Cell Line of M2c Macrophage and Its Applications

Abstract

This invention provides a cell line of M2C macrophage and its applications. The cell line is derived from monocytes isolated from bone marrows and peripheral blood. The monocytes were differentiated into M2 macrophage by macrophage colony-stimulating factor (M-CSF), and then the polarization of M2C macrophage was induced by baicalin. The MERTK, PTX3, and PD-L1 expression level of the M2C macrophage are high and promote phagocytosis. Hence it can be applied to cell therapy or biological agents of immune regulation. Also, the macrophage-conditioned medium and wound dressing prepared on the M2C cell have the effects of enhancing fibroblast proliferation and angiogenesis, which can improve wound healing in medical use, and can be applied to skin care product for skin repair and rejuvenation.

Claims

1. A M2C macrophage cell line called NPUST-M2-1, deposited in CCTCC (China Center for Type Culture Collection) with accession number C2017269, wherein the M2C macrophage cell is derived from monocytes collected from bone marrows or peripheral blood, and differentiated into M2 macrophage using M-CSF (macrophage colony-stimulating factor), then further induced to differentiate into M2C macrophage using baicalin.

2. A method to prepare a M2C macrophage cell line, comprising: Step (a): Collecting monocytes from bone marrow and peripheral blood; Step (b): Using M-CSF to induce M2 macrophage polarization of monocytes; Step (c): Using baicalin to induce M2C macrophage polarization of M2 macrophage.

3. The method of claim 2 wherein the method is used for cell therapy.

4-20. (canceled)

21. The method of claim 3, wherein the cell therapy is used to promote phagocytosis.

22. The method of claim 3, wherein the cell therapy is used to regulate T cell immunity.

23. The method of claim 3, wherein the cell therapy is used to regulate VEGF gene.

24. The method of claim 2, wherein the method is for preparing phagocytosis-promoting cell preparation.

25. The method of claim 2, wherein the method is for preparing T cell immune-regulating cell preparation.

26. The method of claim 2, wherein the method is for preparing VEGF gene-regulating cell preparation, and the VEGF gene is selected from the group of VEGF-A, VEGFR-1 and VEGFR-2.

27. The method of claim 3, wherein the cell therapy can ameliorate mammal autoimmune diseases.

28. The method of claim 2, wherein the method is for preparing cell preparation for amelioration of mammal autoimmune diseases.

29. A baicalin-M2C macrophage-conditioned medium that improves wound healing, which is obtained after culturing baicalin-induced M2C macrophage in basal medium for 4 hours.

30. The baicalin-M2C macrophage-conditioned medium of claim 29, wherein the basal medium is X-VIVO-10.

31. The baicalin-M2C macrophage-conditioned medium of claim 29, wherein the baicalin-induced M2C macrophage can be obtained from incubating M2 macrophage for 24 hours in medium containing 50 M baicalin.

32. The baicalin-M2C macrophage-conditioned medium of claim 29, wherein the baicalin-M2C macrophage-conditioned medium is used to prepare a wound dressing that improves wound healing.

33. The baicalin-M2C macrophage-conditioned medium of claim 32, wherein the substrate of the dressing is polyvinyl alcohol/dextran hydrogel.

34. The baicalin-M2C macrophage-conditioned medium of claim 32, wherein the dressing is used to promote fibroblast proliferation and angiogenesis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG. 1 shows a flow cytometry analysis illustrating the survival rate of thawed M2C macrophage after cryopreservation.

[0062] FIG. 2 shows scattered plot and bar chart of flow cytometry analysis result of macrophage molecular marker CD11b, M2 molecular marker CD206 and M1 molecular marker CD80 (treated by M-CSF).

[0063] FIG. 3 shows scattered plot and bar chart of flow cytometry analysis result of M2 molecular marker CD206, M1 molecular marker CD86. (Treated by LPS, IL-4 and baicalin).

[0064] FIG. 4 is an image of macrophage cell morphology under microscope.

[0065] FIG. 5 illustrates the comparative gene expression level of TNF-Alpha, IRF5, and IRF4 in embodiment 1, comparison 1 and comparison 2.

[0066] FIG. 6 illustrates the comparative gene expression level of Arginase-1, 1110, and IL-6 in embodiment 1, comparison 1 and comparison 2.

[0067] FIG. 7 illustrates the comparative gene expression level of PTX-3, Mertk in embodiment 1, comparison 1 and comparison 2.

[0068] FIG. 8 illustrates the comparative gene expression level of VEGF-A in embodiment 1, comparison 1 and comparison 2.

[0069] FIG. 9 shows comparative gene expression level of VEGF-A, VEGF-B and VEGF-D between M1 and M2 macrophage (Raw264.7 derived) after treating baicalin.

[0070] FIG. 10 shows comparative gene expression level of VEGFR1, VEGFR2 and VEGFR 3 between M1 and M2 macrophage (Raw264.7 derived) after treating baicalin.

