CN1517435A - Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application - Google Patents
Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application Download PDFInfo
- Publication number
- CN1517435A CN1517435A CNA031184928A CN03118492A CN1517435A CN 1517435 A CN1517435 A CN 1517435A CN A031184928 A CNA031184928 A CN A031184928A CN 03118492 A CN03118492 A CN 03118492A CN 1517435 A CN1517435 A CN 1517435A
- Authority
- CN
- China
- Prior art keywords
- cell
- substratum
- leukemia
- radix angelicae
- angelicae sinensis
- 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.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
A culture medium for inducing the leukemia cell to generate dendric cell (DC) is prepared through adding the angelica polyose to the convertional non-serum culture medium of leukemia cell for promoting the function of hematopoietic cell, immunity and the phagocytic function of mononuclear-macrophage, and inducing the differentiation of leukamia cell strain K562. Its advantages are high molecular expression of its antigen to costimulation, and short preparing time (3-5 days).
Description
Technical field
The substratum and cultural method and application, the especially leukemia cell that the present invention relates to a kind of cell induce substratum and cultural method and the application that generates dendritic cell.
Background technology
(dendritic cells is the most important antigen presenting cell that starts T cell initial reaction DC) to dendritic cell, and DC can be used for the immunotherapy of tumour.Marrow hemopoiesis progenitor cell and peripheral blood lymphocytes are all adopted in traditional DC preparation.The preparation method adds grain-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4), tumour necrosis factor (TNF) in the serum culture system is arranged.In addition, in preparation process, also need add the specific antigens that extracts from tumour cell and impact cultivation, complex process, incubation time is long, about 15 days.Also have from the leukemia cell in recent years to prepare DC, this kind method prepares DC itself and carries tumour specific antigen, but the specific reaction of activated T cell and produce antitumor action.Therefore can save to extract tumour antigen in the preparation process and add antigen and impact the step of cultivating, simplify technology.But weak point is to prepare leukemia DC with traditional method, and the antigen costimulatory molecules is expressed low, and the activated T cell effect is poor, and incubation time is still longer, about 13 days.For this reason, shorten incubation time, seek the method that better prepares leukemia DC and seem very important.
Summary of the invention
The object of the present invention is to provide the short leukemia cell of a kind of incubation time to induce substratum and cultural method and the application that generates dendritic cell.
Technical scheme of the present invention is:
The leukemia cell induces the substratum that generates dendritic cell, adds Radix Angelicae Sinensis polysaccharide in leukemia cell's serum free medium of routine.Radix Angelicae Sinensis polysaccharide English is that Angelica Polysaccharide is called for short APS.
More excellent technical scheme has following:
Radix Angelicae Sinensis polysaccharide concentration is 10-800ug/ml.
Radix Angelicae Sinensis polysaccharide concentration is 50-400ug/ml.
The leukemia cell induces the substratum that generates dendritic cell, and the serum free medium serum that adds 5-15% in the IMDM substratum is at thing, and wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, human transferrin, cholesterol, Regular Insulin is formed.
The leukemia cell induces the substratum that generates dendritic cell, uses the concentration of phosphate buffered saline buffer allotment Radix Angelicae Sinensis polysaccharide.
The leukemia cell induces the cell culture processes of the substratum that generates dendritic cell, and leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50-400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 1-10 days.
The leukemia cell induces the cell culture processes of the substratum that generates dendritic cell, places the cell culture incubator of 37 ℃ of routines, 5%CO2 to cultivate, and half amount is changed liquid 2 times weekly, and additional fresh culture and corresponding cytokine were cultivated 3-5 days.
The leukemia cell induces the cell culture processes of the substratum that generates dendritic cell, adds TNF-α after 10 days of cultivation, continues to cultivate collecting cell 3 days.
The leukemia cell induces the substratum that generates dendritic cell to be used for leukemia cell, dendritic cell analyzing and testing.
Principle and advantage of the present invention is:
Growth has promoter action to Radix Angelicae Sinensis polysaccharide to hematopoietic cell, can inducing leukemia cell strain K
562Differentiation can promote immunologic function, strengthens the phagocytic function of mononuclear macrophage, and therefore, Radix Angelicae Sinensis polysaccharide is used to prepare DC and has sufficient theoretical foundation.
