A method for isolating and identifying interstitial cells from the testes of Bactrian camels.

Abstract

This invention provides a method for isolating and identifying interstitial cells from Bactrian camels, belonging to the field of cell isolation technology. A mixture of 0.08–0.12% collagenase IV solution and 0.23–0.27% trypsin-EDTA at a volume ratio of 1:2 is used to digest Bactrian camel testicular tissue blocks. The mixture is then purified by hypotonic treatment to remove excess germ cells, differential adhesion purification to remove a large number of supporting cells, and Percoll density gradient centrifugation to remove a small number of supporting cells. The suspension, obtained from a 34%–60% Percoll gradient, is transferred to a new culture flask to obtain interstitial cells with a purity ≥95%. Identification is performed using morphological observation, immunofluorescence staining, and secretory function testing. The interstitial cells isolated from Bactrian camels using this invention can be used for research on the reproductive performance of Bactrian camels, laying the foundation for understanding the reproductive performance of Bactrian camels.

Description

Technical Field

[0001] This invention relates to the field of cell separation technology, and in particular to a method for separating and identifying interstitial cells from Bactrian camel testes. Background Technology

[0002] The Bactrian camel (Camelus Bactrianus) is an important domestic animal widely distributed in arid regions of Asia, possessing strong adaptability and survival capabilities. Its reproductive system is particularly adept at adapting to environmental changes, making it an ideal subject for studying mammalian reproductive physiology and endocrine regulation. Leydig cells are the main cell type in testicular tissue that secretes male hormones (such as testosterone), playing a crucial role in maintaining male sexual characteristics, sperm production, and reproductive health. Despite the Bactrian camel's immense potential in biological research, research on its testicular interstitial cells is still in its early stages, especially regarding the immaturity of cell isolation and culture techniques.

[0003] Currently, the isolation of testicular interstitial cells mainly relies on conventional enzymatic digestion techniques, commonly used in mammalian models such as mice, rats, cattle, and pigs. However, these methods often face problems during the isolation process, including low cell purity, low cell viability, and unsuitable cell culture environments. This results in the acquisition of cell populations that may contain a large number of impurities such as vascular endothelial cells and fibroblasts, affecting the accuracy and reliability of experimental results. Furthermore, testicular interstitial cells from different species differ in morphology, function, and physiological characteristics, making existing methods difficult to directly apply to Bactrian camels.

[0004] Although some studies have attempted to isolate and culture Bactrian camel testicular interstitial cells, the methods for their isolation and culture have not yet been effectively optimized due to the different physiological characteristics and adaptation mechanisms of Bactrian camels compared to other livestock. To better study the physiological functions, androgen secretion mechanisms, and reproductive health-related diseases of Bactrian camel testicular interstitial cells, there is an urgent need to develop a primary isolation and culture method specifically for Bactrian camel testicular interstitial cells. Summary of the Invention

[0005] In view of this, the present invention provides a method for isolating and identifying interstitial cells of Bactrian camel testes to solve the above problems.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for isolating interstitial cells from the testes of Bactrian camels, comprising the following steps:

[0008] (1) Sample pretreatment: The parenchymal tissue of Bactrian camel testes was cut into pieces of 0.8–1.2 mm. 3The tissue blocks were washed 3-4 times with physiological saline.

[0009] (2) Enzymatic digestion: Mix 0.08-0.12% collagenase IV solution and 0.23-0.27% trypsin-EDTA at a volume ratio of 1:2 to obtain a digestion solution;

[0010] (3) Filtration and washing: The homogenate was sieved, the filtrate was collected, then centrifuged, the precipitate was collected, resuspended in complete culture medium, washed 2-3 times, and an equal volume of complete culture medium containing 10% FBS was added to stop the digestion; the tissue block was mixed with the digestion solution and digested in a water bath at 37°C for 30 min to obtain a homogenate;

[0011] (4) Obtaining single-cell suspension: The digestion solution is sieved, the filtrate is collected, then centrifuged, the precipitate is collected, the cells are resuspended in complete culture medium, and cultured at 37°C and 5% CO2.

