CN101481678B - Natural killer cell and cultivation method thereof - Google Patents
Natural killer cell and cultivation method thereof Download PDFInfo
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Abstract
The invention discloses a natural killer cell and a culturing method thereof. The natural killer cell is a human natural killer cell NKG cell of which the preservation number is CGMCC No. 2901. The NKG cell has strong lethality to tumor cells, strong survival ability in vivo, stable quality and strong tumor-resistant reaction. Cells cultured by the method have fast growing speed, large quantity, even quality, high and stable survival rate. The preparation method has easy operation, controllable quality, high yield and low cost, and realizes mass, scale, homogeneous and streamlined production for cell treating products like medicament. The method for culturing NKG cells in a large scale can ensure cell phenotype, purity, activity, cell density, culturing volume and the like to achieve clinical treatment and ensure that the NKG cells can be applied to clinical treatment for malignant tumors and injection for 1 to 5 patients at the same time. Therefore, the cells, the method for preparing cells and the preparation method for cell liquid preparation have wide application value in treating malignant tumors.
Description
Technical field
The present invention relates to a kind of natural killer cell and cultural method thereof.
Background technology
The cellular immunization treatment that is used for oncotherapy than other cell therapy scheme Zao enter clinical, in scheme on probation, LAK cell, CIK cell and A-NK cell all are anticancer systems that the NK cell is taken as the leading factor, this is because natural killer cell (natural killer, NK) be main natural immunity member, comprise in malignant tumour first defence line and can play a significant role in the defence disease.Compare with the cell (for example T cell) of acquired immune system, the NK cell need not to stimulate in advance or sensitization can start cytotoxic activity, thereby instant natural reaction is provided.The NK cell shows as direct dissolving and two aspects of secrete cytokines to the advantage of killing and wounding of tumour, and its killing tumor cells not only can but also can have been passed through the FAS aglucon by pore-forming protein.The NK cell can produce TNF-α, IFN-γ and IL-1, and these cytokines have very critical role in NK cell antitumor response and transfer T lymphocyte.Because the NK cell has lethal effect and target cell fast widely, prompting NK cell can be used for the treatment of malignant tumour.Studies have shown that, adopt transfer external activation, the amplification effective from body or allogeneic NK cell to treating multiple leukemia and solid tumor.
It is the NK cell that tumour cell is had killing ability by external preparation that the NK cell is used for oncotherapy, pass through a series of processing (as irradiation, cytokine stimulation etc.) back infusion again and give the patient, in patient's body, realize the killing and wounding of tumour cell reached result of treatment.
Obtain sufficient amount, strong, the stay-in-grade NK cell of killing activity, be the key that realizes the clinical application of NK cell.But be difficult in external a large amount of amplifications owing to derive from the NK cell of peripheral blood, and separation and purification NK cell has certain technical difficulty from peripheral blood, be difficult to avoid the lymphocytic pollution of T, can't obtain the NK cell of a large amount of homogeneous and reach clinical required standard, so that the NK cell can't be widely used in is clinical; In addition, in some case, a little less than the cell-mediated antitumor reaction of NK, may with NK cell killing ability, survival ability difference or relevant in vivo to factors such as the tumor locus migration are limited.
Therefore, seek active strong NK cell and to set up external large scale culturing system be NK cell adoptive immunotherapy tumour is needed solution badly in Clinical Application problem.Set up easy handling, extensive cultured continuously of NK clone and preparation technology quality controllable, that output is high, its lytic activity is high, cost is low can solve the bottleneck difficult problem that the cellular immunization treatment technology is faced, and make the cell therapy product might be as medicine mass, mass-producing, homogenization, procedure production.
Summary of the invention
An object of the present invention is to provide a kind of natural killer (NK) cell that is used for the treatment of tumour.
Natural killer provided by the present invention (NK) cell behaviour natural killer cell NKG cell, this cell strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center on February 9th, 2009 and (is called for short CGMCC, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCC No.2901.
Another object of the present invention provides a kind of method of cultivating above-mentioned NKG cell.
The method of the above-mentioned NKG cell of cultivation provided by the present invention is to cultivate the NKG cell with cell culture medium; Described cell culture medium can be that to contain the L-glutaminate that final concentration is 1.5mM-2.5mM, the sodium bicarbonate that final concentration is 2.0g/L-2.4g/L, the ribonucleoside that final concentration is 5.0mg/L-9.0mg/L, the dezyribonucleoside that final concentration is 5.0mg/L-9.0mg/L, the new-born calf serum that final concentration is 8.0%-12.5% (v/v), horse serum, the final concentration that final concentration is 8.0%-12.5% (v/v) be 5.0 * 10
4IU/L-1.25 * 10
5The α of compositions such as the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium.
Described ribonucleoside is the mixture of A and G and C and U; Described dezyribonucleoside is the mixture of dA and dG and dC and dT.
Described cell culture medium can be preferably following substratum: contain the L-glutaminate that final concentration is 2.0mM, final concentration is the sodium bicarbonate of 2.2g/L, final concentration is the ribonucleoside of 7.0mg/L, final concentration is the dezyribonucleoside of 7.0mg/L, final concentration is the new-born calf serum of 10% (volume percent), final concentration is the horse serum of 10% (volume percent), and final concentration is 1.0 * 10
5The α of the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium; Described ribonucleoside is A and G and C and U; Described dezyribonucleoside is dA and dG and dC and dT.
In the described cultivation, described best cultivation can be shakes the pocket type cultivation; The described swing rate that shakes the pocket type cultivation can be 4rpm-10rpm, and amplitude of fluctuation can be 3 °-9 °; Described swing rate is preferably 7rpm, and described amplitude of fluctuation is preferably 7 °.
It is CO in 35 ℃-37 ℃, air that described culture condition can be temperature
2Volume content can be 4.0%-10.0%, CO
2Flow velocity can be 0.10L/min-0.25L/min; Described temperature is preferably 37 ℃, CO in the described air
2Volume content is preferably 5.0%, described CO
2Flow velocity is preferably 0.15L/min.
