CN105112371A - Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation - Google Patents
Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation Download PDFInfo
- Publication number
- CN105112371A CN105112371A CN201510316136.8A CN201510316136A CN105112371A CN 105112371 A CN105112371 A CN 105112371A CN 201510316136 A CN201510316136 A CN 201510316136A CN 105112371 A CN105112371 A CN 105112371A
- Authority
- CN
- China
- Prior art keywords
- cell
- cik
- preparation
- hours
- cell preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a DC-CIK co-culture cell preparation method. According to the invention, mononuclear cells separated from umbilical cord blood are separately differentiated into DC cells and CIK cells, and then the DC cells and the CIK cells are co-cultured; and mature DC cells are obtained through two-stage differentiation of the mononuclear cells separated from umbilical cord blood in virtue of a plurality of factors. The DC-CIK co-culture cell preparation method provided by the invention shortens time needed for obtainment of the mature DC cells by at least two folds and improves DC-CIK cell co-culture efficiency.
Description
Technical field
The present invention relates to a kind of immunocyte to produce and co-culture method, particularly relate to a kind of DC cell and CIK cell is produced and co-culture method, the mononuclearcell that DC cell is separated from Cord blood with the equal Induction and differentiation of CIK cell, and the application in the immunocyte preparation for the treatment of tumour.
Background technology
Activated T cell depends on 2 activation signalses (peptide-MHC-TCR complex body, costimulatory molecules CD80/86 and CD4
+the CD28 molecule of T Expressions In Lymphocytes combines) acting in conjunction, stimulate specific cytotoxic t lymphocytes (cytotoxicTcell, and helper lymphocyte (Thelpercell Tc), Th) breed, excitating organism produces tumour immunity or strengthens immunne response ability.Dendritic cell (Dendriticcell, DC) be the professional antigen presenting cells (antigenpresentingcell that the function that finds so far is the strongest, APC), ripe DC can offer tumour antigen by approach such as II type histocompatibility antigens (MHC-II), effectively resists the Immune escaping mechanism of tumour cell.
Cytokine induced kill cell (cytokine-inducedkillers, CIK) is a class anti-tumor viral effector cell, can be induced in vitro and breed in a large number.DC-CIK cell can be obtained after DC and homology CIK cell Dual culture.DC is the antigen presenting cell that in body, function is the strongest, its picked-up, processing, process antigen, high expression level MHC-I, MHC-II quasi-molecule, costimulatory molecules and adhesion molecule, promote the interaction of T cell signal transduction molecule and the activation of signal path, APC and the T cell of activation can secrete IL-1, IL-2, IFN-γ etc., promote the performance of T cell effector function.Therefore, as joined together to treat malignant tumour with the CIK cell with Effective Anti tumor activity by the DC with powerful tumour antigen submission ability, synergistic antitumor effect can be played undoubtedly.
The many employings of DC preparation method of the prior art contain the substratum cultured continuously 5 days of GM-CSF and IL-4, within the 6th day, add TNF-α and continue cultivation again 2 ~ 3 days.DC cell prepared by this method exists that the cycle is long, yield is low and the shortcoming such as antigen presentation ability.The peripheral blood mononuclear cell of CIK preparation method many employings patient, the many meetings of the CIK cell prepared with this kind of cell exist that fecundity difference, cytoactive are low, CD3
+and CD56
+the problems such as cell proportion is lower.
Summary of the invention
One object of the present invention is to provide a kind of DC cell preparation method, separation is carried out respectively being divided into DC cell from the mononuclearcell of Cord blood, shortens the incubation time of DC cell.
Another object of the present invention is to provide a kind of DC-CIK Dual culture preparation method, by separation after the mononuclearcell of Cord blood carries out being divided into DC cell and CIK cell respectively, then carries out Dual culture.
Another object of the present invention is to provide a kind of DC-CIK Dual culture preparation method, adopts the separation of multiple factor pair to carry out differentiation from the mononuclearcell of Cord blood and produces DC cell, shorten the time of producing ripe DC cell, be beneficial to producing of DC-CIK Dual culture.
A kind of DC cell preparation method provided by the invention, carries out being divided into ripe DC cell (mDC) from the mononuclearcell of Cord blood by being separated respectively.HLA-DR, CD80, CD83 and CD86 phenotype that mDC has accounts for more than 90%.
