Method for enhancing immunoregulation capability of mesenchymal stem cells

Abstract

The invention belongs to the technical field of cell culture, discloses a method for enhancing the immunomodulatory ability of mesenchymal stem cells, and particularly discloses application of a liquid-liquid interface culture system in improving the immunomodulatory ability of stem cells. Wherein the liquid-liquid interface culture system comprises an upper-layer water phase and a lower-layer oil phase; the upper-layer water phase comprises a protein solution or a biocompatible polymer solution, and the lower-layer oil phase comprises a fluorinated solution. The liquid-liquid interface culture system is used for remarkably enhancing the response of the stem cells (mesenchymal stem cells) to the stimulation of the cytokines for the first time, and the liquid-liquid interface can promote the stem cells (mesenchymal stem cells) to respond to the stimulation of the cytokines, so that a better pre-activation effect is achieved, and the immunoregulation ability of the stem cells is improved. For example, under the action of IFN-gamma with different concentrations, the expression of an immunosuppressive protein IDO gene of mesenchymal stem cells on a liquid-liquid interface always shows a higher level.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell culture, and particularly relates to a method for enhancing the immunomodulatory ability of mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cells with functions such as self-renewal, multi-directional differentiation, immunomodulation, and tissue repair, and are widely used in disease treatment, especially in inflammatory diseases and autoimmune diseases. In the field of stem cell therapy, the existing methods for improving the immunomodulatory ability of MSCs in vitro mainly fall into two categories: gene modification and non-gene modification. Gene modification mainly enhances the ability of MSCs to inhibit inflammation and respond to the microenvironment by overexpressing immunomodulatory factors (such as TGF-β, IL-10, IDO, etc.), regulating immune-related signaling pathways (such as NF-κB, JAK-STAT1, Notch pathway, etc.), and epigenetic regulation based on CRISPR / dCas9 editing of DNA methylation or histone modification. However, gene-modified MSCs pose great risks in terms of off-target effects and tumorigenicity, and their safety cannot be guaranteed.

[0003] Non-gene modification methods can avoid the risks brought by gene insertion and have higher safety, making them a better choice by comparison. Non-gene modification is mainly achieved through two strategies: one is the biochemical strategy, which uses cytokines such as TNF-α, INF-γ, and IL-1β to pretreat MSCs to induce MSCs to secrete anti-inflammatory factors and inhibit the proliferation and activation of T cells and NK cells; the other is the pre-activation strategy that mimics the physiological microenvironment of MSCs, which regulates the paracrine function of MSCs by constructing a hydrogel scaffold system loaded with MSCs, thereby improving the therapeutic effect of MSCs.

[0004] Current biochemical factor pre-activation strategies are mostly based on conventional culture conditions, i.e., solid culture dishes. However, solid culture conditions have significant limitations in maintaining the immunomodulatory and paracrine effects of MSCs. Specifically, the static mechanical microenvironment is insufficient to simulate the in vivo dynamic physiological environment of mesenchymal stem cells, making it difficult to synchronously optimize cell metabolic activity and paracrine function. Ultimately, the pre-activation effect of mesenchymal stem cells is limited. Although the three-dimensional hydrogel culture system can solve the problems brought by solid culture dishes, the hydrogel preparation process is complex, difficult to regulate, and the polymer materials used in it cannot be recycled. These problems will ultimately lead to increased culture costs and environmental pollution. Whether it is the traditional two-dimensional solid culture dish or the three-dimensional hydrogel culture system, there are difficulties and cumbersome processes in collecting pre-activated cells. Existing collection methods mostly rely on trypsin or collagenase digestion, which easily causes damage and decreased activity of cell membrane surface proteins, thereby affecting their clinical treatment effects. Biochemical factor pre-activation strategies face high cost problems. The cytokines used in pre-activation are expensive. Taking the most critical cytokine IFN-γ as an example, the price of 1 mg is above 3000 yuan. Therefore, when pre-activating mesenchymal stem cells on a large scale, the dosage of biochemical factors increases, and the cost increases significantly, severely restricting their industrial application.

