CN112442482A

CN112442482A - NKT cell culture method

Info

Publication number: CN112442482A
Application number: CN202011436752.4A
Authority: CN
Inventors: 段海峰; 薛冰华; 于婷婷; 解晶; 刘丽华; 庞如梦; 陆颖; 张超
Original assignee: Beijing Huizhi Chikang Biotechnology Co ltd; Beijing Shuangyin Biotechnology Co ltd
Current assignee: Beijing Huizhi Chikang Biotechnology Co ltd; Beijing Shuangyin Biotechnology Co ltd
Priority date: 2020-12-11
Filing date: 2020-12-11
Publication date: 2021-03-05

Abstract

A method for culturing NKT cells. The application provides a mesenchymal stem cell for expressing IL-2 and IL-21, a method for culturing NKT cells by using the mesenchymal stem cell without adding cytokines, a corresponding cell culture container and application.

Description

NKT cell culture method

Technical Field

The application belongs to the field of cell culture and immunotherapy, and particularly provides a method for culturing NKT cells by using mesenchymal stem cells expressing IL-2 and IL-21 and a corresponding culture tool.

Background

NKT (natural killer T) cells are a distinct subset of T cells with both T cell receptors TCR and NK cell receptors on the cell surface. NKT cells produce cytokines in large quantities. NKT cells can secrete a large amount of IL-4, IFN-gamma, GM-CSF, IL-13 and other cytokines and chemokines to play a role in immune regulation after being stimulated, and the NKT cells are one of bridges for connecting innate immunity and acquired immunity.

NKT cells, which are a group of cells having strong lethality, have a broad-spectrum antitumor effect, have begun to be widely used in clinical treatment, and are low in toxicity. NKT cells obtained by in vitro culture have good antitumor activity and health care function. The conventional method for culturing NKT cells at present is to add activating antibodies and cytokines into the culture medium of peripheral blood mononuclear cells, and obtain a large amount of NKT cells after 2-3 weeks of culture. In the traditional culture process, various factors need to be added, so that the culture process is complex, and the cost is high. Furthermore, the NKT cells obtained by the conventional culture method have low yield, and a large amount of T cells are contained in the immune cells. The presence of large numbers of T cells not only reduces the purity of NKT cells but also affects the proliferation of NKT cells, and there is an urgent need to improve the existing processes for culturing NKT cells.

Disclosure of Invention

In order to solve the problems, the applicant designs a new NKT cell culture idea, and mesenchymal stem cells expressing IL-2 and IL-21 are used as a feeder layer to culture the NKT cells so as to simplify the preparation/control process of a culture medium in the NKT cell culture process and reduce the operation difficulty and the cost; meanwhile, the purity of NKT cells is improved and the killing effect is kept.

In one aspect, the present application provides a mesenchymal stem cell characterized by expressing IL-2 and IL-21.

Further, the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.

Further, the IL-2 sequence is SEQ ID NO.1 or a variant thereof;

further, the IL-21 sequence is SEQ ID No.3 or a variant thereof;

further, the IL-2 and IL-21 gene sequences are SEQ ID NO.2 and SEQ ID NO.4 or variants thereof;

in another aspect, the present application provides an application of the above mesenchymal stem cell in culturing an immune cell.

Further, the immune cell is an NKT cell.

Further, the mesenchymal stem cells are used as feeder cells.

In another aspect, the present invention provides a method for culturing NKT cells, which comprises culturing NKT cells using the above mesenchymal stem cells as a feeder layer.

In another aspect, the present application provides a cell culture vessel characterized by comprising the above mesenchymal stem cells.

Further, the cell culture vessel is a culture flask.

In another aspect, the present application provides the use of the above cell culture vessel for culturing NKT cells.

The mesenchymal stem cells in the present application include mesenchymal stem cells of various sources including, but not limited to, adipose, spinal cord, umbilical cord, muscle, mucosa, etc., and the methods for isolating mesenchymal stem cells can be found in the literature and books related to the field of stem cell therapy and cell culture, such as "basic and clinical mesenchymal stem cells", korean council, scientific press.

The IL-2 and IL-21 gene and protein sequences of the present application can be obtained from databases such as Genbank by those skilled in the art. They can also be obtained according to the description of patent or non-patent documents; the IL-2 and IL-21 gene and protein sequences may be human or mammalian IL-2 and IL-21 native or variant gene and protein sequences.

