CN103981144B - The preparation method of autoserum antigen sensibilization DC CIK cells - Google Patents
The preparation method of autoserum antigen sensibilization DC CIK cells Download PDFInfo
- Publication number
- CN103981144B CN103981144B CN201410095323.3A CN201410095323A CN103981144B CN 103981144 B CN103981144 B CN 103981144B CN 201410095323 A CN201410095323 A CN 201410095323A CN 103981144 B CN103981144 B CN 103981144B
- Authority
- CN
- China
- Prior art keywords
- cik
- antigen
- cell
- autoserum
- 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.)
- Active
Links
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of preparation method of autoserum antigen sensibilization DC CIK cells, the preparation method of the autoserum antigen sensibilization DC CIK cells, including step：A）The preparation of autoserum antigen；B）The preparation of PMNC；C）The separation and culture of DC；D）The induced amplification of CIK cell；E）DC is co-cultured with CIK cell and is prepared DC CIK cells.The DC CIK of the method for the invention culture its proliferation activity to CIK and kill tumor activity and substantially increase compared with DC CIK prepared by cellar culture, and its proliferation times has averagely reached 200 250 times, is 23 times of classical culture protocols；Tumor activity is killed during experiment in vitro and reaches 70 80%, hence it is evident that higher than the DC CIK cells of conventional method culture.This method operation is simple, and condition is easily-controllable, and the requirement to equipment is relatively low, and the DC CIK cells propagation for being obtained is in hgher efficiency.
Description
Technical field
The invention belongs to cell biology, immunotherapy of tumors field, autologous tumor antigens sensitization DC-CIK is directed to use with
Preparation method and antitumor adoptive immunotherapy application.
Background technology
Malignant tumour has turned into the Chinese cause of death first, seriously threatens the health and life of the people, tumour immunity
Treatment is the fourth-largest therapy for being clinically used for oncotherapy, and wherein tumour adoptive immunity cell therapy obtains widely should in China
With what domestic development was most at present is exactly the adoptive feedback therapy of DC-CIK cells.
DC cells are most important antigen presenting cells in body, by antigen uptake, process and offer, and DC cells can be with
Effective inducing antigen-specific CD4 and CD8 T lymphocyte activations, so as to activate body the acquired immune response.Additionally, DC is same
Sample can activate NK cells, strengthen its multiplication capacity and lethal effect, so as to strengthen innate immune reaction, play antitumor jointly
Effect.
CIK cell is Cytokine-induced killer cells, be by human peripheral blood single nucleus cell in vitro with various kinds of cell
The non-principal histocompatibility complex that factor co-incubation is obtained afterwards for a period of time（MHC）Restricted cytotoxic T
Cell, has efficient dissolving toxicity to tumour cell, after DC is co-cultured with CIK cell, can effectively increase the propagation of CIK
Ability and killing activity, are more beneficial for improving the antineoplastic immune curative effect of DC-CIK, are also most commonly used current domestic application
Technology.
DC-CIK mainly includes DC-CIK, the DC-CIK of tumour specific antigen sensitization and full tumour of nonantigenic sensitization
The DC-CIK of antigen sensibilization.Research shows that the DC through the antigen sensibilization and DC without antigen sensibilization can cause the propagation of CIK
Ability and kill tumor activity rising, and by antigen sensibilization DC can preferably cooperate with stimulate CIK cell propagation and activation, tool
Have and preferably kill tumor activity.Therefore, a key influencing factor of DC-CIK curative effects is turned into from suitable antigen sensibilization DC.
The antigen that the current external sensitization of DC is used is mainly known tumour specific antigen, tumor cell line cracking and produces
Thing or tumor specimen pyrolysis product.Because the shortage specific antigen of most of tumours, and tumour are easily mutated and then support
The immune attack of single antigen, therefore the DC clinical practices of specific antigen sensitization is resisted to be very limited.And tumor cell line
In incubation, surface antigen would generally change, and the DC-CIK clinical effectiveness obtained after sensitization DC is not good, and tumour
Sample pyrolysis product is only used for the patient of operation consent specimen taken, and the patient that DC-CIK treatments are carried out in clinical practice is mostly
Postoperative patient, it is difficult to obtain fresh specimens from pri, these all greatly limit the clinical effectiveness of DC-CIK.And due to swollen
The individual difference of knurl patient, even same type of tumour, the tumour antigen for being possessed also differs widely, and artificial synthesized is anti-
Original can not be widely used in the treatment of tumor patient.