Adopt mainly by bovine serum albumin, catalase, human transferrin, cholesterol, the serum free medium serum substitute that Regular Insulin is formed prepares leukemia DC, prepare required time and have the serum cultivation similar, but the DC activated T cell effect ratio that serum-free condition prepares down has serum pref strong, simultaneously, the hepatitis virus that serum may bring, the possibility of AIDS viral infection have been avoided adding.
With do not add the Radix Angelicae Sinensis polysaccharide relatively, add the DC of Radix Angelicae Sinensis polysaccharide group preparation, its antigen costimulatory molecules (CD
80, CD
86) express obviously rising, preparation time only needs 3-5 days, and cytoactive is better than not adding Radix Angelicae Sinensis polysaccharide group.Therefore, Radix Angelicae Sinensis polysaccharide can promote inducing of leukemia DC and maturation, is the good inducer of preparation leukemia DC.
Embodiment
Embodiment 1, leukemia cell induce the substratum that generates dendritic cell, add Radix Angelicae Sinensis polysaccharide in leukemia cell's serum free medium of routine.
Embodiment 2, leukemia cell induce the substratum that generates dendritic cell, and Radix Angelicae Sinensis polysaccharide concentration is 10-800ug/ml.
Embodiment 3, leukemia cell induce the substratum that generates dendritic cell, and Radix Angelicae Sinensis polysaccharide concentration is 50-400ug/ml.
Embodiment 4, leukemia cell induce the substratum that generates dendritic cell, the serum free medium serum that adds 5-15% in the IMDM substratum replaces at thing, wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, human transferrin, cholesterol, Regular Insulin is formed.
Embodiment 5, leukemia cell induce the substratum that generates dendritic cell, use the concentration of phosphate buffered saline buffer allotment Radix Angelicae Sinensis polysaccharide.
Embodiment 6, leukemia cell induce the cell culture processes of the substratum that generates dendritic cell, and leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50-400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 1-10 days.
Embodiment 7, leukemia cell induce the cell culture processes of the substratum that generates dendritic cell, place the cell culture incubator of 37 ℃ of routines, 5%CO2 to cultivate, and half amount is changed liquid 2 times weekly, and additional fresh culture and corresponding cytokine were cultivated 3-5 days.
Embodiment 8, leukemia cell induce the cell culture processes of the substratum that generates dendritic cell, add TNF-α after 10 days of cultivation, continue to cultivate collecting cell 3 days.
Embodiment 9, leukemia cell induce the substratum that generates dendritic cell to be used for leukemia cell, dendritic cell analyzing and testing.
Embodiment 10,
(1), establish serum pref human leukemia cell DC method, get leukaemic's (comprising acute myeloid leukaemia, chronic myelocytic leukemia) medullary cell, isolated mononuclear cell, mirror are observed the leukemia juvenile cell down and are accounted for 80-90% by traditional method.Adjusting cell concn is 1 * 10
6Individual/ml.Cultural method: with IMDM substratum+10% people AB type serum, restock GM-CSF, IL-4, cytokines such as TNF.Place 37 ℃, the cell culture incubator of 5%CO2, saturated humidity to cultivate, change liquid twice with the fresh culture that contains above-mentioned cytokine weekly.
(2), set up the method for preparing human leukemia DC under the serum-free condition: replace at thing (mainly containing bovine serum albumin, catalase, human transferrin, cholesterol, Regular Insulin etc.) with IMDM substratum+10% serum free medium serum.
(3), from Chinese traditional medicine angelica (Minxian County, Gansu product), extract Radix Angelicae Sinensis polysaccharide, purity＞98% is mixed with proper concn with Radix Angelicae Sinensis polysaccharide with phosphate buffered saline buffer PBS, filtration sterilization is standby.
(4), Radix Angelicae Sinensis polysaccharide is added preparation leukemia DC in the serum free culture system of having set up, observe the DC of different concns, the preparation of different incubation time, adopt morphology, cellular immunization phenotype and mixed lymphocyte reacion are observed, and Radix Angelicae Sinensis polysaccharide is to the influence of preparation leukemia DC.
