CN112646771A - Cell line of human follicle cumulus cells or/and granulosa cells and preparation method thereof - Google Patents
Cell line of human follicle cumulus cells or/and granulosa cells and preparation method thereof Download PDFInfo
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- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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- C12N2501/30—Hormones
- C12N2501/31—Pituitary sex hormones, e.g. follicle-stimulating hormone [FSH], luteinising hormone [LH]; Chorionic gonadotropins
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Abstract
The embodiment of the invention discloses a cell line of human follicle cumulus cells or/and parietal granule cells and a preparation method thereof, wherein the cell line comprises the following steps: subjecting human follicle cumulus cells or/and parietal granule cells to first centrifugation to obtain a first solid; adding the gradient centrifugal culture solution into the first solid, dividing the mixture into three layers after second centrifugation, adding phosphate buffer solution into the middle layer of the three layers, performing third centrifugation to obtain a third solid, adding hyaluronidase solution and lysis buffer solution, adding phosphate buffer solution into the fourth solid obtained by fourth centrifugation, performing fifth centrifugation to obtain purified cells, adding the purified cells into the culture solution, and performing subculture to obtain a cell line of human follicle cumulus cells or/and parietal granule cells; the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin; follicle stimulating hormone 0.012-0.02 weight portion; successfully obtains the cell line of human follicle cumulus cells or/and granulosa cells.
Description
Technical Field
The embodiment of the invention relates to the technical field of cellular endocrine, in particular to a cell line of human follicle cumulus cells or/and granulosa cells and a preparation method thereof.
Background
Follicular Granulosa Cells (GCs) are somatic Cells within the follicle and play a crucial role in the growth and development of follicles. As the follicles develop to the antral follicular stage, granulosa cells differentiate into two populations that differ spatially and functionally: cumulus Cells (CCs) surrounding oocytes and parietal granule Cells (Mural Granulosa Cells, MGCs) lining the follicular wall. Cumulus cells and parietal granulosa cells are considered to be the same cells with identical origin and different locations.
However, studies have shown that cumulus cells and parietal granulosa cells differ not only in location within the follicle, but also in function and gene expression. In the ovary, granulosa cells are the primary cell type that provides physical support and microenvironment for oocyte growth, and when the oocyte enters the late stages of growth and development, the follicle forms the sinus cavity. From the development of the secondary follicle until the follicular antral stage is formed, granulosa cells begin to differentiate into granulosa-sparing cells and cumulus cells. Cumulus cells surround oocytes and are in direct contact with the oocytes to form cumulus-oocyte-complexes, the number of which is closely related to the number of oocytes. Cumulus cell numbers around human oocytes have been considered as a useful marker of ovum quality and implantation potential. The parietal granulosa cells are distributed on the follicular wall and are associated with the follicular wall, and mainly play a role in the production of steroids and estrogens. The differentiation of cumulus cells is mediated by the secretion of potent growth factors (OSFs) from Oocytes, whereas the formation of parietal granulosa cells is regulated by FSH secreted from the extrafollicular, anterior pituitary. Cumulus cells are oocyte supporting cells and play an important role in the processes of oocyte growth, development, maturation, ovulation and fertilization. The corona radiata is thought to be the first layer of cumulus cells surrounding the oocyte, with cumulus cells being more compact near the inner layer of the oocyte than the outer layer, expanding concentrically. The cumulus cell directly contacts the oocyte, the cytoplasmic processes penetrate through the zona pellucida, and gap junctions are established with the oocyte to form a cumulus-oocyte-complex, the complex directly affects gene expression and protein synthesis, so that communication between the cumulus cell and the oocyte is realized, and the oocyte is differentiated, expanded and matured, the gap junctions are composed of protein polymers of intercellular channels, and the connexins C x 43 and C x 37 are two proteins most closely related to the protein polymers, and the channels allow ions, small molecules and small metabolites to pass through but do not allow macromolecules such as proteins and nucleic acids to pass through. Gap junctions help cumulus cells to provide energy and nutrition for oocyte growth and development, and promote oocyte maturation by reducing or closing regulatory channels until late development. Parietal granulosa cells are arranged on the inner wall of the follicle, are closely related to the growth, differentiation and maturation of the follicle and the subsequent ovulation and pregnancy maintenance, mainly perform endocrine functions, and are the main source of female estrogen. Parietal granule cells also promote ovulation by secreting proteases, and after ovulation, parietal granule cells gradually transform into progesterone-producing luteal cells, which are involved in a subsequent series of hormonal regulation processes. Under the action of luteinizing hormone, parietal granule cells and inner layer cells of the follicular membrane divide and proliferate, the cells are polygonal, yellow granules and lipid droplets are in cytoplasm, and the yellow granules and lipid droplets are called corpus luteum. Granulosa luteal cells are transformed by follicular granulosa cells, located in the center of the corpus luteum, large in size, lightly stained, and mainly secrete progesterone.
