CN113481151B - In-vitro culture method for chicken small yellow follicular membrane cells for improving cell survival rate - Google Patents
In-vitro culture method for chicken small yellow follicular membrane cells for improving cell survival rate Download PDFInfo
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- 238000012136 culture method Methods 0.000 title claims abstract description 14
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Abstract
The invention discloses an in-vitro culture method of chicken small yellow follicular membrane cells for improving cell survival rate, and relates to the technical field of bioengineering; the method specifically comprises the following steps: mixing the membrane layer of chicken small yellow follicles with collagenase for digestion, and filtering after digestion to obtain cell suspension; centrifuging the obtained cell suspension to obtain a precipitate, namely chicken small yellow follicular membrane cells; mixing and culturing the obtained chicken small yellow follicular membrane cells with a cell culture solution to obtain a chicken small yellow follicular membrane cell line; the in-vitro culture method can obtain a large number of chicken small yellow follicular membrane cells in a short time, has higher survival rate which reaches more than 95 percent, and ensures the successful implementation of subsequent experiments of the chicken small yellow follicular membrane cells.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an in-vitro culture method for chicken small yellow follicular membrane cells, which improves the cell survival rate.
Background
Development of hen ovaries and follicles is a highly coordinated complex physiological process regulated by various endocrine, paracrine and autocrine processes. In sexually mature hen ovaries, follicles exist at various stages of development, at the early stages of follicular development, somatic cells around the oocytes differentiate and tightly bind together, develop into primordial granulosa cells, and thus primordial follicles begin to form. When primordial follicles are selected to develop into primary follicles, the mesenchymal cells differentiate into membranous cell layers. The granulosa cells and the membranous cells are separated by a basal membrane composed of non-cellular components, effectively preventing direct contact of blood with the granulosa cells. The initial stage of the white follicle has only one layer of granulosa cells, and the granulosa cells are divided into two layers from the late stage of the development to the yellow follicle stage, and become multiple layers, and gradually become one layer of granulosa cells when the granulosa cells develop into the grade follicle. And the single-layer membrane cells finally develop into inner and outer membranes with obvious differences in morphology and function. The follicle consists of oocytes, granular cells and membranous cells, wherein the oocytes are wrapped outside the follicle, the follicle can be roughly classified into a pre-grade follicle and a grade follicle according to the size, the development and the ovulation follow a strict grade system, F1 is the follicle to be ovulated, F2, F3 and the like are next, and after F1 is ovulated, one of small yellow follicles with the size of 6-8mm is selected to enter the development stage of the grade follicle. Thus follicles in the 6-8mm developmental state are key points in follicle selection for access to grade follicles. The follicular membrane cells mainly produce luteinizing hormone, have the function of promoting the conversion of cholesterol into sex hormone in vivo, promote follicular maturation under the combined action of the follicle stimulating hormone, secrete estrogen, promote ovarian ovulation, and produce luteum and maintain the function of luteum after the ovarian ovulation. The follicular membrane cells also produce androgens, while the estrogens promote the maturation of the follicles, which then, after the ovulatory period, are expelled from the body to form the function of ova. Thus, the egg cells are ideal models for in vitro research of follicular development regulation and control mechanisms.
In the research report, no report of in-vitro culture of chicken small yellow follicular membrane cells is seen, so that an effective separation, purification and culture method is established, is a precondition for application of chicken follicular membrane cells, has high efficiency in separating membrane cells, high purity and low price, is easy to popularize, and has important significance for further researching the characteristics of poultry germplasm resources and exploring follicular development regulation mechanisms.
Disclosure of Invention
The invention aims to provide an in-vitro culture method of chicken small yellow follicular membrane cells for improving cell survival rate, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides an in vitro culture method of chicken small yellow follicular membrane cells for improving cell survival rate, which specifically comprises:
(1) Mixing the membrane layer of the isolated chicken small yellow follicles with collagenase for digestion, and filtering after digestion to obtain a cell suspension;
(2) Centrifuging the cell suspension obtained in the step (1), wherein the obtained precipitate is chicken small yellow follicular membrane cells;
(3) And (3) mixing and culturing the chicken small yellow follicular membrane cells obtained in the step (2) with a cell culture solution to obtain a chicken small yellow follicular membrane cell line.
