15 minutes, and washed with distilled water to remove floating color; then, differentiated with differentiation solution for 3 minutes; rinsed with tap water twice, each time for 2 minutes; finally, placed in eosin staining solution for 1 minute, washed with distilled water for 3 seconds, Get dehydrated quickly.

Fig. 8 is a diagram of HE staining and identification results of Sertoli cells and Leydig cells in the primary culture stage isolated by the method of the present invention in Example 1 (Scale bar = 50 μm). As shown in Figure 8, the results showed that the cytoplasm of the primary Sertoli cells of pig testis was extended and attached to the bottom of the dish, showing a polygonal shape, and there were multiple obvious nucleoli in the nucleus (see Figure 8-A). However, the primary mesenchymal cells were more round in shape, and there was no obvious multi-nucleolus phenomenon in the nucleus (see Figure 8-B).

9. Immunofluorescence staining identification of the specific markers of the pig testicular Sertoli cells and Leydig cells isolated by the method of the present invention in Example 1.

In order to further identify the isolated cells, immunofluorescent staining was carried out on the expected Sertoli cells and Leydig cells of porcine testis.

Immunofluorescence staining method, the specific steps are: first, take the cells to be stained, fully remove the medium, and rinse with 500 μL of DPBS + 0.3% PVP buffer; then add 500 μL of 4% PFA fixative solution pre-cooled at 4 °C After fixing for 15 minutes, discard the fixative; secondly, add 500 μL DPBS+0.3% PVP buffer solution to rinse the cells three times, each time for 5 minutes; then add 500 μL 0.5% Triton permeabilization solution, and keep at room temperature for 30 minutes; Rinse the cells with buffer three times, 5 min each time; then, add 500 μL 2% BSA, block at room temperature for 2 h; remove the blocking solution, dilute the primary antibody with 2% BSA, add the diluted primary antibody to the cell culture dish, incubate and aspirate Add the antibody again, incubate at 4°C for 12-14 hours; finally, aspirate the primary antibody, add 500 μL DPBS buffer to rinse 3×

15min; add 200μL of secondary antibody working solution, incubate at room temperature for 1h; remove secondary antibody, rinse cells with DPBS buffer for 3×15min, add 200μL of DAPI working solution, incubate at room temperature in the dark for 10min, observe and photograph the test results under an inverted fluorescence microscope .

Fig. 9 is a diagram showing the results of immunofluorescent staining identification of specific markers of the porcine Sertoli cells isolated by the method of the present invention in Example 1 (Scale bar = 250 μm).

Referring to previous studies, Sertoli cells of porcine SCs expressed specific functional proteins such as AMH, ABP, and FASL. We cultured SCs from the first passage (p1) after isolation and used them for the detection of the above proteins. Anti-Müllerian hormone (AMH) is a glycoprotein produced by immature Sertoli cells, which can induce the degeneration of Müllerian ducts and make the mesonephric ducts develop towards the male reproductive tract under the action of androgens. As a marker AMH belongs to the group of secreted protein markers and is also a marker of immature testicular function in humans. The expression of AMH in Sertoli cells is localized extracellularly. Androgen binding protein (ABP) is a glycoprotein (β-globulin) produced by Sertoli cells. ABP binds specifically to testosterone, dihydrotestosterone and 17β-estradiol. ABP maintains a high level of testosterone concentration in tubules and regulates FSH secretion through negative feedback. Like AMH, ABP is mainly expressed extracellularly, but is also present in the plasma membrane. ABP is highly expressed in Sertoli cells and brain at the organ level. The high expression of FASL localized on the cell membrane, cell membrane and nucleus. After routine fixation and antibody incubation and staining, AMH, ABP, FASL, and SCs immunofluorescence staining results showed that the isolated cells were consistent with the expression of SCs cell-specific markers.

Fig. 10 is a diagram showing the results of immunofluorescence staining identification of specific markers of porcine Leydig cells isolated by the method of the present invention in Example 1 (Scale bar = 250 μm).

