Preparation method of immune regulation type stem cell for treating graft versus host disease

Abstract

The invention relates to the technical field of cell engineering, in particular to a preparation method of immune regulation type stem cells for treating graft versus host diseases. The invention provides a preparation method of immune regulation type stem cells for treating graft versus host diseases, which comprises the following steps: (1) cleaning a human fat aspirator, and digesting with collagenase to obtain mononuclear cells; (2) inoculating the mononuclear cells obtained in the step (1) into a differential culture medium, culturing for 2-3 days, and further culturing the adherent cells for 18-22 days to obtain mesenchymal stem cells; and (3) inoculating the mesenchymal stem cells obtained in the step (2) into an induction culture medium for induction culture, and screening to obtain the immune regulation type stem cells. According to the preparation method provided by the invention, the technical problems of unstable source, function attenuation and poor treatment effect of the immune regulation type stem cells in the prior art are solved, and an efficient, safe and standardized new scheme is provided for treatment of GVHD.

Description

Technical Field

[0001] The present invention relates to the technical field of cell engineering, and in particular to a method for preparing an immunomodulatory stem cell for treating graft-versus-host disease. Background Art

[0002] Graft-versus-host disease (GVHD) is a common and severe complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT), which is caused by donor immune cells attacking recipient tissues, and the clinical manifestations are multi-organ injuries such as the skin, liver, and gastrointestinal tract. Although glucocorticoids and immunosuppressive agents (such as calcineurin inhibitors) are the first-line treatment regimens for GVHD, about 30% - 50% of patients develop steroid-resistant GVHD, with a very poor prognosis and a mortality rate as high as 80%. Existing therapies such as monoclonal antibodies (such as anti-CD25 or anti-TNF-α) or mesenchymal stem cells (MSCs) have certain curative effects, but there are problems such as low response rate, unstable curative effect, or potential pro-tumor risk.

[0003] In recent years, immunomodulatory stem cells (such as regulatory T cell-induced stem cells or genetically modified mesenchymal stem cells) have become a research hotspot due to their bidirectional immunoregulatory ability. Such cells can relieve GVHD by secreting anti-inflammatory factors (such as IL-10, TGF-β), inhibiting the proliferation of effector T cells, or inducing immune tolerance. However, traditional preparation methods face the following bottlenecks: cell source limitation: primary MSCs have problems such as poor amplification ability and large differences between batches; insufficient functional stability: in vitro culture easily leads to attenuation of the immunomodulatory function of stem cells; targeting defect: lack of the ability to respond to the specific inflammatory microenvironment of GVHD (such as high expression of IFN-γ or IL-6); standardization challenge: existing processes are difficult to balance cell purity, viability, and large-scale production requirements.

[0004] Therefore, how to develop a method for preparing an immunomodulatory stem cell with a stable source, controllable function, and capable of targeting GVHD lesions is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing an immunomodulatory stem cell for treating graft-versus-host disease.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides a method for preparing an immunomodulatory stem cell for treating graft-versus-host disease, comprising the following steps:

[0008] (1) After washing the human adipose aspirate, digest it with collagenase to obtain mononuclear cells;

[0009] (2) Inoculate the monocytes obtained in step (1) into a differentiation medium and culture for 2 - 3 days, and then continue to culture the adherent cells for 18 - 22 days to obtain mesenchymal stem cells;

[0010] The differentiation medium is based on DMEM medium and includes the following components: 6 - 8% (v / v) human serum albumin, 20 - 30 ng / mL basic fibroblast growth factor, 0.2 - 0.4 mM L-alanine, 0.2 - 0.4 mM L-aspartic acid, 0.3 - 0.5 mM L-proline, 0.3 - 0.5 mM L-serine, 6 - 9 μg / L 4-O-galloylpaeoniflorin, 25 - 35 ng / mL total saponins of Panax notoginseng, 0.2 - 0.4% (v / v) β-mercaptoethanol;

[0011] (3) Inoculate the mesenchymal stem cells obtained in step (2) into an induction medium for induction culture, and after screening, obtain immunomodulatory stem cells.