In conclusion, the follicular cumulus cells and granulosa cells can produce some natural estrogen and anti-cell aging and aging factors, and if the cell lines of the follicular cumulus cells and/or granulosa cells can be cultured in vitro, effective guarantee can be provided for producing the natural estrogen and the anti-cell aging and aging factors. However, to date, there have been no reports on establishing cell lines for human ovarian follicle cumulus cells and parietal granule cells. Human ovarian follicle cumulus cells and parietal granule cells are difficult to culture in vitro: 1) cumulus cells and granulosa cells grow slowly in vitro culture; 2) cumulus cells and granulosa cells easily lose the expression of the original characteristic marker genes during in vitro culture and hardly maintain the original characteristics.
Therefore, how to successfully develop a cell line of human follicle cumulus cells or/and granulosa cells and a preparation method thereof become a technical problem to be solved urgently.
Disclosure of Invention
The embodiment of the invention aims to provide a cell line of human follicle cumulus cells or/and parietal granulosa cells and a preparation method thereof, which can not only accelerate the in vitro culture production, but also can not lose a characteristic marker gene, and meanwhile, the expression of the characteristic marker gene can maintain the original characteristics, so that the cell line of the human follicle cumulus cells or/and granulosa cells can be successfully obtained.
In order to achieve the above object, the present invention provides a method for preparing a cell line of human follicle cumulus cells or/and parietal granule cells, the method comprising:
respectively carrying out first centrifugation on human follicle cumulus cells or/and parietal granule cells to obtain a first solid;
sequentially adding a gradient centrifugal culture solution with the concentration of 75-85% and a gradient centrifugal culture solution with the concentration of 35-45% into the first solid, dividing the mixture into three layers after second centrifugation, and taking the middle layer of the three layers to obtain a second solid;
adding the second solid into a phosphate buffer solution for third centrifugation to perform solid-liquid separation to obtain a third solid;
adding the third solid into a hyaluronidase solution for digestion, adding a lysis buffer for lysis, and performing fourth centrifugation for solid-liquid separation to obtain a fourth solid;
adding the fourth solid into a phosphate buffer solution for fifth centrifugation to perform solid-liquid separation, thereby obtaining purified cells;
adding the purified cells into a culture solution for subculture to obtain a cell line of human follicle cumulus cells or/and parietal granule cells; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin mixed solution; follicle stimulating hormone 0.012-0.02 weight portion.
Further, the rotation speed of the second centrifugation is 280-320g, and the time of the second centrifugation is 13-17 min.
Further, the concentration of the phosphate buffer is (10. + -. 1) mM.
Further, the first centrifugation, the third centrifugation, the fourth centrifugation, and the fifth centrifugation each include: the rotation speed is 280-320g, and the time is 8-12 min.
Further, the digestion time is 3-5 min.
Further, the cracking temperature is 2-6 ℃, and the cracking time is 8-12 min.
Further, the filtration and the adding of the purified cells into a culture solution for subculture comprise:
filtering the purified cells to obtain cells to be inoculated;
inoculating the cells to be inoculated into a culture solution for culture until the confluency is more than or equal to 90%, and then carrying out cell subculture.
Further, the filtration adopts a 70 μm cell filter screen; the ratio of the number of the cells to be inoculated to the volume of the culture solution is (0.8X 10)6-1.2×106) The method comprises the following steps: (9-11) mL.
Furthermore, the generation number of the subculture of the follicle cumulus cells is more than or equal to 15 generations; for the parietal granule cells, the generation number of subculture is more than or equal to 25.
One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:
the preparation method of the cell line of the human follicle cumulus cell or/and parietal granule cell provided by the embodiment of the invention comprises the following steps: respectively carrying out first centrifugation on human follicle cumulus cells or/and parietal granule cells to obtain a first solid; sequentially adding a gradient centrifugal culture solution with the concentration of 75-85% and a gradient centrifugal culture solution with the concentration of 35-45% into the first solid, dividing the mixture into three layers after second centrifugation, and taking the middle layer of the three layers to obtain a second solid; adding the second solid into a phosphate buffer solution for third centrifugation to perform solid-liquid separation to obtain a third solid; adding the third solid into a hyaluronidase solution for digestion, adding a lysis buffer for lysis, and performing fourth centrifugation for solid-liquid separation to obtain a fourth solid; adding the fourth solid into a phosphate buffer solution for fifth centrifugation to perform solid-liquid separation, thereby obtaining purified cells; adding the purified cells into a culture solution for subculture to obtain a cell line of human follicle cumulus cells or/and parietal granule cells; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin mixed solution; follicle stimulating hormone 0.012-0.02 weight portion. In the embodiment of the invention, the cells are separated and purified by using a gradient centrifugate, a hyaluronidase solution, a lysis buffer and phosphate buffered saline to purify the cells and subculture the cells, and a cell mass is dispersed into single cells in the culture, so that the growth of the cells is more facilitated, any one step is omitted, both human cumulus cells and granulosa cells grow slowly in vitro culture, and a cell line of the human cumulus cells or/and granulosa cells is difficult to establish; and the components in the culture solution cooperate with each other to ensure that the human follicle cumulus cells and the parietal granulosa cells can keep the characteristic genes thereof, specifically, the addition of FSH is beneficial to the human follicle cumulus cells and the granulosa cells to keep the characteristic gene expression and the estrogen secretion capacity, and in addition, other growth factors are not added in the culture solution to prevent the original characteristics of the cells from being damaged. Not only can the in vitro culture production be accelerated, but also the characteristic marker gene can not be lost, and meanwhile, the expression of the characteristic marker gene can maintain the original characteristics, and the cell line of the human follicle cumulus cells or/and granulosa cells can be successfully obtained.
