A method for preparing immunomodulatory stem cells for treating graft-versus-host disease
Immunomodulatory stem cells prepared by collagenase digestion of human fat extract and treatment with a specific culture medium have solved the problems of unstable stem cell sources and functional decline, and have achieved a highly effective treatment for graft-versus-host disease.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- FUMEI ZHONGKANG MEDICAL TECHNOLOGY (ZHUHAI HENGQIN) CO LTD
- Filing Date
- 2025-04-03
- Publication Date
- 2026-06-30
Smart Images
Figure BDA0005344644120000081 
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Abstract
Description
Technical Field
[0001] This invention relates to the field of cell engineering technology, and in particular to a method for preparing immunomodulatory stem cells for treating graft-versus-host disease. Background Technology
[0002] Graft-versus-host disease (GVHD) is a common and serious complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT). It is caused by the donor's immune cells attacking the recipient's tissues, clinically manifesting as damage to multiple organs, including the skin, liver, and gastrointestinal tract. Although glucocorticoids and immunosuppressants (such as calcineurin inhibitors) are first-line treatments for GVHD, approximately 30%–50% of patients develop hormone-resistant GVHD, resulting in a very poor prognosis and a mortality rate as high as 80%. Existing therapies, such as monoclonal antibodies (e.g., anti-CD25 or anti-TNF-α) or mesenchymal stem cells (MSCs), have shown some efficacy, but suffer from low response rates, unstable efficacy, and potential tumor-promoting risks.
[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 immunomodulatory capabilities. These cells can alleviate GVHD by secreting anti-inflammatory factors (such as IL-10 and TGF-β), inhibiting effector T cell proliferation, or inducing immune tolerance. However, traditional preparation methods face the following bottlenecks: limited cell source: primary MSCs have poor expansion capacity and large batch-to-batch variability; insufficient functional stability: in vitro culture easily leads to the decline of stem cell immunomodulatory function; targeting defects: lack of responsiveness to GVHD-specific inflammatory microenvironment (such as high expression of IFN-γ or IL-6); standardization challenges: existing processes are difficult to balance cell purity, viability, and large-scale production requirements.
[0004] Therefore, how to develop a method for preparing immunomodulatory stem cells that are stable in origin, functionally controllable, and can target GVHD lesions is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0005] The purpose of this invention is to provide a method for preparing immunomodulatory stem cells for treating graft-versus-host disease.
[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0007] This invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, comprising the following steps:
[0008] (1) After washing the human fat extract, collagenase was used to digest it to obtain mononuclear cells;
[0009] (2) The mononuclear cells obtained in step (1) are seeded in differentiation medium and cultured for 2-3 days. The adherent cells are then cultured for 18-22 days to obtain mesenchymal stem cells.
[0010] The differentiation medium is based on DMEM medium, comprising 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, and 0.2–0.4% (v / v) β-mercaptoethanol;
[0011] (3) The mesenchymal stem cells obtained in step (2) are seeded in an induction culture medium for induction culture, and after screening, immunomodulatory stem cells are obtained.
[0012] Preferably, the cleaning in step (1) involves rinsing the human fat extract with a phosphate buffer solution.
[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 digestion temperature in step (1) is 36-38°C and the time is 35-45 min.
[0015] Preferably, the inoculation density in step (2) is 6–8 × 10⁻⁶. 5 per ml.
[0016] Preferably, the induction medium is based on DMEM medium and includes the following components: 20-30 IU / mL IFN-γ.
[0017] Preferably, the induction culture medium further includes: 8-12 μg / L platelet-derived growth factor, 12-18 μg / ml endothelial growth factor, 12-18 U / ml heparin, 8-12% human serum albumin, 6-9 μg / L 4-O-gallopaeoniflorin, 15-25 ng / mL total saponins of Panax notoginseng, and 2-4 mM glutamine.
[0018] Preferably, the inoculation density in step (3) is 8–12 × 10⁻⁶. 5 per ml.
[0019] Preferably, the induction culture time in step (3) is 1 to 2 days.
[0020] The present invention also provides the application of the immunomodulatory stem cells prepared by the 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] This invention uses human fat extract as a stem cell source and prepares mesenchymal stem cells through collagenase digestion and differentiation culture medium formulation. This method significantly improves the extraction efficiency of monocytes and the proliferation capacity of mesenchymal stem cells.
[0023] The differentiation and induction media of this invention contain unique 4-O-galloylpaeoniflorin and total saponins of Panax notoginseng. The addition of these two components can enhance the proliferation capacity of mesenchymal stem cells and improve the immunomodulatory function of immunomodulatory stem cells.
