CN116496987A - Culture method for improving inflammatory properties of dendritic cells - Google Patents
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0634—Cells from the blood or the immune system
- C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
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Abstract
The invention discloses a culture method for improving the inflammatory property of Dendritic Cells (DC). Belongs to the technical field of cell culture. The invention solves the problems of immature DC and insufficient immunoinflammatory obtained by the existing DC culture method. The invention provides a culture method for promoting DC inflammatory by replacing glucose-free culture medium for 2 days in the later stage of DC culture, which is proved by experiments to be capable of effectively promoting DC inflammatory, obviously up-regulating the expression of DC surface co-stimulatory molecules CD40, CD80, CD86 and pro-inflammatory cytokines IL6, IL12p35, IL12p40 and IL23p19 and down-regulating the expression of anti-inflammatory factor IL10, thereby enhancing the capability of DC mediated T cells for secreting IFNgamma and anti-tumor immune effect thereof.
Description
Technical Field
The invention relates to the technical field of cell culture, in particular to a culture method for improving the inflammatory property of dendritic cells.
Background
Dendritic Cells (DCs) are the most potent antigen presenting cells that take up, process and present antigens to T cells via major histocompatibility complexes to activate them, while up-regulating the expression of surface co-stimulatory molecules CD40, CD80, CD86, etc., promote proliferation of T cells and secrete IL-12, IL-6, IL-23, etcProinflammatory factor induced initial CD4 + T cells and naive CD8 + T cells differentiate towards effector T cells. The differentiated effector T cells can secrete different inflammatory factors to participate in immune responses of various diseases such as tumor, bacterial or fungal infection, autoimmune diseases, asthma and the like of the organism, and the secreted inflammatory factor IFNgamma plays an important role in anti-tumor immune responses of the organism.
In the tumor immune microenvironment, accumulation of lactic acid, various growth factors and cytokines secreted by tumor cells and the like can influence the maturation and activation of DC, inhibit the antigen presenting capability of DC, and reduce the anti-tumor immune effect of DC. DC immunotherapy, which is obtained by culturing DC in vitro and enhancing the antitumor activity of DC and then infusing the DC back into a patient, has the characteristics of high targeting, high safety and long-term action, and has become one of the most widely used immunocyte therapies. However, the in vitro cultured DC is immature DC, is in an immune tolerance state, has weak capability of expressing costimulatory molecules and secreting inflammatory factors, has weak capability of promoting differentiation of effector T cells, and is difficult to effectively exert anti-tumor immune effect.
Thus, how to increase the immunoinflammatory nature of in vitro cultured DCs and to promote their ability to mediate effector T cell differentiation has been a problem that has been urgently addressed by those skilled in the art.
Disclosure of Invention
In view of this, the present invention provides a culture method for enhancing the inflammatory properties of dendritic cells using glucose-free medium during in vitro culture of dendritic cells.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a culture method for improving the inflammatory properties of dendritic cells, comprising the steps of:
step 1: obtaining single bone marrow cells, re-suspending in a complete culture medium, then spreading in a cell culture container, placing in a cell culture box containing 5% carbon dioxide at 37 ℃ for 5 days, and changing fresh culture medium according to the growth condition of cells;
step 2: removing the complete culture medium of the cultured cells in the step 1, adding a glucose-free culture medium, and continuously placing the culture medium in a cell culture box containing 5% carbon dioxide at 37 ℃ for 2 days to obtain the mature dendritic cells with high inflammatory property.
Further, the complete medium is RPMI1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, glutamine.
Further, the glucose-free medium is glucose-free RPMI1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, and glutamine.
Further, final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine in the complete medium were 10ng/ml, 5ng/ml, 10%, 100U/ml, and 2mM, in that order.
Further, the final mass concentration of glucose in the complete medium was 11.11mM.
Further, final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine in the glucose-free medium were 10ng/ml, 5ng/ml, 10%, 100U/ml, and 2mM, in that order.
Further, the RPMI1640 medium is a commercial medium, and the glucose-free RPMI1640 medium does not contain glucose, and other components are consistent with the RPMI1640 medium.