Drawings
In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a flowchart of a method for preparing a cell line of human ovarian follicle cumulus cells or/and parietal granule cells according to an embodiment of the present invention;
FIG. 2 shows the transcriptomics results of a cell line of human ovarian follicle cumulus cells or/and parietal granule cells according to the embodiment of the present invention; wherein, FIG. 2(A) is the FSHR transcription result of the FSHR cell line of the ovarian follicle cumulus cell and/or parietal granule cell; FIG. 2(B) is the result of Luteinizing Hormone Receptor (LHR) transcription of a cell line of follicular cumulus cells or/and parietal granule cells; FIG. 2(C) is the result of transcription of Androgen Receptor (AR) of a cell line of ovarian follicle cumulus cells or/and parietal granule cells; in the abscissa of FIG. 2(A), FIG. 2(B) and FIG. 2(C), C represents a cumulus cell, M represents a parietal granulosa cell, and C0, C1, C3 and C5 are passage 0, passage 1, passage 3 and passage 5, respectively, of the cumulus cell in comparative example 1; C1F, C2F, C4F, C6F, C8F and C10F are passages 1, 2,4, 6, 8 and 10, respectively, of the FSH-added cumulus cells in example 1; m0, M5, M10, M15 and M20 are respectively passage 0, passage 5, passage 10, passage 15 and passage 20 of the parietal granulocytes in example 2;
FIG. 3 shows metabonomics results of a cell line of human ovarian follicle cumulus cells or/and parietal granule cells provided by the present invention; wherein FIG. 3(A) is a hierarchical clustering result of metabolites with significantly different negative ion patterns, FIG. 3(B) is a hierarchical clustering result of metabolites with significantly different positive ion patterns, on the abscissa, ccp10 represents passage 10 of cumulus cells, ccp7 represents passage 7 of cumulus cells, ccp0 represents passage 0 of cumulus cells, MGC-P0 represents passage 0 of parietal granule cells, MGC-P10 represents passage 10 of parietal granule cells, and MGC-P20 represents passage 20 of parietal granule cells;
FIG. 4 shows the results of hormone detection of a cell line of human ovarian follicle cumulus cells or/and parietal granule cells according to an embodiment of the present invention; wherein FIG. 4(A) is the result of hormone (Estradiol Estradiol, progasterone progesterone) detection of a cell line of parietal cell; p0, P10 and P20 in FIG. 4(A) represent passage 0, passage 10 and passage 20, respectively, of the parietal granulocytes in example 2; FIG. 4(B) shows the results of hormone (Estradiol Estradiol, progasterone progesterone) detection in a cell line of ovarian follicle cumulus cells; p0, P7 and P14 in FIG. 4(B) represent passage 0, passage 7 and passage 14, respectively, of the cumulus cell in example 1;
FIG. 5 shows the immunofluorescence detection results of a cell line of human ovarian follicle cumulus cells or/and parietal granule cells according to the embodiment of the present invention; wherein, in FIG. 5, MGC-P0, MGC-P5, MGC-P10, MGC-P15 and MGC-P20 respectively represent the 0 th, 5 th, 10 th, 15 th and 20 th generations of parietal cell in example 2; CC-P0, CC-P5, CC-P10, CC-P12, CC-P13 and CC-P14 represent the 0 th, 5 th, 10 th, 12 th, 13 th and 14 th passages of the cumulus cells in example 1, respectively.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the embodiments of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that the present embodiments and examples are illustrative of the present invention and are not to be construed as limiting the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood in accordance with the meanings commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which embodiments of the invention belong. If there is a conflict, the present specification will control.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions.
In order to solve the technical problems, the embodiment of the invention provides the following general ideas:
1. analysis of causes of technical problems
Human ovarian follicle cumulus cells and parietal granule cells are difficult to culture in vitro: technical problem 1: human cumulus cells and granulosa cells grow slowly in vitro culture; technical problem 2: human cumulus cells and granulosa cells easily lose the expression of the original characteristic marker genes during in vitro culture and hardly maintain the original characteristics.
