CN109207424A - A kind of cultural method of immunocyte - Google Patents

The invention discloses a kind of cultural methods of immunocyte, comprising the following steps: (1) acquires peripheral blood mononuclear cells；(2) above-mentioned cell inoculation is cultivated in the culture bottle containing culture medium；(3) cell after above-mentioned steps (2) are cultivated is imported in culture bag, wherein the volume of culture solution is less than the volume of culture bag；(4) immunocyte is harvested.The present invention provides a kind of cultural method of immunocyte, by control incubation in nutrient solution volume and culture bag volume ratio, improve cell amplification ability and finally obtained cell quantity, realize a large amount of proliferation of immunocyte, the utilization rate for improving culture solution, has saved cost.

A kind of cultural method of immunocyte

Technical field

The present invention relates to field of cell culture more particularly to a kind of cultural methods of immunocyte.

Background technique

Currently, malignant tumour is to seriously endanger one of a kind of disease of human health, the main reason for being death, occur With development mainly since human defensive system loses regulation and control to cancer cell, lead to disequilibrium between body and tumour It is caused.Traditional treatment means include operative treatment, radiotherapy and chemotherapy, studies have shown that operation, radiotherapy, chemotherapy energy Curing tumour is not to kill whole tumour cells, but due to when tumor load is substantially reduced, the immune function of body Restore, to remove minimal disease or the obvious proliferation for inhibiting remaining tumor cells.Immunotherapy of tumors is exactly to pass through artificially Intervene, excitation and the immune system for transferring body enhance anti-tumor immunity, thus control and killing tumor cell.

Adoptive immunotherapy (adoptive cellular immunotherapy, ACI) is current more common one Kind immunotherapy of tumors mode is the immune effector cell for having antitumor action by the amplification of external mass propgation, comes direct It kills tumour or excites a kind of therapeutic modality of body anti tumor immune response.Such cell specifically includes that Lymphokine Killing cell (LAK), tumor infiltrating lymphocyte (TIL), cytokine induced kill cell (CIK), natural kill it is thin Born of the same parents (NK) or the T lymphocyte (such as CAR-T, TCR-T) etc. being transformed through gene technology.

For immune cell therapy, sufficient amount of effector cell is the necessary condition for guaranteeing therapeutic effect, therefore is Enough immunocyte quantity is obtained, technical staff just constantly changes the various aspects of production cell by studying and testing Into improving immunocyte quantity by increasing the dosage of culture solution is most simple and effective method, and technical staff is to make to be immunized Cell quantity reaches treatment and requires, and generally can all reach cell culture bags volume to the use volume of culture solution in single culture bag Maximum value or the narrow fluctuation above and below its maximum value, with promotion culture solution dosage as much as possible.Although this method is one Determine to improve cell quantity in degree, but seriously reduces the culture efficiency of culture solution, the immunocyte quantity finally obtained It is extremely limited, it is ineffective.

Summary of the invention

It for overcome the deficiencies in the prior art, can the purpose of the present invention is to provide a kind of cultural method of immunocyte To effectively improve the culture quantity of immunocyte, the utilization rate of culture solution and the culture quantity of cell are substantially increased.

The purpose of the present invention adopts the following technical scheme that realization:

A kind of cultural method of immunocyte, comprising the following steps:

(1) peripheral blood mononuclear cells is acquired；

(2) above-mentioned cell inoculation is cultivated in the culture bottle containing culture medium；

(3) cell after above-mentioned steps (2) are cultivated is imported in culture bag, wherein the volume of culture solution is less than culture The volume of bag；

(4) immunocyte is harvested.

Further, the volume of culture solution and the ratio of culture bag volume are 0.42-0.55:1 in above-mentioned steps (3).

Further, it is put in 37 DEG C after the cell inoculation in above-mentioned steps (2) is in culture bottle, saturated humidity, 5%CO₂ It is cultivated 3-5 days in incubator.

Further, in the culture bottle in above-mentioned steps (2) and in the culture solution of step (3) added with cell because Son.

Further, cell is imported into culture bag in above-mentioned steps (3), supplements culture solution, continue to cultivate to after the 2-7 days It expands culture.

Further, the cell culture expanded culture in above-mentioned steps (3) is washed to being centrifuged after the 4-14 days Up to immunocyte after washing.