1 material and method
1.1 sample source
6 example chronic phase CML are through bcr/abl fusion gene test positive.
1.2 main medicine and reagent
APS is by Shanghai Univ. of Traditional Chinese Medicine's separation and Extraction, and purity＞95% is mixed with the desired concn working fluid, filtration sterilization.GM-CSF is available from the refined company in first Lingbao City, and IL-4, TNF-α, ametycin are purchased the company in Sigma, and lymphocyte separation medium is purchased the hematology institute in the Chinese Academy of Sciences.FITC-HLA-DR, FITC-CD80, FITC-CD83, FITC-CD86 monoclonal antibody are purchased the company in BD, and perfect medium comprises that IMDM and foetal calf serum purchase the company in Gibco.
1.3 cell cultures
CML patient's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and adjusting cell concn is 1 * 10
6/ ml is suspended in the IMDM substratum that contains 10% foetal calf serum, and adding concentration is 1000u/mlrhGm-CSF, and 1000u/ml rhIL-4 organizes in contrast, and experimental group adds the APS that final concentration is 50-400ug/ml more respectively in above-mentioned culture system.Place 37 ℃, the cell culture incubator of 5%CO2 to cultivate, half amount is changed liquid 2 times weekly, additional fresh culture and corresponding cytokine, cultivate the TNF-α that added the 100ug/ml of final concentration on the 10th day, continue to cultivate 3 days, collecting cell, a part is used the cell phenotype analysis, and all the other are frozen in liquid nitrogen.
1.4 cellular immunization phenotypic evaluation and morphological observation: see our reported method
[2]
1.5APS influence: cultivating sampling in the 1st, 3,5,7,10 day respectively, carrying out the blue dyeing of platform phenol, the living cell counting number to CML-DC propagation.
1.6 mixed lymphocyte reacion
Reach CR patient's peripheral blood after getting healthy people (blood transfusion person) and treatment, separate mononuclearcell, adjust cell concn 2 * 10 with lymphocyte separation medium
6/ ml adds and contains among the IMDM of 5% foetal calf serum, places the plastic culture bottle, leaves standstill 60min at 37 ℃, collects non-adherent cell.It is standby to adjust suitable cell concn.Recovery DC in 37 ℃ of water-baths washs 3 times, and adding and containing final concentration is 25ug/ml mitomycin IMDM, hatches 45min at 37 ℃, and IMDM substratum washing 3 times is adjusted cell concn with containing 10% foetal calf serum IMDM substratum, respectively with 1 * 10
3/ hole, 3 * 10
3/ hole, 1 * 10
4/ hole adds 96 well culture plates, 3 multiple holes of every winding kind, and every hole adds 2 * 10 again
5Individual healthy people or patient (CD) peripheral blood lymphocyte, add MTT (5ug/ml after 96 hours at 37 ℃, 5%CO2 cell cultures and middle cultivation, the 10ul/ hole, Sigmn company), cultivate and finish back adding dimethyl sulfoxide (DMSO) (DMSO) 150ul/ hole, OD value when detecting wavelength 570nm with microplate reader, the result gets average.
2 results
2.1 cell counting and cell survival rate are observed
With GM-CSF, IL-4 for inducing the DC condition substantially, when adding different APS concentration the variation of the counting of culturing cell and surviving rate see Table 1, table 2, as can be seen from the table, when APS concentration is 100ug/ml, the propagation of cell and surviving rate are best, and the best time is when cultivating 3-5 days.
