CN112300990A

CN112300990A - Culture medium for in-vitro amplification and activation of gamma delta-T cells

Info

Publication number: CN112300990A
Application number: CN202010475488.9A
Authority: CN
Inventors: 张顺浪; 林杰良; 杨智雅; 孙境新; 刘威廷; 唐晓艳
Original assignee: Jiya Cell Technology Co ltd
Current assignee: Jiya Cell Technology Co ltd
Priority date: 2019-07-24
Filing date: 2020-05-29
Publication date: 2021-02-02
Also published as: TWI768357B; TWI768356B; TW202118858A; CN112300989A; CN115678847A; TW202104578A

Abstract

The invention relates to a culture medium for in vitro amplification and activation of gamma delta-T cells, which comprises autologous dendritic cell exosomes, can effectively improve the cell amplification multiple and generate the gamma delta-T cells with high toxic activity to cancer cells.

Description

Culture medium for in-vitro amplification and activation of gamma delta-T cells

Technical Field

The invention relates to a culture medium for in vitro amplification and activation of gamma delta-T cells.

Background

Immune cell therapy is a treatment mode in which the immune cells of a patient are proliferated and activated in vitro and then returned to the body. At present, immune cell therapy is gradually applied to cancer therapy, and the key point of success is to understand the characteristics and functions of various immune cells and select the most suitable immune cell types, such as NK cells, T cells and the like, according to the conditions and gene characteristics of cancer patients.

T cells in the human immune system are largely divided into two major classes, Alpha-Beta T cells and Gamma-Delta T (γ. Delta. -T) cells. The gamma delta-T cells are present in peripheral blood in an amount of only 1% to 5% of all T cells in peripheral blood. It distinguishes normal from abnormal cells by recognizing isopentenyl pyrophosphate (IPP) molecules on the cell surface. IPP is an intermediate product of cellular metabolism and its production in cancer cells is increased, especially when p53 gene of cancer cells is mutated. The γ δ -T cells recognizing IPP proliferate and activate and enhance the force to attack tumor cells, which is a special function of γ δ -T cells, which can find and attack cancer cells if other immune cells do not detect cancer cell markers. In general, abnormal cells will possess molecules that are different from other normal cells, and γ δ -T cells will recognize and attack these as cancer cell markers. In addition to IPP molecules, γ δ -T cells can also be identified by markers such as MIC A/b, HMB-PP, intercellular adhesion molecule-1, CD166, etc. Thus, γ δ -T cells can recognize and kill a variety of cancer cells.

One important feature of γ δ -T cells is that they do not require HLA in identifying abnormal cells and are therefore independent of the individual's HLA type. This feature allows γ δ -T cells to be used in any human without producing graft-versus-host disease (GVHD). Many of the clinical applications of cell therapy in humans, which have been carried out in the united states, europe and japan, are currently carried out using γ δ -T cells, and these clinical applications have confirmed that the use of γ δ -T cells is safe and safe.

Since γ δ -T cells are scarce in blood and their proliferative capacity varies from person to person, the challenge of cell therapy with γ δ -T cells is whether it is possible to expand and activate γ δ -T cells in vitro in large quantities and rapidly. In recent years, the use of Zoledronic acid (Zoledronic acid) has been found to proliferate γ δ -T cells in large quantities, and its technology has also been established. In addition to the use of zoledronic acid in culturing γ δ -T cells, the injection of zoledronic acid into cancer patients can find that cancer cells express IPP in greater amounts, thereby increasing the sensitivity of γ δ -T cells to cancer cells. However, zoledronic acid is a drug for treating osteoporosis, and there is still a certain doubt about the use of zoledronic acid in culturing cells or injecting the zoledronic acid into patients, so that the development of a convenient, effective and safe culture medium for in vitro amplification and activation of gamma delta-T cells is still highly needed.

Disclosure of Invention

The invention provides a culture medium for in vitro amplification and activation of gamma delta-T cells, which comprises autologous dendritic cell exosomes with the concentration ranging from 5 mu g/ml to 50 mu g/ml. In a preferred embodiment, the concentration of the autologous dendritic cell exosomes is 25 μ g/ml.