[0005] In recent years, a new type of bio-dynamic material, the liquid-liquid interface system, has been proven by research to have a biomimetic dynamic self-adaptability closer to the natural extracellular matrix than hydrogels. The liquid-liquid interface system usually consists of an upper aqueous phase and a lower fluorinated liquid, and forms a protein nanofilm or fiber sheet layer for MSCs to adhere to, spread on, and grow through the interfacial tension at the two-phase interface. The liquid-liquid interface provides an ideal scenario for simulating the physiological environment of MSCs. Summary of the Invention

[0006] An object of the first aspect of the present invention is to provide an application of a liquid-liquid interface culture system in improving the immunomodulatory ability of stem cells.

[0007] An object of the second aspect of the present invention is to provide a method for improving the immunomodulatory ability of stem cells.

[0008] To achieve the above objects, the technical solutions adopted by the present invention are as follows:

[0009] The first aspect of the present invention provides an application of a liquid-liquid interface culture system in improving the immunomodulatory ability of stem cells;

[0010] Among them, the liquid-liquid interface culture system includes an upper aqueous phase and a lower oil phase;

[0011] The upper aqueous phase includes a protein solution or a biocompatible polymer solution, and the lower oil phase includes a fluorinated liquid.

[0012] In some embodiments of the present invention, the liquid-liquid interface culture system is prepared by the following preparation method: add the lower oil phase and the upper water phase into a container in sequence, incubate to form a protein nanofilm, then wash the upper water phase with PBS, and replace the upper water phase with a culture medium to obtain the liquid-liquid interface culture system.

[0013] In some embodiments of the present invention, the protein includes at least one of lysozyme, serum albumin, whey protein, casein, fibronectin, collagen, laminin, type I collagen, fibrinogen, elastin, vitronectin.

[0014] In some embodiments of the present invention, the biocompatible polymer includes gelatin, polylysine, amyloid fibrils, casein-chitosan complex.

[0015] In some embodiments of the present invention, the concentration of the protein solution or the biocompatible polymer solution is at least 1 mg / mL.

[0016] In some embodiments of the present invention, the concentration of the protein solution or the biocompatible polymer solution is 1 - 5 mg / mL.

[0017] In some embodiments of the present invention, the fluorinated liquid includes at least one of perfluoroalkane, hydrofluoroether, perfluorocyclobutane, perfluoromethylcyclohexane, perfluorodecalin, perfluorooctylpropane, perfluorooctanesulfonic acid, fluorinated telomer alcohol (PFO).

[0018] In some embodiments of the present invention, the fluorinated liquid includes perfluoroalkane (such as FC40, FC70, FC72, FC3283) and hydrofluoroether (HFE7000, HFE7500).

[0019] In some embodiments of the present invention, the volume ratio of the perfluoroalkane to the hydrofluoroether is 1:(1 - 10), such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.

[0020] By controlling the types of proteins and fluorinated liquids and the mixing ratio of the fluorinated liquids, the controllability of the liquid-liquid interface culture system has been achieved.

[0021] In some embodiments of the present invention, the incubation time is 1 - 24 h; further 1 - 10 h; still further 1 - 5 h.

[0022] In some embodiments of the present invention, the method for washing the upper aqueous phase with PBS is as follows: First, remove half of the volume of the upper aqueous phase, then add PBS to the upper aqueous phase, and then remove the upper aqueous phase with the same volume as the added PBS. Repeat this process 3 to 5 times. After washing, the remaining volume of the upper aqueous phase is half of the volume of the protein solution or biocompatible polymer solution during the original incubation.

[0023] In some embodiments of the present invention, add a culture medium to the upper aqueous phase, and then remove the upper aqueous phase with the same volume as the added culture medium. Repeat this process 3 to 5 times. After washing, the remaining volume of the upper aqueous phase is half of the volume of the protein solution or biocompatible polymer solution during the original incubation.

[0024] In some embodiments of the present invention, the stem cells include at least one of mesenchymal stem cells (such as iPS-induced mesenchymal stem cells (iPS-MSC), umbilical cord blood mesenchymal stem cells (UCMSC), bone marrow mesenchymal stem cells (BMMSC), adipose mesenchymal stem cells (ADMSC), hematopoietic mesenchymal stem cells (HSC)), neural stem cells, skin stem cells, muscle stem cells, embryonic stem cells, dental pulp stem cells, bone stem cells, and hematopoietic stem cells.

[0025] In some embodiments of the present invention, the container includes a cell culture vessel, such as a cell culture plate.

