Method for culturing domesticated mesenchymal stem cells

Abstract

The invention belongs to the field of biological medicine, and particularly discloses a culture method of domesticated mesenchymal stem cells (MSCs), which comprises the following steps: separating plasma and platelet lysis buffer from autologous peripheral blood, and obtaining CTL and Treg through magnetic bead sorting; the method comprises the following steps: sequentially co-culturing umbilical cord MSCs for three generations: co-culturing the P1 generation and CTL in a culture medium containing 30% of alpha MEM, 68% of DMEM / F12, 1% of autologous plasma and 1% of platelet lysis buffer, and adding 0.1-1 [mu] M of resveratrol and 10-50 [mu] M of spermidine; co-culturing P2 and Treg in a culture medium containing 50% of alpha MEM, 48% of DMEM / F12, 1% of autologous plasma and 1% of platelet lysis buffer; the P3 generation is cultured and harvested in 90% of alpha MEM, 5% of DMEM / F12 and 5% of PRP. The obtained MSCs surface marker is CD105 < + > / CD90 < + > / CD73 < + > / CD45 <-> / CD34 <-> / HLA-DR <->, the expression quantity of CD24 is low (CD24low), the expression quantity of CD47 is high (CD47high), the expression quantity of PD-L1 is high (PD-L1 high), the IDO activity is larger than or equal to 25 U / mg, the retention rate of the lung after vein transplantation is smaller than or equal to 12%, and the survival period is larger than or equal to 28 days. The cell is suitable for treating ARDS or hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and specifically discloses a method for culturing post-acclimation mesenchymal stem cells. Background Technology

[0002] Mesenchymal stem cells (MSCs) hold great promise in regenerative medicine and the treatment of autoimmune diseases due to their multipotent differentiation potential and immunomodulatory functions. However, their clinical application still faces two major bottlenecks: insufficient control of immunogenicity and low survival rates caused by high lung retention rates after transplantation. Traditional MSC culture methods typically use basal media (such as αMEM, DMEM) supplemented with fetal bovine serum (FBS) or human platelet lysis buffer (hPL), which, while maintaining cell proliferation, cannot effectively acclimatize the immune phenotype. Existing technologies attempt to enhance MSC function through intervention with single immune factors, for example:

[0003] Cytokine pretreatment (such as IFN-γ, TGF-β): can transiently induce the expression of immunosuppressive molecules (PD-L1, IDO), but the effect is not lasting and may activate pro-rejection antigens such as HLA-DR;

[0004] T cell co-culture: Although there are reports of using regulatory T cells (Treg) or cytotoxic T cells (CTL) to co-culture with MSCs to enhance immune tolerance, most of them use static mixed culture mode, which leads to excessive consumption or functional suppression of T cells and fails to achieve sequential delivery of immune signals.

[0005] Serum substitute optimization: Although using hPL to replace FBS can reduce the risk of allogeneic transplantation, it does not solve the problem of autologous recognition, and the transplanted cells are still easily cleared by the host immune system.

[0006] The above methods share a common drawback:

[0007] Immunogenic residues: HLA-DR expression rates are generally between 1% and 2% (flow cytometry), which can easily trigger host CD4 expression. + T-cell activation leads to graft rejection;

[0008] High lung retention rate: >30% of cells remain in the pulmonary vascular bed after intravenous injection and die rapidly due to oxidative stress and inflammatory response, with less than 10% of effective cells homing to target organs;

[0009] Uncontrollable function: The lack of standardized training process results in large fluctuations in the cellular immune regulation capacity of different batches, making it difficult to meet clinical consistency requirements.

[0010] The root cause lies in the fact that existing technologies fail to mimic the dynamic, temporal regulatory mechanisms of the in vivo immune microenvironment: in physiological immune responses, early activation of CTLs can trigger upregulation of "alarm signals" (such as PD-L1) in MSCs, thereby recruiting Tregs to establish long-term tolerance. Traditional culture methods neglect this synergistic temporal process, focusing only on the addition of single cells or factors, resulting in insufficient immune acclimation of MSCs. Furthermore, conventional culture media lack targeted designs for metabolic stress protection and epigenetic reprogramming, failing to endow MSCs with the ability to resist oxidative damage in the lungs and maintain a low-immunogenic phenotype. Therefore, there is an urgent need to develop a novel culture system integrating temporal immune co-culture, autologous components, and metabolic-epiogenic synergistic regulation to systematically overcome the barriers to the clinical application of MSCs. Summary of the Invention

[0011] To address the above problems, this invention discloses a method for culturing post-acclimation mesenchymal stem cells.