Last purpose of the present invention provides a kind of method of liquid preparation of the NKG of preparation cell.
Preparation method provided by the present invention comprises the steps: 1) cultivate described cell with above-mentioned arbitrary described cell culture processes; 2) with gamma-rays the cell that obtains in the described step 1) is carried out irradiation.
Wherein, the dosage of described irradiation can be 800cGy-1200cGy, is preferably 800cGy.
The speed of described irradiation can be 50cGy/min-200cGy/min, is preferably 50cGy/min.
Described liquid preparation specifically can be injection.
The enchylema body preparation that is obtained by aforesaid method also belongs to protection scope of the present invention.
The present invention has adopted the wave bio-reactor that NK cells of human beings is cultivated.The present inventor cultivates the NKG cell with different substratum, observe indexs such as its speed of growth in various substratum, cell state, finally determined to be used for type of culture medium, the serum content of NK cell cultures and added condition such as cytokine kind.On this basis, the contriver has investigated and has used cell bags to cultivate the influence to NK cell proliferation, survival rate, yield and killing activity of additive capacity, CO2 concentration and flow velocity, swing rate and pendulum angle, substratum and the cytokine prolongation and the dosage etc. of cell inoculation density different in the NK cell, type of culture medium, nutritive ingredient; And the yield of NK cell growth curve, cytobiology feature and NK cell product and survival rate, purity and homogeneity or characteristic surface sign studied; Finally determined NKG cell large scale preparation method.
NKG cell of the present invention is strong to the lethality of tumour cell, steady quality, and antitumor reaction is strong.Use the inventive method culturing cell; high cell growth speed, many, the quality homogeneous, active high and stable of quantity; and preparation method of the present invention has easy handling, quality controllable, output is high, cost is low characteristics, realized that cell therapy product is as medicine mass, mass-producing, homogenization, procedure production.Use the technology that the present invention set up the NKG cell is carried out large scale culturing, cell phenotype, purity, activity, cell density, volume of culture etc. all can guarantee to reach clinical application, the clinical treatment that can be used for multiple malignant tumour can be for 1-5 position patient while infusion.Therefore, the preparation method of cell of the present invention, the method for preparing cell and enchylema body preparation has a wide range of applications in treatment malignant tumour field.
Description of drawings
Fig. 1 is the propagation situation of NKG cell.
Fig. 2 is the killing activity of NKG cell to tumour cell.
Fig. 3 is the surface molecular expression of NKG cell.
Fig. 4 is the cell state of NKG cell under different culture medium culturing situations.
Fig. 5 is the growth velocity of NKG cell in different substratum.
Fig. 6 is the growth velocity of NKG cell under different culture condition.
Fig. 7 be 48 hours NKG cell proliferation situations behind the irradiation (
3HR mixes).
Fig. 8 is surface C D3, CD16, CD19, the CD56 developed by molecule situation of NKG cell behind the pre-irradiation.
Fig. 9 is the apoptosis situation of NKG cell.
Figure 10 is the killing activity of NKG cell (800cGy irradiation) to K562 cell, Ho8910 cell.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Lymphocyte separation medium is available from Sigma company, the MACS magnetic bead is available from Miltemyi Biotec company, new-born calf serum (super) is available from Shanghai Excell Biology Product Co., Ltd., horse serum is available from Hyclone company, α-MEM liquid nutrient medium, α-MEM dehydrated medium, 1640 substratum are available from Invitrogen company, and dimethyl sulfoxide (DMSO) is given birth to worker's biotechnology company limited available from Shanghai, and cell bags is available from U.S. Wave Biotech company, fluidic cell antibody is available from BD company
51Cr is available from Chinese isotropic substance main office, and the soft-agar cloning test kit is available from GENMEDSCIENTIFICS company, and nude mice is available from model animal institute of Nanjing University, and the K562 cell is available from the biological product collecting center (ATCC) of USS, and catalog number is CCL-243; The Ho-8910 cell is available from Chinese Academy of Sciences's cell bank, catalog number is TCHu 24, the HepG2 cell is available from the biological product collecting center (ATCC) of USS, catalog number is HB-8065, the Hep2 cell is available from the biological product collecting center (ATCC) of USS, catalog number is CCL-23, and WI-38 cell is available from the biological product collecting center (ATCC) of USS, and catalog number is CCL-75.
Micropipet is available from French Gilson company, low speed centrifuge is available from Sigma company, the MACS magnetic separator is available from Miltemyi Biotec company, 78-1 type magnetic force heating stirrer is pacified general electronic engineering company limited available from Jiangsu, clean work station is available from Jinan City, Shandong Province air purifying and sterilizing instrument factory, AvantiTM J-20 * P type whizzer is available from Beckman company, Ultralow Temperature Freezer is available from Harris Manufacturing CO. company, the CO2 incubator is available from Thermo Forma company, wave bio-reactor (2/10 type) is available from U.S. WaveBiotech company, flow cytometer is available from BD company, the γ calculating instrument available from good photoelectric instrument company, biomicroscope is available from German Carl Zeiss company, and the blood irradiation instrument is available from German BIOBEAM company.
The foundation of embodiment 1, NKG cell strain
One, the separation of cell
1. peripheral blood sample: get the patient's who suffers from non_hodgkin lymphoma peripheral blood 10ml, anticoagulant heparin is agreed through the patient.
2. the separation of peripheral blood mononuclear cell: behind the aseptic PBS solution dilution of 10ml anticoagulation cirumferential blood sample, carry out density gradient centrifugation with lymphocyte separation medium, 2200 rev/mins, 30 minutes, 20 degree; The mononuclearcell of interlayer face in the absorption uses the PBS solution washing 3 times, and 1500 rev/mins, 10 minutes, 20 degree; Use the aseptic PBS solution of 250 μ l re-suspended cell, counting, cell concn is 1.1 * 10
7/ 250 μ l, 4 degree are placed, and are standby.