[0012] Preferably, the washing in step (1) is to rinse the human fat extract with phosphate buffer.

[0013] Preferably, the collagenase in step (1) is type I collagenase or type II collagenase; the concentration of the collagenase is 150 - 200 U / ml.

[0014] Preferably, the temperature of digestion in step (1) is 36 - 38 °C and the time is 35 - 45 min.

[0015] Preferably, the seeding density in step (2) is 6 - 8×10 5 cells / ml.

[0016] Preferably, the induction medium is based on DMEM medium and includes the following component: 20 - 30 IU / mL IFN-γ.

[0017] Preferably, the induction medium further includes: 8 - 12 μg / L platelet-derived growth factor, 12 - 18 μg / ml endothelial cell growth factor, 12 - 18 U / ml heparin, 8 - 12% human serum albumin, 6 - 9 μg / L 4-O-galloylpaeoniflorin, 15 - 25 ng / mL total saponins of Panax notoginseng, 2 - 4 mM glutamine.

[0018] Preferably, the seeding density in step (3) is 8 - 12×10 5 cells / ml.

[0019] Preferably, the time of induction culture in step (3) is 1 - 2 days.

[0020] The present invention also provides the use of the immunomodulatory stem cells prepared by the above method in the preparation of drugs for treating graft-versus-host disease.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention uses human adipose aspirate as the stem cell source, and through collagenase digestion and differentiation medium formulation, to prepare mesenchymal stem cells. This method significantly improves the extraction efficiency of monocytes and the proliferation ability of mesenchymal stem cells.

[0023] The specific 4-O-galloylpaeoniflorin and total saponins of Panax notoginseng are added to the differentiation medium and induction medium of the present invention. The addition of these two components can improve the proliferation ability of mesenchymal stem cells and enhance the immunomodulatory function of immunomodulatory stem cells.

[0024] The immunomodulatory stem cells prepared by the present invention exhibit significant immunosuppressive ability. Experimental data show that the positive rates of its surface markers CD73 / CD90 / CD105 are greater than 99%, and the negativity of CD34 / CD45 / HLA-DR is lower than 0.02%, meeting the relevant standards. Further animal experiments provided by the present invention confirm that these stem cells can effectively alleviate the symptoms of GVHD, significantly reduce the bone marrow chimerism rate of mice, and improve the survival rate of mice.

[0025] The preparation method provided by the present invention solves the technical problems of unstable source, functional attenuation and poor treatment effect of immunomodulatory stem cells in the prior art, and provides a new efficient, safe and standardizable solution for the treatment of GVHD. Detailed Embodiments

[0026] The present invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, comprising the following steps:

[0027] (1) After washing the human adipose aspirate, it is digested with collagenase to obtain monocytes;

[0028] (2) The monocytes obtained in step (1) are inoculated in a differentiation medium and cultured for 2 - 3 days, and then the adherent cells are further cultured for 18 - 22 days to obtain mesenchymal stem cells;

[0029] The differentiation medium is based on DMEM medium and comprises the following components: 6-8% (v / v) human serum albumin, 20-30 ng / mL basic fibroblast growth factor, 0.2-0.4 mM L-alanine, 0.2-0.4 mM L-aspartic acid, 0.3-0.5 mM L-proline, 0.3-0.5 mM L-serine, 6-9 μg / L 4-O-galloylpaeoniflorin, 25-35 ng / mL total saponins of Panax notoginseng, 0.2-0.4% (v / v) β-mercaptoethanol; preferably 7% (v / v) human serum albumin, 22-28 ng / mL basic fibroblast growth factor, 0.3 mM L-alanine, 0.3 mM L-aspartic acid, 0.4 mM L-proline, 0.4 mM L-serine, 7-8 μg / L 4-O-galloylpaeoniflorin, 27-33 ng / mL total saponins of Panax notoginseng, 0.3% (v / v) β-mercaptoethanol; more preferably 7% (v / v) human serum albumin, 24-26 ng / mL basic fibroblast growth factor, 0.3 mM L-alanine, 0.3 mM L-aspartic acid, 0.4 mM L-proline, 0.4 mM L-serine, 8 μg / L 4-O-galloylpaeoniflorin, 29-31 ng / mL total saponins of Panax notoginseng, 0.3% (v / v) β-mercaptoethanol; even more preferably 7% (v / v) human serum albumin, 25 ng / mL basic fibroblast growth factor, 0.3 mM L-alanine, 0.3 mM L-aspartic acid, 0.4 mM L-proline, 0.4 mM L-serine, 8 μg / L 4-O-galloylpaeoniflorin, 30 ng / mL total saponins of Panax notoginseng, 0.3% (v / v) β-mercaptoethanol.