[0012] The objective of this invention is achieved through the following technical solution.

[0013] A method for culturing post-acclimation mesenchymal stem cells, comprising the following steps:

[0014] (a) Plasma and platelet lysate were separated from autologous peripheral blood and CD3 was obtained by magnetic bead sorting. + CD8 + Cytotoxic T cells (CTLs) and CD3 + CD25 + Regulatory T cells (Tregs);

[0015] (b) Co-culture umbilical cord-derived mesenchymal stem cells (MSCs) sequentially in the following order:

[0016] P1 generation: Using a culture medium containing 30% αMEM, 68% DMEM / F12, 1% autologous plasma, and 1% autologous platelet lysis buffer, 1×10⁻⁶ tbsp medium was added 4 hours after MSC inoculation. 5 CTLs were cultured at a density of 1 / mL, and the medium was completely replaced on the 3rd day. The cells were then passaged on the 4th day.

[0017] P2 generation: Using a culture medium containing 50% αMEM, 48% DMEM / F12, 1% autologous plasma, and 1% autologous platelet lysis buffer, 1×10⁻⁶ tbsp medium was added 4 hours after MSC inoculation. 5 Treg cells / mL were cultured, and the medium was completely replaced on the 3rd day. The cells were then passaged on the 4th day.

[0018] P3 generation: Cultured on a medium containing 90% αMEM, 5% DMEM / F12, and 5% autologous platelet lysis buffer until day 4 for harvest.

[0019] Furthermore, in the above-mentioned method for culturing post-acclimation mesenchymal stem cells, the preparation of the platelet lysis buffer includes: centrifuging autologous PRP after 3-5 freeze-thaw cycles to obtain the supernatant.

[0020] Furthermore, in the above-mentioned method for culturing post-acclimation mesenchymal stem cells, the P1 and P2 generation culture media also contain an immunomodulator, wherein the immunomodulator is selected from at least one of 0.1-1 μM resveratrol or 0.5-2 mM metformin.

[0021] Furthermore, in the above-mentioned method for culturing post-acclimation mesenchymal stem cells, at least one of 10-50 μM spermidine or 1-5 mM N-acetylcysteine ​​(NAC) is additionally added to the P1 generation culture medium.

[0022] Furthermore, in the above-mentioned method for culturing post-acclimation mesenchymal stem cells, the glycoprotein expression profile of the MSCs meets the following requirements: CD24 expression downregulated by ≥50%, and CD47 expression upregulated by ≥30% (compared to the non-sequential co-culture group).

[0023] Furthermore, in the above-mentioned method for culturing post-acclimatization mesenchymal stem cells, the lung retention rate of the MSCs after intravenous transplantation is ≤15% within 72 hours, and the survival period is ≥28 days (mouse model).

[0024] This invention also discloses a low-immunogenic mesenchymal stem cell, prepared by any of the above methods, whose surface markers satisfy: CD105. + / CD90 + / CD73 + / CD45 - / CD34 - / HLA-DR - And CD24 expression level is low (CD24 low High expression levels of CD47 (CD47) high High PD-L1 expression (PD-L1) high ).

[0025] The present invention also discloses the application of the above-mentioned low immunogenic mesenchymal stem cells in the preparation of drugs for treating acute respiratory distress syndrome (ARDS) or liver fibrosis.

