CN109679905B - Preparation method of chicken dendritic cells and chicken dendritic cells - Google Patents
Preparation method of chicken dendritic cells and chicken dendritic cells Download PDFInfo
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Abstract
The invention discloses a preparation method of chicken dendritic cells and the chicken dendritic cells, belonging to the technical field of veterinary immunology, wherein mononuclear cells are separated from chicken bone marrow or peripheral blood thereof, basic culture medium is used for culturing for 12-14h, non-adherent cells are removed, induction culture medium is added for continuous culture, 2/3-dose liquid change is carried out by using enhanced induction culture medium after 2 days of culture, half-dose liquid change is carried out by using enhanced induction culture medium after 5 days of culture, immature dendritic cells are obtained on 7 days, and phenotype detection is carried out on the immature dendritic cells for carrying out experiment or cryopreservation. According to the invention, the chicken serum is used for replacing bovine serum, so that harmful stimulation of heterogeneous serum to chicken cells is avoided; the induction culture of the mononuclear cells is carried out by two stages of an induction culture medium and an enhanced culture medium, and the enhanced culture medium is added with the lactoprotein hydrolysate and the chick embryo allantoic fluid, so that the requirement of later cell proliferation on nutrition is met, and the proliferation and differentiation of the cells are promoted.
Description
Technical Field
The invention relates to a preparation method of a cell and a chicken dendritic cell, in particular to a preparation method of a chicken dendritic cell and a chicken dendritic cell, and belongs to the technical field of veterinary immunology.
Background
Dendritic cells DCs, which were first found in the spleen of mice in 1973 and named dendritic cells because they have a unique morphology of dendritic-like processes, were differentiated from hematopoietic stem cells, widely distributed in various tissues including skin dermis and mucosa, and are one of the cells that were first exposed to antigens, and at present, the found antigen-presenting cells mainly include DCs, macrophages and B lymphocytes, but the antigen-presenting ability of DCs is much stronger than that of macrophages and B lymphocytes, and is currently known as the most powerful professional antigen-presenting cell, DCs are able to stimulate the activation and proliferation of naive T lymphocytes, while macrophages and B lymphocytes can only stimulate activated T cells or memory T cells, and thus DCs are the initiator of specific immune responses.
In view of the role of DCs in initiating immune responses, targeting immunotherapy and prophylaxis based on DCs has become a hot research spot at home and abroad, and especially has gained more and more extensive attention in the field of anti-tumor therapy in human medicine and vaccine development in veterinary medicine, and thus, the in vitro culture technology of DCs cells has been receiving more and more attention from researchers in various countries.
Although a great deal of research and optimization has been carried out on the in vitro culture conditions of human-derived DCs and murine-derived DCs, the cost of the in vitro culture of the DCs is still high at present, the quality of the obtained DCs is uneven, and in the aspect of in vitro culture of the chicken DCs, the culture method of the mouse-derived DCs which is along with the prior art is used for carrying out induction culture on chicken bone marrow mononuclear cells into the DCs by using a culture medium containing GM-CSF (50-100ng/ml) and IL-4(50-100ng/ml) with larger concentration.
Therefore, it is necessary to optimize and perfect the existing preparation method of chicken DCs, so as to reduce the preparation cost of the chicken DCs and improve the quality of the chicken DCs.
Disclosure of Invention
The invention mainly aims to provide a preparation method of chicken dendritic cells and the chicken dendritic cells, and improve the purity of the chicken dendritic cells.
The purpose of the invention can be achieved by adopting the following technical scheme:
a method for preparing chicken dendritic cells includes separating mononuclear cells from chicken bone marrow or peripheral blood thereof, culturing in basic culture medium for 12-14h, removing nonadherent cells, adding induction culture medium, culturing for 2 days, changing culture medium by 2/3 times with enhanced induction culture medium, culturing for 5 days, changing culture medium by half, obtaining immature dendritic cells on day 7, performing phenotype detection, and performing experiment or freezing.
The basic culture medium is RPMI-1640 culture medium containing chicken serum;
the induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4 and chicken serum;
the enhanced induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4, hydrolyzed milk protein, chick embryo allantoic fluid and chick serum.
In an induction medium, the concentration of rchGM-CSF is 20-25ng/ml, the concentration of rchIL-4 is 4-6ng/ml, and the volume fraction of chicken serum is 4-6%.
