CN103589685B - A kind of fast preparation method of DC cell - Google Patents
A kind of fast preparation method of DC cell Download PDFInfo
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- CN103589685B CN103589685B CN201310610764.8A CN201310610764A CN103589685B CN 103589685 B CN103589685 B CN 103589685B CN 201310610764 A CN201310610764 A CN 201310610764A CN 103589685 B CN103589685 B CN 103589685B
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Abstract
The present invention relates to cell preparation field, specifically a kind of fast preparation method of DC cell, preparation time compared with the existing technology, is foreshortened to 3 days in 7-9 days by prior art by the present invention; The vigor of DC cell is increased to 88-95% by the 68-75% of prior art, and purity is increased to 80-90% by the 60-65% of prior art; The DC cell adopting the inventive method to prepare has fully matured phenotype, expresses Chemokines CC CR7, and secretion high density IL-12 cytokine also effectively promotes T cell propagation.
Description
Technical field
The present invention relates to cell preparation field, specifically a kind of fast preparation method of DC cell.
Background technology
DC cell, be dendritic cell (Dendritic Cell), it is the professional antigen presenting cells that body immune system function is the most powerful, by picked-up, processing treatment and offer antigen, start antigen-specific immune response, resist in virus infection, tumor invasion at body immune system and play keying action.In recent years, domestic and international multiple research team utilizes the diseases such as antigen-specific DC vaccine therapy tumour all to obtain challenging progress, and part enters clinical experimental stage.
But DC cell quantity is rare, only accounts for 1% of human peripheral blood single nucleus cell, and DC vaccine needs a large amount of ripe DC cell, only allow by being separated autologous patient monocyte at present, further external evoked amplification obtains.Therefore, efficiently a large amount of obtain ripe and the DC cell with function becomes the successful decisive link of antigen-specific DC vaccine therapy disease.
Conventional DC cells in vitro induced amplification scheme is utilize cytokine: GM-CSF and IL-4, differentiation-inducing through 5 ~ 7 days, and and then again through the induced maturation of 2 days, thus obtain ripe and have the DC cell of function, total duration of cultivating was at 7 ~ 9 days.The problems such as conventional DC cells in vitro induced amplification scheme existence operation length consuming time, cell purity are low, cell function disappearance, cause the industrialization of DC vaccine, mass production process slowly, and then are difficult to apply.So, find low, that time-histories the is short functional DC cells in vitro induced amplification mode of cost and seem particularly urgent.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, provide the fast preparation method of the DC cell that a kind of induction time is short, cell purity is high, function is strong.
In order to achieve the above object, the present invention is a kind of fast preparation method of DC cell, it is characterized in that: complete following steps successively: step one: in 3 milliliters of lymphocyte separation mediums, add 5 milliliters of whole bloods with the speed of per minute 1 milliliter, lymphocyte separation medium liquid level is kept not broken through, under room temperature state, adopt 300g centrifugal force, centrifugal 20 minutes, speed-raising and all not strap brakes that slows down in centrifugal process; Step 2: draw cloud and mist layer with dropper, adds 30 milliliters, the physiological saline washing of 0.9% once, under room temperature state, adopts 300g centrifugal force, centrifugal 5 minutes, abandon supernatant; Step 3: with the sorting damping fluid re-suspended cell of 30 milliliters of precoolings, counting cells number, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant; Step 4: every 1 х 10
7individual cell adds the sorting damping fluid of 30 microlitre precoolings, 10 microlitre Fc receptor blocking reagent and 10 microlitre biotin antibody mixed solutions, and mix, after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 10 minutes; Step 5: every 1 х 10
7individual cell adds sorting damping fluid and the micro-magnetic bead of 20 microlitre antibiotin of 30 microlitre precoolings, mixes, and after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 15 minutes; Step 6: the sorting damping fluid adding the precooling of 1-2 microlitre, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant, add the sorting damping fluid re-suspended cell of 500 microlitre precoolings; Step 7: MS post is placed on magnet stand, adds the sorting damping fluid of 500 microlitre precoolings, after liquid drains off completely, adds 500 l of cell suspension, and collect the cell flowed out, the cell of this outflow is the monocyte of CD14+; Step 8: after liquid drains off completely, post washed by the sorting damping fluid adding 500 microlitre precoolings, repeats twice; Step 9: the cell quantity of counting collection, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant; Step 10: regulate cell concn to 0.5-0.6x10 with DC cell culture medium
6individual/milliliter, spreads in 6 orifice plates by cell suspension, 3 milliliters, every hole; Step 11: 6 orifice plates being placed in temperature is 37 degrees Celsius, and gas concentration lwevel is in the incubator of 5%, cultivated after 48 hours, supplemented the hybrid cytokine adding induction DC cell maturation; Step 12: continue cultivation after 24 hours, detect cell quantity, cell viability, cell phenotype and function.