Conventional protein compression technology includes bag filter concentration method, freeze-drying concentration method, dries up concentration method, surpasses at present
Filter membrane concentration method, chemical precipitation method etc., but ultrafiltration membrane concentration method and bag filter concentration method it is more advanced say need cost compared with
Height, is unfavorable for promoting, and other method easily causes the pollution of serum mostly, it is difficult to which, for the culture of cell, we are based on freezing
Melt the principle that can make serum chemistry composition precipitation concentration, on the basis of serum antigen activity is not influenceed, find practical
Autoserum antigen preparation procedure, and and then carry out sensitization DC using these serum, cultivate specific by after antigen sensibilization
DC-CIK cells, for clinic.
The content of the invention
Above-mentioned deficiency it is an object of the invention to overcome prior art, there is provided one kind can obtain high proliferation activity and kill knurl
The preparation method of the self-antigen sensitization DC-CIK cells of activity.
The preparation method of autoserum antigen sensibilization DC-CIK cells, comprises the following steps：
The preparation of autoserum antigen：Peripheral blood 50-100ml, centrifugation patients serum adds final concentration of 0.1-
The bright suppression protease inhibitors of 1.0mg/ml, is well mixed, and is then stored with sterile centrifugation tube, is placed in less than -20 DEG C environment cold
Freeze 24-48h, place 10-16h after taking-up in 0-4 DEG C of environment, draw upper plasma part, and discard, inactivate 20-30min,
Filtering with microporous membrane, obtains autoserum tumour antigen；
Preferably：Albumen treatment conditions after the separation serum after protein concentration are -20 DEG C of freezing 48h, then at 4 DEG C
16h is placed in environment vertically, protein concentrate can be helped by gravity；The inactivation condition is 56 DEG C of inactivation complement components
30min；The miillpore filter is that aperture is 22 μm and following miillpore filter.
The preparation of PMNC：Separated using density-gradient centrifugation method and obtain PMNC
（PBMC）.Suspended separate PBMC using AIM-V serum free mediums, according to 0.5-1 × 108The quantity of/bottle is placed in blake bottle
In, 1-3h is stood in cell culture incubator, drawing not adherent lymphocyte is used for the culture of CIK cell；Attached cell is used for DC's
Culture.
The separation and culture of DC：According to tumor markers measurement result, add final concentration of 5-20%'s after cell attachment
Autoserum carries out DC sensitization, in the TNF that culture 6- adds final concentration of 200-1000ng/ml on the 7th day, obtains
DC to after autoantigen sensitization.
The induced amplification of CIK cell：Above-mentioned non-attached cell is carried out according to CIK cell culture conventional scheme.
DC is co-cultured with CIK cell and is prepared DC-CIK：DC after above-mentioned antigen sensibilization is mixed with foregoing CIK cell
5-8 days, harvest self-antigen sensitization DC-CIK cells.
Cell is constituted：CD3+ cells account for more than 90%, and wherein about in 10-20%, CD3+CD8+ cells exist CD3+CD4+ cells
60-80%, CD3+CD56+ cell are about in 10-20%.
The antitumor adoptive immunity work that can be used for clinic is prepared by the above method another object of the present invention is to prepare
Property cell.
The DC-CIK of above method culture its proliferation activity to CIK and is killed compared with DC-CIK prepared by cellar culture
Tumor activity all substantially increases, and its proliferation times has averagely reached 200-250 times, is 2-3 times of classical culture protocols；Experiment in vitro
When kill tumor activity and reach 70-80%, hence it is evident that higher than the DC-CIK cells of conventional method culture.Using the above method, using autologous blood
Antigen compared with single tumour specific antigen, is prevented effectively from because of tumour cell as my specific tumor antigen in clear
It is mutated the resistant function to specific antigen；Compared with tumor cell line antigen, serum antigen is self-antigen, be effectively prevent
The change of the antigenic structure that cell line is caused in succeeding generations in vitro；In face of postoperative patients or be difficult to obtain tumor specimen
Patient, effectively prevent without the available awkward situation of tumour antigen.During prepared by tumour antigen, appropriate albumen enzyme level
Agent effectively maintains the integrality of serum tumor antigen；Additionally, this method operation is simple, condition is easily-controllable, and equipment is wanted
Ask relatively low, and the DC-CIK cells propagation for being obtained is in hgher efficiency.
Brief description of the drawings
Fig. 1 is the influence of serum cooling time and temperature to protein content after concentration（mg/ml）；
Fig. 2 is influence of the serum dissolution time to protein content after concentration；
Fig. 3 is the cell growth curve in incubation；
Fig. 4 is cultured cells flow cytometer detection result；CD3+ cells account for more than 90%, and wherein CD3+CD4+ cells are about in 10-
, in 60-80%, CD3+CD56+ cells are about in 10-20% for 20%, CD3+CD8+ cell.