[0030] (3) Inoculate the mesenchymal stem cells obtained in step (2) into the induction medium for induction culture, and after screening, immune regulatory stem cells are obtained.

[0031] In the present invention, in step (2), preferably, the monocytes obtained in step (1) are inoculated into the differentiation medium for culture for 3 d, and then the adherent cells are continuously cultured for 19-21 d to obtain mesenchymal stem cells; more preferably, the monocytes obtained in step (1) are inoculated into the differentiation medium for culture for 3 d, and then the adherent cells are continuously cultured for 20 d to obtain mesenchymal stem cells.

[0032] In the present invention, the washing in step (1) is to rinse the human fat extract with phosphate buffer solution.

[0033] In the present invention, the collagenase in step (1) is type I collagenase or type II collagenase; the concentration of the collagenase is 150 - 200 U / ml; preferably 160 - 190 U / ml; more preferably 170 - 180 U / ml; even more preferably 180 U / ml.

[0034] In the present invention, the temperature of digestion in step (1) is 36 - 38 °C, and the time is 35 - 45 min; preferably the temperature of digestion is 37 °C, and the time is 37 - 43 min; more preferably the temperature of digestion is 37 °C, and the time is 39 - 41 min; even more preferably the temperature of digestion is 37 °C, and the time is 40 min.

[0035] In the present invention, the seeding density in step (2) is 6 - 8×10 5 cells / ml; preferably 7×10 5 cells / ml.

[0036] In the present invention, the induction medium is based on DMEM medium and includes the following components: 20 - 30 IU / mL IFN-γ; preferably 22 - 28 IU / mL IFN-γ; more preferably 24 - 26 IU / mL IFN-γ; even more preferably 25 IU / mL IFN-γ.

[0037] In the present invention, the induction medium further includes: 8 - 12 μg / L platelet-derived growth factor, 12 - 18 μg / ml endothelial cell growth factor, 12 - 18 U / ml heparin, 8 - 12% human serum albumin, 6 - 9 μg / L 4-O-galloylpaeoniflorin, 15 - 25 ng / mL total saponins of Panax notoginseng, 2 - 4 mM glutamine; preferably 9 - 11 μg / L platelet-derived growth factor, 13 - 17 μg / ml endothelial cell growth factor, 13 - 17 U / ml heparin, 9 - 11% human serum albumin, 7 - 8 μg / L 4-O-galloylpaeoniflorin, 17 - 23 ng / mL total saponins of Panax notoginseng, 3 mM glutamine; more preferably 10 μg / L platelet-derived growth factor, 14 - 16 μg / ml endothelial cell growth factor, 14 - 16 U / ml heparin, 10% human serum albumin, 8 μg / L 4-O-galloylpaeoniflorin, 19 - 21 ng / mL total saponins of Panax notoginseng, 3 mM glutamine; even more preferably 10 μg / L platelet-derived growth factor, 15 μg / ml endothelial cell growth factor, 15 U / ml heparin, 10% human serum albumin, 8 μg / L 4-O-galloylpaeoniflorin, 20 ng / mL total saponins of Panax notoginseng, 3 mM glutamine.