In described step 2, described cloud and mist layer is mononuclearcell layer.
In described step 10, described DC cell culture medium is that GT551 serum free medium adds 1000 U/mL granulocyte-macrophage colony stimutaing factors and 500 U/mL interleukin-4s.
In described step 11, the hybrid cytokine of described induction DC cell maturation is 1000 U/mL tumour necrosis factors, 10000 U/mL interleukin 1,1000 U/mL Interferon, rabbit r, 1 micromoles per liter prostaglandin E2,500 ng/mL CD40L, 10 ng/mL lipopolysaccharides, 2.5 mcg/ml R848.
Preparation time compared with the existing technology, is foreshortened to 3 days in 7-9 days by prior art by the present invention; The vigor of DC cell is increased to 88-95% by the 68-75% of prior art, and purity is increased to 80-90% by the 60-65% of prior art; The DC cell adopting the inventive method to prepare has fully matured phenotype, expresses Chemokines CC CR7, and secretion high density IL-12 cytokine also effectively promotes T cell propagation.
Accompanying drawing explanation
Fig. 1 is MHC II quasi-molecule and the costimulatory molecules expression level comparison diagram of DC dendritic cell prepared by the present invention and prior art.
Fig. 2 is the Chemokine Receptors CCR7 expression level comparison diagram of DC dendritic cell prepared by the present invention and prior art.
Fig. 3 is the horizontal comparison diagram of secreting leukocytes mesonium 12 of DC dendritic cell prepared by the present invention and prior art.
Fig. 4 is the comparison diagram of DC Dendritic Cells Induced T lymphopoiesis ability prepared by the present invention and prior art.
Embodiment
Now the present invention is described further.
The present invention is a kind of fast preparation method of DC cell, it is characterized in that: complete following steps successively: step one: in 3 milliliters of lymphocyte separation mediums, add 5 milliliters of whole bloods with the speed of per minute 1 milliliter, lymphocyte separation medium liquid level is kept not broken through, under room temperature state, adopt 300g centrifugal force, centrifugal 20 minutes, speed-raising and all not strap brakes that slows down in centrifugal process.
Step 2: slowly draw cloud and mist layer with dropper, when drawing cloud and mist layer, does not bring a lot of parting liquid into, cloud and mist layer is mononuclearcell layer, adds 30 milliliters, the physiological saline washing of 0.9% once, under room temperature state, adopt 300g centrifugal force, centrifugal 5 minutes, abandon supernatant.
Step 3: with the sorting damping fluid re-suspended cell of 30 milliliters of precoolings, counting cells number, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant.
Step 4: every 1 х 10
7individual cell adds the sorting damping fluid of 30 microlitre precoolings, 10 microlitre Fc receptor blocking reagent and 10 microlitre biotin antibody mixed solutions, and mix, after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 10 minutes.
Step 5: every 1 х 10
7individual cell adds sorting damping fluid and the micro-magnetic bead of 20 microlitre antibiotin of 30 microlitre precoolings, mixes, and after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 15 minutes.
Step 6: the sorting damping fluid adding the precooling of 1-2 microlitre, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant, add the sorting damping fluid re-suspended cell of 500 microlitre precoolings.
Step 7: MS post is placed on magnet stand, adds the sorting damping fluid of 500 microlitre precoolings, after liquid drains off completely, adds 500 l of cell suspension, and collect the cell flowed out, the cell of this outflow is the monocyte of CD14+.
Step 8: after liquid drains off completely, post washed by the sorting damping fluid adding 500 microlitre precoolings, repeats twice.
Step 9: the cell quantity of counting collection, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant.