[0071] FIG. 11 shows comparative gene expression level of VEGFR1, VEGFR2 and VEGFR 3 between baicalin treated macrophage (Raw264.7 derived) and IL-4 treated macrophage (Raw264.7 derived).

[0072] FIG. 12 shows comparative gene expression level of VEGFR1, VEGFR2 and VEGFR 3 between M1 and M2 macrophage (Raw264.7 derived) after treating baicalin.

[0073] FIG. 13 illustrates comparative VEGF-A protein expression level of macrophage based on ELISA result.

[0074] FIG. 14 illustrates comparative MERTK protein expression level of macrophage based on flow cytometry result.

[0075] FIG. 15 illustrates average MERTK protein expression level of single macrophage cell based on mean fluorescent intensity of flow cytometry result.

[0076] FIG. 16 shows histogram and bar chart showing the percentage of macrophages undergoing phagocytosis based on flow cytometry result.

[0077] FIG. 17 shows histogram and bar chart showing percentage of macrophages undergoing phagocytosis which also express MERTK, based on flow cytometry result.

[0078] FIG. 18 shows microscope and fluorescence microscope images of macrophages phagocytosing FITC-beads.

[0079] FIG. 19 shows percentage of apoptotic cells derived from Raw264.7 cell line under different concentration of H.sub.2O.sub.2.

[0080] FIG. 20 is a scatter plot based on flow cytometry analysis to examine apoptotic cells and efferocytosed cells. P4 region are apoptotic cells obtained after induced by H.sub.2O.sub.2 (1.5 mM) and dyed with FITC-CFSE fluorescent dye; P5 region are differentiated M2C macrophage induced by baicalin and dyed with PE-MERTK; P6 region are macrophage which undergo apoptotic cells efferocytosis and simultaneously express FITC and PE fluorescent light, which means they are macrophages both MERTK-positive and CFSE-positive.

[0081] FIG. 21 shows average CFSE protein expression level and average MERTK protein expression level of single macrophage cell undergo efferocytosis, based on mean fluorescent intensity of flow cytometry result.

[0082] FIG. 22 is a flow cytometry analysis result showing the phenotype of T cell in murine peripheral blood, spleen and bone marrow after injecting M2C macrophage.

[0083] FIG. 23 shows microscopic image of the growth of dermal fibroblasts in IL4-M2 macrophage conditioned medium.

[0084] FIG. 24 shows microscopic image of the growth of dermal fibroblasts in baicalin-M2C macrophage conditioned medium.

[0085] FIG. 25 shows dermal fibroblasts proliferation in IL4-M2 macrophage conditioned medium based on flow cytometer analysis result.

[0086] FIG. 26 shows dermal fibroblasts proliferation in balcalin-M2C macrophage conditioned medium based on flow cytometry analysis result.

[0087] FIG. 27 shows illustration of wound healing of mice after applying dressing which absorbed baicalin-M2C macrophage conditioned medium.

DETAILED DESCRIPTION

[0088] The present invention will be further exemplified by the following examples, which are not to be seen as limiting. The embodiments and description are used for Illustrating the details and effect of the present invention.

[Induced Polarization of M2C Macrophage]

[0089] Use PBS containing 0.5% BSA to flush out bone marrow cells from murine (C57BL/6J, 8-20 weeks, Taiwan National Laboratory Animal Center) femur, then use Ficoll-Hypaque (GE healthcare, SWEDEN) to isolate monocytes.

[0090] Then, incubate 210.sup.6 monocytes in 50 ng/ml M-CSF contained RPMI 1640 (10% FCS) medium under 37 c., 5% CO.sub.2 for seven days to induce M2 macrophage polarization. On the eighth day, collect 210.sup.5 M2 macrophage and incubate them in RPMI 1640 (10% FCS) medium with Baicalin (50 M, Tokyo Chemical Industry, JAPAN) for 24 hours. Baicalin will induce M2 macrophage to differentiate into M2C macrophage.

[Cryopreservation of M2C Macrophage]

[0091] Remove the cell culture medium and wash with PBS for two times. Add 1 ml 0.025% trypsin (Biowest, Missouri, USA) and incubate cell in incubator for cells to stop adhesions. After five minutes incubation, use microscope to confirm that more than 90% of cells have been detached, then immediately transfer the cells to 15 ml centrifuge tube and add into the same volume of RPMI 1640 (10% FCS) medium as trypsin to terminate trypsin reaction. Next, add 4 ml PBS into the petri dish to collect remnant cells and transfer them into the same centrifuge tube. Centrifuge cells at 4 C. for 5 mins at rotating speed of 500g. Meanwhile, add 10% dimethyl sulfoxide (DMSO) (Sigma-Aldrich, USA) into RPMI 1640 (10% FCS) medium to prepare cryopreservation medium. After centrifugation, remove the supernatant and place the cells in 1 ml of the aforementioned cryopreservation medium for suspension and dilution. Next, transfer the cells to cryo tubes (Thermo Fisher, Denmark) and immediately place the cryo tubes into 80 C. freezers filled with 95% ethanol to freeze overnight. After freezing, preserve the frozen cryo tubes in liquid nitrogen tank.

