CN115505560B - A method for culturing chicken yolk follicles in vitro - Google Patents
A method for culturing chicken yolk follicles in vitro Download PDFInfo
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Abstract
本发明公开了一种体外培养鸡小黄卵泡的方法。涉及家禽卵泡体外培养技术领域。包括将筛选后的小黄卵泡转移到PET细胞培养小室上层，并加入预热好的生长培养基，得培养物；将培养物转移到38.5℃，5％CO2的环境下培养。本发明采用PET细胞小室进行体外卵泡培养，进一步模拟体内环境在体外给予卵泡支撑功能；卵泡在38.5℃，5％CO2的培养箱中培养更接近于鸡体内的温度，模拟真实情况；在DMEM培养基中添加活性VD3，调控体外培养系统类固醇的代谢，促进卵泡的增殖和存活。
The present invention discloses a method for culturing chicken yolk follicles in vitro. It relates to the technical field of poultry follicle in vitro culture. The method comprises transferring the screened yolk follicles to the upper layer of a PET cell culture chamber, adding a preheated growth medium, and obtaining a culture; transferring the culture to a 38.5°C, 5% CO2 environment for culture. The present invention adopts a PET cell chamber for in vitro follicle culture, further simulating the in vivo environment to provide follicle support function in vitro; the follicles are cultured in an incubator at 38.5°C, 5% CO2, which is closer to the temperature in the chicken body and simulates the real situation; active VD3 is added to the DMEM culture medium to regulate the metabolism of steroids in the in vitro culture system and promote the proliferation and survival of follicles.
Description
Technical Field
The invention relates to the technical field of poultry follicle in-vitro culture, in particular to a method for in-vitro culture of chicken small yellow follicles.
Background
Most of the follicles of the laying hens are blocked in the follicular development process, only a few follicles can be further developed and matured through follicular selection, and the small yellow follicular stage is a key stage for further development and accumulation of vitelline substances from a white follicular bank. Unlike mammals, the synthesis and secretion of various steroid hormones in the follicular development process of poultry require the co-participation of granulosa cells and membranous cells of the follicles, which are a continuous and complex process, the follicular development of poultry is subject to various effects of microenvironment growth factors, steroid hormones, etc. related thereto in addition to the granulosa cells and membranous cells of the follicles, which have an important role in the follicular development.
Current follicular cell research is limited primarily to the inability to accurately replicate phenotypic and genetic characteristics in vivo, and conventional methods of in vitro culture of poultry follicular primary granulosa cells and membranous cells are inadequate to reflect the actual changes in follicular development in poultry.
Compared with the conventional cell culture, the in-vitro culture model of the chicken follicles has lower cost, higher efficiency and shorter time consumption, and attempts to simulate the change of the follicles in the chicken in different states, so that the integrity of the tissue is maintained as much as possible to reflect the real process of the chicken follicles in the development process.
Therefore, the establishment of an efficient in-vitro follicular culture model of small yellow follicles has important significance for researching the development of poultry follicles and the mechanism of micro-environmental factors on the regulation and control of the poultry follicles.
Disclosure of Invention
In view of this, the present invention provides a method for in vitro culturing chicken small yellow follicles. Compared with the conventional granular cell or membrane cell culture, the process flow is shown in the figure 1, the interaction among cells is reserved, the real situation of the follicles in the internal environment can be etched more deeply, the defect that the traditional primary follicle cell culture period is long, survival is difficult and pollution is easy is overcome, the gap of in-vitro culture of small yellow follicles of poultry is filled, and a rapid and efficient in-vitro poultry follicle culture model is established.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
A method for in vitro culturing chicken small yellow follicles, comprising the steps of:
1) Transferring the small yellow follicles after screening to the upper layer of a PET cell culture chamber, and adding a preheated growth medium to obtain a culture;
2) The culture was transferred to 38.5℃and incubated in an environment of 5% CO 2.
The beneficial effects are that: the temperature of the culture is increased to be 40.8-41.5 ℃ closer to the temperature in the chicken body.
Preferably: criteria for step 1) screening: the surface of the structure is complete, has no blood stain, and has the diameter of 6-8 mm; PET cell culture chamber: 24 wells with 0.45 μm, each well containing 1 small yellow follicle; preheating: preheating is carried out in a water bath kettle at 37 ℃ for 40min, and the addition amount of the growth medium is as follows: 1mL per well.
