CN111500531A

CN111500531A - Method for in vitro long-term culture of chicken PGCs

Info

Publication number: CN111500531A
Application number: CN202010410510.1A
Authority: CN
Inventors: 杨婷; 常国斌; 仇玲玲; 陈博雯; 孔令琳; 白蒙; 董钊绮; 胡晓丹
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2020-05-15
Filing date: 2020-05-15
Publication date: 2020-08-07
Also published as: AU2021101223A4

Abstract

A method for culturing chicken PGCs in vitro for a long time includes such steps as separating chicken PGCs, purifying and culturing the PGCs, systematically comparing the addition of feeder layer, the treatment of feeder layer and the addition ratio of BR L to culture medium, and culturing chicken PGCs in vitro for a long time.

Description

Method for in vitro long-term culture of chicken PGCs

Technical Field

The invention relates to a method for in vitro long-term culture of chicken PGCs, belonging to the technical field of zootechnics and cytology.

Background

Since Dubois reported in 1969, several studies have been conducted on chicken PGCs, and several basic growth factors and specific growth factors L IF, bFGF and hSCF have been found to maintain PGC survival, proliferation and growth in vitro, and in addition, in combination with growth factors, conditioned medium and feeder cells, PGC can remain proliferative and undifferentiated in vitro for long periods of time, but there are few available methods for long-term culture of PGCs in vitro, which greatly affects long-term studies and utilization of PGC.

Disclosure of Invention

Although many research researches on separation methods, in vitro culture and the like of chicken PGCs are carried out, no systematic and comprehensive culture system is available for in vitro long-term culture of PGCs, which greatly limits research and application of poultry PGCs in aspects of germ cell development, genetic resource protection and the like.

Therefore, the invention provides a method for in vitro long-term culture of chicken PGCs, and the compound culture system can be used for carrying out long-term in vitro culture on PGCs, so that research and application of poultry PGCs in the aspects of germ cell development, genetic resource protection and the like can be fully carried out.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

the chicken PGCs are obtained by separation, and then the PGCs are purified and cultured, systematic comparison is made on the aspects of the PGC culture method, whether a feeding layer is added or not, the feeding layer treatment method, the addition proportion of a BR L conditioned medium and the like, and finally the method for in-vitro long-term culture of the chicken PGCs is determined.

Specifically, the method comprises the following steps:

separating chicken PGCs from gonads of 4.5-5.5 d embryo chicken embryos, and purifying the PGCs by a differential wall-pasting method. The culture methods of PGCs were systematically compared in terms of whether or not a feeder layer was added, the method of treating the feeder layer, the ratio of the addition of the conditioned medium, and the like.

The results showed that PGCs were cultured for about 300 days on feeder cells treated with 20. mu.g/ml mitomycin C for 2 hours and in a complex culture system in factor medium supplemented with 50% BR L conditioned medium, and that PGCs cultured for a long period in this culture system maintained high proliferative capacity without transformation, and their pluripotency, migratory ability and differentiative ability.

The invention has the following beneficial effects:

the method for culturing chicken PGCs in vitro for a long term is obtained through experiments such as respectively treating the feeder layer cells with mitomycin C with various concentrations (10, 15, 20 and 30 mug/ml) for different times (2, 3 and 4h) according to the addition of the feeder layer cells, adding BR L conditioned medium with different proportions (0%, 20%, 40%, 50%, 60% and 80%) into the culture medium, and the like.

In order to fully exert the value of PGC used as a transgenic tool and a bioreactor carrier and simultaneously more conveniently and effectively utilize the chicken gonad PGC, the invention researches the optimal culture system for culturing the chicken gonad PGC for a long time, thereby fully excavating the research value of the chicken gonad PGC and laying a solid foundation for later-stage PGC establishment and even production of transgenic chickens.

