CN117586944B - Method for in-vitro serum-free amplification culture of primordial germ cells of chickens - Google Patents
Method for in-vitro serum-free amplification culture of primordial germ cells of chickens Download PDFInfo
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- C12N5/06—Animal cells or tissues; Human cells or tissues
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Abstract
The invention discloses a method for in vitro serum-free amplification culture of chicken primordial germ cells, which belongs to the technical field of bioengineering and comprises the following steps of: configuration of the culture medium: the culture medium does not contain fetal bovine serum, and the culture medium comprises a plurality of inhibitors; isolation and culture of chicken primordial germ cells: hatching eggs, opening the hatching eggs, placing the hatching eggs in a culture dish, separating gonads, and placing the gonads in a phosphate buffer solution; adding pancreatin digestive juice to perform digestion treatment, performing centrifugal treatment to obtain chicken primordial germ cells, transferring the chicken primordial germ cells into a cell culture plate, adding a prepared culture solution to culture, and performing subculture after 5 d; cell proliferation count: re-suspending the cultured chicken primordial germ cells with PBS, calculating the total cell amount, equally dividing the total cell amount into two groups, repeating each group by three, respectively adding the culture medium without fetal calf serum, culturing for 4-5d, respectively sampling and counting, and calculating the cell concentration and proliferation rate; the purpose of rapidly obtaining a large number of early primordial germ cells in a short period is achieved.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a method for in-vitro serum-free amplification culture of chicken primordial germ cells.
Background
Primordial Germ Cells (PGCs) are germ stem cells with multipotent and reproductive properties, which are precursors to gametes, sperm and ovum, capable of transmitting genetic information within the same generation. The unique migration characteristics of chicken PGCs in blood circulation provide a promising strategy for the production of transgenic chicken models and the protection of poultry species resources; however, this approach is limited by an insufficient number of PGC sources and uncertainty in the molecular mechanisms that regulate PGC formation.
In most animals, the separation of the germ line from the somatic cell line is one of the earliest events in the development process. In avian embryos, PGCs are first found in the extraembryonic region, the zone of onset of development, after about 18 hours of incubation. After 50-55 hours of development, PGCs migrate to the gonads, which in turn produce functional sperm and oocytes. Chicken PGCs can be isolated, cultured and transgenic while maintaining their germline stability. In addition, chicken PGCs can induce differentiation into embryonic germ cells in vitro and promote formation of somatic tissues. The retention of the germ line by PGCs after prolonged culture and genetic modification, and their ability to obtain somatic cells in vitro, provides a new model for developmental biology. The operability of avian embryos enhances the utility of the model, which helps to enter early stages of development and provides a convenient way to reintroduce PGCs into the embryo vasculature. In addition, these properties create new opportunities for manipulating chicken genomes for agricultural and pharmaceutical applications.
At present, less research reports are about the long-term culture of chicken PGCs, and the main components of an in-vitro culture system are as follows: KO-DMEM, fetal Bovine Serum (FBS), chicken serum, nonessential amino acids, glutaMAXTM, pyruvic acid, beta-mercaptoethanol, recombinant human basic fibroblast growth factor (rhFGF), human leukemia inhibitory factor (humanLIF) or human stem cell factor (humanSCF). By utilizing the systems, the success rate of the isolated culture of the chicken primordial germ cells is low, the culture time is short, and the excessively complex components have a certain influence on the subsequent transgenic operation. Positively charged liposomes form complexes with negatively charged phosphate groups of nucleic acids that are endocytosed by the cell. Serum contains a large amount of proteins, and negatively charged proteins may interfere with the adsorption of nucleic acids by cationic liposomes during transfection, affecting transfection efficiency. In addition, when a transfection reagent such as liposome is used, the transfection with serum brings proteins in serum into cells, which causes cytotoxicity, and the transfection efficiency is lowered, so that the transfection with serum is not possible. These transfection reagents require replacement of serum-free medium prior to transfection and replacement with serum medium 4-6 hours after transfection to reduce cytotoxicity caused by transfection.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, and provides a method for in-vitro serum-free amplification culture of chicken primordial germ cells, so as to achieve the purpose of rapidly obtaining a large number of early primordial germ cells in a short period.
