1. What are dendritic cells (DC)?
Dendritic cells (DCs) are the most effective antigen presenting cells (APCs) in the human body. DCs are the only APCs capable of robustly stimulating naïve T cell proliferation. Other types of APC (such as monocytes, macrophages, B cells, etc.) can only stimulate activated or memory T cells. Therefore, DC cells are the initiators of adaptive T cell immune responses and play an extremely important role in tumor immunity. DC cells highly express MHC-I and MHC-II molecules on their surface and have specific surface markers. They take up, process and stimulate T cells to activate antigens, and ultimately determine the differentiation direction of T cells.
2. Source of DC
DC cells are present in very small amounts in the body. It takes a long time to directly isolate DC from the body and the cell yield is very low, which greatly limits the research and application of DC. However, DC can be differentiated and induced from DC precursor cells in different tissues, such as precursor cells in bone marrow fluid, monocytes in peripheral blood and umbilical cord blood.
For humans, since human peripheral blood is the most convenient to obtain and has the largest number of monocytes, the most commonly used method is to induce DC from human peripheral blood mononuclear cells (PBMC). For mice, the most common method is to induce DC from bone marrow cells, namely bone marrow-derived dendritic cells (BMDC).
Figure 1. Schematic diagram of the classic BMDC preparation method
3. BMDC Preparation Method
Common methods for preparing mouse BMDCs are:
3.1 Classical BMDC culture method - Inaba method (modified)
3.2 Large-scale preparation of BMDC - Sons method
3.3 Large-scale preparation of BMDC - Lutz method
3.1 [Classic BMDC culture method] - Inaba method (improved)
【Background】
a. The number of BMDCs obtained by the Inaba method is 5-7 x 106/mouse;
b. The original Inaba method only uses GM-CSF to induce the production of BMDCs. Although the BMDCs obtained have a strong stimulating ability in the mixed lymphocyte reaction, the maturity of DCs is not as good as that of the combined induction of GM-CSF+IL-4. Therefore, the subsequent improved method often uses the combined induction of GM-CSF+IL-4.
【Cultivation steps】
3.1.1 Obtaining Mouse Bone Marrow Cells
1) Mice (6-10 weeks old) were killed by cervical dislocation, all femurs and tibias were removed surgically, and the muscle tissue around the bones was removed as much as possible with scissors and forceps.
[Note] Do not damage the bones.
2) Move the bone to the clean bench and soak it in a sterile culture dish containing 70% alcohol for 2-5 minutes to disinfect and sterilize, then wash it twice with sterile PBS.
3) Move the bone to another new culture dish containing PBS, cut the two ends of the bone with scissors, and then use a syringe to extract PBS. Insert the needle into the bone marrow cavity from both ends of the bone, and repeatedly flush the bone marrow into the culture dish until the bone turns completely white.
4) Collect the bone marrow suspension and filter out small fragments and muscle tissue with a 200-mesh nylon mesh.
5) Centrifuge the filtrate at 1200 rpm for 5 min and discard the supernatant.
6) Add 2 ml of ammonium chloride red blood cell lysis buffer (1x), resuspend the cells, and incubate at room temperature for 3-5 min, up to 10 min.
7) Add 10 ml PBS to neutralize the effect of the lysis buffer, then centrifuge at 1200 rpm for 5 min and discard the supernatant.
8) Wash once with PBS, and then resuspend the cells in RPMI1640 culture medium containing 10% FBS. Mouse bone marrow cells have been obtained.
Preparation of ammonium chloride red blood cell lysis solution:
a. Prepare 10x storage solution as follows: weigh 82.9 g NH4Cl, 10.0g KHCO3 and 0.37g Na2EDTA, dissolve in 1L distilled water, filter with 0.22 μm filter membrane to sterilize, and store at 4℃ for 6 months;
b. Before use, dilute 10x storage solution with sterile distilled water 1:9 to 1x working solution.
[Note] Because ammonium chloride red blood cell lysis solution has a certain harmful effect on bone marrow cells, the hemolysis time should be shortened as much as possible.
3.1.2 Induction of BMDC differentiation
1) Count the mouse bone marrow cells obtained in step 1 and adjust the cell concentration to 0.5-1× 106/ml with RPMI 1640 complete culture medium containing 10% FBS.
2) Plate into a 24-well culture plate, 1 ml of cells per well, add recombinant mouse GM-CSF (20 ng/mL) and IL-4 (10 ng/mL), and culture in a 37℃, 5% CO2 incubator. This is the 0th day of culture.
【Note】
a. Generally, about 4-5x107 bone marrow cells can be harvested from one mouse, so at least 40-50 wells of a 24-well plate can be plated.
b. The concentration ranges of GM-CSF and IL-4 are 20-50 ng/mL and 10-40 ng/mL, respectively.
3) Gently shake the culture plate every 2 days, then replace 3/4 of the volume with fresh culture medium and replenish cytokines.
4) Between the 5th and 8th days, gently blow the culture medium to collect suspended cells and loosely attached cells.
5) Centrifuge at 1200 rpm for 5 min and discard the supernatant.
6) Resuspend the cells in RPMI 1640 complete culture medium containing 10% FBS and count them, then adjust the cell concentration to 1×106/ml, and add recombinant mouse GM-CSF (20 ng/mL) and IL-4 (10 ng/mL).
7 ) Plate the cells into 100mm culture dishes (up to 10ml per dish) or 6-well culture plates (2m/well).
8) Continue to culture in a 37℃, 5% CO2 incubator for 1-2 days.
9) Collect the suspended cells, which are the more mature BMDCs.
【Note】
a. Steps 2.5-2.8 are re-plating steps, the purpose of which is to make the BMDC obtained in step 2.4 more mature.
b. Within 3 hours after re-plating, many spiny adherent cells can be seen migrating from the DC clusters, and after 1 day of culture, these adherent cells will be found to be detached from the bottom of the culture plate, and many typical DCs can be seen floating in the culture medium.
3.1.3 Full maturation of BMDC
[Note] The BMDCs obtained in step 2 are not fully mature DCs. If you want to obtain fully mature DCs, you still need to be induced by LPS, CD40L or TNF-a.
1) Centrifuge the BMDC obtained in step 2.4 or 2.9 at 1200 rpm for 5 min and discard the supernatant.
2) Resuspend the precipitate with RPMI complete culture medium containing recombinant mouse GM-CSF (20 ng/mL) and IL-4 (10 ng/mL), and adjust the cell concentration to 1×106/ml after counting.
3) Add to 24-well culture plates and add maturation inducers such as TNF-α (250 U/mL), LPS (1 μg/mL), or CD40L (1 μg/mL).
4) Culture in a 37℃, 5% CO2 incubator for 2 days.
5) Collect the suspended cells and cells that grow loosely attached to the wall, which are mature dendritic cells.
3.2【BMDC mass production method】-Son method
【Background】
a. This method can obtain 30-40×106 DC/mouse within 7 days, which is 7-10 times that of the Inaba classic method. After DC is centrifuged through 14.5% metrizamide gradient, the purity (i.e. CD11c+/I-Ab+ cells) can reach 85-95%.
b. The endocytosis ability of DC obtained by this method is weaker than that of the Inaba classic method, but the amount of IL-12p70 secreted is similar.
c. DC obtained by this method has stronger stimulation ability in mixed lymphocyte reaction than the Inaba classic method.
d. DC obtained by this method can induce stronger specific T cell response.
e. In summary, the Son method can obtain more and more mature BMDC than the classic method.
【Cultivation steps】
3.2.1 Obtaining mouse bone marrow cells
See the corresponding steps in the Inaba method (modified).
3.2.2 Preparation of large quantities of BMDC
1) Count the mouse bone marrow cells obtained in step 1 and adjust the cell concentration to 2×105/ml with RPMI 1640 complete culture medium containing 10% FBS.
2) Spread into 6-well culture plates, 5ml cells per well, add recombinant mouse GM-CSF (1000 U/mL) and IL-4 (1000 U/mL), and culture in a 37℃, 5% CO2 incubator.
3) On the 4th day of culture, supplement the culture system with recombinant mouse GM-CSF (1000 U/mL) and IL-4 (1000 U/mL).
4) Collect DCs on the 7th day of culture, resuspend with 2-4 ml RPMI 1640 complete culture medium, add to an equal volume of 14.5% (w/v) mepanema, and centrifuge at room temperature for 20 min at 1200xg.
5) Collect the middle layer and wash it three times with RPMI 1640 complete culture medium for later use.
[Note] The DC at this point is an immature BMDC. If you want to further mature it, please skip to step 3.
3.2.3 Full maturity of BMDC
1) Re-plated the BMDCs collected in step 2.4, and added recombinant mouse GM-CSF (1000 U/mL) and IL-4 (1000 U/mL), as well as LPS (1-10 μg/mL) to the culture system
2) Cultured in a 37℃, 5% CO2 incubator for 2 days to obtain mature BMDCs.
3.3【BMDC mass preparation method】-Lutz method
【Background】
a. The Lutz method is similar to the Son method, and both methods can prepare BMDC in large quantities, but the Lutz method is more widely used than the Son method.
b. This method can obtain more BMDC, up to 1-3 x 108 DC/mouse, and the purity can reach 90-95%;
c. The cytokine concentration used in this method is much lower than that of the Son method, only 200 U/mL, and it drops to 30-100 U/mL from the 8th to the 10th day of culture, which can greatly save the reagent cost;
d. The biggest difference between this method and the Inaba classic method and the Son method is that the bone marrow cells are cultured in a bacterial culture dish (Petri dish) instead of a cell culture plate. Inaba explained that the bacterial culture dish is not easy for macrophages in the bone marrow to adhere to the wall, thereby inhibiting the development of macrophages and avoiding the inhibitory effect of macrophages on DC maturation. This may be the main reason why this method can obtain a large number of BMDCs at a lower plating density.
e. However, the culture time of this method is relatively long, requiring 10-12 days. On the one hand, this is to obtain more BMDCs. On the other hand, most granulocytes and lymphocytes are difficult to survive for such a long time, so the purity of the BMDCs finally obtained can be improved;
f. This method only uses GM-CSF for induction culture, and the BMDCs obtained contain both immature and mature DCs. To further improve the maturity, LPS or TNF-α needs to be used for another 1-2 days of induction, and the content of mature DC cells will reach 50-70%.
【Cultivation steps】
3.3.1 Obtaining mouse bone marrow cells
See the corresponding steps in the Inaba method (modified), and note that the hemolysis step should be omitted.
3.3.2 Large-scale preparation of BMDC
1) Count the mouse bone marrow cells obtained in step 1 and adjust the cell concentration to 2×105/ml with RPMI 1640 complete culture medium containing 10% FBS;
2) Spread into 100mm bacterial culture dishes (Petri Dish), 10ml cells per dish, and add recombinant mouse GM-CSF (200 U/mL), and culture at 37℃, 5% CO2 incubator;
[Note] Bacterial culture dishes are used here, not cell culture plates.
3) On the 3rd day, add 10ml of complete culture medium containing 20 ng/mL recombinant mouse GM-CSF to the culture dish;
4) On the 6th and 8th days, change half of the medium, that is, collect the old culture medium, resuspend the cell pellet with complete culture medium containing 20 ng/mL recombinant mouse GM-CSF after centrifugation, and then put the cell suspension back into the original dish;
5) On the 10th day, the cells can be collected, which are BMDCs.
3.3.3 Full maturation of BMDCs
1) Collect the suspended cells by gently blowing the DCs on the 10th day of culture with a pipette, and centrifuge at 300xg for 5 minutes at room temperature;
2) Discard the supernatant, resuspend the cell pellet with 10ml RPMI 1640 complete culture medium, and then spread it on a 100 mm cell culture plate;
3) Add recombinant mouse GM-CSF (100 U/mL) and TNF-α (500 U/mL), or recombinant mouse GM-CSF (100 U/mL) and LPS (1 μg/mL);
4) Continue to culture in a 37℃, 5% CO2 incubator for 1-2 days.
4. Identification of BMDCs
l Morphological observation: Most BMDCs grow in colonies, and the cells have multiple dendritic protrusions, which are more obvious in mature BMDCs.
l Cell phenotype analysis: Flow cytometry was used to detect the expression of CD11c, CD40, CD80, CD86, MHC class II molecules (I-A/I-E) on the surface of DC cells. BMDCs highly express these molecules, and the expression of these molecules in fully mature BMDCs will be further increased.
l Mixed lymphocyte reaction (MLR): BMDCs have strong stimulatory ability, and the higher the maturity, the stronger the stimulatory ability.
5. How to choose a maturation inducer?
LPS, CD40L and TNF-α are commonly used and effective maturation inducers for both human DC and mouse DC. TNF-a has the weakest ability to induce DC maturation among the three. LPS and CD40L are both strong inducers for DC full maturation in vitro. The maturation of DC induced by both is similar, but the induced cytokine spectrum is different. CD40L-induced mature BMDC shows the strongest immunoregulatory ability in vivo, including the generation of protective and therapeutic tumor immune responses. The concentration used to stimulate DC full maturation with LPS is generally 1-10 μg/mL, but in fact 0.1 μg/mL has a very strong effect, but for insurance purposes, 1 μg/mL is generally used. It should be noted that CD40L molecules belong to the TNF ligand family, which is characterized by the fact that they can only function after forming trimers. Therefore, it is best to use recombinant CD40L trimer protein to stimulate DC, which will have a good effect. If CD40L monomers are used to stimulate DC, the maturity is not very high in most cases.
6. Choice of training method
Table 1. Comparison of three common experimental methods for preparing BMDC
|
Parameter |
Classic BMDC culture method - Inaba method |
Large-scale preparation of BMDC - Son method |
Large-scale preparation of BMDC - Lutz method |
|
Number of citations in PubMed |
783 |
24 |
663 |
|
Bone marrow hemolysis, lysis of red blood cells |
YES |
YES |
No |
|
Bone marrow first removes lymphocytes |
YES |
No |
No |
|
Removal of granulocytes during culture |
YES |
No |
No |
|
Initial plating volume of bone marrow cells |
1ml |
15ml |
10ml |
|
Incubator |
24-well cell culture plates |
6-well cell culture plate |
100mm bacterial culture dish |
|
Culture medium |
RPMI 1640+10% FCS |
||
|
Concentration of mouse GM-CSF |
200-1000 U/mL |
The initial concentration added is 125-1000 U/mL On the 4th and 7th days, sufficient amounts of GM-CSF and IL-4 are added to the culture system. |
The initial added concentration is 200 U/mL. 3-100 U/mL after the 10th day |
|
Mouse IL-4 |
Need not |
Need，Concentration is the same as GM-CSF |
Need not |
|
Fluid exchange method |
On the 2nd and 4th day, discard 50%-75% of the old culture medium (which contains cells) and replace with fresh complete culture medium containing sufficient GM-CSF. |
/ |
On the 3rd day, an equal volume of complete culture medium containing GM-CSF was added. On the 6th, 8th and 10th days, half of the medium was replaced. The old culture medium (containing cells) was aspirated, centrifuged, resuspended in fresh complete culture medium containing GM-CSF and then put back. |
|
Culture time before passaging (expansion) |
6 days |
7 days |
10 days |
|
Culture time after passaging (maturation) |
2 days |
None |
1-2 days |
|
Full maturity inducer |
TNF-ɑ（250 U/mL） |
LPS(1-10 μg/mL) |
TNF-ɑ（250 U/mL）or LPS(1 μg/mL) |
|
Time of DC cell harvest |
7-8 days |
7-8 days |
10-13 days |
|
DC Purity |
Day 8：60-70% |
Day 7：85-95% |
Day 10-12：80-90% |
|
DC production/mouse |
(5-7)×106 |
(3-4)×107 |
(1-3)×108 |
7. Recommended DC culture reagents
|
Product Name |
Cat |
Size |
|
91108ES |
5 μg/50 μg/100 μg/500 μg |
|
|
90144ES |
5 μg/50 μg/100 μg/500 μg |
|
|
90621ES |
5 μg/20 μg/50 μg/500 μg |
|
|
94016ES |
25 μg/100 μg/500 μg |