[0024] The immunomodulatory stem cells prepared in this invention exhibit significant immunosuppressive activity. Experimental data show that the positive rate of their surface markers CD73 / CD90 / CD105 is greater than 99%, and the negative rate of CD34 / CD45 / HLA-DR is less than 0.02%, meeting relevant standards. Animal experiments provided by this invention further confirm that these stem cells can effectively alleviate GVHD symptoms, significantly reduce bone marrow chimerism in mice, and improve mouse survival rates.
[0025] The preparation method provided by this invention solves the technical problems of unstable source, functional decline and poor therapeutic effect of immunomodulatory stem cells in the prior art, and provides a new, efficient, safe and standardized solution for the treatment of GVHD. Detailed Implementation
[0026] This 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 fat extract, collagenase was used to digest it to obtain mononuclear cells;
[0028] (2) The mononuclear cells obtained in step (1) are seeded in differentiation medium and cultured for 2-3 days. The adherent cells are then cultured for 18-22 days to obtain mesenchymal stem cells.
[0029] The differentiation medium is based on DMEM medium, comprising 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, and 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, and 0.4 mM L-aspartic acid. 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; 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) The mesenchymal stem cells obtained in step (2) are seeded in an induction culture medium for induction culture, and after screening, immunomodulatory stem cells are obtained.
[0031] In this invention, step (2) preferably involves seeding the mononuclear cells obtained in step (1) into a differentiation medium and culturing them for 3 days, and then continuing to culture the adherent cells for 19 to 21 days to obtain mesenchymal stem cells; more preferably, the mononuclear cells obtained in step (1) are seeded into a differentiation medium and cultured for 3 days, and then continuing to culture the adherent cells for 20 days to obtain mesenchymal stem cells.
[0032] In this invention, the cleaning in step (1) is to rinse the human fat extract with phosphate buffer.
[0033] In this 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; and even more preferably 180 U / ml.
[0034] In this invention, the digestion temperature in step (1) is 36-38°C and the time is 35-45 min; preferably, the digestion temperature is 37°C and the time is 37-43 min; more preferably, the digestion temperature is 37°C and the time is 39-41 min; more preferably, the digestion temperature is 37°C and the time is 40 min.
[0035] In this invention, the inoculation density in step (2) is 6–8 × 10⁻⁶. 5 pcs / ml; preferably 7×10 5 per ml.
[0036] In this 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; and more preferably 25 IU / mL IFN-γ.
[0037] In this invention, the induction culture medium further includes: 8–12 μg / L platelet-derived growth factor, 12–18 μg / ml endothelial growth factor, 12–18 U / ml heparin, 8–12% human serum albumin, 6–9 μg / L 4-O-galloylpaeonidine, 15–25 ng / mL total saponins of Panax notoginseng, and 2–4 mM glutamine; preferably 9–11 μg / L platelet-derived growth factor, 13–17 μg / ml endothelial growth factor, 13–17 U / ml heparin, 9–11% human serum albumin, 7–8 μg / L 4-O-galloylpaeonidine, and 17–23 ng / mL total saponins of Panax notoginseng. / mL total saponins of Panax notoginseng, 3mM glutamine; more preferably 10μg / L platelet-derived growth factor, 14-16μg / ml endothelial growth factor, 14-16U / ml heparin, 10% human serum albumin, 8μg / L 4-O-galloylpaeoniflorin, 19-21ng / mL total saponins of Panax notoginseng, 3mM glutamine; more preferably 10μg / L platelet-derived growth factor, 15μg / ml endothelial growth factor, 15U / ml heparin, 10% human serum albumin, 8μg / L 4-O-galloylpaeoniflorin, 20ng / mL total saponins of Panax notoginseng, 3mM glutamine.
[0038] In this invention, the inoculation density in step (3) is 8–12 × 10⁻⁶. 5Cells / ml; preferably 9–11 × 10⁻⁶ 5 10 × 10⁻⁶ cells / ml; more preferably 10 × 10⁻⁶ cells / ml. 5 per ml.
[0039] In this invention, the induction culture time in step (3) is 1 to 2 days; preferably 2 days.
[0040] The present invention also provides the application of the immunomodulatory stem cells prepared by the method in the preparation of drugs for treating graft-versus-host disease.
[0041] In this invention, the CAS number of -O-galloylpaeoniflorin is 1201580-97-3; and the CAS number 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0043] Example 1
[0044] This invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, comprising the following steps:
[0045] (1) After rinsing the human fat aspirate with phosphate buffer, it was digested with type II collagenase at 36°C for 35 min to obtain mononuclear cells; the concentration of the collagenase was 150 U / ml.