[0026] In some embodiments of the present invention, the liquid-liquid interface culture system enhances the response of stem cells to cytokine stimulation, thereby achieving the purpose of improving the immunomodulatory ability of stem cells.

[0027] The second aspect of the present invention provides a method for improving the immunomodulatory ability of stem cells, comprising the following steps:

[0028] (1) Add a fluorinated liquid as the lower oil phase in a cell culture container, then add a protein solution or a biocompatible polymer solution as the upper aqueous phase. After incubation, a protein nanofilm is formed. Then wash the upper aqueous phase with PBS and replace the upper aqueous phase with a culture medium to obtain a liquid-liquid interface culture system;

[0029] (2) Inoculate stem cells into the upper aqueous phase of the liquid-liquid interface culture system and culture;

[0030] (3) Remove part of the culture medium in the upper aqueous phase and add a culture medium containing cytokines with the same volume, and then culture.

[0031] In some embodiments of the present invention, the protein includes at least one of lysozyme, serum albumin, whey protein, casein, fibronectin, collagen, laminin, type I collagen, fibrinogen, elastin, and vitronectin.

[0032] In some embodiments of the present invention, the biocompatible polymer includes gelatin, polylysine, amyloid fibrils, and casein-chitosan complex.

[0033] In some embodiments of the present invention, the concentration of the protein solution or the biocompatible polymer solution is at least 1 mg / mL.

[0034] In some embodiments of the present invention, the concentration of the protein solution or the biocompatible polymer solution is 1 - 5 mg / mL.

[0035] In some embodiments of the present invention, the fluorinated liquid includes at least one of perfluoroalkane, hydrofluoroether, perfluorocyclobutane, perfluoromethylcyclohexane, perfluorodecalin, perfluorooctylpropane, perfluorooctanesulfonic acid, and fluorinated telomer alcohol (PFO).

[0036] In some embodiments of the present invention, the fluorinated liquid includes perfluoroalkane (such as FC40, FC70, FC72, FC3283) and hydrofluoroether (HFE7000, HFE7500).

[0037] In some embodiments of the present invention, the volume ratio of the perfluoroalkane to the hydrofluoroether is 1:(1 - 10), such as 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, etc.

[0038] In some embodiments of the present invention, the cytokine includes at least one of interferon, tumor necrosis factor, and interleukin.

[0039] In some embodiments of the present invention, the interferon includes at least one of IFN-α, IFN-β, IFN-γ, and IFN-ω.

[0040] In some embodiments of the present invention, the tumor necrosis factor includes TNF-α and / or TNF-β.

[0041] In some embodiments of the present invention, the interleukin includes at least one of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10, and IL-12.

[0042] In some embodiments of the present invention, the cytokine includes IFN-γ and TNF-α.

[0043] In some embodiments of the present invention, in the culture system of (3), the final concentration of the cytokine is 0.1 - 100 ng / mL.

[0044] In some embodiments of the present invention, the final concentration of the cytokine is 0.1 - 50 ng / mL.

[0045] In some embodiments of the present invention, the incubation time in (1) is 1 to 24 h; further 1 to 10 h; still further 1 to 5 h.

[0046] In some embodiments of the present invention, the culture conditions in (2) are 35 to 38 °C, 4% to 6% CO2 for 2 to 10 h; further culture for 2 to 5 h.

[0047] In some embodiments of the present invention, the culture time in (3) is 20 to 72 h; further 24 to 48 h.

[0048] In some embodiments of the present invention, the stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, embryonic stem cells, dental pulp stem cells, bone stem cells, and hematopoietic stem cells.

[0049] In some embodiments of the present invention, the stem cells include at least one of mesenchymal stem cells (iPS-induced mesenchymal stem cells (iPS-MSC), umbilical cord blood mesenchymal stem cells (UCMSC), bone marrow mesenchymal stem cells (BMMSC), adipose mesenchymal stem cells (ADMSC), hematopoietic mesenchymal stem cells (HSC)), neural stem cells, skin stem cells, muscle stem cells, embryonic stem cells, dental pulp stem cells, bone stem cells, and hematopoietic stem cells.

[0050] In some embodiments of the present invention, the culture conditions and the culture medium used in the above method can also be selected according to the cells to be cultured.

[0051] When the cell culture is completed, pure cell suspension can be collected by centrifugation, and the process does not require any biochemical reagents for digestion assistance at all.

