CN103981144A - Preparation method for autologous-serum antigen-sensitized DC-CIK cells - Google Patents
Preparation method for autologous-serum antigen-sensitized DC-CIK cells Download PDFInfo
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Abstract
The invention discloses a preparation method for autologous-serum antigen-sensitized DC-CIK cells. The preparation method for the autologous-serum antigen-sensitized DC-CIK cells comprises the steps: A) preparation of an autologous-serum antigen; B) preparation of peripheral blood mononuclear cells; C) separation and culture of DC; D) induction amplification of CIK cells; and E) preparation of the DC-CIK cells through co-culture of DC cells and CIK cells. Compared with the DC-CIK cells prepared by employing a routine culture method, the DC-CIK cells cultured by employing the method are obviously increased in CIK proliferation activity and tumoricidal activity; the average proliferation multiple is 200-500 times and is 2-3 times of that of the DC-CIK cells cultured by employing the routine culture method; and the tumoricidal activity is 70-80% when an in-vitro experiment is performed, and is obviously higher than that of the DC-CIK cells cultured by employing the routine culture method. The method is simple in operation, easily controllable in conditions and relatively low in equipment requirements, and the cell proliferation efficiency of the obtained DC-CIK cells is relatively high.
Description
Technical field
The invention belongs to cytobiology, immunotherapy of tumors field, relate to preparation method and the application of antitumor adoptive immunotherapy of using autologous tumor antigen sensibilization DC-CIK.
Background technology
Malignant tumour has become first of the Chinese cause of death, the people's health and life in serious threat, immunotherapy of tumors is the clinical the fourth-largest therapy for oncotherapy, wherein tumour adoptive immunity cell therapy is widely used in China, and at present domestic to carry out maximum be exactly that adopting property of DC-CIK cell feeds back therapy.
DC cell is most important antigen presenting cell in body, and by antigen uptake, process and offer, DC cell is inducing antigen-specific CD4 and CD8 T lymphocyte activation effectively, thus activation body the acquired immune response.In addition, DC can activate NK cell equally, strengthens its multiplication capacity and lethal effect, thereby strengthens innate immune reaction, jointly brings into play antitumor action.
CIK cell is cytokine-induced killer cell, be by human peripheral blood single nucleus cell in vitro with cytokine profiles co-cultivation for some time after the restrictive cytotoxic T lymphocyte of non-major histocompatibility complex (MHC) that obtains, tumour cell is had to efficient dissolving toxicity, after DC and CIK co-culture of cells, multiplication capacity and the killing activity of CIK can have effectively been improved, more being conducive to improve the antineoplastic immune curative effect of DC-CIK, is also current domestic application technology the most widely.
DC-CIK mainly comprises DC-CIK, the DC-CIK of tumour specific antigen sensitization and the DC-CIK of full tumour antigen sensitization without antigen sensibilization.Research shows, through the DC of antigen sensibilization and all can be so that the multiplication capacity of CIK and kill tumor activity and raise without the DC of antigen sensibilization, and can better work in coordination with through the DC of antigen sensibilization propagation and the activation that stimulates CIK cell, has and better kills tumor activity.Therefore, select suitable antigen sensibilization DC to become a key influencing factor of DC-CIK curative effect.
The antigen that the external sensitization of DC is used is at present mainly known tumour specific antigen, tumor cell line split product or tumor specimen split product.Due to the shortage specific antigens of most of tumours, and tumour easily suddenlys change and then resists the immune attack of single antigen, so the DC clinical application of specific antigens sensitization is very limited.And tumor cell line is in culturing process; surface antigen can change conventionally; the DC-CIK clinical effectiveness obtaining after sensitization DC is not good; and tumor specimen split product can only be for the patient of specimen taken before performing the operation; the patient who carries out DC-CIK treatment in clinical practice is mostly postoperative patient; be difficult to obtain fresh specimens from pri, these have all limited the clinical effectiveness of DC-CIK greatly.And due to the individual difference of tumour patient, even the tumour of same kind, the tumour antigen having also differs widely, the antigen of synthetic can not be widely used in the treatment of tumour patient.
Conventional protein compression technology comprises dialysis tubing method of enrichment at present, lyophilize method of enrichment, dry up method of enrichment, ultra-filtration membrane method of enrichment, chemical precipitation method etc., but ultra-filtration membrane method of enrichment and dialysis tubing method of enrichment are more advanced, say that the cost needing is higher, be unfavorable for promoting, and additive method easily causes the pollution of serum mostly, be difficult to use in the cultivation of cell, we can make serum chemistry become the concentrated principle of fractional precipitation based on freeze thawing, do not affecting on the basis of serum antigen activity, find practicable autoserum antigen preparation method, and and then with these serum, carry out sensitization DC, cultivate specific DC-CIK cell after antigen sensibilization, for clinical.