In one embodiment, the medium further comprises interleukin-2; in a preferred embodiment, the concentration of the interleukin-2 is between 500IU/ml and 2000 IU/ml; in a more preferred embodiment, the concentration of IL-2 is 1000 IU/ml.

In one embodiment, the medium further comprises zoledronic acid; in a preferred embodiment, the concentration of the zoledronic acid is between 1 μ M and 20 μ M; in a more preferred embodiment, the concentration of zoledronic acid is 5 μ M.

In one embodiment, the culture medium further comprises autologous plasma, human AB serum, or fetal bovine serum; in a preferred embodiment, the autologous plasma, human AB serum or fetal bovine serum is about 10% of the total culture volume.

In one embodiment, the medium further comprises a basal medium; in a preferred embodiment, the basal medium is selected from the group consisting of AIM V, X-VIV015, CellGro SCGM, KBM 501, DMEM and RPM1-1640 medium.

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, the drawings show some, but not all alternative embodiments. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown. The accompanying drawings, which are incorporated in and constitute a part of this specification, help explain the principles of the invention.

Drawings

FIG. 1 is a gamma delta-T cell expansion fold analysis of the invention after addition of autologous dendritic cell exosomes to the culture medium.

FIG. 2 is an analysis of the poisoning efficiency of gamma delta T cells on Daudi cells after adding autologous dendritic cell exosomes to the culture medium according to the present invention.

FIG. 3 is the analysis of the poisoning efficiency of gamma delta T cells on A549 cells after adding autologous dendritic cell exosomes to the culture medium.

Detailed Description

In view of the above problems, the present invention provides a culture medium for in vitro amplification and activation of γ δ -T cells, which is added with autologous dendritic cell exosomes, and is capable of efficiently increasing the amplification factor of γ δ -T cells and producing γ δ -T cells with high cancer cytotoxic activity.

Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present document, including definitions, will control.

As used herein, "about," "approximately," or "approximately" shall generally mean within 20%, preferably within 10%, and more preferably within 5% of a particular value or range. As used herein, the terms "about", "approximately", or "approximately", unless expressly stated otherwise, are intended to be open-ended and are intended to encompass any number of degrees of certainty.

As used herein, the term "γ δ -T cell" refers to a cell surface antigen presenting CD3⁺And expressing TCR V gamma 9 and TCR V delta 2.

In the present invention, the "cell amplification factor" is determined in the following manner: "cell number after 12 days in vitro culture" was divided by "initial γ δ -T cell number isolated from peripheral blood mononuclear cells".

In the present invention, the "cancer cell killing efficiency" is obtained by performing a killing test using γ δ -T cells as effector cells (effector cells) and Daudi cell lines or a549 cell lines as target cells (target cells) at a ratio (E: T ratio) of the effector cells to the target cells of 0.5, 1 or 5, and using the ratio of the target cells to the effector cells as the killing efficiency.

In the present invention, an "exosome" is a minute membrane vesicle secreted by most cells, having a lipid bilayer membrane structure with a diameter of about 40-100 nm. Exosomes were first discovered in 1983, but it has been considered to be only a waste product of cells. Recently, however, it has been discovered that such minute membrane vesicles contain cell-specific proteins, lipids and nucleic acids, which can be transmitted to other cells as signaling molecules to alter the functions of other cells. Recent studies have found that exosomes play important roles in many physiopathologies, such as antigen presentation in immunity, tumor growth and migration, repair of tissue damage, etc., and that exosomes secreted by different cells have different compositions and functions.

Materials and methods

The peripheral blood sample is prepared by collecting whole blood from the arm of a subject according to a plan passed by the ethical committee, placing the whole blood in a sterile blood collection tube, and storing the whole blood at room temperature for subsequent treatment.

The basic culture medium used in the invention can be selected from: commercially available basal media such as CellGro SCGM (CellGenix corporation), KBM 501(Kohjin Bio corporation), AIM-V (Thermo Fisher corporation), X-VIV015(Lonza corporation), DMEM, RPM1-1640, and the like.

The medium of the present invention may contain suitable components such as proteins, cytokines, antibodies, serum, and compounds. The cytokine is sometimes interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-7 (IL-7), interleukin-12 (IL-12), interleukin-15 (IL-15), interleukin-18 (IL-18), or interleukin-21 (IL-21).