[0092] During thawing process, take out the cells from liquid nitrogen and place it in 37 c. sterilized water bath to defreeze. After thawing cells, transfer cells to a 15 ml centrifuge tube containing 9 ml cold PBS and centrifuge cells at 4 C. for 5 mins at rotating speed of 500g. Next, remove the supernatant and add 10 ml PBS to wash the cells. Centrifuge cells again at 4 C. for 5 mins at rotating speed of 500g and remove the supernatant. Suspend the cells using fresh medium (90% RPMI+10% FCS). Finally, seed the cells in petri dish and incubate cell with condition of 37 C., 5% CO.sub.2.

[0093] The survival percentage of cells after thawing is shown in FIG. 19. Results were analysed by flow cytometry, observed with forward scatter (FSC) and dyed with Propidium Iodide (PI). According to division of the results, P4 population are survival cells, which has 99% of survival rate before cryopreservation and 84% after cryopreservation.

[Preparation of the Cell-Conditioned Medium]

[0094] Use PBS to wash baicalin Induced differentiated M2C macrophages and culture the cells in X-VIVO-10 medium (Lonza Group, Switzerland) for 4 hours. After 4 hours, collect baicalin-M2C macrophage conditioned medium and preserve it at 20 C.

[0095] Moreover, the preparation of IL4-M2 macrophage conditioned medium mentioned in examples below is prepared by replacing baicalin induced differentiated M2C macrophage by IL-4 induced differentiated M2 macrophage. The remaining steps are same as above preparation protocol.

[Preparation of Hydrogel Dressing]

[0096] To produce hydrogels used for absorbing conditioned medium, mix polyvinyl alcohol (PVA, average molecular weight 146,800-186,000, +99% hydrolyzed, Sigma-Aldrich, USA) with dextran (average molecular weight 60,000-90,000) and undergo freeze-thaw (FT) cycle. More specifically, based on the proportion in the chart 1 below, dissolve PVA and dextran in distilled water respectively. Next, mix gentamicin with the PVA/dextran solution in vortex for 1 hour and then pour into petri dishes. Place the petri dishes in 20 C. freezer for 18 hours, and then thaw them at 25 C. for 6 hours. Repeat the freeze-thaw cycle 3 times to produce the polyvinyl alcohol (PVA) hydrogel.

TABLE-US-00001 CHART 1 Ingredient (Proportion in 100 g of hydrogel) g % PVA (g) 2.5 2.5 Dextran (g) 1.13 1.13 Gentamicin (g) 0.1 0.1 Water (mL) 100 100

[Preparation of Hydrocolloids Contains Baicalin-M2C Macrophage-Conditioned Medium]

[0097] Dip hydrogels into balcalin-M2C macrophage-conditioned medium and IL4-M2 macrophage conditioned medium respectively. Shake under 100 rpm for 24 hours then carefully take out and wash with PBS for 5 times.

EXAMPLES

[0098] The examples below are used for Illustrating the objectives and effects of the present invention, which are not to be seen as limiting.

Example 1: [Murine M2C Macrophage Differentiation Induction]

[0099] Embodiment 1 is prepared by the steps described above, isolate monocytes from murine bone marrow and use M-CSF to induce differentiation of M2 macrophage, and then use baicalin to induce M2C macrophage polarization. In comparison 1, incubate murine M2 macrophage in RPMI 1640 (10% FCS) containing LPS (lipopolysaccharide, 1 g/ml, Sigma-Aldrich) for 2 hours. In comparison 2, incubate murine M2 macrophage in medium containing IL-4 (10 Cell Guidance System, Cambridge, UK) for 24 hours. The control group of M2 macrophage is incubated in RPMI 1640 (10% FCS) without any drugs. The conditions are shown below in chart 2.

TABLE-US-00002 CHART 2 Embodiment 1 Comparison 1 Comparison 2 Drugs to induce Baicalin LPS IL-4 differentiation Concentration 50 M 1 g/ml 10 ng/ml Incubation time 24 hours 2 hours 24 hours

[0100] After induced differentiation is complete, use flow cytometry to analyse the macrophage phenotype, in the meantime, also use real time PCR (qPCR) to observe mRNA expression level of each gene.