[0026] Compared with existing technologies, the present invention has the following advantages and beneficial effects:

[0027] This invention discloses a method for culturing post-acclimation mesenchymal stem cells (MSCs). This invention simulates the physiological immune microenvironment through sequential CTL→Treg co-culture, combined with autologous components (plasma, PRP) and targeted metabolic regulators (resveratrol, spermidine / NAC), to synergistically achieve the following effects:

[0028] 1. Extremely low immunogenicity: HLA-DR expression ≤0.02% (compared to >1% in traditional methods), avoiding host T cell activation;

[0029] 2. Improved lung homing capacity: CD24↓62% / CD47↑45% inhibited macrophage clearance, and combined with antioxidant protection, reduced lung retention rate to 12% (compared to 45% in the control group);

[0030] 3. High functional stability: IDO activity ≥25U / mg, T cell inhibition rate 78%, with small batch-to-batch variation;

[0031] 4. Significant therapeutic advantages: 82% improvement rate in lung injury in ARDS model, and 58% decrease in hydroxyproline in liver fibrosis model. Attached Figure Description

[0032] Figure 1 These are flow cytometry images of the domesticated MSCs prepared in Example 6 of this invention. Image A: CD90-FITC channel detection (FMO control gated M3), showing 99.29% CD90 positive cells; Image B: CD105-PE channel detection (FMO control gated M4), showing 99.67% CD105 positive cells; Image C: CD73-APC channel detection (FMO control gated M5), showing 99.89% CD73 positive cells; Image D: Mixed PE channel detection of five negative markers (CD34 / CD45 / CD11b / CD19 / HLA-DR) (isotype control gated M3), showing a five-negative expression rate of 99.69%.

[0033] Note: The FMO (Fluorescence Minus One) control is used to exclude fluorescence compensation interference, and the isotype control is used to set the negative background threshold. Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below. However, it should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of the invention. Furthermore, descriptions of well-known structures and technologies are omitted in the following description to avoid unnecessarily obscuring the concept of the invention. All raw materials used in the embodiments of this invention are commercially available.

[0035] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0036] In this invention, the 'glycoprotein expression profile' specifically refers to the expression level patterns of specific transmembrane glycoproteins that are closely related to cellular immune recognition, clearance, and tolerance functions. Wherein:

[0037] CD24 is a glycoprotein anchored to glycosylphosphatidylinositol (GPI). Upon recognition by receptors such as Siglec-10 on macrophages, it transmits an "eat-me" signal, promoting phagocytosis and clearance of the cell. Therefore, downregulation of CD24 expression (compared to the non-sequential co-culture group) is a key negative regulatory phenotype in this invention regarding the resistance of acclimated MSCs to macrophage-mediated in vivo clearance (such as pulmonary vascular retention), rather than a classic biomarker defining the fundamental identity of MSCs.

[0038] CD47 is a widely expressed transmembrane glycoprotein that inhibits phagocytosis by binding to SIRPα (signal regulatory protein α) on the surface of macrophages and transmitting a "don't-eat-me" signal. Therefore, upregulation of CD47 expression (compared to the non-sequential co-culture group) is a core positive regulatory phenotype that enhances the survival and homing ability of acclimatized MSCs in vivo.

[0039] The CD24 expression downregulation ≥50% and CD47 expression upregulation ≥30% are calculated based on the expression levels of umbilical cord MSCs (e.g., P0 generation or Comparative Example 1 cells cultured in basal medium) that have not undergone the sequential co-culture step of this invention.

[0040] Due to the standardized nature of flow cytometry detection procedures (such as FMO / isotype control gating principles and instrument parameter consistency), this invention only uses representative illustrations. Figure 1 The core phenotypic validation is shown, and the remaining quantitative data (such as the MFI changes of CD24 / CD47) are all based on the same experimental system. Their reliability has been verified by multi-dimensional cross-validation in Table 1-5.

[0041] Example 1

[0042] Basic sequential co-cultivation method

[0043] 1.1 Sample Preparation

[0044] Collect 20 mL of peripheral blood from a healthy donor, and anticoagulate with heparin.

[0045] Centrifugation (2000×g, 20min) resulted in the following layers: upper layer plasma, middle layer PRP, and lower layer red blood cells / white blood cells.

[0046] PRP treatment: After the PRP layer was subjected to 3 freeze-thaw cycles (-80℃ / 37℃), it was centrifuged at 10000×g for 10min, and the supernatant was taken as platelet lysis buffer.

[0047] Blood cell sorting: White membrane layer resuspended in PBS, CD3+ CD8 + CTL and CD3 + CD25 + Treg sorting kit, positive rate ≥95%.

[0048] 1.2 Umbilical cord MSCs culture

[0049] P0 generation: Umbilical cord tissue blocks were cultured using the adherent culture method. Culture medium: αMEM 30% + DMEM / F12 68% + autologous plasma 2%.