[0052] The beneficial effects of the present invention are:

[0053] The present invention first provides a liquid-liquid interface culture system that significantly enhances the response of stem cells (mesenchymal stem cells) to cytokine stimulation. The liquid-liquid interface can promote the response of stem cells (mesenchymal stem cells) to cytokine stimulation, achieving a better pre-activation effect to improve the immunomodulatory ability of stem cells. For example, under the action of different concentrations of IFN-γ, the expression of the immunosuppressive protein IDO gene in mesenchymal stem cells on the liquid-liquid interface always shows a higher level.

[0054] Compared with the culture system of traditional solid culture dishes, the present invention uses a liquid-liquid interface culture system to reduce the cytokine dosage by up to 90%. Taking IFN-γ as an example, on the premise of achieving the same pre-activation effect, the liquid-liquid interface system only requires an IFN-γ dosage of 10 ng / mL to achieve the activation effect of 100 ng / mL by the traditional method, breaking through to reduce the unit dose requirement by one order of magnitude. Based on the substantial reduction of cytokine dosage, it is expected to reduce the cytokine cost by 80% - 90% in industrial production applications.

[0055] Compared with the culture system of traditional solid culture dishes, the liquid-liquid interface has significant advantages in the immunomodulatory ability of mesenchymal stem cells. On the liquid-liquid interface, stimulation with low-dose cytokines can effectively improve the immunomodulatory ability of mesenchymal stem cells, manifested as significant increases in the gene and protein expression levels of related immunomodulatory proteins such as TSG6, IDO, COX2, and TGF-β1. At the same time, the immunosuppressive function of the cells lasts for a long time, and significantly inhibits the proliferation of cytotoxic CD8 + T cells after co-culture with T cells for three days, showing better functional stability.

[0056] Compared with three-dimensional hydrogel pre-activation, the present invention uses a liquid-liquid interface culture system to achieve convenient and efficient operation processes and shows the characteristics of environmental friendliness. The liquid-liquid interface culture system adopts a dual-liquid interface culture mode, with easy control of parameters during the culture process, and can also achieve efficient directional enrichment of cells through physical stratification (such as centrifugation) without the assistance of biochemical reagents (such as enzymes), simplifying the operation process. In addition, the recovery rate of the fluorinated liquid in the liquid-liquid interface culture system is as high as over 95%, and it can be reused after purification without burdening the environment. Brief Description of the Drawings

[0057] Figure 1 It is a schematic diagram of the liquid-liquid interface.

[0058] Figure 2 It is a protein membrane diagram of the liquid-liquid interface.

[0059] Figure 3 It is a comparison of the IDO gene expression levels of mesenchymal stem cells on solid culture dishes and the liquid-liquid interface in response to IFN-γ stimulation after pre-activation with different concentrations of IFN-γ.

[0060] Figure 4 It is a comparison of the IDO gene expression levels of mesenchymal stem cells on solid culture dishes pre-activated with high-concentration IFN-γ and mesenchymal stem cells on the liquid-liquid interface pre-activated with low-concentration IFN-γ. In the figure, ns represents no significant difference, and * represents p < 0.05.

[0061] Figure 5Results of gene expression levels of immunosuppressive proteins TSG6, IDO, COX2, and TGF-β1 in mesenchymal stem cells at the liquid-liquid interface on solid culture dishes after pre-activation with 10 ng / mL IFN-γ and 30 ng / mL TNF-α and without pre-activation. In the figure, ns represents no significant difference, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.005, and **** represents p < 0.001.

[0062] Figure 6 Results of the expression levels of immunosuppressive proteins TSG6, IDO, COX2, and TGF-β1 in mesenchymal stem cells at the solid culture dish and liquid-liquid interface after pre-activation with 10 ng / mL IFN-γ and 30 ng / mL TNF-α. In the figure, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.005, and **** represents p < 0.001.

[0063] Figure 7 Proliferation inhibition results of CD8 + T cells by mesenchymal stem cells at the solid culture dish and liquid-liquid interface after pre-activation with 10 ng / mL IFN-γ and 30 ng / mL TNF-α. In the figure, **** represents p < 0.001. Detailed implementation manners

[0064] The content of the present invention will be further described in detail below through specific examples.

[0065] It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0066] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0067] The features and performance of the present invention will be further described in detail below in combination with the examples.

