KR20190032197A - Method of decreasing immunogenicity - Google Patents

Abstract

The present invention relates to an immunogenic reduction method which can be usefully applied to various treatment techniques using stem cells. The immunogenic reduction method comprises a step of culturing a stem cell in a medium (xeno-free medium) which does not include xenoantigen, wherein the xenoantigen is a protein derived from a species different from one from which the stem cell is derived.

Description

Method of decreasing immunogenicity [

A method for producing an immunogenicity-reduced stem cell comprising culturing a stem cell in a xeno-free medium containing no antigen, and a method for producing a stem cell comprising the stem cell prepared by the method .

Human mesenchymal stem cells have characteristics such as proliferation or differentiation depending on the surrounding environment, their ability to regulate immunity, and their secretory action, and they can be used to treat heart diseases, neurological diseases, orthopedic diseases, digestive and immune system diseases It is highly likely to be used as a therapeutic agent for a wide range of intractable diseases. In addition, unlike embryonic stem cells, there are no teratomas, which are relatively safe after implantation, and are widely used in the development of therapeutic agents. In the early days, most of the self-derived cells have been developed to minimize the immune response, but the proportion of using homologous cells capable of mass production is gradually increasing. Thus, the immune response to homologous antigen, which may arise from allogeneic mesenchymal stem cell transplantation, may result in the development of memory immune cells and allogeneic T cells, which in turn may increase the rejection of organs for transplantation. .

Mesenchymal stem cells do not express secondary stimulating molecules such as MHC (major histocompatibility complex) class II and CD40, CD80, CD86, which play an important role in allogeneic antigen recognition, I expression level is also low. Based on this theoretical logic, it is recognized that mesenchymal stem cells do not have immunogenicity or low immunogenicity at allograft. In addition, the mesenchymal stem cells are known to have immunomodulatory effects and clinically can be expected to have therapeutic effects on immune diseases. In some countries, it is already known that graft-versus-host disease (GvHD) Some clinical uses have been approved (Prochymal). On the other hand, in contrast, in some research reports, there is a lack of immunosuppressive effect or an even worse disease outcome when mesenchymal stem cells are applied to animal models of immunological disease, And it is controversial. In addition, among the research reports showing the immunosuppressive effect of mesenchymal stem cells, some of the syngeneic animal models, that is, animal stem cells, were transplanted into the same genetically identical animal, so that the immunogenicity There is a limit to the analysis.

Therefore, in order to verify the immunogenicity of human-derived mesenchymal stem cells, which can occur in allograft, it is necessary to use an allogeneic experimental model, that is, a system for exposing human-derived mesenchymal stem cells to another human's immune system . When an immune response against allogeneic antigens occurs after the administration of allogeneic stem cells, the administered cells may not be able to grow and may be removed to reduce the therapeutic effect. In particular, inflammatory cytokines (IFN-gamma, IL- 17A, etc.) may increase the risk of induction of allogeneic immune responses. Therefore, in order to utilize allogeneic mesenchymal stem cells from allogeneic origin, anticipation of the possibility of induction of immune response by allogeneic antigens, that is, immunogenicity, before administration to the human body is expected to have an important meaning in ensuring safety . The present invention has developed and suggested an optimal strategy for minimizing the immune response that can occur by exposing human mesenchymal stem cells to the immune system of another human in vitro, and a method for optimally maintaining such mesenchymal stem cells.

INDUSTRIAL APPLICABILITY The present invention can reduce the immunogenicity of stem cells as compared with the case where stem cells are cultured in a xeno-free medium immediately after isolation from a living body, for example, in a culture medium (for example, a medium containing a heterologous antigen) And it was completed. In addition, the present invention suggests that culturing the stem cells immediately after isolation from the autologous serum-containing or non-autologous xeno-free medium is the most effective method for solving the problem of cytotoxic T cell activity and immune response. .

One example is a method for reducing the immunogenicity of stem cells or a method for producing immunogenicity-reduced stem cells comprising culturing the stem cells in a medium containing no different antigens. The stem cells may be isolated from the living body. The step of culturing may be performed in vitro. The culturing may be performed immediately after the stem cells are separated from the living body.

The stem cells may be for use for allograft or autologous transplantation.

Another example provides a composition for stem cell transplantation comprising stem cells prepared by the method for producing stem cells with reduced immunogenicity. The composition for stem cell transplantation may be used for transplantation (allograft or autologous transplantation) into an individual of the same species as the individual from which the stem cell is derived.

The subject may be selected from mammals including humans.

The heterologous antigen may be any protein derived from a species different from the species from which the stem cells are derived (for example, a heterologous albumin such as bovine serum albumin (BSA) or a serum or blood containing the heterologous albumin such as FBS (Fetal bovine serum) And the like).

The medium containing no heterologous antigen can be prepared by adding 1 to 10% (v / v), 1 to 8% (w / v) of the autologous serum isolated from the transplant recipient of the stem cell, (v / v), 1 to 6% (v / v), 3 to 10% (v / v), 3 to 8% (v / v), 3-6% (v / v), or 5 to 8% (v / v). The recipient may be a mammal such as a human.

Another example provides a culture medium for reducing immunogenicity of a stem cell, wherein the culture medium for stem cell culture does not contain a heterologous antigen. The culture medium for stem cell culture may be a medium used for culture of stem cells (including nutrients (eg, carbon sources (saccharides), nitrogen sources (amino acids), buffers, vitamins, and inorganic salts necessary for stem cell culture) In one embodiment, the stem cell culture medium is selected from the group consisting of Dulbecco's modified eagle medium (DMEM), minimal essential medium (MEM), alpha-minimal essential medium Eagles ' s basal medium, CMRL (Connaught Medical Research Laboratory) medium, Glasgow minimum essential medium, Ham's F-12 medium, IMDM (Iscove's modified Dulbecco's medium, Liebovitz's L-15 medium, RPMI (Roswell Park Memorial Institute) 1640 medium, Medium 199, Hank's Medium 199, However, In one embodiment, the medium that does not contain the heterologous antigen can be, but is not limited to, serum-free medium (such as CellGro® medium).

The culture medium may contain 1 to 10% (v / v), 1 to 8% (v / v), 1 to 6% (v / v), 3-10% (v / v), 3-8% (v / v), 3-6% (v / v), 5-10% % (v / v). < / RTI >

In the present invention, the stem cell may be an autologous stem cell isolated from a stem cell or a recipient separated from a recipient (recipient) and allogeneic recipients other than recipient.

The stem cells include embryonic stem cells, adult stem cells, induced pluripotent stem cells (iPS cells), and progenitor cells. For example, the stem cells may be at least one selected from the group consisting of embryonic stem cells, adult stem cells, pluripotent stem cells, and pregenerating cells. Embryonic stem cells are stem cells derived from embryonic stem cells that are capable of differentiating into all tissue types. Induced pluripotent stem cells (iPS cells), also called degenerative stem cells, inject cells with differentiation-related genes into the differentiated somatic cells and return them to the cell stage before differentiation to induce pluripotent stem cells to become pluripotent Means cells that have been removed.

