CN113249322A - Culture solution and culture method for DC cells - Google Patents
Culture solution and culture method for DC cells Download PDFInfo
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Abstract
The invention discloses a culture solution of DC cells and a culture method thereof, wherein the culture solution of the DC cells is added in batches according to the growth condition of the DC cells and according to adherent cell culture solution, subsequent first supplemented DC cell culture solution and subsequent second supplemented DC cell culture solution; the adherent cell culture solution comprises: serum-free DC medium, GM-SCF, IL-13; the first subsequent supplementation of DC cell culture broth includes: serum-free DC medium, GM-CSF, IL-4, anti-CD 83 antibody and autologous serum at a concentration of 1%; the subsequent second addition of DC cell culture fluid comprises: TNF-a, vitamin B12, nicotinamide; the culture solution and the culture method of the invention have the advantages that the maturation time of the DC cell culture is fast, the DC cell can mature only in 3 days, the cell number and the purity are high, the antigen presenting capability of the DC cell is strong, and the DC cell culture has excellent anti-tumor effect.
Description
Technical Field
The invention relates to the field of cell culture, in particular to a culture solution of DC cells and a culture method thereof.
Background
Dendritic Cells (hereinafter referred to as DC) are a kind of macrophages, and are also the most powerful Antigen Presenting Cells (APC). DC cells activate helper T cells as well as B cells in humans primarily by phagocytosis of the antigen and expression of a portion thereof on MHC II molecules on the surface of the DC cells. It has been demonstrated that DCs are the only APCs capable of significantly stimulating the proliferation of naive T cells (T cells). The existing DC cell culture methods are many, the prior art generally adopts GM-CSF, IL-4 and TNF-a to culture DC cells in a matching way, the GM-CSF has the function of promoting the differentiation of monocytes to macrophagocyte-like cells in the DC culture, and the expression of MHC class II molecules on the cell surface is improved, so that the antigen presentation function of the cells is enhanced. In addition, GM-CSF can also promote survival of DCs; IL-4 plays a role in the induction of monocytes into DCs by inhibiting the overgrowth of macrophages, thereby directing the differentiation of monocytes in the direction of DCs. If IL-4 is not added to the culture system, the monocytes will differentiate into macrophages. IL-4 also has the ability to decrease the cell surface expression of the CD14 molecule. The reduction in the expression level of CD14 is an important marker for the differentiation of monocytes into DCs. Under the combined action of GM-CSF and IL-4, monocytes can be directionally differentiated into immature DC (immaturity DC), and the DC has strong antigen uptake and processing capacity but weak antigen presenting capacity. TNF-a down-regulates the megakaryolysis and surface Fc receptor expression of immature DCs, abolishing the intracellular MHC class II molecular compartment (class II component), but up-regulates the cell surface expression of MHC class I, class II and B7 family molecules (CD80, CD86, etc.) and differentiates immature DCs into mature DCs (cultured DCs). However, the culture solution still has the problems that the maturation time of DC cells in culture is slow, the DC cells are matured within about 7-14 days, the number and purity of the cells are low, and the like; the present invention solves such problems.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a culture solution of DC cells and a culture method thereof, the culture solution and the culture method of the DC cells have the advantages of fast maturation time of the culture, maturation within 3 days, high cell number and purity, strong antigen presenting capability of the DC cells and excellent anti-tumor effect.
In order to achieve the above object, the present invention adopts the following technical solutions:
a culture solution of DC cells, comprising: adherent cell culture solution, a subsequent first supplemented DC cell culture solution and a subsequent second supplemented DC cell culture solution;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml;
the first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum;
the subsequent second addition of DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, and 1.2-1.7ul of 300U/mL nicotinamide.
The culture solution of the DC cells, the culture solution of the adherent cells includes: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
The culture solution for adherent cells of the aforementioned DC cells further comprises: 50ul of 200mM amino acid.
The aforementioned culture solution of DC cells, the amino acids include: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine.
The aforementioned culture solution for DC cells, the first subsequent supplement of the culture solution for DC cells comprises: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
The aforementioned culture solution for DC cells, and the second subsequent supplement of the culture solution for DC cells comprises: 1.9ul 150U/mL TNF-a, 1.2ul 250U/mL vitamin B12, 1.5ul 300U/mL niacinamide.
A method for culturing DC cells, comprising the following steps:
step one, processing plasma to obtain white blood cells;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml;
step three, adding the subsequent first supplement DC cell culture solution into a culture bottle with adherent cells, culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum;
step four, adding the subsequent second supplemented DC cell culture solution into adherent cell culture solutionCulturing in culture flask for 2 days at 36-38 deg.C and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, 1.2-1.7ul of 300U/mL nicotinamide;
and step four, harvesting the DC cells.
In the third step, the adherent cells are washed with physiological saline for 1-3 times before the culture solution for the subsequent first supplemented DC cells is added into the culture bottle with the adherent cells.
The invention has the advantages that:
the DC cells cultured by the method have quick maturation time, can mature within 3 days, and have high cell number and purity;
according to the invention, different cell culture solutions are added according to various periods of DC cell culture, so that the optimal culture is realized to the greatest extent, and the cell quantity and purity are improved;
the invention discovers that TNF-a, vitamin B12 and nicotinamide all have synergistic effect on increasing the cell number and the antigen presenting capability of DC cells, and have better anti-tumor effect.
Detailed Description
The present invention will be described in detail with reference to the following embodiments.
A method for culturing DC cells, comprising the following steps:
step one, processing plasma to obtain white blood cells;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml; preferably, the adherent cell culture solution further comprises: 50ul of 200mM amino acid; the amino acid is not limited in kind, and any amino acid that can provide cell nutrition can be used in the present invention. The amino acids include: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine. As a preference, the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
Washing adherent cells 1-3 times with physiological saline, adding the subsequent first supplemented DC cell culture solution into a culture bottle with the adherent cells, and culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum; as a preference, the subsequent first supplemented DC cell culture fluid comprises: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
Step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, 1.2-1.7ul of 300U/mL nicotinamide; as a preference, the subsequent second supplemented DC cell culture fluid comprises: 1.9ul 150U/mL TNF-a, 1.2ul 250U/mL vitamin B12, 1.5ul 300U/mL niacinamide.
Step four, harvesting DC cells
The specific procedure is not limited as long as the method capable of harvesting DC cells is within the scope of the present invention.
The following tests were carried out using the samples prepared in the following examples:
example 1:
step one, processing plasma to obtain white blood cells;
1. confirming the sealing of the aspiration tube of the plasma bag. The aspiration tube and scissors were wiped with two different clean sheets dedicated to the sterile room soaked in 70% ethanol.
2. The pipette was grasped with forceps, the front end of the tubing was cut open and the flow rate was controlled with forceps to transfer plasma to a 50ml tube. The plasma volume transferred was recorded.
3. The tubes were centrifuged (400g, 4 ℃) for 30 minutes to remove platelets.
4. The supernatant was poured into a new 50ml tube.
5. The tube was placed in a constant temperature water bath (56 ℃ C.), and incubated for 30 minutes.
6. The tube was centrifuged (400g, 4 ℃) for 60 minutes to remove precipitated fibrin.
7. The supernatant (autologous serum) was poured into a new 50ml tube. The tube is labeled with the patient's name, date of preparation, and refrigerated (4 ℃).
8. Confirm the aspiration tube seal of the PBMC bag. The PBMC bags were suspended. The aspiration tube and scissors were wiped with two different strips of clean paper specially soaked in 70% ethanol sterile room.
9. Transfer cells to 50ml tubes. The capacity of the transfer is recorded.
10. Calculating the number of required 50ml test tubes, and adding 15ml of lymphocyte separation solution 1077 into all the test tubes;
11. enough new 50ml tubes were prepared to dilute the sample 2-fold. 25ml hanks buffer was poured into all 50ml tubes prepared. Transfer 25ml of the cell suspension to each tube using a 10ml pipette. To all tubes 30ml of diluted cell suspension was transferred.
12. All tubes (400g, room temperature) were centrifuged for 40 min.
13. And sucking up and discarding the upper liquid until the distance from the liquid surface to the white blood cells at the bottom layer is 1 cm.
14. A new 50ml tube was prepared. The leukocytes were collected cautiously using a 10ml pipette.
Step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.4ul of GM-SCF at a concentration of 800U/ml, 6.9ul of IL-13 at a concentration of 500U/ml, and 50ul of 200mM L-glutamine.
1. The leukocytes were collected in every two tubes and transferred to a new 50ml tube.
2. T75 cell culture flasks (DC flasks) were prepared. And (5) marking the date. 10ml of room temperature ALyS505N-0 medium, 9.4ul of GM-SCF at a concentration of 800U/ml, 6.9ul of IL-13 at a concentration of 500U/ml were added to each flask.
3. To the cell tube was added 10ml of ALyS505N-0 using a 10ml pipette. The cells were resuspended. 1ml of a penicillin-streptomycin double-mixed solution and 50ul of 200mM L-glutamine were added.
4. 5ml of cell suspension was transferred to each DC flask.
5. The cover of the culture flask is buckled. The bottle was gently shaken to homogenize the cell suspension. The cells were cultured in an incubator for 2 hours (37 ℃ C., 5% CO 2).
Washing adherent cells 1-3 times with physiological saline, adding the subsequent first supplemented DC cell culture solution into a culture bottle with the adherent cells, and culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum;
the first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 9.8ul of GM-CSF at a concentration of 800U/ml, 7.2ul of IL-4 at a concentration of 500U/ml, 2.9ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
After the two hour incubation of adherent cell culture was completed, the cells were microscopically secured to the bottom of the flask.
Gently shake the flask back and forth 10 times to suspend the non-adherent cells, and transfer the non-adherent cell suspension to a 50ml tube.
Rapidly adding the subsequent first supplement DC cell culture solution into each DC culture bottle, and culturing in an incubator at 36-38 deg.C for 20 hr with 3-8% CO2。
Step four, the following stepAdding the secondary-supplemented DC cell culture solution into a culture bottle with adherent cells, culturing for 2 days at 36-38 deg.C and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, 1.2-1.7ul of 300U/mL nicotinamide;
the subsequent second addition of DC cell culture fluid comprises: 1.7ul 150U/mL TNF-a, 1.4ul 250U/mL vitamin B12, 1.2ul 300U/mL niacinamide.
After the culture of the subsequent first DC cell culture fluid is finished, rapidly adding the subsequent second DC cell culture fluid to each DC culture bottle, and culturing for 2 days at 36-38 ℃ in an incubator with 3-8% CO2。
Step four, harvesting DC cells
1. The harvested cell culture flasks were confirmed.
2. Ensure endotoxin detection as negative.
3. An ice box was prepared to buffer the cells and ice-cold RPMI1640 was prepared.
4. After shaking 15ml of buffer was added, the cell suspension was transferred to a 50ml tube. Transfer 10ml RPMI into the flask with a 10ml pipette and wash the flask 5 times. The cell suspension was transferred to a 50ml tube and kept on ice for a while.
5. After all cells were harvested into tubes, the tubes were centrifuged for 5 minutes (400g, 4 ℃). After the centrifugation is finished, the upper layer liquid is removed by suction. A50 ml tube was suspended with a small amount of RPMI 1640. Transfer all cell suspensions in tubes to one of the tubes and add RPMI1640 up to 50 ml. Samples were taken for FACS testing.
6. The number of DC cells and the survival rate were calculated.
7. Centrifuge tube for 5 minutes (1500rpm, 4 ℃ C.)
8. Preparation of cryovials patient name, cell name (DC), cell number and date were marked on all cryovials.
9. Preparation of CELL cryo-fluid (CELL GRO DC broth, 20% autologous serum, 10% DMSO) was suspended on ice.
10. After centrifugation of the cell tube for 5 minutes (400g, 4 ℃), the supernatant was decanted into a sterile tube.
11. Performing sterility testing
12. The cells were plated out on ice.
13. Add the freezing fluid to the cell tube and mix well with a pipette.
14. And (5) freezing and storing.
Example 2:
step one, processing plasma to obtain white blood cells;
same as example 1;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.9ul of GM-SCF at a concentration of 800U/ml, 6.3ul of IL-13 at a concentration of 500U/ml, and 50ul of 200mM L-glutamine.
The procedure was as in example 1 except that the adherent cell culture solution was different;
washing adherent cells 1-3 times with physiological saline, adding the subsequent first supplemented DC cell culture solution into a culture bottle with the adherent cells, and culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 10.3ul of GM-CSF at a concentration of 800U/ml, 6.7ul of IL-4 at a concentration of 500U/ml, 3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
The same as example 1 except that the culture broth of the subsequent first-fed DC cells was different;
step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 2.2ul 150U/mL TNF-a, 0.9ul 250U/mL vitamin B12, 1.2ul 300U/mL niacinamide.