[0038] In the present invention, the seeding density in step (3) is 8 - 12×10 5cells / ml; preferably 9-11×10 5 cells / ml; more preferably 10×10 5 cells / ml.

[0039] In the present invention, the time of the induction culture in step (3) is 1-2 d; preferably 2 d.

[0040] The present invention also provides the use of the immunomodulatory stem cells prepared by the described method in the preparation of drugs for treating graft-versus-host disease.

[0041] In the present invention, the CAS of -O-galloylpaeoniflorin is 1201580-97-3; the CAS of total saponins of Panax notoginseng is 88105-29-7.

[0042] The technical solutions provided by the present invention will be described in detail below in conjunction with examples, but they should not be construed as limiting the protection scope of the present invention.

[0043] Example 1

[0044] The present invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, and the steps are as follows:

[0045] (1) After rinsing human adipose aspirate with phosphate buffer, digesting with type II collagenase at 36 °C for 35 min to obtain monocytes; the concentration of the collagenase is 150 U / ml.

[0046] (2) Inoculating the monocytes obtained in step (1) at 6×10 5 cells / ml in a differentiation medium and culturing for 2 d, and then continuing to culture the adherent cells for 18 d to obtain mesenchymal stem cells;

[0047] The differentiation medium is based on DMEM medium and is added with the following components: 6% (v / v) human serum albumin, 20 ng / mL basic fibroblast growth factor, 0.2 mM L-alanine, 0.2 mM L-aspartic acid, 0.3 mM L-proline, 0.3 mM L-serine, 6 μg / L 4-O-galloylpaeoniflorin, 25 ng / mL total saponins of Panax notoginseng, 0.2% (v / v) β-mercaptoethanol;

[0048] (3) Inoculating the mesenchymal stem cells obtained in step (2) at 8×10 5 cells / ml in an induction medium for induction culture for 1 d, and after screening (detecting immunomodulatory stem cells by flow cytometry, and the results show that the positive rates of cell surface markers CD73 / CD90 / CD105 are greater than 99%, and the negative rates of CD34 / CD45 / HLA-DR are lower than 0.02%), immunomodulatory stem cells are obtained.

[0049] The induction medium uses DMEM medium as the basal medium and adds the following components: 20 IU / mL IFN-γ, 8 μg / L platelet-derived growth factor, 12 μg / ml endothelial cell growth factor, 12 U / ml heparin, 8% human serum albumin, 4-O-galloylpaeoniflorin 6 μg / L, 15 ng / mL total saponins of Panax notoginseng, 2 mM glutamine.

[0050] Example 2

[0051] The present invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, and the steps are as follows:

[0052] (1) After rinsing human adipose aspirate with phosphate buffer, digest it with type I collagenase at 38 °C for 45 min to obtain monocytes; the concentration of the collagenase is 200 U / ml.

[0053] (2) Inoculate the monocytes obtained in step (1) at 8×10 5 cells / ml in the differentiation medium and culture for 3 d, and then continue to culture the adherent cells for 22 d to obtain mesenchymal stem cells;

[0054] The differentiation medium uses DMEM medium as the basal medium and adds the following components: 8% (v / v) human serum albumin, 30 ng / mL basic fibroblast growth factor, 0.4 mM L-alanine, 0.4 mM L-aspartic acid, 0.5 mM L-proline, 0.5 mM L-serine, 9 μg / L 4-O-galloylpaeoniflorin, 35 ng / mL total saponins of Panax notoginseng, 0.4% (v / v) β-mercaptoethanol;

[0055] (3) Inoculate the mesenchymal stem cells obtained in step (2) at 12×10 5 cells / ml in the induction medium and induce for 2 d. After screening (detecting immunomodulatory stem cells by flow cytometry, the results show that the positive rate of cell surface markers CD73 / CD90 / CD105 is greater than 99%, and the negativity of CD34 / CD45 / HLA-DR is lower than 0.02%), immunomodulatory stem cells are obtained.