3.NK the separation and purification of cell: in above-mentioned cell solution, add the non-specific mouse IgG of 20 μ l, 4 degree, 30 minutes, with the sealing non-specific binding.In cell solution, add 100 μ l CD56 MicroBeads, the vibration mixing, 4 degree, 30 minutes, per 10 minutes mixings were once.Use MACS sorter sorting cells, collect the CD56 positive NK cells.Use the PBS solution washing 2 times, 1500 rev/mins, 10 minutes, 20 degree; Use complete α-MEM liquid nutrient medium re-suspended cell, adjusting concentration is 1.0 * 10
5/ ml, 37 degree, 5%CO2 cultivates; Above-mentioned cell detects through flow cytometer, and the CD56 positive cell accounts for more than 95%.
4. cloning grown cell enlarged culturing: the NK cell of above-mentioned purifying is contained 5 cells through limiting dilution to every milliliter, add in 96 orifice plates 100 μ l/ holes, be equivalent to every hole and contain 0.5 cell, nutrient solution is complete α-MEM liquid nutrient medium, 37 degree, and 5%CO2 cultivates; In time add IL-2 solution, its concentration is maintained about 100U/ml.Observe clone's formation situation, cultivate hole, 10 days rear sections and cell clone occurs, select single clone hole, when treating that cell grows to sufficient amount, picking is the cell of cloning growth, change over to and carry out enlarged culturing in 24 orifice plates, change in the culturing bottle after the cell proliferation and cultivate, the cell colony that acquired character is stable, wherein 1 strain cell continuous passage was cultivated 184 days, go down to posterity after the freeze-stored cell recovery and cultivated 69 days, cell growing state, cell state etc. are all good, with this strain cell called after NKG cell.
Two, the biological characteristics of NKG cell
1.NKG the multiplication capacity analysis of cell: the time-effect relationship of NKG cell proliferation is observed in cell counting: add 10 in 6 orifice plates
5The cell of cells/ml and complete α-MEM liquid nutrient medium were counted once in per two days and half amount is changed liquid, and cultured continuously 10 days, result show that the NKG cell has rate of propagation (Fig. 1) faster.
2.NKG cell is to the killing activity analysis of tumour cell: utilize
51The Cr release experiment detects the killing activity of NKG cell to K562 cell, HepG2 cell and Ho8910 cell.Experimental technique is as follows: the use Sodium chromate (
51Cr) labels targets cell, i.e. K562 cell, HepG2 cell and Ho8910 cell: 37 ℃ of water-baths, 1 hour, rocked once the mixing cell in per 5～10 minutes.Use the RPMI-1640 washed cell afterwards, 3000rpm, 10 minutes, 20 ℃, totally 3 times.Use RPMI-1640 that cell concn is adjusted into 1 * 10
5/ ml, standby.4 hours
51Cr release experiment: in 96 hole circle floor cells culture plates, add
51The target cell of Cr mark, every hole add 100 μ l.According to the concentration of the ratio of effector cell and target cell (imitate target than) equalizing effect cell (NKG cell), and add the NKG cell in each hole, volume is 100 μ l/ holes.The nature release aperture does not add the RPMI-1640 that the effector cell only adds 100 μ l, adds 100 μ l 2%Triton-X 100 in the maximum release aperture.Four multiple holes are put in each experiment.Of short duration centrifugal after, 37 ℃, 5% CO
2, cultivated 4 hours.1000rpm, 10 minutes, centrifugal culture plate, every hole sucking-off 100 μ l supernatant liquors were measured the per minute radioactive activity (cpm value) in the supernatant liquor on the γ calculating instrument in detector tube.The calculating of killing activity: represent: cytotoxicity (%)=[(experimental group cpm-nature release group cpm)/(maximum release group cpm-nature release group cpm)] * 100% with cytotoxicity per-cent.The result shows that NKG all has very strong killing activity (Fig. 2) to K562 cell, HepG2 cell and Ho8910 cell.
3.NKG the mensuration of cell phenotype: use flow cytometer to detect the expression of NKG cell surface molecule, the result shows, NKG cell surface CD56 high positive, CD3 is negative, CD16 is negative, CD19 is negative, CD25 is positive, CD48 is positive, the high positive of CD54, the CD69 positive, the CD95 positive (Fig. 3).
This cell name is people's natural killer cell NKG cell, this cell strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center and (has been called for short CGMCC on 02 09th, 2009, address: Datun Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), preserving number is CGMCCNo.2901.
The preparation of the cultural method of embodiment 2, NKG cell and the agent of NKG injection cell
One, culture condition gropes
The deactivation of new-born calf serum, horse serum: the new-born calf serum, the horse serum that will not have bacterium, mycoplasma and virus pollution place 56 ℃ of waters bath with thermostatic control 30 minutes, and the deactivation complement is put in 4 ℃ of refrigerators afterwards and preserves, and is standby.
Following each substratum of preparation:
1) preparation of α-MEM liquid nutrient medium fully
Get α-MEM liquid nutrient medium and (contain L-glutaminate, sodium bicarbonate, ribonucleoside and dezyribonucleoside) 800ml, add following material respectively, in the bracket is the final concentration of this material in substratum: new-born calf serum (10%, v/v), horse serum (10%, v/v), human interleukin-12 (1.0 * 10
5IU/L).
2) preparation of α-MEM dehydrated medium fully
Get α-1 bag in MEM pulvis (10.2g contains L-glutaminate, ribonucleoside and dezyribonucleoside), join in the beaker that the 950ml tri-distilled water is housed, magnetic stirrer is dissolved it fully, adds the NaHCO of 2.2g
3, continue stirring and dissolving, with the HCl adjust pH 7.2 of 1M, add tri-distilled water to 1,000ml, 0.22 μ m membrane filtration degerming, 4 ℃ of preservations after the packing.Getting substratum 800ml before the use, add following material respectively, is the final concentration of this material in substratum in the bracket: new-born calf serum (10%, v/v), horse serum (10%, v/v), human interleukin-12 (1.0 * 10
5IU/L).