Drawings

FIG. 1 is a schematic diagram showing the isolation and culture of PGCs in the present invention;

in the figure: A-D are morphologies of the cultured PGCs;

a is that PGCs are separated at the 27 th embryonic stage (C arrow), and are subjected to primary culture together with chick embryo gonad stromal cells (B arrow) and blood cells (A arrow);

b is PGC aggregation for 3 days of primary culture (C arrow);

c is the growth of PGCs which are difficult to maintain by gonadal stromal cells after 7 days of primary culture (C arrow);

d is subculture of PGCs on mitomycin C treated feeder cells (C arrow);

e is the morphology of STO cells;

f is the morphology of BR L cells;

FIG. 2 shows a complex culture system for PGCs;

in the figure: a is the form of PGC with feeder cells and the form of PGC 5 days after no addition of bFGF;

b is the cell proliferation rate of PGC with feeder cells and 5 days after the addition of bFGF;

c is that when the cell fusion degree reaches 60%, mitomycin C with various concentrations (10, 15, 20 and 30g/ml) is respectively used for treating feeder layer cells for 2, 3 and 4 hours, and CCK-8 is used for detecting the influence of the mitomycin C treatment on cell proliferation and vitality;

d is the growth curve of STO cells treated with mitomycin C at different concentrations;

e morphology of mitomycin C treated STO cells at different concentrations;

f is the cell proliferation rate of PGCs supplemented with BR L Conditioned Medium (CM) and 5 days later without BR L conditioned medium;

g is a conditioned medium which is added into the culture medium in a proportion of 0%, 20%, 40%, 50%, 60% and 80%, and the proliferation rate and the activity of the PGCs are detected by CCK-8;

FIG. 3 shows that PGC cell lines maintain high proliferation potency without transformation

In the figure: a is a growth curve of PGCs separately cultured by the culture system;

b is ZW type chicken PGCs (female parent) metaphase (left) chromosome and karyotype (right), and the chromosome karyotype is still diploid;

C-E cell cycle analysis of freshly isolated PGCs (C), thawed PGCs (D), and long-term cultured PGCs (E);

f is the colony forming ability of PGCs; colony forming ability of hela cells (positive control) (top) and PGCs (bottom) at 1, 3, 5, 7 days was measured by soft agar method;

g is the number of clones measured by the soft agar method under a stereomicroscope after crystal violet staining;

h is RT-PCR analysis of telomerase reverse transcriptase in PGCs cultured in vitro (cultured for 15, 68, 180 and 268 days and thawed PGCs);

FIG. 4 shows that PGCs cultured for a long period of time have high pluripotency and differentiation potential;

in the figure, A-D are immunocytochemical analyses of cultured PGCs, and 180 days after the culture, immunohistochemical staining was performed with antibodies against SSEA-1(A), C-kit (B), cDAZ L (C) and Sox2(D), respectively

E is RT-PCR analysis of in vitro cultured PGCs (cultured for 15, 68, 180, 268 days and thawed) POUV, NANOG, DAZ L;

f is migration of the cultured PGCs to the crescent area of the reproductive system; approximately 3000 PGCs cultured for 200 days were labeled with PKH26 and then transferred to the subgerminal cavity of embryonic blastoderms; labeled cells (red) were detected in the crescentic region of reproduction;

g is gonad migration of the cultured PGCs; PKH 26-labeled PGCs on the left, and controls on the right; approximately 3000 PGCs cultured for 230 days were labeled with PKH26 and then injected into the blood vessels of recipient embryos at stages 14-17; detection of labeled cells in the embryonic gonads (red);

h isolating cells from gonads of PGCs labeled with PKH 26;

i is the formation of Embryoid Bodies (EB) of PGCs cultured for 248 days, and 2 mu mol/L of RA is used to induce PGCs cultured for a long time, and after 7 days, simple embryoid bodies (upper) grow and can continue to grow for 14 days (lower).

Detailed Description

A method for in vitro long-term culture of chicken PGCs comprises the following specific steps:

1. materials and methods

1.1 isolation, purification and culture of PGCs

Fertilized eggs (Babcock B300 strain; Yangzhou Rui agricultural technologies, Inc.) were incubated at 37 ℃ and 70% relative humidity. After 120H incubation, gonadal tissue was extracted from embryos at about H & H27 stage under a stereomicroscope with fine forceps, PGCs were isolated by enzymatic digestion, and cells were further purified by differential adherence.

