CN105624111A - Culture reagent for culturing and amplifying DC-CIK cells with high proliferation force and high cytotoxic activity - Google Patents
Culture reagent for culturing and amplifying DC-CIK cells with high proliferation force and high cytotoxic activity Download PDFInfo
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Abstract
The invention discloses a culture reagent for culturing and amplifying DC-CIK (Dendritic Cells-Cytokine Induced Killer) cells with high proliferation force and high cytotoxic activity. The culture reagent comprises a DC cell culture reagent, a CIK cell culture reagent and a co-culture reagent; the DC cell culture reagent comprises a DC cell culture reagent A and a DC cell culture reagent B, the DC cell culture reagent A contains GM-SCF and IL-4, and the DC cell culture reagent B contains GM-SCF, IL-4, TNF-alpha and MDC; the CIK cell culture reagent comprises a CIK cell culture reagent A and a CIK cell culture reagent B, the CIK cell culture reagent A contains INF-gamma, and the CIK cell culture reagent B contains OKT-3, IL-l alpha and IL-2; the co-culture reagent contains a compound SchinlignanF and IL-2. According to the DC-CIK cell culture reagent and a culture method thereof disclosed by the invention, the DC-CIK cells with the high proliferation force and high lethality can be obtained.
Description
Technical field
The invention belongs to field of cell culture, it is specifically related to a kind of cultivation reagent cultivating amplification high proliferation power, high cytotoxic activity DC-CIK cell.
Background technology
Malignant tumour is serious threat people's life and health, after resection operation, radiotherapy, chemotherapy, tumor vaccine cells treatment is clinical the 4th big therapy for oncotherapy, it is a kind of emerging, tumor treatment model with significant curative effect, is the anticancer novel method for the treatment of of a kind of autoimmunization. It returns, after using biotechnology and biotechnological formulation that the immunocyte gathered in patient body is carried out vitro culture and amplification, the method being passed in patient body, excites, enhancing body autoimmune function, thus reaches the object for the treatment of tumour. Existing immune cell therapy has LAK cell therapy, til cell therapy, DC cell therapy, CIK cell therapy and DC-CIK conjoint therapy occur in succession, oncotherapy plays very important effect.
Dendritic cell (Dendriticcells, DC) it is the professional antigen presenting cell that body function is the strongest, it can absorb efficiently, processing treatment and pass in antigen, immature DC has stronger transfer ability, ripe DC can effectively activate initial type T cell, is in startup, regulates and controls and maintains the key link of immunne response. DC cell be in known body function the most by force, uniquely can activate the professional antigen presenting cells of tranquillization T cell, be start, regulation and control and maintain the key link of immunne response. By the DC cell of a large amount of external activation culture load tumour antigen, return after cell quantity reaches some amount and it is defeated by patient, body can be induced to produce strong anti tumor immune response.
CIK cell, namely cytokine-induced killer cell cell (Cytokine-InducedKiller, CIK) is a kind of novel immunologically competent cell, and CIK multiplication capacity is strong, and cytotoxicity is strong, has certain immunological characteristic. Owing to this cell expresses CD3 and CD56 two kinds of membrane protein molecules simultaneously, therefore being also called NK cell (natural killer cell) sample T lymphocyte, with the anti-tumor activity that T lymphocyte is powerful, and the non-MHC of NK cell is restricted kills knurl advantage.
DC and CIK Dual culture can promote propagation and the immunologic function of CIK cell and DC cell simultaneously. Apply DC-CIK cell after chemotherapy can effectively grow by inhibition tumor cell, even make cases of complete remission; And collective's function of immune system is not produced harm by the anti-tumour effect of DC-CIK cell, when currently understanding relatively less to tumour specific antigen, application DC-CIK cell has important clinical meaning as the assisting therapy of tumor chemoradiotherapy and Post operation.
Summary of the invention
It is an object of the invention to provide and a kind of cultivate amplification high proliferation power, the cultivation reagent of high cytotoxic activity DC-CIK cell and cultural method, to obtain the DC-CIK cell of high proliferation power, High Fragmentation power.