For variants of a protein or gene sequence, on the one hand the person skilled in the art can consult relevant literature and databases to obtain variants known to be useful; on the other hand, variants can be designed by themselves based on the relevant knowledge in protein and gene science about the prediction of protein or gene function, which retain the basic function of the original protein or gene and have desired properties, such as expression properties, stability properties, effect properties, etc. The variant has a certain identity with the parent sequence, such as more than 99%, more than 95%, more than 90%, more than 85%, more than 80%, more than 75%. Identity of 70% or more.

The cell culture methods and media of the present application can be used with reference to such literature or books in the art, e.g., the "guidelines for basic and specific applications of animal cell culture", r.

The cell culture container in the present application may be a culture product, a culture dish, a culture tube, a culture cup, or the like, which is known in the art, or may be a culture apparatus composed of a container and other apparatuses.

The method has the main advantages that NKT cells can be expanded economically, simply, efficiently and massively without adding cytokines, and the killing activity of the NKT cells is not influenced

Drawings

FIG. 1 is a map of a plasmid carrying IL2 and IL21 genes;

FIG. 2 shows the morphology of Treg cells during culture;

FIG. 3 is a flow cytometry assay for NKT cell content (CD3 and CD56 positive cells) after expansion;

FIG. 4 shows the amount of NKT cell proliferation;

FIG. 5 shows the killing efficiency of NKT cells against HepG2 cells.

SAMPLE 1 and 2 are two separate SAMPLEs of different origin.

Detailed Description

Example 1 preparation of IL2-IL21 fusion Gene and recombinant plasmid thereof

(1) Carrier: the vector pCDH-EF1, 3ug was digested with BamHI and EcoRI, and directly purified and recovered. The size of the vector is 7100bp,

(2) PCR IL2 fragment, original plasmid pCDH-EF1-sumIL2-Fc (existing), template 100ng

With GXL enzyme

Primer F：EF1-BamHI-Kozak-ATG-F，Tm＝72℃

Primer R：sumIL2Fc-T2A-R，Tm＝62℃

Gel recovery of about 1260p fragments

(3) PCR T2A fragment, original plasmid containing T2A fragment (available), 100ng template

Using Ex Taq enzyme

Primer F：sumIL2Fc-T2A-F，Tm＝60℃

Primer R：T2A-spAAT-R，Tm＝57℃

Gel recovery of about 94bp fragment

(4) PCR T2A-spAAT fragment, pCDH-EF1-sumIL2-Fc (existing), template 100ng

Using Ex Taq enzyme

Primer F：T2A-spAAT-F(335)，Tm＝78℃

Primer R：spAAT-IL21-R(336)，Tm＝79℃

Recovering about 103bp fragments from gel

(5) PCR IL21 fragment, original plasmid Sequence 51 (Zhongmeitai and synthetic) as template, 100ng template

Using Ex Taq enzyme

Primer F：spAAT-IL21-F(337)，Tm＝55℃

Primer R：IL21-SalI-WPRE-R(338)，Tm＝59℃

Gel recovery of 473bp fragments

(6) Recombinant ligation of the vector, IL2 fragment, T2A fragment, T2A-spAAT fragment and IL21 fragment with Mimetai and 2X mix

(7) Transformation of DH5a, ampicillin resistance

(8) Sequencing primer: general primer PEF-F forward sequencing and self-designed pCDH-DOWN reverse sequencing

(9) After correct sequencing, the strain is preserved, and the quality is improved greatly

In the above steps, the agarose is purchased from biogest, the DNA electrophoresis marker is purchased from tiangen biochemical technology (beijing) limited, the PCR amplification system is purchased from daintily physician's biotechnology (beijing) limited, the DNA recovery kit is purchased from tiangen biochemical technology (beijing) limited, the restriction enzymes BamHI and EcoRI are purchased from NEB, the T4 DNA ligase is purchased from puro maige (beijing) biotechnology limited, the escherichia coli e.

Wherein the amino acid sequence of the IL-2 fused with the Fc fragment and provided with mutation is SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO. 2; the amino acid sequence of the IL-21 is SEQ ID NO.3, and the nucleotide sequence is SEQ ID NO. 4.

Example 2 preparation of recombinant lentivirus carrying IL2-IL21

(1) 1 frozen 293T cell (purchased from ATCC) was rapidly placed in a 37 ℃ water bath from liquid nitrogen until ice disappeared, added dropwise to a 15ml centrifuge tube containing 5ml of a pre-warmed medium, centrifuged at 1200rpm for 3min, the supernatant discarded, the cells were re-suspended with 293T medium (10% FBS +1mM sodium pyruvate +2mM glutamine + 1% non-essential amino acid + DMEM) and inoculated into a 150mM petri dish, 37 ℃ with 5% CO₂And (5) culturing at saturated humidity. In the culture process, when the confluency of cells reaches more than 90%, subculturing, removing the old culture medium, adding 5ml of sterilized PBS solution, slightly shaking, washing the cells, removing the PBS solution, adding 2ml of 0.25% Trypsin-EDTA digestive juice, and digesting for 1-2min until the cells are completely digested; the digestion was stopped by adding serum-containing medium, the cell suspension was centrifuged at 1200rpm for 3min, and the centrifuged cells were resuspended in medium. Cells were seeded at 1.2X 10 per coated 150mm dish⁷The cells were used for packaging lentiviruses at 37 ℃ with 5% CO₂Saturated humidity culture, 20ml medium/dish.