Ripe DC cell first adopts the factor differentiation such as giant cells G CFS (GM-CSF), IL-4 (IL-4) and FLT3L (Flt3L) to obtain immature DC cell (iDC), and then uses tumor necrosis factor alpha (TNF-α), interleukin-11 β (IL-1 β), interleukin 6 (IL-6) and prostaglandin E2 (PGE2) to act on the ripe DC cell of iDC acquisition.
Another kind of DC cell preparation method provided by the invention, first adopt GM-CSF, IL-4 to be separated with Flt3L and within 24 hours ~ 48 hours, obtain iDC from the mononuclearcell of Cord blood, then use TNF-α, IL-1 β, IL-6 and PGE224 hour ~ within 48 hours, make iDC become mDC.
DC cell preparation method provided by the invention, GM-CSF consumption is 20ng/ml ~ 30ng/ml, as: but be not limited only to 20ng/ml, 21ng/ml, 22ng/ml, 23ng/ml, 24ng/ml, 25ng/ml, 26ng/ml, 27ng/ml, 28ng/ml, 29ng/ml and 30ng/ml.
DC cell preparation method provided by the invention, IL-4 consumption is 10ng/ml ~ 20ng/ml, as: but be not limited only to 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml, 15ng/ml, 16ng/ml, 17ng/ml, 18ng/ml, 19ng/ml and 20ng/ml.
DC cell preparation method provided by the invention, Flt3L consumption is 45ng/ml ~ 55ng/ml, as: but be not limited only to 45ng/ml, 46ng/ml, 47ng/ml, 48ng/ml, 49ng/ml, 50ng/ml, 51ng/ml, 52ng/ml, 53ng/ml, 54ng/ml and 55ng/ml.
DC cell preparation method provided by the invention, TNF-α consumption is 95ng/ml ~ 105ng/ml, as: but be not limited only to 95ng/ml, 96ng/ml, 97ng/ml, 98ng/ml, 99ng/ml, 100ng/ml, 101ng/ml, 102ng/ml, 103ng/ml, 104ng/ml and 105ng/ml.
DC cell preparation method provided by the invention, IL-1 β consumption is 5ng/ml ~ 15ng/ml, as: but be not limited only to 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml and 15ng/ml.
DC cell preparation method provided by the invention, IL-6 consumption is 5ng/ml ~ 15ng/ml, as: but be not limited only to 5ng/ml, 6ng/ml, 7ng/ml, 8ng/ml, 9ng/ml, 10ng/ml, 11ng/ml, 12ng/ml, 13ng/ml, 14ng/ml and 15ng/ml.
DC cell preparation method provided by the invention, PGE2 consumption is 0.3 μ g/ml ~ 3 μ g/ml, as: but be not limited only to 0.3 μ g/ml, 0.4 μ g/ml, 0.5 μ g/ml, 0.6 μ g/ml, 0.7 μ g/ml, 0.8 μ g/ml, 0.9 μ g/ml, 1 μ g/ml, 2 μ g/ml and 3 μ g/ml.
Another kind of ripe DC cell provided by the invention can obtain as follows, and it comprises:
By 4 × 10
6individual/ml ~ 5 × 10
6the mononuclearcell that individual/ml takes from Cord blood is placed in 37 DEG C, 5%CO
2sucking-off suspension cell after 2 hours is hatched in incubator, cleaning, adds factor GM-CSF to 25ng/ml in substratum, adds IL-4 to 16ng/ml and add Flt3L to 50ng/ml to break up 24 hours ~ 48 hours, period keeps GM-CSF, IL-4 and Flt3L final concentration to stablize, and obtains iDC cell;
Re-use 100ng/mlTNF-α, 10ng/mlIL-1 β, 10ng/mlIL-6 and 1 μ g/mlPGE2 to act on iDC cell and obtain mDC cell in 24 hours ~ 48 hours.
The DC cell adopting DC cell preparation method provided by the invention to produce can carry out Dual culture with CIK cell, and obtained DC-CIK co-cultured cell preparation, is used for the immunotherapy of tumour as medicine.
A kind of DC-CIK cell preparation method provided by the invention, by separation after the mononuclearcell of Cord blood carries out being divided into DC cell and CIK cell respectively, then carry out Dual culture, the DC-CIK co-cultured cell of acquisition has CD3+ and CD56+ immunophenotype.
Mononuclearcell provided by the invention adopts Ficoll density gradient centrifugation to be separated, and after erythrocyte cracked liquid cracking, with physiological saline cleaning, obtains after low-speed centrifugal.