[0068] Example 1

[0069] A method for enhancing the immunomodulatory ability of mesenchymal stem cells (hMSCs), comprising the following steps:

[0070] (1) Liquid preparation: Mix two fluorinated liquids, FC40 and HFE7100, in a volume ratio of 1:3 (V FC40 :V HFE7100Perform sufficient mixing to prepare a mixed fluorinated liquid, which serves as the lower liquid phase at the liquid-liquid interface. Weigh an appropriate amount of lysozyme protein and dissolve it in PBS to prepare a protein solution with a protein concentration of 1 mg / mL, which serves as the upper liquid phase at the liquid-liquid interface;

[0071] (2) Preparation of the liquid-liquid interface: Add 1 mL of the mixed fluorinated liquid from step (1) to a 24-well plate as the lower liquid phase. The fluorinated liquid should completely immerse the bottom of the well plate and form a horizontal liquid surface. Then, add an equal volume (i.e., 1 mL) of the protein solution from step (1) as the upper aqueous phase. Incubate statically for 3 h. After static incubation is completed, the liquid-liquid interface is obtained ( Figure 1 ). Remove 500 μL of the upper liquid phase, and wash the aqueous phase 3 times with PBS and α-MEM medium containing 10% FBS respectively. Finally, replace the upper aqueous phase with the medium;

[0072] (3) Cell seeding and culture: Remove 500 μL of the upper liquid phase, evenly add 500 μL of the mesenchymal stem cell suspension to the upper aqueous phase, and culture it at 37 °C and 5% CO2;

[0073] (4) Pre-activate mesenchymal stem cells with cytokines: 2 h after the mesenchymal stem cells are seeded in step (3), remove 500 μL of the upper liquid phase, and then add 500 μL of the medium containing IFN-γ and TNF-α to activate hMSCs with IFN-γ and TNF-α at final concentrations of 10 ng / mL and 30 ng / mL respectively, and act for 24 h;

[0074] (5) Cell collection and fluorinated liquid recovery: After removing most of the medium, dilute the medium with PBS multiple times, then transfer all the liquid in each well to a centrifuge tube and centrifuge. The centrifugation conditions are 300 rcf, 5 min, and 25 °C. After centrifugation, the fluorinated liquid converges to the lower layer, and the cells are concentrated in the upper aqueous phase. Collect the aqueous phase to obtain pre-activated mesenchymal stem cells (denoted as L-L-2); Recover the fluorinated liquid in the lower liquid phase after centrifugation, filter and sterilize it with a microporous filter membrane, and it can be used for the next culture.

[0075] Example 2

[0076] A method for enhancing the immunomodulatory ability of mesenchymal stem cells (hMSCs), compared with Example 1, the only difference is that the final concentration of IFN-γ is 0.1 ng / mL.

[0077] Example 3

[0078] A method for enhancing the immunomodulatory ability of mesenchymal stem cells (hMSCs), compared with Example 1, the only difference is that the final concentration of IFN-γ is 1 ng / mL.

[0079] Example 4

[0080] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that the final concentration of IFN-γ is 5 ng / mL.

[0081] Example 5

[0082] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that IFN-γ and TNF-α are not used to preactivate the mesenchymal stem cells.

[0083] Example 6

[0084] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs) includes the following steps:

[0085] (1) Liquid preparation: Use FC40 as the lower liquid phase of the liquid-liquid interface. Weigh an appropriate amount of lysozyme protein and dissolve it in PBS to prepare a protein solution with a protein concentration of 1 mg / mL as the upper liquid phase of the liquid-liquid interface;

[0086] (2) Preparation of the liquid-liquid interface: Add 1 mL of the mixed fluorinated liquid in step (1) to a 24-well plate as the lower liquid phase. The fluorinated liquid should completely immerse the bottom of the well plate and form a horizontal liquid surface, and then add an equal volume (i.e., 1 mL) of the protein solution in step (1) as the upper aqueous phase. Incubate statically for 3 h. After the static incubation is completed, remove 500 μL of the upper liquid phase, and wash the aqueous phase 3 times with PBS and culture medium respectively. Finally, replace the upper aqueous phase with culture medium;

[0087] (3) Cell seeding and culture: Remove 500 μL of the upper liquid phase, evenly add 500 μL of the mesenchymal stem cell suspension to the upper aqueous phase, and culture it at 37 °C and 5% CO2;

