One-way and two-way MLR assays were used to determine the immunogenic properties of ASCs after cell isolation and expansion in different culture conditions:
FBS, HS and chemically defined XF/SF conditions. The MLR assay was used to obtain information with respect to the immunogenic and suppressive properties of ASCs. Also the signaling protein secretion of ASCs was characterized through MLR assay. HS (PAA Laboratories) medium (10%) was chosen to serve as a constant environment for MLR cultures because of significantly decreased proliferation and metabolic activity of PBMCs when cultured in XF/SF condition (Figure 8).
Therefore, prior to MLR assays, ASCs isolated and expanded in three different culture conditions, FBS, HS (Lonza), and XF/SF conditions, received the same treatment of medium change and were allowed to adjust in HS medium (PAA Laboratories) for 24 hours prior to co-culture. The same five patient ASC lines were used for MLR assays and for flow cytometric analyses, whereas the protein secretion studies were performed with four different ASC lines.
Figure 8. The proliferation of PBMCs under different culture media (FBS, HS and XF/SF conditions) at 1 and 5 day time points, assessed by the PreMix WST-1 Cell Proliferation Assay. Significantly lower proliferation of PBMCs was observed under XF/SF conditions as compared to FBS- and HS- supplemented media at day 5.
0 0,1 0,2 0,3 0,4 0,5
FBS HS XF/SF FBS HS XF/SF
DAY 1 DAY 5
Relative cell proliferation
Proliferation of PBMCs
4.7.1 One-way mixed lymphocyte reaction (MLR) immunogenicity assay One-way MLRs were performed as described previously by McIntosh et al.
(McIntosh, 2011). PBMCs, acting as responder cells, were seeded on 96-well plates at cell density of 2.5x105 cells/cm2. Three different donor PBMCs were used and co- cultured with three different stimulator populations: 1) autologous PBMCs (baseline control response), 2) allogeneic BPMCs (positive control response), both plated at 1.0x104 cells per well and 3) the ASC test population, plated at 0.5x104, 1.0x104 and 2.0x104 cells per well (Figure 9). Stimulator PBMCs and test ASCs were irradiated with γ-rays (40 Gy) prior to the co-culture to inhibit the proliferation of the stimulator cells. ASCs in medium alone were plated as control cultures. In addition, control cultures of PBMCs alone were added as well as PBMCs supplemented with mitogen phytohemagglutinin (PHA, 1 mg/mL) to activate the responder PBMC lines to serve as a maximal positive control response. Quadruplicate reactions were performed from each treatment, and the cultures were incubated at 37° C in 5%
CO2 for 5 days in HS medium (PAA Laboratories).
Figure 9. Schematic illustration of one-way MLR assay. Autologous PBMCs (blue) represent a baseline control response, allogeneic BPMCs (red) a positive control response and ASCs (yellow) a test population in one-way MLR assay. PBMCs, peripheral blood mononuclear cells; ASCs, adipose stem cells.
4.7.2 Two-way MLR immunosuppression assay
Two-way MLRs were also performed as described previously by McIntosh et al.
(McIntosh, 2011). Two different MLR combinations were formed from three different responder PBMCs based on their cross-reactivity and HLA dissimilarities that were observed during pre-tests (data not shown). For each MLR combination, cells from two different donors were mixed in equal amounts to activate the proliferative response of each PBMC line. A total number of 2.5x105 PBMCs were seeded per well on 96-well plates. After initiating the MLRs, test ASCs were added to the reactions at cell densities of 0.5x104, 1.0x104 or 2.0x104 cells per well (Figure 10). Control wells containing only MLR combinations without ASCs, and ASCs alone were also seeded. Quadruplicate reactions were performed from each treatment group, and the cultures were incubated at 37°C in 5% CO2 for 5 days in HS (PAA Laboratories) medium.