DC-CIK cell preparation method provided by the invention, DC cell and CIK cell carry out Dual culture by 1: 10 ~ 100, and incubation time is 7 days ~ 10 days, and IL-2 concentration (95ng/ml ~ 105ng/ml) keeps stable.
A kind of CIK cell provided by the invention can obtain as follows, and it comprises:
By 4 × 10
6individual/ml ~ 5 × 10
6the mononuclearcell that individual/ml takes from Cord blood is placed in 37 DEG C, 5%CO
2obtain suspension cell after hatching 2 hours in incubator, substratum is diluted to cell concn 1 × 10
6individual/ml ~ 3 × 10
6individual/ml, adds cytokine IFN-γ to 20 ~ 30ng/ml, is placed in 37 DEG C, 5%CO
2in incubator, add Anti-CD3mAb to 95 ~ 105ng/ml next day, add IL-2 to 95 ~ 105ng/ml, add IL-1 α to 3ng/ml ~ 8ng/ml, and in 37 DEG C, 5%CO
2continue cultivation in incubator 4 days ~ 6 days, period IL-2 concentration keeps stable.
Substratum used in the present invention is substratum based on serum-free lymphocyte culture fluid, and the fostering requirement according to cell (as: DC cell and CIK cell) also adds 0.5v/v ~ 1v/v% umbilical cord blood.Serum-free lymphocyte culture fluid by commercial nutrient solution, as: but be not limited only to X-VIVO
tM10 (Lonza company of the U.S.) and X-VIVO
tM15 (Lonza companies of the U.S.) etc.
The beneficial effect that technical solution of the present invention realizes:
DC preparation method provided by the invention, first adopt GM-CSF, IL-4 and Flt3L factor to break up and obtain immature DC cell, and then the DC cell of the maturation described in described immature DC cell acquisition is acted on by TNF-α, IL-1 β, IL-6 and PGE2, only need cost 2 days ~ 4 day time, what improve ripe DC cell produces efficiency.With existing cultural method (ImmunologyLetters, 2002,83,101 ~ 109; ClinicalImmunology, 2007,125,257 ~ 267; Chinese Clinical rehabilitation [J], 2006,10 (9), 40 ~ 42) need to compare for more than 7 days, the time shortens greatly.
DC-CIK preparation method provided by the invention, improves the efficiency that monocyte is induced to DC, also uses Anti-CD3mAb to coordinate the combinations of factors induced amplification CIK cell such as IFN-γ, IL-2 and IL-1 α.In addition, by this kind of fast m DC with derive from the CIK cell that same cord blood mononuclearcell induces and carry out Dual culture, increase substantially CIK cell multiplication capacity and kill tumor activity, the immunocyte preparation preparing clinical treatment tumour needs from Cord blood fast can be realized.
Accompanying drawing explanation
Figure 1A is the 1st day DC cell (10 × 40) under inverted microscope, and cell is single spherical;
Figure 1B is the 2nd day DC cell (10 × 40) under inverted microscope, and part projection appears in cell;
Fig. 1 C is the 4th day DC cell (10 × 40) under inverted microscope, and cell colony grows and obviously has projection;
Fig. 1 D is the 4th day DC cell (10 × 40) under inverted microscope, and cell colony grows and obviously has projection.
Fig. 2 A is the 1st day CIK cell (10 × 40) under inverted microscope, and cell is single spherical;
Fig. 2 B is the 3rd day CIK cell (10 × 20) under inverted microscope, and cell shape is irregular, colony growth, starts amplification;
Fig. 2 C is the 7th day CIK cell (10 × 20) under inverted microscope, and cell increases in a large number;
Fig. 3 is the ratio of CD80 positive cell in FCM analysis mDC;
Fig. 4 is the ratio of CD86 positive cell in FCM analysis mDC;
Fig. 5 is the ratio of HLA-DR positive cell in FCM analysis mDC;
Fig. 6 is the ratio of CD83 positive cell in FCM analysis mDC;
Fig. 7 is that FCM analysis cultivates CD3 in the 7th day CIK cell
+and CD56
+cell proportion;
Fig. 8 is that FCM analysis cultivates CD3 in the 14th day CIK cell
+and CD56
+cell proportion.