Table 1 is with the variation (* 10 of APS concentration culturing cell number
6/ ml)
Incubation time (my god)
Grouping APS (ug/ml)
0 1 3 5 7 10
1 0 1.00 1.10 0.93 0.82 0.43 0.18
2 50 1.00 1.01 1.08 0.80 1.02 0.83
3 100 1.00 1.02 1.53 2.02 1.91 1.83
4 200 1.00 0.89 1.02 1.12 1.23 1.45
5 400 1.00 1.03 1.02 1.34 1.82 1.35
The variation (%) of cell survival rate when table 2 is cultivated with APS concentration
Incubation time (my god)
Grouping APS (ug/ml)
0 1 3 5 7 10
1 0 100 95 82 62 45 35
2 50 100 90 90 83 70 60
3 100 100 96 94 90 89 87
4 200 100 94 76 86 78 63
5 400 100 92 91 87 66 48
2.2 morphological observation
6 routine CML BMNCs, after cultivating the cell volume increase can appear partly through GM-CSF, IL-4 combined induction, and stretch out outstanding, prolongation along with incubation time, total cell quantity reduces, most of necrocytosis, but remaining cell adds TNF-α 10 days the time again when cultivating, the DC increasing proportion of dendron sample occurs.And cultured cells behind the adding APS is cultivated the cell that next day promptly occurred being dendritic processes, and occurring DC form cell proportion to cultivation in the time of 3-5 days obviously increases, and cell growth state is better than not adding APS, and total cellular score also increases.Total cellular score does not reduce yet when cultivating 10 days.
2.3 cell phenotype changes
Because when find adding 100ug/ml APS from dynamic observe, it is best that cell proliferation and surviving rate reach, therefore, we select to add the DC cell that 100ug/ml APS organizes and make phenotype analytical, and with do not add APS compare (seeing Table 3).From table 3, can point out: add the APS group, cell DC correlation table and the developed by molecule rate obviously raises, and after cultivation first day just begin to rise, and it is the most obvious wherein to raise in the time of 3-5 days.As CD83 is 40.6%, and CD80 44.6%, and CD86 50.6%, and HLA-DR 60.3%; And add APS group, and it is not obvious that DC correlation table and molecule raise, and CD83 just is 18.6% during by the 10th day, and CD80 14.1%, and CD86 15.1%, and HLA-DR 21.8%.At this moment, continue to cultivate 3 days add TNT-α again in not adding APS group after, then the ratio of CD83 can be elevated to 36.4%, but most of necrocytosis in the culture system, total cellular score obviously reduces.
Table 3 APS changes (%) to the cell phenotype of cultivating DC
Cell table incubation time (my god)
APS(ug/ml)
Type 01357 10
CD83 0 1.2±0.4 1.8±0.8 4.6±2.3 6.8±2.1 9.1±1.9 18.6±2.4
CD83 100 1.2±0.4 19.3±1.9 36.4±5.2 40.6±7.0 39.8±6.6 35.5±10.1
CD80 0 3.4±1.1 2.9±1.1 41.1±1.3 8.3±2.9 10.1±2.3 14.1±3.4
CD80 100 3.4±1.1 16.2±2.2 42.3±6.4 44.6±5.9 42.1±7.0 39.7±12.1
CD86 0 2.8±0.9 3.4±1.2 5.3±1.8 9.1±2.2 11.8±2.3 15.1±3.7
CD86 100 2.8±0.9 17.3±3.4 39.4±4.2 50.6±11.6 48.1±10.6 45.1±7.6
HLA-DR 0 6.6±2.2 7.6±2.5 18.4±2.9 19.6±2.4 20.8±7.2 21.8±3.1
HLA-DR 100 6.6±2.2 20.1±4.1 45.1±6.3 60.3±11.8 55.4±14.8 49.3±10.1
2.4 APS stimulates lymphopoietic influence to CML-DC
We collect the APS group CML-DC that do not add that cultivated the 10th day and are used for mixed lymphocyte reacion (seeing Table 4) with the CML-DC that the collection cultivation added APS on the 5th day, results suggest: two groups of CML-DC organize strong so can stimulate from body or allogeneic lymphopoiesis but the energy force rate of adding APS group inductive CML-DC activated lymphocyte propagation does not add APS.
