CN112941032A

CN112941032A - Method for culturing memory T cells with modified MSCs

Info

Publication number: CN112941032A
Application number: CN202110201797.1A
Authority: CN
Inventors: 段海峰; 薛冰华; 于婷婷; 解晶; 张超; 庞如梦; 刘丽华; 陆颖
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: 2021-02-23
Filing date: 2021-02-23
Publication date: 2021-06-11

Abstract

A method of culturing memory T cells with modified MSCs, wherein the modified MSCs express IL-15 and IL-7; IL15 was mutated to increase activity; also provided are cell culture flasks prepared using the aforementioned MSCs.

Description

Method for culturing memory T cells with modified MSCs

Technical Field

The present application is in the field of cell culture technology, and in particular, provides methods for culturing memory T cells with modified MSCs, wherein the modified MSCs express IL-15 and IL-7.

Background

The memory T cell (Tcm) is a T cell which is generated after a juvenile T cell is activated by an antigen, has long-term memory, can home to a lymph node to receive antigen re-stimulation, and can continuously generate a large number of cloned effector memory T cell (Tem) cells carrying the same antigen under the re-stimulation of the antigen. The biomarker of the memory T cells is cells which are double positive for CD62L and CD45ROa, which represents that the memory T cells can return to lymph nodes through lymph shielding and are in a state of being activated by antigen.

The memory T cell has the following characteristics: first, because it expresses CD62L +, it is more receptive to antigenic information presented by APC for secondary activation in lymph nodes; secondly, the memory T cells can be activated by tumor antigens to directly kill tumors after being returned to the body; thirdly, the memory T cell has self-renewal and replication ability, and long survival time in vivo, and can play a role in long-term anti-tumor; fourth, the number of T cells that can be returned can be efficiently amplified in vitro.

The currently known method for preparing memory T cells is to isolate primary naive T cells from peripheral blood, and to activate them first with the CD3 monoclonal antibody and IL-2. After 24 hours of activation, a medium comprising IL-7, IL-15 and IL-21 was added for culture. The cells were harvested after 6-9 days of culture. Due to the proliferation characteristics of the memory T cell subsets, the proportion of the memory T cell subsets is rapidly reduced along with the prolonging of the culture time in the in vitro culture. Therefore, the short-term culture process cannot ensure that a sufficient number of target cells are obtained, and increasing the culture number of initial seed cells sharply increases the production cost. Therefore, how to obtain memory T cells by using an economical and efficient method is a technical problem to be solved at present.

Disclosure of Invention

In response to the above problems, applicants have developed a method of culturing memory T cells with modified MSCs, wherein the modified MSCs express IL-15 and IL-7; IL15 was mutated to increase activity; also provided are cell culture flasks prepared using the aforementioned MSCs.

In one aspect, the present application provides a modified MSC cell, wherein the MSC cell expresses IL-15 and IL-7.

Further, the protein sequences of the IL-15 and the IL-7 are SEQ ID NO.1 or SEQ ID NO.3 and SEQ ID NO.5 or variants thereof, respectively.

Further, the fusion gene sequence of IL-15 and IL-7 in the MSC is SEQ ID NO. 7.

Further, the MSC is a fat MSC or an umbilical cord MSC.

In another aspect, the present application provides a cell culture flask comprising the aforementioned MSC cells.

Further, the MSC cells in the culture flask are fixed to form a feeder layer.

Further, the immobilization was performed using absolute ethanol.

In another aspect, the present application provides methods of culturing memory T cells using the above-described MSCs or culture flasks.

In another aspect, the present application provides a method of preparing the aforementioned MSC cell, comprising: constructing IL-15 and IL-7 vectors; preparing lentivirus particles; the MSC cells were infected.

The molecular biology techniques used in the construction of MSC cells in this application are conventional techniques well known to those skilled in the art, and can be obtained from works such as molecular cloning, sambrook, scientific press, and the like, and can be consulted with relevant literature or tool software using existing primer design, protein function prediction, and the like.