Further, the single bone marrow cells in step 1 were plated at a density of 1X 10 6 /ml。
Further, the preparation method of the single bone marrow cells in the step 1 is as follows:
(1) Taking femur and tibia of a mouse, sucking PBS (phosphate buffered saline) by using a syringe, flushing bone marrow cells into a centrifuge tube, centrifuging at a rotating speed of 400g for 5 minutes, and discarding supernatant;
(2) After the PBS is used for resuspension of the cells, the cells are filtered through a 40 mu m filter membrane, and the supernatant is discarded after centrifugation at 400g for 5 minutes;
(3) Lysing erythrocytes mixed in bone marrow cells with an erythrocyte lysate, and washing with PBS 1 time after stopping the lysis;
(4) The resulting bone marrow cells were resuspended and counted in complete medium for plating.
Further, the specific operation of replacing the fresh medium in the step 1 is as follows: half of the volume of the original complete medium in the cell culture vessel was removed, followed by the addition of an equivalent amount of freshly prepared complete medium pre-warmed at 37 ℃.
Further, the specific operation of replacing the culture medium in the step 2 is as follows: the original complete medium in the cell culture vessel was removed entirely, and an equal amount of freshly prepared glucose-free medium, pre-warmed at 37 ℃, was added.
Compared with the prior art, the invention has the beneficial effects that:
the invention solves the problems of immature DC and insufficient immunoinflammatory obtained by the existing DC culture method. The invention provides a culture method for promoting DC inflammatory by replacing glucose-free culture medium for 2 days in the later stage of DC culture, which is proved by experiments to be capable of effectively promoting DC inflammatory, obviously up-regulating the expression of DC surface co-stimulatory molecules CD40, CD80, CD86 and pro-inflammatory cytokines IL6, IL12p35, IL12p40 and IL23p19 and down-regulating the expression of anti-inflammatory factor IL10, thereby enhancing the capability of DC mediated T cells for secreting IFNgamma and anti-tumor immune effect thereof.
In addition, the promotion of glycolysis rise following DC activation to promote cytokine expression is a consensus of research in the art, and glucose-free culture has also been reported to reduce the expression of inflammatory molecules in DCs. The present invention shows that the length of time of culture in glucose-free medium is critical for the transition of DC inflammatory properties. The expression levels of the inflammatory factors Il12p35 and Il12p40 were indeed reduced in DCs cultured in the late stage of in vitro culture with glucose-free medium for 1 day. The invention is used for culturing the DC in vitro for 2 days by replacing glucose-free culture medium at the later stage, the inflammatory performance of the DC is obviously enhanced, and the expression of inflammatory factors IL6, IL12p35, IL12p40 and IL23p19 is obviously up-regulated. The present invention was made to overcome the inherent thinking of the researchers in the field.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the results of flow cytometry detection of CD40, a DC costimulatory molecule in example 1 of the present invention;
FIG. 2 shows the results of flow cytometry detection of CD80, a DC costimulatory molecule in example 1 of the present invention;
FIG. 3 shows the results of flow cytometry detection of CD86, a DC costimulatory molecule in example 1 of the present invention;
FIG. 4 shows the number of living cells in example 1 of the present invention;
FIG. 5 shows the results of a fluorescent quantitative PCR assay for the relative expression level of DC cytokines in example 2 of the present invention, wherein a is the relative expression level of IL6, b is the relative expression level of IL12p35, c is the relative expression level of IL12p40, d is the relative expression level of IL23p19, and e is the relative expression level of IL 10;
FIG. 6 is a DC-mediated CD4 in example 3 of the present invention + The detection result of the secretion of IFNgamma by the T cells is shown in the specification, wherein a is the detection result of flow cytometry, and b is a percentage statistical graph;
FIG. 7 is a DC-mediated CD8 in example 3 of the present invention + The detection result of the secretion of IFNgamma by the T cells is shown in the specification, wherein a is the detection result of flow cytometry, and b is a percentage statistical graph;
FIG. 8 is the experimental results of DC-immunized subcutaneous tumor model mice in example 4 of the present invention, wherein a is a working pattern diagram, b is a tumor growth curve, and c is a survival curve of the mice.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The methods used in the respective examples are conventional methods unless otherwise specified.