Embodiment
Technical scheme of the present invention is described in detail below in conjunction with accompanying drawing.The embodiment of the present invention is only in order to illustrate technical scheme of the present invention and unrestricted, although with reference to preferred embodiment to invention has been detailed description, those of ordinary skill in the art is to be understood that, can modify to the technical scheme of invention or equivalent replacement, and not departing from the spirit and scope of technical solution of the present invention, it all should be encompassed in right of the present invention.
The preparation of embodiment 1 umbilical cord blood and mononuclearcell (CBMNC)
Mononuclearcell adopts Ficoll density gradient centrifugation to be separated, and after erythrocyte cracked liquid cracking, with physiological saline cleaning, obtain after low-speed centrifugal, its concrete steps are as follows:
1, aseptic collection full-term normal delivery fetus is broken the Cord blood 80ml after navel, citrate anticoagulation, by the phosphate buffered saline(PBS) (PBS+2mMEDTA) of concentration 2mMEDTA by 1: 1 dilution Cord blood;
2, add
-PaquePremium centrifugate (GEHealthcare) separation of C BMNC, in 18 DEG C ~ 20 DEG C, 400G is centrifugal, 30min, draws upper plasma and CBMNC layer respectively;
3, the blood plasma collected is put into 56 DEG C of water-baths 30 minutes, 4 DEG C 30 minutes, deactivation complement.Centrifugal 10 minutes of 3000rpm, moves to-20 DEG C of packing in new centrifuge tube frozen for subsequent use by supernatant;
4, use PBS+2mMEDTA solution washing CBMNC1 time, 20 DEG C, 400G, centrifugal 10 minutes, abandons supernatant;
5, precipitation erythrocyte cracked liquid (ACK) cracking 10min, centrifuge washing 2 times (the 1st time: room temperature 400G, 10min; 2nd time: room temperature, 60G ~ 100G, 10min), removing thrombocyte and separating medium;
6, X-VIVO is used
tM15 serum-free mediums (Lonza company of the U.S.) are resuspended carries out cell counting under microscope, uses lymphocyte culture fluid X-VIVO
tM15 regulate cell density to become 4 × 10
6individual/ml ~ 5 × 10
6the cell suspension of individual/ml.
Embodiment 2 mononuclearcell Differentiation Induction in vitro DC
1, by 4 × 10
6individual/ml ~ 5 × 10
6the cell suspension of individual/ml spreads into 6 orifice plates (non-tissue culture is used), and every hole 2ml, is placed in 37 DEG C, 5v/v%CO
2in incubator;
2, after hatching 2 hours, light 6 orifice plates that shake, sucking-off suspension cell;
3, X-VIVO is used
tM15 serum-free mediums (containing 0.5v/v ~ 1v/v% umbilical cord blood) clean 6 orifice plate 1 ~ 2 time, add factor GM-CSF (25ng/ml), IL-4 (16ng/ml) and Flt3L (50ng/ml) combine differentiation 24 hours ~ 48 hours to attached cell;
4, change liquid every other day, and make GM-CSF, IL-4 and Flt3L concentration stabilize, produce iDC;
5, ripe stimulating factor TNF-α (100ng/ml), IL-1 β (10ng/ml), IL-6 (10ng/ml), PGE2 (1 μ g/ml) is used to act on 24 hours ~ 48 hours and obtain mDC again;
6, get the DC cell being just separated the attached cell after adherent 2 hours, having cultivated the 48th hour I and the 96th hour respectively, examine under a microscope cellular form, see Figure 1A ~ 1D.
Embodiment 3iDC and mDC immunophenotype detect
Get the DC cell being just separated the attached cell after adherent 2 hours, cultivating the 48th hour and the 96th hour respectively, after washing 2 times with PBS+2mMEDTA solution, respectively get 1 × 10
5individual/ml, adds in corresponding streaming pipe respectively.Add monoclonal antibody to be detected and comprise each 10 μ l of HLA-DR, CD80, CD83, CD86 and CD14 antibody, 4 DEG C of lucifuges hatch 10 minutes, 1 time is washed with PBS, and be resuspended in the PBS of 400 μ l, adopt flow cytometer FACSCalibur (BD company) to detect, the results are shown in Figure 3 ~ Fig. 6 and table 1
The values of immunophenotyping (%) of table 1 different time DC
In surface antigen, CD83 is film marker molecule specific to ripe DC cell, CD80 and CD86 is the costimulatory molecules of DC cell, and HLA-DR is the molecules of immunization stimulus of DC cell.Result as shown in Table 1 can be found out, the monocyte be just separated, high expression level CD14 molecule, differentiation is combined after 48 hours with cytokine GM-CSF, IL-4 and Flt3L, be divided into iDC, phenotype CD80, HLA-DR, CD86 express more than 55%, low expression CD83 molecule.Induce stimulation 48 hours through TNF-α, IL-1 β, IL-6 and PGE2 again, be divided into ripe DC cell, phenotype CD80, CD86, HLA-DR and CD83 express and all increase, and account for more than 90%.Cells show goes out typical ripe DC cellular form, and suspension cell increases, and volume becomes large, and surface grows the antenna of a large amount of spiculation, and shared by DC, ratio constantly raises, and can obtain the DC cell of highly purified maturation thus fast.