[0012] (5) Hypotonic purification: After culturing for 48 hours, the culture medium was aspirated, and Tris-HCl at a concentration of 19-21 mM was added until the cells were covered. The cells were treated with hypotonic solution for 3.5-4.5 min. Then the hypotonic solution containing Tris-HCl was aspirated, the cells were washed with complete culture medium, and fresh complete culture medium was added to continue culturing.

[0013] (6) Differential adhesion purification: After the cells purified by hypotonic treatment were cultured for another 24 hours, the non-adherent cell suspension was aspirated and transferred to a new culture dish for further culture.

[0014] (7) When the cell fusion reaches 80% to 90%, digest with 0.05% trypsin once; the first digestion time is 1 to 2 minutes, and the time of each subsequent digestion is 1 / 2 of the previous time; place the digested cells in a new culture dish for culture.

[0015] (8) When the cell fusion reaches 80-90%, digest with 0.25% trypsin for 1-2 minutes, and then stop digestion with an equal volume of whole culture medium; centrifuge at 1200 rpm for 5 minutes and discard the supernatant.

[0016] (9) Primary culture: The purified cells were cultured at 37°C and 5% CO2.

[0017] (10) Subculture: When the cell confluence reaches 80% to 90%, subculture is performed. The culture medium is discarded and the cells are washed 2 to 3 times with PBS. 0.05% trypsin-EDTA is added and incubated at 37°C for 1 to 2 minutes. When the cells become round and the gaps between them increase, some cells are detached and complete culture medium is added to stop digestion. Then, the liquid is aspirated into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes. The precipitate is washed and placed back into a culture dish for culture. After purification by Percoll density gradient centrifugation, the suspension between 34% and 60% of the Percoll gradient is transferred to a new culture flask to obtain mesenchymal cells with a purity of ≥95%.

[0018] Preferably, the Bactrian camel testes are taken from male Bactrian camels aged 3 to 5 years.

[0019] Preferably, in step (2), the tissue block is mixed with the digestive liquid at a volume ratio of 1:4.5 to 5.5; the rotation speed of the oscillation digestion is 450 to 550 rpm.

[0020] Preferably, the mesh size of the sieve in steps (3) and (4) is 100 mesh.

[0021] Preferably, step (4) involves resuspending the cells to a concentration of 4.5–5.5 × 10⁻⁶ cells. 6 per mL.

[0022] Preferably, the complete culture medium consists of: DMEM high glucose medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin.

[0023] This invention also provides a method for identifying Bactrian camel testicular interstitial cells. Cell identification is performed by observing cell morphology, RT-PCR, and immunofluorescence staining to identify specific markers. The secretory function is confirmed by ELISA, and the purified cells are confirmed to be testicular interstitial cells.

[0024] Preferably, the immunofluorescence staining is performed using CYP11A1, 3βHSD, and AR cell markers as specific antibodies, and cell identification is performed using a fluorescence microscope. If strong positive fluorescence is observed, the cells are identified as Bactrian camel testicular interstitial cells.

[0025] Preferably, the RT-PCR is performed by detecting the mRNA levels of testicular Sertoli cell markers CYP11A1, 3βHSD, and AR. If bright bands appear at 224bp, 175bp, and 218bp, they are identified as Bactrian camel testicular interstitial cells.

[0026] This invention also provides the application of Bactrian camel testicular interstitial cells obtained by the aforementioned separation method in the study of Bactrian camel reproductive performance.

[0027] By adopting the above technical solution, the present invention has the following beneficial effects:

[0028] This invention utilizes specific enzyme digestion and density gradient centrifugation with differential adhesion to efficiently isolate interstitial cells from Bactrian camel testicular tissue, avoiding the cell contamination problems common in conventional methods and significantly improving cell purity and viability.

[0029] This invention significantly improves cell survival and proliferation by optimizing culture conditions and adjusting the composition and conditions of the culture medium, thus effectively ensuring the stability of cell culture.

[0030] The isolation and culture technology provided by this invention has strong stability and reproducibility, which can ensure that different experimenters obtain consistent experimental results at different time points. It is suitable for large-scale cell culture and long-term cell storage (up to 6 passages).