3) complete 1640 culture medium preparation
Get 1 bag in 1640 pulvis (10.4g), join in the beaker that the 950ml tri-distilled water is housed, magnetic stirrer is dissolved it fully, adds the NaHCO of 2.0g
3, continue stirring and dissolving, with the HCl adjust pH 7.2 of 1M, add tri-distilled water to 1,000ml, 0.22 μ m membrane filtration degerming, 4 ℃ of preservations after the packing.Getting substratum 800ml before the use, add following material respectively, is the final concentration of this material in substratum in the bracket: new-born calf serum (10%, v/v), horse serum (10%, v/v), human interleukin-12 (1.0 * 10
5IU/L).
With the NKG cell with 2 * 10
4The density of individual/ml is inoculated in respectively in the above-mentioned substratum of 500ml, cultivates with cell bags, and culture condition is 37 ℃, 5.0%CO
2, CO
2Flow velocity 0.15L/min, 7rpm/7 °.Per therebetween 72 hours add 2.5ml concentration is 2.0 * 10
4The human interleukin-12 solution of IU/ml, the observation of cell speed of growth and state.
(A, C, E gathered picture under 100 times of mirrors on the 3rd day for the inoculation back to the growth conditions of NKG cell in three kinds of substratum as shown in Figure 4; B, D, F gather picture down for the 7th day 100 times of mirrors in inoculation back.A, B are the NKG cell of complete 1640 culture medium culturing; C, D are the NKG cell that complete α-MEM dehydrated medium is cultivated; E, F are the NKG cell that complete α-MEM liquid nutrient medium is cultivated), show that NKG cell state in three kinds of substratum is all better.
Make the growth curve of NKG cell in three kinds of substratum, as shown in Figure 5.Show that the speed of growth is faster than in complete 1640 dehydrated mediums and complete α-MEM dehydrated medium in complete α-MEM liquid nutrient medium, difference has significance.
The above results shows that α-MEM liquid nutrient medium is suitable for the growth of NKG cell fully.
Two, preparation injection
(1) cultivates with cell culture medium I
Cell culture medium I: be that to contain the L-glutaminate that final concentration is 2.0mM, the sodium bicarbonate that final concentration is 2.2g/L, the ribonucleoside that final concentration is 7.0mg/L, the dezyribonucleoside that final concentration is 7.0mg/L, the new-born calf serum that final concentration is 10% (v/v), horse serum, the final concentration that final concentration is 10% (v/v) be 1.0 * 10
5The α of the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium; Described ribonucleoside is that (mass ratio of 4 kinds of ribonucleoside is 1: 1: 1: 1) for A, G, C and U; Described dezyribonucleoside is that (mass ratio of 4 kinds of dezyribonucleosides is 1: 1: 1: 1) for dA, dG, dC and dT.
Preparation process following (being applicable to 1 routine patient's infusion):
1, the static cultivation of NKG cell: with the NKG cell with 2 * 10
4The density of/ml is inoculated among the cell culture medium I, uses the 250ml culturing bottle, and 37 ℃, 5%CO
2, cultivate.
2, the cultivation of NKG cell in the cell bags:
After the NKG cell that is in logarithmic phase in the step 1 washed (800rpm, 10min, 25 ℃) twice with aseptic Hanks, with 2 * 10
4The density of/ml is inoculated in the cell bags of 2.0 liters of sizes that 500ml cell culture medium I is housed, and uses the wave bio-reactor to shake pocket type and cultivates, and culture condition is as follows: 37 ℃, 5.0%CO
2, CO
2Flow velocity: 0.15L/min, 7rpm/7 ° (swing rate/amplitude of fluctuation).
3, the interpolation of substratum and human interleukin-12:
When cell density reaches 1.2-1.6 * 10
5During/ml (inoculation back the 3rd day), interpolation 200ml cell culture medium I and 2.5ml concentration are 2.0 * 10 in cell bags
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 5.0%CO
2, CO
2Cultivate under flow velocity: 0.15L/min, 7rpm/7 ° the condition; When cell density reaches 7.0-8.5 * 10
5During/m (inoculation back the 6th day) in cell bags, add 300ml cell culture medium I and 3.5ml concentration is 2.0 * 10
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 5.0%CO
2, CO
2Cultivate under flow velocity: 0.15L/min, 7rpm/7 ° the condition.
3 repetitions are established in test.
Add up the speed of growth of cell during this time, make the growth curve (Fig. 6) of cell.The result shows that when using the aforesaid method culturing cell, the cell growth is rapid, and the cell quantity that obtains is many.
When the density of cell reaches 1.1 * 10 approximately
6Individual/ml (inoculation back 7-8 days) detected cell growth state, and expected blue dyeing counting with platform.Cell state is good as a result, and platform expects that the painted cell count of blue dyeing less than 10%, shows the effective of the inventive method culturing cell.
4, the collection of NKG cell and radiation treatment:
(1) irradiation dose gropes
The density of cell reaches 1.1 * 10 approximately in step 3
6Individual/ml (inoculation back 7-8 days), collecting cell is in the aseptic centrifugal bottle of 1000ml, the 500ml/ bottle, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, add serum-free α-MEM liquid nutrient medium re-suspended cell, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, repeated washing one time; Add serum-free α-MEM liquid nutrient medium re-suspended cell, adjust cell concn to 2 * 10
9Individual/L, the 500ml/ packing.
Cell is carried out various dose irradiation, and experimental procedure is as follows: use the blood irradiation instrument that the NKG cell of results is carried out irradiation, dosage is respectively 0cGy, 100cGy, 200cGy, 400cGy, 800cGy, 1600cGy, and speed is 50cGy/min; Cell uses behind the irradiation
3The H method of mixing detects its propagation situation, and the result shows that as shown in Figure 7 the NKG cell is behind the various dose x ray irradiation x, and proliferation rate has decline in various degree, and the NKG cell is no longer bred behind the 800cGy dosage irradiation, shows, 800cGy dosage irradiation is suitable for the NKG cell most.