Prepared by embodiment 4CIK cell
1, after hatching 2 hours in Example 2, the suspension cell of sucking-off is cultivated for CIK;
2, X-VIVO is used
tM15 serum free mediums (0.5v/v% ~ 1v/v% umbilical cord blood) are resuspended, and cell concn is 1 × 10
6individual/ml ~ 3 × 10
6individual/ml spreads into culturing bottle, adds cytokine IFN-γ (25ng/ml), is placed in 37 DEG C, 5%CO
2in incubator;
3, add Anti-CD3mAb (100ng/ml), IL-2 (100ng/ml), IL-1 α (5ng/ml) next day, be placed in 37 DEG C, 5%CO
2cultivation is continued 4 days ~ 6 days in incubator;
4, fluid infusion in every 2 days ~ 3 days or go down to posterity, and keep IL-2 concentration stabilize.
Embodiment 5DC cell and CIK cell Dual culture
1, the mDC cultivating maturation is collected, be assigned in experimental group CIK cell with 1: 10 ~ 1: 100, and add GM-CSF to 25ng/ml, continue Dual culture and within 7 ~ 10 days, obtain ripe DC-CIK cell, fluid infusion in every 2 days ~ 3 days therebetween or go down to posterity, keeps IL-2 concentration 100ng/ml to stablize simultaneously;
2, set independent CIK cell to test as a control group simultaneously;
3, cultivation CIK cell, the DC-CIK co-cultured cell of the 1st, 7 and 14 days is got respectively, basis of microscopic observation cellular form, as shown in Figure 5, the cell of the 1st day obviously presents the spherical of mononuclearcell, and the 3rd day starts obvious colony growth, within the 5th day, to be added by DC in CIK after Dual culture, there is exponential amplification in cell, partial shape changes the irregular shape of a hoof gradually into, and visible a large amount of cloning cluster is formed, and sees Fig. 2 A ~ Fig. 2 C.
The amplification of embodiment 6CIK cell and DC-CIK co-cultured cell
Get the CIK/DC-CIK cell of the 1st, 7 and 14 day of cultivation respectively, Trypan Blue, the sum of living cell counting under inverted microscope, result is see table 2.
The number of amplification of table 2 cell
The detection of embodiment 7CIK cell and DC-CIK co-cultured cell immunophenotype
Get the DC-CIK cell of cultivation the 7th and 14 days respectively, after washing 2 times with PBS+2mMEDTA solution, respectively get 1 × 10
5individual/ml, adds in corresponding streaming pipe respectively.Add monoclonal antibody to be detected and comprise each 10 μ l of CD3, CD56 antibody, 4 DEG C of lucifuges hatch 10 minutes, wash 1 time with PBS, and are resuspended in the PBS of 400 μ l, adopt flow cytometer FACSCalibur to detect, and the results are shown in Figure 7, Fig. 8 and table 3.
Table 3CD3+ and CD56+ cell proportion (%,
n=3)
7 days and 14 days CD3+ and CD56+ cell percentages two groups compare and are P < 0.001.
As can be seen here, along with the prolongation of incubation time, in CIK cell and DC-CIK co-cultured cell, the per-cent of CD3+ and CD56+ cell rises all gradually.CD3+ and CD56+ cell proportion rising more obvious than CIK cell in DC-CIK coculture.
Embodiment 8IFN-γ content detection
The cells and supernatant of get cultivation respectively the 7th day, 11 days and 14 days, carries out IFN-γ assay by ELISA kit specification sheets, the results are shown in Table 4.
Table 4IFN-γ secretory volume (pg/ml,
n=3)
In the nutrient solution supernatant of 7 days, 11 days and 14 days, IFN-γ content two groups compares and is P < 0.001.