[0046] (2) The mononuclear cells obtained in step (1) are divided into 6×10 5 Mesenchymal stem cells were seeded at a density of 1 / ml in differentiation medium and cultured for 2 days. The adherent cells were then cultured for another 18 days to obtain mesenchymal stem cells.
[0047] The differentiation medium was based on DMEM medium, supplemented 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, and 0.2% (v / v) β-mercaptoethanol;
[0048] (3) Take the mesenchymal stem cells obtained in step (2), and mix them at a ratio of 8×10⁻⁶. 5 Immunomodulatory stem cells were seeded at a density of 1 / ml in induction medium and induced for 1 day. After screening (immunomodulatory stem cells were detected by flow cytometry, and the results showed that the positive rate of cell surface markers CD73 / CD90 / CD105 was greater than 99%, and the negative rate of CD34 / CD45 / HLA-DR was less than 0.02%), thus obtaining immunomodulatory stem cells.
[0049] The induction medium was based on DMEM medium, with the following components added: 20 IU / mL IFN-γ, 8 μg / L platelet-derived growth factor, 12 μg / ml endothelial growth factor, 12 U / ml heparin, 8% human serum albumin, 6 μg / L 4-O-gallopaeoniflorin, 15 ng / mL total saponins of Panax notoginseng, and 2 mM glutamine.
[0050] Example 2
[0051] This invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, comprising the following steps:
[0052] (1) After rinsing the human fat aspirate with phosphate buffer, it was digested with type I collagenase at 38°C for 45 min to obtain mononuclear cells; the concentration of the collagenase was 200 U / ml.
[0053] (2) The mononuclear cells obtained in step (1) are divided into 8×10 5 Mesenchymal stem cells were seeded at a density of 1 / ml in differentiation medium and cultured for 3 days. The adherent cells were then cultured for another 22 days to obtain mesenchymal stem cells.
[0054] The differentiation medium was based on DMEM medium, supplemented with 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, and 0.4% (v / v) β-mercaptoethanol.
[0055] (3) The mesenchymal stem cells obtained in step (2) are processed at a ratio of 12 × 10⁻⁶. 5 Immunomodulatory stem cells were seeded at a density of 1 / ml in induction medium and induced for 2 days. After screening (immunomodulatory stem cells were detected by flow cytometry, and the results showed that the positive rate of cell surface markers CD73 / CD90 / CD105 was greater than 99%, and the negative rate of CD34 / CD45 / HLA-DR was less than 0.02%), thus obtaining immunomodulatory stem cells.
[0056] The induction medium was based on DMEM medium, with the following components added: 30 IU / mL IFN-γ, 12 μg / L platelet-derived growth factor, 18 μg / ml endothelial growth factor, 18 U / ml heparin, 12% human serum albumin, 4-O-gallopaeoniflorin 9 μg / L, 25 ng / mL total saponins of Panax notoginseng, and 4 mM glutamine.
[0057] Example 3
[0058] This invention provides a method for preparing immunomodulatory stem cells for treating graft-versus-host disease, comprising the following steps:
[0059] (1) After rinsing the human fat aspirate with phosphate buffer, it was digested with type II collagenase at 37°C for 40 min to obtain mononuclear cells; the concentration of the collagenase was 180 U / ml.
[0060] (2) The mononuclear cells obtained in step (1) are divided into 7×10 5 Mesenchymal stem cells were seeded at a density of 1 / ml in differentiation medium and cultured for 3 days. The adherent cells were then cultured for another 20 days to obtain mesenchymal stem cells.
[0061] The differentiation medium was based on DMEM medium, 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, and 0.3% (v / v) β-mercaptoethanol;
[0062] (3) The mesenchymal stem cells obtained in step (2) are processed at a ratio of 10×10⁻⁶. 5 Immunomodulatory stem cells were seeded at a density of 1 / ml in induction medium and induced for 2 days. After screening (immunomodulatory stem cells were detected by flow cytometry, and the results showed that the positive rate of cell surface markers CD73 / CD90 / CD105 was greater than 99%, and the negative rate of CD34 / CD45 / HLA-DR was less than 0.02%), thus obtaining immunomodulatory stem cells.
[0063] The induction medium was based on DMEM medium, with the following components added: 10 μg / L platelet-derived growth factor, 15 μg / ml endothelial growth factor, 15 U / ml heparin, 10% human serum albumin, 8 μg / L 4-O-gallopaeoniflorin, 20 ng / mL total saponins of Panax notoginseng, and 3 mM glutamine.
[0064] Comparative Example 1
[0065] The other methods are the same as in Example 3, except that 4-O-galloylpaeoniflorin was not added to the differentiation medium and induction medium.
