A method for isolating, culturing and identifying chicken gonadal somatic cells

Abstract

This invention discloses a method for the isolation, culture, and identification of chicken sexual gonadal cells, belonging to the field of biotechnology. The invention selects chicken embryos hatched to 18.5 days old, isolates the gonads after PCR sex identification, and uses 0.25% trypsin-EDTA digestion followed by filtration through a 70μm filter to obtain a single-cell suspension with a survival rate >90% and free from PGC contamination. A sex-specific culture system is established, allowing cells to be stably passaged and maintain a sex-specific phenotype. Through morphological, qRT-PCR, immunofluorescence, and Western blot multidimensional identification, accurate identification of male testicular Sertoli cells and female ovarian granulosa cells is achieved. This method is simple to operate, has good reproducibility, and produces high cell viability and purity. It can provide a stable gonadal microenvironment for the in vitro directed differentiation of chicken primordial germ cells, significantly improving gamete induction efficiency. It is suitable for research on avian reproductive and developmental mechanisms, transgenic breeding, and germplasm resource preservation.

Description

Technical Field

[0001] This invention belongs to the field of avian reproductive biology and cell culture technology, specifically relating to a method for isolating, culturing and identifying chicken sexual gland cells. Background Technology

[0002] Advances in avian reproductive development and assisted reproductive technologies heavily rely on stable, high-purity gonadal support cell systems. The in vitro proliferation, directed differentiation, and gametogenesis of chicken primordial germ cells (PGCs) all require the supportive microenvironment provided by gonadal cells, including extracellular matrix, signaling molecules, and nutrient supply. Current research indicates that DMRT1 regulates male gonadal development, while FOXL2 maintains female ovarian fate; the RSPO1 / WNT4 / β-catenin pathway participates in the proliferation and differentiation of female gonadal cells and synergistically regulates germ cell reprogramming with the FOXL2 / CYP19A1 / ERα axis.

[0003] However, existing technologies have obvious drawbacks: 1) There is a lack of standardized isolation and purification protocols for chicken gonadal cells. Traditional single digestion with collagenase and trypsin can easily cause cell damage, with a survival rate of only 70%–80%, and it is difficult to remove PGC contamination. 2) The sex-specific culture system is not perfect, and the cells cannot maintain their morphology and function for a long time, making stable passage impossible; 3) The identification methods are limited, relying mainly on morphological observation and lacking multi-dimensional verification at the gene and protein levels, so cell purity and sex specificity cannot be guaranteed.

[0004] Publicly available patents, such as CN112852715A, focus only on PGC culture and do not optimize the isolation and purification of gonadal cells. This makes it difficult to construct a gonadal microenvironment that closely resembles that in vivo, resulting in low efficiency of in vitro induced differentiation of PGCs and limiting their application in avian transgenic breeding and germplasm resource preservation.

[0005] To address the aforementioned deficiencies, this invention provides a method for isolating, culturing, and identifying chicken sexual gland cells with high survival rate, high purity, stable passage capability, and a complete identification system. Summary of the Invention

[0006] Technical Problem Solved: To address the aforementioned technical problems, this invention provides a method for the isolation, culture, and identification of chicken sexual gland cells, solving the problems of low isolation efficiency, poor survival rate, heavy PGC contamination, and incomplete identification in existing technologies. The method achieves: optimized enzymatic digestion and filtration steps, resulting in a cell survival rate ≥90% and no PGC residue; the establishment of a sex-specific culture system that allows for stable passage and maintenance of sex-specific phenotypes; the construction of a multi-dimensional identification system encompassing morphology, genes, and proteins to ensure cell purity and function; and the provision of reliable somatic cell support for the in vitro directed differentiation of PGCs, improving gamete induction efficiency and serving poultry breeding and germplasm preservation.

[0007] Technical solution: A method for isolating, culturing, and identifying chicken gonadal cells, comprising the following steps: S1. Cell Isolation: Chicken embryos hatched to 18.5 days were selected, and their sex was determined by PCR (ZZ male, ZW female). Gonads were isolated under a stereomicroscope, and 50 pairs of each sex were collected. The cells were washed 3-5 times with PBS. 500 μL of preheated 0.25% trypsin-EDTA was added, and the cells were digested in a 37°C water bath for 2 min. The digestion was repeated 3-4 times by pipetting and dispersing until the tissue was completely dispersed. 800 μL of DMEM containing 10% FBS was added to stop the digestion. The cells were centrifuged at 700×g for 5 min, and the supernatant was discarded. The cells were resuspended in PBS and centrifuged again. The cells were resuspended in DMEM containing 10% FBS and filtered through a 70 μm nylon mesh. The cells were counted with trypan blue, and the cell viability was >90%.

[0008] S2, Cell culture: at 6×10 5 Inoculate each well with sex-specific culture medium and incubate at 37°C with 5% CO2. After 6 h, wash with PBS to remove suspended impurities and replace with sex-specific culture medium. Male Support cells: α-MEM + 10% FBS + 10 ng / mL IGF-1; Female granulocytes: M199+25 ng / mL FSH; Change half the medium every 2–3 days; passage with 0.25% trypsin-EDTA digestion at a ratio of 1:3 to 1:4; cells adhere to the wall and maintain good viability and typical morphology even after passage to P2 generation.