[0031] This invention utilizes techniques such as RT-PCR, immunofluorescence staining, and ELISA to accurately identify isolated mesenchymal cells, ensuring high purity of the cell population, eliminating interference from other cell types, and further enhancing the reliability of subsequent experiments. Attached Figure Description

[0032] Figure 1 This refers to the 1-15 day culture and purification process of mesenchymal cells.

[0033] Figure 2 The mRNA expression levels of CYP11A1, HSD3β, and AR in interstitial cells, supporting cells, and miscellaneous cells of the bihumped testis were measured.

[0034] Figure 3 The results of IF detection of interstitial cell markers CYP11A1, HSD3β and AR in bihumped testes.

[0035] Figure 4 The secretion levels of T, DHT, and E2 in the interstitial cells of the bihumped testis relative to those in the supporting cells and other cells. Detailed Implementation

[0036] The main reagents and experimental equipment used in this invention are as follows:

[0037] 1. Main reagents

[0038] Basic culture medium: DMEM / F12 (1:1) medium;

[0039] Complete culture medium: DMEM / F12 (high glucose) + 10% fetal bovine serum (FBS, Gibco, 141C) + 100 U / mL penicillin + 100 μg / mL streptomycin;

[0040] Digestive solution: 0.05% collagenase IV, 0.25% trypsin-EDTA (1:1 mixture);

[0041] Hypotonic solution: 0.075 mol / L NaCl solution; 2 mL 20 mM (mmol / L, pH 7.4) Tris-HC1 hypotonic solution for 5 min, followed by two washes with PBS at 37°C.

[0042] Identification reagents: specific antibodies (CYP11A1, 3βHSD and AR), monoclonal antibodies, DAPI and fluorescent secondary antibodies, etc.

[0043] Percoll stock solution (White Shark, 65455-52-9) was used to prepare Percoll solutions at concentrations of 21%, 26%, 34%, and 60%. The cells in the 34%–60% concentration gradient were mesenchymal cells. The different Percoll solutions are shown in Table 1.

[0044] Table 1 Percoll solutions at different concentration gradients

[0045] Gradient solutions (different concentrations) Percoll stock solution PBS dilution 21％ 210μL × 4 = 840μL 790 μL (PBS) × 4 = 3160 μL 26％ 260μL × 4 = 1040μL 740 μL (PBS) × 4 = 2960 μL 34％ 340μL × 4 = 1360μL 660μL (PBS) × 4 = 2640μL 60％ 600μL × 4 = 2400μL 400 μL (PBS) × 4 = 1600 μL

[0046] 2. Experimental equipment

[0047] Clean bench, CO2 incubator (37℃, 5% CO2), inverted microscope, centrifuge, fluorescence microscope, etc.;

[0048] Sterile consumables: culture dishes, centrifuge tubes, pipettes and tips, surgical scissors, forceps, etc.

[0049] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0050] Example 1

[0051] I. Material Selection and Pretreatment

[0052] 1. Sample Collection: Bactrian camel testes were obtained from Pingshan Lake in Zhangye. Complete testes from male camels (4 years old) were obtained through castration surgery and placed in an ice bath of physiological saline containing penicillin-streptomycin. The samples were then brought back to the laboratory within 6 hours. During collection, it is crucial to ensure tissue freshness and avoid prolonged storage at low temperatures, which could lead to decreased cell viability. Care must also be taken to prevent contamination from other materials.

[0053] 2. Pretreatment: In a laminar flow hood, wipe the surface of the testis with 75% alcohol more than three times, then transfer it to a sterile culture dish; rinse repeatedly with PBS containing penicillin-streptomycin, then use ophthalmic scissors to remove the outer tunica albuginea, surface connective tissue, and blood vessels, preserving the seminiferous tubule-rich parenchyma; cut the parenchyma into 1mm pieces. 3Wash the small pieces with saline solution three times, 5 minutes each time, to remove blood and impurities.

[0054] II. Enzymatic Digestion

[0055] 1. Digestion: Since the interstitial cells are located between the various seminiferous tubules and are arranged relatively loosely, during digestion, add 3 times the volume of a mixture of 0.1% collagenase IV solution and 0.25% trypsin-EDTA (1:2 mixture) to the tissue block, and digest in a 37°C water bath with shaking for 30 minutes at a speed of 500 rpm. During this period, gently blow the tissue block twice with a pipette every 10 minutes.