For the above technical problem 1, the applicant has found through research that cumulus cells and granulosa cells collected from waste human follicular fluid cannot be directly cultured when mixed with erythrocytes, metabolites and various impurities, so that the human cumulus cells and granulosa cells are easily polluted and grow slowly during in vitro culture;
for the above technical problem 2, the loss of expression of a specific gene may be caused by the lack of related hormones or components during the culture;
2. exploration of solution
With respect to the above technical problem 1, the present applicant has tried various methods including: collecting cumulus cells and parietal granule cells in follicular fluid separated from cumulus cell-ovum-complex before intracytoplasmic sperm microinjection, directly inoculating the cumulus cells and the parietal granule cells in a culture dish for subculture, but obtaining a cell line of the cumulus cells or/and the parietal granule cells is difficult; in the embodiment of the invention, the cells are separated and purified by using a gradient centrifugate, a hyaluronidase solution, a lysis buffer and phosphate buffered saline to purify the cells and subculture the cells, and a cell mass is dispersed into single cells in the culture, so that the growth of the cells is more facilitated, and any one step is omitted, so that the cumulus cells and parietal granulosa cells grow slowly in vitro culture and are difficult to culture into a cell line of the follicle cumulus cells or/and the parietal granulosa cells;
for the above technical problem 2, the culture solution used in the embodiment of the present invention includes, by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin mixed solution; follicle stimulating hormone 0.012-0.02 weight portion; the components in the culture solution cooperate with each other to ensure that the follicle cumulus cells and the parietal granulosa cells can keep the characteristic genes thereof, specifically, the addition of FSH is beneficial to the cumulus cells and the granulosa cells to keep the characteristic gene expression and the estrogen secretion capacity thereof, and in addition, other growth factors are not added to prevent the original characteristics of the cells from being damaged.
According to an exemplary embodiment of the present invention, there is provided a method for preparing a cell line of human ovarian follicle cumulus cells or/and parietal granule cells, as shown in fig. 1, the method comprising:
s1, respectively carrying out first centrifugation on the human follicle cumulus cells or/and the parietal granule cells to obtain a first solid;
sources of human ovarian follicle cumulus cells or/and parietal granulosa cells can include: collecting parietal granulosa cells in the patient's waste follicular fluid after clinical assisted reproductive aspiration and cumulus cells isolated from cumulus cell-ovum-complex prior to intracytoplasmic sperm microinjection: under a solid microscope, collecting cumulus cells and wall granular cells into two round-bottom test tubes by using a Pasteur pipette, wherein the cumulus cells under the microscope are clear in color, loose and cloudy, and the wall granular cells are dark in color and sink to form lumps or slices;
the purpose of the first centrifugation is: to remove supernatant such as follicular fluid to obtain impurities such as cell precipitate;
as an alternative embodiment, the rotation speed of the first centrifugation is 280-320g, and the time of the first centrifugation is 8-12 min. If the rotating speed of the first centrifugation is less than 280g and the time is less than 8min, impurities are difficult to remove; if the rotating speed of the first centrifugation is more than 320g, the time is more than 12min, which is not beneficial to the survival of cells;
s2, sequentially adding a gradient centrifugal culture solution with the concentration of 75-85% and a gradient centrifugal culture solution with the concentration of 35-45% into the first solid, dividing the mixture into three layers after second centrifugation, and taking the middle layer of the three layers to obtain a second solid;
the second centrifugation is to remove red blood cells;
as an alternative embodiment, the concentration of 35-45% of the gradient centrifugation culture solution is preferably 40%; too high or too low concentration is not favorable for the separation and purification of cells; the preferable concentration of the gradient centrifugal culture solution with the concentration of 75-85 percent is 80 percent; the gradient centrifugal culture solution with the concentration of 35-45 percent and the gradient centrifugal culture solution with the concentration of 75-85 percent are solutions in the gradient culture kit; in particular, the gradient centrifugate and the culture broth can be purchased from products sold by companies.
As an alternative embodiment, a round-bottom test tube is taken, 1.0mL of 80% and 40% gradient centrifugate is added from bottom to top respectively, cell sediment after supernatant is discarded, and the action is slow and gentle;
as an alternative embodiment, the rotation speed of the second centrifugation is 280-320g, and the time of the second centrifugation is 13-17 min. Specifically, centrifugation at 300g for 15min can be selected. After centrifugation, the tube is divided into three layers, red blood cells can be seen to sink at the bottom of the tube, the cells are seen between the liquid levels of the first layer and the second layer, and the cells are sucked out to a 1.5mL EP tube;
s3, adding the second solid into a phosphate buffer solution for third centrifugation to perform solid-liquid separation to obtain a third solid;
the phosphate buffer solution plays a role in washing;
as an alternative embodiment, the concentration of the phosphate buffer is (10. + -. 1) mM. Phosphate buffer in this concentration range is useful for maintaining the activity of the cells and is centrifuged to remove impurities;
s4, adding the third solid into a hyaluronidase solution for digestion, adding a lysis buffer for lysis, and performing fourth centrifugation for solid-liquid separation to obtain a fourth solid;
as an alternative embodiment, the digestion is carried out for a period of 3 to 5 min. Too long digestion time is not beneficial to cell survival and cell damage, and too short digestion time is not beneficial to cell mass dispersion into single cells; the concentration of the hyaluronidase solution is (1 +/-0.2) mg/mL, the cell mass is easily not completely separated due to too low concentration, and the cells are easily damaged due to too high concentration;
as an alternative embodiment, the Lysis Buffer comprises one of 1 × RBC Lysis buffers for the purpose of removing red blood cells.