Method for separating peripheral blood mononuclear cells from blood sample

7.5-8ml of blood was drawn into a blood collection tube containing anticoagulant heparin and Ficoll-Hypaque reagent, plus a polyester gel barrier to separate the two fluids. Separating blood collecting tube at 1800g at room temperatureHeart 20 min. After centrifugation is complete, plasma fractions are collected for subsequent cell culture and 5 to 10mm plasma layers are left on the interface, operating without disturbing the cell layer. Then, a Peripheral Blood Mononuclear Cell (PBMC) layer of the interface was collected by a pipette into a 15ml conical tube, the PBMC was washed with 10ml of Phosphate Buffered Saline (PBS) and the conical tube was inverted 5 times, and then centrifuged at 400g for 5 minutes. After repeating the washing step twice, the cells were resuspended in 5ml of PBS. Counting the number of cells, 1.3X10 can be isolated from 1ml of whole blood⁶The PBMC of (1). Finally, the proportion and the phenotype of the gamma delta-T cells in the PBMCs are confirmed by a flow cytometer.

Method for amplifying and activating gamma delta-T cells in vitro

After centrifugation of the peripheral blood mononuclear cell suspension in a 15ml conical tube at 400g for 5 minutes at room temperature, the supernatant was discarded. Cell culture medium was prepared by adding interleukin-2 (IL-2) and zoledronic acid (Zomet) to final concentrations of 1000IU/ml and 5. mu.M, respectively. Wherein zoledronic acid is added in liquid form, and 50. mu.l of zoledronic acid (concentration 4mg/5ml) is added per 30ml of the culture medium. The cell pellet was then resuspended in culture medium and adjusted to 1X10 per ml of culture medium⁶A cell. Using 24-well cell culture plates, add 1X10 per well⁶The cells are cultured. If large-scale culture is required, the culture medium can be 0.5x10 per square centimeter⁶The cell density was adjusted in accordance with the surface area of the culture dish or the culture flask used as a principle. Autologous plasma, human AB serum, fetal bovine serum, or autologous dendritic cell exosomes were then added to make up about 10% of the total culture volume (equivalent to a 24-well cell culture plate with 100 μ l cells per well). And (3) placing the cell culture plate in a cell culture box with the temperature of 37 ℃ and the content of 5% CO2 for culturing for 24-48 hours. Cell density was maintained at 0.5x10 per ml⁶～2x10⁶Every 2 to 3 days, fresh medium containing 1000IU/ml interleukin-2 is added into each cell, and the cells are transferred to a new culture dish or a new culture bottle to be cultured according to the degree of cell expansion when necessary. Serum concentration in the medium was maintained at least at 1% during the culture. Cells were harvested on day 12 and run in flowThe number, phenotype and function of gamma delta-T cells were confirmed by the cytometer.

Analysis of cell surface antigens by flow cytometry

At 2x10⁵Cells/200. mu.l the amplified and activated cells were placed in a 96-well plate, and 3. mu.l of a fluorescence-labeled antibody was added to react at 4 ℃ for 15 minutes, followed by washing 3 times with PBS, adding 400. mu.l of PBS to suspend the cells, and analyzing the fluorescence-labeling on the cell surface by flow cytometry. The fluorescence labeled antibody comprises anti-CD3 antibody, anti-TCR V gamma 9 antibody and anti-TCR V delta 2 antibody.

Gamma delta-T cell virus cancer cell killing capability test mode

The amplified and activated gamma delta-T cells are used as action cells (effector cells), and Daudi cell strains (lymph cancer cell strains) or A549 cell strains (lung cancer cell strains) are used as target cells (target cells). After the affected cells and the target cells were cultured in a mixed manner at 0.5:1, 1:1 or 5:1, they were reacted for 4 hours, and then the number of apoptotic cells was measured by staining with 7-AAD.