[0056] 2. Filtration and washing: Filter the digestion solution through a 100-mesh cell sieve, collect the filtrate, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the precipitate in complete culture medium, wash 3 times to remove residual collagenase and trypsin; add an equal volume of complete culture medium containing 10% FBS to terminate the digestion.

[0057] 3. Obtaining a single-cell suspension: Filter through a 100-mesh cell sieve, collect the filtrate, centrifuge at 1000 rpm for 5 min, and discard the supernatant; resuspend the pellet in complete culture medium and adjust the cell concentration to approximately 5 × 10⁻⁶ cells / mL. 6 Cells / mL were incubated in a 37°C incubator with 5% CO2.

[0058] III. Purification of Mesenchymal Cells

[0059] 1. Hypotonic purification (removal of excess germ cells): After culturing for 48 hours, aspirate the culture medium and add 2 mL of pre-cooled 20 mM (mmol / L, pH 7.4) Tris-HC1 (enough to cover the cells). Treat with hypotonic solution for 4 minutes. Spermatogenic cells are sensitive to hypotonicity and will rupture and die. Then immediately aspirate the hypotonic solution containing Tris-HC1, wash 3 times with complete culture medium, and add fresh complete culture medium to continue culturing.

[0060] 2. Differential adhesion purification (removal of a large number of supporting cells): Single-cell suspensions were seeded into culture dishes and incubated in a CO2 incubator for 24 hours (37℃, 5% CO2). v During this period, a large number of fibroblasts and other miscellaneous cells adhered rapidly, while supporting cells and mesenchymal cells adhered more slowly. Using a 1 mL pipette, the unadhered cell suspension was gently aspirated and transferred to a new culture dish for continued culture. At this time, the culture dish contained a small number of supporting cells and a large number of mesenchymal cells.

[0061] 3. When the cell confluence reaches 80%–90%, digest the cells twice using a low concentration (0.05%) of trypsin. The first digestion takes 1 minute, and the second digestion takes half the time of the first. Since the mesenchymal cells have weaker adhesion than supporting cells, they are digested first and then placed in a new culture dish. Once the cell confluence reaches 80%–90%, this is designated as generation P0.

[0062] 4. Density gradient centrifugation purification (removal of a small number of supporting cells): ① Prepare Percoll separation solution with volume fractions of 21%, 26%, 34%, and 60% using PBs containing 1% penicillin and antibiotics, and filter through a 0.22 μm filter membrane. ② Digest two flasks of 90%–100% confluent P0 generation adherent cells with 0.25% trypsin for 1 min, and terminate digestion with an equal volume of complete culture medium. ③ Centrifuge at 1200 rpm for 5 min, discard the supernatant, and add 1 mL of each of the prepared Percoll gradient solutions to 10 mL sterile centrifuge tubes in descending order of density. Gently add the cell suspension to be separated to the top layer of the gradient and centrifuge at 2200 rpm for 25 min. ④ Transfer the suspension between the 34% and 60% Percoll gradients (mesenchymal cells) to a new culture flask, add complete culture medium, gently pipette to mix, and observe under a microscope.

[0063] 5. Primary culture and subculture

[0064] (1) Primary culture: The purified cells were cultured in a 37°C, 5% CO2 incubator. The cell status was observed every 5 days. The culture medium was changed and dead cells were removed. The medium was changed every 2 days thereafter.

[0065] (2) Passaging: When the cell confluence reaches 80%–90%, passaging is performed: the culture medium is aspirated, and the cells are washed three times with PBS; 0.05% trypsin-EDTA is added, and the cells are incubated at 37°C for 2 minutes (the degree of digestion can be judged by the cell morphology); when the cells become rounded and the gaps between them increase under a microscope, and some cells detach, complete culture medium is added to stop the digestion; then the liquid is aspirated into a centrifuge tube and centrifuged at 1000 rpm for 5 minutes; after repeated washing, the cells are resuspended in a new culture dish and cultured. At this time, the purity of the mesenchymal cells is ≥95%, which is recorded as generation P1. Mesenchymal cells can be passaged up to 6 times.