[0088] (4) Preactivate mesenchymal stem cells with cytokines: 2 h after the mesenchymal stem cells are seeded in step (3), remove 500 μL of the upper liquid phase, and then add 500 μL of the culture medium containing IFN-γ and TNF-α to activate hMSCs with IFN-γ and TNF-α at final concentrations of 10 ng / mL and 30 ng / mL for 24 h;

[0089] (5) Cell collection and recovery of fluorinated liquid: After removing most of the culture medium, dilute the culture medium with PBS multiple times, then transfer all the liquid in each well to a centrifuge tube and centrifuge. The centrifugation conditions are 300 rcf, 5 min, and 25 °C. After centrifugation, the fluorinated liquid converges to the lower layer, and the cells are concentrated in the upper aqueous phase. Collect the aqueous phase to obtain preactivated mesenchymal stem cells (denoted as L-L-1); Recover the fluorinated liquid in the lower liquid phase after centrifugation, and filter and sterilize it with a microporous filter membrane for the next culture.

[0090] Example 7

[0091] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 6 in that IFN-γ and TNF-α are not used to preactivate the mesenchymal stem cells.

[0092] Example 8

[0093] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that the volume ratio of the fluorinated liquid FC40 to HFE7100 is 1:2.

[0094] Example 9

[0095] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that the volume ratio of the fluorinated liquid FC40 to HFE7100 is 1:4.

[0096] Example 10

[0097] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that the volume ratio of the fluorinated liquid FC40 to HFE7100 is 1:9.

[0098] Example 11

[0099] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Example 1 in that the protein is serum protein, whey protein, gelatin, amyloid fibrils, casein-chitosan complex, polylysine.

[0100] Comparative Example 1

[0101] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs) includes the following steps: directly inoculating the same number of mesenchymal stem cells as in Example 1 in a common 24-well plate, and culturing under the conditions of 37 °C and 5% CO2. The process of preactivating the mesenchymal stem cells with cytokines is the same as in Example 1. Collect the cells to obtain the preactivated mesenchymal stem cells (denoted as the TCP control group).

[0102] Comparative Example 2

[0103] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Comparative Example 1 in that the final concentration of IFN-γ is 0.1 ng / mL.

[0104] Comparative Example 3

[0105] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Comparative Example 1 in that the final concentration of IFN-γ is 1 ng / mL.

[0106] Comparative Example 4

[0107] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Comparative Example 1 in that the final concentration of IFN-γ is 5 ng / mL.

[0108] Comparative Example 5

[0109] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Comparative Example 1 in that the final concentration of IFN-γ is 100 ng / mL.

[0110] Comparative Example 6

[0111] A method for enhancing the immunomodulatory ability of human mesenchymal stem cells (hMSCs), which is only different from Comparative Example 1 in that IFN-γ and TNF-α are not used to preactivate mesenchymal stem cells.

[0112] Effect Example

[0113] 1. Characterization of the protein film at the liquid-liquid interface by atomic force microscopy

[0114] The protein film at the liquid-liquid interface in Example 1 was transferred onto a silicon wafer, and then the silicon wafer was clamped out with tweezers and dried at room temperature to obtain a test sample. The protein film was characterized and imaged using a Dimension Icon atomic force microscope (Bruker). The protein film was gently scratched with tweezers, and the thickness of the protein film was measured by measuring the height difference between the protein film and the substrate.

[0115] The results are as Figure 2 shown, and the thickness of the protein film is 1.26 ± 0.08 nm.

[0116] 2. Determination of the gene expression level of cell immunosuppressive proteins

[0117] The total RNA of the preactivated mesenchymal stem cells cultured in Examples 1 to 7 and Comparative Examples 1 to 6 was extracted using the phenol-chloroform method, and then the total RNA was further purified using the magnetic bead method. Referring to the method of the reverse transcription kit (TransGen Biotech, AE341), the RNA was reverse transcribed into cDNA. Referring to the reference " Carvalho, M. R. R., Sousa, T., Alencar-Silva, T., et al. Mesenchymal stem cells immunomodulation: The road to IFN-γ licensing and the path ahead. Cytokine & Growth Factor Reviews, 2019, 47(32 - 42).; Ji Y, et al. Substrate stiffness affects the immunosuppressive and trophic function of hMSCs via modulating cytoskeletal polymerization and tension. Biomaterials Science, 2019, 7(5292–300). The expression levels of the cell immunosuppressive proteins IDO, TSG6, PTGS2, and TGF-β1 genes were measured using a real-time fluorescence quantitative PCR instrument.