The IL-15 and IL-7 sequences in the present application are not limited to the species listed in the present application, and various sequences that are inferior to the animal interleukin sequences and are commonly known to those skilled in the art from the Genbank et al website for use in the methods of the present application, and variant sequences can be obtained from the above sequences with the current knowledge and functional prediction methods/software in the protein field. The variant sequence shares a certain sequence identity with the parent sequence, such as more than 70%, more than 80%, more than 90%, more than 95%, more than 99%, and has substantially the same function as the parent sequence.

The cell culture bottle in the present application is not limited to a bottle-shaped container, and other shapes of cell culture tools, such as a plate shape, a cup shape, a culture tank, etc., are also within the scope of the present application.

One skilled in the art can obtain MSCs from suitable sources including, but not limited to, fat, bone marrow, umbilical cord, etc., or from desired functions, by procedures such as physical manipulation, suspension, dissociation, centrifugation, etc., as described in reference to cytological relevant laboratory kits.

The invention has the advantages that the memory T cells can be expanded economically, simply, conveniently, efficiently and massively without adding cytokines.

Drawings

FIG. 1 is a plasmid map expressing pCDH-EF1-IL15mut-IL 7;

FIG. 2 is a plasmid map expressing pCDH-EF1-RaIL15-IL 7;

FIG. 3 shows the effect of different feeder layers on the expansion of memory T cells;

FIG. 4 is a flow assay of genotype T cell phenotypes;

FIG. 5 shows the killing efficiency of memory T cells against Raji cells.

Detailed Description

Example 1 fusion Gene of IL15 and IL7 and recombinant plasmid thereof

Agarose used in the experiment was purchased from Biowest, DNA electrophoresis marker was purchased from tiangen biochemical technology (beijing) ltd, PCR amplification system was purchased from daidzein technologies (beijing) ltd, DNA recovery kit was purchased from tiangen biochemical technology (beijing) ltd, restriction enzymes BamHI and EcoRI were purchased from NEB, T4 DNA ligase was purchased from promo maige (beijing) biotechnology ltd, escherichia coli e.

(1) Construction method of pCDH-EF1-IL15mut-IL7 vector

A. Carrier: the vector pCDH-EF1, 3ug, 3h was digested with BamHI and EcoRI, purified and recovered directly at 37 ℃. Remarking: the vector should be enzymatically cleaved and ready for use. The vector size was approximately 7100 bp.

PCR IL15mut fragment, original plasmid pcDNA3.4-dmuIL15-Fc, template 100ng

Annealing with Ex Taq enzyme at 55 deg.C

Primer F：EF1-BamHI-Kozak-ATG-F，Tm＝63℃

Primer R：IL15mut-T2A-R，Tm＝60℃

Recovering about 450bp fragments from glue

PCR T2A fragment, original plasmid with T2A fragment, 100ng template

Annealing with Ex Taq enzyme at 52 deg.C

Primer F：IL15mut-T2A-F，Tm＝60℃

Primer R：T2A-IL7-R，Tm＝57℃

Gel recovery of about 98bp fragments

PCR IL7 fragment, original plasmid pcDNA3.4-IL7-Fc as template, 100ng of template

Annealing with Ex Taq enzyme at 53 deg.C

Primer F：T2A-IL7-F，Tm＝58℃

Primer R：IL7-EcoRI-WPRE-R，Tm＝59℃

Gel recovery of 550bp fragment

F. Colony PCR primers for recombination and connection of vector, IL15mut fragment, T2A fragment and IL7 fragment by using Mimetai and 2X mix

F：EF1-BamHI-Kozak-ATG-F(314)，Tm＝69℃

R：T2A-IL7-R(318)，Tm＝72℃

Fragment size: 525bp

G. Sequencing primer: EF1 universal primer PEF-F

H. After correct sequencing, the strains, large quality-improving particles are preserved, and the plasmid map is shown in figure 1.