Step 10: regulate cell concn to 0.5-0.6x10 with DC cell culture medium
6individual/milliliter, cell suspension is spread in 6 orifice plates, 3 milliliters, every hole, DC cell culture medium is that GT551 serum free medium that precious biotechnology (Dalian) company limited produces adds granulocyte-macrophage colony stimutaing factor that 1000 U/mL send Portec Inc. to produce and the interleukin-4 that 500 U/mL send Portec Inc. to produce.
Step 11: 6 orifice plates being placed in temperature is 37 degrees Celsius, and gas concentration lwevel is in the incubator of 5%, cultivated after 48 hours, supplemented the hybrid cytokine adding induction DC cell maturation.A kind of part of the hybrid cytokine of described induction DC cell maturation to be prostaglandin E2,500 ng/mL CD40L, 10 ng/mL lipopolysaccharides, 2.5 mcg/ml R848, R848 that Interferon, rabbit r, 1 micromoles per liter Sigma-Aldrich that interleukin 1,1000 U/mL that tumour necrosis factor, 10000 U/mL that 1000 U/mL send Portec Inc. to produce send Portec Inc. to produce send Portec Inc. to produce produce be TLR7/8.
Step 12: continue cultivation after 24 hours, detect cell quantity, cell viability, cell phenotype and function.
In the present invention, lymphocyte separation medium is that Chemical Reagent Co., Ltd., Sinopharm Group produces, and using monocyte sorting test kit in the 4th, the 5th step is that German Mei Tian Ni company produces, and sorting damping fluid is that German Mei Tian Ni company produces.
Conventional DC cell preparation needs 7-9 days, and the present invention prepares DC cell only needs 3 days, and experimental period shortens greatly.
The usual vigor of DC cell prepared by ordinary method is 68-75% and purity is 60-65%, and vigor and purity are all lower, and DC cell viability prepared by the present invention is 88-95% and purity is 80-90%, and vigor and purity all significantly improve.
See Fig. 1, wherein left side one is classified as Isotype control figure, centre one is classified as MHC II quasi-molecule and the costimulatory molecules expression level figure of conventional DC cell culture method, right side one is classified as MHC II quasi-molecule and the costimulatory molecules expression level figure of DC cell culture method of the present invention, compared with conventional DC cell preparation method, DC cell prepared by the present invention has fully matured phenotype
See Fig. 2, wherein left side is Isotype control figure, centre is the Chemokine Receptors CCR7 expression level figure of conventional DC cell culture method, right side is the Chemokine Receptors CCR7 expression level figure of DC cell culture method of the present invention, compared with conventional DC cell preparation method, DC cell expressing Chemokines CC CR7 prepared by the present invention.
See Fig. 3, the horizontal comparison diagram of secreting leukocytes mesonium 12, wherein left side is secreting leukocytes mesonium 12 level of conventional DC cell culture method, right side is secreting leukocytes mesonium 12 level of DC cell culture method of the present invention, compared with conventional DC cell preparation method, DC emiocytosis high density interleukin 12 cytokine prepared by the present invention.
See Fig. 4, the DC Dendritic Cells Induced T lymphopoiesis that wherein the first behavior adopts conventional DC cell culture method to prepare the 4th day can be tried hard to, the DC Dendritic Cells Induced T lymphopoiesis that second behavior DC cell culture method of the present invention is prepared the 4th day can be tried hard to, the third line is that the DC Dendritic Cells Induced T lymphopoiesis adopting conventional DC cell culture method to prepare the 6th day can be tried hard to, fourth line is that the DC Dendritic Cells Induced T lymphopoiesis that DC cell culture method of the present invention is prepared the 6th day can be tried hard to, compared with conventional DC cell preparation method, the effective inducer T lymphocyte propagation of DC cell prepared by the present invention.