The beneficial effects are that: compared with mammal, the ovary of poultry has follicles with different continuous sizes in each period and does not need synchronous estrus, and the materials are convenient to obtain and are ideal models for researching the development of follicles;
The PET cell culture chamber was used as a permeable scaffold for follicular culture.
Furthermore, the follicle is positioned on the upper layer filter membrane of the culture chamber, the pore diameter is close to the diameter of the follicle, so that the in-vivo follicle supporting effect of connective tissue connected with the ovary in vitro can be simulated, and the complexity and the dynamics in the development process of the follicle can be summarized;
penicillin-streptomycin solution, ITS broth supplement, and fetal bovine serum FBS are all volume ratios.
Preferably: adding humidity balancing liquid into gaps of holes of the PET cell chamber, wherein the humidity balancing liquid is as follows: 20. Mu.L of sterilized PBS.
Preferably: growth medium: DMEM medium containing 1% penicillin-streptomycin solution, 1% its broth supplement, 5% fetal bovine serum FBS and 1000nM active VD 3.
The beneficial effects are that: the DMEM culture medium is added with 1% of diabody, so that the possibility of pollution in the sampling and transferring process is reduced;
Active VD3 has the functions of promoting the proliferation of follicular cells and the synthesis of estrogen and progestogen, makes up the defect of an in vitro culture system compared with an in vivo development follicular system, and promotes the proliferation and survival of the follicular cells.
Preferably: time of incubation in step 2): 72h.
Preferably: step 2) changing the growth medium once every 24 hours, and collecting the replaced growth medium for measuring the content of estrogen and progestogen when the growth medium is changed.
Preferably: step 2) observing the growth condition of follicles and the consumption condition of a culture medium every 12 hours, and carrying out cross shaking: the frequency is 20-30 times per minute, and the shaking is 10 times.
The beneficial effects are that: ensuring that tissues fully contact with gas and simulating the dynamic environment in the body.
The invention also provides the use of any of the methods described above in co-culture with granulosa cells or membranous cells.
Further: the cell laying at the lower layer realizes the co-culture with granular cells or membranous cells, and better simulates the action and function of cells in the follicular development process.
The invention also provides chicken small yellow follicles cultured by any one of the methods.
Compared with the prior art, the invention discloses a method for in vitro culturing chicken small yellow follicles, which has the following technical effects:
1) The small yellow follicles are separated by adopting a pure mechanical method under the aseptic condition, so that the integrity of the follicles is reserved to the greatest extent, and the influence on the structural integrity and development potential of the follicles is reduced.
2) In vitro follicular culture was performed using 24-well 0.4 μm PET cell chambers, further simulating in vivo environment giving follicular support function in vitro.
3) The follicles were cultured in an incubator at 38.5 ℃ with 5% co 2 closer to the temperature in the chicken body, simulating the real situation.
4) Active VD3 is added into the DMEM culture medium, so that the metabolism of steroid in an in-vitro culture system is regulated and controlled, and the proliferation and survival of follicles are promoted.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the flow of in vitro culture of small yellow follicles.
FIG. 2 is a drawing showing small yellow follicles of 6-8 mm diameter provided by the present invention.
FIG. 3 is a diagram showing the culture of small yellow follicles in a PET cell culture chamber.
Fig. 4 is a diagram showing the morphology of follicles detected by HE staining provided by the present invention.
FIG. 5 is a diagram showing the proliferation of Brdu detected by the present invention with or without active VD3 follicle.
FIG. 6 is a graph showing the trend of the estrogen content in the culture solution with or without the activity VD3 provided by the invention.
FIG. 7 is a graph showing the trend of the progestogen content in the active or inactive VD3 culture medium provided by the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The embodiment of the invention discloses a method for in vitro culture of chicken small yellow follicles.