Among them, 293T medium (10% FBS +1mM sodium pyruvate +2mM glutamine + 1% nonessential amino acids + DMEM) was derived from Gibco, PBS solution was purchased from Gibco, and Trypsin-EDTA digest solution was purchased from Gibco.

(2) 2 hours before transfection, the 293T cell culture medium is replaced by 18ml of DMEM medium, 1ml of preheated DMEM medium is added into a sterilized centrifuge tube A, a mixture of the envelope plasmid PMD.2G, the packaging plasmid PSPAX and the recombinant plasmid prepared in the example 1 is added according to the mass ratio of 1:2:3, and the mixture is blown and uniformly mixed; adding 1ml of preheated DMEM culture medium into the sterilized centrifugal tube B, then adding 162 mu l of PEI as a transfection reagent, and uniformly mixing; tube A and B were incubated at room temperature for 5min; the liquid in tube B was added dropwise to tube A, mixed well and incubated at room temperature for 10min to form DNA-transfection reagent complexes. Transferring the DNA-transfection reagent complex to 293T cells with a pre-changed solution, mixing, and performing 5% CO at 37 deg.C₂And (5) culturing at saturated humidity. After 6-8h of incubation, the medium containing the transfection mixture was aspirated, and 20ml of preheated DMEM medium containing 5% FBS was added to each dish of cells, at 37 deg.C and 5% CO₂And (5) culturing at saturated humidity. After the medium change, the supernatants were collected for 24h and 48h respectively and stored at 4 ℃ and 20ml of fresh medium was changed.

Wherein DMEM medium is purchased from Gibco, enveloped plasmid PMD.2G and packaging plasmid PSPAX are purchased from Addgene, the transfection reagent PEI is purchased from Polysciences, and FBS is purchased from Bioind.

(3) Centrifuging the collected supernatant at 4 deg.C and 3500rpm for 15min, discarding the precipitate, and filtering with filter membrane with pore diameter of 0.45 μm. The filtered recombinant lentivirus is mixed with 5X PEG, placed at 4 ℃ for 24 hours, centrifuged at 4 ℃ and 3000rpm for 30min, the supernatant is discarded, and the precipitate is resuspended in 500. mu.l DMEM medium.

Example 3 preparation of IL2-IL21 modified mesenchymal Stem cells

The mesenchymal stem cells are umbilical cord mesenchymal stem cells, and are separated by adopting an umbilical cord tissue block climbing method according to the following steps:

placing in PBS buffer solution (containing 200U/ml penicillin and 200U/ml streptomycin) for normal parturition, washing residual hematocele in umbilical vein and umbilical artery with syringe, and cutting umbilical cord tissue into pieces of 1mm³Filtering small umbilical cord tissues by using a 200-mesh filter screen, collecting umbilical cord tissue blocks on the filter screen, and removing the small umbilical cord tissue blocks to obtain tissue blocks with the diameter of 1-1.5 mm; directly inoculating the tissue block into culture flask, and placing at 37 deg.C and 5% CO₂Standing for 1-2 hours in a saturated humidity incubator; after the tissue block adheres firmly, the alpha-MEM culture solution containing 10% fetal calf serum is added, and the mixture is placed at 37 ℃ and 5% CO₂Continuing culturing in a saturated humidity incubator, and adding 0.25% trypsin (containing trypsin) when the mesenchymal stem cells of the umbilical cord tissue proliferate to about 80% of the full culture flask0.01% EDTA) to obtain primary cells.

MSC is isolated and cultured by the umbilical cord tissue block climbing method, a small amount of cells climb out around the umbilical cord tissue after 72 hours, and after about 7 days, the cells are free from the tissue and gradually form clone.

Selecting P3 generation cells, digesting with 0.05% trypsin, washing twice with PBS, labeling 5 × 10 with mouse anti-human CD11b-PE, CD45-PE, HLA-DR-PE, CD73-PE, CD90-PE, CD105-PE, CD34-FITC and CD19-FITC antibodies respectively⁵And (3) placing the mesenchymal stem cells at room temperature in a dark place for 30min, washing the mesenchymal stem cells twice by PBS, fixing the mesenchymal stem cells by 4% paraformaldehyde, and detecting cell surface markers by a flow cytometer. And (4) freezing the qualified cells in a liquid nitrogen tank, recovering when used and performing post-treatment.