According to previous studies, the surface of porcine Leydig cells LCs positively expressed HSD3B1, IGF1, INSL3 and other surface-specific antigens. We used HSD3B1, IGF1, INSL3 and other primary antibodies for immunofluorescent staining of LCs cells under in vitro culture conditions. HSDB3B1 is a protein marker highly expressed in endoplasmic reticulum, mitochondria and nucleus. HSDB3B1 is expressed in both fetal and adult mouse LCs. IGF1 is a hormone marker highly expressed in the plasma membrane and extracellular, and INSL3 is also a hormone marker. INSL3 is mainly highly expressed extracellularly and slightly expressed in the nucleus. During the experiment, we single-stained each primary antibody, and the results are shown in Figure 10. The results of LCs immunofluorescence staining showed that the isolated cells were consistent with the expression of LCs cell-specific markers.

In summary, the morphological characteristics and marker expression of the isolated Sertoli and mesenchymal cells were identified. The results were similar to the previous research reports, Sertoli cells were polygonal cells in morphology, and the nuclei contained a large number of nucleoli. Compared with the previously reported separation of supporting and mesenchymal cells, HE staining results showed better cell morphology and structure, indicating that the isolated cells had good cell viability. In addition, there are relatively abundant lipid droplet structures in the Sertoli cells separated by the method of the present invention, and the diameter of the lipid droplet can be close to the size of the nucleus. In addition, lipid droplets observed under a phase-contrast microscope appear brighter orange-red; while under a non-phase-contrast microscope, lipid droplets appear dark red to cyan in color. The fine lipid droplet structure observed in interstitial cells is also consistent with previous reports.

At the same time, in order to further identify the identity of the isolated cells, protein markers such as AMH, ABP, and FASL were detected. Among them, AMH is a secretory protein, a marker of immature Sertoli cells, and its content in cells is relatively low. ABP is a glycoprotein (β-globulin) produced by Sertoli cells that specifically binds to testosterone, dihydrotestosterone, and 17β-estradiol. ABP in the seminiferous tubules can help Sertoli cells maintain a high level of testosterone concentration, and regulate FSH secretion through negative feedback. ABP is also a secretory protein, but it also has a higher content in the cell membrane. Because of its higher expression in testicular tissue and brain, ABP can be used as a marker for testis and brain. Furthermore, ABP and AMH of Sertoli cells demonstrated the immature state of Sertoli cells. During testicular development, the immune privilege features in testicular tissue are critical to the spermatogenesis process, in which FASL plays a key role in supporting the maintenance of cellular immune privilege function. The expression level of FASL in the Sertoli cells isolated in this study is higher, which further proves that the obtained cells belong to Sertoli cells.

Moreover, since there are relatively few reports on the establishment of in vitro culture of porcine immature mesenchymal cells, in the present invention, markers of immature mesenchymal cells of other species were selected to study and judge the expression of porcine genes, including HSD3B1, IGF1, INSL3 . Among them, HSD3B1 is expressed in both fetal and adult mouse LCs, which can regulate the lipid metabolism of mouse Leydig cells and promote ketone synthesis. HSD3B1 protein is mainly distributed in endoplasmic reticulum, mitochondria and nucleus, which is consistent with the detection results of the present invention. IGF1, as a possible regulator of the activity of Leydig cells, can also be used as a marker of Leydig cells. In addition, as a secreted protein, INSL3 is mainly secreted outside the cell after expression, and also has a certain concentration in the nucleus. INSL3 plays a vital role in the process of testicular descent. The above markers collectively indicate that the cells obtained by the method of the present invention have typical characteristics of Leydig cells.

The present invention is a synchronous separation method of pig testicular Sertoli cells and testicular interstitial cells, which uses two types of commercial enzyme preparations containing collagenase P and Collagenase/Dispase to carry out the first enzyme digestion, and then physically separates to obtain testicular interstitial cells Cells; then a second enzymatic digestion with two types of commercial enzyme preparations containing trypsin and DNase I, and physical separation after digestion to obtain Sertoli cells. The process of the invention is simple and efficient, and can realize synchronous separation of pig testicular Sertoli cells and testicular interstitial cells, and the separated cells are relatively uniform and stable in subsequent culture without any purification measures. Moreover, in the usual process of separating and raising interstitial cells, there will be more spermatogenic cells at all levels remaining, so the Tris-HCl solution hypotonic treatment method is usually used to remove the remaining spermatogenic cells, and the method of the present invention is used to separate testicular interstitial cells. , a more uniform mesenchymal cell population can be obtained without the need for hypotonic treatment with Tris-HCl solution.