Further, the immunocyte is one of CIK cell, til cell, NK cell.

Compared with prior art, the beneficial effects of the present invention are: the present invention provides a kind of cultural method of immunocyte, lead to The ratio for crossing nutrient solution volume and culture bag volume in control incubation improves the amplification ability of cell and finally obtained thin Born of the same parents' quantity realizes a large amount of proliferation of immunocyte, improves the utilization rate of culture solution, saved cost.

Detailed description of the invention

Fig. 1 is the cell quantity statistical result datagram of the embodiment of the present invention 1 to 6 and comparative example 1 to 4.

Specific embodiment

In the following, being described further in conjunction with specific embodiment to the present invention, it should be noted that is do not collided Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.

1 Fiber differentiation CIK cell of embodiment

(1) it is anticoagulant to extract patient 50ml peripheral blood addition heparin sodium, is sub-packed in two 50ml centrifuge tubes and is centrifuged, from Mental and physical efforts 800G, 4 DEG C, acceleration 9, deceleration 9 is centrifuged 15min, draws upper plasma after centrifugation, be put in 4 DEG C of preservations；By two Haemocyte in centrifuge tube respectively with normal saline dilution to 35ml, slowly filling in equipped with 15ml lymphocyte separation medium from It is centrifuged in heart pipe, 4,20 DEG C of centrifugal force 800G, acceleration 4, deceleration centrifugation 17min, centrifugation terminate, and draw peripheral blood list A nucleus is put in 50ml centrifuge tube, with normal saline dilution to 50ml and is put in centrifuge centrifugation, centrifugal force 300G, acceleration 7, deceleration 5,4 DEG C of centrifugation 8min, wash cell twice；

(2) by the cell inoculation after washing with the pre-coated 75cm for having CD3 monoclonal antibody and RetroNectin of PBS²Culture bottle It is interior, and 30ml GT-T551 culture medium is added, wherein CD3 monoclonal antibody is 15ul/ bottles, RetroNectin is 60ul/ bottles, PBS is 10ml/ bottles, IL-2 1000IU/ml, IFN-r 1000IU/ml, gentamicin 160IU/ml, autologous plasma 5% will Above-mentioned culture bottle is put in 37 DEG C, saturated humidity, 5%CO₂It is cultivated 4 days in incubator；

(3) cell is averagely imported in the cell culture bags that two volumes are 1800ml, and supplement is self containing 2% respectively To 75ml, culture expands culture cell to after the 6th day the GT-T551 culture solution that blood plasma and IL-2 are 1000IU/ml, Supplement the volume for the GT-T551 culture solution culture solution into each culture bag for being 1000IU/ml containing 2% autologous plasma and IL-2 For 750ml, the ratio of nutrient solution volume and culture bag volume is 0.42:1, cultivates to after the 12nd day, cultured cell is led Enter Centrifuge Cup to be centrifuged, 5,4 DEG C of centrifugal force 400G, acceleration 7, deceleration centrifugation 8min, be collected after centrifugation immune thin Born of the same parents are in 50ml centrifuge tube and in centrifugal force 300G, acceleration 7,5,4 DEG C of centrifugation 8min of deceleration, and centrifuge washing twice, is added Cell liquid is imported normal saline bag by 2.5% human serum albumin.

2 Fiber differentiation CIK cell of embodiment

The difference of embodiment 2 and embodiment 1 are as follows: nutrient solution volume is 900ml in each culture bag in above-mentioned steps (3), Nutrient solution volume and the ratio of culture bag volume are 0.5:1.

3 Fiber differentiation CIK cell of embodiment

The difference of embodiment 3 and embodiment 1 are as follows: nutrient solution volume is 990ml in each culture bag in above-mentioned steps (3), Nutrient solution volume and the ratio of culture bag volume are 0.55:1.

4 Fiber differentiation CIK cell of embodiment

The difference of embodiment 4 and embodiment 2 are as follows: the culture solution used is ALyS505N, remaining is and embodiment 2 is identical.