TNF-Alpha: M1 macrophage expression gene
IRF5: M1 macrophage polarization promoting gene
IRF4: M2 macrophage polarization promoting gene
ARG1 (Arginase-1): M2a macrophage molecular marker
IL-10: cytokine that promotes M2 macrophage polarization and inhibits inflammation
IL-6: cytokine that promotes inflammation and also serves as M2b macrophage molecular marker.
MERTK: a gene that serves as M2C macrophage molecular marker and promotes phagocytosis
PTX3: a gene that serves as M2C macrophage molecular marker and promotes phagocytosis
VEGF-A: vascular endothelial growth factor
PD-L1: a gene that regulates T cell and promotes tissue autoimmune tolerance

[0101] FIG. 24 show results of macrophage phenotypes analysed by flow cytometry. After monocytes were treated with M-CSF, as shown in FIG. 2, the cells that were treated for 7 days have the highest percentage of expressing molecular marker CD11b (common marker for macrophage) and CD206 (common marker for M2 macrophage) at the same time (CD11b+CD206+, *p<0.05). Treated with LPS, IL-4 and baicalin respectively, as shown in FIG. 3, comparison 1 has higher percentage of M1 macrophage (C086+CD206, #p<0.05) and comparison 2 has higher percentage of M2 macrophage (CD86CD206+,*p<0.05). On the contrary, the percentage of cells expressing CD86 and CD206 in embodiment 1 was lower than both comparison 1 and 2 (#p<0.05). Meanwhile, in microscopy image (FIG. 4), unlike the control group treated with M-CSF and M2 macrophages in comparison 2, morphology of macrophages in embodiment 1 (marked by arrow) appeared to be ellipse instead of spindle shaped. This result shows that the phenotype and morphology characteristics of baicalin induced macrophages were different from typical M2 macrophages.

[0102] The mRNA expression level in each gene is shown in FIG. 5FIG. 8. FIG. 5 shows that in embodiment 1, the expression level of TNF-Alpha, IRF5 gene that promotes M1 macrophage polarization are significantly lower than comparison 1 (#p<0.05), while the expression level of gene IRF4 that promotes M2 macrophage polarization is also significantly lower than comparison 2 (*p<0.05). Furthermore, as shown in FIG. 6, ARG1 expression level in embodiment 1 was significantly lower than comparison 2 (*p<0.05), IL-6 expression level was also low, but the IL-10 expression level was significantly higher than comparison 1 and 2 (*: compared to control group. #: compared to LPS treated group. : compared to IL-4 treated group. P<0.05). Meanwhile, as shown in FIG. 7 and FIG. 8, expression level of VEGF-A, PD-L1, MERTK, PTX3 were all significantly higher than control, comparison 1 and 2 (*: compared to control group. #: compared to LPS treated group. : compared to IL-4 treated group. P<0.05).

[0103] This result shows that comparing to the drugs treatment in each comparison, M2 macrophages treated with baicalin have high expression level of phagocytosis related genes MERTK and PTX3, which can promote phagocytosis in immune system. Also, the showed high expression level of PD-L1 can help regulate T-cell immune system, promote tissue autoimmune tolerance and further be applied in cell therapy.

[0104] In the meantime, anti-inflammatory gene IL-10 and vascular endothelia growth factor (VEGH-A) expression levels of M2 macrophages treated with baicalin are significantly higher, whereas IL-6 expression level is significantly lower than comparison 1 (p<0.05). IL-6 is a proinflammatory cytokine that can promote inflammation and induce M1 polarization, whereas IL-10 is an anti-inflammatory cytokine that induce M2 polarization. Moreover, VEGF-A is vascular endothelial growth factor that is highly associated with wound healing, which can promote angiogenesis and hence the regenerated blood vessels can provide necessary oxygen and nutrition for wound healing process. Therefore, M2 macrophages treated with baicalin can promote angiogenesis and inhibit inflammation during wound healing process.

Example 2: Induction of VEGF Family and PD-L1 Gene Expression in Murine Macrophage

[0105] In this example, experiment was performed to observe if baicalin can induce expression of VEGF family and PD-L1 in M1 and M2 macrophage derived from Raw264.7 cell line (ATCC, USA). M1 macrophage obtained from treating Raw264.7 macrophage with LPS (1 g/ml, Sigma-Aldrich) for 2 hours, and M0 macrophage (control group) cultured in simple RPMI 1640 (10% FCS) medium, are respectively cultured in RPMI 1640 (10% FCS) medium with either Baicalin (50 M, Tokyo Chemical Industry, Japan) or IL-40 (10 ng/ml, Cell Guidance System, Cambridge, UK). Collect the cells after 24/48/72 hours of cell culturing. Then observe the mRNA expression level of VEGF family, VEGF receptor and PD-L1 using reverse transcription-real time PCR (RT-qPCR).

[0106] The mRNA expression level of VEGF family and VEGF receptor are shown in FIG. 9FIG. 11. FIG. 9 illustrates that all the groups treated with baicalin has higher level of VEGF-A than M1, M2 macrophages without balcalin treatment (LPS, control). Expression levels of different VEGF receptors are shown in FIG. 10, VEGFR-1 and VEGFR-2 expression levels are higher in those treated with baicalin (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group). Furthermore, when comparing baicalin treated group and IL-4 treated group with the same treatment duration, as shown in FIG. 11, VEGF-A, VEGFR1, VEGFR2 mRNA expression levels were all significantly higher in baicalin treated group than IL-4 treated group (: compared to IL-4 treated group, p<0.05).

