CN106190973A - A kind of novel NKT cell culture processes - Google Patents

A kind of novel NKT cell culture processes Download PDF

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CN106190973A
CN106190973A CN201610532223.1A CN201610532223A CN106190973A CN 106190973 A CN106190973 A CN 106190973A CN 201610532223 A CN201610532223 A CN 201610532223A CN 106190973 A CN106190973 A CN 106190973A
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cell
nkt
humanized
monoclonal antibody
final concentration
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CN106190973B (en
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徐榕
王立燕
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Beijing Jingda Biotechnology Co., Ltd.
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Beijing Tongli Marine Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2302—Interleukin-2 (IL-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere

Abstract

The invention discloses a kind of novel NKT cell culture processes, the method includes NKT primitive cell culture and NKT Subculture, described NKT primitive cell culture is to use PERIPHERAL BLOOD MONONUCLEAR CELL inoculation serum-free medium, and first day of described successive transfer culture adds the startup factor humanized CD3 monoclonal antibody, humanized CD28 monoclonal antibody and IL 2 in serum-free medium；Amplification factor IL 2 and IL 7 is added subsequently in follow-up amplification liquid.The NKT cell that NKT cell culture processes disclosed by the invention is cultivated, cell state is good, growth rate is fast, proliferation times is high, it is possible to obtain the NKT effector lymphocyte of higher purity, and the NKT Cell killing efficacy obtained is also apparently higher than existing cultural method.

Description

A kind of novel NKT cell culture processes
Technical field
The present invention relates to technical field of cell biology, be specifically related to a kind of novel NKT cell culture processes.
Background technology
Cellular immunotherapy is a kind of emerging treatment technology.Cellular immunotherapy therapy is that collection human autoimmune is thin Born of the same parents, through In vitro culture so that it is quantity becomes Radix Achyranthis Bidentatae to increase, and targeting killing ability strengthens, and feeds back to human body the most again and kills Pathogen in blood and tissue, cancerous cell, the cell of sudden change, break immune tolerates, and activates and the immunocompetence of enhancing body, Take into account treatment and the double effects of health care.Cellular immunotherapy can recover and strengthen the immunologic surveillance of tumor patient self and kill Tumor function, kills after operation in patients and the tumor cell of internal remaining after chemicotherapy effectively, reach to treat tumor, prevention of recurrence with Transfer and the final purpose effecting a radical cure tumor, have the advantages such as high specificity, side effect be light.
NKT cell is a very important class cell mass in immune cell therapy.NKT (natural killer T) cell It is a group cell surface existing φt cell receptor TCR, has again the special T cell subgroup of NK cell receptor, substantial amounts of IL-4 can be secreted With the IFN-γ factor, it is the important cells group of immunomodulating and cell killing effect.There is after NKT cell activation NK cell sample Cytotoxic activity, the cell sensitive target cell of solubilized NKT, main effects molecule is perforin, FasL and IFN-γ.Mesh Front NKT cell kills a class cell mass of target cell as having strength, has extremely important in tumor vaccine cells is treated Effect.The NKT cell obtained through In vitro culture can well play immunotherapy of tumors and health-care effect.
In NKT cell routine is cultivated, directly train by certain cell density mainly by culture bottle or culture bag Supporting, concrete cultural method is: Nostoc commune Vanch bottle is coated with being coated liquid (Anti-CD3 albumen)；Peripheral blood separation mononuclearcell (PBMC), through magnetic bead sorting obtain NKT cell, be seeded to coated after culture bottle in；Cultivate 14 days and collect NKT cell.
But conventional flask cultivates NKT cell, part cell is easy to adherent.When cell proliferation is cultivated, substantial amounts of Packed cell is easy to be sunken to bottom, is easily caused NKT cell and can not be fully contacted culture fluid, slows down cell proliferation, and easily Make pockets of cell death.
Summary of the invention
In order to solve the problems referred to above, it is an object of the invention to provide a kind of novel NKT cell culture processes.This cultivation Method uses the mononuclearcell that obtains from patient's autologous peripheral blood cell separation, through manipulation in vitro and relevant cell factor thereof Activation is stimulated to make it be transformed into efficient NKT cell, for the lethal effect of tumor cell.