(2) irradiation
The density of cell reaches 1.1 * 10 approximately in step 3
6Individual/ml (inoculation back 7-8 days), collecting cell is in the aseptic centrifugal bottle of 1000ml, the 500ml/ bottle, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, add serum-free α-MEM liquid nutrient medium re-suspended cell, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, repeated washing one time; Add serum-free α-MEM liquid nutrient medium re-suspended cell, adjust cell concn to 2 * 10
9Individual/L, the 500ml/ packing.Packaged cell is used the irradiation of γ irradiation instrument, speed: 50cGy/min, time: 16min, total dose: 800cGy obtains the agent of NKG injection cell.
5, NKG injection cell agent effect detection
(1) uses Flow cytometry NKG cell surface CD3, CD16, CD19, CD56 developed by molecule.Fluidic cell antibody is respectively: anti-people CD3, CD16, CD19, CD56 antibody.
The result as shown in Figure 8, phenotype is CD3-CD16-CD19-CD56+ behind the NKG cell pre-irradiation, shows that cell phenotype is stable in the injection cell agent of the inventive method preparation.
(2) Flow cytometry NKG apoptosis situation
The result as shown in Figure 9, the NKG cell of living accounts for more than 90% of total cell count, expects that with platform the result of blue staining conforms to, and shows that viable cell quantity is many in the injection cell agent of the inventive method preparation, is beneficial to the raising therapeutic efficiency.
(3) NKG tumorigenicity detects behind the irradiation
External soft-agar cloning test method: (available from GENMED SCIENTIFICS company) experimentizes with test kit, and cell concn in the injection is adjusted to 2.5 * 10
3Individual/ml, get the 3ml cell solution in the aseptic centrifuge tube of 50ml, 800rpm, 10min, 20 ℃, abandon supernatant after centrifugal, add and support the liquid re-suspended cell among the 3.375ml, and add the clone's liquid of 1.125ml and the recombinated interleukin-2 solution (2.0 * 10 of 25 μ l
4IU/ml), mixing is added to above-mentioned cell suspension in 12 orifice plates that are covered with gel, the 1.5ml/ hole, and mixing gently, room temperature was placed after 2 hours, 12 orifice plates was put into incubator, 37 ℃, 5.0% CO
2Cultivate that every hole adds the low liquid 0.5ml of supporting that contains the 100IU/ml recombinated interleukin-2,37 ℃, 5.0% CO after 12 hours
2Cultivate, added recombinated interleukin-2 in per 72 hours and hanged down foster liquid; Cultivate after 14 days, observe cell clone formation situation in 12 orifice plates.The result shows that the acellular clone of each Kong Zhongjun forms after cultivating in 14 days.Test is through 3 repetitions, unanimities as a result.(in support liquid, clone's liquid, low to support liquid be to provide in the test kit).
Tumor after being inoculated into nude mice test: according to " three ones of Chinese pharmacopoeia, version required to detect the tumorigenicity of the NKG cell of the inventive method preparation to nude mice in 2005.Use the Hep2 cell as positive control cell, injection volume is 1.0 * 10
6Cell/mouse, back subcutaneous injection, volume injected 0.2ml; Use WI-38 cell as negative control cell, injection volume is 1.0 * 10
7Cell/mouse, back subcutaneous injection, volume injected 0.2ml; Use NKG cell in the injection of the present invention as experimental cell, injection volume is 1.0 * 10
7Cell/mouse, back subcutaneous injection, volume injected 0.2ml; The inoculation back is observed the mouse back tubercle and is formed situation, and in inoculation the 3rd week of back, tumor nodule all appears in 12 nude mice backs of positive group, and the increase of carrying out property, and each 12 nude mice of negative group and experimental group are until inoculation 12 weeks of back, and tubercle does not all appear in the back.Inoculating cell after 12 weeks is put to death nude mice, uses tumor cell invasion situation in the Flow cytometry peripheral blood, and histopathology detects tumor cell invasion situation in the internal organs such as liver, spleen.The result shows, does not all detect the NKG cellular infiltration in experimental mice peripheral blood and each internal organs.Experiment is through 3 repetitions, unanimities as a result.
Above result shows that there is not tumorigenicity in the NKG injection cell agent of the inventive method preparation.
(4)
51The Cr release experiment detects the killing activity of NKG cell to K562 cell and Ho8910 cell
Experimental technique is as follows: the use Sodium chromate (
51Cr) labels targets cell, i.e. K562 cell and Ho8910 cell: 37 ℃ of water-baths, 1 hour, rocked once the mixing cell in per 5～10 minutes.Use the RPMI-1640 washed cell afterwards, 3000rpm, 10 minutes, 20 ℃, totally 3 times.Use RPMI-1640 that cell concn is adjusted into 1 * 10
5/ ml, standby.4 hours
51Cr release experiment: in 96 hole circle floor cells culture plates, add
51The target cell of Cr mark, every hole add 100 μ l.According to the concentration of the ratio of effector cell and target cell (imitate target than) equalizing effect cell (NKG cell), and add the NKG cell in each hole, volume is 100 μ l/ holes.The nature release aperture does not add the RPMI-1640 that the effector cell only adds 100 μ l, adds 100 μ l 2%Triton-X100 in the maximum release aperture.Four multiple holes are put in each experiment.Of short duration centrifugal after, 37 ℃, 5% CO
2, cultivated 4 hours.1000rpm, 10 minutes, centrifugal culture plate, every hole sucking-off 100 μ l supernatant liquors were measured the per minute radioactive activity (cpm value) in the supernatant liquor on the γ calculating instrument in detector tube.The calculating of killing activity: represent: cytotoxicity (%)=[(experimental group cpm-nature release group cpm)/(maximum release group cpm-nature release group cpm)] * 100% with cytotoxicity per-cent.
3 repetitions are established in experiment.The result as shown in figure 10, the killing activity of imitating the target ratio and be 1: 1 o'clock NKG cell is about 25%, the killing activity of 2: 1 o'clock NKG cells is about 40%, the killing activity of 5: 1 o'clock NKG cells is about 60%, the killing activity of 10: 1 o'clock NKG cells is about 70%, the killing activity rate of 20: 1 o'clock NKG cells is between 80%-90%, and the result shows that NKG all has very strong killing activity to K562 cell and Ho8910 cell.
(5) in vivo test
Experimental procedure:
Set up nude mice ovarian cancer ascites model: use the Ho8910 cell to give nude mice (3-4 age in week, female, BALB/c source) lotus knurl, 5 * 10 by the mode of abdominal injection
6Cell/mouse after inoculating cell 3-4 week, ascites occurs in the nude mice abdominal cavity, and occur becoming thin, One's spirits are drooping, the movable symptom that reduces, the emaciation situation finally appears, mouse is all death in 6 weeks after ascites occurring.
NKG cell therapy mice with tumor: get NKG injection cell of the present invention agent, by 3.33 * 10
4Cell/g (body weight) dosage is given the nude mice (mouse of inoculation Ho8910 cell after 3 weeks) that has formed ascites, and through abdominal injection NKG cell, volume is 100 μ l/ mouse, for three days on end, and 1 time/day; Set up control group simultaneously, abdominal injection serum-free α-MEM liquid nutrient medium, volume are 100 μ l/ mouse, for three days on end, and 1 time/day.Observe mouse active situation and lifetime.
Repetition, unanimity are as a result established in experiment 3 times.The result shows that the NKG cell therapy can improve the life quality of mice with tumor, prolongs lifetime.The NKG cell therapy group mouse mental status is significantly better than control group, the activity of treatment group mouse more than control group, occur cachectic time delay, contrast group leader 5-6 week lifetime.
(6) exogenous factors such as bacterium, virus and mycoplasma detect
According to " three ones of Chinese pharmacopoeia, exogenous factor pollution condition such as bacterium, virus and mycoplasma in the NKG injection cell agent of version requirement detection the inventive method in 2005 preparation.
The result shows that exogenous factors such as bacterium, virus and mycoplasma are all negative in the different batches NKG cell.
More than every result show that the NKG injection cell agent of the inventive method preparation is used for the treatment of the malignant tumour security and validity meets the requirements.
(2) cultivate with cell culture medium II
Cell culture medium II: be that to contain the L-glutaminate that final concentration is 1.5mM, the sodium bicarbonate that final concentration is 2.0g/L, the ribonucleoside that final concentration is 5.0mg/L, the dezyribonucleoside that final concentration is 5.0mg/L, the new-born calf serum that final concentration is 8.0% (v/v), horse serum, the final concentration that final concentration is 8.0% (v/v) be 5.0 * 10
4The α of the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium; Described ribonucleoside is that (mass ratio of 4 kinds of ribonucleoside is 1: 1: 1: 1) for the mixture of A, G, C and U; Described dezyribonucleoside is that (mass ratio of 4 kinds of dezyribonucleosides is 1: 1: 1: 1) for the mixture of dA, dG, dC and dT.
Preparation process following (being applicable to 2-5 example patient infusion):
1, the static cultivation of NKG cell: with the NKG cell with 2 * 10
4The density of/ml is inoculated among the cell culture medium II, uses the 250ml culturing bottle, and 37 ℃, 5%CO
2, cultivate.
2, the cultivation of NKG cell in the cell bags:
After the NKG cell that is in logarithmic phase in the step 1 washed (800rpm, 10min, 25 ℃) twice with aseptic Hanks, with 2 * 10
4The density of/ml is inoculated in the cell bags of 10 liters of sizes that 2000ml cell culture medium II is housed, and uses the wave bio-reactor to shake pocket type and cultivates, and culture condition is as follows: 37 ℃, 4.0%CO
2, CO
2Flow velocity: 0.10L/min, 4rpm/3 ° (swing rate/amplitude of fluctuation).
3, the interpolation of substratum and human interleukin-12:
When cell density reaches 1.2-1.6 * 10
5During/ml (inoculation back the 3rd day), interpolation 2000ml cell culture medium II and 5ml concentration are 2.0 * 10 in cell bags
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 4.0%CO
2, CO
2Cultivate under flow velocity: 0.10L/min, 4rpm/3 ° the condition; When cell density reaches 6.0-8.5 * 10
5During/m (inoculation back the 6th day) in cell bags, add 1000ml cell culture medium II and 10ml concentration is 2.0 * 10
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 4.0%CO
2, CO
2Cultivate under flow velocity: 0.10L/min, 4rpm/3 ° the condition.
3 repetitions are established in test.
Add up the speed of growth of cell during this time, make the growth curve (Fig. 6) of cell.The result shows that when using the aforesaid method culturing cell, the cell growth is rapid, and the cell quantity that obtains is many.
When the density of cell reaches 1.1 * 10 approximately
6Individual/ml (inoculation back 7-8 days) detected cell growth state, and expected blue dyeing counting with platform.Cell state is good as a result, and platform expects that the painted cell count of blue dyeing less than 10%, shows the effective of the inventive method culturing cell.
4, the collection of NKG cell and radiation treatment:
The density of cell reaches 1.1 * 10 approximately in step 3
6Individual/ml (inoculation back 7-8 days), collecting cell is in the aseptic centrifugal bottle of 1000ml, the 500ml/ bottle, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, add serum-free α-MEM liquid nutrient medium re-suspended cell, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, repeated washing one time; Add serum-free α-MEM liquid nutrient medium re-suspended cell, adjust cell concn to 2 * 10
9Individual/L, the 500ml/ packing.Packaged cell is used the irradiation of γ irradiation instrument, speed: 50cGy/min, time: 16min, total dose: 800cGy obtains the agent of NKG injection cell.
5, NKG injection cell agent effect detection
(1) uses Flow cytometry NKG cell surface CD3, CD16, CD19, CD56 developed by molecule.
Phenotype was CD3-CD16-CD19-CD56+ after the result showed NKG cell pre-irradiation, showed that cell phenotype is stable in the injection cell agent of the inventive method preparation.