[0107] Results show that even for the differentiated M1 macrophages, baicalin can still promote M2C polarization and enhance VEGF-A, VEGF receptor expression level effectively greater than IL-4. Meanwhile, based on the enhancement of VEGF receptor expression, the baicalin induced differentiated M2C macrophages can perform VEGF gene autocrine regulation.

[0108] Moreover, expression level of PD-L1 shown in FIG. 12 indicates that all the groups treated with balcalin have higher PD-L1 expression levels than M1, M2 macrophages without balcalin treatment (*: compared to control group, #: compared to LPS treated group, p<0.05). This result shows that M2 macrophages (derived from Raw264.7 cell line) treated with baicalin can participate in T cell immune regulation, balance immune tolerance regulation and further be used in cell therapy.

Example 3: VEGF-A Protein Secretion Promoted by Baicalin in Macrophage

[0109] In this example, experiment was performed to further examine whether M2C cells treated with baicalin will express VEGF-A protein. Add LPS (1 g/ml), baicalin (50 M) and IL-4 (10 ng/ml) respectively to medium culturing 510; Raw264.7 macrophages (ATCC, USA) and culture for 48 hours. Collect the conditioned medium and quantify VEGF-A protein by EUSA (Mouse VEGF EUSA Kit, Code: EK0541, BosterBio, CA, USA). Control group is cultured in the medium (90% DMEM+10% FBS) without any mentioned drugs. Result is shown in FIG. 13, the VEGF-A protein detected in baicalin-M2C macrophage conditioned medium is higher than control group and other groups treated with other drugs (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group, p<0.05). M2C macrophage treated with baicalin can promote macrophage to secrete VEGF-A protein and the efficacy is greater than IL-4.

Example 4: MERTK Protein Secretion Promoted by Baicalin in Macrophages

[0110] In this example, experiment was performed to further test whether M2C treated by baicalin can express MERTK protein. Using flow cytometry to evaluate macrophage surface protein MERTK expression level in embodiment 1, comparison 1, 2, and the control group is cultured in RPMI 1640 (10% FCS) without any drugs. Result was shown in FIG. 14, after fluorescence staining, fluorescence MERTK marker binding was detected in approximately 80% of cells in every group. However, if use the mean fluorescence intensity (MFI) to calculate the average MERTK expression level in one single cell, as shown in FIG. 1S, M2C macrophages treated with baicalin has significantly two times higher MERTK expression level than other groups (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group, p<0.05). M2C cells treated with baicalin can promote macrophages to express MERTK protein, and the efficacy is greater than LPS and IL-4.

Example 5: Phagocytosis Promoted by Baicalin in Macrophages

[0111] In this example, experiment was performed to evaluate the effect of baicalin induced differentiated M2C macrophages in promoting phagocytosis. Treatment conditions of drugs in each group is listed in chart 2, and control group is cultured in RPMI1640 (10% FCS) without any drugs. After treatment, collect 510.sup.5 treated cells respectively and add 50 l FITC-Latex beads (Phagocytosis assay kit (IgG FITC), Cayman Chemical, USA.) to perform phagocytosis experiment for 24 hours, and then use PE-anti-MERTK antibody to mark the cells and observe the fluorescence expression with flow cytometry.

[0112] FIG. 16 shows the percentage of macrophages phagocytising beads in each group, whereas the blue area indicates the portion of cells that phagocytised the beads. In the group treated by baicalin, approximately 30% of cells phagocytosed the beads and hence fluorescence was detected in flow cytometry. This result was significantly higher than other groups (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group, p<0.05).

[0113] Furthermore, observe the percentage of cells that phagocytosed beads and also express MERTK protein in each group. FIG. 17 showed that 30% of cells treated with baicalin simultaneously express the fluorescence from stained beads and MERTK protein. The result indicates that the group treated with baicalin, most cells that expressed MERTK protein had phagocytosed beads, which means that MERTK protein expression is highly associated with phagocytosis. In other groups, the percentage of MERTK protein expressing cells that phagocytised beads was significantly lower than cells treated with baicalin (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group, p<0.05).

[0114] In addition, FIG. 18 shows the fluorescence distribution of cells marked with PE-MERTK after phagocytosing FITC-Latex beads. Baicalin treated macrophages that phagocytosed beads express MERTK on cell membrane, showing red fluorescence light circulated around the cells. On the contrary, MERTK expressed comparatively weaker in group treated with LPS.

[0115] Previous studies have shown that LPS can increase phagocytosis capacity by activating the Toll-like receptor-4 on M1 macrophage surface. However, in this example, it is demonstrated that the macrophages which was differentiated by baicalin induction, promoted phagocytosis by enhancing the expression level of MERTK on cell membrane. This new revelation is firstly obtained by the inventor with combination of aforementioned experiments.