For achieving the above object, the present invention adopts the following technical scheme that
Provide firstly a kind of novel NKT cell culture processes, the method includes that NKT primitive cell culture and NKT are thin Born of the same parents' successive transfer culture, wherein, described NKT primitive cell culture is to use PERIPHERAL BLOOD MONONUCLEAR CELL inoculation serum-free medium, institute State first day of successive transfer culture in serum-free medium, add that to start the factor humanized CD3 monoclonal antibody, humanized CD28 mono- Resist and IL-2；Amplification factor IL-2 and IL-7 is added subsequently in follow-up amplification liquid.
Preferably, the inoculum concentration of described mononuclearcell inoculation serum-free medium is 0.5-2 × 106Individual/mL.
Preferably, the final concentration of 50-500ng/mL of humanized CD3 monoclonal antibody, the final concentration of humanized CD28 monoclonal antibody Final concentration of 2000-5000IU/mL for 50-500ng/mL and IL-2.
Preferably, described amplification liquid is serum-free medium, the final concentration of 500-1000IU/mL, IL-7 of described IL-2 Final concentration of 10-50ng/mL.
Preferably, the serum-free medium of NKT primitive cell culture adds IFN-γ, this IFN-γ final concentration of 500-2000IU/mL's.
Preferably, the cultivation cycle of described successive transfer culture is more than 14 days.
The NKT cell cultivated 14 days carry out tumor cell fragmentation test (kill select cell line be: MCF-7, mammary gland The cell line of cancer), use the method for CCK-8 test kit to carry out fragmentation test, NKT cell i.e. can reach the killing rate of the 14th day More than 80%.
Mononuclearcell described in cultural method of the present invention can be by peripheral blood isolated.
In some embodiments, the preparation method of described mononuclearcell is that peripheral blood adds normal saline dilution, slow Slowly joining in the centrifuge tube containing lymphocyte separation medium, the blood making dilution is clear with lymphocyte separation medium layering, 500- 800g is centrifuged 20-30min.
Further, the invention provides a kind of antitumor adoptive immunity cell, described antitumor adoptive immunity cell is Obtained by above-mentioned preparation method.
Further, the invention provides a kind of biological product, described biological product contain by above-mentioned preparation method The NKT cell obtained.
Beneficial effect
The invention discloses the cultural method of a kind of novel NKT cell, the NKT cell that the method is cultivated, cell state Well, growth rate is fast, proliferation times is high, it is possible to obtain the NKT effector lymphocyte of higher purity, the NKT Cell killing efficacy obtained Also apparently higher than existing cultural method.
Accompanying drawing explanation
Fig. 1 cultivates front streaming result figure；
Fig. 2 cultivate 14 days after streaming result figure；
Fig. 3 cultivates front cell photo；
Fig. 4 cultivate 14 days after cell cultivate photo；
The lethal effect of Fig. 5 NKT cells against tumor cells.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment The conventional means that technological means used by is well known to those skilled in the art.
Experimental apparatus, reagent and consumptive material are as follows:
NKT cell must be to prepare under the super-clean environment meeting GMP condition.Cultivating relevant material to cell must be Aseptic, without pyrogen, and in strict accordance with operating instruction use, except having the situation of specified otherwise.Experiment instrument is such as Shown in table 1, experiment agents useful for same is as shown in table 2.
Table 1 experiment instrument
Title Article No./model Producer
Biohazard Safety Equipment BOOSERIES A2 Thermo
Horizontal low speed centrifuge X3 Thermo
CO2 gas incubator FORMA SERIES II WATER JACKET Thermo
Inverted microscope XDS-113 Chongqing broadcasting and TV
Blood cell counts device Z318427 Because of medical treatment
Blood cell counting plate Z319023 Because of medical treatment
Pipettor 26300 eppendorf
Heat-sealing machine GZR-Ⅲ Suzhou City's medical apparatus
-20 DEG C of refrigerators BCD-322W Rich Xihua home electric company limited
4 DEG C of refrigerators BCD-322W Rich Xihua home electric company limited
Biochemical cultivation case Blue pard Shanghai Yiheng Scientific Instruments Co., Ltd
Table 2 experiment consumptive material
Embodiment 1: the separation of mononuclearcell
In the case of Voluntary Blood Donors is voluntary, inventor gathers people vein peripheral blood 50mL, adds 10mL normal saline Dilution, is slowly added in the centrifuge tube containing lymphocyte separation medium 15mL, makes the blood of dilution divide with lymphocyte separation medium Layer is clear, 500g-800g centrifugal force, centrifugal 20-30min, extracts middle tunica albuginea confluent monolayer cells, obtains mononuclearcell, add physiology Saline cleans twice, and counting, cell concentration is 2 × 107Individual～4 × 107Individual.