In the enhanced induction culture medium, the concentration of rchGM-CSF is 30-40ng/ml, the concentration of rchIL-4 is 4-6ng/ml, the concentration of hydrolyzed milk protein is 1-3mg/ml, the volume fraction of chick embryo allantoic fluid is 1-3%, and the volume fraction of chick serum is 4-6%;
wherein the preparation of chick embryo allantoic fluid comprises: freezing and thawing 12-15 days old SPF chick embryo allantoic fluid at-70 deg.C for 1-2 times, centrifuging 1200g for 30min, collecting supernatant, and filtering for sterilization to obtain chick embryo allantoic fluid for preparing enhanced induction culture medium.
Isolating mononuclear cells from chicken bone marrow or its peripheral blood includes: preparing chicken peripheral blood mononuclear cells and bone marrow mononuclear cells;
wherein, the preparation of the chicken peripheral blood mononuclear cells comprises the following steps: mixing peripheral blood of chicken with anticoagulant according to the proportion of 4: 1, then mixing with a rinsing solution, 1: 1, diluting, adding the diluted solution above the liquid level of the mononuclear cell separation solution, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the peripheral blood mononuclear cells;
wherein the preparation of bone marrow mononuclear cells comprises: flushing out the mononuclear cells from the bone marrow of the chicken by using a flushing liquid, then adding the mononuclear cells above the liquid level of a mononuclear cell separation liquid, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the bone marrow mononuclear cells.
The anticoagulant for preparing the chicken peripheral blood mononuclear cells comprises sodium chloride, EDTA and glucose, and the flushing fluid comprises the sodium chloride, the EDTA, glutamine and the glucose.
The concentration of sodium chloride in the anticoagulant is 6-9g/L, the concentration of EDTA is 8-14g/L, and the concentration of glucose is 8-12 g/L;
the concentration of sodium chloride in the flushing fluid is 6-9g/L, the concentration of EDTA is 1-3g/L, the concentration of glutamine is 1-3g/L, and the concentration of glucose is 8-12 g/L.
Separating mononuclear cells from chicken bone marrow or its peripheral blood, and adjusting the concentration of mononuclear cells to 3-5 xl 0 before culturing in basic culture medium 6 /ml。
The cryopreservation method of the dendritic cells comprises the following steps:
centrifuging the prepared dendritic cells, removing supernatant, adding freezing medium, and adjusting cell concentration to l0 6 Putting the mixture into a freezing storage box, freezing and storing the mixture for 16 to 24 hours at the temperature of minus 70 ℃, and transferring the mixture into a liquid nitrogen tank for long-term storage;
the frozen stock solution is RPIM-1640 cell culture medium containing 10% DMSO, 8% chicken serum and 5mg/ml hydrolyzed milk protein.
A chicken dendritic cell is prepared by the preparation method of the chicken dendritic cell.
The invention has the beneficial technical effects that: compared with the existing mode of culturing the chicken dendritic cells, the preparation method of the chicken dendritic cells and the chicken dendritic cells provided by the invention have the advantages that the chicken serum is used for replacing bovine serum, so that the harmful stimulation of heterogeneous serum to the chicken cells is avoided; the induction culture of the mononuclear cells is carried out by using an induction culture medium and an enhanced culture medium in two stages, and the enhanced culture medium is added with lactoprotein hydrolysate and chick embryo allantoic fluid, so that the requirement of later cell proliferation on nutrition is fully met, and the proliferation and differentiation of the cells are promoted.
Detailed Description
In order to make the technical solutions of the present invention clearer and clearer to those skilled in the art, the present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.
