Cell culture and cell line authentication
PBMCs: Whole blood samples collected from research subjects were mixed thoroughly with an equal volume of Hank’s balanced salt solution (HBSS) and then carefully and slowly added to an equal volume of Ficoll Hypaque. After centrifugation at 1731 ×g for 15 minutes, mononuclear cells in the MNC layer were harvested using a drop pipette and mixed thoroughly with an equal volume of HBSS. For cell counting, 10 µL of the cell suspension was mixed with 90 µL of 10× trypan blue, and the remaining cell suspension was centrifuged at 623 ×g for 5 minutes. After discarding the supernatant, the resulting cell pellet was resuspended in Gibco™ AIM V™ Medium and incubated at 37°C with 5% CO2 and 21% O2 .
hUCMSCs: The identity of the MSCs isolated from Wharton’s jelly of the umbilical cord was verified by characterizing their surface markers and functions. hUCMSCs were expanded and cultured prior to cryopreservation. Cells from the second passage were used for subsequent experiments. For culture, hUCMSCs were seeded at an initial density of 3000 cells/cm2 in alpha Minimum Essential Medium (α-MEM; Invitrogen, Life Technologies Corporation, Gaithersburg, MD, USA) containing 5% UltraGRO (Helios Bioscience, AventaCell BioMedical Corporation, Atlanta, GA, USA) and 1% penicillin-streptomycin at 37°C in an incubator (Thermo Scientific, Waltham, MA, USA) with 5% CO2 and 21% O2. TrypLE (Gibco, Life Technologies Corporation, Waltham, MA, USA) was used to harvest cells during subculture .
For authentication, MSCs were immunolabeled with mouse anti-human antibodies against the following antigens: CD34, CD45, CD29, CD31, CD44, CD90, HLA-A, HLA-B, HLA-C, HLA-DR (BD Biosciences, San Jose, CA, USA), CD105 (AbD Serotec, Oxford, UK), CD73, CD117, and CD184 (BD Pharmingen, San Diego, CA, USA)[22, 21]. The immunolabeled cells were incubated with anti-mouse fluorescein isothiocyanate (FITC)- or phycoerythrin-conjugated IgG as the secondary antibodies, followed by flow cytometry analysis (BD Biosciences).
Evaluation of cell growth rate
Harvested cells were diluted in culture medium and mixed thoroughly with an equal volume of trypan blue (1:1) for cell counting.
PBMC suppression assay
Co-culture of hUCMSCs and PBMCs: Before the experiment, the cells were cultured at 37°C with 5% CO2. The PBMCs were divided into the phytohemagglutinin (PHA) and control groups. PHA was added to the former at a final concentration of 5 µg/mL. For co-culture, the PBMCs were divided into two experimental groups, i.e., the PBMC control and hUCMSCs co-culture groups. PBMCs grown in suspension were harvested and co-cultured with stably growing, adherent hCMSCs in Transwell culture plates. Following co-culture, the PBMCs were isolated by centrifugation and subject to cell counting to calculate and compare the number of cells [22, 23].
In co-cultures of exogenous IL-6 and PBMCs, the PBMCs were exposed to different concentrations of exogenous IL-6 (2.5, 5, 25, 50, 100, 250, 500, and 1000 ng/ml), and cell counting was performed on days 0, 1, 2, 4, and 6.
Enzyme-linked immunosorbent assay (ELISA)
IL-6 levels in cell culture supernatant were analyzed using the ELISA MAX™ Deluxe Set Human IL-6 kit (BioLegend, San Diego, CA, USA). After terminating the TMB reaction with stop reagents, the optical density of the reaction mixture was measured at 450 and 570 nm (OD450 and OD570, respectively) within 15 minutes to obtain the experimental result .
Analysis of the effects of IL-6-neutralizing antibody on PBMCs
PBMCs (PHA-stimulated cells): The PBMCs were divided into five groups: the PBMC-control group and four groups of PBMC-UCMSC co-cultures exposed to different concentrations (1000, 316, 100, and 31.6 ng/mL) of IL-6-neutralizing antibody. Adherent cultures of hUCMSCs, as well as cell harvesting and cell counting of PBMCs, were carried out as described above. Then, the PHA-stimulated PBMCs were co-cultured with hUCMSCs in Transwell chambers and exposed to different concentrations (1000, 316, 100, and 31.6 ng/mL) of IL-6 neutralizing antibody for 0, 1, 2, 3, and 4 days. The media in the wells and inserts were mixed and collected for cell counting to observe the growth of cells and determination the IL-6 concentration in the culture medium.
Determination of the effects of tocilizumab (TCZ) on PBMCs
PBMC were divided into four groups. The two groups were treated as control groups without any pretreatment. One group was co-cultured with UCMSC, and the other group was not co-cultured with UCMSC. The commonly used clinical immunosuppressive drug TCZ was selected as the pretreatment of the experimental group. 100 µg tocilizumab (TCZ) was pretreated for 5 hours and 10 hours. At each time point, there is a group co-cultivating with UCMSC. The other group did not add UCMSC to the co-culture. The co-culture of PBMC and UCMSC was prepared and processed as described above, and after 72 hours of culture, the number of PBMC cells was counted.
Cells were lysed in TRI Reagent (Sigma) and shaken with 1-bromo-3-chloropropan (BCP) for 15 seconds prior to centrifugation. After centrifugation, the aqueous layer was mixed gently with isopropanol and allowed to stand. After centrifugation, the resulting RNA pellet was washed with 75% ethanol and centrifuged again. The washed RNA pellet was then air-dried or vacuum-dried and reconstituted with an appropriate amount of DEPC water or 0.5% SDS. The quality of the extracted RNA was assessed based on the A260/A280 ratio, and its concentration was determined based on the A260 value .
Reverse transcription-polymerase chain reaction (RT-PCR) and Polymerase chain reaction (PCR)
Total RNA (3 µl containing >1 μg) was subject to RT-PCR using GoScript™ Reverse Transcriptase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Then, the concentration of the reverse-transcribed cDNA was measured . Primers (shown below) were designed according to the experimental requirements. The concentration of DNA template was adjusted accordingly and added to reaction mixtures containing the following pairs of forward and reverse primers:
IL-6_forward (5’-CTGGATTCAATGAGGAGACTTGC-3’) and IL-6_reverse (5’-GGACAGGTTTCTGACCAGAAG-3’), IL-6R_forward (5’-AAGGACCTCCAGCATCACTGTGTCA-3’) and IL-6R_reverse (5’- CCTTCAGAGCCCGCAGCTTCCACGT-3’), GAPDH_forward (5’-ATCAAGAAGGTGGTGAAGCAGG-3’) and GAPDH_reverse (5’-GCAACTGTGAGGAGGGGAGATT-3’), along with DNA Taq polymerase, PCR buffer, and nucleotides.
The PCR cycling conditions consisted of 30 cycles (for IL-6 and GAPDH) or 35 cycles (for IL-6R) of 95°C for 30 seconds, 55°C for 30 seconds, and 72°C for 30 seconds, to amplify the targeted DNA fragments .
All experiments were repeated three times, and the data are presented as mean ± standard error of the mean (SEM). The inhibitory effect of hUCMSCs is illustrated in graphs. Multiple comparisons were performed using the Kruskal-Wallis test with Bonferroni correction. Reductions in cell proliferation, i.e., the number of PBMCs between the control and experimental groups, were compared using the Wilcoxon rank-sum test. Differences with P values less than 0.05 were considered statistically significant.