PGCs were placed on a feeder layer of mitomycin C-treated STO and cultured in an incubator at 37 ℃ with 5% CO2 and 60-70% relative humidity, the medium consisted of KO-DMEM, and 10% Fetal Bovine Serum (FBS), 2.5% Chicken Serum (CS), 2mM L-glutamine, 1 × MEM nonessential amino acids, 1 × nucleosides, 1 × HEPES, 0.1mM β -mercaptoethanol, 5ng/ml human stem cell factor (hSCF), 10ng/ml basic fibroblast growth factor (bFGF) and 10ng/ml mouse leukemia inhibitory factor (L IF) were added to the medium.

1.2 treatment of feeder layers

In the incubator, STO was cultured in DMEM medium containing 10% FBS. To determine the optimal concentration for feeder cells, we treated STO 2, STO 3, and STO 4h with mitomycin C at various concentrations (10, 15, 20, and 30. mu.g/ml) after 60% confluence, and then examined cell viability using CCK-8 assay. To prevent excessive toxicity of mitomycin C, STO was subcultured on complete medium for at least 24h, after which the inactivated STO was used as a feeder layer for PGC.

1.3 preparation of conditioned Medium

BR L was cultured in KO-DMEM medium containing 10% FBS, 2mM L-glutamine, the medium was changed within 24h to remove debris and waste from the cells after passaging, conditioned medium was collected, filtered, and then added to PGC medium after 2 days of medium change, and to test the optimum addition ratio of BR L conditioned medium, we then added 0%, 20%, 40%, 50%, 60%, 80% conditioned medium, respectively, and tested the viability of the cultured PGCs with CCK-8.

1.4 cryopreservation and thawing of PGCs

The cultured PGCs were collected and resuspended in cryoprotectant containing 10% DMSO and 90% FBS, each tube was frozen 2 × 10⁶-1×10⁷The individual cell cryovials were placed in a freezing container with isopropanol and stored overnight in a freezer at-80 ℃. The sample in the cryovial was heated in a water bath at 37 ℃ and then shaken until the ice crystals disappeared. The antifreeze was diluted 10-fold in 1 minute.

1.5 cell viability assay

To measure cell viability, cell counting kit 8(CCK-8) was used according to the instructions, cells were seeded into 96-well plates, after approximately 24h of culture, treated according to the instructions CCK-8(10 μ l) was added per well, incubated for 1.5 hours, and then cell viability was measured, absorbance was measured at 450nm with a Microplate reader, cell viability was calculated according to the formula (control OD-treated OD)/(control OD-blank OD) × 100%, assay was repeated 3 times.

1.6 immunofluorescence

Cultured PGCs were seeded into 24-well plates with treated feeder layers and incubated for about 24 h.cells were fixed in freshly prepared 4% paraformaldehyde-PBS for 20min, then washed 3 times with Phosphate Buffered Saline (PBS), then samples were treated with 0.5% Triton X-100 permeabilization buffer for 20min after 3 washes with PBS, cells were blocked with 1% bovine serum albumin blocking buffer for 30min, then incubated with primary antibody (anti-SSEA-1, c-kit, Sox2, cDAZ L) diluted to 5-20 μ g/m L in BSA at 4 ℃ overnight protected from light, after 3 washes with PBS containing 0.05% Tween-20 (PBS-T), cells were incubated with secondary antibody conjugated with fluorescent dye for 1h at room temperature, finally cells were counterstained with DAPI for 10min and analyzed under fluorescent microscope.

1.7 cell cycle analysis

To analyze cell cycle and apoptosis of freshly isolated PGCs, thawed PGCs and long-term cultured PGCs, a cell cycle and apoptosis assay kit (Beyotime) was used according to the instructions. Harvested cells were fixed with 70% ethanol overnight and then incubated with RNase for 5 min. After addition of Propidium Iodide (PI) at 37 ℃ away from light for 30min, the DNA content was analyzed using a FACSCalibur flow cytometer over 24 h.

1.8 Soft agar assay

1.2% melted agar was cooled to 37 ℃ and mixed with 2 × DMEM containing 20% fetal bovine serum as the basal layer, the final concentration of agarose being 0.6%. cells suspended in 0.3% agarose (1000 cells/m L) were then seeded onto the basal layer (an appropriate amount of medium was added to keep moist). 7d the colony formation was observed under a microscope, and the visible colonies were stained with crystal violet and counted under a stereomicroscope.

