Macrophage Inflammation Model in vitro
RAW 264.7 murine macrophage-like cell line was purchased from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). Cells were cultured in petri dishes (Thermo Fisher Scientific, LabServ, China) using Dulbecco’s modified Eagle medium with high glucose (H-DMEM, Gibco, USA), supplemented with 10% heat-inactivated FBS and 100 U/mL penicillin/streptomycin in a humidified air with 5% CO2 at 37 ℃. The medium was replaced after every 2 days until cells reached 70-80% confluency. An inflammatory model in vitro was set up by using lipopolysaccharide (LPS, Merck KGaA, Darmstadt, Germany) to induce RAW 264.7 cells at a concentration of 100 ng/mL (denoted as LPS group).
Culture of hUCMSCs
For 2 D culture, the hUCMSCs were seeded at a density of 5×104 cells / mL in a T-75 cell culture flask (Scheme 1) and the medium was replaced every three days. hUCMSCs on the third passage (P3) were adopted for the subsequent experiments. All the numbers and diameter of cells were counted by the automatic counting cell analyzer (CountStar®Regel S3, Ruiyu, China).
For 3 D suspension culture, 1.5 L bioreactors (Guoqiang, China) was used in this study, equipped with dissolved oxygen (DO) probe (Mettler, Toledo) and a pH probe (Mettler, Toledo). The vessels were filled with α-MEM medium and 10% FBS ( 600 mL), hUCMSCs (1.35 × 105 cells/mL) and Cytodex3 microcarrier (1.8 g, 10 cells/bead). The cells were then inoculated at 37 ℃ with air and 40% DO by bottomspace gassing. The reactors was agitated at 55 rpm for 120 hours to provide the 3 D suspension cultured cells.
The P3 hUCMSCs were stained with fluorescent-labeled monoclonal antibodies, including positive markers (APC CD105, FITC CD73 and FITC CD90) and negative ones (FITC CD34, FITC CD45 and FITC HLA-DR). All the markers were purchased from BD Biosciences (USA). IgG was used as an isotype control and cultured under the same condition. The stained cells were sorted into defined populations using fluorescent-activated cell sorting (FACS, Beckman Coulter, CA, USA) to produce a pure population with the appropriate cell marker profile.
Multilineage Differentiation Assays
Adipogenesis. Cells were seeded in 6-well plates at 2.0×105 per well and cultured in the presence of adipogenic supplements, consisting of α-MEM, 10% FBS, dexamethasone (1 µM), insulin (10 µg/ mL), L-ascorbic acid 2-phospate (100 µg/mL), 3-isobutyl-1-methyl-xantine (0.5 mM) and indomethacin (20 uM). On day 14, cells were stained with oil red O (Merck KGaA, Germany) at room temperature for 15 minutes, and the cells were visualized under Invitrogen EVOS FL Auto Cell Imaging System.
Osteogenesis. Cells were seeded in 6-well plates at 2.0×105 per well and cultured in growth medium to 80-90% confluency. After that, the medium was replaced with osteogenic induction medium, consisting of H-DMEM medium, L-ascorbic acid 2-phospate (50 µg/mL), sodium β-glycerophosphate (10 mM) and dexamethasone (100 nM). Moreover, the fresh differentiation medium was replaced every 2-3 days for three weeks. Then the cells were fixed in 4% paraformaldehyde for 30 min at 4 ℃ and the calcium nodules were stained by 0.1% Alizarin Red at 37 ℃ for 30 minutes.
Preparation of Conditioned Medium Derived from hUCMSCs
Six-well transwell co-culture system (Corning, USA) with 8 µm pore filters was used to co-culture RAW 264.7 cells and the hUCMSCs, where RAW-264.7 cells (1.0×106) and hUCMSCs (1.0×105) were seeded into the upper and lower units of the chamber, respectively. The control group was added only with RAW 264.7 cells (1.0×106) to the upper chamber. When the hUCMSCs reached 8090% confluency, they were washed three times with PBS and the medium was replaced with serum-free α-MEM medium. After further culturing for 48 h, the conditioned medium (2 D hUCMSCs©) was collected by centrifuge (2000 g for 10 min at 4 ℃) and stored at -80°C until usage. A similar method was used to obtain 3 D hUCMSCs©.
Cell Viability Assay
Cell viability was tested using MTT assay. Firstly, RAW 264.7 cells were seeded in a 96-well plate at a density of 1.0×104 cells per well. After incubation for 12 h to allow cells to attach on the wall, the growth medium was removed and the cells were washed 3 times with PBS. Then fresh growth medium and the conditioned medium were added. After further culturing for 24 h, MTT (5 mg/mL, 20 µL) was added and incubated for 4 h at 37 ℃ in a humidified atmosphere with 5% CO2. MTT was removed and dimethyl sulfoxide (150 µL) was added to dissolve the formazan crystals. The absorbance of colored solution was recorded at 570 nm to calculate the cell viability using a standard method described in the previous report .