The preparation method of chicken dendritic cells provided by this embodiment includes separating mononuclear cells from chicken bone marrow or peripheral blood thereof, culturing for 12-14h with a basal medium, removing nonadherent cells, adding an induction medium to continue culturing, performing 2/3 times of liquid change with an enhanced induction medium after culturing for 2 days, performing half of liquid change with the enhanced induction medium after culturing for 5 days, obtaining immature dendritic cells on day 7, performing phenotype detection on the immature dendritic cells, and performing experiments or cryopreservation;
wherein the basic culture medium is RPMI-1640 culture medium containing chicken serum;
the induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4 and chicken serum, the concentration of the rchGM-CSF is 20-25ng/ml, the concentration of the rchIL-4 is 4-6ng/ml, and the volume fraction of the chicken serum is 4-6%;
the enhanced induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4, hydrolyzed milk protein, chick embryo allantoic fluid and chick serum, the concentration of the rchGM-CSF is 30-40ng/ml, the concentration of the rchIL-4 is 4-6ng/ml, the concentration of the hydrolyzed milk protein is 1-3mg/ml, the volume fraction of the chick embryo allantoic fluid is 1-3%, and the volume fraction of the chick serum is 4-6%;
wherein the preparation of chick embryo allantoic fluid comprises: freezing and thawing 12-15 day-old SPF chick embryo allantoic fluid at-70 deg.C for 1-2 times, centrifuging 1200g for 30min, collecting supernatant, and filtering for sterilization to obtain chick embryo allantoic fluid for preparing enhanced induction culture medium.
Isolation of mononuclear cells from chicken bone marrow or its peripheral blood includes: preparing chicken peripheral blood mononuclear cells and preparing bone marrow mononuclear cells;
wherein, the preparation of the chicken peripheral blood mononuclear cells comprises the following steps: mixing peripheral blood of chicken with anticoagulant according to the ratio of 4: 1, then mixing with a rinsing solution, 1: 1, diluting, adding the diluted solution above the liquid level of the mononuclear cell separation solution, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the peripheral blood mononuclear cells;
wherein, the preparation of the bone marrow mononuclear cell comprises the following steps: flushing out the mononuclear cells from the bone marrow of the chicken by using a flushing liquid, then adding the mononuclear cells above the liquid level of a mononuclear cell separation liquid, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the bone marrow mononuclear cells.
The anticoagulant during preparation of the chicken peripheral blood mononuclear cells comprises sodium chloride, EDTA and glucose, the flushing fluid comprises sodium chloride, EDTA, glutamine and glucose, the concentration of the sodium chloride in the anticoagulant is 6-9g/L, the concentration of the EDTA is 8-14g/L, and the concentration of the glucose is 8-12 g/L;
the concentration of sodium chloride in the flushing fluid is 6-9g/L, the concentration of EDTA is 1-3g/L, the concentration of glutamine is 1-3g/L, and the concentration of glucose is 8-12 g/L.
Separating mononuclear cells from chicken bone marrow or its peripheral blood, and adjusting the concentration of mononuclear cells to 3-5 xl 0 before culturing in basic culture medium 6 /ml。
The cryopreservation method of the dendritic cells comprises the following steps: centrifuging the prepared dendritic cells, removing supernatant, adding freezing medium, and adjusting cell concentration to l0 6 Putting the mixture into a freezing storage box, freezing and storing the mixture for 16 to 24 hours at the temperature of minus 70 ℃, and transferring the mixture into a liquid nitrogen tank for long-term storage; the frozen stock solution is RPIM-1640 cell culture medium containing 10% DMSO, 8% chicken serum, and 5mg/ml hydrolyzed milk protein.
The method for separating bone marrow mononuclear cells in the present example was: flushing the mononuclear cells from the bone marrow cavity of the chicken by using flushing fluid, then adding the mononuclear cells above the liquid level of a mononuclear cell separation solution, centrifuging for 30-40 min at 500g, and collecting the bone marrow mononuclear cells for induction culture.
The preparation method of the washing liquid in the embodiment comprises the following steps: adding 6-9g of sodium chloride, 1-3g of EDTA, 1-3g of glutamine and 8-12g of glucose into 1000ml of deionized water, completely dissolving, and filtering and sterilizing to obtain the flushing fluid.
The method for separating peripheral blood mononuclear cells in the present example includes: mixing peripheral blood of chicken with anticoagulant according to the ratio of 4: 1, then mixing with a rinsing solution, 1: 1, diluting, adding the diluted solution above the liquid level of a mononuclear cell separation solution, centrifuging for 30-40 min at 500g, and collecting peripheral blood mononuclear cells for induction culture.
The preparation method of the anticoagulant in the embodiment comprises the following steps: adding 6-9g of sodium chloride, 8-14g of EDTA and 8-12g of glucose into 1000ml of deionized water, completely dissolving, and filtering for sterilization to obtain the anticoagulant.