Fig. 5 is the killing effect in vitro of the cell to A549 cells of culture.
Specific embodiment
The present invention is illustrated with example below, but the present invention is not intended to be limited thereto.All unreceipted specific bars in Examples below
The experimental technique of part, being the method for observing a usual practice and producer's operating instruction is carried out.
The liquaemin anti-freezing of conventional Christmas Patients with Advanced Lung Cancer peripheral blood 50-100ml, 5-15IU/ml, rotating speed 800g, from
The heart 10 minutes, collects upper plasma part（About 1/3 liquid level）, add the bright suppression protease of final concentration of 0.1-1.0mg/ml
Inhibitor, is well mixed, and is then stored with sterile centrifugation tube, is placed in less than -20 DEG C environment freezing 24-48h（Referring to Fig. 1）, take
10-16h is placed after going out in 0-4 DEG C of environment（Referring to Fig. 2）；56 DEG C of inactivation 30min, 0.22 μm of filtering with microporous membrane is obtained certainly
Body blood serum tumor antigen.
Density-gradient centrifugation method separates PBMC, the haemocyte of physiological saline equimultiple dilution precipitation, human lymphocyte separating liquid
With the blood for diluting according to 1:2 ratio is added in centrifuge tube, rotating speed 700g, is centrifuged 20 minutes, careful to draw tunica albuginea layer, with life
Reason salt water washing 2 times, is centrifuged 8 minutes by 1500 revs/min, that is, obtain PMNC.
Suspended separate PBMC using AIM-V serum free mediums, according to 1 × 108The quantity of/bottle is placed in 75cm2Culture
In bottle, 2h is stood in cell culture incubator, gently rock blake bottle, drawing not adherent lymphocyte is used for the culture of CIK cell, patch
Parietal cell is used for the culture of DC.
DC is cultivated and sensitization：According to shown in table 1, antigen is divided into 3 classes of high and low, middle expression.
The antigen presentation of table 1 is classified
| Tumour reference material measurement result sum R | Antigen presentation group T high | Middle antigen presentation group | Low antigen presentation group |
| R | T >=10 times normal value | 5 times of normal value≤10 times of T ＜ | 5 times of normal values of T ＜ |
| Sensitized serum concentration | 5%-10% | 10%-15% | 15%-20% |
The tumour reference material result that the present embodiment is used：CEA 149.7ng/ml（Normal 0-3.8 ng/ml）,CA125
16.04U/ml（Normal 0-35 U/ml）,CA199 22.87 U/ml（Normal 0-27 U/ml）, tumor markers measurement result is
" more than 10 times of normal values ", final concentration of 5-20% is separately added into for the 3rd, 5 days in attached cell culture, preferably 5-10% from
Body serum carries out DC sensitization（Referring to Fig. 3）, the TNF of final concentration of 200-1000ng/ml is added within the 7th day in culture,
The actually used concentration of this patient is 800ng/ml, obtains the ripe DC after autoantigen sensitization.
The induced amplification of CIK cell：Above-mentioned non-attached cell is carried out according to CIK cell culture conventional scheme.
DC is co-cultured with CIK cell and is prepared DC-CIK：DC after above-mentioned antigen sensibilization is mixed into training with foregoing CIK cell
Support, 7 days or so, harvest self-antigen sensitization DC-CIK cells（Referring to Fig. 3）.
Compared with DC-CIK prepared by cellar culture, its proliferation activity to CIK and kill tumor activity and all substantially increase, its increasing
Grow multiple and averagely reached 200-250 times, be 2-3 times of classical culture protocols；Tumor activity is killed during experiment in vitro and reaches 70-80%,
Apparently higher than the DC-CIK cells of conventional method culture.
It is special with single tumour using antigen in autoserum as my specific tumor antigen using the above method
Specific Antigen is compared, and is prevented effectively from the resistant function to specific antigen because of tumor cell mutations；With tumor cell line antigen phase
Than serum antigen is self-antigen, effectively prevent the change of the antigenic structure that cell line is caused in succeeding generations in vitro；Face
To postoperative patients or the patient for being difficult to obtain tumor specimen, effectively prevent without the available awkward situation of tumour antigen.In tumour
During prepared by antigen, appropriate protease inhibitors effectively maintains the integrality of serum tumor antigen；Additionally, this
Method operation is simple, and condition is easily-controllable, and the requirement to equipment is relatively low, and the DC-CIK cells propagation for being obtained is in hgher efficiency, has
Effect improves the curative effect of clinic.