S5, adding the fourth solid into a phosphate buffer solution for fifth centrifugation to separate solid from liquid, and obtaining purified cells;
as an alternative embodiment, the third centrifugation, the fourth centrifugation, and the fifth centrifugation each comprise: the rotation speed is 280-320g, and the time is 8-12 min.
The concentration of the phosphate buffer was (10. + -. 1) mM. Preferably, the concentration of phosphate buffer is 10mM, i.e., 1 XPBS; at the moment, phosphate buffer solution is added to mainly play a role in washing;
s6, adding the purified cells into a culture solution for subculture to obtain a cell line of human follicle cumulus cells or/and parietal granule cells; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin mixed solution; 0.012-0.02 parts of follicle-stimulating growth hormone.
In this embodiment, the final concentration of FSH is too high to maintain cell characteristics and too low to allow cell growth; more preferably, the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 10 parts of fetal bovine serum; 1 part of streptomycin mixed solution; 0.016 part of FSH;
as an alternative embodiment, the filtration of the purified cells and the adding of the filtered cells into a culture solution for subculture comprises:
filtering the purified cells to obtain cells to be inoculated;
inoculating the cells to be inoculated into a culture solution for culture until the confluency is more than or equal to 90%, and then carrying out cell subculture.
In this embodiment, the purpose of the filtration is to remove bacterial microorganisms; one of the difficulties in establishing cell lines is that cells are slow to produce and are prone to contamination. Therefore, the whole process from the collection of the cells to the inoculation and culture of the cells requires special attention to the aseptic operation, and bacterial microorganisms are removed to prevent pollution; specifically, the filtration adopts a 70 μm cell filter screen;
in the prior art, the standard for measuring the passability of cells is 80-90% of the confluent culture field of cell growth, and our experiments show that when the cells are P0, the cell growth time is prolonged, so that the cells grow to more than 90% of the confluent culture field, which is more favorable for long-term culture of the cells. When the cells are inoculated for the first time, the number of the inoculated cells is slightly increased, and the growth of the cells is more favorable.
As an alternative embodiment, the ratio of the number of cells to be seeded to the volume of the culture broth is (0.8X 10)6-1.2×106) The method comprises the following steps: (9-11) mL. Increasing the number of cells seeded slightly more favours cell growth.
As an alternative embodiment, the liquid is changed 24 hours after the cells are initially inoculated or passaged, and then the liquid is changed every 48 hours, so that the cells which are suspended and dead can be eliminated in time, and the cell viability can be better preserved.
For the cumulus cells, the generation number of subculture is more than or equal to 15 generations; for the parietal granule cells, the generation number of subculture is more than or equal to 25.
The embodiment of the invention also provides a cell line of human follicle cumulus cells or/and parietal granule cells prepared by the method; meanwhile, the embodiment of the invention adopts experiments such as gene transcriptomics, metabonomics, hormone detection, morphology and the like to verify that the cell line of the human follicle cumulus cells or/and parietal granule cells has no essential change with the follicle cumulus cells or/and parietal granule cells in morphology and function, gene and metabonomics and has no loss of characteristic markers.
A cell line of human ovarian follicle cumulus cells or/and parietal granule cells and a preparation method thereof will be described in detail below with reference to examples and comparative experimental data.
Instrument equipment and reagent consumables:
cell incubator (three gas incubator): 37 ℃ and 5.0% CO2，5％O2(ii) a Olympus solid body microscope; nikon inverted microscope; pasteur pipette, disposable aseptic syringe, round bottom test tube, cell filter screen (70 μm), freezing tube; gradient centrifugation culture (40% and 80%), ready-to-use hyaluronidase, eBioscience 1X RBC Lysis Buffer, 1 XPBS Buffer, Dulbecco's Modified Eagle Medium (DMEM basic (1X)), penillin Streptomyces, extra Fetal Bovine Serum (FBS), menotrophin for injection (FSH: LH ═ 1: 1), Dimethylsulfoxide (DMSO), 0.25% Trypsin-EDTA (1X);
example 1 cell line of human ovarian follicle cumulus cells and method for preparing the same
This example provides a method for preparing a cell line of human ovarian follicle cumulus cells, which comprises the following steps:
(1) source and culture of cumulus cells:
collecting cumulus cells separated from the cumulus cell-ovum-complex before intracytoplasmic sperm microinjection: under a solid microscope, collecting cumulus cells into a round-bottom test tube by using a Pasteur pipette, wherein the cumulus cells under the microscope are clear in color and loose and cloudy;
collecting cumulus cells, centrifuging for 10min at 300g, and removing supernatant;
③ separating the cells by using gradient centrifugal culture solution (40 percent and 80 percent), taking a round bottom test tube, adding 1.0mL of 80 percent and 40 percent gradient centrifugal solution from bottom to top respectively, discarding the cell sediment after supernatant, paying attention to slow and gentle movement, and centrifuging for 15min at 300 g.