5 Fiber differentiation til cell of embodiment

(1) fresh tumor tissue is put in the culture solution containing pake purpke dual anti-and gentamicin and is impregnated 10 minutes, then taken Tumor tissues are put in six orifice plates out, are wiped out fat and necrotic tissue with eye scissors, remaining tumor tissues turn after being cut to fragment Move on to 50ml centrifuge tube, be resuspended with culture solution to being centrifuged after 30 milliliters, centrifugal force 500G, 4 DEG C of centrifugation 5min, remove after centrifugation on Clearly, it is resuspended with culture solution to 10-20ml, and Collagenase I, II, IV (the use of concentration being 0.5mg/ml) is added, centrifuge tube is put into Rotation vortex mixer simultaneously rotates 1-2h, and cell is resuspended to 50ml with physiological saline after rotation, is centrifuged after mixing, centrifugal force 500G, 4 DEG C of centrifugation 5min, remove supernatant after centrifugation, and appropriate physiological saline is added according to cell quantity and mixes, with 100um's Cell strainer filtration cell, is centrifuged again, and centrifugal force 500G, 4 DEG C of centrifugation 5min remove supernatant after centrifugation, by cell physiology Salt water is resuspended to 5ml, and Ficol the and 5ml cell of Ficol, 5ml75% of 5ml100% is sequentially added in the centrifuge tube of 15ml Suspension is then centrifuged for, centrifugal force 800G, and 20 DEG C of centrifugation 17min take out mononuclearcell after centrifugation, is transferred to 15ml centrifugation It manages and is resuspended to 15ml, wash twice, centrifugal force 500G, 40 DEG C of centrifugation 5min；

(2) cell is resuspended in containing in the X-VIVO culture solution that autologous plasma is 2% and IL-2 is 6000IU/ml, is turned 24 orifice plates are moved to, are put in 37 DEG C, saturated humidity cultivates in 5%CO2 incubator, carries out half amount within every 2-3 days in incubation and changes Liquid, cell are transferred to 75cm after reaching certain amount²Culture bottle in cultivated, to be transferred to CD3 mono- after continuing culture 3 days Anti- and RetroNectin pre-coated culture bottle is cultivated, and culture 4 days is continued；

(3) cell is averagely imported in the cell culture bags that two volumes are 1800ml, and supplement contains self blood respectively Slurry is that the X-VIVO culture solution that 2% and IL-2 is 6000IU/ml carries out expansion training to cell after continuing culture 2 days to 90ml It supports, the X-VIVO culture solution that supplement is 2% containing autologous plasma and IL-2 is 6000IU/ml to final volume 900ml, culture solution is whole Volume and the ratio of culture bag volume are 0.5:1, continue culture 4 days, and cultured cell importing Centrifuge Cup is centrifuged, from Mental and physical efforts 400G, 4 DEG C of centrifugation 8min, acceleration 7, deceleration 5, are collected after centrifugation cell in 50ml centrifuge tube and wash twice, add Enter 2.5% human serum albumin, cell liquid is imported into normal saline bag.

6 Fiber differentiation NK cell of embodiment

(1) it is anticoagulant to extract patient 50ml peripheral blood addition heparin sodium, is sub-packed in two 50ml centrifuge tubes and is centrifuged, from Mental and physical efforts 800G, 4 DEG C, acceleration 9, deceleration 9 is centrifuged 15min, draws upper plasma after centrifugation, be put in 4 DEG C of preservations；By two Haemocyte in centrifuge tube respectively with normal saline dilution to 25ml, slowly filling in equipped with 15ml lymphocyte separation medium from It is centrifuged in heart pipe, 4,20 DEG C of centrifugal force 800G, acceleration 4, deceleration centrifugation 17min, centrifugation terminate, and draw peripheral blood list A nucleus is put in 50ml centrifuge tube, with normal saline dilution to 50ml and is put in centrifuge centrifugation, centrifugal force 300G, acceleration 7, deceleration 5,4 DEG C of centrifugation 8min, wash cell twice；

(2) by the cell inoculation after washing in 75cm²In culture bottle, and the GT-T561 culture solution of 40ml is added, wherein certainly Body blood plasma is 5%, IL-2 200IU/mL, IL-15 50ng/mL, IL-21 25ng/mL, gentamicin 160IU/mlL, And the trophocyte (2 × 10 after pipe recovery is added⁶A K562 cell), in incubation, appropriate supplement contains daily or every other day There is the GT-T561 culture solution that autologous plasma is 2%, IL-2 200IU/mL, IL-15 50ng/mL, IL-21 are 25ngmL, trains It supports to the 5th day and cell is transferred to 175cm²In culture bottle, and fluid infusion is to 150ml, culture to fluid infusion in the 6th day to 300ml；