Further, the preparation method of the membrane layer of the isolated chicken small yellow follicles comprises the following steps: removing the granular layer of the chicken small yellow follicles to obtain the membrane layer of the chicken small yellow follicles.
Further, the collagenase is a type II collagenase.
Further, the membrane layer of the isolated chicken small yellow follicles is the membrane layer of the broken chicken small yellow follicles.
Further, the cell culture broth was prepared from fetal bovine serum, chicken serum, DMEM, and diabodies at a ratio of 2: 8:0.001.
Further, the diabodies are penicillin and streptomycin.
Further, the penicillin working concentration is 100U/ml; the working concentration of the streptomycin is 0.1mg/ml.
The invention discloses the following technical effects: the in-vitro culture method can obtain a large number of chicken small yellow follicular membrane cells in a short time, has higher survival rate which reaches more than 95 percent, and ensures the successful implementation of subsequent experiments of the chicken small yellow follicular membrane cells.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments 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.
In order that the above-recited objects, features and advantages of the present invention will become more apparent, a more particular description of the invention will be rendered by reference to specific embodiments thereof.
Example 1 in vitro culture method of chicken small yellow follicular membrane cells for increasing cell survival rate
First, preparation of culture solution
1Wt% of diabody and 10% of chicken serum were added to DMEM basal medium.
Second, the hen is sterilized and small yellow follicles are taken out
Selecting a hen which enters the egg laying peak period and continuously lays eggs, filling 75% alcohol in a large 2000ml beaker, soaking the killed hen in the beaker, taking out after 2-3 minutes, opening the abdominal cavity by using a sterilizing scalpel, taking out the whole ovarian tissue by using a sterilizing dissecting scissors, separating small yellow follicles with the size of 6-8mm and placing the small yellow follicles in a sterilizing culture dish.
Third, stripping connective tissue and trimming membranous tissue
After the connective tissue layer of the small yellow follicle is stripped by using elbow tweezers, the elbow tweezers are reversely fixed to the small yellow follicle, after the small yellow follicle is sheared by using ophthalmic scissors, yolk is gently extruded, the small yellow follicle is further sheared by using the ophthalmic scissors, the inner layer of the follicle membrane is turned out, the membrane layer is clamped by using the tweezers, the particle layer is rinsed clean in a culture dish containing sterilized PBS, and 5-7 membrane layers (inner layer and outer layer) of the small yellow follicle are taken according to the method. The membrane layer is washed three times by D-Hank's, and the membrane layer is sheared into a viscous state in a culture dish by elbow scissors to be digested.
Fourth step, digestion and purification
Adding 10ml of 2% type II collagenase into a viscous membrane tissue culture dish, fully mixing follicular membrane tissues and type II collagenase by a pipettor, transferring the type II collagenase containing follicular membrane tissues into a 50ml centrifuge tube together, sealing the sealing membrane, putting the centrifuge tube into a constant temperature incubator at 38 ℃ for digestion, shaking uniformly every 5 minutes, taking out the incubator after 15 minutes, stopping digestion, placing a 200-mesh and 400-mesh filter screen on a culture dish with the diameter of 10cm for filtration, pouring the digestion liquid into the 200-mesh filter screen, removing undigested tissues, filtering the filtered cell suspension by 400-mesh, adding the filtered cell suspension into a new centrifuge tube, centrifuging for 7 minutes at 1200 turns, sucking the supernatant by the pipettor, adding D-Hanks for blowing and cleaning, centrifuging for 4 minutes at 1000 turns after resuspension, sucking the supernatant, and obtaining the lower-layer sediment.
Fifth step, cell plating and differential adherence
5Ml of cell culture solution is added into a centrifuge tube of the obtained cells, the cells are blown and resuspended, the action is gentle when blown so as not to damage the cells, the cell suspension is transferred into a cell culture dish with the diameter of 10cm, and the cell culture solution is added to 10ml and then is placed into an incubator for normal culture.
50Ml cell culture broth preparation: 10ml chicken serum, 40ml DMEM and 5ul of diabody (penicillin working concentration 100U/ml, streptomycin working concentration 0.1 mg/ml).