Resuscitating the pre-frozen P3 mesenchymal stem cells to a 150mm culture dish in 20ml serum-free medium at 37 deg.C and 5% CO₂And (5) culturing at saturated humidity. After the revived cells were confluent, the cells were digested with 0.05% trypsin, the digestion was stopped with serum-containing medium, the cell suspension was centrifuged at 800rpm for 5min, and the centrifuged cells were resuspended in mesenchymal stem cell serum-free medium (purchased from Bioind). Cells were seeded 2X 10 per 150mm dish⁶Cells, the medium from which the cells were aspirated the next day after inoculation was discarded, replaced with serum-free α -MEM medium, 20ml medium/dish, 16 μ l Polybrene (purchased from Sigma) was added, and recombinant IL2-IL21 lentivirus (titer 1X 10) was added at a multiplicity of infection of 40MOIs (titer 1X 10)⁸U/ml), cultured at 37 ℃ and 5% CO2 saturated humidity for 7 h. After 7 hours, the virus-containing α -MEM medium (purchased from Gibco) was discarded and replaced with serum-free medium, and the culture was continued at 37 ℃ under 5% CO2 saturated humidity for 3 days.

After the cells were confluent, cell supernatants were collected and assayed for the expression of IL2 and IL21 factors by ELISA. Then washing the cells with PBS, digesting the cells with 0.05% trypsin, terminating digestion with serum-containing medium, centrifuging the cell suspension at 800rpm for 5min, resuspending the centrifuged cells with serum-free medium, and passaging at a passage ratio of 1:6, wherein the serum-free medium is at 37 deg.C and 5% CO₂Culturing for 3 days to obtain the recombinant mesenchymal stem cells stably expressing IL2 and IL 21.

Example 4 culturing of NKT cells Using IL2-IL21 modified mesenchymal Stem cells

(1) Preparation of IL2-IL21 feeder layer cells

The cells cultured in example 4 to P5 passage were selected, and when the confluency of the cells reached 90% or more, mitomycin C at a working concentration of 20ug/ml was added to the cell culture solution for 2 hours, followed by washing three times with PBS, digesting the cells with 0.05% trypsin, terminating the digestion with serum-containing medium, and freezing the cells as required.

(2) Preparation of IL2-IL21 feeder layer-coated culture flask

And (3) thawing the frozen IL2-IL21 feeder layer cells into a cell culture bottle by using an MSC culture medium, adjusting the cell density to reach 80% confluence after the cells are attached to the wall and stretched, and culturing overnight at 37 ℃ and 5% CO2 saturated humidity. The next day, the cells were washed three times with PBS, and then treated with absolute ethanol for 5 minutes. Then, the absolute ethanol was discarded, the cells were washed three times with PBS, and finally a small amount of PBS was added to the coated flask. The coated culture flask is placed at 4 degrees for standby. The cells in the non-fixed coated flask can not be treated by absolute ethyl alcohol, and the feeder cells are revived on the day before the NK cells are cultured.

(3) NKT cell expansion

Separation of autologous plasma: transferring the anticoagulated peripheral blood into a 50ml centrifuge tube, centrifuging for 20min at 800g, and sucking the upper plasma into another centrifuge tube.

Inactivation of autologous plasma: inactivating the plasma in a 56 deg.C water bath for 30min, and centrifuging the inactivated plasma in a centrifuge at 400g for 8 min.

Isolation of peripheral blood mononuclear cells: adding physiological saline which is 1-1.5 times of the volume of the sucked plasma into the blood, and blowing and stirring the mixture by a pipette to be uniformly mixed. 20ml of lymphocyte separation liquid is added into a 50ml centrifuge tube, then blood with the same volume is slowly added into the upper layer of the separation liquid, and 800g of the blood is centrifuged for 20min (after centrifugation, the blood sample is divided into 4 layers from top to bottom, namely, the first layer is a plasma layer, the second layer is a peripheral blood lymphocyte layer, the third layer is a separation liquid layer, and the fourth layer is a red blood cell layer). Carefully sucking the second layer of peripheral blood lymphocyte layer into a new centrifuge tube, adding physiological saline to 45ml, uniformly blowing, and centrifuging for 8min at 400 g. After the centrifugation, the supernatant was discarded, the cells were resuspended in 45ml of physiological saline, and 400g of the cells were centrifuged for 8min after counting.