The invention is a synchronous separation method of pig testicular Sertoli cells and testicular interstitial cells, which is simple and convenient to operate, and the isolated pig testicular Sertoli cells and testicular interstitial cells have high uniformity, good follow-up culture stability, and high separation efficiency; at the same time It can be effectively controlled in terms of cell contamination, separation, purification and identification, cytoplasmic quantity and quantity, and cost of the separation method, which is conducive to industrial promotion and application.

The above is an exemplary description of the invention. Obviously, the specific implementation of the present invention is not limited by the above-mentioned method, as long as the method concept and technical solution of the present invention are used for this non-substantial improvement, or the concept of the invention is not improved. And technical solutions that are directly applied to other occasions are within the protection scope of the present invention.

Claims (9)

1. A method for synchronously separating porcine testicular sertoli cells and testicular interstitial cells is characterized by comprising the following steps:

s1, disinfecting pig testicles:

A. placing testis from immature pig from farm in 50mL centrifuge tube, and washing each testis by reversal shaking with 30mL iodophor; cleaning testis with 30mL 75% ethanol, and shaking for 1-2min; finally washing the testis with PBS solution containing 2% of P/S streptomycin, and shaking for 1-2min;

B. repeating the steps for three times;

s2, testis tissue separation:

fixing the disinfected testis in a cell culture dish by using a large forceps, removing the attached seminal vesicle by an operation, and repeating the step A in the step S1 for cleaning and disinfection; fixing testis in a 100mm culture dish with tweezers, horizontally cutting on the white membrane, stripping off the white membrane with two tweezers, and wetting testis tissue with 2% double-antibody-containing PBS; placing 0.2-0.5 g of testis tissue in each portion into 1 culture dish with 60mm, adding 1mL of tissue digestive juice a in total, and fully organizing testis into uniform pulp by using ophthalmic scissors;

s3, first enzyme digestion:

continuously and respectively adding 4mL of tissue digestive juice a into each culture dish with the thickness of 60mm in the step S2, fully and uniformly mixing the tissue homogenate with the tissue digestive juice a by using a Pasteur pipette, and transferring the mixture into a 15mL centrifuge tube; performing first digestion at 34 deg.C under centrifugation condition of 210g for 45min; transferring the centrifuged supernatant into a first centrifugal tube, and respectively carrying out next-step treatment on the supernatant and the lower-layer tissue;

s4, first physical separation:

A. firstly, filtering cells in supernatant of the first centrifugal tube in the step S3 by using 100-mesh and 200-mesh stainless steel sieves in sequence; next, cells on 100-mesh and 200-mesh sieves were collected in a 15mL centrifuge tube using a DMEM/F-12 solution containing 10% FBS, and centrifugally rinsed 3 times with a DMEM/F-12 medium solution; then, centrifuging for 15min at 34 ℃ under the condition of 210g centrifugation, wherein the collected cell population contains the leydig cells; finally, the cell population containing the leydig cells was resuspended in a 10% FBS-containing DMEM/F-12 solution, and the suspension was placed in a petri dish and cultured after labeling;

B. firstly, placing stainless steel sieves of 40 meshes, 100 meshes, 200 meshes and 400 meshes in sequence, and sieving the lower-layer tissues in the step S3 in sequence; secondly, backflushing with DMEM/F-12 solution containing 10% FBS, collecting the cells and tissues trapped on the 200-mesh and 400-mesh sieves into a 60mm petri dish, and finally, collecting into a 15mL centrifuge tube II and centrifuging to remove the supernatant;

s5, second enzyme digestion:

adding the tissue digestive juice B into each centrifuge tube II in the step B of S4, removing the supernatant, centrifuging and digesting at 34 ℃ under the condition of 300g for 1h; removing the supernatant after centrifugation, adding a DMEM medium containing FBS into the cell sediment immediately, and resuspending the cells to stop digestion;

s6, second physical separation:

collecting the cells suspended after resuspension in step S5, sieving with 500 mesh sieve, back-washing with 10% FBS-containing DMEM/F-12 solution, collecting the cell population retained on the 500 mesh sieve, i.e., the cells containing testicular support cells;

then rinsing the cells 3 times with a culture medium, resuspending the above cell population containing testicular-supporting cells with a 10% FBS-containing DMEM/F-12 solution, and inoculating into a culture dish, and culturing after labeling;

s7, cell identification:

and respectively carrying out morphological and structural identification on the cell group containing the testicular interstitial cells cultured in the culture dish in the step A of S4 and the cell group containing the testicular supporting cells cultured in the culture dish in the step S6, thereby realizing the whole process of the synchronous separation method of the pig testicular supporting cells and the testicular interstitial cells.