(2) pCDH-EF1-RaIL15-IL7 vector construction method

PCR Ra fragment, original plasmid sequence29, template 100ng

Annealing with Ex Taq enzyme at 56 deg.C

Primer F：Primer 2EF1-BmaH-IL15Ra-F，Tm＝63℃

Primer R：IL15 mut-R，Tm＝61℃

Recovering about 450bp fragments from glue

PCR IL15mut-IL7 fragment, original plasmid pCDH-EF1-IL15mut-IL7, template 100ng

Annealing with Ex Taq enzyme at 56 deg.C

Primer F：IL15 mut-F，Tm＝61℃

Primer R：IL7-EcoRI-WPRE-R，Tm＝70℃

Recovering 954bp fragment from glue

D. Recombinant ligation with Mimetita and 2X mix, vector, Ra fragment and IL15mut-IL7 fragment

E. Primers for colony PCR: f: EF1-BamHI-Kozak-ATG-F (314), Tm 69 ℃; r: IL15 mut-R (331), Tm 61 ℃; fragment size: 450bp

F. Sequencing primer: EF1 universal primer PEF-F and self-made pCDH-Down

G. After correct sequencing, the strains, large quality-improving particles were preserved, and the plasmid map is shown in FIG. 2.

The amino acid sequence of the IL-15 is SEQ ID NO.1, and the nucleotide sequence is SEQ ID NO. 2; the amino acid sequence of the RaIL15 is SEQ ID NO.3, and the nucleotide sequence is SEQ ID NO. 4; the amino acid sequence of the IL-15 is SEQ ID NO.5, and the nucleotide sequence is SEQ ID NO. 6; the fusion gene sequence is SEQ ID NO. 7.

Example 2 preparation of recombinant lentivirus carrying IL15-IL7

(1) 1 frozen 293T cell (purchased from ATCC) was taken out from liquid nitrogen, quickly placed in a 37 ℃ water bath until ice blocks disappeared, added dropwise to a 15ml centrifuge tube containing 5ml of pre-heated medium, centrifuged at 1200rpm for 3min, the supernatant was discarded, and 293T medium (10% FBS +1mM sodium pyruvate +2mM glutamine + 1% non-essential amino acids +) was usedDMEM) resuspended cells were inoculated into a 150mm dish at 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; incubating tube A and tube B at room temperature for 5 min; 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 IL15-IL7 modified mesenchymal Stem cells

The mesenchymal stem cells are umbilical cord mesenchymal stem cells, are separated by adopting an umbilical cord tissue block climbing method and are carried out 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% CO₂And (3) continuing culturing in a saturated humidity incubator, and adding 0.25% trypsin (containing 0.01% EDTA) for digestion when the mesenchymal stem cells of the umbilical cord tissue proliferate to about 80% of the confluent culture flask, thereby obtaining 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.

Reviving pre-frozen P3 generation mesenchymal stem cellsTo a 150mm dish, 20ml of serum-free medium was incubated at 37 ℃ and 5% CO2 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 IL15-IL7 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 alpha-MEM medium (purchased from Gibco) was discarded and replaced with serum-free medium at 37 ℃ with 5% CO₂The incubation was continued for 3 days at saturated humidity.

After the cells were confluent, cell supernatants were collected and assayed for the expression of IL5 and IL7 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 IL15 and IL 7.

Example 4 culture of memory T cells with modified MSCs

(1) Preparation of IL15-IL7 feeder layer

Selecting and culturing cells until the P5 generation, adding mitomycin C with the working concentration of 20ug/ml into a cell culture solution when the confluence degree of the cells reaches above 90%, treating for 2h, then washing with PBS for three times, digesting the cells with 0.05% trypsin, stopping digestion with a serum-containing culture medium, and freezing the cells according to the required amount.