As can be seen here, along with the prolongation of incubation time, in CIK cell and DC-CIK cell culture medium supernatant, the content of IFN-γ rises gradually, and in DC-CIK cell culture medium supernatant, the content of IFN-γ obviously raises than simple CIK.
Embodiment 9 is killed tumor activity and is detected
Get the cultivation CIK cell of 14 days and DC-CIK cell action effect cell respectively, human chronic polymorpho nuclear leukemia cells K562 (from Medical College, Shanghai Communication Univ.) is target cell, and effect target ratio is 10: 1 and 20: 1, the results are shown in Table 5.
The killing activity of table 5 couple K562 (%,
n=3)
The killing activity two groups of different effect target comparison K562 compares and is P<0.001.
As can be seen here, the effect target 10 ~ 20: 1 is than in scope, and CIK cell and DC-CIK co-cultured cell all have stronger lethal effect to K562 cell, and lethal effect strengthens with the increase of effect target ratio.At same effect target than in situation, the killing activity of DC-CIK co-cultured cell to K562 cell is significantly higher than simple CIK cell.
Claims (25)
1. a DC cell preparation method, is characterized in that carrying out respectively being divided into ripe DC cell from the mononuclearcell of Cord blood by being separated.
2. DC cell preparation method according to claim 1, it is characterized in that first being broken up by GM-CSF, IL-4 and Flt3L factor obtaining immature DC cell in 24 hours ~ 48 hours, and then act on described immature DC cell 24 hours ~ 48 hours by TNF-α, IL-1 β, IL-6 and PGE2, the DC cell of the maturation described in acquisition.
3. DC cell preparation method according to claim 2, is characterized in that described GM-CSF consumption is 20ng/ml ~ 30ng/ml.
4. DC cell preparation method according to claim 2, is characterized in that IL-4 consumption is 10ng/ml ~ 20ng/ml.
5. DC cell preparation method according to claim 2, is characterized in that described Flt3L consumption is 45ng/ml ~ 55ng/ml.
6. DC cell preparation method according to claim 2, is characterized in that described TNF-α consumption is 95ng/ml ~ 105ng/ml.
7. DC cell preparation method according to claim 2, is characterized in that described IL-1 β consumption is 5ng/ml ~ 15ng/ml.
8. DC cell preparation method according to claim 2, is characterized in that described IL-6 consumption is 5ng/ml ~ 15ng/ml.
9. DC cell preparation method according to claim 2, is characterized in that described PGE2 consumption is 0.3 μ g/ml ~ 3 μ g/ml.
10. DC cell preparation method according to claim 1, is characterized in that the DC cell step of the maturation described in obtaining comprises:
By 4 × 10
6individual/ml ~ 5 × 10
6the mononuclearcell that individual/ml takes from Cord blood is placed in 37 DEG C, 5%CO
2sucking-off suspension cell after 2 hours is hatched in incubator, cleaning attached cell, factor GM-CSF is added to concentration 25ng/ml in substratum, add IL-4 to concentration 16ng/ml and add Flt3L to concentration 50ng/ml and break up 24 hours ~ 48 hours, period keeps GM-CSF, IL-4 and Flt3L final concentration to stablize, and obtains immature DC cell;
Re-use the DC cell that 100ng/mlTNF-α, 10ng/mlIL-1 β, 10ng/mlIL-6 and 1 μ g/mlPGE2 act on the maturation described in the acquisition in 24 hours ~ 48 hours of described immature DC cell.
11. DC cell preparation methods according to claim 10, is characterized in that described substratum is for substratum based on serum-free lymphocyte culture fluid.
12. DC cell preparation methods according to claim 10, is characterized in that described substratum is for substratum based on serum-free lymphocyte culture fluid, also adds umbilical cord blood to concentration 0.5v/v ~ 1v/v%.
13. 1 kinds obtain the application of DC cell in the immunotherapy medicaments of preparation tumour according to the method described in claim 1 ~ 12.
14. 1 kinds obtain DC cell and CIK cell Dual culture according to the method described in claim 1 ~ 12.
1: 10 ~ 100 Dual culture are pressed for 15. 1 kinds according to the method acquisition DC cell described in claim 1 ~ 12 and CIK cell.
16. 1 kinds of DC-CIK co-cultured cell preparation methods, is characterized in that the DC cell that the method described in claim 1 ~ 12 obtained and are separated the CIK cell be divided into from the mononuclearcell of Cord blood and carry out Dual culture, obtain DC-CIK co-cultured cell.