Fourthly, after the centrifugation is finished, the erythrocyte can be seen to sink at the bottom of the tube, the cell can be seen between the first layer liquid level and the second layer liquid level, and the cell is sucked out to a 1.5mL EP tube;
adding 1 Xphosphate buffered saline (PBS), centrifuging for 10min to form precipitate;
sixthly, removing the supernatant, adding a proper amount of hyaluronidase, mixing, and placing in an incubator for digestion for 3-5 min;
seventhly, adding a proper amount of 1 XRBC lysis buffer solution, placing the mixture into an upper layer refrigerator for refrigeration for 10min, centrifuging the mixture by a micro centrifuge for 10min to form precipitates, discarding supernate after centrifugation is finished, and repeating the step if more red blood cells exist;
(viii) washing with 1ml of Phosphate Buffered Saline (PBS), and using a microcentrifuge for 10 minutes to form a precipitate, discarding the supernatant, and washing twice in total;
ninthly, adding 1mL of culture solution into the purified cells, after counting by a hemocyte counter, selecting a culture dish with a proper size according to the number of the cells, filtering the cells to the culture dish by using a cell filter screen, and adding proper culture; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 10 parts of fetal bovine serum; 1 part of streptomycin mixed solution; follicle stimulating hormone 0.016 parts.
(2) Passage of cells:
firstly, culturing cells, changing liquid 24 hours after inoculation, changing liquid every 48 hours, and carrying out cell passage until the cells are observed under a microscope and basically spread in a culture dish (more than 90 percent);
adding a proper amount of trypsin according to the size of the culture dish;
placing the cells in an incubator for digestion for 3-5min until the cells are observed to be dispersed and become single under a mirror;
adding culture solution with the same amount as pancreatin to stop digestion, transferring the liquid in the dish into a 15mL conical tube, and centrifuging for 10min at 1350 rpm;
fifthly, centrifuging, then removing the supernatant, adding 1mL of culture solution, counting by a hemocytometer, selecting a proper culture dish according to the number of cells, carrying out subculture on a part of cells, and freezing and storing a part of cells (frozen cell sap: FBS: DMSO: 5: 4: 1, and storing in a freezer at-80 ℃); subculturing to 15 generations to obtain cell lines of follicle cumulus cells of different generations.
Example 2 cell line of human follicular wall granulosa cells and method for preparing the same
This example provides a method for preparing a cell line of granulosa cells on the follicular wall of a human, comprising the following steps:
(1) source and culture of parietal granulosa cells:
collecting parietal granule cells in the waste follicular fluid of a patient after clinical assisted reproduction and ovum taking: under a solid microscope, respectively collecting the wall granular cells into a round-bottom test tube by using a Pasteur pipette, wherein the granular cells under the tube are dark in color and sink to form lumps or sheets;
collecting parietal granule cells, centrifuging for 10min at 300g, and removing supernatant;
③ separating the cells by using gradient centrifugal culture solution (40 percent and 80 percent), taking a round bottom test tube, adding 1.0mL of 80 percent and 40 percent gradient centrifugal solution from bottom to top respectively, discarding the cell sediment after supernatant, paying attention to slow and gentle movement, and centrifuging for 15min at 300 g.
Fourthly, after the centrifugation is finished, the erythrocyte can be seen to sink at the bottom of the tube, the cell can be seen between the first layer liquid level and the second layer liquid level, and the cell is sucked out to a 1.5mL EP tube;
adding 1 Xphosphate buffered saline (PBS), centrifuging for 10min to form precipitate;
sixthly, removing the supernatant, adding a proper amount of hyaluronidase, mixing, and placing in an incubator for digestion for 3-5 min;
seventhly, adding a proper amount of 1 XRBC lysis buffer solution, placing the mixture into an upper layer refrigerator for refrigeration for 10min, centrifuging the mixture by a micro centrifuge for 10min to form precipitates, discarding supernate after centrifugation is finished, and repeating the step if more red blood cells exist;
(viii) washing with 1ml of Phosphate Buffered Saline (PBS), and using a microcentrifuge for 10 minutes to form a precipitate, discarding the supernatant, and washing twice in total;
ninthly, adding 1mL of culture solution into the purified cells, after counting by a hemocyte counter, selecting a culture dish with a proper size according to the number of the cells, filtering the cells to the culture dish by using a cell filter screen, and adding proper culture; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 10 parts of fetal bovine serum; 1 part of streptomycin mixed solution; follicle stimulating hormone 0.016 parts;
(2) passage of cells:
firstly, culturing cells, changing liquid 24 hours after inoculation, changing liquid every 48 hours, and carrying out cell passage until the cells are observed under a microscope and basically spread in a culture dish (more than 90 percent);
adding a proper amount of trypsin according to the size of the culture dish;
placing the cells in an incubator for digestion for 3-5min until the cells are observed to be dispersed and become single under a mirror;
adding culture solution with the same amount as pancreatin to stop digestion, transferring the liquid in the dish into a 15mL conical tube, and centrifuging for 10min at 1350 rpm;
fifthly, centrifuging, then removing the supernatant, adding 1mL of culture solution, counting by a hemocytometer, selecting a proper culture dish according to the number of cells, carrying out subculture on a part of cells, and freezing and storing a part of cells (frozen cell sap: FBS: DMSO: 5: 4: 1, and storing in a freezer at-80 ℃); subculturing to 15 generations to obtain cell lines of parietal granule cells with different generations.