(2) Preparation of IL15-IL7 feeder layer-coated culture flask

Resuscitated and cryopreserved by MSC culture mediumIL15-IL7The feeder layer cells are put into a cell culture bottle, the cell density is adjusted to reach 80 percent confluence degree after the cells are attached to the wall and stretched, and the cells are cultured overnight at 37 ℃ and 5 percent CO2 saturated humidity. The next day, the cells were washed with PBSNext, the cells were 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.

(3) Memory T 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 memory T cells: the memory T cell activation flask was rinsed 2 times with 10ml of physiological saline (liquid was rinsed to the entire bottom surface without impacting the flask coating with liquid). Taking 3-7 multiplied by 107 cells, suspending the cells by 45ml 1640 medium, transferring the cells to a culture flask, and adding 3500 ten thousand units of recombinant human IL2, 2.25ug of CD3 monoclonal antibody and 5ml of inactivated autologous plasma. 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.

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

As shown in figure 3, the mesenchymal stem cells modified by IL15mut-IL7 and RaIL15-IL7 are used as a feeder layer to culture the memory T cells, the expansion effect is better than that of the traditional factor adding method, and as shown in figure 4, the flow type phenotypic analysis also shows that the mesenchymal stem cells modified by IL15mut-IL7 are used as the memory T cells cultured by the feeder layer, and the proportion of cells with double positive of CD62L and CD45Ra is high. Namely, the method in the embodiment can efficiently and quickly expand a large number of memory T cells.

(4) Killing effect of memory T cells

Using Raji cells (human lymphoma cell line) as target cells, using memory T cells of a control group (traditional factor culture) and an experimental group (the patent method) as effector cells, inoculating the target cells into a 96-well plate according to the density of 5000 cells/ml, wherein each well is 100 microlitres, adding the effector cells into the target cells according to the effective target ratio of 1:1, 5:1, 10:1 and 20:1, simultaneously adding CCK8 (Bycyunnan) reagent, placing the target cells in 5% CO₂And continuously culturing for 4h in an incubator at 37 ℃, reading at the wavelength of 450nm, and calculating the killing efficiency. The results are shown in fig. 5, and the results show that the cultured NK cells of the present invention have good tumor killing activity compared with the control group.

Sequence listing

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tttttaaaag acatatctgt gatgctaata aggaaggtat gtttttattc cgtgctgctc 720

gcaagttgag gcaatttctt aaaatgaata gcactggtga ttttgatctc cacttattaa 780

aagtttcaga aggcacaaca atactgttga actgcactgg ccaggttaaa ggaagaaaac 840

cagctgccct gggtgaagcc caaccaacaa agagtttgga agaaaataaa tctttaaagg 900

aacagaaaaa actgaatgac ttgtgtttcc taaagagact attacaagag ataaaaactt 960

gttggaataa aattttgatg ggcactaaag aacac 995

Claims

1. A modified MSC cell, wherein said MSC cell expresses IL-15 and IL-7.

2. The MSC cell according to claim 1, wherein the protein sequences of IL-15 and IL-7 are SEQ ID No.1 or SEQ ID No.3 and SEQ ID No.5 or variants thereof, respectively.

3. The MSC cell according to claim 2, wherein the fused gene sequence of IL-15 and IL-7 in the MSC is SEQ ID No. 7.

4. The MSC cell according to any of claims 1 to 3, which is an adipose MSC or an umbilical cord MSC.

5. A cell culture flask comprising the aforementioned MSC cells.

6. The cell culture flask of claim 5, wherein the MSC cells in the cell culture flask are immobilized to form a feeder layer.

7. The cell culture flask of claim 6, wherein the fixing is performed using absolute ethanol.

8. A method of culturing memory T cells using the MSC cells according to any one of claims 1 to 3 or the cell culture flask according to any one of claims 5 to 7.

9. A method of preparing the MSC cell according to any one of claims 1 to 3, comprising: constructing IL-15 and IL-7 vectors; preparing lentivirus particles; the MSC cells were infected.