In the examples, the test materials not mentioned are all conventional commercial materials, and the test methods not mentioned are conventional test methods, for example, penicillin-streptomycin solution (diabody); gibco, lot number: 15140-122
DMEM medium; gibco, lot number: c11995500BT
ITS broth supplement (100 x); sigma lot number: i3146
Fetal bovine serum FBS (FetalBovineSerum); gibco, lot number 10099-133
A PET cell culture chamber; LABSELECT; lot number: 14341
Active VD3 (powder) (Calcitriol); MCE; lot number: HY-10002
5-Bromodeoxyuridine: brdU; MCE, lot number: HY-15910
General tissue fixative: a Boerf organism; lot number B0038
Muscle fixing liquid: a Boerf organism; lot number: b0007
(Sterile) PBS: 16g of NaCl and 0.48g of Na 2HPO412H2O5.8g,KCl0.2g,KH2PO4; adjusting pH to 7.2, fixing volume to 2L, sterilizing under high pressure after aseptic filtration, and adding 1% penicillin-streptomycin solution into sterilized PBS (1% diabody is added into PBS used in test);
Dissecting the culture medium: subpackaging DMEM culture medium into 50ml centrifuge tubes under aseptic condition, adding 1% penicillin-streptomycin solution (preserving at 4deg.C refrigerator for transferring small yellow follicles after separation), placing the centrifuge tubes filled with culture medium and follicles into ice box to carry to cell house after sampling);
active VD3 working fluid: 10mg of active VD3 powder stored in a refrigerator with the temperature of-80 ℃ is added with absolute ethyl alcohol under the condition of light shading to prepare 10mM of active VD3 mother liquor, the mother liquor is diluted (0-1000 nM) as working solution for standby according to the requirement, and the mother liquor is stored for one month at the temperature of-20 ℃ and is stored for 6 months at the temperature of-80 ℃ in the refrigerator;
Growth medium: taking 5mL of sterile FBS, adding 1mL of penicillin-streptomycin solution and 1mL of ITS culture medium supplement, and adding 93mLDMEM basic culture medium and 10 μl of active VD3 mother liquor to prepare 1000nM active VD3 culture medium, wherein the culture medium is ready to be prepared;
Small yellow follicles are obtained from isolated small yellow follicles or prepared by the following method:
1) The jugular vein of the sea-blue brown laying hen (35-45 weeks old) with regular egg producing period is selected to bleed and kill, the abdominal cavity is rapidly dissected to remove the whole complete ovary, the surface blood stain (blood and grease on the surface of the follicle are taken out as much as possible) is rinsed by precooled PBS under aseptic condition, small yellow follicles with the diameter of 6-8 mm are picked up according to the diameter of the follicles and placed in a 50mL sterile centrifuge tube, and the sterile centrifuge tube is temporarily preserved by using an anatomical culture medium (DMEM culture medium containing 1% double antibody).
2) The follicles were placed in an ice box to be carried to the cell house, and transferred to an ultra clean bench sterilized for more than 30 minutes for manipulation. Rinsing small yellow follicles in a culture dish for 3-5 times by using precooled sterile PBS, clamping one end of the follicles connected with an ovary matrix by using sterile pointed cell forceps for macroscopic blood stains, slightly scraping the blood stains on the surfaces of the follicles by using other forceps, slightly cross-shaking the culture dish during rinsing to clean the blood stains on the surfaces of the follicles as much as possible, and finally shearing redundant connective tissues on the follicles by using ophthalmic scissors, so that the whole-process action is gentle and the structural integrity of the follicles is maintained.
Hematoxylin-eosin (H-E) staining:
1) Preparation of paraffin sections: and after the culture is finished, placing the small yellow follicles in a general tissue fixing solution for fixing for 24 hours, and then replacing the small yellow follicles with a muscle fixing solution for fixing for 24 hours. Washing and dewatering, transparentizing, wax dipping, embedding, slicing (selecting the largest surface of the follicle) and baking the slices are carried out according to a conventional method.
2) Hematoxylin-eosin (H-E) staining: dewaxing, soaking, nuclear dyeing, color separation, bluing, mass dyeing, dewatering, transparency and sealing.