Preparation and purification of autologous dendritic cell exosomes

Autologous dendritic cell exosomes (Autologous dendritic cell exosomes) were prepared by replacing the dendritic cell culture medium cultured up to the fifth day with fresh cell culture medium, and adding Granulocyte-colony stimulating growth factor (GM-CSF) and interleukin-4 and continuing to culture the dendritic cells for 24 hours. The cultured medium was collected and centrifuged at 300g and 1000g for 10 minutes, respectively, followed by filtration through a filter having a pore size of 0.45 μm to remove cells and debris. The filtered medium was concentrated by centrifugation at 1000g for 45 minutes using a Centricon Plus-70 Millipore filter, then the dendritic cell exosomes were isolated from the medium by ultracentrifugation at 100,000g for one hour, washed twice with PBS, centrifuged at 1000g for 25 minutes through an Amicon Ultra-15 filter, and finally the dendritic cell exosomes were resuspended in 200. mu.L of PBS. Quantification of dendritic cell exosomes was performed using BCA protein assay kit (Thermo Scientific).

The amplified and activated gamma delta-T cells obtained by the method can be mixed with a proper excipient for storage, and the excipient can be a phosphate buffer solution, and finally, the pharmaceutical composition is prepared.

Examples

Example 1 culture results of addition of autologous dendritic cell exosomes to γ δ -T cell culture media

FIG. 1 is a graph showing the fold expansion analysis of γ δ -T cells after adding autologous dendritic cell exosomes to the culture medium. From FIG. 1, it was found that the cell expansion ratio was 6,236-fold at day 12 of culture using a medium containing 25. mu.l/ml of autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a 3,754 fold cell expansion.

Example 2 poisoning test of cultured γ δ -T cells of the present invention on Daudi cells

FIG. 2 is a graph showing the poisoning efficiency of γ δ -T cells on Daudi cells after adding autologous dendritic cell exosomes to the culture medium. From FIG. 2, it was found that the γ δ -T cell poisoning efficiency was 78.3% in the case of the E: T ratio of 5, when cultured using a medium containing 25 μ l/ml of autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a γ δ -T cell cytotoxic efficiency of 64.4%.

Example 3 poisoning test of cultured γ δ -T cells of the present invention on a549 cells

FIG. 3 is the analysis of the poisoning efficiency of gamma delta T cells on A549 cells after adding autologous dendritic cell exosomes to the culture medium. From FIG. 3, it was found that γ δ -T cell poisoning efficiency was 58.9% in the case of the E: T ratio of 5 and addition of zoledronic acid, cultured using the medium containing 25 μ l/ml of autologous dendritic cell exosomes; the control group (without autologous dendritic cell exosomes) had a γ δ -T cell cytotoxic efficiency of 43.7%.

In conclusion, the autologous dendritic cell exosomes are added into the gamma delta-T cell culture medium, so that the amplification rate of the gamma delta-T cells and the poisoning activity on Daudi and A549 cancer cells can be effectively improved.

The foregoing description of the embodiments of the present invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Modifications or variations are possible in light of the above teachings.

Alternatives to the embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description and examples. Some references, which may include patents, patent applications, and various publications, are cited and discussed herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety as if each had been individually incorporated for the same degree of completeness.

Claims

1. A culture medium for in vitro expansion and activation of γ δ -T cells comprising autologous dendritic cell exosomes at a concentration ranging from 5 μ g/ml to 50 μ g/ml.

2. The culture medium according to claim 1, wherein the concentration of autologous dendritic cell exosomes is 25 μ g/ml.

3. The culture medium of claim 1, further comprising interleukin-2.

4. The culture medium of claim 3, wherein the concentration of interleukin-2 is between 500IU/ml and 2000 IU/ml.

5. The culture medium of claim 4, wherein the concentration of interleukin-2 is 1000 IU/ml.

6. The culture medium of claim 1, further comprising zoledronic acid.

7. The culture medium of claim 6, wherein the concentration of zoledronic acid is between 1 μ M and 20 μ M.

8. The culture medium of claim 7, wherein the concentration of zoledronic acid is 5 μ M.

9. The culture medium of claim 1, further comprising autologous plasma, human AB serum, or fetal bovine serum, wherein the autologous plasma, human AB serum, or fetal bovine serum is about 10% of the total culture volume.

10. The culture medium according to any one of claims 1 to 9, further comprising a basal medium; wherein the basal medium is selected from the group consisting of AIM V, X-VIV015, CellGro SCGM, KBM 501, DMEM and RPM1-1640 medium.