[0050] P1 generation:

[0051] Culture medium: αMEM 30% + DMEM / F12 68% + autologous plasma 1% + platelet lysis buffer 1%;

[0052] Inoculation with MSCs (1×10) 4 cells / cm 2 Add CTL (1×10) after 4 hours. 5 (cells / mL);

[0053] D3: Complete medium replacement (removal of T cells); D4: Passage.

[0054] P2 generation:

[0055] Use a culture medium containing 50% αMEM, 48% DMEM / F12, 1% autologous plasma, and 1% autologous platelet lysis buffer;

[0056] MSCs were inoculated, and Treg (1×10⁻⁶) was added 4 hours later. 5 (cells / mL);

[0057] Complete fluid replacement on day 3, passage on day 4;

[0058] P3 generation: culture medium (αMEM 90% + DMEM / F12 5% + PRP 5%), harvest cells on D4.

[0059] Example 2

[0060] Optimization scheme with added resveratrol

[0061] Based on Example 1, 0.5 μM resveratrol was added to the P1 / P2 generation culture medium, and the remaining steps were the same as in Example 1.

[0062] Example 3

[0063] Optimization scheme with the addition of metformin

[0064] Based on Example 1, 1 mM metformin was added to the P1 / P2 generation culture medium, and the remaining steps were the same as in Example 1.

[0065] Example 4

[0066] Anti-pulmonary retention regimen with added spermidine

[0067] Based on Example 2, 30 μM spermidine was added to the P1 generation culture medium, and the remaining steps were the same as in Example 2.

[0068] Example 5

[0069] Anti-lung retention regimen with added NAC

[0070] Based on Example 3, 2 mM NAC was added to the P1 generation culture medium, and the remaining steps were the same as in Example 3.

[0071] Example 6

[0072] Overall optimal solution

[0073] Based on Example 1:

[0074] P1 / P2 generation added 0.5μM resveratrol + 30μM spermidine;

[0075] The culture medium remains unchanged for generation P3;

[0076] The rest is the same as in Example 1.

[0077] Comparative Example 1

[0078] T cell-free co-culture

[0079] Only P1 / P2 generation basal culture medium was used (without CTL / Treg addition), otherwise the same as in Example 1.

[0080] Comparative Example 2

[0081] Both P1 and P2 generations added CTL (1×10) 5 (cells / mL), no Treg, otherwise the same as in Example 1.

[0082] Comparative Example 3

[0083] Add only Treg

[0084] Both P1 and P2 generations were treated with Treg (1×10⁻⁶). 5 (cells / mL), no CTL, otherwise the same as in Example 1.

[0085] Comparative Example 4

[0086] Timing Reversal (Treg→CTL)

[0087] P1 generation adds Treg, P2 generation adds CTL, and the rest is the same as in Example 1.

[0088] Comparative Example 5

[0089] Conventional additives comparison

[0090] P1 generation was supplemented with 50 μg / mL vitamin C (replacing spermidine / NAC), and the rest was the same as in Example 6.

[0091] Comparative Example 6

[0092] Commercial culture medium control

[0093] The P1 / P2 generations used commercially available MSC culture medium (ScienCell, #7501) containing 10% FBS and no autologous blood components; otherwise, it was the same as in Example 1.

[0094] Test Example 1

[0095] Immunophenotypic and glycoprotein expression profiling analysis

[0096] 1. Test Objective

[0097] Validation of classic MSC markers (CD105) + / CD90 + / CD73 + The expression levels of CD45- / CD34- / HLA-DR- and function-related glycoproteins (CD24, CD47, PD-L1) were investigated to clarify the synergistic effect of sequential co-culture and additives.

[0098] 2. Testing Principle

[0099] Classic MSC markers (ISCT standard):

[0100] ≥95% positive for CD105, CD90, and CD73 confirms MSC status;

[0101] CD45 and CD34 negative (≥95%) exclude hematopoietic cell contamination;

[0102] HLA-DR negative (≤0.05%) indicates low immunogenicity.

[0103] Functional glycoproteins:

[0104] CD24 (“Eat Me” signal): Downregulation reduces macrophage phagocytosis;

[0105] CD47 (“Don’t eat me” signal): Upregulates and inhibits macrophage clearance;

[0106] PD-L1 (immune checkpoint): High expression enhances immune tolerance.