(3) cell culture was expanded culture to the 7th day, first by the trophocyte cell (5 × 10 after pipe recovery⁶It is a K562 cell) it is added in culture bottle, cell is averagely imported again after mixing in the cell culture bags that two volumes are 1800ml, and It is supplemented respectively containing the GT- that autologous plasma is 2%, IL-2 200IU/mL, IL-15 50ng/mL, IL-21 are 25ng/mL For T561 culture solution to final volume 800ml, the ratio of culture solution final volume and culture bag volume is 0.44:1；Culture was to the 12nd day Afterwards, cultured cell importing Centrifuge Cup is centrifuged, centrifugal force 400G, 4 DEG C of centrifugation 8min, acceleration 7, deceleration 5, from Cell is collected after the heart in 50ml centrifuge tube and to wash twice, and 2.5% human serum albumin is added, cell liquid is imported into normal saline bag ?.

Comparative example 1

Comparative example 1 provides a kind of cultural method of immunocyte and culture solution of the difference in step (3) of embodiment 1 Total dosage is under conditions of being 1500ml, and the ratio of nutrient solution volume and culture bag volume is 0.83:1, remaining and is implemented Example 1 is identical.

Comparative example 2

Comparative example 2 provides a kind of cultural method of immunocyte and the culture of embodiment 2 being distinguished as in step (3) The total dosage of liquid is under conditions of being 1800ml, and the ratio of nutrient solution volume and culture bag volume is 1:1, remaining is and embodiment 6 is identical.

Comparative example 3

Comparative example 3 provides a kind of cultural method of immunocyte and the culture of embodiment 3 being distinguished as in step (3) The total dosage of liquid is 2000ml, and the ratio of nutrient solution volume and culture bag volume is 1.1:1, remaining is and embodiment 3 is identical.

Comparative example 4

Comparative example 4 provides a kind of cultural method of immunocyte and the culture of embodiment 4 being distinguished as in step (3) The total dosage of liquid is under conditions of being 1800ml, and the ratio of nutrient solution volume and culture bag volume is 1:1, remaining is and embodiment 4 is identical.

The cell amplification ability and cell quantity and embodiment 1 of Statistics Implementation example 1 to 6 and comparative example 1 to 4 respectively To the 4 cell growth rate relative to comparative example 1 to 4, the results are shown in Table 1.

Table 1

Same immunocyte is come it can be seen from the statistical data of embodiment 1 to 6 in upper table 1 and attached drawing 1 It says, in the case where total culture solution dosage is constant, changes the proportionate relationship of nutrient solution volume and culture bag volume, the expansion of cell Energization power and cell quantity can change, although with the reduction of nutrient solution volume and culture bag volume fraction, the expansion of cell Energization power improves, but the dosage due to reducing culture solution in each culture bag, in the case where identical culture solution It needs to be divided in cell in more culture bags, on the one hand increases the triviality of operation, need to take more time, it is another Aspect causes the waste of culture bag, increases cost.Currently preferred nutrient solution volume and the ratio of culture bag volume are 0.42- On the one hand 0.55:1 improves the amplification ability of cell, on the other hand guarantees the efficiency of cell culture, into one in the range Walk save the cost.

Allogenic cell is in the equal situation of culture solution dosage it can be seen from the comparison of embodiment 1 to 4 and comparative example 1 to 4 Under, when nutrient solution volume and culture bag volume are equal or when being slightly larger than culture bag volume, the amplification ability of cell and final Obtained cell quantity is not as good as when nutrient solution volume is less than culture bag volume.Illustrate to show using method provided by the invention Write improve immunocyte amplification ability and finally obtained immunocyte culture quantity, while with the condition of culture of cell, training Environment, cultural method, culture level are supported without direct relation, i.e., experimenter is by improving cell cultivation level or changing cell training Method etc. is supported come after promoting cell quantity, the method that can still provide according to the present invention reasonably selects nutrient solution volume and culture bag The ratio of volume further increases cell quantity.

The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto, The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention Claimed range.