Progenitor cells have the ability to differentiate into specific types of cells similar to stem cells, but they are more specific and targeted than stem cells, and unlike stem cells, the number of divisions is finite. The pre-developmental cells may be pre-developmental cells derived from mesoderm, but are not limited thereto. In this specification, pregenerating cells are included in the category of stem cells, and unless otherwise stated, 'stem cells' are interpreted as including the entire developmental cells.

Adult stem cells are stem cells extracted from umbilical cord blood (umbilical cord blood) or adult bone marrow, blood, nerve, etc., which means primitive cells just before being differentiated into cells of specific organs. The adult stem cells may be at least one selected from the group consisting of hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and the like. The adult stem cells may be mammals, such as human adult stem cells. Adult stem cells are difficult to proliferate and tend to differentiate easily. Instead of using various kinds of adult stem cells, they can be used for various long-term regeneration needed in actual medicine. In addition, they can differentiate according to the characteristics of each organ And can be advantageously applied to the treatment of incurable diseases / incurable diseases.

In one example, the adult stem cells may be mesenchymal stem cells, such as human mesenchymal stem cells. Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into various types of cells such as osteoblasts, chondrocytes, myocytes, adipocytes, stromal cell). The mesenchymal stem cells are non-marrow stem cells such as placenta, umbilical cord blood, adipose tissue, adult muscle, corneal stroma, dental pulp, May be selected from pluripotent cells derived from non-marrow tissues.

The method of reducing the immunogenicity of stem cells provided in the present invention is a method of reducing the immunogenicity of stem cells by using the autologous serum of the recipient so that rejection does not occur in transplant recipients, The present invention can be applied particularly advantageously to allografts using stem cells derived from allogeneic non-recipient organisms since the problem of reduced stem cell production caused by culturing stem cells without using serum can be solved.

The present invention has been designed to reduce the incidence of immunogenicity by detecting differences in immunogenicity that may occur during homologous or autologous transplantation according to the culture method of mesenchymal stem cells. The method proposed here is as follows.

Immunogenicity tests on allogeneic mesenchymal stem cells were compared with cytotoxic immune responses by T cells induced by allogeneic antigen stimulation (constituted of donor stem cells + recipient immune system).

1. Preparation of mesenchymal stem cells: Differences in culture method

1) Cultured in DMEM medium containing 10% FBS with time difference before allogeneic antigen stimulation immunity, cultured with xeno-free culture medium for mesenchymal stem cells, and used as donor cells for allogeneic antigen challenge

2. Immediately after isolation from adipose tissue, mesenchymal stem cells cultured in xeno-free medium

3. Culture of allogeneic antigen challenge: xeno-free medium containing 4-5% fetal calf serum

4. Analysis of cytotoxic immune response compared the viability of mesenchymal stem cells.

5. Production of Granzyme B, a substance that induces cytotoxic immune response,

6. Optimal culture method to reduce potential immunogenicity of allogeneic or autologous mesenchymal stem cells

7. Optimal culture method for inducing proliferation of allogeneic or autologous mesenchymal stem cells by reducing immunogenicity.

Methods for evaluating immunogenicity of mesenchymal stem cells are summarized as follows:

1) Evaluation of immunogenicity of existing mesenchymal stem cells has been limited to the production of antibody against mesenchymal stem cells of allogeneic mesenchymal cells, and the test for the activity of simple T cells.

2) In general, the MLR assay used in in vitro immunogenicity assessment is to evaluate the proliferation of T cells after reacting the recipient's immune cells with the donor's irradiated immune cells. That is, the MLR test can assess the proliferation of T cells by inserting 3H-thymidine into the T cells or using the CFSE label, but it is impossible to analyze the proliferation and differentiation of the T cells.

3) The immunogenicity assay provided herein can quantitatively analyze the difference in immunogenicity according to the culture method through the analysis of the cytotoxic immune response of antigen-specific T cells and the production of cytotoxic substances according to allogeneic antigen stimulation .

In the analysis of cytotoxic immune response, the immunogenicity of T cells through the analysis of cell survival rate of mesenchymal stem cells is quantitatively analyzed for differences in immunogenicity according to the culture method.

There is no known method for evaluating the immunogenicity of allogeneic mesenchymal stem cells using the cytotoxic immunoassay using the allogeneic antigen stimulating immunity.

The immune system plays an important role in protecting our body from pathogens, but plays an important role in allogeneic transplant rejection. In particular, both CD4 T cells and CD8 T cells can recognize allogeneic HLA antigens and non-HLA antigens. The recognition of T cells in allogeneic cells, tissues, or organ transplantation is done through direct pathway and indirect pathway. In general, direct pathway (HLA recognition of allogeneic cell surface) is associated with acute rejection And the indirect pathway (recognition of allogeneic HLA peptides presented by autologous antigen presenting cells) is associated with direct and chronic rejection.

The present inventors have proposed 'Allogeneic antigen stimulation assay' as an in vitro immunogenicity assay for mesenchymal stem cells. This evaluation method was performed by using the CFSE-MLR technique together with FACS and ELISPOT analysis to analyze the antigen-specific activated CD4 T memory cell and CD4 T cell subset of allogeneic mesenchymal stem cells, It was a valuation method designed to make the most of the advantages. Hereinafter, the present invention describes whether or not inducing a cytotoxic immune response against mesenchymal stem cells to which T cells activated by allogeneic antigen stimulation to allogeneic mesenchymal stem cells have been induced Evaluation method for verification. Therefore, we aimed to evaluate immunogenicity by evaluating the cytotoxic immune response suggested in the present invention together with the activity of the antigen-specific T cell and the evaluation of the memory cell as previously described for allogeneic mesenchymal stem cells. These results are also expected to help improve the understanding of partial prediction and analysis of clinical outcomes.

This is one of the methods designed to evaluate the in vitro immunogenicity of human mesenchymal stem cells at the present scientific level. Therefore, in the field of research and development, appropriate tests are carried out in consideration of the type of cells used, clinical administration conditions, Design should be done. The methods provided herein are intended to assist in evaluating immunogenicity to researchers or licensors who develop therapeutic agents using allogeneic mesenchymal stem cells.

When human allogeneic mesenchymal stem cells are immunogenic, they induce an adaptive immune response when administered to recipients, resulting in transplant rejection, in which the transplanted cells are removed in vivo . This rejection is mainly due to the cell mediated immunity mediated by the recipient CD4 T and CD8 T cells. The cell-mediated immune response is induced by stimulation of the recipient's T lymphocytes with direct alloantigen recognition or indirect alloantigen presentation stimulation of the mesenchymal stem cells of the treated mesenchymal stem cells (See Figure 1, "T-cell stimulation pattern of recipients for allogeneic antigens"):

[Figure 1] T-cell stimulation pattern of recipient for allogeneic antigen

T cell activation is mediated by 1) cleavage of naive T cells that have never been exposed to antigen, 2) T cell differentiation (CD4 + T cells differentiate into Th1, Th2, and Th17), 3) memory T cells T cells, and finally induce delayed-type hypersensitivity (DTH), cytotoxic T lymphocyte (CTL) responses, and allogeneic antibodies to B cells, Generation results in a variety of subsequent immune responses that result in graft removal. In particular, the activity of CD8 T cells in response to allergen-mediated direct pathways associated with acute rejection may induce cytotoxic immune responses to target cells, killing target cells (Figures 1 and 2). Thus, the presence or absence of immunogenicity can be assessed by measuring the cytotoxic immune response of recipient T cells to allogeneic mesenchymal stem cells (see Figure 2, "Cytotoxic T lymphocyte (CTL) Reference)

[Figure 2] Mechanism of cytotoxic T lymphocyte (CTL) -induced death of target cells

In addition to the analysis of the effector T cell subtypes (Th1, Th2, Th17) differentiated through activation by allogeneic antigen stimulation using ELISPOT and the subsequent screening of the resulting memory T cells using FACS, In this specification, a method for evaluating a target cell death reaction by the cytotoxic immune response induced by the activation of such immune cells is proposed.