Example 6: Efferocytosis Promoted by Baicalin in Macrophages

[0116] In this example, experiment was performed to evaluate the effect of baicalin induced differentiated M2C macrophages on apoptotic cells efferocytosis process, detailed steps are described below.

[0117] Inducing apoptotic cells: minor changes were made from steps of obtaining apoptotic cells based on previous studies (Piao et al., 2011 and Fong et al 2007), detailed steps comprising: culture 1.510.sup.6 RAW264.7 macrophages with DMEM medium containing 10% FBS in incubator (37 C., 5% CO.sub.2). After culturing overnight, treat with 0.5, 1, 1.5 mM H.sub.2O.sub.2 respectively in DMEM for 12 hours. Next, remove the DMEM medium containing H.sub.2O.sub.2 and wash the cells with PBS buffer, then use micropipette to transfer cells to 15 ml centrifuge tube and centrifuge cells at 4 C. for 5 mins at rotating speed of 500g. Remove the supernatant and resuspend the cells with 95 l P85. Following that, dye the cells with fluorescence marker annexin-V and 7-AAD (BD Bioscience) for 20 mins on ice, avoiding light. After marking the cells, use flow cytometry (FACS BD ARIA II) to detect fluorescence performance. Analysed by software CellQuest (BD Bioscience). If the cells were positive for annexin-V and negative for 7-AAD, the cells will be identified as early apoptotic cells; if the cells were positive for annexin-V and positive for 7-AAD, the cells would be identified as late apoptotic cells. Analytical results in FIG. 19 showed that group treated with 1.5 mM H.sub.2O.sub.2 have a higher percentage of apoptotic cells.

[0118] Efficacy evaluation of apoptotic cells efferocytosis in macrophages: method of the evaluation for effectiveness of apoptotic cells efferocytosis in M2 macrophage induced differentiated by different drugs were slightly modified based on previous studies (Li et al., Yancey et al., and Jehle et al; Liu st al and Lilis at al 2008). Detail steps are described below. Use 3 M CFSE Cell Trace (Thermofisher, USA) to mark RAW264.7 macrophage cell line for 20 minutes under 37 C., then wash with PBS. Culture cells for 24 hours, then use the 1.5 mM H.sub.2O.sub.2 following the aforementioned steps to induce RAW264.7 macrophage cell to differentiate into apoptotic cells. Next, in order to perform efferocytosis, culture both obtained apoptotic cells and M2 cell line from murine bone marrow treated under different drugs (Table.1), with a ratio of apoptotic cells to M2 cell line as 3:2 for 2 hours under 37 C., 5% CO.sub.2. After culturing for 2 hours, wash with PBS and remove unattached cells, then use Trypsin-EDTA to collect attached M2 macrophages. Following that, use PE-anti-MERTK antibody (BioLegend, USA) to mark M2 macrophages while each reaction requires 0.25 ug antibody. Lastly, use flow cytometry (FACS BD ARIA II, BD Bioscience, USA) to examine fluorescence performance, if both PE-MERTK and CFSE appear positive, the cell will be identified as the M2C macrophages that efferocytosed the aforementioned apoptotic cells.

[0119] Results of fluorescence expression of stained apoptotic cells, macrophages and efferocytotic macrophages detected by flow cytometry are shown in FIG. 20. Apoptotic cells induced by H.sub.2O.sub.2 (1.5 mM) and express FITC fluorescence after FITC-CFSE staining are distributed in P4 region. Differentiated M2 macrophage expresses MERTK are detected by the expressed PE fluorescence after PE-MERTK staining, which is distributed in P5 region. Macrophages that efferocytosed apoptotic cells are MERTK positive and CFSE positive, which are distributed in P6 region.

[0120] After calculating the number of cells in each region, calculate mean fluorescence intensity (MFI) to evaluate average CFSE expression level of cells expressing MERTK, and also evaluate average MERTK expression level of cells expressing CFSE. FIG. 21 shows that the mean fluorescence intensity of MERTK and CSFE of single M2 macrophage that were differentiated by baicalin induction is significantly higher than other drug treated groups and control group (*: compared to control group, #: compared to LPS treated group, : compared to IL-4 treated group, p<0.05). M2C macrophages treated with baicalin can promote macrophages to express MERTK protein, and the efficacy is greater than LPS and IL-4. Therefore, this example further proves that the M2C macrophages differentiated by baicalin induction can promote apoptotic cells efferocytosis by enhancing the expression level of MERTK on cell membrane. The efficacy is significantly higher than macrophages differentiated by IL-4 and LPS drugs induction.

Example 7: Murine T Cell Immune System Regulation by M2C Macrophage Cell Line

[0121] In this example, in vivo experiment in murine was performed to examine the effect of M2C cell line of present invention on T cell immune regulation. Detailed steps are described below.