Sixth step, counting and plating
2Ml of cell culture solution is added into a centrifuge tube, membrane cells are blown and resuspended, 10 mu L of cell suspension is added into a 200ul PCR tube, 10 mu L of trypan blue cell reactive dye is added, and after uniform mixing, the cell counting plate is dripped for counting. According to the counting result, the cell suspension is plated according to the density of 10 5-106, then a proper amount of cell culture solution (2 mL of each six-hole plate and 200 mu L of each 96-hole plate) is added for gentle blowing and beating uniformly, and the uniformity of the plating is required.
The results were averaged by repeating the experiment three times.
Results: by using the extraction method of the embodiment 1, a large amount of membrane cells can be quickly released in a short time, the survival rate is high and reaches more than 95%, and the subsequent test of the membrane cells is ensured to be smoothly carried out.
Comparative example 1
The chicken small yellow follicular cells were extracted and cultured in the same manner as in example 1, except that the membrane layer in the third step was only an inner layer. The results were averaged by repeating the experiment three times.
Results: with the extraction method of comparative example 1, the time to release the membrane cells was longer, and the average survival rate was 52%.
Comparative example 2
The chicken small yellow follicular cells were extracted and cultured in the same manner as in example 1, except that the membrane layer in the third step was only the outer layer. The results were averaged by repeating the experiment three times.
Results: with the extraction method of comparative example 2, the time to release the membrane cells was longer, and the average survival rate was 41%.
Comparative example 3
The chicken small yellow follicular membrane cell extraction and culture method was the same as in example 1, except that 50ml of cell culture solution was prepared: 10ml of fetal bovine serum, 40ml of DMEM and 5ul of diabody. The results were averaged by repeating the experiment three times.
Results: with the extraction method of comparative example 3, the time to release the membrane cells was long, and the average survival rate was 21%.
Comparative example 4
The chicken small yellow follicular membrane cell extraction and culture method was the same as in example 1, except that 50ml of cell culture solution was prepared: 5ml fetal bovine serum, 5ml chicken serum, 40ml DMEM and 5ul of diabody. The results were averaged by repeating the experiment three times.
Results: with the extraction method of comparative example 4, the time to release the membrane cells was longer, and the average survival rate was 59%.
The above embodiments are only illustrative of the preferred embodiments of the present invention and are not intended to limit the scope of the present invention, and various modifications and improvements made by those skilled in the art to the technical solutions of the present invention should fall within the protection scope defined by the claims of the present invention without departing from the design spirit of the present invention.
Claims (2)
1. An in vitro culture method for chicken small yellow follicular membrane cells for improving cell survival rate is characterized by comprising the following steps:
(1) Mixing the membrane layer of the isolated chicken small yellow follicles with type II collagenase for digestion, and filtering after digestion to obtain a cell suspension, wherein the digestion temperature is 38 ℃ and the time is 15min;
the membrane layer of the chicken small yellow follicles is an inner layer and an outer layer;
(2) Centrifuging the cell suspension obtained in the step (1), wherein the obtained precipitate is chicken small yellow follicular membrane cells;
(3) Mixing and culturing the chicken small yellow follicular membrane cells obtained in the step (2) with a cell culture solution to obtain a chicken small yellow follicular membrane cell line, wherein the cell culture solution is prepared by configuring chicken serum, DMEM and diabody in a volume ratio of 2:8:0.001;
The double antibodies are penicillin and streptomycin, the working concentration of the penicillin is 100U/ml, and the working concentration of the streptomycin is 0.1mg/ml.
2. The in vitro culture method according to claim 1, wherein the preparation method of the membranous layer of chicken small yellow follicles is as follows: removing the granular layer of the isolated chicken small yellow follicles to obtain the membrane layer of the chicken small yellow follicles.
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| CN114672450B (en) * | 2022-01-26 | 2022-11-11 | 中山大学附属第一医院 | Method for separating and purifying theca cells |
| CN116087531B (en) * | 2023-04-11 | 2023-06-20 | 北京大学第三医院（北京大学第三临床医学院） | Follicular membrane cell specific marker and application thereof |
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