1.9RT-PCR analysis

Total RNA of the cultured PGCs was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA) (cultured for 15, 68, 180 and 268 days and thawed), and the First Strand cDNA library was reverse transcribed using Transcriptor First-Strand cDNA Synthesis and the target cDNA was amplified by RT-PCR. The reaction involved 35 cycles (95 ℃ 20s, 60 ℃ 40s, 72 ℃ 1 min). GAPDH expression was used as a reference to normalize expression levels.

1.10 karyotyping

The cultured PGC was mixed with colchicine (final concentration 0.25. mu.g/ml) at 37 ℃ in 5% CO₂After 4h, the cells were harvested, centrifuged and resuspended in 0.075M KCl preheated to 37 ℃. After incubation at 37 ℃ for 15min, the cells were pelleted again, and the cells were incubated with 3: 1 methanol: fix with acetic acid and wash 3 times with fixative. Finally, the cell suspension was dropped onto a cooled glass slide and stained with Giemsa.

1.11 gonadal migration test

Long-term cultured PGCs were used for migration assays. To determine the migration of PGCs into the crescentic region of reproduction, cells were labeled with PKH26 red fluorescent cell linker minikit (Sigma), 3000 cells per egg were transferred into the subgerminal cavity of the stage X recipient embryo. Incubation of the Parafilm-sealed recipient eggs was continued for 24h until stage 6, and the crescentic area of the in vitro embryos was observed under a fluorescent microscope. PKH 26-labeled PGCs were injected into the dorsal aorta of stage 14-17 embryos to analyze PGC migration to the gonads. It was sealed with Parafilm and incubated to stage 30 and the gonads were observed under a fluorescent microscope.

1.12RA Induction assay

PGCs cultured by induction with Retinoic Acid (RA) were differentiated into germ cells to test their differentiation ability, and the PGCs were cultured in a feeder-free complete medium containing 2. mu.M/M L RA, and their morphological changes were observed by an inverted microscope.

1.13 statistical analysis

All results are expressed as mean values. Statistical analysis and graphical representation were performed using GraphPad Prism 6 software. P values less than 0.05 were considered significant, and P values less than 0.01 were considered very significant.

As can be seen from FIGS. 1 and 2, the chicken PGCs were cultured in vitro for a long period in a complex culture system of STO feeder cells treated with mitomycin C at 20. mu.g/ml for 2 hours and factor medium supplemented with 50% BR L conditioned medium.

As can be seen from FIGS. 3 and 4, PGCs cultured in vitro for a long period of time still have high proliferative capacity without transformation, and still have high pluripotency and differentiation capacity.

Claims

1. A method for long-term in vitro culture of chicken PGCs is characterized in that chicken PGCs are obtained by separation, and then the PGCs are purified and cultured; as for the culture of PGCs, the feeder layer is treated and a conditioned medium is added, and PGCs can be cultured in the complex culture system for a long period of time, maintain high proliferation ability without transformation, and maintain pluripotency, migration ability and differentiation ability.

2. The method of claim 1, wherein the step of isolating and purifying the PGCs comprises:

incubating the fertilized egg at 37 ℃ and 70% relative humidity; after 120H incubation, gonadal tissue was extracted from H & H27 stage embryos using fine forceps under a stereomicroscope, PGCs were isolated by enzymatic digestion, and cells were further purified by differential adherence.

3. The method of claim 2, wherein the culturing of the PGCs comprises:

placing PGCs on a feeder layer of mitomycin C-treated STO, and culturing in an incubator at 37 deg.C, 5% CO2, and 60-70% relative humidity;

the culture medium is composed of KO-DMEM, and 10% fetal calf serum, 2.5% chicken serum, 2mM L-glutamine, 1 × MEM nonessential amino acids, 1 × nucleosides, 1 × HEPES, 0.1mM β -mercaptoethanol, 5ng/ml human stem cell factor, 10ng/ml basic fibroblast growth factor and 10ng/ml mouse leukemia inhibitory factor are added into the culture medium;

in subculture, the medium in which the PGCs were cultured was changed every day, and the cells were dissociated with Accutase and passaged onto newly treated feeder cells.

4. The method of claim 3, wherein the feeder layer is treated by: feeder cells were treated with 20. mu.g/ml mitomycin C for 2 h.

5. The method of claim 4, wherein the ratio of BR L conditioned medium to total medium is 50% BR L conditioned medium.