[0118] The comparison results of the IDO gene expression levels of mesenchymal stem cells on solid culture dishes (i.e., Comparative Examples 1 - 4) and liquid-liquid interfaces (i.e., Examples 1 - 4) pre-activated with 0.1, 1, 5, and 10 ng / mL IFN-γ in response to IFN-γ stimulation are as Figure 3 shown. Under the action of different concentrations of IFN-γ, the expression of the immunosuppressive protein IDO gene in mesenchymal stem cells on the liquid-liquid interface always showed a higher level than that on the solid culture dish, indicating that compared with the traditional solid culture dish, the liquid-liquid interface can enhance the response of mesenchymal stem cells to cytokine stimulation. The liquid-liquid interface can promote the response of mesenchymal stem cells to cytokine stimulation and achieve a better pre-activation effect.

[0119] The comparison results of the IDO gene expression levels of mesenchymal stem cells on solid culture dishes pre-activated with high-concentration IFN-γ and mesenchymal stem cells on liquid-liquid interfaces pre-activated with low-concentration IFN-γ are as Figure 4 shown. On the premise of achieving the same pre-activation effect, the liquid-liquid interface system only requires an IFN-γ dosage of 10 ng / mL to achieve the activation effect of 100 ng / mL of the traditional method (solid culture dish), breaking through to reduce the unit dose requirement by one order of magnitude. This suggests that the utilization efficiency of cytokines on the liquid-liquid interface is significantly improved. Based on the liquid-liquid interface culture technology, the dosage of cytokines is greatly reduced (the cytokine dosage is reduced by up to 90%), and it is expected to reduce the cytokine cost by 80% - 90% in industrial production applications.

[0120] The results of the gene expression levels of mesenchymal stem cell immunosuppressive proteins TSG6, IDO, COX2, and TGF-β1 on solid culture dishes (Comparative Example 1 and Comparative Example 6) and liquid-liquid interfaces (Example 1 and Example 5) after pre-activation with or without 10 ng / mL IFN-γ and 30 ng / mL TNF-α are as Figure 5 shown.

[0121] 3. Determination of the expression level of cell immunosuppressive proteins

[0122] Refer to the method of the intracellular fixation and permeabilization buffer kit (Invitrogen, 88-8824-00) to fix and permeabilize the pre-activated mesenchymal stem cells cultured in Example 1, Example 6, and Comparative Example 1, respectively. At room temperature, the cells were stained with the corresponding antibodies of cell immunosuppressive proteins IDO, TSG6, PTGS2, and TGF-β1, respectively. After washing the cells, the fluorescence intensity of the cells was detected using a flow cytometer.

[0123] The results of the expression levels of mesenchymal stem cell immunosuppressive proteins TSG6, IDO, COX2, and TGF-β1 on solid culture dishes and liquid-liquid interfaces after pre-activation with 10 ng / mL IFN-γ and 30 ng / mL TNF-α are as Figure 6 shown. On the liquid-liquid interface, the use of low-dose cytokine stimulation can effectively improve the immunomodulatory ability of mesenchymal stem cells, and the related immunosuppressive proteins TSG6, IDO, COX2, and TGF-β1 are significantly increased in protein expression.

[0124] 4. T cell proliferation inhibition experiment

[0125] The collected MSCs (i.e., the pre-activated mesenchymal stem cells cultured in Example 1, Example 6, and Comparative Example 1) were seeded into 96-well plates. T cells were labeled with CFSE, and after counting, they were added to the 96-well plates at 10 5 / well to co-culture with MSCs. After 3 days of co-culture, the T cells were collected and labeled with a flow antibody against CD8 + T cells. The proliferation index of each group of cells was analyzed to characterize the immunosuppressive effect of MSCs.

[0126] The results of the inhibition of CD8 + T cell proliferation by mesenchymal stem cells on solid culture dishes and liquid-liquid interfaces after pre-activation with 10 ng / mL IFN-γ and 30 ng / mL TNF-α are as Figure 7 shown. Compared with the traditional solid culture dish, the immunosuppressive function of mesenchymal stem cells on the liquid-liquid interface lasts longer, and significantly inhibits the proliferation of cytotoxic CD8 + T cells after 3 days of co-culture with T cells, showing better functional stability.