Figure 10. Schematic illustration of two-way MLR assay. Two different MLR combinations were formed from allogeneic PBMCs (blue and red; red and orange). MLRs 1 and 2 were co-cultured with allogeneic ASCs (yellow) to evaluate the suppressive potential of ASCs on MLR proliferative response. PBMCs, peripheral blood mononuclear cells; ASCs, adipose stem cells.
4.7.3 Bromodeoxyuridine (BrdU) ELISA
On day 4 of the MLRs, 10 mM bromodeoxyuridine (BrdU) was added to mono- and co-cultures, and the cells were incubated for additional 16 hours at 37°C. BrdU is a pyrimidine analogue that instead of thymine is incorporated into the DNA of dividing cells during the incubation. On day 5, PBMC proliferation was assessed by BrdU enzyme-linked immunosorbent assay (ELISA) (Roche Applied Science, Penzberg, Germany, https://www.roche-applied-science.com) according to the
manufacturer’s instructions. Briefly, cells were fixed, permeabilizated and the DNA was denatured, followed by antibody binding to the incorporated BrdU. Detector anti-BrdU monoclonal antibody was incubated for one hour. After washes, HP- conjugated goat anti-mouse antibody was added and bound the detector antibody.
The HP catalyzes the conversion of the substrate tetra-methylbenzidine (TMB) from a colorless solution to a blue solution. Finally, the reaction was terminated with sulfuric acid (H2SO4) and color intensity was determined at 450 nm with a microplate reader. The intensity of the color is proportional to the amount of dividing cells in the sample.
4.7.4 Quantitative protein secretion in mono- and co-cultures
Signaling protein secretion was analyzed using a two-way MLR assay. Four different donor PBMC lines were used as responder cells, in two different MLR combinations.
PBMCs from two donors were mixed in equal amounts to activate the MLR, and a total cell number of 8.0x105 PBMCs was seeded per well on 24-well plate. After initiating the MLRs, ASCs were added at densities of 3.0x104 cells per well either in direct co-culture or using a semipermeable membrane inserts to prevent direct cell- cell contacts between ASCs and PBMCs. When using the inserts, PBMCs were pipetted into the inserts (pore size, 0.4 mm; ThinCert, Greiner Bio-One, Frickenhausen, Germany) and ASCs were seeded on the bottom of the wells. Cell culture supernatants from mono- and co-cultures were collected on day 5 and stored in -20°C until analysis. Cytokines and chemokines secreted by the cells were analyzed using Cytometric Bead Arrays (CBA): Human Chemokine Kit, Human Th1/Th2/Th17 cytokine kit and Human TGF-β1 Single Plex Flex Set (BD Biosciences). IDO, Galectin-1 and Galectin-3 were analyzed using colorimetric ELISA assays: ELISA kit for IDO (Cloud-Clone Corporation, USCN Life Science Inc.), and Human Galectin-1 and-3 Quantikine ELISA Kits (R&D Systems). A list of the analyzed signaling proteins is presented in Table 11. Each colorimetric ELISA reaction was done in triplicate, and averages of the parallel reactions were then taken into account in statistical analysis. CBA output data were analyzed using FCAP Array software version 3.0 (BD Biosciences) according to the manufacturer’s instructions.
Table 11. Cell signaling proteins analyzed in study III.
Signaling protein Used method Manufacturer
CXCL8/IL-8 CCL5/RANTES CXCL9/MIG CCL2/MCP1 CXCL10/IP-10
CBA, Human Chemokine Kit
BD Biosciences, Franklin Lakes, NJ, USA
IL-2 IL-4 IL-6 IL-10 TNF-α IFN-γ IL-17A
CBA,Human Th1/Th2/Th17
Cytokine Kit BD Biosciences
TGF-β1 Single Plex Flex Set BD Biosciences
IDO ELISA kit Cloud-Clone Corporation, Uscn Life
Science Inc., Wuhan, China
Galectin-1 ELISA kit R&D Systems Inc., Minneapolis, MN,
USA
Galectin-3 ELISA kit R&D Systems