Summary of the invention
The object of the invention is to overcome the above-mentioned deficiency of prior art, provide a kind of and can obtain high proliferation activity and kill the preparation method of the self antigen sensitization DC-CIK cell of tumor activity.
The preparation method of autoserum antigen sensibilization DC-CIK cell, comprises the following steps:
The preparation of autoserum antigen: peripheral blood 50-100ml, centrifugation patients serum, adding final concentration is the bright proteinase inhibitor that presses down of 0.1-1.0mg/ml, and mixes, subsequently with aseptic centrifuge tube storage, be placed in-20 ℃ of following freezing 24-48h of environment, after taking-up, in 0-4 ℃ of environment, place 10-16h, draw upper plasma part, and discard, deactivation 20-30min, filtering with microporous membrane, obtains autoserum tumour antigen;
Preferred: after described separation of serum, the albumen treatment condition of albumen after concentrated be-20 ℃ of freezing 48h, vertically place subsequently 16h in 4 ℃ of environment, can be by gravity, help protein concentrate; Described deactivation condition is 56 ℃ of deactivation Complement component 3 0min; Described millipore filtration is that aperture is 22 μ m and following millipore filtration.
The preparation of peripheral blood mononuclear cell: adopt the separated peripheral blood mononuclear cell (PBMC) that obtains of density gradient centrifugation.Adopt the AIM-V serum free medium separated PBMC that suspends, according to 0.5-1 * 10
8the quantity of/bottle is placed in culturing bottle, and standing 1-3h in cell culture incubator draws not adherent lymphocyte for the cultivation of CIK cell; Attached cell is for the cultivation of DC.
Separated and the cultivation of DC: according to tumor markers measurement result, after cell attachment, adding final concentration is that the autoserum of 5-20% carries out DC sensitization, at cultivation 6-, within the 7th day, adding final concentration is the tumour necrosis factor of 200-1000ng/ml, obtains the DC after autoantigen sensitization.
The induction amplification of CIK cell: above-mentioned not attached cell carries out according to CIK cell cultures conventional scheme.
DC and CIK co-culture of cells are prepared DC-CIK: by the DC after above-mentioned antigen sensibilization and aforementioned CIK cell co-cultivation 5-8 days, and results self antigen sensitization DC-CIK cell.
Cell forms: CD3+ cell accounts for more than 90%, and wherein CD3+CD4+ cell is about 10-20%, and CD3+CD8+ cell is at 60-80%, and CD3+CD56+ cell is about 10-20%.
Another object of the present invention is to preparation can be for clinical antitumor adoptive immunity active cells by aforesaid method preparation.
The DC-CIK that the DC-CIK of aforesaid method cultivation is prepared with cellar culture compares, and it is to the proliferation activity of CIK and kill all obviously increases of tumor activity, and its propagation multiple has on average reached 200-250 doubly, is 2-3 times of traditional cultural method; During experiment in vitro, kill tumor activity and reach 70-80%, the DC-CIK cell of cultivating apparently higher than ordinary method.Use aforesaid method, adopt antigen in autoserum as my specific tumour antigen, to compare with single tumour specific antigen, effectively avoid because of the resistant function of tumour cell sudden change to specific antigens; With tumor cell line antigen, compare, serum antigen is self antigen, the change of the antigenic structure of effectively having avoided cell strain to go down to posterity in vitro causing in process; In the face of hand postoperative patient or be difficult to obtain the patient of tumor specimen, effectively avoided the awkward situation that can use without tumour antigen.In the process of preparing at tumour antigen, appropriate proteinase inhibitor has effectively kept the integrity of serum tumor antigen; In addition, present method operation is simple, and condition is easily controlled, lower to the requirement of equipment, and obtain DC-CIK cell proliferation efficiency higher.
Accompanying drawing explanation
Fig. 1 is serum freezing time and the impact (mg/ml) of temperature on protein content after concentrated;
Fig. 2 is the impact of serum dissolution time on protein content after concentrated;
Fig. 3 is the cell growth curve in culturing process;
Fig. 4 is culturing cell flow cytometer detection result; CD3+ cell accounts for more than 90%, and wherein CD3+CD4+ cell is about 10-20%, and CD3+CD8+ cell is at 60-80%, and CD3+CD56+ cell is about 10-20%.
Fig. 5 is the killing effect in vitro of cultured cells to A549 cell.
Embodiment
With example, the present invention is described below, but the present invention is not limited.The experimental technique of all unreceipted actual conditionses in example below, is the method for observing a usual practice and producer's operation instructions is carried out.