[0056] The induction medium uses DMEM medium as the basal medium and adds the following components: 30 IU / mL IFN-γ, 12 μg / L platelet-derived growth factor, 18 μg / ml endothelial cell growth factor, 18 U / ml heparin, 12% human serum albumin, 4-O-galloylpaeoniflorin 9 μg / L, 25 ng / mL total saponins of Panax notoginseng, 4 mM glutamine.

[0057] Example 3

[0058] The present invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, which comprises the following steps:

[0059] (1) After rinsing human adipose aspirate with phosphate buffer, digest it with type II collagenase at 37 °C for 40 min to obtain mononuclear cells; the concentration of the collagenase is 180 U / ml.

[0060] (2) Inoculate the mononuclear cells obtained in step (1) at 7×10 5 cells / ml in a differentiation medium and culture for 3 d, and then continue to culture the adherent cells for 20 d to obtain mesenchymal stem cells;

[0061] The differentiation medium is based on DMEM medium and supplemented with the following components: 7% (v / v) human serum albumin, 25 ng / mL basic fibroblast growth factor, 0.3 mM L-alanine, 0.3 mM L-aspartic acid, 0.4 mM L-proline, 0.4 mM L-serine, 8 μg / L 4-O-galloylpaeoniflorin, 30 ng / mL total saponins of Panax notoginseng, 0.3% (v / v) β-mercaptoethanol;

[0062] (3) Inoculate the mesenchymal stem cells obtained in step (2) at 10×10 5 cells / ml in an induction medium and induce culture for 2 d. After screening (detecting immunomodulatory stem cells by flow cytometry, the results show that the positive rates of cell surface markers CD73 / CD90 / CD105 are greater than 99%, and the negative rates of CD34 / CD45 / HLA-DR are lower than 0.02%), immunomodulatory stem cells are obtained.

[0063] The induction medium is based on DMEM medium and supplemented with the following components: 10 μg / L platelet-derived growth factor, 15 μg / ml endothelial cell growth factor, 15 U / ml heparin, 10% human serum albumin, 8 μg / L 4-O-galloylpaeoniflorin, 20 ng / mL total saponins of Panax notoginseng, 3 mM glutamine.

[0064] Comparative Example 1

[0065] Other methods are the same as those in Example 3, except that 4-O-galloylpaeoniflorin is not added to the differentiation medium and the induction medium.

[0066] Experimental Example 1

[0067] 1. Select 6-week-old NCG mice, male, with a body weight range of 25 ± 5 g. Purchased from Cyagen Biosciences (Suzhou) Inc.

[0068] 2. Breeding conditions for NCG mice: Free access to food and water; environmental temperature 25 ± 2°C, environmental relative humidity 55 ± 5%.

[0069] 3. Preparation of animal models:

[0070] (1) A total of 12 mice were selected, divided into a model group of 9 mice and a blank control group of 3 mice (not irradiated). The 9 mice in the model group were subjected to model establishment treatment;

[0071] (2) After irradiating the NCG mice in the model group with 1.75 Gy of radiation for 6 hours, use a syringe to transplant 5×10 6 hPBMC (human peripheral blood mononuclear cells, purchased from Saisi'ao) into the tail vein of the mice.

[0072] (3) Experimental grouping:

[0073] Experimental group: After irradiation and transplantation of hPBMC, on the 2nd, 5th, and 8th days, inject 1.5×10 6 immunomodulatory stem cells prepared by the method of Example 3 into the tail vein of the mice;

[0074] Control 1: After irradiation and transplantation of hPBMC, on the 2nd, 5th, and 8th days, inject 1.5×10 6 immunomodulatory stem cells prepared by the method of Control 1 into the tail vein of the mice;

[0075] GVHD group: Only inject the same amount of normal saline as the experimental group on the 2nd, 5th, and 8th days after irradiation and transplantation of hPBMC;

[0076] Blank control group: Only inject hPBMC, without irradiation;

[0077] During the experiment, measure the body weight of the mice on the 1st, 2nd, 5th, 8th, 11th, 13th, and 14th days respectively, and count the survival rate of the mice on the 0th, 8th, 11th, 13th, 14th, 17th, and 19th days respectively. The results are shown in Table 1 and Table 2. On the 14th day, take the bone marrow of the mice and use flow cytometry to detect the infiltration of human CD45-positive cells in the bone marrow. The results are shown in Table 3.