Table 4 APS stimulates lymphopoietic influence (OD) to CML-DC
Incubation time (my god)
Grouping APS (ug/ml)
1 * 10
3/ hole 3 * 10
3/ hole 1 * 10
4/ hole
From the body lymphocyte
From body lymphocyte 0 0.32 ± 0.10 0.53 ± 0.14 0.81 ± 0.17
Normal people's lymph is thin by 100 0.48 ± and 0.12 0.81 ± 0.11 1.19 ± 0.18
Born of the same parents 0 0.36 ± 0.15 0.67 ± 0.18 0.89 ± 0.16
Normal people's lymph is thin by 100 0.50 ± and 0.20 0.93 ± 0.14 1.34 ± 0.20
Born of the same parents
3 discuss
CML patient's leukemia cell can induce the generation dendritic cell.Because CML-DC carries tumour specific antigen,, then can activate special specific T-cells immune response, thereby reach the effect of the white cell of specific killing if it has the similar antigen function of normal DC.But it is lower that the CML-DC antigen costimulatory molecules that adopts previous methods to cultivate is expressed, and it is longer to cultivate induction time, and therefore, it stimulates the t cell responses limited ability, is difficult to short-term simultaneously and obtains mature C ML-DC.And incubation time prolongs, and then cell survival rate obviously reduces.
Radix Angelicae Sinensis polysaccharide (APS) is the enrich blood important effective constituent of key medicine Radix Angelicae Sinensis of traditional Chinese medicine and pharmacy.APS has promoter action to hematopoiesis, and can break up by inducing leukemia cell strain K562; APS also can raise immunity, can strengthen the phagocytic function of mononuclear macrophage.We are on the basis that in the past adds IL-4, GM-CSF inducing culture CML-DC, add the APS cultivation again and induce CML-DC, the result shows: ability of cell proliferation and surviving rate with do not add the APS group and obviously improve, be that 100ug/ml, cultivation were best in the time of 3-5 days with APS concentration.Cell phenotype is analyzed: add cell CD83, CD86, CD80, HLA-DR expression ratio showed increased that the APS group was cultivated the 1st day, and be elevated to the peak in the time of 3-5 days.The mixed lymphocyte reacion prompting adds the CML-DC that the APS group induces, and stimulates the lymphopoiesis ability to be better than and does not add the APS group.This result of study proves: APS can promote inducing of CML-DC and maturation, and the expression that can improve the antigen costimulatory molecules, strengthens the ability of CML-DC activated T cell.
Embodiment 11,
1,2% Radix Angelicae Sinensis polysaccharide injection normal mouse and (60 irradiations of 4Gy cobalt) anemia mice are got and are respectively organized the mouse bone marrow cells cultivation, observe the influence that Radix Angelicae Sinensis polysaccharide generates monokaryon-scavenger cell.
The injection Radix Angelicae Sinensis polysaccharide is to the influence (/ 5 * 10 of grain-unicellular granulocyte colony formation
4BMC)
Group n control group (the n Radix Angelicae Sinensis polysaccharide group of X ± the S) (P of X ± S)
Normal mice 20 108.7 ± 16.0 20 146.7 ± 15.7＜0.01
Anaemia mouse 15 17.5 ± 1.6 14 39.5 ± 3.8＜0.01
The proof Radix Angelicae Sinensis polysaccharide can promote granulocyte, monocyte to generate.
2, Radix Angelicae Sinensis polysaccharide promotes proliferation of bone marrow cells (mtt assay)
Radix Angelicae Sinensis polysaccharide concentration/mgmL-1
Divide into groups 1 0.5 0.25 0
Control group (solubilizing agent) 0.107 ± 0.007
Radix Angelicae Sinensis polysaccharide group 0.767 ± 0.501 0.697 ± 0.546 0.842 ± 0.601
3, Radix Angelicae Sinensis polysaccharide is to the synthetic influence of bone marrow nucleated cell DNA (3H-TdR labelling method)
Drug level
Divide into groups 1 0.5 0.25 0
Control group (solubilizing agent) 80.41 ± 48.21
Radix Angelicae Sinensis polysaccharide group 881.87 ± 376.23 981.39 ± 481.05 686.03 ± 193.22
4, Radix Angelicae Sinensis polysaccharide is to the influence of mouse dcs.