[0009] S3. Cell identification: S3-1, Morphological Identification Observation under an inverted microscope: Male supporting cells are arranged in long spindle shapes and cords; female granule cells are cuboidal and tightly connected; HE staining: female cell nuclei are large and loose, while male cell nuclei are long and dense with chromatin; Oil Red O staining: female granule cells have abundant lipid droplets, while male supporting cells have fewer lipid droplets. S3-2, Molecular biological identification Total RNA was extracted using the Trizol method and reverse transcribed using HiScript III RT SuperMix; qRT-PCR was performed using ChamQ SYBR MasterMix; GAPDH was used as an internal control. −ΔΔCt Analysis showed that female cells highly expressed RSPO1, FOXL2, CYP19A1, and FSHR; male cells highly expressed DMRT1, SOX9, and AMH. S3-3, Protein Level Identification Immunosorbent assay (IPA) was performed with 4% PFA, permeabilized with 0.5% Triton X-100, and blocked with 10% goat serum. Primary antibodies (Anti-DMRT1, Anti-SOX9, Anti-AMH, Anti-FOXL2, Anti-CYP19A1, Anti-FSHR, 1:200) were incubated overnight at 4°C. Secondary antibodies (Alexa Fluor 488 / 594, 1:500) were incubated at room temperature for 1–2 h. DAPI nuclear staining was performed. Immunofluorescence showed that SOX9 / FOXL2 nuclear localization was clear, AMH specifically labeled Supporting cells, CYP19A1 specifically labeled granulocytes, and DAZL negativity confirmed the absence of PGC contamination. Western blot was used to verify protein expression and β-catenin nuclear translocation.

[0010] Beneficial effects: High cell viability and purity: survival rate >90%, significantly higher than the traditional 70%-80%; no PGC contamination (VASA / DAZL negative); The culture system is stable: it can be stably passed to P2-P3 generations, maintaining sex-specific gene expression (female RSPO1 expression is 3-5 times higher, P<0.05). Comprehensive and reliable identification: triple verification of morphology, genes, and proteins, with objective and reproducible results; Significant application value: It significantly improves the in vitro induced microenvironment of PGCs, increasing gamete differentiation efficiency by more than 30%; it is simple to operate, low in cost, and has good reproducibility (n=3, P<0.05), making it suitable for research on avian reproductive mechanisms, transgenic breeding, and long-term preservation of germplasm resources. Attached Figure Description

[0011] Figure 1 This is a diagram of the gonadal tissue of a male (A) and female (B) chicken embryo, with a scale bar of 25 μm. Figure 2 Morphological images of male supporting cells (A) and female granule cells (B) from generation P0 to P2, scale bar 25 μm; Figure 3 This is a bar chart showing the expression of sex-specific genes in male and female cells using qRT-PCR. *P<0.05; Figure 4These are cell immunofluorescence identification images. In Figure A, males are labeled with SOX9 / AMH; in Figure B, females are labeled with FOXL2 / CYP19A1; in Figure C, DAPI stains the nuclei blue; and in Figure D, MERGE merged image. The scale bar is 25 μm. Detailed Implementation

[0012] The present invention will be described in detail below with reference to specific embodiments: Example 1: Isolation and Culture of Chicken Sexual Gonadal Cells Materials: Chicken embryos incubated for 18.5 days (Jiangsu Provincial Poultry Research Institute); CHD1 primers for PCR sex determination. The steps are as follows: 1) Take chicken embryos and wash them three times with PBS; separate the gonads under a stereomicroscope (e.g., Figure 1 (As shown), 50 pairs were collected for each gender; 2) Wash with PBS by pipetting 3-5 times; add 500 μL of 0.25% trypsin-EDTA, digest at 37℃ for 8-10 min, pipetting 3-4 times during digestion until the tissue is dispersed; 3) Add 800 μL of DMEM containing 10% FBS to terminate digestion; centrifuge at 700×g for 5 min and discard the supernatant; 4) After resuspending in PBS, centrifuge again; resuspend in DMEM containing 10% FBS and filter through a 70 μm filter. 5) Trypan blue count showed a cell viability of 95%. 6) Using 6×10 5 Seeds were inoculated into 12-well plates; after culturing at 37°C and 5% CO2 for 6 h, the suspension cells were discarded and replaced with sex-specific culture medium. 7) Change half the medium every 2-3 days; use 0.25% trypsin-EDTA digestion and passage at a ratio of 1:3-1:4; Figure 2 As shown, P2 generation cells maintain typical morphology: males are spindle-shaped and females are cubic.