Example 3
In the embodiment 3 of the invention, the steps are the same as those in the embodiment 1 except that the culture solution is slightly different; specifically, the culture solution comprises the following components in parts by volume: 100 parts of DMEM; 5 parts of fetal bovine serum; 0.5 part of streptomycin mixed liquor; follicle stimulating hormone 0.012 parts.
Example 4
In the embodiment 4 of the invention, the steps are the same as those in the embodiment 1 except that the culture solution is slightly different; specifically, the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM; 15 parts of fetal bovine serum; 1.5 parts of streptomycin mixed liquor; follicle stimulating hormone 0.02 part.
Comparative example 1
The culture solution used in this comparative example 1 was not supplemented with FSH, and the other steps were the same as in example 1; cumulus cell passage 0, cumulus cell passage 1, cumulus cell passage 3 and cumulus cell passage 5 were obtained.
Comparative example 2
The culture solution used in comparative example 2 was supplemented with 0.01 part of FSH, and the other steps were the same as in example 1.
Comparative example 3
The culture solution used in comparative example 3 was supplemented with 0.03 parts of FSH, and the other steps were the same as in example 1.
Experimental example 1
The cell line of the human follicle cumulus cell obtained in example 1 and the cell line of the follicle wall granulosa cell obtained in example 2 were subjected to transcriptomics, metabolomics, hormone detection, and morphological test verification.
1. Cumulus cell (C) and parietal granule cell (M) transcriptomics comparison
The comparison of transcriptomics between cumulus cells (C) in example 1 and parietal granule cells (M) in example 2 is shown in fig. 2, and it can be seen from fig. 2 that the transcription profiles of follicle-stimulating hormone Receptor (FSHR), Luteinizing Hormone Receptor (LHR), and Androgen Receptor (AR) are not different and substantially changed in different generations of cumulus cells in example 1;
in comparative example 1, since FSHR, LHR and AR were not added, the transcription was inferior to that of example 1;
in comparative example 2, since FSH was added in an amount of 0.01 parts, which is less than the range of 0.012 to 0.02 parts in the examples of the present invention, cell growth was slow;
in comparative example 3, since FSH was added in an amount of 0.03 parts, which is greater than the range of 0.012 to 0.02 parts in the examples of the present invention, the cells lost their characteristics;
the results of comparative example 1, comparative example 3 and example 1 show that the addition of 0.012-0.02 parts of FSH in the examples of the invention can maintain the cell characteristics and simultaneously is beneficial to cell growth;
in example 2, FSHR, LHR and AR transcription did not differ greatly among follicular wall granulosa cells of different passage numbers, and they did not change substantially.
2. Cumulus Cell (CC) and parietal granule cell (MGC) metabolomics comparison
Results of comparison of the Cumulus Cell (CC) in example 1 and the parietal granule cell (MGC) metabolomics in example 2 are shown in fig. 3, from which Myo-inositol can be known in fig. 3; l-maleic acid (L-Malic acid), 5-L-Glutamyl-alanine (L-Glutamyl-L-alanine), Pyruvaldehyde (methylglyoxal), Leukotriene C4 (Leukotriene C4), 12-Oxo-2,3-dinor-10,15-phytodienoic acid (12-Oxo-2, 3-bis-10, 15-phytodienoic acid), L-Iditol (L-Iditol), L-histadine (L-Histidine), Succinate (Succinate), (-) -Tylocerebin (-) -sinone, Ordok-Tyr isoleucine-tyrosine, Phe-Tyr phenylalanine-tyrosine, DL-2, 4-diaminobutyrine acid DL-2,4-Diaminobutyric acid, Thymine Thymine, N6, n6, N6-trimethy-L-lysine N6, N6, N6-Trimethyl-lysine, L-Serine L-alanine, N-Acetyl-L-alanine, Gly-His glycine-histidine, 1-stearyl-2-hydroxy-sn-glycero-3-phospholine 1-Stearoyl-2-hydroxy-sn-propanetriyl-3-choline phosphate, Inosine, trans-2-Hydroxycinnamic acid, 1-stearyl-sn-glycerol 3-phospholine 1-Stearoyl-sn-propanetriyl 3-choline phosphate, L-Tyrosine, Dopamine, Metabolites such as DL-Phenylalanine, L-Carnosine, phenylactic acid Phenyllactic acid and DL-O-tyrosine have small difference in cumulus cells of different generations and do not change essentially. The metabolites are not greatly different in different generations of parietal granule cells and do not change substantially.