Outline of cytotoxic immune response assay for allogeneic mesenchymal stem cells Evaluation items Analysis principle Analysis method Interpret results Of mesenchymal stem cells Survival rate viability) The survival rate of surviving mesenchymal stem cells after antigen-specific T cell cytotoxic immune response Cell survival analysis of mesenchymal stem cells:
CCK-8 assay Identification of cytotoxic immune response of mesenchymal stem cells by antigen-specifically activated CD3 T cells in test group with allogeneic antigen stimulation Of mesenchymal stem cells  Apoptosis and dead cell Cell death rate of mesenchymal stem cells due to cytotoxic immune response of antigen-specific T cells Cell death of mesenchymal stem cells:
Apoptosis and dead cell assay Identification of cytotoxic immune response of mesenchymal stem cells by antigen-specifically activated CD3 T cells in test group with allogeneic antigen stimulation

The target cell death response was evaluated by viability or apoptosis and dead cell after long term immune response of mesenchymal stem cells administered to allogeneic antigen stimulation.

The technology provided in the present invention is a cell culture technique for reducing the immunogenicity of stem cells. By lowering the immunogenicity of stem cells, the possibility of inducing an immune response such as a rejection reaction is reduced, which is useful for various therapeutic techniques using stem cells Can be applied.

In addition, there is a concern that mesenchymal stem cells of the same species may develop immunogenicity due to the heterologous antigen of the cell itself at allograft. In addition, the increase in immunogenicity according to the culture method may significantly lower the clinical use of mesenchymal stem cells. However, a culture that can lower this immunogenicity is expected to help increase the survival rate of mesenchymal stem cells. In addition, it can be applied to clinical use of autologous mesenchymal stem cells.

Figure 1 shows the results of cytotoxic immune response (cell viability test) of T-cells to mesenchymal stem cells in the non-differentiated test group (XF-MSC) and the differentiated antigen challenge test group (FBS-MSC).
Figure 2 shows stem cell survival at day 7 after stimulation in an allogeneic antigen challenge study against ADSC.
Figure 3 shows stem cell survival at day 14 after stimulation in an allogeneic antigen challenge study against ADSC.
FIG. 4 shows the effect of culturing conditions of ADSC on cytokines secretion of T cells in allogeneic antigen stimulation experiments.
FIG. 5 shows the results of measurement of the effect of ADSC culture conditions on the viability of stem cells in the allogeneic antigen stimulation (ADAS) of allogeneic antigen stimulation (ADS) at 21 days.
Figure 6 shows the effect of inflammatory cytokine exposure histories, including IFN-gamma, on the viability of stem cells in ADSC allogeneic antigen stimulation of ADSCs
FIG. 7 is a graph showing the results of an autologous human serm. And the effect of FBS (BSA) was confirmed by T cell differentiation through ELISPOT analysis.
FIG. 8 is a graph showing changes in the autologous human serm. The effect of FBS (BSA) was measured by measuring the degree of memory T cell proliferation using CFSE labeling (# of CFSE-low memory T cell).
9 shows the degree of proliferation of mesenchymal stem cells in a medium containing autologous serum.

Hereinafter, the present invention will be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the present invention. The embodiments described below may be modified without departing from the gist of the invention.

Example 1. Allogeneic Antigen Stimulation

1.1. Reagents and equipment

end. reagent

StemPro®Accutase: Life Technologies, A11105-01

Fetal Bovine Serum: Gibco, Mesenchymal Cell Qualified 12664-025,

Certified 16000044

Penycillin-streptomycin: Gibco, 15140-122

Glutamax: Gibco, 35050-079

ACK Lysis Buffer: Gibco, A10492-01

Blood collection tube: BD Vacutainer 367955 (Silicone coated),

367874 (Heparin coated)

CFSE: Sigma, 21888

Ficoll: GE healthcare, 17-1440-02

4% paraformaldehyde (in PBS): Santa Cruz, 30525-89-4

Human CD3 T cell isolation kit - Negative isolation (Pan T cell isolation kit)

Miltenyi, 130-096-535

Human CD3 T cell isolation kit Positive isolation: Miltenyi, 130-050-101

Human CD4 T cell isolation kit: Miltenyi, 130-096-533

Human CD8 T cell isolation kit: Miltenyi, 130-096-495

LS separation column: Miltenyi, 130-042-401

Xenofree-media: CellGro®, 24803-0500 (Human MSC medium)

Cellstart: Life Technologies, A10142-01

ELISPOT:

ELISPOT kit: Human IFN-gamma set (BD, 551849), Human IL-17A kit (Mabtech, 3520-2A), Human Granzyme B kit (Mabtech, 3485-2A)

ELISPOT plate: Millipore, S2EM004M99

BCIP / NBT substrate: Sigma (B5655)

I. equipment

Flow cytometry (Canto II)

Gamma irradiator (10 Gy)

Macs separator, Miltenyi

Centrifugal separator

microscope

ELISPOT Reader: Autoimmun Diagnostika GmbH

1.2. Allogeneic Antigen Stimulation Method

The allogeneic antigen challenge test is performed through several steps. This example is a pretreatment step that must be performed before evaluating the allogeneic immune response of human mesenchymal stem cells using FACS, ELISPOT, and cytotoxic immune response.

(1) Separation of serum from human blood

Human autologous serum was used instead of FBS for cell culture for allogeneic antigen stimulation in which the allogeneic type antigen of the test mesenchymal stem cell (donor) was reacted with the human immune system (recipient's immune system). To obtain the autologous serum, blood was collected from the recipient (5 healthy male and female, 20 to 40 years old, no disease state), and the blood was coagulated and the blood was coagulated by placing it in a silicone coated- After centrifugation, serum was collected.

(2) Separation of PBMC (Peripheral blood mononuclear cells) from blood

For the purpose of isolating PBMCs, use heparin coated-tubes to prevent blood clotting when collecting blood from recipients (five healthy men and women aged 20 to 40 years, no disease status). The process of separating PBMC from blood by centrifugation after Ficoll treatment is schematically shown in Figure 3 below:

[Figure 3] A schematic diagram of PBMC separation using Ficoll

The specific process is as follows:

1) Blood was collected from the recipient in an amount of 15 to 20 ml, and diluted 3-fold with PBS.

2) 3 ml of Ficoll was put into a 15 ml tube, 7 ml of diluted blood was slowly poured, and the tube was centrifuged at 400 × g and 20 ° C. for 30 minutes.