Cell Apoptosis Assay
Cell apoptosis of RAW 264.7 cells was conducted by flow cytometry according to the manufacturer’s instructions of Annexin V-FITC Apoptosis Detection Kit (Dojindo, Tokyo, Japan). RAW 264.7 cells were firstly seeded in a 6-well plate (5.0×105 cells per well). After 24 h incubation, the cells were harvested and washed twice with iced PBS. After removing supernatants, the cells were further treated with trypsin for the appropriate time and terminated with the culture medium. Cells were pelleted by centrifugation (1000 rpm, 3 min) and then washed twice. 10X diluted Annexin V Solution was added to re-suspend the cells (cell concentration of 1.0×106 cells/mL). Annexin V (5 µL), FITC conjugate and PI solution (5 µL) were respectively added to cell suspension (100 µL) and then the mixed solution was incubated for 15 min free of light. Further dilution using Annexin V solution (400 µL) was required before flow cytometry analysis (Accuri C6,BD Biosciences, USA).
Measurement of Nitric Oxide (NO) Production
RAW 264.7 cells were seeded in 96-well culture plates and incubated for 24 h, then the nitric oxide (NO) concentration in the supernatants of cultured RAW 264.7 cells was measured using a NO assay kit (Beyotime Institute of Biotechnology, China). The concentration of NO was obtained by reading the absorbance value at 540 nm, where sodium nitrite (NaNO2) was used as an external standard.
Total RNA Extraction and Quantitative RT-PCR Analysis
Total RNA was extracted from cells using a Total RNA Kit (Invitrogen™ TRIzol™, Thermo, USA) according to the manufacturer’s instructions. Then cDNA was synthesized with PrimeScript™ RT reagent Kit (Takara, Japan). Real-time PCR was performed with SYBR Premix Ex Taq™ II (Tli RNaseH Plus) Kit (TAKARA, Japan) in the CFX96 touch qPCR system (Bio-Rad, USA). The primer sense and antisense sequences are listed in Table 1. The 2−ΔΔCq method was used to calculate the relative expression level for each gene  and normalized to the internal control Housekeeping β-actin gene.
Enzyme-linked Immunosorbent Assay (ELISA)
The concentration of IL-6 was measured via a mouse IL-6 ELISA Kit (R&D Systems, Minneapolis, MN, USA) in the supernatants obtained from RAW 264.7 cells. Absorbance at 450 nm was read on a microplate reader (BioTek, Winooski, Vermont, USA). All the experiments were performed in triplicate and the average value was used to minimize errors. Following the similar method, the concentrations of other cytokines were respectively evaluated.
Western Blotting Analysis
The cytoplasmic protein of RAW 264.7 cells was extracted using a Cytoplasmic Protein Extraction Kit (Beyotime, China). After the extraction, the protein concentrations were determined by Bicinchoninic acid Protein Assay. Then western blot analysis was carried out by applying polyclonal rabbit antibodies such as Erk MAPK, phospho-Erk MAPK, p38 MAPK, phospho-38 MAPK, JNK MAPK, phospho-JNK MAPK, STAT3, phospho-STAT3 and β-actin antibodies. All these antibodies were purchased from Cell Signaling Technology Inc (MA, USA). The secondary antibodies were bought from Signalway Antibody (Signalway Antibody LLC, USA). Protein bands were separated by SDS-polyacrylamide gel electrophoresis (SDS-Page), and the SDS-Page was electroblotted onto PVDF membranes (GE Healthcare, Chicago, IL, USA) and visualized by ECL detection kits (Beyotime, China). Western blot quantification was achieved with Image J software.
ALI Mice Model
Male BLAB-c mice with a bodyweight of ~ 20 g (x5 per group) were employed and the acute liver injury mice model was established by continuously CCl4 injection via tail vein. The serum ALT and AST levels in the CCl4-induced mice were assessed to ensure the ALI mice model. After induction for 24 h, the hUCMSCs or the conditioned medium was transplanted into the ALI mice by tail vein. mRNA or cytokines in serum were respectively assessed according to the aforementioned methods.
All data are representative experiments performed in three independent experiments. Data were expressed as mean value ± standard deviation (SD). The statistical significance of the mean values was compared by one-way ANOVA (analysis of variance) and Student's t-test using GraphPad Prism version 8.0.2 software (GraphPad Software Inc., San Diego, USA). *p < 0.05 vs. control group, **p < 0.01 vs. control group, ***p < 0.001 vs. control group; ##p < 0.01, ###p < 0.001 vs. control group.