Claims (2)
1. the fast preparation method of a DC cell, it is characterized in that: complete following steps successively: step one: in 3 milliliters of lymphocyte separation mediums, add 5 milliliters of whole bloods with the speed of per minute 1 milliliter, lymphocyte separation medium liquid level is kept not broken through, under room temperature state, adopt 300g centrifugal force, centrifugal 20 minutes, speed-raising and all not strap brakes that slows down in centrifugal process; Step 2: draw cloud and mist layer with dropper, adds 30 milliliters, the physiological saline washing of 0.9% once, under room temperature state, adopts 300g centrifugal force, centrifugal 5 minutes, abandon supernatant; Step 3: with the sorting damping fluid re-suspended cell of 30 milliliters of precoolings, counting cells number, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant; Step 4: every 1 х 10
7individual cell adds the sorting damping fluid of 30 microlitre precoolings, 10 microlitre Fc receptor blocking reagent and 10 microlitre biotin antibody mixed solutions, and mix, after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 10 minutes; Step 5: every 1 х 10
7individual cell adds sorting damping fluid and the micro-magnetic bead of 20 microlitre antibiotin of 30 microlitre precoolings, mixes, and after mixing, being placed in temperature is in the refrigerator of 4 degrees Celsius, takes out after 15 minutes; Step 6: the sorting damping fluid adding the precooling of 1-2 microlitre, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant, add the sorting damping fluid re-suspended cell of 500 microlitre precoolings; Step 7: MS post is placed on magnet stand, adds the sorting damping fluid of 500 microlitre precoolings, after liquid drains off completely, adds 500 l of cell suspension, and collect the cell flowed out, the cell of this outflow is the monocyte of CD14+; Step 8: after liquid drains off completely, post washed by the sorting damping fluid adding 500 microlitre precoolings, repeats twice; Step 9: the cell quantity of counting collection, under 4 Celsius Conditions, adopts 300g centrifugal force, centrifugal 10 minutes, abandons supernatant; Step 10: regulate cell concn to 0.5-0.6x10 with DC cell culture medium
6individual/milliliter, spreads in 6 orifice plates by cell suspension, 3 milliliters, every hole; Step 11: 6 orifice plates being placed in temperature is 37 degrees Celsius, and gas concentration lwevel is in the incubator of 5%, cultivated after 48 hours, supplemented the hybrid cytokine adding induction DC cell maturation; Step 12: continue cultivation after 24 hours, detect cell quantity, cell viability, cell phenotype and function,
In described step 10, described DC cell culture medium is that GT551 serum free medium adds 1000 U/mL granulocyte-macrophage colony stimutaing factors and 500 U/mL interleukin-4s,
In described step 11, the hybrid cytokine of described induction DC cell maturation is 1000 U/mL tumour necrosis factors, 10000 U/mL interleukin 1,1000 U/mL Interferon, rabbit r, 1 micromoles per liter prostaglandin E2,500 ng/mL CD40L, 10 ng/mL lipopolysaccharides LPS, 2.5 mcg/ml R848.
2. the fast preparation method of a kind of DC cell according to claim 1, is characterized in that: in described step 2, and described cloud and mist layer is mononuclearcell layer.
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| CN104998260A (en) * | 2015-07-08 | 2015-10-28 | 深圳爱生再生医学科技有限公司 | DC cell-based HPV virus vaccine preparation method |
| CN105734016A (en) * | 2016-03-14 | 2016-07-06 | 上海安集协康生物技术股份有限公司 | DC (dendritic cell) preparation method |
| CN105861438A (en) * | 2016-05-19 | 2016-08-17 | 上海君微生物科技有限公司 | Improvement method for rapidly preparing dendritic cells |
| CN106011063A (en) * | 2016-06-24 | 2016-10-12 | 安徽未名细胞治疗有限公司 | DC cell culture medium and preparing method thereof |
| CN107299083A (en) * | 2017-08-29 | 2017-10-27 | 上海莱馥医疗科技有限公司 | A kind of method that efficient high-purity prepares maturation DC cells |
| 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 |
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| CN102210861B (en) * | 2011-01-13 | 2013-09-04 | 中国人民解放军第四军医大学 | Multi-epitope peptide-loaded DC (dendritic cell) therapeutic vaccine for HCV (hepatitis C viruses) |
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Effective date of registration: 20160711 Address after: 200023, room 6, No. 39, Lane five, 202 Li Qiao Road, Shanghai, Huangpu District Patentee after: Shanghai Junwei Biotechnology Co., Ltd. Address before: 200032, room 13, building 805, West 130, Dongan Road, Shanghai, Xuhui District Patentee before: Fudan University |