The same as example 1 except that the subsequent second supplemented DC cell culture broth was different;
step four, harvesting DC cells
Same as in example 1.
Example 3:
step one, processing plasma to obtain white blood cells;
same as example 1;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
The procedure was as in example 1 except that the adherent cell culture solution was different;
washing adherent cells 1-3 times with physiological saline, adding the subsequent first supplemented DC cell culture solution into a culture bottle with the adherent cells, and culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
The same as example 1 except that the culture broth of the subsequent first-fed DC cells was different;
step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.9ul 150U/mL TNF-a, 1.2ul 250U/mL vitamin B12, 1.5ul 300U/mL niacinamide.
The same as example 1 except that the subsequent second supplemented DC cell culture broth was different;
step four, harvesting DC cells
Same as in example 1.
Comparative example 1: the other steps were the same as in example 3, and the subsequent addition of DC cell culture medium was carried out in one portion without adding it twice.
Step one, processing plasma to obtain white blood cells;
same as example 1;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
The procedure was as in example 1 except that the adherent cell culture solution was different;
washing adherent cells 1-3 times with physiological saline, adding the culture solution for subsequently supplementing DC cells into a culture bottle with the adherent cells, culturing for 3 days at 36-38 ℃ and 3-8% CO2；
Subsequent supplementation of the DC cell culture broth includes: 15mL of serum-free DC medium, 10.1ul of GM-CSF at a concentration of 800U/mL, 6.9ul of IL-4 at a concentration of 500U/mL, 3.1ul of anti-CD 83 antibody at a concentration of 500U/mL and 0.15mL of autologous serum at a concentration of 1%, 1.9ul of 150U/mL TNF-a, 1.2ul of 250U/mL of vitamin B12, 1.5ul of 300U/mL of nicotinamide.
The same as example 1 except that the culture broth of the subsequent first-fed DC cells was different;
step three, harvesting DC cells
Same as in example 1.
Comparative example 2: the other examples were identical to example 3, with the formulation of the subsequent second supplemented DC cell culture medium being different and lacking vitamin B12.
Step one, processing plasma to obtain white blood cells;
same as example 1;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
The procedure was as in example 1 except that the adherent cell culture solution was different;
step three, washing the adherent cells 1-3 times by using physiological saline, and adding the culture solution of the DC cells supplemented with the first supplement DC cells into the adherent cellsCulturing in culture flask at 36-38 deg.C for 20 hr and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
The same as example 1 except that the culture broth of the subsequent first-fed DC cells was different;
step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.9ul 150U/mL TNF-a, 1.5ul 300U/mL nicotinamide.
The same as example 1 except that the subsequent second supplemented DC cell culture broth was different;
step four, harvesting DC cells
Same as in example 1.
Comparative example 3: the other steps are the same as in example 3, with the subsequent second supplement of DC cell culture fluid formulated differently and lacking nicotinamide.
Step one, processing plasma to obtain white blood cells;
same as example 1;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
The procedure was as in example 1 except that the adherent cell culture solution was different;
washing adherent cells 1-3 times with physiological saline, adding the subsequent first supplemented DC cell culture solution into a culture bottle with the adherent cells, and culturing for 20h at 36-38 ℃ and 3-8% CO2；
The first subsequent supplementation of DC cell culture broth includes: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
The same as example 1 except that the culture broth of the subsequent first-fed DC cells was different;
step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The subsequent second addition of DC cell culture fluid comprises: 1.9ul 150U/mL TNF-a, 1.2ul 250U/mL vitamin B12.
The same as example 1 except that the subsequent second supplemented DC cell culture broth was different;
step four, harvesting DC cells
Same as in example 1.
Test one: the number of cells detected in the plasma of two different patients for each sample of the example is shown in A plasma and B plasma, respectively, in Table 1.