[0066] Example 2. Identification of interstitial cells in the testes of Bactrian camels

[0067] 1. Morphological identification

[0068] After 5 days of primary culture, mesenchymal cells began to adhere to the wall in large numbers, taking the form of round, oval, triangular and polygonal shapes, with relatively large cell bodies (approximately 20 μm in diameter), forming cell islands, and distinct nuclei (round or oval, containing 1-2 nucleoli), and abundant cytoplasm.

[0069] 2. Detection of mRNA expression levels of CYP11A1, HSD3β, and AR in interstitial cells of Bactrian camel testes

[0070] Mesenchymal cells from generation P1 were collected and RNA was extracted. Using contaminated cells and supporting cells from the same batch as controls, single-stranded cDNA was synthesized using a reverse transcription kit. The mRNA levels of testicular supporting cell marker molecules (CYP11A1, HSD3β, and AR) were detected by RT-PCR. Primer information is shown in Table 2.

[0071] Table 2 PT-PCR Primer List

[0072]

[0073] The reaction system consisted of 25 μL: 10 μL of 2×M5 Hiperplus PCRMix, 1 μL each of forward and reverse primers, 2 μL of cDNA, and 11 μL of RNase-free water. The reaction program was: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 15 s; 55℃ annealing for 45 s; for a total of 45 cycles. Results showed that compared with the control group (mixed cells), the mRNA levels of CYP11A1, HSD3β, and AR in mesenchymal cells were significantly higher than those in the control group (P<0.01). Compared with Sertoli cells, both CYP11A1 and HSD3β mRNA were expressed with significant differences (P<0.05), while AR expression showed no significant difference (androgen receptors are also present in Sertoli cells). Figure 2 The above preliminary results indicate that the cell is a mesenchymal cell.

[0074] 3. Immunofluorescence staining (IF) to identify specific protein expression

[0075] Based on known marker molecules (CYP11A1, HSD3β, and AR) in testicular interstitial cells, cell morphology and marker molecule expression were detected using IF (instantaneous immunoassay). The steps are as follows:

[0076] 1. Slide preparation: Place a sterile coverslip into a culture dish, inoculate the purified cells, and culture until confluence reaches 70%;

[0077] 2. Fixation: Discard the culture medium, wash 3 times with PBS (5 min each time); add 4% paraformaldehyde and fix at room temperature for 30 min;

[0078] 3. Blocking: After washing with PBS, add blocking solution containing 5% bovine serum albumin (BSA) and block at room temperature for 1 hour;

[0079] 4. Primary antibody incubation: Add monoclonal antibodies (1:200 dilution) of mesenchymal cell-specific markers (CYP11A1, 3βHSD, and AR) and incubate overnight at 4°C;

[0080] 5. Secondary antibody incubation: After washing three times with PBS, add fluorescently labeled secondary antibody (1:200 dilution) and incubate at room temperature in the dark for 1 hour;

[0081] 6. Nuclear staining: Add DAPI staining solution and stain at room temperature in the dark for 5 minutes;

[0082] 7. Observation: Under a fluorescence microscope, the cytoplasm of supporting cells shows green fluorescence (CYP11A1, 3βHSD, and AR), and the cell nucleus shows blue fluorescence (DAPI staining); calculate the proportion of positive cells, and a positive rate of ≥90% is considered as successful purification.

[0083] The results showed that CYP11A1, HSD3β, and AR proteins were strongly expressed, and these three proteins were mainly located in the cytoplasm with a purity greater than 95%. Figure 3 This indicates that the purity of the mesenchymal cells is ≥95%.

[0084] 4. ELISA detection of testicular interstitial cell secretion function in Bactrian camels

[0085] To further investigate the function of interstitial cells, the supernatant of culture medium containing both contaminated cells and cells purified for the first time (testicular Sertoli cells) was collected. The function of secreting hormones (T, DHT, and E2) was detected using ELISA. The results showed that the levels of T, DHT, and E2 in the supernatant of Bactrian testicular interstitial cells were significantly higher than those in contaminated cells (P<0.01); compared with Sertoli cells, the level of T in the supernatant of interstitial cells was significantly higher than that in Sertoli cells (P<0.01), while the secretion levels of DHT and E2 did not differ significantly. Figure 4 The above results demonstrate that interstitial cells have the level to secrete relevant hormones.