Embodiment 2 cell is cultivated
One, the preparation of NKT primary cell
1, by patient's 50-100mL peripheral blood, uniformly divide to 2 pipes, rule with a little former blood of pipette, extract (about 300 μ L) Or instillation plate carries out examining bacterium.Then, under room temperature, 865g, centrifugal 15 minutes (9,7, it is intended that rise 9 falls 7, lower same).
2, upper plasma being proceeded to centrifuge tube, 56 DEG C of inactivation 30min, 865g, 10min are centrifugal, take supernatant standby.
After using isopyknic normal saline to mix with hemocyte precipitation, carefully it is added to the 15mL of 50mL centrifuge tube subpackage On Ficoll layer, layering is made to keep clear.Room temperature 865g, centrifugal 20 minutes (9,3).
3, draw PERIPHERAL BLOOD MONONUCLEAR CELL (PBMC) layer, exhaust the cellular layer of two liquid level junctions as far as possible, add 5mL physiology Saline piping and druming mixing, after add normal saline dilution to 45mL.700g, centrifugal 8 minutes (9,7).Same procedure washed cell again.
After supernatant discarded, resuspended with 5mL serum-free medium, constant volume 20mL, draws a small amount of cell counting.
4, the cell that 20mL has been mixed by kind bottle joins 225cm2In culture bottle, with 10mL serum-free medium clean from Heart pipe, is incorporated in culture bottle, and it is 30-50mL that visual cell quantity adds culture medium final volume, adjusts cell density and is 0.5-2×106Individual/mL.Adding the IFN-γ of 500-2000IU/mL, patients blood plasma 5-10mL, residue blood plasma 4 DEG C seals and preserves Standby.
5, after labelling culture bottle sidewall, CO is put it into2Cultivate in incubator.Check and keep incubator state As follows, temperature: 37 ± 0.5 DEG C, humidity: more than 90%, CO2Concentration: 5%.
6, just being put by the blood agar plate having added sample into 35 DEG C of constant temperature biochemical cultivation cases, date and cultivation put in record Article.It is inverted after overnight, retains 48 hours.
Calculate cell density and cell quantity
Cell concentration (individual/mL)=N/4 × dilution general times × 104
Cell quantity (individual)=cell concentration (individual/mL) × cell suspension cumulative volume (mL)
Two, NKT Subculture
1, amplification is started
Cultivating the 1st day: culture medium is serum-free medium 50mL, inoculating cell 3 × 107Individual；Add and start the factor Different final concentration gradients is set, as humanized CD3 monoclonal antibody (0-500ng/mL), humanized CD28 (0-500ng/mL) and IL-2 (0-5000IU/mL), is specifically shown in Table 3.And add the date on bottle lateral marks, put into CO2Continue in incubator Cultivating, the cell photo before cultivation is as shown in Figure 3.
Expand liquid is formulated in addition (0-1000IU) IL-2 and (0-50ng/mL) IL-7 in 1000mL culture medium, and it is eventually The gradient of concentration is arranged as shown in table 4.
2, amplification for the first time, predominantly fluid infusion operation, culture medium is expanded to 100-150mL, adds inactivation blood plasma 10mL
2.1 cells are cultivated the 4th day, basis of microscopic observation situation without exception, cell well-grown, and culture fluid turns yellow, permissible Start amplification.If growing general or poor, it is considered to decrement amplification or postponement amplification.
Cell culture medium is put to room temperature, with 50 milliliters of centrifuge tube (frame)/pipets/inspection bacterium plate/Tissue Culture Flask by 2.2 Safety cabinet is put in the lump after surface sterilization.