2. The method of claim 1, wherein the ratio of the tissue digestive juice a is: 6mg of P-type collagenase, 5.5mg of Dispase collagenase, 5mL of HBSS + phenol red and 5mL of PBS buffer solution.

3. The method for synchronously separating the sertoli cells and leydig cells of swine testis according to claim 1, wherein the ratio of the tissue digestive fluid b is: 0.25% Trypsin,45.00mL (5 mL); DNase I,5.00mg (1 mg/mL); DMEM,9mL (90%); FBS,1mL (10%).

4. The method of claim 1, wherein the 10% FBS in DMEM/F-12 is 10% FBS in DMEM/F-12 medium, wherein 90% DMEM/F-12 medium and 10% FBS in volume are present.

5. The method for synchronously separating the porcine testicular supporting cells and the testicular interstitial cells according to claim 4, wherein the DMEM/F-12 medium is a liquid medium of 1.

6. The method of claim 1, wherein the step S7 of identifying the morphology and structure of the cells comprises microscopic examination, HE staining, oil red O staining, and immunofluorescence staining.

7. The method for synchronously separating the porcine testicular sertoli cells and the testicular interstitial cells of claim 6, wherein the HE staining method comprises the following steps:

firstly, taking cells to be stained, fully removing a culture medium, and rinsing the cells by using 1000 mu L of PBS buffer solution; treating with 95%,85% and 75% ethanol for 2min each time; secondly, soaking the fabric in distilled water for 2min, dyeing the fabric with hematoxylin dye liquor for 15min, and washing off loose color by distilled water; then, differentiation is carried out for 3min by using a differentiation solution; washing with tap water for 2 times, each for 2min; and finally, placing the red dye solution for 1min, slightly washing the red dye solution for 3s by using distilled water, and quickly dehydrating.

8. The method for synchronously separating the sertoli cells and leydig cells of swine testis as claimed in claim 6, wherein the oil red O staining method comprises the following steps:

firstly, removing a cell culture medium, washing twice by PBS, and adding red O staining fixing solution for 25min; then discarding the stationary liquid, and washing with distilled water for 2 times;

then adding 60% isopropanol for immersion washing for 5min, discarding 60% isopropanol, adding newly prepared oil red O staining solution for immersion washing for 15min, discarding staining solution, and washing with water for 4 times until no excess staining solution exists;

finally, adding Mayer hematoxylin staining solution, re-staining cell nuclei for 2min, removing the staining solution, and washing with water for 4 times; adding oil red O buffer solution for 1min, discarding, adding distilled water to cover the cells, and observing under an inverted microscope.

9. The method for synchronously separating the sertoli cells and leydig cells of a pig according to claim 6, wherein the immunofluorescence staining method comprises the following steps:

first, the cells to be stained are taken, the medium is removed thoroughly and rinsed once with 500 μ L of DPBS +0.3% pvp buffer; adding 500 μ L of 4% PFA fixative pre-cooled at 4 deg.C, fixing for 15min, and removing the fixative;

secondly, add 500 μ L DPBS +0.3% PVP buffer to rinse the cells three times, 5min each time; adding 500 μ L of Triton solution 0.5% (v/v) and keeping at room temperature for 30min; then 500. Mu.L of DPBS +0.3% (w/w) PVP buffer solution was added to rinse the cells for three times, 5min each time;

then, 500. Mu.L of 2% BSA was added and blocked at room temperature for 2h; removing blocking solution, diluting the primary antibody with 2% BSA, adding the diluted primary antibody into a cell culture dish, incubating, sucking away the antibody, adding again, and incubating at 4 deg.C for 12-14 h;

finally, the primary antibody is absorbed completely, and 500 mu L of DPBS buffer solution is added for rinsing for 3 multiplied by 15min; adding 200 mu L of secondary antibody working solution, and incubating for 1h at room temperature; the secondary antibody was removed, the cells were rinsed with DPBS buffer for 3 × 15min, 200 μ L DAPI working solution was added, incubated at room temperature for 10min in the dark, and the test results were observed and photographed under an inverted fluorescence microscope.

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