[0107] 3. Testing Methods

[0108] Harvest MSCs from each group (Examples 1-6, Comparative Examples 1-6) and wash with PBS;

[0109] Antibody incubation (protected from light, 25°C, 30 min):

[0110] Classic biomarkers: Anti-human CD105-BV510, Anti-human CD90-PerCP-Cy5.5, Anti-human CD73-APC-Cy7, Anti-human CD45-FITC, Anti-human CD34-PE-Cy7 (BioLegend);

[0111] Functional biomarkers: anti-human HLA-DR-FITC, anti-human CD24-PE, anti-human CD47-APC, anti-human PD-L1-PerCP (BioLegend / eBioscience);

[0112] CytoFLEX flow cytometry detection, FlowJo analysis:

[0113] Classic biomarker: Co-positive rate (CD105) + / CD90 + / CD73 + ), co-negative rate (CD45) - / CD34 - );

[0114] Functional biomarkers: HLA-DR + Positive rate, CD24 MFI change rate (based on Comparative Example 1), CD47 MFI change rate (based on Comparative Example 1), PD-L1 + Positive rate.

[0115] The results are shown in Table 1 and... Figure 1 Table 1 shows the statistical results (mean ± standard deviation) of each embodiment and comparative example after at least three independent experiments. Figure 1 The diagram shown is a representative flow cytometry result from a single experiment in Example 6 (the same batch of cells as in Example 6 in Table 1), used to visually verify classic biomarkers and negative expression;

[0116] Quantitative analysis of functional biomarkers (CD24 / CD47 / PD-L1) is based on... Figure 1 The statistical values ​​of the parallel flow cytometry tests (see Test Methods for details) are shown in Table 1.

[0117] Table 1. Immunophenotypic and glycoprotein expression profiling analysis

[0118]

[0119] Note: The CD24 / CD47 change rate was calculated based on Comparative Example 1;

[0120] *p<0.05,**p<0.01 vs Example 6 (ANOVA+Tukey test).

[0121] 5. Conclusion

[0122] Classic biomarker consistency: MSCs from all groups met the ISCT criteria (CD105). + / CD90 + The presence of CD73+ > 95% and CD45- / CD34 -> 95% confirms that its basic identity is mesenchymal stem cells.

[0123] Functional marker synergistic optimization:

[0124] Necessity of sequential co-cultivation:

[0125] Example 1 (CTL→Treg sequential) enables HLA-DR + The rate decreased to 0.08% (compared to 1.8% in Comparative Example 1), but was lower than the combined approach (Example 6, 0.02%).

[0126] Timing reversal (Comparative Example 4) leads to PD-L1 + The rate decreased to 50% (vs. 88% in Example 6), proving that the CTL→Treg order is not interchangeable.

[0127] Key role of additives:

[0128] Spermine (Example 4) significantly downregulated CD24 (↓58% vs Comparative Example 1), while NAC (Example 5) significantly upregulated CD47 (↑42% vs Comparative Example 1);

[0129] Resveratrol (Example 2) enhances PD-L1 + The rate reached 78% (vs. 70% in Example 1).

[0130] Breakthrough in comprehensive solutions:

[0131] like Figure 1 As shown, flow cytometry analysis of the MSCs obtained in Example 6 confirmed that:

[0132] Figure A (CD90-FITC-H): The FMO control settling point (M3) showed only 0.71% nonspecific background signal and a CD90 positivity rate of 99.29%.

[0133] Figure B (CD105-PE-H): FMO control (M4) background 0.33%, CD105 positivity rate 99.67%;

[0134] Figure C (CD73-APC-H): FMO control (M5) background 0.11%, CD73 positivity rate 99.89%;

[0135] Figure D (five-negative mixed detection): background of isotype control (M3) 0.31%, expression rate of CD34 / CD45 / CD11b / CD19 / HLA-DR five-negative expression rate 99.69%.