The materials and sources used in the following examples are as follows:
RPMI1640 medium (Gibco);
glucose-free RPMI1640 medium (Gibco);
fetal bovine serum (Gibco);
penicillin-streptomycin (Gibco);
glutamine (Gibco);
GM-CSF(R&D)；
IL-4(NOVUS)；
LPS(Sigma)；
anti-CD 11c, anti-CD 11b, anti-CD 40, anti-CD 80, anti-CD 86, fixable Viability Dye, etc. streaming antibodies (Invitrogen);
Trizol(Invitrogen)；
reverse transcription kit (Takara);
Ficoll(GE)；
initial CD4 + T cell sorting kit (StemCell);
initial CD8 + T cell sorting kit (StemCell);
PMA(Sigma)；
Ionomycin(Sigma)。
the complete medium used in the examples below was RPMI1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, glutamine, and final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine were 10ng/ml, 5ng/ml, 10%, 100U/ml, and 2mM, in that order.
The glucose-free medium used in the examples below was glucose-free RPMI1640 medium containing GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, glutamine, and the final concentrations of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine were 10ng/ml, 5ng/ml, 10%, 100U/ml, and 2mM, in that order.
Example 1
Experiments in which the glucose-free medium was changed at different culture times in the DC culture affected the expression of the DC surface co-stimulatory molecules and the number of cells.
Upregulation of the expression of the DC surface costimulatory molecules CD40, CD80, CD86 plays an important role in the DC's promotion of differentiation of naive T cells into effector T cells.
1. The experimental method and the grouping are as follows:
(1) Bone marrow cells were cultured at 1X 10 6 The density per ml was resuspended in complete medium, plated in 24-well plate cell culture plates with 1ml per well.
(2) Dividing the cultured cells into 6 groups, wherein group 1 is cultured according to conventional culture method, namely 7 days with complete medium; group 2 was cultured for 1 day with the glucose-free medium replaced on day 6 of culture; group 3 was cultured for 2 days with replacement of glucose-free medium at day 5 of culture; group 4 was cultured for 3 days with the glucose-free medium replaced on day 4 of culture; group 5 was cultured for 4 days with replacement of glucose-free medium on day 3 of culture; group 6 was cultured for 5 days with the glucose-free medium replaced on day 2 of culture.
(3) All 6 groups of cells were stimulated on day 7 with 100ng/ml final LPS or their control solution PBS for 16 hours.
(4) Cells were collected in 1.5ml tubes and viable cells were counted.
(5) The supernatant was discarded after centrifugation at 400g for 5 min.
(6) Preparing a mixed solution containing anti-CD 11c, anti-CD 11b, anti-CD 40, anti-CD 80, anti-CD 86, fixable Viability Dye and other streaming antibodies, adding 50ul of the streaming antibody mixed solution into each tube to reselect cells, and incubating for 30 minutes in ice in a dark place.
(7) Excess antibody was washed by adding 1ml PBS buffer to each tube, and the supernatant was discarded after centrifugation at 400g for 5 min.
(8) Cells were reselected by adding 500 μl PBS buffer per tube and transferred to flow tubes.
(9) Detection of cells Using flow cytometry Fixable Viability Dye - The cells are living cells, CD11c + CD11b + The biscationic cells were DCs whose expression levels were reflected by detection of the fluorescence intensity of CD40, CD80, CD86 in the DCs.
The experimental results are shown in FIGS. 1 to 4.
The results of flow cytometry detection of CD40 of the DC co-stimulatory molecule are shown in FIG. 1, and the results show that the average fluorescence intensity of CD40 in the conventional culture DC is 906 and 1386 in the PBS group and the LPS-stimulated group, respectively, and the average fluorescence intensity of CD40 in the DC cultured for 1 day, 2 days, 3 days, 4 days and 5 days before cell harvest is 897, 1165, 1490, 1652 and 1579 in the PBS group and 900, 2323, 3025, 3831 and 2853 in the LPS-stimulated group.
The above data indicate that the expression of CD40 in DC could not be improved by culturing in glucose-free medium for 1 day, and that the expression of CD40 in DC could be significantly improved by culturing in glucose-free medium for 2 days and more, as compared with conventional culture. And, the expression of CD40 in DCs cultured in glucose-free medium for 3 days, 4 days, and 5 days was further enhanced as compared to 2 days in glucose-free medium.