2.3 back out culture bottle, 50mL centrifuge tube, cell culture medium bottle cap successively.The expansion of 100mL is measured with 50mL centrifuge tube Increase liquid, pour in culture bottle, add inactivation blood plasma 10mL.Cover the bottle cap of culture bottle and culture medium, jog culture bottle successively, make Culture medium mixes.Bottle lateral marks is cultivated algebraically P1, date, puts into incubator and continue to cultivate.
3, second time amplification, predominantly bagging operations, cultivating system is expanded to 500mL by 100-150mL, adds residue Blood plasma (about 20mL)
3.1 cells are cultivated the 6th day, basis of microscopic observation situation without exception, cell well-grown, and culture fluid turns yellow, permissible Start amplification.If growing general or poor, it is considered to decrement amplification or postponement amplification.
3.2 tighten bottle cap, from CO2After taking out Tissue Culture Flask, with other material requested in the lump surface sterilization in incubator, Move into Biohazard Safety Equipment.
3.3 pat bottle wall, make cell come off from the bottom of bottle as far as possible, add inactivation blood plasma about 20mL, tighten bottle cap, and jog mixes It is even,
3.4 open culture bag packaging, carry out labelling, indicate operation item title, date etc..
3.5 back out mouth of pipe lid, and lid is carefully upside down in clean area, prevent from passing through above it.
The mouth of pipe and the 50mL syringe nozzle extracting inner core are connected by 3.6, and in bottle, whole cell suspension are poured in bag, and Add in bag amplification liquid to 500mL, finally the mouth of pipe lid of culture bag is built, tightens.
3.7, culture bag sample tap the pipe top of pin (can), extract a small amount of cell suspension with 5mL syringe and add to Examine bacterium on blood plate, and carry out labelling.Then culture bag is put into and cell culture incubator continues cultivate.
4, third time amplification, predominantly fluid infusion operation, cultivating system is expanded to 1000mL by 500mL
4.1 cells are cultivated the 8th day, basis of microscopic observation situation without exception, cell well-grown, and culture fluid turns yellow, permissible Start amplification.If growing general or poor, it is considered to decrement amplification or postponement amplification.
4.2 from CO2After taking out cell culture bags, with other material requested in the lump surface sterilization in incubator, move into biology Safety cabinet.
4.3 back out mouth of pipe lid, and lid is carefully upside down in clean area, prevent from passing through above it.
The mouth of pipe and the 50mL syringe nozzle extracting inner core are connected by 4.4, add amplification liquid 500mL, finally will in bag The mouth of pipe lid of culture bag is built, and tightens.Labelling culture bag number P3 and third time amplification date.
4.5, culture bag sample tap the pipe top of pin (can), extract a small amount of cell suspension with 5mL syringe and add to Examine bacterium on blood plate, and carry out labelling.Then culture bag is put into and cell culture incubator continues cultivate.
5, the 4th amplification, predominantly fluid infusion operation, cultivating system is expanded to 2000mL by 1000mL
5.1 cells are cultivated the 11st day, basis of microscopic observation situation without exception, cell well-grown, and culture fluid turns yellow, permissible Start amplification.If growing general or poor, it is considered to decrement amplification or postponement amplification.
Culture bag is taken out from incubator by 5.2, after the alcohol disinfecting of surface, moves into Biohazard Safety Equipment, turns on cell and cultivates The bag capping of the mouth of pipe also connects with the 50mL syringe nozzle extracting inner core, adds amplification liquid 1L, fully mix training in bag Support the cell suspension in bag, and 2L cell suspension is all assigned in 2 cell culture bags, labelling culture bag number P4 and the 4th expansion Increase the date.Then 2 culture bag are put into and incubator continues cultivate.
Three, results
1, the 14th day harvest bag inner cell suspension, the photo after the cultivation of this experimental cell is as shown in Figure 4.
This is operated material therefor, and including cell culture bags, Tissue Culture Flask, 250 milliliters of centrifuge tubes (frame), surface disappears Safety cabinet is moved into after poison.
2, adjust safety cabinet hook position, make hook be in position convenient to operate, culture bag is vertically mentioned, the company of flicking Adapter root, makes liquid in pipe flow back to culture bag, at least reserves 3 centimetres of dead bands at far-end, with mosquito forceps by dead for this section folder.