[0127] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant art. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of a liquid-liquid interface culture system in improving the immunoregulatory ability of stem cells; in, The liquid-liquid interface culture system comprises an upper aqueous phase and a lower oil phase; The upper aqueous phase comprises a protein solution or a biocompatible polymer solution, and the lower oil phase comprises a fluorinated liquid.

2. The use according to claim 1, characterized in that: The liquid-liquid interface culture system is prepared by the following preparation method: adding a lower oil phase and an upper water phase in a container in sequence, forming a protein nanofilm after incubation, washing the upper water phase with PBS, and replacing the upper water phase with a culture medium to obtain a liquid-liquid interface culture system.

3. The use according to claim 1 or 2, characterized in that: The protein comprises at least one of lysozyme, serum albumin, whey protein, casein, fibronectin, collagen, laminin, type I collagen, fibrinogen, elastin, and vitronectin; and / or, the biocompatible polymer comprises at least one of gelatin, polylysine, amyloid fiber, and casein-chitosan complex; Preferably, the concentration of the protein solution or biocompatible polymer solution is at least 1 mg / mL.

4. The use according to claim 1 or 2, characterized in that: The fluorinated liquid includes at least one of perfluoroalkane, hydrofluoroether, perfluorocyclobutane, perfluoromethylcyclohexane, perfluorodecalin, perfluorooctylpropane, and perfluorooctane sulfonic acid; preferably, the fluorinated liquid includes perfluoroalkane and hydrofluoroether; Preferably, the volume ratio of the perfluoroalkane to the hydrofluoroether is 1:(1-10).

5. A method for improving the immunoregulatory ability of stem cells, comprising the following steps: (1) adding a fluorinated liquid as a lower oil phase into a cell culture container, and then adding a protein solution or a biocompatible polymer solution as an upper aqueous phase, forming a protein nanofilm after incubation, and then washing the upper aqueous phase with PBS, and replacing the upper aqueous phase with a culture medium to obtain a liquid-liquid interface culture system; (2) inoculating stem cells into the upper aqueous phase of the liquid-liquid interface culture system and culturing; (3) Part of the culture medium in the upper aqueous phase is removed, and an equal volume of culture medium containing cytokines is added for culturing.

6. The method according to claim 5, characterized in that The protein comprises at least one of lysozyme, serum albumin, whey protein, casein, fibronectin, collagen, laminin, type I collagen, fibrinogen, elastin, and vitronectin; and / or, the biocompatible polymer comprises gelatin, polylysine, amyloid fiber, and casein-chitosan complex; Preferably, the concentration of the protein solution or biocompatible polymer solution is at least 1 mg / mL.

7. The method according to claim 5, characterized in that The fluorinated liquid includes at least one of perfluoroalkane, hydrofluoroether, perfluorocyclobutane, perfluoromethylcyclohexane, perfluorodecalin, perfluorooctylpropane, and perfluorooctane sulfonic acid; Preferably, the fluorinated liquid includes perfluoroalkane and hydrofluoroether; Preferably, the volume ratio of the perfluoroalkane to the hydrofluoroether is 1:(1-10).

8. The method according to any one of claims 5 to 7, characterized in that: The cytokine includes at least one of interferon, tumor necrosis factor and interleukin; Preferably, the interferon includes at least one of IFN-α, IFN-β, IFN-γ and IFN-ω; Preferably, the tumor necrosis factor includes TNF-α and / or TNF-β; Preferably, the interleukin includes at least one of IL-1β, IL-2, IL-4, IL-6, IL-7, IL-9, IL-10 and IL-12.

9. The method according to any one of claims 5 to 7, characterized in that: The stem cells include at least one of mesenchymal stem cells, neural stem cells, skin stem cells, muscle stem cells, embryonic stem cells, dental pulp stem cells, bone stem cells and hematopoietic stem cells.

10. The method according to claim 9, characterized in that The incubation time in (1) is 1 to 24 h; and / or, the culture conditions in (2) are 35 to 38° C., 4% to 6% CO2 for 2 to 10 h; and / or, the culture time in (3) is 20 to 72 h; and / or, in the culture system of (3), the final concentration of the cytokine is 0.1 to 100 ng / mL.

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