The basic culture medium of this example was: RPMI-1640 cell culture medium containing 4-6% chicken serum.
The induction medium of this example was: RPMI-1640 cell culture medium containing rchGM-CSF (20-25ng/ml), rchIL-4(4-6ng/ml) and 4-6% chicken serum.
The enhanced induction medium of this example was: RPMI-1640 cell culture medium containing rchGM-CSF (30-40ng/ml), rchIL-4(4-6ng/ml), hydrolyzed milk protein (1-3mg/ml), 1-3% chick embryo allantoic fluid, 4-6% chick serum.
The cell cryopreservation solution of this example was: RPMI-1640 medium containing 10% DMSO, 8% chicken serum, 5mg/ml hydrolyzed milk protein.
The preparation method of the chick embryo allantoic fluid comprises the following steps: freezing and thawing 12-15 days old SPF chick embryo allantoic fluid at-70 deg.C for 1-2 times, centrifuging 1200g for 30min, collecting supernatant, and filtering for sterilization to obtain chick embryo allantoic fluid for preparing enhanced induction culture medium.
In this embodiment, the method for preparing dendritic cells comprises the following steps:
step 1: preparing a dendritic cell culture medium, including preparing a basic culture medium, preparing an induction culture medium and preparing an enhanced culture medium;
step 2: preparing mononuclear cells, including preparing bone marrow mononuclear cells and preparing peripheral blood mononuclear cells;
and step 3: induction culture of mononuclear cells
And 4, step 4: collecting, detecting and freezing dendritic cells.
In this embodiment, the specific implementation process of step 1 is as follows:
adding chicken serum with volume fraction of 4-6% into RPMI-1640 culture medium, and preparing basic culture medium;
adding rchGM-CSF with the mass fraction of 20-25ng/ml, rchIL-4 with the mass fraction of 4-6ng/ml and chicken serum with the volume fraction of 4-6% into an RPMI-1640 culture medium to prepare an induction culture medium;
aseptically collecting allantoic fluid of SPF chick embryo incubated to 12-15 days old, freezing and thawing at-70 deg.C for 1-2 times, centrifuging at 1200g for 30min, collecting supernatant, and filtering for sterilization to obtain enhanced induction culture medium.
Adding rchGM-CSF with the mass fraction of 30-40ng/ml, rchIL-4 with the mass fraction of 4-6ng/ml, lactoprotein hydrolysate with the mass fraction of 1-3mg/ml, chick embryo allantoic fluid with the volume fraction of 1-3% and chick serum with the volume fraction of 4-6% into an RPMI-1640 culture medium, and filtering and sterilizing to obtain an enhanced induction culture medium.
In this embodiment, the specific implementation process of step 2 is as follows:
collecting peripheral blood of the chicken through a wing vein, and mixing the blood with an anticoagulant 4: 1, and preparing anticoagulant according to the proportion of 1: 1, diluting the anticoagulation blood by using a flushing fluid, adding the diluted anticoagulation blood onto the liquid surface of the mononuclear cell separation liquid, centrifuging for 30-40 min at 500g, and collecting a white atomized layer, namely the peripheral blood mononuclear cells.
Taking thighbone and shinbone of 2-4 weeks old chicken, flushing out bone marrow with flushing fluid, adding above the liquid level of mononuclear cell separation liquid, centrifuging for 30-40 min at 500g, and collecting white vaporous layer to obtain bone marrow blood mononuclear cells.
Adding 6-9g of sodium chloride, 8-14g of EDTA and 8-12g of glucose into 1000ml of deionized water, and after completely dissolving, filtering and sterilizing to obtain the anticoagulant.
Adding 6-9g of sodium chloride, 1-3g of EDTA, 1-3g of glutamine and 8-12g of glucose into 1000ml of deionized water, completely dissolving, and filtering and sterilizing to obtain the flushing fluid.
In this embodiment, the specific implementation process in step 3 is as follows:
washing the collected peripheral blood mononuclear cells or bone marrow mononuclear cells with a basic culture medium for 2 times, adjusting cell concentration to 3-5 × l0 6 And/ml, culturing in a cell culture box with 5% CO2 and 38 ℃ for 12-14h, removing suspended cells, replacing a basic culture medium with an induction culture medium, continuously culturing until the culture medium is changed by 2/3 amount and half amount respectively on day 2 and day 5, performing induction culture on day 7 to obtain immature dendritic cells, collecting the cells, detecting, and using the cells for the next experiment or freezing.