Behind the 5% Radix Angelicae Sinensis polysaccharide subcutaneous injection normal mouse, control experiment shows:
1. spleen weight increases; 2. acini lienalis germinal center habituation; 3. dendritic cell increases; 4. abdominal cavity monokaryon-scavenger cell significantly strengthens the chicken red blood cell phagocytic activity.Embodiment 11, below be one group of technical parameter:
Radix Angelicae Sinensis polysaccharide concentration is 2ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 1000ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 1500ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 800ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 50ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 25ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 400ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 200ug/ml.All the other are with embodiment 10.
Radix Angelicae Sinensis polysaccharide concentration is 500ug/ml.All the other are with embodiment 10.
Add 5% serum free medium serum at thing in the IMDM substratum, wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, and human transferrin, cholesterol, Regular Insulin is formed.All the other are with embodiment 10.
Add 15% serum free medium serum at thing in the IMDM substratum, wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, and human transferrin, cholesterol, Regular Insulin is formed.All the other are with embodiment 10.
Add 12% serum free medium serum at thing in the IMDM substratum, wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, and human transferrin, cholesterol, Regular Insulin is formed.All the other are with embodiment 10.
Leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 1 day.All the other are with embodiment 10.
Leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and adjusting cell concn is 5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 3 days.All the other are with embodiment 10.
Leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and adjusting cell concn is 3 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 300ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 10 days.All the other are with embodiment 10.
Leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50-400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 4 days.All the other are with embodiment 10.
Leukaemic's anti-freezing marrow separates mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50-400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 5 days.All the other are with embodiment 10.
Claims (9)
1, the leukemia cell induces the substratum that generates dendritic cell, it is characterized in that: add Radix Angelicae Sinensis polysaccharide in leukemia cell's serum free medium of routine.
2, leukemia cell according to claim 1 induces the substratum that generates dendritic cell, and it is characterized in that: Radix Angelicae Sinensis polysaccharide concentration is 10-800ug/ml.
3, leukemia cell according to claim 1 and 2 induces the substratum that generates dendritic cell, and it is characterized in that: Radix Angelicae Sinensis polysaccharide concentration is 50-400ug/ml.
4, leukemia cell according to claim 1 induces the substratum that generates dendritic cell, it is characterized in that: the serum free medium serum that adds 5-15% in the IMDM substratum replaces at thing, wherein serum free medium serum is for will be by bovine serum albumin the owner, catalase, human transferrin, cholesterol, Regular Insulin is formed.
5, leukemia cell according to claim 1 induces the substratum that generates dendritic cell, it is characterized in that: the concentration of using phosphate buffered saline buffer allotment Radix Angelicae Sinensis polysaccharide.
6, leukemia cell according to claim 1 induces the cell culture processes of the substratum that generates dendritic cell, it is characterized in that: leukaemic's anti-freezing marrow, separate mononuclearcell through lymphocyte separation medium, and the adjustment cell concn is 0.5-5 * 10
6/ ml is suspended in the IMDM substratum, and adding final concentration is the Radix Angelicae Sinensis polysaccharide of 50-400ug/ml, places conventional cell culture incubator to cultivate, and routine is changed liquid and conventional fresh culture and the corresponding cytokine of replenishing, and cultivates 1-10 days.
7, the leukemia cell who states according to claim 6 induces the cell culture processes of the substratum that generates dendritic cell, it is characterized in that: place the cell culture incubator of 37 ℃ of routines, 5%CO2 to cultivate, half amount is changed liquid 2 times weekly, additional fresh culture and corresponding cytokine were cultivated 3-5 days.
8, the leukemia cell who states according to claim 6 induces the cell culture processes of the substratum that generates dendritic cell, it is characterized in that: add TNF-α after 10 days of cultivation, continue to cultivate collecting cell 3 days.