The results of flow cytometry detection of CD80 from DC costimulatory molecules are shown in FIG. 2, and show that the average fluorescence intensity of CD80 in normal culture DC is 1776 and 3586 in PBS group and LPS stimulated group respectively, and the average fluorescence intensity of CD80 in DC cultured for 1 day, 2 days, 3 days, 4 days and 5 days before cell harvest is 1377, 2806, 3344, 2774 and 2317 in PBS group, 2111, 4251, 5140, 5739 and 4072 in LPS stimulated group.
The above data indicate that 1 day of culture in glucose-free medium reduces the expression of CD80 in DCs, but 2 days and more of culture in glucose-free medium significantly increases the expression of CD80 in DCs, as compared to conventional culture. Also, CD80 expression in DCs cultured in the glucose-free medium for 3 days and 4 days was further enhanced after LPS stimulation compared to 2 days in the glucose-free medium.
The results of flow cytometry detection of CD86 as the DC co-stimulatory molecule are shown in FIG. 3, and the detection results show that the average fluorescence intensity of CD86 in the conventional culture DC is 810 and 1608 in PBS group and LPS stimulated group respectively, and the average fluorescence intensity of CD86 in the DC cultured for 1 day, 2 days, 3 days, 4 days and 5 days before cell harvest is 681, 2834, 4244, 3811 and 2619 in PBS group and 1271, 4999, 7190, 9495 and 5979 in LPS stimulated group.
The above data indicate that 1 day of culture in glucose-free medium reduces the expression of CD86 in DCs, and 2 days and more of culture in glucose-free medium significantly increases the expression of CD86 in DCs, as compared to conventional culture. And, the expression of CD86 in DCs cultured in glucose free medium for 3 days and 4 days was further enhanced compared to 2 days in glucose free medium.
5. As a result of the number of living cells, as shown in FIG. 4, in the in vitro culture of DC, the number of living cells obtained in the culture in the glucose-free medium was significantly reduced compared with the conventional culture, and the degree of the reduction in the number of cells was proportional to the length of time in the culture in the glucose-free medium in which the number of living cells obtained in the culture in the glucose-free medium was less than half that in the conventional culture for 3 days, 4 days or 5 days.
Example 2
Experiments in which the expression of DC cytokines was affected by changing glucose-free medium at different culture times in DC culture.
IL-6, IL-12 and IL-23 are pro-inflammatory cytokines secreted by DC, and the expression level of the pro-inflammatory cytokines can reflect the intensity of DC inflammatory; IL-10 is an anti-inflammatory factor secreted by DCs, and can inhibit the inflammation of DCs and promote the immune tolerance of DCs. IL-6 coding gene is IL6; IL-12 two subunits by IL12p35 and IL12p40 coding, IL-23 two subunits by IL23p19 and IL12p40 coding gene IL10.
1. The experimental method and the grouping are as follows:
(1) Bone marrow cells were cultured at 1X 10 6 The density per ml was resuspended in complete medium, plated in 24-well plate cell culture plates with 1ml per well.
(2) Dividing the cultured cells into 4 groups, wherein group 1 is cultured according to conventional culture method, namely 7 days with complete medium; group 2 was cultured for 1 day with the glucose-free medium replaced on day 6 of culture; group 3 was cultured for 2 days with replacement of glucose-free medium at day 5 of culture; group 4 was incubated for 3 days with replacement of glucose-free medium on day 4 of incubation.
(3) All 4 groups of cells were stimulated on day 7 with LPS at a final concentration of 100ng/ml for 5 hours.
(4) Cells were collected in 1.5ml tubes without RNase and centrifuged at 400g for 5 min and the supernatant was discarded.
(5) Mu.l Trizol was added to each tube, RNA was extracted and reverse transcribed into cDNA strands using a reverse transcription kit.
(6) The relative expression amounts of Il6, il12p35, il12p40, il23p19, il10 were detected by a fluorescent quantitative PCR instrument, and the reference gene Hprt was used as a reference.
2. The experimental results are shown in fig. 5, and the fluorescent quantitative PCR detection results show that:
(1) Compared with the conventional cultured DC, the expression level of IL12p35 and IL12p40 in the DC cultured in the glucose-free medium for 1 day is reduced, and the expression levels of IL6, IL23p19 and IL10 are not changed obviously.