In this embodiment, the specific implementation process in step 4 is as follows:
the cells are gently blown down, stained with a flow antibody against CD11c, and then put on a machine for flow detection, and the positive rate of CD11c molecules on the cell surface is analyzed for the next experiment or cell cryopreservation.
The cell cryopreservation process comprises centrifuging 250g cells for 10min, adding cryopreservation solution, and adjusting cell concentration to l0 6 Putting the mixture into a freezing storage box, standing overnight at-70 ℃, and transferring the mixture into a liquid nitrogen tank for long-term storage.
The present embodiment is described in further detail below with reference to specific embodiments, which are only used for explaining the present embodiment and should not be construed as limiting the scope of the present embodiment. The examples were carried out under conventional conditions without specifying the specific conditions.
Example 1:
preparation of chicken peripheral blood mononuclear cell-derived dendritic cells
Experimental materials:
SPF chickens from Beijing Meiliya
rchGM-CSF is derived from Abcam
rchIL-4 was derived from Kingfisher
Fluorescently labeled anti-CD 11c antibody was derived from eBioscience
The RPMI-1640 culture medium is derived from Verwendung
The experimental method comprises the following steps:
1. sucking 1ml of anticoagulant (containing 8mg/ml of sodium chloride, 11mg/ml of EDTA and 8mg/ml of glucose) by using a syringe, aseptically collecting 4ml of blood of the SPF chicken through a fin vein, and fully and uniformly mixing the anticoagulant with the blood to obtain anticoagulant; adding 5ml of washing solution (containing 9mg/ml of sodium chloride, 2mg/ml of EDTA, 2mg/ml of glutamine and 10mg/ml of glucose) into the anticoagulated blood, and fully mixing; and then 4ml of diluted anticoagulated blood is added above the liquid level of 5ml of mononuclear cell separating medium in a 10ml centrifuge tube, 500g of the diluted anticoagulated blood is centrifuged for 30min, and a mist white second layer is taken, namely the peripheral blood mononuclear cells.
2. Peripheral blood mononuclear cells were washed 2 times with a basal medium (RPMI-1640 containing 4% chicken serum) to adjust the cell concentration to 5 xl 0 6 Adding the culture medium into a 6-hole cell culture plate for culture, after culturing for 14 hours, slightly shaking the cell culture plate, and discarding a basic culture medium, thereby removing suspended cells; then adding 3ml of induction culture medium (containing 20ng/ml of rchGM-CSF, 5ng/ml of rchIL-4 and 4% of chicken serum RPMI-1640) to continue culturing, after 2 days of culturing, gently sucking out 2ml of induction culture medium and discarding, adding 2ml of enhanced induction culture medium (containing 30ng/ml of rchGM-CSF, 4ng/ml of rchIL-4, 1mg/ml of lactoprotein hydrolysate, 1% of chicken embryo allantoic fluid and 5% of chicken serum RPMI-1640) to continue culturing, after 3 days of culturing, carrying out half-volume liquid change by using the enhanced induction culture medium, and after 2 days of continuing culturing, obtaining the immature dendritic cells.
3. The dendritic cells in the induction culture were gently aspirated to adjust the cell concentration to l0 6 And/ml, 0.1ml of cells are taken, anti-CD 11c antibody is added, the cells are stained for 30min at room temperature, the cells are washed twice and then are put on a computer for flow detection, and the ratio of the cells which are positive to CD11c is calculated.
4. In order to illustrate the advantages of the method for preparing DCs in this example, a control group of DCs cells prepared by the conventional method was also provided. The anticoagulant used in the method is normal saline containing 1% EDTA, the diluent is normal saline, the basic culture medium is RPMI-1640 culture medium containing 10% fetal calf serum, and the induction culture medium is RPMI-1640 culture medium containing rchGM-CSF (50ng/ml), rchIL-4(10ng/ml) and 10% fetal calf serum; the induction procedure is that half amount of liquid is changed by using an induction culture medium at intervals of 2 days, and after the induction culture is finished on the 7 th day, cells are collected for detection; the remaining conditions were the same as in the method of this example.