[0086] As can be seen from the above embodiments, the present invention provides a method for isolating and identifying interstitial cells from Bactrian camels. The interstitial cells from Bactrian camels isolated according to the present invention have high purity and viability.

[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for isolating interstitial cells from the testes of Bactrian camels, characterized in that, Includes the following steps: (1) Sample pretreatment: the parenchymal tissue of the testis of Bactrian camel was cut into 0.8-1.2mm tissue blocks, washed with normal saline for 3-4 times; 3 ; (2) Enzymatic digestion: Mix 0.08-0.12% collagenase IV solution and 0.23-0.27% trypsin-EDTA at a volume ratio of 1:2 to obtain a digestion solution; mix the tissue block with the digestion solution and digest in a water bath at 37°C for 30 min to obtain a homogenate; (3) Filtration and washing: Sieve the homogenate, collect the filtrate, then centrifuge, collect the precipitate, resuspend it with complete culture medium, wash 2-3 times, and add an equal volume of complete culture medium to stop digestion. The complete culture medium consists of: DMEM high glucose medium + 10% fetal bovine serum + 100 U / mL penicillin + 100 μg / mL streptomycin; (4) Obtaining single-cell suspension: The digestion solution is sieved, the filtrate is collected, then centrifuged, the precipitate is collected, the cells are resuspended in complete culture medium, and cultured at 37°C and 5% CO2. In steps (3) and (4), the sieve mesh size is 100 mesh; (5) Hypotonic purification: After culturing for 48 hours, the culture medium was aspirated, and Tris-HCl with a concentration of 19-21 mM was added until the cells were covered. The cells were treated with hypotonic solution for 3 minutes. Then the hypotonic solution containing Tris-HCl was aspirated, the cells were washed with complete culture medium, and fresh complete culture medium was added to continue culturing. (6) Differential adhesion purification: After the cells purified by hypotonic treatment were cultured for another 24 hours, the non-adherent cell suspension was aspirated and transferred to a new culture dish for further culture. (7) When the cell fusion reaches 80% to 90%, digest twice with 0.05% trypsin; the first digestion time is 1 to 2 minutes, and the subsequent digestion time is 1 / 2 of the first time; place the digested cells in a new culture dish for culture. (8) When the cell confluence reaches 80-90%, digest with 0.25% trypsin for 1-2 min, and then stop digestion with an equal volume of complete culture medium; centrifuge at 1200 rpm for 5 min and discard the supernatant; purify by Percoll density gradient centrifugation, and transfer the suspension between 34% and 60% of the Percoll gradient to a new culture flask. (9) Primary culture: The purified cells were cultured at 37°C and 5% CO2. (10) Passage culture: When the cell confluence reaches 80% to 90%, passage is performed. The culture medium is discarded and the cells are washed 2 to 3 times with PBS. 0.05% trypsin-EDTA is added and incubated at 37°C for 1 to 2 minutes. When the cells become round and the gaps between them increase, some cells are detached and complete culture medium is added to stop digestion. Then, the liquid is aspirated and placed in a centrifuge tube, centrifuged at 1000 rpm for 5 minutes, the precipitate is washed, and the cells are placed back in a culture dish for culture to obtain mesenchymal cells with a purity of ≥95%.

2. The separation method according to claim 1, characterized in that, The testes of the Bactrian camels were taken from male Bactrian camels aged 3 to 5 years.

3. The separation method according to claim 1, characterized in that, In step (2), the tissue block and digestion solution are mixed at a volume ratio of 1:3; the rotation speed of the oscillation digestion is 450-550 rpm.

4. The separation method according to claim 1, characterized in that, Step (4) Resuspend the cells to a concentration of 4.5–5.5 × 10⁻⁶ cells. 6 per mL.

5. The application of Bactrian camel testicular interstitial cells obtained by the isolation method according to any one of claims 1 to 4 in the study of Bactrian camel reproductive performance.

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