(2) Compared with the conventional cultured DC, the expression level of IL6, IL12p40, IL12p35 and IL23p19 in the DC cultured in the glucose-free medium for 2 days or 3 days is obviously increased, and the expression level of IL10 is obviously reduced.
(3) There was no significant difference in the expression levels of Il6, il12p40, il12p35, il23p19, il10 in the DCs cultured in the glucose-free medium for 2 days and in the DCs cultured in the glucose-free medium for 3 days.
The above data indicate that, in the late stage of the culture of DC, the treatment with glucose-free medium for 2 or 3 days significantly enhances the expression of pro-inflammatory factors IL6, IL12p35, IL12p40, and IL23p19 in DC and inhibits the expression of anti-inflammatory factor IL10 in DC, but that increasing the treatment time on the basis of 2 days of the culture with glucose-free medium does not further alter the expression level of cytokines in DC. This demonstrates that the method of the invention significantly enhances the inflammatory properties of DCs.
Example 3
Experiments in which the change of glucose-free medium at different culture times in DC culture affected DC-mediated T cell differentiation.
DC energy induced initial CD4 + T cell and naive CD8 + T cells differentiate towards effector T cells. The differentiated effector T cells can secrete cytokines such as IFNgamma and play an important role in the anti-tumor immune response of the organism.
1. Experimental method and grouping of cultured DCs:
(1) Bone marrow cells were cultured at 1X 10 6 Density per ml resuspended in complete cultureIn the medium, 1ml was plated per well in 24-well plate cell culture plates.
(2) Dividing the cultured cells into 4 groups, wherein group 1 is cultured according to conventional culture method, namely 7 days with complete medium; group 2 was cultured for 1 day with the glucose-free medium replaced on day 6 of culture; group 3 was cultured for 2 days with replacement of glucose-free medium at day 5 of culture; group 4 was incubated for 3 days with replacement of glucose-free medium on day 4 of incubation.
(3) All 4 groups of cells were harvested on day 7, centrifuged at 400g for 5 min and the supernatant was discarded and the cells resuspended in 1ml PBS.
(4) 1ml of Ficoll is added to the bottom of a 15ml centrifuge tube, resuspended cells are carefully spread on the Ficoll, centrifuged at 800g for 5 minutes, the speed of centrifugation is set to the minimum value of "1", and the speed of deceleration is set to "0".
(5) The middle layer of viable cells were removed by centrifugation, washed once with PBS, resuspended in T cell medium, and counted.
2.DC-CD4 + Experimental method of T cell co-culture:
(1) By means of initial CD4 + T cell sorting kit for sorting initial CD4 from OT-II mice + T cells, the cells obtained by sorting were resuspended in T cell medium and counted.
(2) 2.5X10 cells per well were added to 48 well cell culture plates 4 DC and 2.5X10 5 Initial CD4 + T cells were co-cultured and 5. Mu.g/ml OVA was added to the co-culture system 323-339 And 100ng/ml LPS.
(3) On day 6 of co-culture, cells were harvested, stimulated with 1. Mu.M ionomycin and 50ng/mL PMA for 5 hours, and then CD4 was detected by intracellular staining by flow cytometry + Expression of ifnγ in T cells.
3.DC-CD8 + Experimental method of T cell co-culture:
(1) By means of an initial CD8 + T cell sorting kit sorting of naive CD8 from OT-I mice + T cells, the cells obtained by sorting were resuspended in T cell medium and counted.
(2) 2.5X10 cells per well were added to 48 well cell culture plates 4 DC and 2.5X10 5 Co-culturing the initial T cells, and adding 10ng/ml OVA into the co-culture system 257-264 。
(3) On day 4 of co-culture, cells were harvested, stimulated with 1. Mu.M ionomycin and 50ng/mL PMA for 5 hours, and then CD8 was detected by intracellular staining by flow cytometry + Expression of ifnγ in T cells.
DC mediated CD4 + The results of the detection of ifnγ secretion by T cells are shown in fig. 6, for CD4 co-cultured with DCs cultured in conventional culture and in glucose-free medium for 1 day, 2 days and 3 days + The proportion of ifnγ secreted by T cells was 10.2%, 7.93%, 14.7%, 14.4%, respectively. The statistical results further showed that the DC-mediated CD4 was cultured in glucose-free medium for 1 day compared to the conventionally cultured DC + The T cells secrete IFNgamma in a reduced proportion, and the cells are cultured for 2 days or 3 days in a glucose-free culture medium to mediate CD4 + The proportion of ifnγ secreted by T cells increases significantly. However, compared to DCs cultured in glucose-free medium for 2 or 3 days, they mediate CD4 + The proportion of ifnγ secreted by T cells was not significantly different. The data show that the method can significantly enhance DC-mediated CD4 + T cells secrete ifnγ.