[0136] The above results are consistent with the data in Table 1, jointly demonstrating that the MSCs obtained in this invention not only meet the basic ISCT criteria (triple positivity > 95%), but also, through a sequential co-culture system, inhibit the expression of the immunogenic marker HLA-DR to near the detection limit (≤ 0.31%), which is significantly superior to the traditional method (Comparative Example 1: HLA-DR). + 1.8%

[0137] Classic logo meets the standard (CD105) + / CD90 + / CD73 + 99.2%, CD45- / CD34- 99.5%; optimal functional marker (HLA-DR) + 0.02%, CD24↓62%, CD47↑45%, PD-L1 + (88%), providing a molecular basis for low immunogenicity and high anti-clearance ability.

[0138] Test Example 2

[0139] IDO enzyme activity and immune regulation function

[0140] 1. Test Objective

[0141] To verify IDO activity (≥20 U / mg) and T cell inhibitory capacity, and to clarify the synergistic enhancement of immunomodulatory function by sequential co-culture and additives.

[0142] 2. Testing Principle

[0143] IDO activity: Catalyzes the breakdown of tryptophan into kynurenine; its activity is positively correlated with immunosuppression.

[0144] T-cell suppression assay: to evaluate the ability of MSCs to inhibit T-cell proliferation and reflect clinical efficacy.

[0145] 3. Testing Methods

[0146] 3.1 IDO Activity Detection

[0147] Reagent kit: MAK367 (Sigma), follow the instructions;

[0148] Calculation: Definition of unit enzyme activity: 1 nmol kynurenine / mg protein produced in 1 hour.

[0149] 3.2 T cell inhibition experiment

[0150] Isolate healthy donor CD3 + T cells (CFSE marker);

[0151] Co-culture with MSCs (1:10 ratio) activates T cells with anti-CD3 / CD28 antibody;

[0152] The dilution of CFSE was detected by flow cytometry after 72 hours, and the T cell proliferation inhibition rate was calculated.

[0153] The results are shown in Table 2.

[0154] Table 2 IDO enzyme activity and T cell inhibitory function

[0155] Group IDO activity (U / mg) T cell proliferation inhibition rate (%) Example 1 18.2±1.5 55±4 Example 2 21.3±1.8 68±5 Example 3 19.7±1.6 62±4 Example 4 23.1±2.0 72±6 Example 5 22.5±1.9 70±5 Example 6 25.3±2.1 78±4 Comparative Example 1 5.2±0.8** 20±3** Comparative Example 4 12.4±1.2** 45±4** Comparative Example 5 15.6±1.4** 50±5**

[0156] Note: **p<0.01 vs Example 6; Data association PD-L1 high Phenotypes (Table 1)

[0157] in conclusion:

[0158] Resveratrol (Example 2) significantly enhanced IDO activity (21.3 U / mg vs. 18.2 U / mg in Example 1);

[0159] The combined approach (Example 6) achieved IDO activity of 25.3 U / mg and T cell inhibition rate of 78%, combined with PD-L1 in Test Example 1. high The 88% phenotype confirmed that it enhanced immune tolerance through a metabolic-immune checkpoint synergistic mechanism, which was significantly better than the time-reversed group (Comparative Example 4: IDO 12.4U / mg, inhibition rate 45%).

[0160] Test Example 3

[0161] Lung retention rate and in vivo survival time

[0162] 1. Purpose of the test

[0163] Verify lung retention rate (≤15%) and survival period (≥28 days).

[0164] 2. Testing Principle

[0165] In vivo imaging: Near-infrared dye DiR-labeled MSCs for dynamic tracking of lung retention;

[0166] Quantitative analysis of human-derived cells: Flow cytometry detection of peripheral blood CD105 + Cells, assessing long-term survival.

[0167] 3. Testing Methods

[0168] Cell labeling: MSCs from each group were incubated with DiR (1 μg / mL, MedChemExpress) for 20 min;

[0169] Animal model: NOD / SCID mice (6-8 weeks old), intravenously injected with 5×10 5 MSCs;

[0170] Live imaging:

[0171] Time points: 0h, 24h, 72h, 7d, 14d, 28d;

[0172] Instrument: IVIS Spectrum (PerkinElmer), excitation 745nm / emission 800nm;

[0173] Quantitative analysis: Region of Interest (ROI) was used to analyze lung fluorescence intensity and calculate retention rate (D72 h intensity / D0 intensity × 100%).

[0174] Long-term survival testing:

[0175] Peripheral blood was collected on day 28, and human MSCs (CD105) were detected by flow cytometry using dual-labeled anti-human CD105-PE (BioLegend, 323204) and anti-human CD90-APC (BioLegend, 328114). + CD90 + (Cell percentage) is used to calculate long-term survival rate.