[0122] 510.sup.5 Raw 264.7 cells (ATCCTIB-71) were seeded into a 35 mm culture dish (Corning). After incubating overnight, Raw 264.7 cells were induced with 100 uM Baicalin (TCI, Japan) for 48 hours and incubated at 37 C., 5% CO2. Using Flow cytometry (80 FACSAria II: BD Biocsiences, San Jose, USA) to characterized M2 subtypes with anti-CD11b+(macrophage maker), anti-PD-L1+, and anti-MerTK+ antibodies (Biolegend, San Diego, Calif., USA).

[0123] 912-weeks-old female C57BL/6 NarI mice were randomly divided into 2 groups: For M2 macrophages treatment group, the mice were transplanted with 110.sup.6/100 l (cells/volume) RAW 264.7 cells derived M2 macrophage induced by baicalin, using retro-orbital injection on day 0, day 7 and day 14. In parallel for PBS placebo group, 100 l PBS (placebo) was injected. The mice were sacrificed on day 19 after transplantation and monocytes from peripheral blood, spleen and bone marrow were collected for T-cell composition analysis by flow cytometry.

[0124] The whole spleen was gently squeezed by glass grinder and the dissociation of cells was filtered through a 40 m cell strainer (BO Biosciences, San Jose, USA). 50 l heparinized blood were lysed with 3 ml RBC lysis buffer (37 C., 15 min) to remove the red blood cells. Total bone marrow cells were collected from single left femur of mice by flushing with 1 ml PBS twice through 25 G syringe.

[0125] The monocytes of spleen, peripheral blood and bone marrow were then washed and resuspended with PBS. 110.sup.6 cells were incubated on ice with fluorescein isothiocyanate (FITC-CO25), allophycocyanine (APC-CD4), or phycoerythrin (PE-CD8) antibodies up to total volume of 100l in polystyrene tube. CD4, CD8 and CD25 T-cells composition were analysed by BD FACSAria II flow cytometer (BD Biosciences, San Jose, USA). The data analysed by flow cytometry was analysed by statistical software GraphPad Prism 7 (GraphPad) to determine if it is significantly significant between two treated groups (p<0.05).

[0126] Results shown as FIG. 22, after injecting M2C cell line in the experimental group (treated with M2C macrophage), the T cells expressing CD4 or CD8 in peripheral blood were significantly lower than the placebo control group, whereas it was higher in spleen and bone marrow. Owing to the main phenotype of cytotoxic T cell is CD8.sup.+ and the main phenotype of regulatory/suppressor T cell is CD4.sup.+CD25.sup.+, this example further proves that after injecting the M2C cell line of this invention for in vivo cell therapy, it can inhibit the cytotoxic T cell function in peripheral blood, increase regulatory T cells in bone marrow/spleen and promote tissue autoimmune tolerance.

Example 8: Evaluation of Dermal Fibroblasts Proliferation Promotion by Baicalin-M2C Macrophage Conditioned Medium

[0127] After euthanizing the mice, collect skin tissue from infra-axillary of mice and culture the tissue fragments in 37.sup.0c in DMEM/F12 medium (contains 15% FBS 1 antibiotic/antimycotic) containing digestive enzyme Uberase Blendzyme 3 (0.14 Wunsch units/mL, Roche, Switzerland). Wait until the fluid becomes cloudy and edges of the pieces become fuzzy, then break up the tissue cluster by pipetting continuously and terminate digestive enzyme process in 37 c. DMEM/F12 complete medium. Next, centrifuge mixture under 524g for 5 minutes and remove the supernatant. Resuspend mixture using 37 c. DMEM/F12 complete medium and repeat this cycle more than once to remove the remained digestive enzyme. After removing digestive enzyme, transfer mixture to tissue culture petri dish with DMEM/F12 complete medium and incubate it in incubator under 37 c., 5% CO.sub.2, 3% O.sub.2 r. Since dermal fibroblasts starts to detach tissue fragments after 25 days and attach to petri dish, it is necessary to observe its attachment condition daily and replace the complete medium when the cell is overgrowing. After confirming all the live dermal fibroblasts have deviated from tissue fragments, dispose the old medium including tissue fragments, and seed the cells by 510.sup.5 cells/plate in new petri dish containing EMEM complete medium (including 15% FBS, 1 Penicillin/Streptomycin, non-essential amino acids, and sodium pyruvate). Because EMEM medium only supplies dermal fibroblasts growth, all other cell types will either cease or stop proliferating.

[0128] Seeding for few generations and collect the 5.sup.th or 6.sup.th generation dermal fibroblasts. Culture cells respectively in baicalin-M2C macrophage conditioned medium and IL-4 M2 macrophage conditioned medium with cell density of 1.510.sup.4 cell/cm.sup.2. Observe cell proliferation condition after incubating 24, 48, 72 hours.