The above-mentioned purpose of the present invention is achieved by technical scheme below:
A kind of DC-CIK cell culture reagent, comprises DC cell culture reagent, CIK cell cultivation reagent and Dual culture reagent; Described DC cell culture reagent comprises DC cell culture reagent A and DC cell culture reagent B, and DC cell culture reagent A comprises GM-SCF and IL-4, and DC cell culture reagent B comprises GM-SCF, IL-4, TNF-�� and MDC; Described CIK cell is cultivated reagent and is comprised CIK cell cultivation reagent A and CIK cell cultivation reagent B, and CIK cell is cultivated reagent A and comprised INF-��, and CIK cell is cultivated reagent B and comprised OKT-3, IL-1 �� and IL-2; Described Dual culture pack is containing compound S chinlignanF and IL-2.
Further, described DC-CIK cell culture reagent also comprises the serum free medium containing blood plasma.
Further, in described serum free medium, the volumn concentration of blood plasma is 5%��15%.
Further, in described DC cell culture reagent A, the concentration of GM-SCF is the concentration of 100��500U/mL, IL-4 is 100��500U/mL.
Further, in described DC cell culture reagent B, the concentration of GM-SCF is the concentration of 100��300U/mL, IL-4 is 200��400U/mL, and the concentration of TNF-�� is the concentration of 300��500U/mL, MDC is 5��15ng/mL.
Further, described CIK cell is cultivated in reagent A, and the concentration of INF-�� is 500��900U/mL.
Further, described CIK cell is cultivated in reagent B, the concentration of OKT-3 be 60��100ng/mL, IL-1 �� concentration be the concentration of 100��200U/mL, IL-2 be 600��800U/mL.
Further, in described Dual culture reagent, the concentration of IL-2 is 100��500U/mL.
Further, in described Dual culture reagent, the concentration of compound S chinlignanF is 20��40 �� g/mL.
Utilize above-mentioned DC-CIK cell culture reagent to cultivate a method for DC-CIK cell, comprise the steps:
Step S1, carries out cell cultures by single core cell, obtains attached cell and suspension cell;
Step S2, adopts DC cell culture reagent A to cultivate described attached cell, cultivates after 4��6 days, adopts DC cell culture reagent B to cultivate 24��48h, obtains DC cell; Adopt CIK cell to cultivate reagent A and cultivate described suspension cell, cultivate after 1��3 day, adopt CIK cell to cultivate reagent B and continue cultivation 7��8 days, continue culturing process added IL-2 every 3 days, obtain CIK cell;
Step S3, mixes the DC cell in step S2 and CIK cell, adopts described Dual culture reagent to cultivate 5��8 days, obtains DC-CIK cell.
The advantage of the present invention:
The DC-CIK cell that cultivation reagent provided by the invention can turn out proliferative ability height, lethality is strong.
Embodiment
The essentiality content of the present invention is described further below in conjunction with embodiment, but does not limit protection domain of the present invention with this. Although the present invention being explained in detail with reference to better embodiment, it will be understood by those within the art that, it is possible to the technical scheme of the present invention is modified or equivalent replacement, and do not depart from essence and the scope of technical solution of the present invention.
The preparation method of the compounds of this invention SchinlignanF is see document: Isolationandanti-hepatitisBvirusactivityofdibenzocyclooc tadienelignansfromthefruitsofSchisandrachinensis, Phytochemistry, 116 (2015), 253-261.
Embodiment 1:DC-CIK cell cultures
(1) single core cellular segregation
1, periphery blood or Cord blood 40mL, centrifugal 10 minutes of 500��800g, draw upper plasma, after 56 DEG C of deactivation 30min, and the centrifugal 5min of 2000��3000g, supernatant blood plasma 2��8 DEG C is for subsequent use;
2, lower floor's blood, the physiological saline adding 2 times of volumes is resuspended, the blood by after dilution: Ficoll parting liquid is 2:1, the blood after dilution is slowly joined Ficoll parting liquid upper strata, makes layering clear;
3, the centrifugal 15��30min of 500��700g, tunica albuginea layer in the middle of drawing, is single core cell.
(2) DC cellular segregation is cultivated
1, the single core cell obtained by the centrifugal 5min of 200��400g, cleans twice after adding normal saline dilution;
2, serum free medium (containing 5% autologous plasma) re-suspended cell is added, by 2��8 �� 106/ mL inoculates and leaves standstill 1��5h in T75 culturing bottle, obtains attached cell;
3, attached cell adds physiological saline and gently washes 1 time, adds the serum free medium containing 5% autologous plasma, and to add factor GM-SCF be 300U/mL, IL-4 simultaneously is that 300U/mL cultivates;
4, to add GM-SCF after three days be 300U/mL, IL-4 is 300U/mL, changes liquid after being cultured to 4��6 days, and to add GM-SCF be 20U/mL, IL-4 simultaneously be 30U/mL, TNF-�� be 400U/mL, MDC is 10ng/mL, cultivates 36h, obtains object DC cell.
(3) CIK cell is cultivated
1, the cell not pasting wall in DC cell cultures the 2nd step is transferred in another T75 culturing bottle and counts, and according to count results, adjustment cell density is 5��15 �� 105/ mL carries out CIK cultivation;
2, within the 1st day, cytokine INF-�� 700U/mL is added, autologous plasma 5%; Within 2nd day, add cytokine OKT-380ng/mL, IL-1 �� 150U/mL, IL-2700U/mL;
3, add serum free medium (autologous plasma adding 5% on the 1st, 4 day) every 3 days, maintain cell density 5��20 �� 105/ mL, and add the IL-2 of 700U/mL.
(4) DC cell and CIK cell Dual culture
CIK is cultured to 7 days, DC cell and CIK cell mixed culture, adds the compound S chinlignanF of the IL-2 and 30 �� g/mL of 300U/mL simultaneously, cultivates and obtain DC-CIK cell after 7 days.
Embodiment 2: embodiment 1 contrasts, does not only add SchinlignanF when DC cell and CIK cell Dual culture
(1) single core cellular segregation
1, periphery blood or Cord blood 40mL, centrifugal 10 minutes of 500��800g, draw upper plasma, after 56 DEG C of deactivation 30min, and the centrifugal 5min of 2000��3000g, supernatant blood plasma 2��8 DEG C is for subsequent use;
2, lower floor's blood, the physiological saline adding 2 times of volumes is resuspended, the blood by after dilution: Ficoll parting liquid is 2:1, the blood after dilution is slowly joined Ficoll parting liquid upper strata, makes layering clear;
3, the centrifugal 15��30min of 500��700g, tunica albuginea layer in the middle of drawing, is single core cell.
(2) DC cellular segregation is cultivated
1, the single core cell obtained by the centrifugal 5min of 200��400g, cleans twice after adding normal saline dilution;
2, serum free medium (containing 5% autologous plasma) re-suspended cell is added, by 2��8 �� 106/ mL inoculates and leaves standstill 1��5h in T75 culturing bottle, obtains attached cell;
3, attached cell adds physiological saline and gently washes 1 time, adds the serum free medium containing 5% autologous plasma, and to add factor GM-SCF be 300U/mL, IL-4 simultaneously is that 300U/mL cultivates;
4, to add GM-SCF after three days be 300U/mL, IL-4 is 300U/mL, changes liquid after being cultured to 4��6 days, and to add GM-SCF be 20U/mL, IL-4 simultaneously be 30U/mL, TNF-�� be 400U/mL, MDC is 10ng/mL, cultivates 36h, obtains object DC cell.
(3) CIK cell is cultivated
1, the cell not pasting wall in DC cell cultures the 2nd step is transferred in another T75 culturing bottle and counts, and according to count results, adjustment cell density is 5��15 �� 105/ mL carries out CIK cultivation;
2, within the 1st day, cytokine INF-�� 700U/mL is added, autologous plasma 5%; Within 2nd day, add cytokine OKT-380ng/mL, IL-1 �� 150U/mL, IL-2700U/mL;
3, add serum free medium (autologous plasma adding 5% on the 1st, 4 day) every 3 days, maintain cell density 5��20 �� 105/ mL, and add the IL-2 of 700U/mL.
(4) DC cell and CIK cell Dual culture
CIK is cultured to 7 days, and DC cell and CIK cell mixed culture, add the IL-2 of 300U/mL simultaneously, cultivates and obtains DC-CIK cell after 7 days.
Embodiment 3: effect example
Measure quantity and the survival rate of embodiment 1 and embodiment 2 Dual culture DC-CIK cell after 7 days respectively, and the lethality to K562 tumour cell.
Tumor-killing experiment process is specific as follows:
Get lethal cell (effector cell) and tumour cell (target cell K562) respectively, by effector cell: target cell is that 20:1 tests, and namely gets target cell 1 �� 105, effector cell gets 2 �� 10 respectively6, Dual culture 4h.
Do 1 group of target cell natural apoptosis simultaneously
After detecting target cell death by microplate reader, the protein ingredient of secretion is identified, determine target cell natural apoptosis amount and each group kill and wound rear target cell death amount, calculate: kill and wound the cell concentration that the dead target cell amount of group subtracts natural apoptosis, then divided by target cell total amount, obtain killing-efficiency value.
The results are shown in following table.
| Group | The quantity of DC-CIK cell | DC-CIK cell survival rate (%) | Killing-efficiency value (%) |
| Embodiment 1 | 2.3��1010 | 96.2 | 92.7 |
| Embodiment 2 | 5.9��109 | 94.9 | 72.9 |
The above results shows, DC-CIK cell culture reagent provided by the invention and cultural method, it is possible to obtain the DC-CIK cell of high proliferation power, High Fragmentation power.
The effect of above-described embodiment is to illustrate the essentiality content of the present invention, but does not limit protection scope of the present invention with this. It will be understood by those within the art that, it is possible to the technical scheme of the present invention is modified or equivalent replacement, and do not depart from essence and the protection domain of technical solution of the present invention.
Claims (10)
1. a DC-CIK cell culture reagent, it is characterised in that: comprise DC cell culture reagent, CIK cell cultivation reagent and Dual culture reagent; Described DC cell culture reagent comprises DC cell culture reagent A and DC cell culture reagent B, and DC cell culture reagent A comprises GM-SCF and IL-4, and DC cell culture reagent B comprises GM-SCF, IL-4, TNF-�� and MDC; Described CIK cell is cultivated reagent and is comprised CIK cell cultivation reagent A and CIK cell cultivation reagent B, and CIK cell is cultivated reagent A and comprised INF-��, and CIK cell is cultivated reagent B and comprised OKT-3, IL-1 �� and IL-2; Described Dual culture pack is containing compound S chinlignanF and IL-2.
2. DC-CIK cell culture reagent according to claim 1, it is characterised in that: also comprise the serum free medium containing blood plasma.
3. DC-CIK cell culture reagent according to claim 2, it is characterised in that: in described serum free medium, the volumn concentration of blood plasma is 5%��15%.
4. DC-CIK cell culture reagent according to claim 1, it is characterised in that: in described DC cell culture reagent A, the concentration of GM-SCF is the concentration of 100��500U/mL, IL-4 is 100��500U/mL.
5. DC-CIK cell culture reagent according to claim 1, it is characterized in that: in described DC cell culture reagent B, the concentration of GM-SCF is the concentration of 100��300U/mL, IL-4 is 200��400U/mL, the concentration of TNF-�� is the concentration of 300��500U/mL, MDC is 5��15ng/mL.
6. DC-CIK cell culture reagent according to claim 1, it is characterised in that: described CIK cell is cultivated in reagent A, and the concentration of INF-�� is 500��900U/mL.
7. DC-CIK cell culture reagent according to claim 1, it is characterised in that: described CIK cell is cultivated in reagent B, the concentration of OKT-3 be 60��100ng/mL, IL-1 �� concentration be the concentration of 100��200U/mL, IL-2 be 600��800U/mL.
8. DC-CIK cell culture reagent according to claim 1, it is characterised in that: in described Dual culture reagent, the concentration of IL-2 is 100��500U/mL.
9. DC-CIK cell culture reagent according to claim 1, it is characterised in that: in described Dual culture reagent, the concentration of compound S chinlignanF is 20��40 �� g/mL.
10. one kind utilizes the method for the arbitrary described DC-CIK cell culture reagent cultivation DC-CIK cell of claim 1��9, it is characterised in that, comprise the steps:
Step S1, carries out cell cultures by single core cell, obtains attached cell and suspension cell;
Step S2, adopts DC cell culture reagent A to cultivate described attached cell, cultivates after 4��6 days, adopts DC cell culture reagent B to cultivate 24��48h, obtains DC cell; Adopt CIK cell to cultivate reagent A and cultivate described suspension cell, cultivate after 1��3 day, adopt CIK cell to cultivate reagent B and continue cultivation 7��8 days, continue culturing process added IL-2 every 3 days, obtain CIK cell;
Step S3, mixes the DC cell in step S2 and CIK cell, adopts described Dual culture reagent to cultivate 5��8 days, obtains DC-CIK cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106635983A (en) * | 2016-10-19 | 2017-05-10 | 天津普瑞赛尔生物科技有限公司 | Method for culturing and cryopreservation of CIK cells by applying serum-free lymphocyte culture medium and obtained 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 |
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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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