[0176] The results are shown in Table 3.

[0177] Table 3. Lung retention rate and in vivo survival time

[0178] Group 72-hour lung retention rate (%) <![CDATA[D28 CD105 + CD90 + Surviving cells (%) Example 1 20±3 5.1±0.7 Example 2 18±2 5.8±0.8 Example 3 19±3 5.5±0.6 Example 4 14±2 6.9±0.9 Example 5 13±3 7.2±0.8 Example 6 12±3 8.7±0.9 Comparative Example 1 42±5** 1.0±0.3** Comparative Example 4 35±4** 2.5±0.5** Comparative Example 5 28±4** 3.1±0.6** Comparative Example 6 45±5** 1.2±0.3**

[0179] in conclusion

[0180] Spermine (Example 4) and NAC (Example 5) reduced lung retention rates to 14% and 13%, respectively, which were significantly better than the untreated group (Example 1, 20%).

[0181] The comprehensive treatment (Example 6) achieved a lung retention rate of 12% and D28 CD105 + CD90 + Cell viability was 8.7%, and it bound CD24. low (↓62%) and CD47 high The (↑45%) phenotype (Table 1) confirms that it significantly improves in vivo survival by inhibiting macrophage clearance.

[0182] Test Example 4

[0183] Disease treatment model validation

[0184] 1. Purpose of the test

[0185] To verify its efficacy in the treatment of ARDS and liver fibrosis.

[0186] 2. Testing Principle

[0187] ARDS model: LPS induces lung injury, MSCs reduce inflammatory infiltration;

[0188] Liver fibrosis model: CCl4 induces fibrosis, MSCs inhibit collagen deposition.

[0189] 3. Testing Methods

[0190] 3.1 ARDS model (mouse)

[0191] C57BL / 6 mice were administered LPS (5 mg / kg) via intratracheal instillation;

[0192] MSCs (1×10⁻⁶) were injected intravenously 6 hours later. 6 (cells / each);

[0193] Test after 72 hours:

[0194] Neutrophil count in bronchoalveolar lavage fluid (BALF) (Diff-Quik staining);

[0195] H&E pathological score of lung tissue (0-4 points: normal → severe damage).

[0196] 3.2 Liver fibrosis model (rat)

[0197] SD rats were subcutaneously injected with 40% CCl4 (2 mL / kg, twice a week for 8 weeks);

[0198] D56 intravenous injection of MSCs (2×10) 6 (cells / each);

[0199] D70 test:

[0200] Liver hydroxyproline content (alkaline hydrolysis method, μg / mg tissue);

[0201] α-SMA immunohistochemical positive area (ImageJ quantification).

[0202] The test results are shown in Tables 4 and 5.

[0203] Table 4 ARDS model data

[0204]

[0205]

[0206] Table 5. Data from the liver fibrosis model

[0207] Group Hydroxyproline (μg / mg) α-SMA+ area (%) Example 6 1.8±0.3 8.2±1.5 Comparative Example 1 3.9±0.5** 25.6±3.0** Comparative Example 4 2.7±0.4** 15.3±2.2** Model control group 4.3±0.6** 32.7±3.1**

[0208] The conclusions can be drawn from Tables 4 and 5.

[0209] Comprehensive treatment plan (Example 6) for ARDS:

[0210] Neutrophil count decreased by 65% ​​(vs. saline group), and lung injury improvement rate was 82%.

[0211] Its low immunogenicity (HLA-DR) - 0.02% and high PD-L1 high (88%) Synergistically inhibited inflammatory infiltration, with an effect 37% higher than control group 2 (CTL co-culture only);

[0212] Comprehensive treatment plan (Example 6) for liver fibrosis:

[0213] Hydroxyproline decreased by 58% (vs. model group), α-SMA + The area decreased to 8.2%;

[0214] High CD47 high (↑45%) Inhibits macrophage clearance and promotes MSC homing to the liver to exert a repair effect, with significantly better results than the time-reversed group (comparative example 4: hydroxyproline only ↓37%).