Primary culture of NKT cells: the NKT cell activation flask was washed 2 times with 10ml of physiological saline (liquid was washed to the entire bottom surface without impacting the coating layer of the flask with liquid). Take 3 to 7X 10⁷The cells were resuspended in 45ml 1640 medium and transferred to a culture flask, and 3500 ten thousand units of recombinant human IL2, 2.25ug of CD3 monoclonal antibody and 5ml of inactivated autologous plasma were added. The bottle cap of the culture bottle is unscrewed and then placed in a 5% CO2 incubator at 37 ℃ for 3 days (72 hours) for amplification and passage.

NKT cell expansion: on day 4, cells were removed, the cell suspension was blown down evenly in a clean bench, counted and then expressed at 4X 10⁵The cell density of each ml was passaged in MD, and 700U/ml of recombinant human IL2 was added depending on the cell amount. Passages were then performed every two days, with flow phenotyping occurring on days 12-14.

The culture results of NKT cells are shown in FIGS. 2 to 4: the method of the present application allows for rapid expansion of high purity NKT cells without the addition of cytokines.

(4) Evaluation of killing Effect of NKT cells

HepG2 cells (human liver cancer cells) are used as target cells, NKT is used as effector cells, the target cells are inoculated into a 96-well plate according to the density of 5000 cells/ml, each well is 100 mu l, the effector cells are added into the target cells according to the effective target ratio of 1:1, 5:1, 10:1 and 20:1, CCK8 (Biyuntian) reagent is added at the same time, the target cells are placed in an incubator with the concentration of 5% CO2 and the temperature of 37 ℃ for continuous culture for 4h, and the reading is carried out at the wavelength of 450nm to calculate the killing efficiency. The results are shown in fig. 5, and show that the NKT cells cultured by the method of the present application have good tumor killing activity.

Sequence listing

<110> Beijing Shuangyin Biotechnology GmbH Beijing Huzhi Chikang Biotechnology GmbH

<120> NKT cell culture method

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<170> SIPOSequenceListing 1.0

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Leu Asn Gly Ile Asn Asn Tyr Lys Asn Pro Lys Leu Thr Arg Met Leu

50 55 60

Thr Ala Lys Phe Tyr Met Pro Lys Lys Ala Thr Glu Leu Lys His Leu

65 70 75 80

Gln Cys Leu Glu Glu Glu Leu Lys Pro Leu Glu Glu Val Leu Asn Leu

85 90 95

Ala Gln Ser Lys Asn Phe His Phe Asp Pro Arg Asp Val Val Ser Asn

100 105 110

Ile Asn Val Phe Val Leu Glu Leu Lys Gly Ser Glu Thr Thr Phe Met

115 120 125

Cys Glu Tyr Ala Asp Glu Thr Ala Thr Ile Val Glu Phe Leu Asn Arg

130 135 140

Trp Ile Thr Phe Cys Gln Ser Ile Ile Ser Thr Leu Thr Gly Gly Gly

145 150 155 160

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ser Lys

165 170 175

Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly

180 185 190

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

195 200 205

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu

210 215 220

Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

225 230 235 240

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg

245 250 255

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

260 265 270

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu

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Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

290 295 300

Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu

305 310 315 320

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

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Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

340 345 350

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

355 360 365

Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His

370 375 380

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu

385 390 395 400

Gly

<210> 2

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<212> DNA

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atgccgtctt ctgtctcgtg gggcatcctc ctgctggcag gcctgtgctg cctggtccct 60

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Claims

1. A mesenchymal stem cell, characterized by expressing IL-2 and IL-21.

2. The mesenchymal stem cell of claim 1, wherein the mesenchymal stem cell is an umbilical cord mesenchymal stem cell.

3. Mesenchymal stem cell according to claim 1 or 2, wherein the IL-2 sequence is SEQ ID No.1 or a variant thereof.

4. Mesenchymal stem cell according to any of claims 1 to 3, wherein the IL-21 sequence is SEQ ID No.3 or a variant thereof.

5. Mesenchymal stem cells according to any of claims 1 to 4, wherein the IL-2 and IL-21 gene sequences are SEQ ID No.2 and SEQ ID No.4 or variants thereof.

6. Use of mesenchymal stem cells according to any one of claims 1 to 5 in the culture of immune cells.

7. The use according to claim 6, wherein the immune cells are NKT cells.

A method for culturing NKT cells, which comprises culturing NKT cells using the mesenchymal stem cells according to any one of claims 1 to 5 as a feeder layer.

9. A cell culture container comprising the above mesenchymal stem cell.

10. Use of a cell culture vessel according to claim 9 for culturing NKT cells.