The aim of the invention is realized by the following technical scheme: a method for in vitro serum-free amplification culture of chicken primordial germ cells, comprising the following steps:
s1, preparation of culture medium without fetal bovine serum
The culture medium does not contain fetal bovine serum, and comprises KO-DMEM, an inhibitor, chicken serum, antibiotics, sodium pyruvate, glutamine, nucleosides, beta-mercaptoethanol, heparin sodium, recombinant human FGF, nonessential amino acids, B-27 and human ActivinA;
S2, separation and culture of chicken primordial germ cells
Hatching eggs, sterilizing eggshells, opening the eggs, placing the eggs in a culture dish (purchased from biosharp, BS-90-D bacterial culture dish), separating gonads under a split microscope, and placing the gonads in a 1.5mL centrifuge tube containing 100 mu L of phosphate buffer solution;
adding pancreatin digestive juice into the centrifuge tube for digestion treatment, and centrifuging after stopping digestion to obtain chicken primordial germ cells;
Transferring the chicken primordial germ cells into a 24-hole cell culture plate, adding the culture medium without fetal calf serum for culturing, changing the liquid by half a day, and subculturing after 5 d;
s3, cell proliferation count
The cultured chicken primordial germ cells are resuspended by PBS, the total cell amount is calculated after counting by a cell counter, the total cell amount is divided into two groups, each group is divided into three times, the freshly prepared culture medium without fetal bovine serum is respectively added, and after 4-5d of culture, the sample is respectively counted, and the cell concentration and proliferation rate are calculated.
The invention improves and improves the culture medium for culturing the primordial germ cells of the chickens in vitro, and cultures the primordial germ cells of the chickens by the culture medium, so that the reproduction of the germ cells in vitro is faster, the cell state is better, the culture cost is reduced, and a large number of early primordial germ cells can be obtained in a short period.
As one of the possible examples, the medium comprises KO-DMEM, 1% chicken serum, 1% diabody, 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1 Xnonessential amino acids, 1X B-27, 25ng/mL human ActivinA, 5. Mu. Mol/LROCK inhibitor, 1. Mu. Mol/LGSK-3 inhibitor, 0.2. Mu.M (. + -.) -bupstatin. The dual antibody is a penicillin and streptomycin mixed solution, the storage solution is 100X (10,000 units/mL penicillin and 10,000 mug/mL streptomycin are contained), and the working concentration is 1X, namely 100 units/mL penicillin and 100 mug/mL streptomycin. The nonessential amino acids comprise 7 amino acids (L-alanine, L-glutamic acid, L-asparagine, L-aspartic acid, L-proline, L-serine, glycine), the concentration of each amino acid in the 100X stock solution is 10mM, the commercial stock solution of glutamine, nucleoside, B-27, nonessential amino acids and the like is 100X, and when the nonessential amino acids are used, only 1% of stock solution is needed to be added into the working solution, namely, the stock solution is diluted 100 times. The culture medium comprises the following components in percentage by volume. The combined use of the ROCK inhibitor, the GSK-3 inhibitor and the (+ -) -bupstatin reduces the use of cytokines, does not use fetal bovine serum, increases the use of apoptosis-related inhibitors and promotes proliferation. ROCK (rho-associated protein kinase) is a kinase of the AGC (PKA/PKG/PKC) serine-threonine kinase family. Ras homologous gene family member a (RhoA) and ROCK mediate a number of pathophysiological signals including induction of cytoskeletal recombination, cell migration, adhesion, proliferation differentiation, tissue contraction and growth, and the like. Y-27632 used in the present invention is an effective and selective ATP competitive inhibitor of Rho-associated kinase (ROCK), and Y-27632 can inhibit the separation-induced apoptosis in multi-generation cultured human embryonic stem cells and improve cloning efficiency. Glycogen synthase kinase-3 (glycogensynthasekinase-3, GSK-3) is a multifunctional serine/threonine protein kinase, is an important component in various intracellular signal transduction pathways, and is involved in various important physiological processes such as intracellular glycometabolism, cell proliferation, cell differentiation, cell apoptosis and the like. GSK-3 inhibitor (CHIR-99021) used in the invention can promote cell proliferation and enhance self-renewal of mouse and human embryo stem cells. Blebbbistatin ((S) - (-) -Blebbbistatin) is a cell permeability inhibitor acting on myosin IIATPase, and can promote directional migration of corneal endothelial cells and accelerate wound healing, and better preserve cell connection integrity and barrier function, and tissue cell migration. The invention does not use fetal bovine serum, increases the inhibitors of three apoptosis and migration related pathways, reduces the consumption of cytokines, and compared with the single signal pathway for inhibiting apoptosis, inhibits apoptosis and differentiation from different pathways and promotes the proliferation and migration of PGC.
As one example, the hatching egg is cultured at 8℃with a relative humidity of 60% and a hatching time of 7d.
As one example, in the digestion treatment, the concentration of the digestive juice is 0.25% (wt), that is, 0.25g of pancreatin is contained in 100ml of the stock solution of the digestive juice, the digestion temperature is 37℃and the digestion time is 20 minutes.
As one example, the centrifugation conditions after digestion treatment were 2500rpm for 3min.
As one example, the culture temperature in the cell culture plate was 37℃and the culture was performed under an atmosphere containing 5% CO 2 for 3 days.