17. DC-CIK co-cultured cell preparation methods according to claim 16, is characterized in that described mononuclearcell adopts Ficoll density gradient centrifugation to be separated, after erythrocyte cracked liquid cracking, with physiological saline cleaning, obtain after low-speed centrifugal.
18. DC-CIK co-cultured cell preparation methods according to claim 16, is characterized in that described DC cell and CIK cell obtain by cultivating respectively from the attached cell of same cord blood mononuclearcell and suspension cell.
19. DC-CIK co-cultured cell preparation methods according to claim 16, is characterized in that described DC cell and CIK cell carry out Dual culture by 1: 10 ~ 100, and incubation time is 7 days ~ 10 days, keep IL-2 concentration 95ng/ml ~ 105ng/ml to stablize.
20. DC-CIK co-cultured cell preparation methods according to claim 16, is characterized in that the DC-CIK co-cultured cell obtained has CD3+ and CD56+ immunophenotype.
21. DC-CIK co-cultured cell preparation methods according to claim 16, is characterized in that the step of the CIK cell described in obtaining comprises:
By 4 × 10
6individual/ml ~ 5 × 10
6the mononuclearcell that individual/ml takes from Cord blood is placed in 37 DEG C, 5%CO
2obtain suspension cell after hatching 2 hours in incubator, substratum is diluted to cell concn 1 × 10
6individual/ml ~ 3 × 10
6individual/ml, adds cytokine IFN-γ to 20 ~ 30ng/ml, is placed in 37 DEG C, 5%CO
2in incubator, add Anti-CD3mAb to 95 ~ 105ng/ml next day, add IL-2 to 95 ~ 105ng/ml, add IL-1 α to 3 ~ 8ng/ml, and in 37 DEG C, 5%CO
2continue cultivation in incubator 4 days ~ 6 days, period IL-2 concentration keeps stable.
22. DC-CIK co-cultured cell preparation methods according to claim 21, is characterized in that described substratum is for substratum based on serum-free lymphocyte culture fluid.
23. DC-CIK co-cultured cell preparation methods according to claim 22, is characterized in that described substratum is for substratum based on serum-free lymphocyte culture fluid, also adds umbilical cord blood to concentration 0.5v/v ~ 1v/v%.
24. according to the application of the DC-CIK co-cultured cell preparation method described in claim 16 ~ 23 in the immunotherapy medicaments of preparation tumour.
25. 1 kinds of DC-CIK co-cultured cell application in the immunotherapy medicaments of preparation tumour of producing according to the method described in claim 16 ~ 23.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510316136.8A CN105112371A (en) | 2015-06-10 | 2015-06-10 | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510316136.8A CN105112371A (en) | 2015-06-10 | 2015-06-10 | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN105112371A true CN105112371A (en) | 2015-12-02 |
Family
ID=54660486
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510316136.8A Pending CN105112371A (en) | 2015-06-10 | 2015-06-10 | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105112371A (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969727A (en) * | 2015-12-29 | 2016-09-28 | 山东省齐鲁细胞治疗工程技术有限公司 | Method for culturing cord blood lymphocyte DC-CIK |
| CN106119198A (en) * | 2016-06-24 | 2016-11-16 | 安徽未名细胞治疗有限公司 | A kind of method of effective acquisition DC cell |
| CN106635984A (en) * | 2016-11-16 | 2017-05-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Human umbilical cord blood immune cell bank establishing method |
| CN108642013A (en) * | 2018-05-02 | 2018-10-12 | 中航（宁夏）生物股份有限公司 | From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale |
| CN108853143A (en) * | 2018-07-04 | 2018-11-23 | 盛春华 | Immunocyte preparation, preparation method and the application for treating psoriasis |
| CN109402055A (en) * | 2018-11-12 | 2019-03-01 | 广州航华生物医药科技有限公司 | A kind of DC-CIK cell culture kit and its cultural method |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321581A (en) * | 2011-09-13 | 2012-01-18 | 深圳市博泰生物医学科技发展有限公司 | Preparation method of ascites tumor cell sensitized DC-CIK |
| CN104073467A (en) * | 2013-03-27 | 2014-10-01 | 上海宇研生物技术有限公司 | Preparing method of human DCCIK immunocompetent cell |
-
2015