The conventional Patients with Advanced Lung Cancer peripheral blood 50-100ml that gathers, the heparin sodium anti-freezing of 5-15IU/ml, rotating speed 800g, centrifugal 10 minutes, collect upper plasma part (approximately 1/3 liquid level), adding final concentration is the bright proteinase inhibitor that presses down of 0.1-1.0mg/ml, mix, with aseptic centrifuge tube storage, be placed in-20 ℃ of following freezing 24-48h(of environment referring to Fig. 1 subsequently), after taking-up, in 0-4 ℃ of environment, place 10-16h(referring to Fig. 2); 56 ℃ of deactivation 30min, 0.22 μ m filtering with microporous membrane, obtains autoserum tumour antigen.
The separated PBMC of density gradient centrifugation, the hemocyte of the doubly dilution precipitation such as physiological saline, the blood of human lymphocyte parting liquid and dilution adds in centrifuge tube according to the ratio of 1:2, rotating speed 700g, centrifugal 20 minutes, carefully draws tunica albuginea layer, with physiological saline washing 2 times, 1500 revs/min, centrifugal 8 minutes, obtain peripheral blood mononuclear cell.
Adopt the AIM-V serum free medium separated PBMC that suspends, according to 1 * 10
8the quantity of/bottle is placed in 75cm
2culturing bottle in, standing 2h in cell culture incubator, rocks culturing bottle gently, draws not adherent lymphocyte for the cultivation of CIK cell, attached cell is for the cultivation of DC.
DC cultivates and sensitization: according to shown in table 1, and high and low, middle expression 3 classes that antigen is divided into.
The classification of table 1 antigen presentation
| Tumour standard substance measurement result sum R | High antigen presentation group T | Middle antigen presentation group | Low antigen presentation group |
| R | T >=10 times normal value | 10 times of 5 times of normal value≤T < | 5 times of normal values of T < |
| Sensitization serum-concentration | 5%-10% | 10%-15% | 15%-20% |
The tumour standard substance result that the present embodiment is used: the normal 0-3.8 ng/ml of CEA 149.7ng/ml(), the normal 0-35 U/ml of CA125 16.04U/ml(), the normal 0-27 U/ml of CA199 22.87 U/ml(), tumor markers measurement result is " surpassing 10 times of normal values ", in the 3rd of attached cell cultivation, within 5 days, adding respectively final concentration is 5-20%, the autoserum that is preferably 5-10% carries out DC sensitization (referring to Fig. 3), in cultivation, within the 7th day, adding final concentration is the tumour necrosis factor of 200-1000ng/ml, the actual working concentration of this patient is 800ng/ml, obtain the ripe DC after autoantigen sensitization.
The induction amplification of CIK cell: above-mentioned not attached cell carries out according to CIK cell cultures conventional scheme.
DC and CIK co-culture of cells are prepared DC-CIK: by the DC after above-mentioned antigen sensibilization and aforementioned CIK cell co-cultivation, and about 7 days, results self antigen sensitization DC-CIK cell (referring to Fig. 3).
The DC-CIK preparing with cellar culture compares, and it is to the proliferation activity of CIK and kill all obviously increases of tumor activity, and its propagation multiple has on average reached 200-250 doubly, is 2-3 times of traditional cultural method; During experiment in vitro, kill tumor activity and reach 70-80%, the DC-CIK cell of cultivating apparently higher than ordinary method.
Use aforesaid method, adopt antigen in autoserum as my specific tumour antigen, to compare with single tumour specific antigen, effectively avoid because of the resistant function of tumour cell sudden change to specific antigens; With tumor cell line antigen, compare, serum antigen is self antigen, the change of the antigenic structure of effectively having avoided cell strain to go down to posterity in vitro causing in process; In the face of hand postoperative patient or be difficult to obtain the patient of tumor specimen, effectively avoided the awkward situation that can use without tumour antigen.In the process of preparing at tumour antigen, appropriate proteinase inhibitor has effectively kept the integrity of serum tumor antigen; In addition, present method operation is simple, and condition is easily controlled, lower to the requirement of equipment, and obtain DC-CIK cell proliferation efficiency higher, effectively improved clinical curative effect.
The DC-CIK cell of the patient-specific of preparing by above technology has following biological characteristics:
Cell forms: CD3+ cell accounts for more than 90%, and wherein CD3+CD4+ cell is about 10-20%, and CD3+CD8+ cell is at 60-80%, CD3+CD56+ cell about 10-20%(referring to Fig. 4).
Cell proliferation multiple: compare with conventional DC-CIK culture technique, use autoserum antigen sensibilization DC, the DC-CIK ability of cell proliferation of cultivation obviously increases, because individual patients is different, general propagation multiple at 200-250 doubly, is 2-3 times of ordinary method.