DC mediated CD8 + The results of the detection of ifnγ secretion by T cells are shown in fig. 7, for CD8 co-cultured with DCs cultured in conventional culture and in glucose-free medium for 1 day, 2 days and 3 days + The proportion of ifnγ secreted by T cells was 17.2%, 14.9%, 25.0%, 25.4%, respectively. The statistical results further showed that the DC-mediated CD8 was cultured in glucose-free medium for 1 day compared to the conventionally cultured DC + The T cells secrete IFNgamma in a reduced proportion, and the cells are cultured for 2 days or 3 days in a glucose-free culture medium to mediate CD8 + The proportion of ifnγ secreted by T cells increases significantly. However, compared to DCs cultured in glucose-free medium for 2 or 3 days, they mediate CD8 + The proportion of ifnγ secreted by T cells was not significantly different. The data show that the method can significantly enhance DC-mediated CD8 + T cells secrete ifnγ.
Example 4
Experiments for influencing the anti-tumor effect of the DC after the late stage of the DC culture are carried out by replacing the culture medium without glucose for 2 days.
From the above examples, it is clear that the glucose-free medium culture for 1 day does not enhance the inflammatory properties of DCs and their ability to mediate ifnγ secretion by T cells, and that the glucose-free medium culture for 2 days and 3 days enhances the inflammatory properties of DCs and their ability to mediate ifnγ secretion by T cells, but that the glucose-free medium culture for 2 days and 3 days does not significantly differ from the inflammatory properties of DCs and their ability to mediate ifnγ secretion by T cells. Also, in the in vitro culture of DC, increasing the culture time of the glucose-free medium decreases the number of living cells, and the number of living cells obtained by culturing the glucose-free medium for 3 days or more is less than half that of conventional culture. Therefore, the invention adopts a culture method of replacing glucose-free culture medium for 2 days at the later stage of DC culture, and can ensure that more cell numbers can be obtained as much as possible under the condition of improving DC inflammation and functions thereof to carry out in vivo function experiments.
1. Experimental method and grouping of cultured DCs:
(1) Bone marrow cells were cultured at 1X 10 6 The density per ml was resuspended in complete medium, plated in 24-well plate cell culture plates with 1ml per well.
(2) Dividing the cultured cells into 2 groups, wherein group 1 is cultured according to conventional culture method, namely 7 days with complete medium; group 2 was cultured for 2 days with the glucose-free medium replaced at day 5 of culture.
(3) Both groups of cells were added on day 7 with final concentration of 10ng/ml OVA 257-264 And LPS at a final concentration of 100ng/ml for 12 hours.
(4) Cells were harvested, centrifuged at 400g for 5 min and the supernatant was discarded and resuspended in 1ml PBS.
(5) 1ml of Ficoll is added to the bottom of a 15ml centrifuge tube, resuspended cells are carefully spread on the Ficoll, centrifuged at 800g for 5 minutes, the speed of centrifugation is set to the minimum value of "1", and the speed of deceleration is set to "0".
(6) The middle layer of viable cells were removed by centrifugation, washed once with PBS, and the cells were resuspended in PBS and counted.
Experimental method for dc immunized subcutaneous tumor model mice:
(1) Wild-type mice were subcutaneously injected with melanoma cells B16-OVA, 2X 10 per mouse 5 Individual B16-OVA melanoma cells.
(2) 1 day after tumor cell inoculation, in situ subcutaneous injection into OVA 257-264 DC incubated with LPS, 2X 10 mice each were injected 5 And DC.
(3) 4 days after tumor cell inoculation, the in situ subcutaneous injection of allogeneic DCs was continued, 2X 10 injections per mouse 5 And DC.
(4) Observing the growth of tumor in mice, measuring the size of tumor, calculating the tumor area, and reaching or exceeding 200mm in tumor area 2 Mice were euthanized and survival curves were recorded.
Experimental results of DC immunized subcutaneous tumor model mice as shown in fig. 8, the growth of tumors was slower, tumors were smaller, and mice survived longer in the DC immunized mice cultured for 2 days in the glucose-free medium compared to the conventional cultured DCs. This shows that the invention can significantly enhance the anti-tumor immune effect of DC, so that the DC can more effectively inhibit the growth of subcutaneous tumor of mice and promote the survival of mice.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A culture method for improving the inflammatory properties of dendritic cells, comprising the steps of:
step 1: obtaining single bone marrow cells, re-suspending in a complete culture medium, then spreading in a cell culture container, placing in a cell culture box containing 5% carbon dioxide at 37 ℃ for 5 days, and changing fresh culture medium according to the growth condition of cells;
step 2: removing the complete culture medium of the cultured cells in the step 1, adding a glucose-free culture medium, and continuously placing the culture medium in a cell culture box containing 5% carbon dioxide at 37 ℃ for 2 days to obtain the mature dendritic cells with high inflammatory property.
2. The method of claim 1, wherein the complete medium is RPMI1640 medium comprising GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, glutamine.
3. The method of claim 1, wherein the glucose-free medium is glucose-free RPMI1640 medium comprising GM-CSF, IL-4, fetal bovine serum, penicillin-streptomycin, glutamine.
4. A method of culturing for enhancing the inflammatory properties of dendritic cells according to claim 2, wherein the final concentration of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine in the complete medium is 10ng/ml, 5ng/ml, 10%, 100U/ml and 2mM in this order.
5. A method of culturing for enhancing the inflammatory properties of dendritic cells according to claim 2 wherein the final mass concentration of glucose in the complete medium is 11.11mM.
6. A method of culturing for enhancing the inflammatory properties of dendritic cells according to claim 3, wherein the final concentration of GM-CSF, IL-4, fetal bovine serum, penicillin, streptomycin, glutamine in the glucose-free medium is 10ng/ml, 5ng/ml, 10%, 100U/ml and 2mM in this order.
7. The method of claim 1, wherein the single bone marrow cells in step 1 are plated at a density of 1X 10 6 /ml。
8. The method of claim 1, wherein the single bone marrow cells in step 1 are prepared by the following steps:
(1) Taking femur and tibia of a mouse, sucking PBS (phosphate buffered saline) by using a syringe, flushing bone marrow cells into a centrifuge tube, centrifuging at a rotating speed of 400g for 5 minutes, and discarding supernatant;
(2) After the PBS is used for resuspension of the cells, the cells are filtered through a 40 mu m filter membrane, and the supernatant is discarded after centrifugation at 400g for 5 minutes;
(3) Lysing erythrocytes mixed in bone marrow cells with an erythrocyte lysate, and washing with PBS 1 time after stopping the lysis;
(4) The resulting bone marrow cells were resuspended and counted in complete medium for plating.
9. The method for improving inflammatory conditions of dendritic cells according to claim 1, wherein the replacing fresh medium in step 1 is performed by: half of the volume of the original complete medium in the cell culture vessel was removed, followed by the addition of an equivalent amount of freshly prepared complete medium pre-warmed at 37 ℃.
10. The culture method for improving the inflammatory properties of the dendritic cells according to claim 1, wherein the specific operation of replacing the culture medium in the step 2 is as follows: the original complete medium in the cell culture vessel was removed entirely, and an equal amount of freshly prepared glucose-free medium, pre-warmed at 37 ℃, was added.
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| KR20190087777A (en) * | 2018-01-17 | 2019-07-25 | 파미셀 주식회사 | Method for producing monocyte derived from bone marrow |
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| KR20190087777A (en) * | 2018-01-17 | 2019-07-25 | 파미셀 주식회사 | Method for producing monocyte derived from bone marrow |
Non-Patent Citations (2)
| Title |
|---|
| ANNA S. BUDIKHINA ET AL: "Aerobic glycolysis is dispensable for pro-inflammatory cytokine production by dendritic cells", IMMUNOLOGIYA, vol. 41, no. 1, pages 31 - 41 * |
| SIMON J. LAWLESS ET AL: "Glucose represses dendritic cell-induced T cell responses", NATURE COMMUNICATIONS, vol. 8, pages 1 - 14 * |
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