[0078] Table 1 Changes in mouse body weight

[0079]

[0080] - indicates mouse death.

[0081] Table 2 Number of surviving mice

[0082]

[0083] Table 3 Statistical table of mouse bone marrow chimerism rate

[0084]

[0085] The results of body weight monitoring, survival rate investigation in Tables 1 - 3 and the statistics of murine bone marrow chimerism rate show that on the 14th day, the body weight and survival rate of the GVHD group were significantly lower than those of the blank control group, and the bone marrow chimerism rate of the GVHD group was significantly higher than that of the public control group. This indicates that the injection of hPBMC led to graft-versus-host disease and the model was successfully established.

[0086] However, the body weight and survival rate of the experimental group mice were significantly increased compared with those of the GVHD group, and the bone marrow chimerism rate was significantly decreased. This shows that immunomodulatory stem cells can improve the survival rate of GVHD mice and alleviate GVHD by reducing the infiltration of hPBMC in the bone marrow.

[0087] The above are only the preferred embodiments of the present invention. It should be pointed out that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing an immunomodulatory stem cell for treating graft-versus-host disease, characterized in that, It includes the following steps: (1) After washing the human fat aspirate, digest it with collagenase to obtain monocytes; (2) Inoculate the monocytes obtained in step (1) into a differentiation medium and culture for 2 - 3 days, then continue to culture the adherent cells for 18 - 22 days to obtain mesenchymal stem cells; The differentiation medium is based on DMEM medium and includes the following components: 6 - 8% (v / v) human serum albumin, 20 - 30 ng / mL basic fibroblast growth factor, 0.2 - 0.4 mM L-alanine, 0.2 - 0.4 mM L-aspartic acid, 0.3 - 0.5 mM L-proline, 0.3 - 0.5 mM L-serine, 6 - 9 μg / L 4-O-galloylpaeoniflorin, 25 - 35 ng / mL total saponins of Panax notoginseng, 0.2 - 0.4% (v / v) β-mercaptoethanol; (3) Inoculate the mesenchymal stem cells obtained in step (2) into an induction medium for induction culture, and after screening, obtain immunomodulatory stem cells.

2. The preparation method according to claim 1, characterized in that, The washing in step (1) is to rinse the human fat aspirate with phosphate buffer.

3. The preparation method according to claim 1, characterized in that, The collagenase in step (1) is type I collagenase or type II collagenase; the concentration of the collagenase is 150 - 200 U / ml.

4. The preparation method according to claim 1, characterized in that, The temperature of the digestion in step (1) is 36 - 38 °C and the time is 35 - 45 min.

5. The preparation method according to claim 1, characterized in that, The inoculation density described in step (2) is 6-8×10 5 cells / ml.

6. The preparation method according to claim 1, characterized in that, The induction medium is based on DMEM medium and includes the following component: 20 - 30 IU / mL IFN-γ.

7. The preparation method according to claim 6, characterized in that, The induction medium also includes: 8 - 12 μg / L platelet-derived growth factor, 12 - 18 μg / ml endothelial cell growth factor, 12 - 18 U / ml heparin, 8 - 12% human serum albumin, 6 - 9 μg / L 4-O-galloylpaeoniflorin, 15 - 25 ng / mL total saponins of Panax notoginseng, 2 - 4 mM glutamine.

8. The preparation method according to claim 1, characterized in that, The inoculation density described in step (3) is 8 - 12×10 5 cells / ml.

9. The preparation method according to claim 1, characterized in that, The time of the induction culture in step (3) is 1 - 2 days.

10. Use of the immunomodulatory stem cells prepared by the method according to any one of claims 1 - 7 in the preparation of a drug for treating graft-versus-host disease.

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