3) Carefully remove the supernatant and transfer the mononuclear cell layer with PBMC to a new 15 ml tube. Fill with PBS, centrifuge at 400 × g and 20 ° C for 10 min, remove the supernatant, PBMC were washed.

4) PBMC obtained by removing the supernatant was resuspended with 1 ml of PBS and stored at 4 ° C until the experiment.

(3) Isolation of T cells from PBMC

Two separate fractions were separated from the prepared recipient PBMCs by 1) T cells and 2) T cell-depleted PBMCs, respectively.

Negative selection is the most effective method for isolating T cells from PBMC cells, since beads used to capture T cells with beads (positive selection) can affect the allogeneic antigen-stimulating immune response T cells were isolated.

Since T-cell separation-PBMC separation is aimed to remove T cells, T cells (CD3 T, CD4 T and CD8 T cells) are captured and removed using beads as a positive selection method, and the remaining cell fraction T cell depletion-PBMC). The purpose of this procedure is to allow B cells in the T cell removal-PBMC to act as antigen-presenting cells.

end. Negative selection: T cell capture from PBMC

1) Human Pan T cell isolation kit (Miltenyi, 130-096-535), Human CD4 T cell isolation kit (Miltenyi, 130-096-533) and Human CD8 T cell isolation kit (Miltenyi, 130-096-495) CD3 T cells, CD4 T cells, and CD8 T cells from the PBMC prepared in Example 1.2 (2), respectively.

2) The volume was adjusted by adding PBS to 40 μl per 10 ⁷ cells of PBMC.

3) 10 μl of Pan T cell biotin-antibody cocktail was added per 10 ⁷ cells of PBMC.

4) Weakly mixed and kept on ice for 10 minutes.

5) 30 [mu] l PBS was added per 10 ⁷ cells of PBMC.

6) Add 20 μl of Pan T cell microbead cocktail (using 20 μl of Human CD4 T cell microbead cocktail or 20 μl of Human CD8 T cell microbead cocktail, respectively) per PBMC 10 ⁷ cells, and mix for 10 minutes. (Each cell is individually reacted for individual isolation).

7) 1 ml of PBS was added to float the cells.

8) A Macs LS column was attached to a magnetic separator and the column was washed with 3 ml of cold PBS.

9) 1 ml of cell suspension was added to the LS column.

10) 3 ml of PBS was added to the column.

11) CD3 T cells (or CD4 T cells, CD8 T cells) that had passed through the column were centrifuged at 400 xg for 5 minutes at 4 ° C.

12) 1 ml of non-heterogeneous culture medium (CellGenix GmbH; containing 4-5% recipient serum) was added to isolated CD3 T cells (or CD4 T cells, CD8 T cells) and stored at 4 ° C until the experiment.

* T cells are separated into CD3 T, CD4 T, or CD8 T cells, respectively, and used according to the purpose of the experiment.

I. Positive selection: From PBMC  T cell removed PBMC  (Td-PBMC, T cell-depleted PBMC)

1) To remove T cells from PBMC isolated from blood, a human CD3 T positive isolation kit (Miltenyi, 130-050-101) was used. Alternatively, mouse anti-human CD3 T and anti-mouse bead conjugated antibodies can be isolated in stages.

2) PBS was added to a 15 ml cornical tube to a volume of 80 μl per 10 ⁷ cells of PBMC.

3) Twenty microliters of CD3 T cell microbeads were added per 10 ⁷ cells of PBMC, mixed and stored in ice for 15 minutes.

4) 1 ml of PBS was added to float the cells.

5) Macs LS column was attached to a magnetic separator and washed with 3 ml of cold PBS.

6) 1 ml of cell suspension was added to LS column.

7) 3 ml of PBS was added to the column.

8) T cell depleted-PBMC cells passed through a column were centrifuged at 400 xg for 5 minutes at 4 ° C.

9) Separated T cell depleted-PBMC cells were inoculated with 1 ml of non-heterogeneous culture medium (containing 4-5% human serum) and stored at 4 ° C until the experiment.

(4) T cell removal-PBMC (T-cell depleted PBMC) was irradiated with radiation

T cell removal-It is a process of losing the ability of B cells to function as antigen-presenting cells by irradiating 1,000,000 rad radiation to PBMC. When it is above 2,000 rad, the function of B cells as antigen presenting cells is remarkably decreased.

Example 1.2 (3) above b. (Td-PBMC) was irradiated at 1,000 rad. Removal of irradiated T cells-1 ml of non-heterogeneous culture medium (containing 4-5% recipient serum) was added to PBMC and stored at 4 ° C until the experiment. Removal of T cells to be used for repeated stimulation-PBMCs were stored in liquid nitrogen with 10% DMSO (a non-heterogeneous culture medium containing 10% fetal bovine serum).

(5) CFSE labeling of T cells

Example 1.2 (3). , T cells were labeled with CFSE to evaluate whether the recipient T cells were differentiated by antigen stimulation of mesenchymal stem cells. CFSE expresses fluorescence by forming strong covalent bonds with living primary amines. The detection principle is that fluorescence sensitivity decreases as CFSE content decreases as cell proliferation proceeds. In the following examples, antigen-specific T cell proliferation was evaluated by the presence or absence of CFSE-low T and CFSE-low memory T cells during FACS analysis.

First, 10 ml of PBS was added to the prepared recipient CD3 T (or CD4 T, or CD8 T) cells, and the cells were washed by centrifugation at 400 xg for 5 minutes at 4 ° C. CD3 T (or CD4 T, or CD8 T) cells were suspended in 1 ml of PBS. 10 ml of 100 uM CFSE was added to the suspension of CD3 T cells on a clean bench, immediately mixed with 1 ml of PBS, and the final concentration of CFSE was adjusted to 0.5 uM. Tube was kept at room temperature for 5 minutes with light blocked. The Xeno-free media was then filled and washed by centrifugation at 400xg and 4 ° C for 5 minutes. This washing process was performed twice using xeno-free media or PBS. 1 ml of xeno-free media was added to CFSE-labeled CD3 T (or CD4 T, or CD8 T) cells and kept on ice until the experiment.

(6) Allogeneic antigen stimulation for cytotoxic immune response assessment

Allogeneic antigen stimulation for cytotoxic immune response assessment and comparison of Granzyme B production was performed by direct stimulation (see Figure 1). This is based on previous findings that direct stimulation methods are more effective in immunogenicity analysis than indirect stimulation methods. Allogeneic antigen stimulation lasts for 14-24 days (cell viability analysis), with the exchange of culture medium every 7 days, as in Example 1.2 (3). Lt; RTI ID = 0.0 > PBMC < / RTI > was added. In order to remove the immune response inducers such as FBS in the allogeneic antigen stimulation, in this test procedure, xeno-free mesenchymal stem cells containing 4-5% of autologous serum Allogeneic antigen stimulation experiments were performed under the condition of exclusive culture medium (CellGenix).

The stimulation conditions and methods of allogeneic antigens for the evaluation of cytotoxic immune responses are as follows.

Direct dimorphic antigen stimulation (Direct allogeneic antigen stimulation) cellular composition

: T cell removal-PBMC + T (CD3 T, CD4 T, or CD8 T) + allogeneic mesenchymal stem cells

1) One day before the experiment, allogeneic mesenchymal stem cells (human normal abdomen or breast adipose-derived stem cells (ADSC)) were plated on a 12-well plate at 1 × 10 ³ cells / The next day, a direct allogeneic antigen challenge experiment was prepared.

2) CFSE-labeled CD4 T or CD8 T, CD3 T (1.8 x 10 ⁵ cells in the case of CD3 T, 1.2 x 10 ⁵ cells in the case of CD4 T, 6 x in 12-well plate the Td-PBMC); 10 ⁴ cells. example 1.2 5 ready) cells and irradiated T-cells removed in -PBMC (1x10 ⁵ cells. example 1.2 (4) prepared in Respectively. Mesenchymal stem cells (XF-ADSC), which were cultured with immune cells in a non-heterogeneous medium, were used as an experimental control for cell survival assay (i.e., xenogeneic ADSCs (XF-ADSC) ADSCs + immune cell comparisons). As an experimental control for the cell death rate test, an experimental group in which only allogeneic mesenchymal stem cells were cultured in a non-heterogeneous medium without containing immune cells (CD3 T and T cell removal-PBMC) was used (i.e., non-heterologous ADSCs + Comparison of cell-free, heterologous ADSCs).

3) 2) was prepared to a final culture volume of 1 ml / well in a non-heterogeneous culture medium containing 4 ~ 5% of autologous serum and 1 ng / ml of human recombinant IL-2.

4) The cells were cultured for 7 days and the medium was changed. At the time of medium change, 50% of the supernatant was removed from the supernatant, and the supernatant was carefully removed without any shaking to prevent the floating cells on the bottom from disappearing. The non-heterogeneous culture medium described in 3) was filled to 1 ml / well.

5) When replacing the culture medium at 7-day intervals, the irradiated T cells removed in liquid nitrogen-PBMC were further thawed and washed (same number of cells as in day 0, 1x10 ⁵ cells).

6) The cytotoxic immune response of T cells using direct allogeneic antigen stimulation to mesenchymal stem cells was evaluated by microscopic observation of mesenchymal stem cell morphology between 14 and 24 days after the reaction, And cell viability and cell death were examined. (Analysis time point is when microscopic observation of morphologic damage of mesenchymal stem cells due to allogeneic antigen stimulation (compared with control group) and time-difference analysis may be necessary for optimal analysis time. , And the analysis of the memory cells proceeded on the 21st day.

Allogeneic mesenchymal stem cells can be administered repeatedly with a single dose at the time of allogeneic antigen challenge, depending on the purpose of the experiment. In the case of repeated administration, allogeneic mesenchymal stem cells can be administered even in the 7th day as in the 0th day in the experimental method described above.

Example 2. Assessment of T cell cytotoxic immune response

In this step, the cytotoxic immune response of T cells (using CD3 T, CD4 T, or CD8 T cells) to allogeneic mesenchymal stem cells after allogeneic antigen stimulation is evaluated by the cell survival rate.

In general, to assess cell survival and death, various evaluation methods ranging from traditional pigment excretion methods (eg, trypan blue), which utilize structural damage of cells due to death, to methods for evaluating functional degeneration of cells by cell death . In this example, cell viability was measured as one of the methods for evaluating cytotoxicity due to an immune response.

One of the most widely used cytotoxicity assays based on functional activity of cells is Microculture Tetrazolium Test, which is called MTT assay. It is used to measure the activity of dehydrogenase (mitochondrial dehydrogenase) contained in mitochondria in cells. (See Figure 4, "Principles of living cell measurement", below).

[Figure 4] Principles of living cell measurement

However, this MTT assay has limitations in that experimental results are somewhat present in the process of dissolving the cells and the resulting insoluble crystals in order to measure the result, since the resultant coloring matter produced by living cells is insoluble in water.

In this embodiment, the cell survival rate was measured using a chromogenic-based evaluation method called Cell Counting Kit-8 (CCK-8), which is commonly used in the present invention. CCK-8 is based on the reaction of colorless WST-8 with orange-colored WST-8 Formazan through oxidation due to electron mediator. It is more sensitive than conventional methods, and the generated coloring agent is water- And it is advantageous in terms of low experimental variability according to.

2.1. Reagents and equipment

(1) Reagent

CCK-8 (Cell Counting Kit-8, Cell Survival Analysis): Dojindo Molecular

Technologies, CK04-11

(2) Equipment

microscope

ELISA Reader (Filter: 450 nm)

2.2. Test Methods

(1) Cell survival analysis

1) It was observed microscopically whether mesenchymal stem cell injury was induced between 14 and 24 days after the allogeneic antigen stimulation.

2) When damaged mesenchymal stem cells were observed, the 12-well plate was shaken gently to suspend T cell and Td-PBMC cells, and the supernatant was removed. To remove the floating cells, they were carefully washed once or twice with 400 μl of PBS (warmed in a 37 ° C water bath). At this time, be careful not to let the attached mesenchymal stem cells fall.

3) To the 12-well plate from which the floating cells had been removed, 0.3 ml of the DMEM culture medium was added, and 30 μl of CCK-8 was added thereto, followed by 3 to 4 hours of reaction in a CO ₂ incubator.

4) 100 μl of each sample in a 12-well plate was transferred to a 96-well plate and dispensed.

5) The cell viability of mesenchymal stem cells was evaluated by analyzing the absorbance at 450 nm using an ELISA reader.

Example 3. Evaluation of granzyme B-secreted T cells using ELISPOT

In this example, the cytotoxic immune response was evaluated by generation of Granzyme B of activated CD3 T cells (or CD8 T) upon allogeneic antigen stimulation of mesenchymal stem cells using ELISPOT.

In this example, allogeneic dendritic cell stimulation (direct pathway) was repeatedly applied to mesenchymal stem cells as in the following FIG. 5 (ELISPOT-based allogeneic antigen stimulation test), and ELISPOT The reaction was carried out and analyzed on day 17 after 3 days:

[Figure 5] Overview of allogeneic antigen stimulation test using ELISPOT

In FIG. 5, allogeneic antigen stimulation (AAS) means that the mesenchymal stem cells and the recipient immune system are mixed and reacted for allogeneic antigen stimulation. Cytokine is an additional test condition for adding cytokines or reflecting the elevated inflammatory state of the patient during the administration of allogeneic mesenchymal stem cells. The ADSC can be administered once (0d) or twice (0d, 7d). In this example, ELISPOT was performed using a commercial kit (Mabtech, BD bioscience).

Analysis process

1) Allogeneic antigen stimulation tests were performed as in steps 1) to 5) of Example 1. An ELISPOT test was performed on the 14th day after the reaction.

2) On the day before the ELISPOT experiment (on the 13th day), 100 μl of 30% ethanol was added to a 96-well plate for ELISPOT, allowed to stand for 30 seconds, and discarded. After washing three times with PBS, PBS was added one more time to prevent the plate from drying and then removed. Then, 100 μl of the capture-antibody (diluted with PBS according to the manufacturer's instructions) was added to each well of the cytokine to be analyzed, and reacted overnight at 4 ° C. This procedure was performed one day before the ELISPOT experiment (day 13).

3) On the 14th day, the capture-antibody attached to the plate was removed from the ELISPOT plate and washed three times with PBS (see Fig. 5).

4) In the case of 1) allogeneic antigen-stimulated group, all of them were collected using acutase and centrifuged to remove supernatant. To this, 1 ml of the new culture was added and the cells were suspended and Td-PBMC (T cell depleted PBMC) was thawed. The cell mixture was added to the ELISPOT 96-well filter plate (PVDF) prepared in 3) in triplicate or quadruplicate, and incubated for 3 days in an incubator. At this time, 10% of the cells harvested after allogeneic antigen stimulation were subjected to flow cytometry and left for analysis of antigen-specific T cells (CFSE-low T cells). CFSE-low T cells were analyzed by flow cytometry after attaching anti-CD3, anti-CD4, and anti-CD8 antibodies (BioLegend).

5) On the ELISPOT plate, allogeneic antigen stimulation was performed for 3 days (day 17), and the culture solution was removed and washed twice with 200 μl of distilled water. The cells were then washed three times with PBS (Washing buffer I) containing 0.05% Tween-20.

6) Detection antibody (biotin conjugated-Ab) was diluted in PBS according to the manufacturer's instructions, added to the plate in an amount of 100 μl, and reacted at room temperature for 2 hours.

7) Washing with Washing buffer I (PBS containing 0.05% Tween-20) three times.

8) Streptavidin-ALP (Alkaline phosphatase, Mabtech) was diluted 1,000-fold with PBS, and 100 μl of each was added to the plate and incubated for 1 hour at constant temperature.

9) Washing buffer I was washed 4 times, and PBS was washed 2 times.

10) 100 μl of BCIP / NBT substrate (Mabtech or Sigma) solution was added to each well. Spot formation was observed for 5 to 60 minutes, preventing overdevelop. This creates a high background which makes data analysis difficult.

11) The plate was washed twice with distilled water to stop the reaction, and the plate was dried at constant temperature.

12) The spot was analyzed with an ELISPOT reader (Autoimmun Diagnostika GmbH).

13) Cytokine spots secreted from antigen-specific T cells (CFSE-low T) were analyzed (eg, spot number / 1x10 ² CFSE-low T cells)

Example 4. Example of immunogenicity reduction test

This example shows a series of evaluation examples conducted to verify the immunogenicity reduction method using the cytotoxic immune response of T cells according to the allogeneic antigen stimulation reaction of the mesenchymal stem cells provided herein do.

First, in order to evaluate the cytotoxic immune response, the cell viability measurement method using CCK-8 uses a method of detecting the chromogenic reaction generated by the dehydrogenase activity in intracellular mitochondria, and it has a higher sensitivity and reproducibility than the cell death rate test There are advantages. However, since the remaining floating cells except for the mesenchymal stem cells to be evaluated before the chromogenic reaction after the allogeneic antigen stimulation reaction are washed, the remaining cells may be adhered. Therefore, all of the cells from the antigen stimulation reaction to the coloring reaction The difference in the cell composition in each reaction system may affect the reaction result, which may cause confusion in the interpretation of the result. Therefore, the cell viability test can be said to be suitable for evaluating the relative cytotoxic immune response difference between the test subjects when the antigen stimulation reaction proceeds under the same conditions. In the present invention, the same donor mesenchymal stem cells are used for the allogeneic antigen stimulation using the recipient's immune cells. However, the present invention is compared with the case where the heterologous antigen (FBS in the culture medium) Cell viability test was performed for the purpose.

Analysis of cytotoxic immune response following allogeneic antigen challenge Test group Target cell Allogeneic Antigen Stimulation Reaction Conditions Analysis method Experience with heterologous antigen exposure Donor antigen Recipient immune cell Serum (5 wt%) 1. Experimental group with heterologous antigen (Transfer from FBS containing medium to non-heterogeneous medium) Human mesenchymal stem cells has exist Recipient sera Cell viability test 2. Non-heterogeneous test group None (non heterogeneous medium) Human mesenchymal stem cells has exist Recipient sera

(In the above table, the non-heterogeneous medium is CellGenix GmbH (serum-free medium), and the mesenchymal stem cells of the non-heterogeneous test group are cultured using donor serum before the allogeneic antigen stimulation It is effective for cell proliferation but can be cultured under serum-free conditions when uncertain).

The experimental conditions of the analysis examples using the cell viability test presented in this specification are summarized in Table 2.

4.1. Preparation of Test Groups

4.1.1. Experimental group of heterologous antigen (FBS-MSC; comparative example)

Mesenchymal stem cells were isolated from adipose tissue and subcultured in DMEM medium containing 10% FBS. Cells were washed with PBS and replaced with a non-heterogeneous culture medium 14-1 days before or at the day of allogeneic type antigen stimulation experiment. Cells cultured in this manner were harvested at the day before the allogeneic antigen stimulation experiment (for experiments in which the date of the culture medium was not 0d) or on the same day , and compared with the control group under the same conditions between the same experiments. Subsequently, in the case of allogeneic heterologous antigen stimulation, the medium composition was the same as that of the heterologous antigen test group and the non-heterologous test group, and the recipient immune cells were added (see Example 1).

4.1.2. Non-heterogeneous test group (XF-MSC)

Immediately after the mesenchymal stem cells were isolated from the adipose tissue, cells cultured in a heterogeneous medium were used for allogeneic antigen stimulation. The medium used in this reaction was a donor-derived medium supplemented with donor serum, and a recipient immune cell was added to induce a homologous antigen-stimulating reaction (see Example 1).

4.2. Cell survival analysis

(TdPBMC + T cell: recipient immune system; XF-MSC; non-heterogeneous test group; FBS-T cell; MSC, heterologous antigen (FBS) experience test group, ***: p <0.001). FIG. 1 A shows an image obtained by observing the degree of cell proliferation under a microscope. It was observed that the cells of the heterologous antigen test group (FBS-MSC) exhibited a considerable increase in damage compared to the non-heterogeneous test group (XF-MSC) . FIG. 1B is a graph showing the results of comparing cell survival rate with CCK-8 on the 14th day of allogeneic antigen stimulation. As compared with the non-heterogeneous test group (XF-MSC) (P <0.001), which was about 75% of the mean cell survival rate.

The results of the above experiments show that the cell viability assay using CCK-8 is suitable for measuring the cytotoxic immune response due to allogeneic antigen stimulation, and even if full-scale cell proliferation is performed in a non-heterogeneous medium, It is suggested that mesenchymal stem cells, which have been exposed to the same heterologous antigen, may have increased immunogenicity compared to the non-heterogeneous medium in the whole cycle. Therefore, the use of non - heterogeneous media in the whole cell processing process from tissue collection may be helpful in lowering immunogenicity.

(One) ADSC Allogeneic antigen  During stimulation ADSC  Effect of culture conditions on viability: Cell viability after 7 days of allogeneic antigen stimulation

The cultivation of ADSCs can be cultured under various culture conditions depending on the experimental conditions. In particular, the culture medium containing FBS is highly cultured, and the effect of such culture conditions on the immunogenicity test of ADSCs was investigated. Thus, ADSCs were cultured under three conditions that could be performed in the laboratory as follows: immunogenicity testing with allogeneic antigen stimulation was performed:

In other words, ADSCs were cultured in 1) Xeno-free medium (XF-MSC) and 2) DMEM containing FBS immediately after isolation from adipose tissue, washed with PBS 4 days before allogeneic antigen stimulation, (-3d XF), 3) cultured in DMEM containing FBS, washed with PBS 2 days before allogeneic antigen stimulation and replaced with xeno-free media (-1d XF).

Allogeneic antigen stimulation (AAS) was performed using three ADSCs cultured in this manner. The serum used was recipient serum (recipient autologous serum, T cell and PBMC donor).

The results of cell viability measurement on day 7 after allogeneic antigen challenge to ADSC are shown in FIG. (CD4 ⁺ T + CD8 ⁺ T cells, present in the blood), CD8 ⁺ T (purity: 99.9%), and CD3 ⁺ T CD4 ⁺ T (purity: 100%) cells. As shown in Fig. 2, the -1d XF MSC showed a slight decrease in cell viability compared to the Xeno-free (XF, non-heterogeneous) MSC by CD3 T cells. However, the -1d XF MSC showed a significant decrease in cell viability compared to the XF MSC by CD8 T cells. However, compared to XF MSC, CD4 T cells did not show cytotoxicity in both -3d XF MSC and -1d XF MSC. These results suggest that CD8 T cells may exhibit cytotoxicity against the -1d XF MSC that had been cultured in DMEM containing FBS rather than XF MSC. That is, as shown in FIG. 2, -1d XF MSC induces the cytotoxic action of CD8 T cells as compared to XF MSC.

(2) ADSC Allogeneic antigen  During stimulation ADSC  Effect of culture conditions on Viability: Allogeneic antigen stimulation 14 days after cell viability test

Cell viability was checked on day 14 after allogeneic antigen challenge to ADSC cells and the results are shown in FIG. As shown in Fig. 3, CD-1 T cells show that -1d XF MSC and -3d XF MSC have significantly decreased cell viability as compared to Xeno-free (XF) ADSC. This is a phenomenon that did not occur on day 7 after allogeneic antigen challenge (FIG. 2), and cytotoxicity by T cells can be observed on day 14. However, it is necessary to identify which T cells contribute to this cytotoxicity. CD8 T cells show that the cell viability of the -3d XF MSC as well as the -1d XF MSC is significantly lower than that of the XF ADSC. However, CD4 T cells did not show cytotoxicity for both -3d XF MSC and -1d XF MSC as compared with XF ADSC at 14days as well as 7days after allogeneic antigen stimulation stimulation. These results suggest that CD3 T cells and CD8 T cells may exhibit cytotoxicity against -1d XF MSC and -3d XF MSC, which had been cultured in DMEM containing FBS rather than XF ADSC cells. However, CD4 T cells did not show cytotoxicity after 14 days of allogeneic antigen stimulation. This shows that cytotoxicity is caused by antigen-specific CD8 T cells. These results indicate that ADSCs cultured in FBS can further increase cytotoxic effects compared to Xeno-free ADSCs. As shown in FIG. 3, MSCs cultured in -1d XF and -3d XF induce cytotoxic effects of CD8 T cells compared with Xeno-free MSCs. However, CD4 T cells do not have direct cytotoxic action.

(3) ADSC  Culture conditions Allogeneic antigen  Effect of antigen-specific T cell activation on stimulation: AAS 17days  on ELISPOT  (Generation of Granzyme B Causing Cytotoxic Immune Response by CD3 T Cells)

Specificity of antigen-specific CD3 T cells producing granzyme B secreted by allogeneic antigen stimulation experiments using ADSC prepared according to the three culture conditions of ADSC (① XF ADSC, ② -3d XF MSC, ③ -1d XF MSC) Was verified by ELISPOT. The results are shown in Fig. FIG. 4 shows the effect of culturing conditions of ADSC on the secretion of cytokines in the allogeneic antigen stimulation experiment, and shows the results of two experiments averaged, and the ELISPOT figure represents one of them. As shown in FIG. 4, antigen-specific CD3 T cells producing granzyme B occurred more frequently in -1d XF MSC and -3d XF MSC than in XF-ADSC. In addition, XF ADSC also shows that CD3 T cells induce the production of granzyme B. This indicates that XF ADSC has also been shown to be cytotoxic in response to CD3 T cells (data not shown). In addition, IFN-gamma and IL-17A produced from antigen-specific CD3 T cells were similar between the three culturing conditions, XF ADSC, -3d XF MSC, and -1d XF MSC. As shown in Figure 4, ADSCs cultured in -1d XF and -3d XF increase the production of Granzyme B by CD8 T cells compared to Xeno-free ADSC. Production of IFN-gamma and IL-17A was similarly induced in all conditions of ADSC.

(4) ADSC Allogeneic antigen  During stimulation ADSC  Effect of culture conditions on viability: Allogeneic antigen stimulation 21 days after cell viability test

The cell viability was checked on day 21 after allogeneic antigen challenge to ADSC cells (cell viability test by CD3 T cell, -14d XF MSC to 0d XF MSC) is shown in FIG. In FIG. 5, it can be seen how the schedule change from 14 days to 0 day of the allogeneic antigen stimulation experiment (DMEM to FBS-free DM to non-diabetic) affects cytotoxicity. FIG. 5 shows that the -14d XF MSC showed the highest survival rate after 21 days of allogeneic antigen stimulation, and the cytotoxicity of T cells was increased (-3d XF, -1d XF, and 0d XF) . Thus, these results show that the cell survival rate can be most stable when the ADSCs are cultured in media containing heterologous antigens and then cultured in a non-heterogeneous form, rather than cultured in a non-heterogeneous medium.

(5) ADSC Allogeneic antigen  During stimulation ADSC IFN Including -gamma Inflammatory cytokine  Effect of exposure history on Viability

(IFN-gamma; 50 ng / ml, 34-8319-85), interleukin (IL), and IL-17 stimulated with a combination of inflammatory cytokine IFN-gamma and IL-17A and IL-23 4 days before allogeneic antigen stimulation IL-23 (10 ng / ml, 14-8239-63; eBioscience)) allogeneic antigens of ADSC, HLA-HBC and HLA-DR The degree of expression and the cell viability were measured and shown in Fig.

As shown in Fig. 6, the degree of expression of allogeneic antigens of inflammatory cytokine-treated ADSC, i.e., HLA-HBC and HLA-DR, was increased as compared with the Xeno-free MSC without treatment of the inflammatory cytokine, Respectively. These results demonstrate that the absence of exposure to inflammatory cytokine environments, including IFN-gamma, by ADSC can reduce antigenicity and decrease survival due to increased immunogenicity. In addition, this phenomenon can be applied even when the mesenchymal stem cells are clinically administered to the patient.

Example 5. MSC Influence of heterologous serum contained in the medium upon allogeneic antigen stimulation : Analysis of IFN-gamma production by CD3 T cells

Autologous human serum (4-5% autologous human serum vs. The effect of FBS (10%) and BSA was confirmed in terms of T cell differentiation (IFN-gamma production) through ELISPOT analysis, and the results are shown in FIG. The results of FIG. 7 show that heterologous sera such as FBS and BSA induce the activity of human immune T cells, while those of autologous serum-containing non-heterogeneous medium do not induce such immune T cell activity.

Example 6. MSC Influence of serum contained in medium upon stimulation with allogeneic antigens : Specific memory T cell generation assay for heterologous serum

Autologous human serum (4-5% autologous human serum vs. The effect of BSA was measured by the degree of memory T cell proliferation (# of CFSE-low memory T cell) using CFSE labeling, and the result is shown in FIG. The results of FIG. 8 show that induction of the production of heterologous serum antigen-specific human immuno-memory T cells such as FBS and BSA is not induced in the non-heterogeneous medium containing autologous human serum Show.

Example  7. Non-heterogeneous medium ( Xeno -free media) contrast Autologous serum  include In a heterogeneous medium  Mesenchymal stem cell proliferation test

The problem with this medium is that it significantly reduces the proliferation of MSCs, which is a major limitation in the clinical application of mesenchymal stem cells including allografts and autologous transplantation. . However, the use of heterologous sera, which are commonly used in the culture of MSCs, causes an increase in immunogenicity in allografts, and it also causes the heterologous serum to cause immunogenicity in autologous transplantation. In the example,

Therefore, in this example, a method for lowering or eliminating immunogenicity and using autologous serum at 5% to 10% using medium for mesenchymal stem cells without heterologous material is proposed.

The degree of proliferation of mesenchymal stem cells when autologous serum was separated and added to a non-heterogeneous medium treated with a filter (0.45um) was measured and shown in FIG. As shown in FIG. 9, the proliferation rate of mesenchymal stem cells cultured in the autologous serum-containing medium was significantly increased compared to that of autologous serum-free medium. In other words, the mesenchymal stem cells of autogenous serum-free mesenchymal stem cells may be able to reduce or eliminate immunogenicity concerns, but they are limited quantitatively for clinical use. Is predicted.

Assessment Considerations

This study of allogeneic antigen stimulation using human-derived allogeneic mesenchymal stem cells and recipient's immune cells can be used to assess the immunogenicity of mesenchymal stem cells and to determine the possible induction of immune response in clinical applications Is the evaluation method. Analysis of cytotoxic immune responses by antigen-specific T cells on stem cells will aid in the evaluation of immunogenicity and understanding of this response. Although there is an experimental limitation that it is difficult to predict all of the in vivo reactions clinically using the in vitro experimental results, there is a limitation in the current science level that the reactions occurring in vivo are limited And can help in analyzing and interpreting results. However, since the cells used in this study, particularly the recipient immune cells, have a great influence on the experimental results depending on the cell conditions, the following considerations must be taken into consideration in carrying out the experiments for evaluation.

1) The recipient immune system (T cell-depleted PBMC, CD3 T cells) used for the allogeneic antigen stimulation reaction is the most important factor in securing the quality of the experimental results as a subject of cytotoxic immune response. It is required to conduct experiments by securing the purity of the isolated cells and the freshness of the cells. Since the purity of isolated cells is related to the proficiency of the experimenter, the experimenter should always thoroughly establish the experimental plan in advance and be familiar with the process. To ensure freshness of the cells, the cells should be separated from the recipient and stored at 4 ° C in a medium containing autologous serum. Minimize the incubation time until allogeneic antigen stimulation.

2) In the cell viability test, since the activity of all the cells in the reaction vessel is measured sensitively, differences in cell composition in the reactor may affect the experimental results. Although the floating cells are washed before the chromogenic reaction, it is difficult to completely wash the added cells in this process, and there are cases in which the cells are strongly adherent depending on the type of the cells. Therefore, it is recommended to evaluate the cell death rate rather than the cell survival rate when comparing different cell compositions when inducing allogeneic antigen stimulation.

3) In the analysis example of this specification, the mesenchymal stem cell with heterologous antigen experience showed significant cell survival rate compared to the cell without heterologous condition after 14-21 days from induction of allogeneic antigen stimulation. Since the cytotoxic immune response induction time may vary depending on the degree of the immune response, in the actual evaluation experiment, the state of the cell is closely examined with a microscope during the homotypic antigen stimulation reaction period, And the reaction may be delayed in some cases. Therefore, it is necessary to induce and evaluate the reaction by appropriately changing the culture medium as described in the experimental method for a sufficient period of time. In addition, if there is an available negative and positive control group, it is determined whether the reaction is cytotoxic immune response of the recipient immune cells according to the immunogenicity of the subject cell, and whether the immune cells of the recipient have proper activity It is also helpful to check.

4) Assessment of cytotoxic immune response induced by allogeneic antigen stimulation using the cell survival rate presented herein is an in vitro assay for the evaluation of immunogenicity against allogeneic mesenchymal stem cells. Therefore, the actual situation in which the cells are administered to the living body depends on a variety of conditions such as the amount of the administered cells, the site of administration of the cells, the immune system state of the recipient, etc. Therefore, Is limited in determining whether a meaningful quantitative immune response will be induced, and the presence or effect of immunogenicity can be assessed. However, it is believed that the present invention will be helpful as a basis for the immunological analysis and interpretation of clinical results. In addition, the evaluation method proposed in the present invention may be useful for comparative comparative evaluation such as optimization of a process for minimizing the generation of immunogenicity in the course of developing therapeutic agents using mesenchymal stem cells of the same species.

5) As shown in the above analysis example, even when the heterogeneous medium is used for the full-scale cell proliferation, if the heterogeneous medium containing FBS is used for tissue harvesting, stem cell isolation, and initial culture, Cytotoxic immune response was significantly higher than that of the control group. Therefore, in the course of developing a therapeutic agent using mesenchymal stem cells of allogeneic species, it is considered to be possible to minimize the immunogenicity by eliminating the process of exposing cells to heterologous antigens as much as possible.

Claims (11)

Culturing the stem cells in a xeno-free medium containing no heterologous antigen,
Wherein the heterologous antigen is a protein derived from a species different from the one from which the stem cell is derived,
A method for producing stem cells with reduced immunogenicity. The method according to claim 1, wherein the heterologous antigen is at least one selected from the group consisting of albumin derived from a species different from the stem cell-derived individual, and serum and blood containing the same. 2. The method of claim 1, wherein the entity is a human. 4. The method according to any one of claims 1 to 3, wherein said stem cells are for use in allograft or autotransplantation. 5. The method of claim 4, wherein the non-heterogeneous medium further comprises an autologous serum derived from an allograft recipient. 6. The method of claim 5, wherein the content of autologous serum is 1 to 10% (v / v) based on the total volume of the non-heterogeneous medium. 7. The method of claim 6, wherein the autologous serum content is 1-8% (v / v) based on the total volume of the non-heterogeneous medium. A composition for allogeneic stem cell transplantation comprising the stem cell prepared by the method of claim 4. 9. The composition of claim 8, wherein the stem cells have been cultured in a non-heterogeneous medium further comprising autologous serum derived from recipients of allografts. 10. The composition of claim 9, wherein the autologous serum content is 1 to 10% (v / v) based on the total volume of the non-heterogeneous medium. 11. The composition of claim 10, wherein the autologous serum content is 1-8% (v / v) based on the total volume of the non-heterogeneous medium.

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