The results of the above experiment repeated 3 times are shown in table 1:
TABLE 1 Positive rate of dendritic cells derived from chicken peripheral blood mononuclear cells prepared by the method of the present example and the conventional method
The positive rate of DCs prepared by the method of the embodiment is 87.07% which is far higher than 71.31% of the existing method, and the dosage of the cell factors (rchGM-CSF, rchIL-4) is saved by 50% compared with the existing method.
Example 2:
preparation of bone marrow monocyte-derived dendritic cells
Experimental materials:
the 3-week-old broiler chickens come from ordinary state
rchGM-CSF is derived from Abcam
rchIL-4 was derived from Kingfisher
Fluorescently labeled anti-CD 11c antibody was derived from eBioscience
RPMI-1640 medium from Verwendung
The experimental method comprises the following steps:
1. taking the tibia and the femur of a chicken under an aseptic condition, sucking 8ml of washing liquid (containing 9mg/ml of sodium chloride, 1.5mg/ml of EDTA, 2mg/ml of glutamine and 11mg/ml of glucose) by using a syringe to wash the marrow cavity of the tibia or the femur back and forth for 3 times, centrifuging 250g of the obtained washing liquid for 10min, re-suspending the precipitate by using 4ml of the washing liquid, adding the precipitate onto the liquid surface of 5ml of mononuclear cell separation liquid in a 10ml centrifuge tube, centrifuging 500g for 40min, and taking a mist white second layer to obtain the bone marrow mononuclear cells.
2. The bone marrow mononuclear cells were washed 2 times with a basal medium (RPMI-1640 containing 5% chicken serum) to adjust the cell concentration to 3 xl 0 6 Adding the culture medium into a 6-hole cell culture plate for culture, after 12 hours of culture, slightly shaking the cell culture plate, and discarding a basic culture medium, thereby removing suspended cells; then adding induction medium (containing rchGM-CSF 25ng/ml, rchIL-4 6ng/ml, etc.),RPMI-1640 with 5% chicken serum), and changing the culture medium with enhanced induction medium (containing rchGM-CSF 30ng/ml, rchIL-4 ng/ml, hydrolyzed milk protein 2mg/ml, allantoic fluid 1% of chick embryo, and RPMI-1640 with 5% chicken serum) for 2/3 times after 2 days; and continuously culturing for 3 days, performing half-amount liquid change by using an enhanced induction culture medium, and culturing for 2 days to obtain the immature dendritic cells.
3. Dendritic cells were gently aspirated from 2 wells to adjust the cell concentration to l0 6 And/ml, 0.1ml of cells are taken, anti-CD 11c antibody is added, the cells are stained for 30min at room temperature, the cells are washed twice and then are put on a computer for flow detection, and the ratio of the cells which are positive to CD11c is calculated.
4. In order to illustrate the advantages of the method for preparing DCs in this example, a control group of DCs cells prepared by the prior art method was also set. The used flushing fluid is physiological saline, the basal culture medium is RPMI-1640 culture medium containing 10% fetal calf serum, and the induction culture medium is RPMI-1640 culture medium containing rchGM-CSF (50ng/ml), rchIL-4(10ng/ml) and 10% fetal calf serum; the induction procedure is that half amount of liquid is changed by using an induction culture medium at intervals of 2 days, and after the induction culture is finished on the 7 th day, cells are collected for detection; the remaining conditions were the same as in the method of this example.
The results of the above experiment repeated 3 times are shown in table 2:
TABLE 2 Positive Rate of dendritic cells derived from chicken bone marrow mononuclear cells prepared by the method of the present example and the conventional method
The positive rate of DCs prepared by the method of the embodiment is 90.02% which is far higher than 76.16% of that of the existing method, and the dosage of the cell factors (rchGM-CSF, rchIL-4) is saved by 50% compared with that of the existing method.
In this example, the chicken dendritic cells provided in this example were the chicken dendritic cells prepared by the above-described method for preparing chicken dendritic cells.
In summary, in the preparation method of chicken dendritic cells provided in this embodiment, the induction culture of mononuclear cells is performed in two stages by using the induction culture medium and the enhanced culture medium, and the lactoprotein hydrolysate and the chick allantoic fluid are added to the enhanced culture medium, which not only fully satisfies the requirement of late cell proliferation on nutrition, but also promotes the proliferation and differentiation of cells.