9, the leukemia cell who states according to claim 6 induces the substratum that generates dendritic cell to be used for leukemia cell, dendritic cell analyzing and testing.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03118492 CN1264974C (en) | 2003-01-17 | 2003-01-17 | Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 03118492 CN1264974C (en) | 2003-01-17 | 2003-01-17 | Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1517435A true CN1517435A (en) | 2004-08-04 |
| CN1264974C CN1264974C (en) | 2006-07-19 |
Family
ID=34284900
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 03118492 Expired - Fee Related CN1264974C (en) | 2003-01-17 | 2003-01-17 | Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1264974C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101155914B (en) * | 2005-04-08 | 2013-03-06 | 阿戈斯治疗公司 | Dendritic cell compositions and methods |
| CN105219712A (en) * | 2015-11-16 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of NKT cell culture medium and NKT cell culture processes |
| CN112029724A (en) * | 2020-09-17 | 2020-12-04 | 和泓尚医(成都)生物科技有限公司 | In-vitro culture method for accelerating dendritic cell maturation and application thereof |
| CN114517181A (en) * | 2021-12-26 | 2022-05-20 | 大连博格林生物科技有限公司 | Serum-free culture medium for human T lymphocytes and preparation method and application thereof |
-
2003
- 2003-01-17 CN CN 03118492 patent/CN1264974C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101155914B (en) * | 2005-04-08 | 2013-03-06 | 阿戈斯治疗公司 | Dendritic cell compositions and methods |
| CN105219712A (en) * | 2015-11-16 | 2016-01-06 | 广州赛莱拉干细胞科技股份有限公司 | A kind of NKT cell culture medium and NKT cell culture processes |
| CN112029724A (en) * | 2020-09-17 | 2020-12-04 | 和泓尚医(成都)生物科技有限公司 | In-vitro culture method for accelerating dendritic cell maturation and application thereof |
| CN114517181A (en) * | 2021-12-26 | 2022-05-20 | 大连博格林生物科技有限公司 | Serum-free culture medium for human T lymphocytes and preparation method and application thereof |
| CN114517181B (en) * | 2021-12-26 | 2023-11-24 | 大连博格林生物科技有限公司 | Serum-free medium for human T lymphocyte, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1264974C (en) | 2006-07-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wu et al. | Development of dendritic cell system | |
| CN1784488A (en) | Generation of dendritic cells from monocytic dendritic precursor cells with GM-CSF in the absence of additional cytokines | |
| CN105062968B (en) | A kind of DC-CIK cell culture reagent and its cultural method | |
| CN1541113A (en) | Maturation of antigen-presenting cells using activated T cells | |
| CN1346403A (en) | Method and apparatus or maintenance and expansion of hemopoietic stem cells and/or progenitor cells | |
| CN1452655A (en) | Method of culturing mesenchymal stem cells | |
| EP3770251A1 (en) | Method for producing natural killer cells | |
| CN104815323B (en) | A kind of dendritic cell tumor vaccine and preparation method thereof | |
| CN1282485C (en) | Dendron shaped cell tumour vaccine for loading withered heat shock tumour cell, its preparation method and application | |
| CN1990044A (en) | Preparation and use of human body immunocompetent cell DCCIK anti-tumor cell preparation | |
| CN1264974C (en) | Culture media for leukemia cell inducel production of dendrite cell and its culturing method and application | |
| AU2020103625A4 (en) | Applications of bacillus calmette-guerin (bcg) polysaccharide and nucleic acid in in-vitro culture of cytokine-induced-killer (cik) cells and preparation of antitumor drugs | |
| CN1703506A (en) | Procedure for the large-scale t-lymphocytes culture in a homogeneous system | |
| CN115896016B (en) | Culture composition and application thereof in culturing immune cells | |
| CN1553812A (en) | Autologous T-cell vaccines materials and method | |
| CN1844149A (en) | Monoclonal antibody against human 4-1BBL and its use | |
| CN1706495A (en) | New type of cell vaccine with heteroimmune cell as cell vector and its prepn process | |
| CN105039254B (en) | A kind of immunocyte and preparation method thereof | |
| CN1313151C (en) | Preparation of high-performance noncellular vaccine and usage | |
| CN105219723A (en) | A kind of test kit for activating cancer of the stomach specific immune response | |
| CN105219727A (en) | A kind of test kit for activating colorectal cancer specific immune response | |
| CN1609195A (en) | Dendritic cell culturing method and kit | |
| CN114790445B (en) | Preparation method and application of CD4-CD8-NKT cell | |
| CN103602634A (en) | Preparation method of DCs, and application of DCs in preparing anti-tumor cell preparation | |
| CN106148281A (en) | A kind of preparation method of Tlr7a induction-enhanced DC NK cell |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |