CN106701678A

CN106701678A - Method for efficient preparation of CIK cell

Info

Publication number: CN106701678A
Application number: CN201510797763.8A
Authority: CN
Inventors: 谢兵; 秦检
Original assignee: Chongqing Dingsheng Biotechnology Co Ltd
Current assignee: Chongqing Dingsheng Biotechnology Co Ltd
Priority date: 2015-11-18
Filing date: 2015-11-18
Publication date: 2017-05-24

Abstract

The invention relates to the field of biotechnology, in particular to a method for efficient preparation of a CIK cell. The method is characterized by adopting the following preparation steps: preliminary separation of lymphocytes; twice washing with lotion; lymphocyte concentration regulation; culture: adding the concentration regulated lymphocytes into a cell bottle coated with CD3 antibody to conduct culture for 24h, then adding 1ml of an IFN-gamma reagent and 1ml of an IL-1beta reagent to perform culture for 48h to obtain the CIK cell; enlarged culture; and collection. Compared with the prior art, the method provided by the invention adopts coating with the CD3 antibody firstly, then adds IFN-gamma, also breaks the routine and uses IL-1beta, through coating, the cell can contact the antibody more lastingly, and a better activation effect can be achieved with a small dosage. The CIK cell prepared by the method provided by the invention has cytotoxic activity obviously higher than that of existing CIK cells by more than 30%, the problems of poor reproductive capacity and low cell viability of CIK cells are solved, and the cost is greatly lowered.

Description

A kind of efficient method for preparing CIK cell

Technical field

The present invention relates to biological technical field, specifically a kind of efficient method for preparing CIK cell.

Background technology

It is many in preparation method in the past first to add IFN-r to activate using in human peripheral blood single nucleus cell, after 24 hours, again plus other cell factors such as CD3, PHA, IL-2, IL -1 α, Amplification Culture after 3 days, CIK cell fertility that this method is prepared is poor, cytotoxic activity is low, and low cost.

The content of the invention

The purpose of the present invention is to overcome the deficiencies in the prior art, and that breaks through routine employs different cell factor addition sequences, i.e. using CD3 antibody is first coated with, the order of INF- γ is used afterwards；Using different washing lotions with entering, and with IL-1 β substitute IL-1 α stimulates the cell to increase.

To achieve the above object, a kind of efficient method for preparing CIK cell is designed, it is characterised in that use following preparation process：（1）Initial gross separation lymphocyte：People's White Blood Cells Concentrate is taken, employment lymphocyte separation medium separates lymphocyte with 2000rpm, is centrifuged 20 minutes, then first suctions out upper plasma, and inactivation is standby, then draw white ring layer of the lower floor rich in lymphocyte；（2）Wash twice：Washing lotion is added in the solution rich in lymphocyte drawn, then is centrifuged 10 minutes with 1500rpm, abandon supernatant, take the cell of precipitation, complete to wash for the first time；Washing lotion is added in the cell of the precipitation for obtaining again, is centrifuged 10 minutes with 1500rpm, abandon supernatant, take the cell of precipitation, i.e. lymphocyte, so as to complete the washing twice to lymphocyte, and counted；（3）Regulation lymphocyte concentration：Lymphocyte concentration to 2~3 × 10 is adjusted with minimal medium⁶Individual/ml；（4）Culture：The lymphocyte that concentration will be regulated adds the cell culture in glassware 24 hours for being coated with CD3 antibody, adds IFN-γ reagent 1ml, IL-1 β reagents 1ml and cultivates 48 hours to obtain CIK cell；（5）Amplification Culture：The CIK cell that growth vigor is 100% is transferred to 175cm²In Tissue Culture Flask, culture is enlarged with CIK culture mediums, every 48~72 hours Amplification Cultures once, to 15-20 days；（6）Collect：CIK cell by cell viability more than 98% is collected and collected, and is then washed as follows：The CIK cell for collecting will be collected to be centrifuged 10 minutes with 1500rpm, supernatant is abandoned and taken bottom precipitation cell；Add washing lotion to repeat above-mentioned washing 2~3 times in sedimentation cell is obtained again, the sedimentation cell of acquisition is filtered with 200 mesh filters, is taken and is filtered down CIK cell, count CIK cell sum and cell viability；（7）CIK cell is suspended in the physiological saline of 100ml 0.9%, 2 × 10 are added⁵The albumin of the IL-2 and 10ml of unit, after being well mixed, seals to obtain final finished.

Described washing lotion is the heparin of addition 5U/ml in 0.9% physiological saline of 500ml, and the gentamicin for adding 80 U/ml is mixed.

Described minimal medium is using addition heparin 5 U/ml, the U/ml of gentamicin 80, anphotericin 30ng/ml in GT-T551 culture mediums 500ml, add the standby upper plasma of above-mentioned inactivation to be constituted, the percent by volume that the upper plasma for being added is accounted in minimal medium is 1%.

Described CIK culture mediums add the IL-2 of 1000 U/ml to be constituted by using in the minimal medium of 500ml.

Described CD3 ACs are 2ug/ml, are first coated in cell bottle bottom with coating buffer.

The concentration of described IFN-γ reagent is 1000 U/ml.

The concentration of described IL-1 β reagents is 500 U/ml.

The present invention is compared with the existing technology, using first coating CD3 antibody, INF- γ are added afterwards, and break the normal procedure using IL-1 β, the more longlasting contact antibody of cell is caused by coating, the few activation effect of consumption is more preferable, adding IL-1 β can strengthen stimulation, and the cell phenotype after cell harvesting is detected that CD3+CD56+ expresses more preferably, while amplification times improve 2 times, reduce because heavy dose uses the toxic and side effect produced by cell factor；CIK cell prepared by the present invention, cytotoxic activity is apparently higher than existing CIK cell more than 30%, and cytotoxic activity can be maintained 2~3 weeks, CIK cell highest killing rate appears in the 12nd~15 day of culture, cell it is lethal stronger, Clinical practice effect more preferably, solves the problems, such as that CIK cell fertility is poor, cytoactive is low, and greatly reduce cost.

Specific embodiment

The present invention is further described with reference to example.

Embodiment

Following preparation method is used in the present invention：

First, initial gross separation lymphocyte：

People's White Blood Cells Concentrate is taken, employment lymphocyte separation medium separates lymphocyte with 2000rpm, is centrifuged 20 minutes, then first suctions out upper plasma, and inactivation is standby, then draw white ring layer of the lower floor rich in lymphocyte.

2nd, wash twice：

Washing lotion is added in the solution rich in lymphocyte being drawn to, then is centrifuged 10 minutes with 1500rpm, abandon supernatant, draw the cell of precipitation, complete to wash for the first time；Washing lotion is added in the cell of the precipitation drawn again, is centrifuged 10 minutes with 1500rpm, abandon supernatant, the cell of precipitation, i.e. lymphocyte are drawn, so as to complete, to the washing twice rich in lymphocyte solution, to obtain lymphocyte, and count；Described washing lotion is to add heparin 5 U/ml, again plus gentamicin 80U/ml is mixed in 0.9% physiological saline of 500ml.

3rd, lymphocyte concentration is adjusted：

Lymphocyte concentration to 2~3 × 10 is adjusted with minimal medium⁶Individual/ml；Minimal medium is that the U/ml of heparin 5, the U/ml of gentamicin 80, anphotericin 30ng/ml are added in the GT-T551 culture mediums of 500ml, add the standby upper plasma of above-mentioned inactivation to be constituted, the percent by volume that the upper plasma for being added is accounted in minimal medium is 1%.

4th, cultivate：

The coating buffer that the CD3 antibody pH value of 2ug/ml is 7.4 is first coated in cell bottle bottom, then the lymphocyte that concentration will be regulated adds the cell culture in glassware 24 hours for being coated with CD3 antibody, add the IFN-γ reagent that concentration is 1000 U/ml, concentration is the IL-1 β reagents culture 48 hours of 500 U/ml.

5th, Amplification Culture：

The CIK cell that growth vigor is 100% is transferred to 175cm²In Tissue Culture Flask, culture is enlarged with CIK culture mediums, every 48~72 hours Amplification Cultures once, to 15-20 days；CIK culture mediums add the IL-2 of 1000 U/ml to be constituted by using in the minimal medium of 500ml.

6th, CIK cell is collected：

CIK cell by cell viability more than 98% is collected and collected, and is then washed as follows：Collection collects CIK cell, is centrifuged 10 minutes with 1500rpm, abandons supernatant and takes bottom precipitation cell；Washing lotion repeat above-mentioned washing 2~3 times final sedimentation cell for obtaining afterwards is added in sedimentation cell again, is filtered with 200 mesh filters, the CIK cell that acquisition is filtered down from filter, and count the CIK cell sum and cell viability of acquisition.

7th, the CIK cell after filtering is suspended in 100ml physiological saline, adds 2 × 10⁵The albumin of the IL-2 and 10ml of unit, after being well mixed, sealing gets product.

Claims

1. it is a kind of it is efficient prepare CIK cell method, it is characterised in that use following preparation process：（1）Initial gross separation lymphocyte：People's White Blood Cells Concentrate is taken, employment lymphocyte separation medium separates lymphocyte with 2000rpm, is centrifuged 20 minutes, then first suctions out upper plasma, and inactivation is standby, then draw white ring layer of the lower floor rich in lymphocyte；（2）Wash twice：Washing lotion is added in the solution rich in lymphocyte drawn, then is centrifuged 10 minutes with 1500rpm, abandon supernatant, take the cell of precipitation, complete to wash for the first time；Washing lotion is added in the cell of the precipitation for obtaining again, is centrifuged 10 minutes with 1500rpm, abandon supernatant, take the cell of precipitation, i.e. lymphocyte, so as to complete the washing twice to lymphocyte, and counted；（3）Regulation lymphocyte concentration：Lymphocyte concentration to 2~3 × 10 is adjusted with minimal medium⁶Individual/ml；（4）Culture：The lymphocyte that concentration will be regulated adds the cell culture in glassware 24 hours for being coated with CD3 antibody, adds IFN-γ reagent 1ml, IL-1 β reagents 1ml and cultivates 48 hours to obtain CIK cell；（5）Amplification Culture：The CIK cell that growth vigor is 100% is transferred to 175cm²In Tissue Culture Flask, culture is enlarged with CIK culture mediums, every 48~72 hours Amplification Cultures once, to 15-20 days；（6）Collect：CIK cell by cell viability more than 98% is collected and collected, and is then washed as follows：The CIK cell for collecting will be collected to be centrifuged 10 minutes with 1500rpm, supernatant is abandoned and taken bottom precipitation cell；Add washing lotion to repeat above-mentioned washing 2~3 times in sedimentation cell is obtained again, the sedimentation cell of acquisition is filtered with 200 mesh filters, is taken and is filtered down CIK cell, count CIK cell sum and cell viability；（7）CIK cell is suspended in the physiological saline of 100ml 0.9%, 2 × 10 are added⁵The albumin of the IL-2 and 10ml of unit, after being well mixed, seals to obtain final finished.

2. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：Described washing lotion is the heparin of addition 5U/ml in 0.9% physiological saline of 500ml, and the gentamicin for adding 80 U/ml is mixed.

3. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：Described minimal medium is using addition heparin 5 U/ml, the U/ml of gentamicin 80, anphotericin 30ng/ml in GT-T551 culture mediums 500ml, add the standby upper plasma of above-mentioned inactivation to be constituted, the percent by volume that the upper plasma for being added is accounted in minimal medium is 1%.

4. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：Described CIK culture mediums add the IL-2 of 1000 U/ml to be constituted by using in the minimal medium of 500ml.

5. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：Described CD3 ACs are 2ug/ml, are first coated in cell bottle bottom with coating buffer.

6. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：The concentration of described IFN-γ reagent is 1000 U/ml.

7. a kind of efficient method for preparing CIK cell as claimed in claim 1, it is characterised in that：The concentration of described IL-1 β reagents is 500 U/ml.