The patient-specific DC-CIK cells prepared by above technology have following biological characteristics：
Cell is constituted：CD3+ cells account for more than 90%, and wherein about in 10-20%, CD3+CD8+ cells exist CD3+CD4+ cells
60-80%, CD3+CD56+ cell are about in 10-20%（Referring to Fig. 4）.
Cell proliferation times：Compared with conventional DC-CIK culture techniques, using autoserum antigen sensibilization DC, culture
DC-CIK ability of cell proliferation substantially increases, and because individual patients are different, universal proliferation times, at 200-250 times, are conventional sides
2-3 times of method.
Cytotoxic activity：Compared with conventional DC-CIK culture techniques, with human lung carcinoma cell line A549 as target cell, lung is gathered
Cancer patients serum prepares specificity DC-CIK cells, and according to different effect targets than cell mixing, 24 hourly average killing activities are such as
Shown in table 2, hence it is evident that better than conventional culture methods（Referring to Fig. 5）.（n=10）
The routine of table 2 and sensitization DC-CIK cytotoxic activity results contrast tables
Above content is to combine concrete condition detailed description of the invention, it is impossible to assert specific implementation of the invention with regard to office
It is limited to these explanations, for the common researcher of specific field belonging to the present invention, is not departing from the feelings of specific pattern of the invention
Under condition, some simple deductions can also be made and replaced, should all be considered as belonging to protection scope of the present invention.
Claims (5)
1. the preparation method of autoserum antigen sensibilization DC-CIK cells, comprises the following steps：
A）The preparation of autoserum antigen；
B）The preparation of PMNC；
C）The separation and culture of DC；
D）The induced amplification of CIK cell；
E）DC is co-cultured with CIK cell and is prepared DC-CIK cells；
Described A）To take peripheral blood 50-100ml, centrifugation patients serum adds final concentration of 0.1-1.0mg/ml to step
Bright suppression protease inhibitors carry out protein concentration, then stored with sterile centrifugation tube, be placed in less than -20 DEG C environment freezing 24-
48h, places 10-16h in 0-4 DEG C of environment after taking-up, draw upper plasma part and discard；56 DEG C of inactivation complement component 20-
30min, filtering with microporous membrane obtains autoserum tumour antigen；
The step C）It is according to tumor markers measurement result, A the step of final concentration of 5-20% is added after cell attachment）
The autoserum antigen for preparing carries out DC sensitization, adds final concentration of 200-1000ng/ml's within the 6th to the 7th day in culture
TNF, obtains the DC after autoantigen sensitization.
2. the preparation method of autoserum antigen sensibilization DC-CIK cells as claimed in claim 1, it is characterized in that：The separation
Albumen treatment conditions after serum after protein concentration are -20 DEG C of freezing 48h, then place 16h vertically in 4 DEG C of environment；It is described
Inactivation condition is 56 DEG C of inactivation Complement component 3 0min；The miillpore filter is that aperture is 22 μm and following miillpore filter.
3. the preparation method of autoserum antigen sensibilization DC-CIK cells as claimed in claim 1, it is characterized in that：Described B）
Step is to separate to obtain PMNC using density-gradient centrifugation method（PBMC）；Using AIM-V serum free mediums
Suspend separate PBMC, according to 0.5-1 × 108The quantity of/bottle is placed in blake bottle, and 1-3h is stood in cell culture incubator, is drawn
Not adherent lymphocyte is used for the culture of CIK cell；Attached cell is used for the culture of DC.
4. the preparation method of autoserum antigen sensibilization DC-CIK cells as claimed in claim 1, it is characterized in that：Described E）
Step is that the DC after above-mentioned antigen sensibilization is mixed 5-8 days with foregoing CIK cell, harvests self-antigen sensitization DC-CIK thin
Born of the same parents.