(2) Flow cytometry NKG apoptosis situation
The result shows, the NKG cell of living accounts for more than 90% of total cell count, expects that with platform the result of blue staining conforms to, and shows that viable cell quantity is many in the injection cell agent of the inventive method preparation, is beneficial to the raising therapeutic efficiency.
(3) NKG tumorigenicity detects behind the irradiation
External soft-agar cloning test: method is with consistent described in the experiment ().The result shows that the acellular clone of each Kong Zhongjun forms after cultivating in 14 days.Test is through 3 repetitions, unanimities as a result.
The tumor after being inoculated into nude mice test: method is with consistent described in the experiment ().The result shows, does not all detect the NKG cellular infiltration in experimental mice peripheral blood and each internal organs.Experiment is through 3 repetitions, unanimities as a result.
Above result shows that there is not tumorigenicity in the injection cell agent of the inventive method preparation.
(4)
51The Cr release experiment detects the killing activity of NKG cell to K562 cell and Ho8910 cell
Method is with consistent described in the experiment ().Repetition, unanimity are as a result established in experiment 3 times.
The result: the killing activity of imitating the target ratio and be 1: 1 o'clock NKG cell is about 25%, the killing activity of 2: 1 o'clock NKG cells is about 40%, the killing activity of 5: 1 o'clock NKG cells is about 60%, the killing activity of 10: 1 o'clock NKG cells is about 70%, the killing activity rate of 20: 1 o'clock NKG cells is between 80%-90%, and the result shows that NKG all has very strong killing activity to K562 cell and Ho8910 cell.
(5) in vivo test
Method is with consistent described in the experiment ().Repetition, unanimity are as a result established in experiment 3 times.
The result shows that the NKG cell therapy can improve the life quality of mice with tumor, prolongs lifetime.The NKG cell therapy group mouse mental status is significantly better than control group, the activity of treatment group mouse more than control group, occur cachectic time delay, contrast group leader 5-6 week lifetime.
(6) exogenous factors such as bacterium, virus and mycoplasma detect
According to " three ones of Chinese pharmacopoeia, exogenous factor pollution condition such as bacterium, virus and mycoplasma in the NKG injection cell agent of version requirement detection the inventive method in 2005 preparation.
The result shows that exogenous factors such as bacterium, virus and mycoplasma are all negative in the different batches NKG cell.
More than every result show that the NKG injection cell agent of the inventive method preparation is used for the treatment of the malignant tumour security and validity meets the requirements.
(3) cultivate with cell culture medium III
Cell culture medium III: be that to contain the L-glutaminate that final concentration is 2.5mM, the sodium bicarbonate that final concentration is 2.4g/L, the ribonucleoside that final concentration is 9.0mg/L, the dezyribonucleoside that final concentration is 9.0mg/L, the new-born calf serum that final concentration is 12.5% (v/v), horse serum, the final concentration that final concentration is 12.5% (v/v) be 1.25 * 10
5The α of the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium; Described ribonucleoside is that (mass ratio of 4 kinds of ribonucleoside is 1: 1: 1: 1) for the mixture of A, G, C and U; Described dezyribonucleoside is that (mass ratio of 4 kinds of dezyribonucleosides is 1: 1: 1: 1) for the mixture of dA, dG, dC and dT.
Preparation process following (being applicable to 2-5 example patient infusion):
1, the static cultivation of NKG cell: with the NKG cell with 2 * 10
4The density of/ml is inoculated among the cell culture medium III, uses the 250ml culturing bottle, and 37 ℃, 5%CO
2, cultivate.
2, the cultivation of NKG cell in the cell bags:
After the NKG cell that is in logarithmic phase in the step 1 washed (800rpm, 10min, 25 ℃) twice with aseptic Hanks, with 2 * 10
4The density of/ml is inoculated in the cell bags of 10 liters of sizes that 2000ml cell culture medium III is housed, and uses the wave bio-reactor to shake pocket type and cultivates, and culture condition is as follows: 37 ℃, 10.0%CO
2, CO
2Flow velocity: 0.25L/min, 10rpm/9 ° (swing rate/amplitude of fluctuation).
3, the interpolation of substratum and human interleukin-12:
When cell density reaches 1.2-1.6 * 10
5During/ml (inoculation back the 3rd day), interpolation 2000ml cell culture medium III and 12.5ml concentration are 2.0 * 10 in cell bags
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 10.0%CO
2, CO
2Cultivate under flow velocity: 0.25L/min, 10rpm/9 ° the condition; When cell density reaches 6.0-8.5 * 10
5During/ml (inoculation back the 6th day) in cell bags, add 1000ml cell culture medium III and 25ml concentration is 2.0 * 10
4The human interleukin-12 solution of IU/ml continues at 37 ℃, 10.0%CO
2, CO
2Cultivate under flow velocity: 0.25L/min, 10rpm/9 ° the condition.
3 repetitions are established in test.
Add up the speed of growth of cell during this time, make the growth curve (Fig. 6) of cell.The result shows that when using the aforesaid method culturing cell, the cell growth is rapid, and the cell quantity that obtains is many.
When the density of cell reaches 1.1 * 10 approximately
6Individual/ml (inoculation back 7-8 days) detected cell growth state, and expected blue dyeing counting with platform.Cell state is good as a result, and platform expects that the painted cell count of blue dyeing less than 10%, shows the effective of the inventive method culturing cell.
4, the collection of NKG cell and radiation treatment:
The density of cell reaches 1.1 * 10 approximately in step 3
6Individual/ml (inoculation back 7-8 days), collecting cell is in the aseptic centrifugal bottle of 1000ml, the 500ml/ bottle, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, add serum-free α-MEM liquid nutrient medium re-suspended cell, 800rpm, 20 ℃, centrifugal 10min, centrifugal back supernatant discarded, repeated washing one time; Add serum-free α-MEM liquid nutrient medium re-suspended cell, adjust cell concn to 2 * 10
9Individual/L, the 500ml/ packing.Packaged cell is used the irradiation of γ irradiation instrument, speed: 200cGy/min, time: 6min, total dose: 1200cGy obtains the agent of NKG injection cell.