The beneficial effects of the invention are as follows:
The existing in-vitro culture system is more suitable for proliferation and separation culture of male chicken primordial germ cells, and improves the cell proliferation rate; the invention can keep the undifferentiated state and good biological property of the original germ cells of the female chickens under the condition of not using the fetal bovine serum; simple components and lower cell culture cost.
The invention reduces the use and the dosage of cytokines, increases the apoptosis inhibitor and promotes the proliferation and migration of cells.
The invention does not need to be replaced between serum and serum-free when carrying out transgene, reduces the workload and improves the transfection efficiency.
The primordial germ cells obtained by the invention can be used for preparing transgenic, animal cloning and chimeric chicken.
Drawings
FIG. 1 is a diagram of primordial germ cells of chickens after 4 days of culture in the medium system of the present application;
FIG. 2 is a graph showing the effect of the culture solution on proliferation of primordial germ cells of chicken after 4 days of culture in the culture medium system of the present application;
FIG. 3 is a graph showing the effect of the culture solution on proliferation of primordial germ cells of chickens after 1, 4, 7, and 11 days of culture in the culture medium system of the application;
FIG. 4 is a graph showing the effect of the culture broth on proliferation of primordial germ cells in chickens after 1,4, 7, 11 days of incubation with inhibitors alone in the medium system of the present application;
description of the drawings: BR is the microscopic bright field mode, COUNT is the counting mode.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following description.
Example 1
A method for in vitro serum-free amplification culture of chicken primordial germ cells, comprising the following steps:
s1, preparation of culture medium without fetal bovine serum
The culture medium does not contain fetal bovine serum, and comprises KO-DMEM, 1% chicken serum, 1% double antibody (penicillin and streptomycin), 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1 Xnonessential amino acids, 1XB-27, 25ng/mL human ActivinA, 0.2 mu M (+ -) -bupstatin, 5 mu mol/LROCK inhibitor, and 1 mu mol/LGSK-3 inhibitor;
S2, separation and culture of chicken primordial germ cells
Hatching eggs at the temperature of 8 ℃ and the relative humidity of 60%, wherein the hatching time is 7d, sterilizing eggshells, opening the hatching eggs, placing the hatching eggs in a culture dish, separating gonads under a split microscope, and placing the separated gonads in a 1.5mL centrifuge tube containing 100 mu L of phosphate buffer solution;
Adding pancreatin digestive juice with concentration of 0.25% into the centrifuge tube, digesting for 20min at 37 ℃, and centrifuging for 3min at 2500rpm after stopping digestion to obtain chicken primordial germ cells;
Transferring the chicken primordial germ cells into a 24-hole cell culture plate, adding a freshly prepared culture solution, culturing for 3d at 37 ℃ in an atmosphere containing 5% CO 2, performing half-volume liquid exchange every other day, and performing subculture after 5 d;
s3, cell proliferation count
The cultured chicken primordial germ cells are resuspended by PBS, the total cell amount is calculated after counting by a cell counter, the total cell amount is divided into two groups, each group is divided into three times, the freshly prepared culture medium without fetal bovine serum is respectively added, and after 4-5d of culture, the sample is respectively counted, and the cell concentration and proliferation rate are calculated.
Comparative example 1
A method for in vitro serum-free amplification culture of chicken primordial germ cells, comprising the following steps:
S1, preparation of culture medium containing fetal bovine serum
The culture medium comprises KO-DMEM, 5% chicken serum, 15% FBS, 1% double antibodies (penicillin and streptomycin), 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1X nonessential amino acid, 1XB-27, 25ng/mL human ActivinA, 6ng/mLSCF, 25ng/mLLIF;
S2, separation and culture of chicken primordial germ cells
Hatching eggs at the temperature of 8 ℃ and the relative humidity of 60%, wherein the hatching time is 7d, sterilizing eggshells, opening the hatching eggs, placing the hatching eggs in a culture dish, separating gonads under a split microscope, and placing the separated gonads in a 1.5mL centrifuge tube containing 100 mu L of phosphate buffer solution;
Adding pancreatin digestive juice with concentration of 0.25% into the centrifuge tube, digesting for 20min at 37 ℃, and centrifuging for 3min at 2500rpm after stopping digestion to obtain chicken primordial germ cells;
transferring the chicken primordial germ cells into a 24-hole cell culture plate, adding the freshly prepared culture medium of the comparative example, culturing for 3d at 37 ℃ in an atmosphere containing 5% CO 2, performing half-volume liquid exchange every other day, and performing subculture after 5 d;
s3, cell proliferation count
The cultured chicken primordial germ cells are resuspended by PBS, the total cell amount is calculated after counting by a cell counter, the total cell amount is divided into two groups, each group is divided into three times, the freshly prepared culture medium without fetal bovine serum is respectively added, and after 4-5d of culture, the sample is respectively counted, and the cell concentration and proliferation rate are calculated.