Cytotoxic activity: compare with conventional DC-CIK culture technique, take human lung carcinoma cell line A549 as target cell, gather Serum of Patients with Lung Cancer and prepare specificity DC-CIK cell, according to different effect targets, compare cell mixing, 24 hourly average killing activities are as shown in table 2, are obviously better than cellar culture method (referring to Fig. 5).（n=10）
Table 2 routine and sensitization DC-CIK cytotoxic activity result comparison sheet
Above content is in conjunction with particular case detailed description of the invention; can not assert that specific embodiment of the invention is just confined to these explanations; common researchist for specific field under the present invention; in the situation that not departing from the concrete pattern of the present invention; can also make some simple deductions and replacement, all should be considered as belonging to protection scope of the present invention.
Claims (7)
1. the preparation method of autoserum antigen sensibilization DC-CIK cell, comprises the following steps:
A) preparation of autoserum antigen;
B) preparation of peripheral blood mononuclear cell;
C) the separated and cultivation of DC;
D) induction of CIK cell amplification;
E) DC and CIK co-culture of cells are prepared DC-CIK cell.
2. the preparation method of autoserum antigen sensibilization DC-CIK cell as claimed in claim 1, it is characterized in that: described A) step is for getting peripheral blood 50-100ml, centrifugation patients serum, adding final concentration is that 0.1-1.0mg/ml bright pressed down proteinase inhibitor to carry out albumen concentrated, subsequently with aseptic centrifuge tube storage, be placed in-20 ℃ of following freezing 24-48h of environment, after taking-up, in 0-4 ℃ of environment, place 10-16h, draw upper plasma part and discard; Deactivation 20-30min, filtering with microporous membrane, obtains autoserum tumour antigen.
3. the preparation method of autoserum antigen sensibilization DC-CIK cell as claimed in claim 2, is characterized in that: after described separation of serum, the albumen treatment condition of albumen after concentrated are-20 ℃ of freezing 48h, vertically place subsequently 16h in 4 ℃ of environment; Described deactivation condition is 56 ℃ of deactivation Complement component 3 0min; Described millipore filtration is that aperture is 22 μ m and following millipore filtration.
4. the preparation method of autoserum antigen sensibilization DC-CIK cell as claimed in claim 1, is characterized in that: described B) step is for adopting the separated peripheral blood mononuclear cell (PBMC) that obtains of density gradient centrifugation; Adopt the AIM-V serum free medium separated PBMC that suspends, according to 0.5-1 * 10
8the quantity of/bottle is placed in culturing bottle, and standing 1-3h in cell culture incubator draws not adherent lymphocyte for the cultivation of CIK cell; Attached cell is for the cultivation of DC.
5. the preparation method of autoserum antigen sensibilization DC-CIK cell as claimed in claim 1, it is characterized in that: described C) step is according to tumor markers measurement result, after cell attachment, adding final concentration is that the autoserum of 5-20% carries out DC sensitization, in cultivation, within the 6th to the 7th day, adding final concentration is the tumour necrosis factor of 200-1000ng/ml, obtains the DC after autoantigen sensitization;
6. the preparation method of autoserum antigen sensibilization DC-CIK cell as claimed in claim 1, is characterized in that: described E) step, for by the DC after above-mentioned antigen sensibilization and aforementioned CIK cell co-cultivation 5-8 days, is gathered in the crops self antigen sensitization DC-CIK cell.
7. the preparation method of the autoserum antigen sensibilization DC-CIK cell as described in claim 1-6 any one, is characterized in that: the groups of cells of described DC-CIK cell becomes CD3
+cell accounts for more than 90%, wherein CD3
+cD4
+cell is at 10-20%, CD3
+cD8
+cell is at 60-80%, CD3
+cD56
+cell is at 10-20%.
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| CN106676067A (en) * | 2015-11-10 | 2017-05-17 | 成都嘉泰美康生物科技有限公司 | Culture method of efficient and specific i-DC/CIK (individual DC/CIK) cells |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105062968A (en) * | 2015-09-14 | 2015-11-18 | 广州赛莱拉干细胞科技股份有限公司 | DC-CIK (Dendritic cell-Cytokine Induced Killer) cell culture reagent and culture method thereof |
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| CN106676067A (en) * | 2015-11-10 | 2017-05-17 | 成都嘉泰美康生物科技有限公司 | Culture method of efficient and specific i-DC/CIK (individual DC/CIK) cells |
| 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 |
| CN109535241A (en) * | 2018-12-18 | 2019-03-29 | 北昊干细胞与再生医学研究院有限公司 | DC-CIK co-cultured cell and preparation method thereof, sensitising antigens and application |
| CN109535241B (en) * | 2018-12-18 | 2021-01-08 | 北昊干细胞与再生医学研究院有限公司 | DC-CIK (dendritic cell-cytokine induced killer) co-culture cell, preparation method thereof, sensitizing antigen and application |
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