[0066] Experimental Example 1
[0067] 1. Six-week-old male NCG mice, weighing between 25±5g, were selected and purchased from Cyagen (Suzhou) Biotechnology Co., Ltd.
[0068] 2. Housing conditions for NCG mice: free access to food and water; ambient temperature 25±2℃, relative humidity 55±5%.
[0069] 3. Animal model preparation:
[0070] (1) A total of 12 mice were selected and divided into a model group (n=9) and a blank control group (n=3) (without radiation exposure). The 9 mice in the model group were subjected to model treatment;
[0071] (2) After irradiating the model group NCG mice with 1.75 Gy rays for 6 hours, 5 × 10⁶ g of ray were transplanted into the tail vein of the mice using a syringe. 6 One hPBMC (human peripheral blood mononuclear cells, purchased from TheoSys).
[0072] (3) Experimental Groups:
[0073] Experimental group: After radiation irradiation and hPBMC transplantation, mice were injected intravenously with 1.5 × 10⁻⁶ mcg of thrombocytopenic porpoise (hPBMCs) on days 2, 5, and 8. 6 Immunomodulatory stem cells prepared by the method in Example 3;
[0074] Comparative Example 1: After irradiation with radiation and transplantation of hPBMCs, mice were injected intravenously with 1.5 × 10⁻⁶ g of irradiated protein (rPBMCs) on days 2, 5, and 8. 6 Immunomodulatory stem cells prepared by the method in Comparative Example 1;
[0075] GVHD group: On days 2, 5, and 8 after radiation irradiation and hPBMC transplantation, only the same amount of physiological saline as the experimental group was injected.
[0076] Blank control group: only injected with hPBMC, without radiation exposure;
[0077] During the experiment, the body weight of mice was measured on days 1, 2, 5, 8, 11, 13, and 14, and the survival rate of mice was recorded on days 0, 8, 11, 13, 14, 17, and 19. The results are shown in Tables 1 and 2. On day 14, bone marrow was collected from the mice, and flow cytometry was used 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 that the mouse has died.
[0081] Table 2 Number of surviving mice
[0082]
[0083] Table 3. Statistics on chimerism rate of mouse bone marrow
[0084]
[0085] The results of weight monitoring, survival rate survey, and bone marrow chimerism rate statistics in Tables 1-3 show that on day 14, the 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 open control group. This indicates that the injection of hPBMCs led to graft-versus-host disease and the model was successfully established.
[0086] The experimental group mice showed significantly higher body weight and survival rate compared to the GVHD group, and a significantly lower bone marrow chimerism rate. This indicates that immunomodulatory stem cells can improve the survival rate of GVHD mice and alleviate GVHD by reducing hPBMC infiltration in the bone marrow.
[0087] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A method for preparing immunomodulatory stem cells for treating graft-versus-host disease, characterized in that, Includes the following steps: (1) After washing the human fat extract, it was digested with collagenase to obtain mononuclear cells; (2) The mononuclear cells obtained in step (1) are seeded in differentiation medium and cultured for 2-3 days. The adherent cells are then cultured for 18-22 days to obtain mesenchymal stem cells. The differentiation medium is based on DMEM medium, comprising 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, and 0.2-0.4% (v / v) β-mercaptoethanol; (3) The mesenchymal stem cells obtained in step (2) are seeded in induction culture medium for induction culture, and after screening, immunomodulatory stem cells are obtained; The induction medium is based on DMEM medium and includes the following components: 20-30 IU / mL IFN-γ, 8-12 μg / L platelet-derived growth factor, 12-18 μg / mL endothelial growth factor, 12-18 U / mL heparin, 8-12% human serum albumin, 6-9 μg / L 4-O-gallopaeoniflorin, 15-25 ng / mL total saponins of Panax notoginseng, and 2-4 mM glutamine.
2. The preparation method according to claim 1, characterized in that, The cleaning in step (1) involves rinsing the human fat extract 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 150~200U / ml.
4. The preparation method according to claim 1, characterized in that, The digestion temperature in step (1) is 36~38℃ and the time is 35~45min.
5. The preparation method according to claim 1, characterized in that, The inoculation density in step (2) is 6~8×10 5 per ml.
6. The preparation method according to claim 1, characterized in that, The inoculation density in step (3) is 8~12×10 5 per ml.
7. The preparation method according to claim 1, characterized in that, The induction culture time in step (3) is 1~2 days.
8. The use of immunomodulatory stem cells prepared by the method according to any one of claims 1 to 5 in the preparation of drugs for treating graft-versus-host disease.