[0013] Example 2: qRT-PCR identification of sex-specific gene expression 1) Take 1×10 6 Cells were washed twice with PBS; 1 mL of Trizol was added for lysis, and the cells were incubated at room temperature for 5 min. 2) Add 200 μL of chloroform, shake for 15 s; centrifuge at 4℃ and 10000 rpm for 10 min; 3) Take the aqueous phase, add an equal volume of isopropanol to precipitate RNA; wash with 75% ethanol, air dry, and dissolve in enzyme-free water; 4) NanoDrop detection concentration: 5-10 μg; take 1 μg of RNA for reverse transcription; 5) Reverse transcription system: 1 μg RNA + 4 μL 4×gDNA wiper Mix, 42℃ for 2 min; then add 4 μL 5×HiScript III qRT SuperMix, 37℃ for 15 min, 85℃ for 5 s; 6) qRT-PCR system: 2×Master Mix 5 μL + cDNA 1 μL + primer 0.2 μL + ddH2O to make up the volume; 7) Reaction conditions: 95℃ for 30 s; 40 cycles (95℃ for 10 s, 60℃ for 30 s, 72℃ for 15 s).

[0014] The results are as follows Figure 3 As shown: RSPO1, FOXL2, CYP19A1, and FSHR were significantly highly expressed in female cells (P < 0.001); DMRT1, SOX9, and AMH were significantly highly expressed in male cells.

[0015] Example 3: Indirect immunofluorescence identification 1) Wash cells twice with PBS; fix with 4% PFA at room temperature for 15-20 min; 2) Permeabilize with 0.5% Triton X-100 for 10-15 min; block with 10% goat serum for 1 h; 3) Incubate with primary antibodies (Anti-SOX9, Anti-FOXL2, 1:200) overnight at 4°C; 4) Wash with PBST 4-5 times; use secondary antibody (Alexa Fluor 488 / 594, 1:500) at room temperature in the dark for 1-2 hours. 5) Stain with DAPI for 5-10 min; mount with anti-fluorescence quenching mounting solution.

[0016] The results are as follows Figure 4 As shown: male cells SOX9 showed green nuclei positive; female cells FOXL2 showed red nuclei positive; the signal was specific and there was no cross-reactivity.

[0017] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for isolating, culturing, and identifying chicken sexual gland cells, characterized in that, Includes the following steps: S1. Cell isolation: Chicken embryos hatched to 18.5 days were selected, and the gonads were isolated after sex identification by PCR. The embryos were digested in stages with 0.25% trypsin-EDTA, and the digestion was terminated with DMEM containing 10% FBS. After centrifugation and resuspension with PBS, the cells were filtered through a 70 μm nylon mesh to obtain a single-cell suspension with a cell viability of >90%. S2. Cell culture: After inoculating cells for 6 hours, replace with sex-specific medium, change half of the medium every 2-3 days, and passage at a ratio of 1:3-1:

4. S3. Cell identification: Multi-dimensional identification is performed through morphology, molecular biology, and protein levels to confirm cell type, sex specificity, and absence of PGC contamination.

2. The method for isolating, culturing, and identifying chicken gonadal cells according to claim 1, characterized in that, The digestion process in step S1 is as follows: digest in a 37℃ water bath for 2 min, repeatedly pipetting and digesting 3-4 times until the tissue is completely dispersed; after digestion, centrifuge at 700×g for 5 min and discard the supernatant.

3. The method for isolating, culturing, and identifying chicken gonadal cells according to claim 1, characterized in that, In step S1, 50 pairs of gonads were collected from each sex; the gonads were washed with PBS 3-5 times; after filtration, they were counted using trypan blue.

4. The method for isolating, culturing, and identifying chicken hermaphroditic gonadal cells according to claim 1, characterized in that: In step S2, cells are at a rate of 6 × 10 5 One sample per well was inoculated into a 12-well plate and cultured at 37°C with 5% CO2. After 6 h, the plate was washed with PBS to remove suspended impurities.

5. The method for isolating, culturing, and identifying chicken sexual gland cells according to claim 1, characterized in that, The sex-specific culture medium in step S2 is: Male Support cells: α-MEM + 10% FBS + 10 ng / mL IGF-1; Female granulocytes: M199+25 ng / mL FSH.

6. The method for isolating, culturing, and identifying chicken sexual gland cells according to claim 1, characterized in that, The morphological identification in step S3 includes: observing cell morphology under an inverted microscope, HE staining, and Oil Red O staining; male supporting cells are long spindle-shaped and arranged in cords, with fewer lipid droplets; female granular cells are cuboidal, tightly connected, and rich in lipid droplets.

7. The method for isolating, culturing, and identifying chicken hermaphroditic gonadal cells according to claim 1, characterized in that, In step S3, molecular biological identification was performed using qRT-PCR: female cells highly expressed RSPO1, FOXL2, CYP19A1, and FSHR; male cells highly expressed DMRT1, SOX9, and AMH.

8. The method for isolating, culturing, and identifying chicken sexual gland cells according to claim 1, characterized in that, Protein level identification in step S3 included immunofluorescence and Western blot; immunofluorescence showed clear nuclear localization of SOX9 / FOXL2, AMH-specific labeling of supporting cells, CYP19A1-specific labeling of granulocytes, and DAZL negativity confirmed the absence of PGC contamination.

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