3. Comparison of Cumulus Cell (CC) and parietal granulosa cell (MGC) hormone detection
The results of comparison of hormone detection of Cumulus Cells (CC) in example 1 and parietal granule cells (MGC) in example 2 are shown in fig. 4, and it can be seen from fig. 4 that Estradiol (Estradiol) is not very different in cumulus cells of different generations and progestasterone (Progesterone) is not very different in cumulus cells of different generations; estradiol (Estradiol) is not very different in different generations of parietal granulosa cells, and Progesterone (Progesterone) is not very different in different generations of parietal granulosa cells.
4. Cumulus Cell (CC) and parietal granulosa cell (MGC) morphological changes
The immunofluorescence results of Cumulus Cells (CC) in example 1 and parietal granule cells (MGC) in example 2 are shown in fig. 5, and it can be seen from fig. 5 that the morphology of the parietal granule cells in different generations is not very different; the morphologic difference of the follicular cumulus cells of different generations is not great.
In conclusion, the embodiment of the invention successfully cultures the follicular wall granulosa cells to 25 generations and the follicular cumulus cells to 15 generations, and tests and verifications such as transcriptomics, metabonomics, hormone detection, morphology and the like are carried out on the cells, so that the cells are proved to have no characteristic change and no loss of characteristic markers. Thus, the present example successfully established cell lines of follicular cumulus cells and parietal granule cells in vitro.
Finally, it should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the embodiments of the invention.
It will be apparent to those skilled in the art that various modifications and variations can be made in the embodiments of the present invention without departing from the spirit or scope of the embodiments of the invention. Thus, if such modifications and variations of the embodiments of the present invention fall within the scope of the claims of the embodiments of the present invention and their equivalents, the embodiments of the present invention are also intended to encompass such modifications and variations.
Claims (10)
1. A method for preparing a cell line of human ovarian follicle cumulus cells or/and parietal granule cells, the method comprising:
respectively carrying out first centrifugation on human follicle cumulus cells or/and the parietal granule cells to obtain a first solid;
sequentially adding a gradient centrifugal culture solution with the concentration of 75-85% and a gradient centrifugal culture solution with the concentration of 35-45% into the first solid, dividing the mixture into three layers after second centrifugation, and taking the middle layer of the three layers to obtain a second solid;
adding the second solid into a phosphate buffer solution for third centrifugation to perform solid-liquid separation to obtain a third solid;
adding the third solid into a hyaluronidase solution for digestion, adding a lysis buffer for lysis, and performing fourth centrifugation for solid-liquid separation to obtain a fourth solid;
adding the fourth solid into a phosphate buffer solution for fifth centrifugation to perform solid-liquid separation, thereby obtaining purified cells;
adding the purified cells into a culture solution for subculture to obtain a cell line of human follicle cumulus cells or/and parietal granule cells; wherein the culture solution comprises the following components in parts by volume: 100 parts of alpha-MEM or DMEM; 5-15 parts of fetal bovine serum; 0.5-1.5 parts of streptomycin mixed solution; follicle stimulating hormone 0.012-0.02 weight portion.
2. The method for preparing a cell line of human ovarian follicle cumulus cells or/and parietal granule cells as claimed in claim 1, wherein the rotation speed of the second centrifugation is 280-320g, and the time of the second centrifugation is 13-17 min.
3. The method of claim 1, wherein the phosphate buffer is present in a concentration of (10 ± 1) mM.
4. The method of claim 1, wherein the first centrifugation, the third centrifugation, the fourth centrifugation, and the fifth centrifugation each comprise: the rotation speed is 280-320g, and the time is 8-12 min.
5. The method of claim 1, wherein the digestion is performed for 3-5 min.
6. The method of claim 1, wherein the lysis temperature is 2-6 ℃ and the lysis time is 8-12 min.
7. The method according to claim 1, wherein the filtration of the purified cells and the subculture of the cells in a culture medium comprises:
filtering the purified cells to obtain cells to be inoculated;
inoculating the cells to be inoculated into a culture solution for culture until the confluency is more than or equal to 90%, and then carrying out cell subculture.
8. The method of claim 7, wherein the filtration is performed using a 70um cell strainer; the ratio of the number of the cells to be inoculated to the volume of the culture solution is (0.8X 10)6-1.2×106) The method comprises the following steps: (9-11)mL。
9. The method for producing a cell line of human ovarian follicle cumulus cells or/and parietal granule cells according to claim 1, wherein the generation number of the subculture of the ovarian follicle cumulus cells is not less than 15 generations; for the parietal granule cells, the generation number of subculture is more than or equal to 25.
10. A cell line derived from human ovarian follicle cumulus cells or/and parietal granule cells, prepared by the method of any one of claims 1-9.
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