4, the most softly press culture bag so that it is in cell suspend mixing as far as possible.250mL centrifuge tube is marked, In case obscuring.
5, back out centrifuge tube bottle cap, put to clean area on the right side of safety cabinet, make operation hereafter try one's best without going past bottle The side of covering.Centrifuge tube scale oriented manipulation person, it is simple to observe.
6, backing out mouth of pipe lid, take off and hold mosquito forceps, left hand is fixing near mosquito forceps near-end connecting tube, and end is in Directly over the centrifugal mouth of pipe.Open mosquito forceps, and control mouth of pipe direction and flow rate of liquid simultaneously, collect cell suspension in 250mL from In heart pipe.Trim centrifuge tube, builds lid, tightens.700g, centrifugal 8 minutes.
7, cell precipitation uses brine 2 times, can be not only used for the Function detection test in downstream.
Four, interpretation:
1, variable concentrations and the impact starting the factor pair cultivation cell number of 14 days of kind
The total cellular score of initially-separate is: 3 × 107Individual.
After cultivating 14 days, the cell number change of each group as shown in table 3, by data analysis in table understand 30-50mL without Being simultaneously introduced the startup factor of the CD3 monoclonal antibody in three-type-person source, humanized CD28 and IL-2 in serum free culture system liquid, its NKT is thin Born of the same parents' expanding effect is better than addition one or two of which starts the factor.Inventor finds when the these three added through many experiments Starting the factor in more than table during the highest addition, the NKT cell number of cultivation is not significantly increased, but cost can increase very Many, it is thus determined that addition is humanized CD3 monoclonal antibody 500ng/mL, humanized CD28 monoclonal antibody 500ng/mL, IL-2 5000IU/mL, is that NKT cell cultivates optimized culture scheme.
The addition of the table 3 initial start factor
2, the amplification procedure of NKT cell expands interpolation IL-7 and IL-2 in liquid and can significantly increase the cell number of amplification Amount.
The total cellular score of initially-separate is: 3 × 107Individual
Startup factor final concentration in the interpolation in first day cultivated is: the CD3 monoclonal antibody of 500ng/mL, the CD28 of 500ng/mL Monoclonal antibody, under the conditions of 5000IU/mL IL-2, adds amplification factor IL-7 and IL-2 to amplification liquid.Such as analysis of experimental data in table 4 Understand, be simultaneously introduced IL-7 and IL-2 amplification factor and to be substantially better than and be not added with or the situation of single addition IL-7 or IL-2, and When IL-7 and the IL-2 final concentration added is respectively the final concentration of 10-of the final concentration of 500-1000IU/mL, IL-7 of IL-2 50ng/mL, the NKT cell amplification effect obtained is best, it is possible to reach 1010The order of magnitude.
Table 4 expands liquid and adds activation factor amount
3, streaming result
Before cultivating, streaming result figure is shown in accompanying drawing 1, and after cultivating 14 days, streaming result figure is shown in that accompanying drawing 2, comparison diagram 1 and Fig. 2 understand, Illustrate by this method so that we the required effector lymphocyte increase that obtains in number and percentage.For cell number (start the factor is the group of amplification optimum: the CD3 monoclonal antibody of 500ng/mL, the CD28 monoclonal antibody of 500ng/mL, 5000IU/mL IL- 2；Amplification liquid is: 1000IU IL-2,50ng/mL IL-7, serum-free medium) carry out flow cytometer detection, data see table 5:
The method of streaming phenotype analytical: take the cell of the 0th, 14 days respectively, sucks 1.5mL EP pipe, often pipe containing about 1.0 × 106Cell, 2000rpm is centrifuged supernatant discarded after 3 minutes, and cell is resuspended in 100 μ L PBS, addition fluorescent-labeled antibody (BD, APC-CD56, FITC-CD3), 4 DEG C of lucifuges hatch 30 minutes；Wash 2 times with PBS, supernatant discarded；With 0.3mL PBS by cell Resuspended, carry out detection with flow cytometer BD FACSAria III and analyze.NKT(CD3+CD56+) streaming phenotype the 14th day time can Reach 87.7%.