5, NKG injection cell agent effect detection
(1) uses Flow cytometry NKG cell surface CD3, CD16, CD19, CD56 developed by molecule.
Phenotype was CD3-CD16-CD19-CD56+ after the result showed NKG cell pre-irradiation, showed that cell phenotype is stable in the injection cell agent of the inventive method preparation.
(2) Flow cytometry NKG apoptosis situation
As a result, the NKG cell of living accounts for more than 90% of total cell count, expects that with platform the result of blue staining conforms to, and shows that viable cell quantity is many in the injection cell agent of the inventive method preparation, is beneficial to the raising therapeutic efficiency.
(3) NKG tumorigenicity detects behind the irradiation
External soft-agar cloning test method: method is with consistent described in the experiment ().The result shows that the acellular clone of each Kong Zhongjun forms after cultivating in 14 days.Test is through 3 repetitions, unanimities as a result.
The tumor after being inoculated into nude mice test: method is with consistent described in the experiment ().The result shows, does not all detect the NKG cellular infiltration in experimental mice peripheral blood and each internal organs.Experiment is through 3 repetitions, unanimities as a result.
Above result shows that there is not tumorigenicity in the injection cell agent of the inventive method preparation.
(4)
51The Cr release experiment detects the killing activity of NKG cell to K562 cell and Ho8910 cell
Method is with consistent described in the experiment ().Repetition, unanimity are as a result established in experiment 3 times.
The result: the killing activity of imitating the target ratio and be 1: 1 o'clock NKG cell is about 25%, the killing activity of 2: 1 o'clock NKG cells is about 40%, the killing activity of 5: 1 o'clock NKG cells is about 60%, the killing activity of 10: 1 o'clock NKG cells is about 70%, the killing activity rate of 20: 1 o'clock NKG cells is between 80%-90%, and the result shows that NKG all has very strong killing activity to K562 cell and Ho8910 cell.
(5) in vivo test
Method is with consistent described in the experiment ().Repetition, unanimity are as a result established in experiment 3 times.
The result shows that the NKG cell therapy can improve the life quality of mice with tumor, prolongs lifetime.The NKG cell therapy group mouse mental status is significantly better than control group, the activity of treatment group mouse more than control group, occur cachectic time delay, contrast group leader 5-6 week lifetime.
(6) exogenous factors such as bacterium, virus and mycoplasma detect
According to " three ones of Chinese pharmacopoeia, exogenous factor pollution condition such as bacterium, virus and mycoplasma in the NKG injection cell agent of version requirement detection the inventive method in 2005 preparation.
The result shows that exogenous factors such as bacterium, virus and mycoplasma are all negative in the different batches NKG cell.
More than every result show that the NKG injection cell agent of the inventive method preparation is used for the treatment of the malignant tumour security and validity meets the requirements.
Claims (11)
1. deposit number is people's natural killer cell NKG cell of CGMCC No.2901.
2. a method for preparing the described cell of claim 1 is to cultivate cell described in the claim 1 with cell culture medium; Described cell culture medium is that to contain the L-glutaminate that final concentration is 1.5mM-2.5mM, the sodium bicarbonate that final concentration is 2.0g/L-2.4g/L, the ribonucleoside that final concentration is 5.0mg/L-9.0mg/L, the dezyribonucleoside that final concentration is 5.0mg/L-9.0mg/L, the new-born calf serum that final concentration is 8.0%-12.5% (volumn concentration), horse serum, the final concentration that final concentration is 8.0%-12.5% (volumn concentration) be 5.0 * 10
4IU/L-1.25 * 10
5The α of the human interleukin-12 of IU/L-MEM liquid nutrient medium; Described final concentration is the concentration of each material in described cell culture medium.
3. method according to claim 2, it is characterized in that: the final concentration of described L-glutaminate is 2.0mM, the final concentration of described sodium bicarbonate is 2.2g/L, the final concentration of described ribonucleoside is 7.0mg/L, the final concentration of described dezyribonucleoside is 7.0mg/L, the final concentration of described new-born calf serum is 10% (volume percent), and the final concentration of described horse serum is 10% (volume percent), and the final concentration of described human interleukin-12 is 1.0 * 10
5IU/L.
4. according to claim 2 or 3 described methods, it is characterized in that: described best cultivation is cultivated for shaking pocket type; The described swing rate that shakes the pocket type cultivation is 4rpm-10rpm, and amplitude of fluctuation is 3 °-9 °.
5. method according to claim 4 is characterized in that: the described swing rate that shakes the pocket type cultivation is 7rpm, and amplitude of fluctuation is 7 °.
6. according to claim 2 or 3 described methods, it is characterized in that: described culture condition is that temperature is CO in 35 ℃-37 ℃, air
2Volume content is 4.0%-10%, CO
2Flow velocity is 0.10L/min-0.25L/min.
7. method according to claim 6 is characterized in that: described culture condition is that temperature is CO in 37 ℃, air
2Volume content is 5.0%, CO
2Flow velocity is 0.15L/min.
8. a method for preparing the liquid preparation of the described cell of claim 1 comprises the steps: 1) cultivate described cell with the described method of claim 2; 2) with gamma-rays the cell that obtains in the described step 1) is carried out irradiation;
The dosage of described irradiation is 800cGy-1200cGy;
The speed of described irradiation is 50cGy/min-200cGy/min.
9. method according to claim 8 is characterized in that: the dosage of described irradiation is 800cGy; The speed of described irradiation is 50cGy/min.
10. according to Claim 8 or 9 described methods, it is characterized in that: described liquid preparation is an injection.
11. the enchylema body preparation that obtains by arbitrary described method among the claim 8-10.
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| Noboru Suzuki et al.Natural killer lines and clones with apparent antigen specificity.《J Exp Med.》.1990,第172卷(第2期),457-462. * |
| 徐彤等.人外周血NK细胞纯化、扩增及克隆化的初步探索.《上海免疫学杂志》.2000,第20卷(第3期),148-151. * |
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