[0215] The following conclusions can be drawn from the test data:

[0216] 1. Necessity of sequential co-culture: Reversing the time sequence (Comparative Example 4) resulted in a 38% decrease in PD-L1 positivity rate and a 51% decrease in IDO activity;

[0217] 2. Synergistic effect of additives:

[0218] Spermine / NAC reduced lung retention rate to 12-13% (compared to 20% in the no-addition group);

[0219] Resveratrol increased IDO activity to 25.3 U / mg (compared to 18.2 U / mg in the no-addition group);

[0220] 3. Overall advantages of the solution: Example 6 achieved HLA-DR 0.02%, CD47↑ 45%, lung retention rate 12%, and D28 survival rate 8.7%, which is significantly better than the comparison ratios (such as the commercial culture medium group with a lung retention rate of 45%).

[0221] 4. Disease treatment verification: In ARDS and liver fibrosis models, the neutrophil clearance rate and collagen deposition inhibition rate of Example 6 were both increased by more than 30%.

[0222] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Therefore, any changes and modifications made to the embodiments described herein based on the innovative concept of the present invention, or equivalent structural or procedural transformations made using the content of the present invention specification, directly or indirectly applying the above technical solutions to other related technical fields, are all included within the scope of protection of the present invention patent.

Claims

1. A method for culturing post-acclimation mesenchymal stem cells, characterized in that, Includes the following steps: (a) Plasma and platelet lysate were separated from autologous peripheral blood and CD3 was obtained by magnetic bead sorting. + CD8 + Cytotoxic T cells (CTLs) and CD3 + CD25 + Regulatory T cells (Tregs); (b) Co-culture umbilical cord-derived mesenchymal stem cells (MSCs) sequentially in the following order: P1 generation: Using a culture medium containing 30% αMEM, 68% DMEM / F12, 1% autologous plasma, and 1% autologous platelet lysis buffer, 1×10⁻⁶ tbsp medium was added 4 hours after MSC inoculation. 5 CTLs were cultured at a density of 1 / mL, and the medium was completely replaced on the 3rd day. The cells were then passaged on the 4th day. P2 generation: Using a culture medium containing 50% αMEM, 48% DMEM / F12, 1% autologous plasma, and 1% autologous platelet lysis buffer, 1×10⁻⁶ tbsp medium was added 4 hours after MSC inoculation. 5 Treg cells / mL were cultured, and the medium was completely replaced on the 3rd day. The cells were then passaged on the 4th day. P3 generation: Cultured on a medium containing 90% αMEM, 5% DMEM / F12, and 5% autologous platelet lysis buffer until day 4 for harvest.

2. The method according to claim 1, characterized in that, The preparation of the platelet lysis buffer includes: centrifuging autologous PRP after 3-5 freeze-thaw cycles to obtain the supernatant.

3. The method according to claim 1, characterized in that, The P1 and P2 generation culture media also contain an immunomodulator, which is selected from at least one of 0.1-1 μM resveratrol or 0.5-2 mM metformin.

4. The method according to claim 1, characterized in that, The P1 generation culture medium is supplemented with at least one of 10-50 μM spermidine or 1-5 mM N-acetylcysteine ​​(NAC).

5. The method according to claim 1, characterized in that, The surface HLA-DR expression level of the harvested MSCs was ≤0.05%, and the indoleamine 2,3-dioxygenase (IDO) activity was ≥20 U / mg protein.

6. The method according to claim 1, characterized in that, The glycoprotein expression profiles of the MSCs met the following criteria: CD24 expression was downregulated by ≥50%, and CD47 expression was upregulated by ≥30% (compared to the non-sequential co-culture group).

7. The method according to claim 1, characterized in that, The lung retention rate of the MSCs after intravenous transplantation was ≤15% within 72 hours, and the survival period was ≥28 days (mouse model).

8. A type of low-immunogenic mesenchymal stem cell, characterized in that, Prepared by the method described in any one of claims 1-7, the surface markers of which satisfy CD105 + / CD90 + / CD73 + / CD45- / CD34- / HLA-DR-, and low CD24 expression (CD24 low High expression levels of CD47 (CD47) high High PD-L1 expression (PD-L1) high ).

9. The use of the low immunogenicity mesenchymal stem cells of claim 8 in the preparation of drugs for treating acute respiratory distress syndrome (ARDS) or liver fibrosis.

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