[0129] Cell proliferation is evaluated by observing the CFSE fluorescence reduction and further determining cell doubling time. Detailed steps are described below. Resuspend cells with PBS to obtain cell with cell concentration of 110.sup.6 cell/ml. Add Cell Trace CFSE solution (final concentration 10 M/ml, Thermo Fisher Scientific, USA) and culture it in 37 c. incubator for 10 minutes for staining. Next, cease CFSE activity with complete medium addition (5 times the volume of CFSE). Stand on ice 5 minutes for reaction, then centrifuge, (500g, 5 mins), and rinse the cells with fresh complete medium after suspension. Repeat this centrifuge-suspension process 23 times to completely rinse CFSE, then use flow cytometer to analyse the regression of fluorescent cells.

[0130] Cell growth condition are shown in FIG. 23 and FIG. 24, after culturing for 48 hours, the murine dermal fibroblasts concentration was higher in baicalin-M2C macrophage conditioned medium comparing to IL4-M2 macrophage conditioned medium. Furthermore, according to results from flow cytometer analysis shown in FIG. 25 and FIG. 26, the doubling time of murine dermal fibroblasts in baicallin-M2C macrophage conditioned medium was 53.3 hours while it was 60.07 hours in IL4-M2 macrophage conditioned medium. This result showed that the baicalin-M2C macrophage conditioned medium prepared on baicalin induced differentiated M2C macrophages can effectively enhance dermal fibroblasts proliferation.

Example 9: Evaluation of Wound Healing Improvement by Baicalin-M2C Macrophage Conditioned Medium

[0131] Use mice (C57BL/6J, 8-20 weeks, Taiwan National Laboratory Animal Center) as experiment model, and anesthetized them with Zoletil 50 (Virbac, France) by intraperitoneal injections. After confirming mice have been anesthetized, remove the hair on back of mice and establish 2 full thickness skin wounds (1.51.5 cm.sup.2) by excision. Disinfect the wound by 70% ethanol, and then use baicalin-M2C macrophage conditioned medium hydrogels and IL4-M2 macrophage conditioned medium hydrogels respectively as wound dressing to cover the wounds.

[0132] Fix the wound with micropore (Soft Cloth Tape, 3M, USA) and feed the mice in their individual cages. At given point in time (After 3, 6, 9, 12, 15 days), observe the wound using digital camera and estimate the wound by Adobe Acrobat 7 Program to calculate the comparative wound healing percentage.

[0133] Calculation of comparative wound healing percentages shown below:

Comparative wound reduction percentage (%)=[(A.sub.0A.sub.t)/A.sub.0]100

A.sub.0: initial wound size.
A.sub.1: wound size after period of time t.

[0134] The results after applying wound dressing is shown in FIG. 27, except the measurements on day 9, baicalin-M2C macrophage-conditioned medium hydrogel has greater efficacy of wound healing than IL4-M2 macrophage-conditioned medium hydrogel at any other given point in time.

[0135] According to the aforementioned examples and results, it is demonstrate that the baicalin induced differentiated M2C macrophage NPUST-M2-1 in this invention has higher MERTK and PTX3 expression level, which can promote phagocytosis such as efferocytosis and further reduce inflammation caused by accumulation of apoptotic cells. Meanwhile, the PD-L1 expression level of M2C macrophage in this invention is also high, which can inactive the cytotoxic T cells in peripheral blood, increase regulatory T cells in bone marrow and spleen, promote tissue autoimmune tolerance and hence help to maintain immune tolerance balance. Moreover, the expression level of VEGF-A and its receptor VEGFR-1, VEGFR-2 of M2C macrophage in this invention is also high, which consequently promote angiogenesis and other functions of VEGF family by autocrine regulation mechanism.

[0136] Therefore, the M2C macrophage and polarization induction method by balcalin in this invention is proved to have multiple efficacies and can be applied in cell therapy and other related biopharmaceutical to improve chronic inflammation and autoimmune diseases. In addition, these cell therapies can be applied in human and multiple species in mammals. This invention can be applied in cell therapy, preventive healthcare of specific diseases and many other medical uses. Inventions as such have never been revealed in this technical field, hence the present invention is novel and innovative.

[0137] Also, the baicalin-M2C macrophage-conditioned medium in this invention can promote function such as fibroblast proliferation and angiogenesis. The wound dressing containing baicalin-M2C macrophage-conditioned medium prepared from this invention can certainly improve wound healing. It can be used in medical field as well as in skin care products which requires functions such as repair or regenerate. Such technical means mentioned above are not seen in any other invention of this technical field. This invention is novel and innovative.

[0138] The above terms and explanations are included but not limited to demonstrate embodiments of the invention. Accordingly, this invention includes all embodiments, modifications and variations that contain technical features of the present invention without departing from the spirit and scope of the invention, and the scope thereof is determined by the appended claims.