Example 1
Method for in-vitro culture of chicken small yellow follicles
1) The prepared growth medium is placed in a water bath kettle with the temperature of 37 ℃ for preheating in advance, small yellow follicles with complete structure and no blood stain on the surface are selected and separated from a culture dish (see figure 2), and the small yellow follicles are transferred to the upper layer of a PET cell culture cell with the diameter of 24 holes and the diameter of 0.45 mu m (a humidity balance liquid is added into gaps of each hole of the PET cell, and the humidity balance liquid: 20 μl of sterilized PBS), 1 small yellow follicle per well was placed and 1mL of growth medium (DMEM medium containing 1% penicillin-streptomycin solution, 1% its medium supplement, 5% fetal bovine serum FBS and 1000nM active VD 3) was added (fig. 3A, B). All cultures were transferred to a cell incubator at 38.5 ℃,5% co 2, and incubated for 72 hours, with replacement of the growth medium every 24 hours (replacement, collection of the growth medium under replacement for estrogen and progestogen content determination), observation of follicular growth and medium consumption at 12 hours intervals, and shaking (cross shaking: frequency 20-30 times per minute, shaking 10 times) to simulate the dynamic environment in vivo.
2) The follicular medium was collected with a sterile 1.5mLEP tube at each change of fluid (growth medium) for determination of estrogen and progestin content, and 20 μg/mL BrdU was added to the growth medium at 48 hours of culture to examine proliferation of small yellow follicles in an in vitro culture environment.
Operating requirements (emphasis)
1) Aseptic manipulation is required throughout the process.
2) The follicular isolation and transfer were controlled to be within 40-60 minutes (4-6 hen ovaries).
3) The follicles were taken out of the incubator for no more than 10 minutes each time.
4) The medium was changed every 24 hours and the shaking was performed every 12 hours.
5) Incubator temperature 38.5deg.C, 5% CO 2 conditions were used for cultivation.
The technical effects are as follows:
Survival and growth of small yellow follicles
And selecting follicles with normal morphology, complete structure and clean diameter of 6-8 mm, and culturing in vitro for 72 hours. The small yellow follicles can maintain normal form in the in-vitro culture process (figure 4), the in-vitro proliferation of the small yellow follicles is detected by BrdU, and the proliferation of the in-vitro follicles is promoted by adding 1000nM active VD3 working liquid under the same condition (figure 5), so that the survival and the growth of the small yellow follicles in the in-vitro culture environment are successfully realized. 24. The concentration changes of estrogen and progestogen which are main functional products of follicular synthesis in the culture solution are detected at 48 and 72 hours, and the addition of the culture medium with the activity of VD3 of 1000nM under the same conditions can significantly improve the content of estrogen and progestogen and show obvious promotion effect with the increase of time (figures 6 and 7).
In addition, the invention repeatedly tests, and the test result is successful, thus realizing the purpose of the invention.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (3)
1. A method for in vitro culturing chicken small yellow follicles, comprising the steps of:
1) Transferring the small yellow follicles after screening to the upper layer of a PET cell culture chamber, and adding a preheated growth medium to obtain a culture;
2) Transferring the culture to 38.5 ℃ and culturing in the environment of 5% CO 2;
Criteria for screening as described in step 1): the surface of the structure is complete, has no blood stain, and has the diameter of 6-8 mm; the PET cell culture chamber: 24 wells with 0.45 μm, each well containing 1 small yellow follicle; the preheating: preheating is carried out in a water bath kettle at 37 ℃ for 40min, and the adding amount of the growth medium is as follows: 1mL per well;
and adding humidity balance liquid into gaps of holes of the PET cell chamber, wherein the humidity balance liquid is as follows: 20. Mu.L of sterilized PBS;
The growth medium: DMEM medium containing 1% penicillin-streptomycin solution, 1% its broth supplement, 5% fetal bovine serum FBS and 1000nM active VD 3;
step 2) the time of incubation: 72h;
Step 2) changing the growth medium once every 24 hours, and collecting the replaced growth medium for measuring the content of estrogen and progestogen when the growth medium is changed.
2. The method for in vitro culturing chicken small yellow follicles according to claim 1, wherein step 2) is performed by observing the growth of follicles and the consumption of medium every 12 hours, and performing cross shaking, wherein the cross shaking: the frequency is 20-30 times per minute, and the shaking is 10 times.
3. Use of the method according to any one of claims 1-2 for co-cultivation with granulosa cells or membranous cells.
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| 活性VitD3调控蛋鸡卵泡发育和产蛋性能的机制;程漫漫;博士电子期刊;20231215(第12期);第1-142页 * |
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