5. the preparation method of the autoserum antigen sensibilization DC-CIK cells as described in claim any one of 1-4, it is characterized in that：
The groups of cells of the DC-CIK cells turns into CD3+Cell accounts for more than 90%, wherein CD3+CD4+Cell is in 10-20%, CD3+CD8+Carefully
Born of the same parents are in 60-80%, CD3+CD56+Cell is in 10-20%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410095323.3A CN103981144B (en) | 2014-03-17 | 2014-03-17 | The preparation method of autoserum antigen sensibilization DC CIK cells |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410095323.3A CN103981144B (en) | 2014-03-17 | 2014-03-17 | The preparation method of autoserum antigen sensibilization DC CIK cells |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103981144A CN103981144A (en) | 2014-08-13 |
| CN103981144B true CN103981144B (en) | 2017-06-06 |
Family
ID=51273314
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410095323.3A Active CN103981144B (en) | 2014-03-17 | 2014-03-17 | The preparation method of autoserum antigen sensibilization DC CIK cells |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103981144B (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105062968B (en) * | 2015-09-14 | 2019-01-15 | 广州赛莱拉干细胞科技股份有限公司 | A kind of DC-CIK cell culture reagent and its cultural method |
| CN105194649A (en) * | 2015-09-14 | 2015-12-30 | 山东景源生物科技有限公司 | Separation method and application of active glycoprotein composition in serum of tumor patient |
| 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 |
| 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 |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321577B (en) * | 2011-01-26 | 2013-09-11 | 深圳市中美康士生物科技有限公司 | Preparation method of antitumor adoptive immune cells and prepared immune cells |
| CN102657853A (en) * | 2012-04-27 | 2012-09-12 | 蔡建辉 | Preparation and application of tumor specific killer cells serving as source of initial thymus (T) cells |
| CN103386123A (en) * | 2012-05-08 | 2013-11-13 | 刘江秋 | Method and application of MEK used as costimulatory factor for preparing novel tumor vaccine |
-
2014
- 2014-03-17 CN CN201410095323.3A patent/CN103981144B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN103981144A (en) | 2014-08-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103981144B (en) | The preparation method of autoserum antigen sensibilization DC CIK cells | |
| CN105238754B (en) | A kind of high proliferation power and the extracorporeal culturing method of High Fragmentation power NK cell | |
| CN103756963A (en) | Method used for in vitro proliferation of NK cells | |
| CN103966164B (en) | A kind of hemizygote CAPRI cell preparation method | |
| CN104498434B (en) | A kind of preparation method of a large amount of BMDCs, gained BMDC | |
| CN105316287A (en) | Method for long-term storage and resuscitation culture of adult peripheral blood mononuclear cell | |
| CN102676455B (en) | Preparation method for dendritic cell of umbilical cord blood source and dendritic cell vaccine | |
| EP1944360A1 (en) | Method for culturing dendritic cells (DC) and cytokine-induced killer cells (D-CIK) and applications thereof | |
| CN104593326A (en) | Method for preparing enhanced DC-CIK cell induced by traditional Chinese medicines and application of enhanced DC-CIK cells induced by traditional Chinese medicines | |
| CN103301449A (en) | Preparation method of large-scale culture dendritic cell vaccine and application thereof | |
| CN105969727A (en) | Method for culturing cord blood lymphocyte DC-CIK | |
| CN106222141B (en) | NK cell culture fluids and cell culture processes | |
| CN111662870A (en) | Application of BCG polysaccharide nucleic acid in CIK cell in-vitro culture and preparation of tumor medicine | |
| CN104262459A (en) | Acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide, and vaccine and pharmaceutical application of acute monocytic leukemia-associated antigen MLAA-34 epitope polypeptide | |
| CN102861107B (en) | DC-CIK (dendritic cells-cytokine-induced killers) cell treatment composition | |
| CN106754704B (en) | Method for inducing and expanding immune cells in vitro | |
| CN105296421B (en) | The T cell and preparation method of a kind of activation of bispecific antibody and application | |
| CN104480069A (en) | Method of carrying out isolated culture on immune cells by virtue of peripheral blood | |
| CN105219711A (en) | The culture system of a kind of CIKs cell and DC-CIKs cell | |
| CN105219713A (en) | For high proliferation power, the High Fragmentation power NK cell of tumour adoptive immunity | |
| CN105969731B (en) | A method of High Fragmentation activity til cell is largely prepared using pernicious Pleural effusions | |
| CN105296422B (en) | A kind of NK cell culture compositions and its cultural method | |
| CN105505872B (en) | Method and composition for sensitizing NK cells | |
| CN105087489B (en) | A kind of DC cell culture reagents and its cultural method | |
| CN106479973B (en) | A kind of external IAK immunocyte cultural method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB02 | Change of applicant information |
Address after: 518057, Guangdong Futian Road, Shenzhen, Futian District Futian Free Trade Zone, Fuzhou hi tech plant, seven floor B workshop, office space Applicant after: Unification health biotech inc of Shenzhen Address before: 518057, Nanshan District science and technology zone, Guangdong, Shenzhen Province, 13 North Road, 4 Thunis Road, 406, B404 Applicant before: Unification health bio tech ltd, Shenzhen |
|
| COR | Change of bibliographic data | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CB03 | Change of inventor or designer information | ||
| CB03 | Change of inventor or designer information |
Inventor after: Huang Hao Inventor after: Zou Chang Inventor after: Luo Xiaoling Inventor before: Huang Hao Inventor before: Zou Chang |