Table 5 flow cytometer detection result
4, the lethal effect of NKT cells against tumor cells
The take the logarithm MCF-7 cell strain of trophophase is as target cell, and to adjust cell density be 8 × 105Cells/mL,
Every hole takes 50 μ L and is laid in 96 well culture plates.Taking cell prepared by this method adjusts density to be 4 × 106cells/mL、8 ×106cells/mL、1.6×107Cells/mL, adds in 96 well culture plates, every hole 50 μ L, make effect target than respectively 5:1,10: 1,20:1, often group sets three multiple holes.Vaccination ways is as follows:
Experimental group (a value): MCF-7 (50ul)+NKT (50ul)
Matched group (b value): MCF-7 (50ul)+serum-free medium (50ul)
(c value): NKT (50ul)+serum-free medium (50ul)
Blank group (d value): serum-free medium (100ul)
The cell that will have inoculated, is placed in 37 DEG C, 5%CO2Under the conditions of co-culture 24 hours after, every hole add 10 μ LCCK-8 examination Agent, shakes up, 37 DEG C, 5%CO2Under the conditions of continue cultivate 3 hours, microplate reader 450nm wavelength survey absorbance (A450).Count as the following formula Calculate killing activity: kill tumor efficiency=[1-(a value-c value-d value)/(b value-d value)] × 100%.
Result shows that NKT cell prepared by this method has higher killing activity to MCF-7 cell strain, when effect target ratio is When 10:1,20:1, the killing rate of the NKT cell of the 14th day i.e. can reach more than 80%, and the killing of NKT cells against tumor cells is made With as shown in Figure 5.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.

Claims (10)

1. a novel NKT cell culture processes, the method includes NKT primitive cell culture and NKT Subculture, its Being characterised by, described NKT primitive cell culture is to use PERIPHERAL BLOOD MONONUCLEAR CELL inoculation serum-free medium, and described subculture is trained First day supported adds the startup factor humanized CD3 monoclonal antibody, humanized CD28 monoclonal antibody and IL-2 in serum-free medium； Amplification factor IL-2 and IL-7 is added subsequently in follow-up amplification liquid.
2. the method for claim 1, it is characterised in that the inoculum concentration of described mononuclearcell inoculation serum-free medium For 0.5-2 × 106Individual/mL.
3. the method for claim 1, it is characterised in that the final concentration of 50-500ng/ of described humanized CD3 monoclonal antibody ML, the final concentration of 2000-5000IU/mL of final concentration of 50-500ng/mL and IL-2 of humanized CD28 monoclonal antibody.
4. method as claimed in claim 3, it is characterised in that the optimum of described humanized CD3 monoclonal antibody is final concentration of 500ng/mL, the final concentration of 5000IU/mL of optimum of final concentration of 500ng/mL and IL-2 of optimum of humanized CD28 monoclonal antibody.
5. the method for claim 1, it is characterised in that described amplification liquid is serum-free medium, the end of described IL-2 Concentration is the final concentration of 10-50ng/mL of 500-1000IU/mL, IL-7.
6. the method for claim 1, it is characterised in that add in the serum-free medium of described NKT primitive cell culture Enter IFN-γ, the final concentration of 500-2000IU/mL of this IFN-γ.
7. the method for claim 1, it is characterised in that the cultivation cycle of described successive transfer culture is more than 14 days.
8. the method for claim 1, it is characterised in that the preparation method of described mononuclearcell is that peripheral blood adds life Reason saline dilution, is slowly added in the centrifuge tube containing lymphocyte separation medium, makes blood and the lymphocyte separation medium of dilution Layering is clear, and 500-800g is centrifuged 20-30min.
9. an antitumor adoptive immunity cell, it is characterised in that described antitumor adoptive immunity cell is by claim 1 ～8 cells of obtaining of preparation method described in any one.
10. biological product, it is characterised in that described biological product contain by making described in claim 1～8 any one The NKT cell that Preparation Method obtains.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108690830A (en) * 2017-04-11 2018-10-23 上海尚泰生物技术有限公司 A kind of method of efficient amplification NKT cells
CN115369086A (en) * 2022-02-22 2022-11-22 北京景达生物科技有限公司 Culture scheme for NK cell amplification culture

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