The above description is only a further embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can substitute or change the technical solution of the present invention and its idea within the scope of the present invention.
Claims (7)
1. A preparation method of chicken dendritic cells is characterized in that anticoagulant and flushing liquid are used for separating mononuclear cells from chicken bone marrow or peripheral blood thereof, the mononuclear cells are cultured for 12-14 hours by using a basic culture medium, non-adherent cells are removed, an induction culture medium is added for continuous culture, 2/3 liquid changing is carried out by using an enhanced induction culture medium after 2 days of culture, half liquid changing is carried out by using the enhanced induction culture medium after 5 days of culture, immature dendritic cells are obtained on the 7 th day, and are subjected to phenotype detection for carrying out experiments or cryopreservation;
the anticoagulant comprises sodium chloride, EDTA and glucose, and the flushing fluid comprises sodium chloride, EDTA, glutamine and glucose;
the basic culture medium is RPMI-1640 culture medium containing chicken serum;
the induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4 and chicken serum;
the enhanced induction culture medium is RPMI-1640 cell culture medium containing rchGM-CSF, rchIL-4, hydrolyzed milk protein, chick embryo allantoic fluid and chick serum.
2. The method according to claim 1, wherein the rchGM-CSF concentration is 20 to 25ng/ml, the rchll-4 concentration is 4 to 6ng/ml, and the volume fraction of chicken serum is 4 to 6% in the induction medium.
3. The method of claim 1, wherein in the enhanced induction medium, the rchGM-CSF is in a concentration of 30-40ng/ml, the rchll-4 is in a concentration of 4-6ng/ml, the lactoprotein hydrolysate is in a concentration of 1-3mg/ml, the allantoic fluid of chick embryos is in a volume fraction of 1-3%, and the serum of chick embryos is in a volume fraction of 4-6%;
wherein the preparation of chick embryo allantoic fluid comprises: freezing and thawing 12-15 day-old SPF chick embryo allantoic fluid at-70 deg.C for 1-2 times, centrifuging 1200g for 30min, collecting supernatant, and filtering for sterilization to obtain chick embryo allantoic fluid for preparing enhanced induction culture medium.
4. The method of claim 1, wherein isolating mononuclear cells from bone marrow of the chicken or peripheral blood thereof comprises: preparing chicken peripheral blood mononuclear cells and bone marrow mononuclear cells;
wherein, the preparation of the chicken peripheral blood mononuclear cells comprises the following steps: mixing peripheral blood of chicken with anticoagulant according to the ratio of 4: 1, then mixing with a rinsing solution, 1: 1, diluting, adding the diluted solution above the liquid level of the mononuclear cell separation solution, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the peripheral blood mononuclear cells;
wherein the preparation of bone marrow mononuclear cells comprises: flushing out the mononuclear cells from the bone marrow of the chicken by using a flushing liquid, then adding the mononuclear cells above the liquid level of a mononuclear cell separation liquid, centrifuging for 30-40 min by 500g, and collecting a white and vaporous second layer, namely the bone marrow mononuclear cells.
5. The method of claim 1, wherein the concentration of sodium chloride in the anticoagulant is 6-9g/L, the concentration of EDTA is 8-14g/L, and the concentration of glucose is 8-12 g/L;
the concentration of sodium chloride in the flushing fluid is 6-9g/L, the concentration of EDTA is 1-3g/L, the concentration of glutamine is 1-3g/L, and the concentration of glucose is 8-12 g/L.
6. The method for preparing chicken dendritic cells according to claim 1, wherein mononuclear cells are isolated from chicken bone marrow or peripheral blood thereof, and the concentration of mononuclear cells is adjusted to 3 to 5 xl 0 before culturing in a basal medium 6 /ml。
7. The method for preparing dendritic cells of chicken as claimed in claim 1, wherein the method for cryopreserving dendritic cells comprises:
centrifuging the prepared dendritic cells, removing supernatant, adding freezing medium, and adjusting cell concentration to l0 6 Putting the mixture into a freezing storage box, freezing and storing the mixture for 16 to 24 hours at the temperature of minus 70 ℃, and transferring the mixture into a liquid nitrogen tank for long-term storage;
the frozen stock solution is RPIM-1640 cell culture medium containing 10% DMSO, 8% chicken serum and 5mg/ml hydrolyzed milk protein.
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