TABLE 1 cell number detection Table
| A plasma cell number | B plasma cell number | |
| Example 1 | 2.14x10^7/ml | 2.07x10^7/ml |
| Example 2 | 2.08x10^7/ml | 2.10x10^7/ml |
| Example 3 | 2.19x10^7/ml | 2.18x10^7/ml |
| Comparative example 1 | 1.74x10^7/ml | 1.78x10^7/ml |
| Comparative example 2 | 1.32x10^7/ml | 1.35x10^7/ml |
| Comparative example 3 | 1.40x10^7/ml | 1.42x10^7/ml |
As is clear from the results of Table 1, the number of cells and the purity thereof were significantly higher than those of the conventional methods by the method of the present invention; and the culture solution is added in batches to increase the number and purity of cells; TNF-a has synergistic effect with vitamin B12 and nicotinamide in increasing cell number.
And (2) test II: FACS flow cytometry identified the number of cells detected in each sample from two different bags of patient plasmA, as indicated by the plasmA from example N-A and example N-B, respectively, in Table 2.
TABLE 2FACS flow cytometer identification results
From the results of table 2, it can be seen that: the antigen presenting ability effect of the invention is obviously higher than that of the conventional method, and the culture solution is added in batches to have certain gain effect on the antigen presenting ability; NF-a, vitamin B12 and nicotinamide all have synergistic effect on the antigen presenting capability of DC cells, and especially have excellent promoting effect on HLA-DR and CD 86.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (8)
1. A culture solution for DC cells, comprising: adherent cell culture solution, a subsequent first supplemented DC cell culture solution and a subsequent second supplemented DC cell culture solution;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml;
the subsequent first supplementation of DC cell culture fluid comprises: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum;
the second subsequent supplemented DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, and 1.2-1.7ul of 300U/mL nicotinamide.
2. The culture solution of DC cells according to claim 1, wherein said adherent cell culture solution comprises: 20ml of ALyS505N-0 medium, 9.7ul of GM-SCF at a concentration of 800U/ml, and 6.6ul of IL-13 at a concentration of 500U/ml.
3. The culture solution of DC cells according to claim 1 or 2, wherein said culture solution of adherent cells further comprises: 50ul of 200mM amino acid.
4. A culture solution of DC cells according to claim 3, wherein the amino acids comprise: one or more of L-glutamine, L-arginine, L-aspartic acid or L-phenylalanine.
5. A culture of DC cells according to claim 1, wherein the first subsequent supplemented culture of DC cells comprises: 15ml of serum-free DC medium, 10.1ul GM-CSF at a concentration of 800U/ml, 6.9ul IL-4 at a concentration of 500U/ml, 3.1ul anti-CD 83 antibody at a concentration of 500U/ml and 0.15ml of 1% autologous serum.
6. A culture of DC cells according to claim 1, wherein the second subsequent supplemented culture of DC cells comprises: 1.9ul 150U/mL TNF-a, 1.2ul 250U/mL vitamin B12, 1.5ul 300U/mL niacinamide.
7. A method for culturing DC cells, comprising the steps of:
step one, processing plasma to obtain white blood cells;
step two, culturing white blood cells by using adherent cell culture solution, and removing the suspension liquid after culturing for 2 hours to leave adherent cells;
the adherent cell culture solution comprises: 20ml of serum-free DC medium, 9.4-9.9ul of GM-SCF at a concentration of 800U/ml, 6.3-6.9ul of IL-13 at a concentration of 500U/ml;
step three, adding the subsequent first supplement DC cell culture solution into a culture bottle with adherent cells, culturing for 20h at 36-38 ℃ and 3-8% CO2；
The subsequent first supplementation of DC cell culture fluid comprises: 15ml of serum-free DC medium, 9.8-10.3ul of GM-CSF at a concentration of 800U/ml, 6.7-7.2ul of IL-4 at a concentration of 500U/ml, 2.9-3.4ul of anti-CD 83 antibody at a concentration of 500U/ml and 0.13-0.17ml of 1% autologous serum;
step four, adding the subsequent second supplemented DC cell culture solution into a culture bottle with adherent cells, and culturing for 2 days at 36-38 ℃ and 3-8% CO2；
The second subsequent supplemented DC cell culture fluid comprises: 1.7-2.2ul of 150U/mL TNF-a, 0.9-1.4ul of 250U/mL vitamin B12, 1.2-1.7ul of 300U/mL nicotinamide;
and step four, harvesting the DC cells.
8. The method of claim 7, wherein in step three, the adherent cells are washed 1-3 times with saline before the first subsequent additional DC cell culture medium is added to the culture flask with the adherent cells.
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