Comparative examples 2-4, on the basis of comparative example 1, the medium was replaced with the following medium:
preparation of Medium without separate addition of three inhibitors of fetal bovine serum
The culture medium comprises KO-DMEM, 1% chicken serum, 1% diabody, 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1 Xnonessential amino acid, 1XB-27, 25ng/mL human ActivinA and 5 mu mol/LROCK inhibitor;
The culture medium comprises KO-DMEM, 1% chicken serum, 1% diabody, 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1 Xnonessential amino acid, 1XB-27, 25ng/mL human ActivinA, and 1 mu mol/LGSK-3 inhibitor;
The culture medium comprises KO-DMEM, 1% chicken serum, 1% diabody, 1.2mM sodium pyruvate, 1X glutamine, 1X nucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1X nonessential amino acid, 1XB-27, 25ng/mL human ActivinA, 0.2 mu M (+ -) -bupstatin;
As a result, as shown in FIGS. 1 to 4, it was found that the proliferation rate of the primordial germ cells of chickens cultured in the examples was significantly higher than that of the comparative examples, and the effect of the present invention could not be achieved by using three inhibitors alone without adding fetal bovine serum. The chicken primordial germ cells cultured by the culture method are round, have bright edges and large volume, can be generally seen to be connected together or in strings, accords with the morphological characteristics of the female chicken primordial germ cells, and is beneficial to the proliferation of the chicken primordial germ cells in vitro and keeps the undifferentiated state and good biological characteristics.
In conclusion, the invention can effectively promote the proliferation efficiency of the chicken primordial germ cells in vitro and can well maintain the biological characteristics of the chicken primordial germ cells. Only a small amount of chicken serum is used, so that the interference for the subsequent cell transfection operation is reduced, and the transfection efficiency is improved to a certain extent.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (5)
1. A method for in vitro serum-free amplification culture of chicken primordial germ cells, which is characterized in that; the method comprises the following steps:
s1, preparation of culture medium without fetal bovine serum
The culture medium does not contain fetal bovine serum, and comprises KO-DMEM, an inhibitor, chicken serum, diabody, sodium pyruvate, glutamine, nucleoside, beta-mercaptoethanol, heparin sodium, recombinant human FGF, nonessential amino acid, B-27 and human ActivinA; the inhibitor is (+ -) -bupivastatin, a ROCK inhibitor and a GSK-3 inhibitor;
S2, separation and culture of chicken primordial germ cells
Hatching the hatching eggs, sterilizing the eggshells, opening the hatching eggs, placing the hatching eggs in a culture dish, separating gonads under a split microscope, and placing the gonads in a phosphate buffer solution;
Adding pancreatin digestive juice into phosphate buffer solution for digestion treatment, and centrifuging after stopping digestion to obtain chicken primordial germ cells;
Transferring the chicken primordial germ cells into a cell culture plate, adding a freshly prepared culture solution for culture, performing half liquid exchange every other day, and then performing subculture;
s3, cell proliferation count
The cultured chicken primordial germ cells are resuspended by PBS, the total cell amount is calculated after counting by a cell counter, the total cell amount is divided into two groups, each group is divided into three times, the freshly prepared culture medium without fetal bovine serum is respectively added, the sample is respectively sampled and counted after culturing, and the cell concentration and proliferation rate are calculated.
2. The method for in vitro serum-free expansion culture of chicken primordial germ cells according to claim 1, wherein the method comprises the following steps: the culture medium comprises KO-DMEM, 1% (V/V) chicken serum, 1% (V/V) double antibody, 1.2mM sodium pyruvate, 1 Xglutamine, 1 Xnucleoside, 0.1mM beta-mercaptoethanol, 100mg/mL heparin sodium, 4ng/mL recombinant human FGF, 1 Xnonessential amino acids, 1X B-27, 25ng/mL human ActivinA, 5 mu mol/L ROCK inhibitor, 1 mu mol/L GSK-3 inhibitor, and 0.2 mu M (+ -) -bupstatin.
3. The method for in vitro serum-free expansion culture of chicken primordial germ cells according to claim 1, wherein the method comprises the following steps: in the digestion treatment, the concentration of the digestion solution was 0.25% (wt), the digestion temperature was 37℃and the digestion time was 20min.
4. The method for in vitro serum-free expansion culture of chicken primordial germ cells according to claim 1, wherein the method comprises the following steps: the centrifugation conditions after digestion were 2500rpm, 3min.
5. The method for in vitro serum-free expansion culture of chicken primordial germ cells according to claim 1, wherein the method comprises the following steps: the culture temperature in the cell culture plate was 37℃and the culture was carried out under an atmosphere containing 5% CO 2 for 3d.
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