- 2015-06-10 CN CN201510316136.8A patent/CN105112371A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321581A (en) * | 2011-09-13 | 2012-01-18 | 深圳市博泰生物医学科技发展有限公司 | Preparation method of ascites tumor cell sensitized DC-CIK |
| CN104073467A (en) * | 2013-03-27 | 2014-10-01 | 上海宇研生物技术有限公司 | Preparing method of human DCCIK immunocompetent cell |
Non-Patent Citations (2)
| Title |
|---|
| 易良才等: "Flt3-L与 GM-CSF体外诱导急性髓系白血病患者DC功能比较研究", 《中国肿瘤生物治疗杂志》 * |
| 袁开长等: ""自体血清+鸡尾酒法"诱导外周血 DC 成熟的研究", 《中国临床神经外科杂志》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105969727A (en) * | 2015-12-29 | 2016-09-28 | 山东省齐鲁细胞治疗工程技术有限公司 | Method for culturing cord blood lymphocyte DC-CIK |
| CN106119198A (en) * | 2016-06-24 | 2016-11-16 | 安徽未名细胞治疗有限公司 | A kind of method of effective acquisition DC cell |
| CN106635984A (en) * | 2016-11-16 | 2017-05-10 | 沈阳细胞治疗工程技术研发中心有限公司 | Human umbilical cord blood immune cell bank establishing method |
| CN108642013A (en) * | 2018-05-02 | 2018-10-12 | 中航（宁夏）生物股份有限公司 | From being detached in Cord blood after CD34 candidate stem cells expand culture, induction prepares Dendritic Cells method to one kind on a large scale |
| CN108853143A (en) * | 2018-07-04 | 2018-11-23 | 盛春华 | Immunocyte preparation, preparation method and the application for treating psoriasis |
| CN109402055A (en) * | 2018-11-12 | 2019-03-01 | 广州航华生物医药科技有限公司 | A kind of DC-CIK cell culture kit and its cultural method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105112371A (en) | Preparation method for DC-CIK cells originated from umbilical cord blood mononuclear cells and preparation | |
| JP5358683B2 (en) | Method of growing natural killer cells | |
| CN104357394B (en) | Culture method of autologous peripheral blood lymphocyte DC-CIK (Dendritic Cell- Cytokine-induced Killer) | |
| CN107460168B (en) | The amplification cultivation method of natural killer cells culture substrate and natural killer cells | |
| CN106434556B (en) | A kind of method of external evoked amplification I type NKT cell | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN102676454B (en) | Preparation method for CIK (cytokine induced killer) cell of umbilical cord blood source | |
| CN107475196A (en) | The amplification cultivation method of NK culture matrix and NK | |
| CN104845934B (en) | Bleeding of the umbilicus CD34+Derived from hematopoietic precursor cells Dendritic Cells is prepared on a large scale method | |
| CN102268405A (en) | Method for auto NK (Natural Killer) cell in-vitro activation and amplification culture and special culture medium thereof | |
| CN105349489A (en) | Culture method of CIK cell | |
| CN104498434A (en) | Preparation method of large number of dendritic cells and obtained dendritic cells | |
| CN109825473A (en) | A kind of cultural method of the autologous peripheral blood lymphocyte using the stimulation of TLR7 agonist | |
| CN107446888B (en) | The application of NK cell culture mediums, cultural method and the two | |
| CN112410294A (en) | Amplification culture method of peripheral blood CIK cells | |
| JP2014030375A (en) | Method for obtaining monocytes or nk cells | |
| CN102861107B (en) | DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition | |
| Yi et al. | Adherent and non-adherent dendritic cells are equivalently qualified in GM-CSF, IL-4 and TNF-α culture system | |
| CN104109653B (en) | Method using human peripheral DNT cells are expanded without animal blood serum cultivating system on a large scale | |
| CN105543171A (en) | Method for amplifying regulatory T cells | |
| CN103173410B (en) | Composition and method for stimulating dendritic cell maturation | |
| CN104762261A (en) | Tumor infiltrating lymphocytes separation method | |
| CN109957543A (en) | Utilize the method for Cord blood massive amplification Cord Blood Natural Killer Cells: Impact | |
| Delirezh et al. | Generation of mature monocyte-derived dendritic cells in the presence of heparin and monocyte conditioned medium: phenotypic and functional comparison | |
| CN107586758B (en) | In-vitro inducer and inducing method for stem cell-like memory T cells |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20151202 |
|
| WD01 | Invention patent application deemed withdrawn after publication |