Macrophage Polarization for Immunomodulation and Tailoring Local Biological Response
3.2. Materials and Methods
3.2.1. Silk processing and fibroin isolation
Silkworm cocoons from Bombyx mori were purchased from Tajima Shoji Co., LTD (Yokohama, Japan) (Rockwood et al., 2011). Cocoons were cut into small pieces and boiled in 0.02 M Na2CO3 (Sigma-Aldrich, St. Louis, MO) for 30 min. After boiling, fibers were washed using ultrapure water. Degummed fibers were dried in a fume hood overnight. The dried fibers were dissolved in 9.3 M LiBr (Sigma-Aldrich, St. Louis, MO) solution at 60 ºC for 4 h (20% w/v) and dialyzed against deionized water for 3 days to remove the LiBr. Post-dialysis, solution was centrifuged (4000g, 20 min, 4 ºC) to remove insoluble particulates. To determine the concentration of regenerated silk fibroin solution, a gravimetric method was adopted and silk solution was stored at 4 ºC for further use.
3.2.2. Silk hydrogel preparations
3.2.2.1. Silk-PEG (SP) hydrogel preparation
PEG MW 400 g mol-1 (Sigma-Aldrich, Kollisolv PEG E 400) liquid was used for SP hydrogel preparation with the technique previously described (Wang et al., 2015a).
PEG-400 was dissolved in ultrapure distilled water (Invitrogen, USA), mixed homogenously to prepare 80% (v/v) PEG solution, filtered through 0.22 µm filter and stored at room temperature (~25 ºC).
Figure 3.1. Schematic representation of silk hydrogel preparation. Covalent cross- linking of tyrosine residues on silk protein chains in enzymatically formed SH hydrogels (A). Sonication-induced assembly of silk solution into hydrogels (B). Silk- PEG gel formation with β-sheet structure (C).
To prepare 8% and 1% SP (w/v) hydrogels, 16% and 2% (w/v) of silk was gently mixed to equal volume of 80% (v/v) PEG-400 solution (1:1) in Lobind Microcentrifuge
tubes (Fisher Scientific, USA), separately. The mixture was incubated at 37 ºC until gelation. For cell culture studies, sterile silk and PEG solutions were used.
3.2.2.2. Silk-HRP (SH) hydrogel preparation
Silk-HRP hydrogel was fabricated as previously described, with some modifications (Partlow et al., 2014). Lyophilized HRP type VI (Sigma-Aldrich, St. Louis, MO) powder was mixed with ultrapure distilled water to prepare a stock of 1 mg/mL.
Similarly, 1% (v/v) hydrogen peroxide (H2O2, Sigma-Aldrich, St. Louis, MO) stock solution was prepared in ultrapure distilled water. Enzyme stock solution of 20 µL was mixed with 1 mL of silk solution followed by addition of 20 µL H2O2 stock solution to initiate gelation. A similar protocol was followed for 8% and 1% SH hydrogel preparation. The solutions were mixed by gentle pipetting until gelation.
3.2.2.3. Silk-sonicated (SS) hydrogel preparation
SS hydrogels were prepared by sonicating 2 mL of silk solution (8% and 1%) in a 15 mL tube (Fisher Scientific) using a Branson 450 Sonifier (Branson Digital Sonifier, Model 450 power supply). The solution was sonicated at 10% amplitude for 20 s and left at 37 ºC for gelation which could be monitored visually (Soffer et al., 2008).
3.2.3. Cytokine loading on hydrogels and in vitro release
Recombinant human cytokines (IFN-γ and IL-4, Peprotech, Rocky Hill, NJ, USA) were encapsulated in all hydrogel variants during gelation. For release studies, 20 µL hydrogels containing 1 µg of cytokine (IFN-γ or IL-4) was incubated in Lobind Microcentrifuge tubes in 1 mL phosphate buffered saline (PBS) (Gibco, ThermoFisher Scientific, pH 7.4) and placed on a shaker (Reeves et al., 2015). PBS was replenished at specified time points (day 1, 3, 5, 7, 14, 21). Collected samples were stored at -80 ºC for further enzyme-linked immunosorbent assay (Human ELISA Development Kit, Peprotech, Rocky Hill, NJ, USA) for IFN-γ and IL-4, following manufacturer’s instruction.
3.2.4. In vitro cytotoxicity of hydrogels with human mesenchymal stem cell (hMSCs)
Human mesenchymal stem cells (hMSCs, P0) were isolated from fresh bone marrow aspirate (Lonza, Basel, Switzerland) as described previously (Altman et al., 2002).
Cells were cultured in high glucose Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic (100 U/mL penicillin and 100 mg/mL streptomycin, Gibco, Thermo Fisher Scientific), L-glutamine and non- essential amino acids (Life Technologies, Grand Island, NY). Cells were maintained in T175 flasks at optimal density and expanded in an incubator at 37 ºC and 5% CO2. Cell culture medium was replenished two times per week. Cells were cultured to 90%
confluence and trypsin passaged. For surface seeding, the three hydrogels types (100 µL) with a final silk concentration of 5% (w/v) were prepared in a 48 well plate and allowed to solidify in an incubator at 37 ºC for 1 h. Cells at passage 2 were used for seeding at a density of 5 × 104 cells (in 100 µL) per well and placed in an incubator for 1 h prior to flooding the wells with 1 mL media.
For cell encapsulation in SH hydrogels, the following steps were utilized (Partlow et al., 2014; Zhao et al., 2016). Silk, HRP and H2O2 were mixed and allowed to pre-gel for approximately 20 min, at which time 4 × 106 per mL cell suspension was mixed with silk in 1:1 volume ratio. Then 100 μL of the cell suspension in the pre-gelled silk solution was added to 48 well and allowed to gel for an additional 20 min and 1 mL media added to the wells. For cell encapsulation in 5% SP hydrogels, 500 µL of 10%
silk was mixed with 500 µL of 80% (v/v) PEG. Cells were mixed to achieve a final cell concentration of 2 × 106 cells per mL and 100 μL of hydrogel was added to 48 well plates. For SS gel, 2 mL of silk solution (5% w/v) was sonicated at 10% amplitude for 20 s in a laminar flow hood and final cell concentration was maintained. Cell pellets, containing 2 × 106 cells, were dissolved in 100 μL of silk and further mixed with 900 μL of silk 8% (w/v). Then 100 μL of solution was added to 48 well plates. All cell culture studies were performed in an incubator (37 ºC, 5% CO2).
Cytotoxicity and metabolic activity of the hydrogel encapsulated cells was determined after 1, 4 and 7 days of culture via AlamarBlue assay (Life Technologies, Grand Island, NY) following to the manufacturer's instructions. AlamarBlue, a non-toxic dye, measures the viability of the same batch of seeded cells at different time points. Briefly, cells were incubated in medium supplemented with 10% (v/v) AlamarBlue dye for 5 h at 37 ºC with 5% CO2. After incubation, the absorbance of 100 μL of medium from each sample was read at 570/600 nm in a multiplate reader. Non-seeded wells supplemented with 10% AlamarBlue dye were used as negative control.
Imaging of the surface seeded cells was performed with Calcein AM (Sigma-Aldrich, USA) staining following the manufacturer's instructions. Live cells convert calcein AM
to calcein and fluoresce green showing viability (Wang et al., 2015a). Briefly, hydrogels were washed twice with PBS and incubated with 4 μM of Calcein AM, prepared in incomplete media (DMEM without FBS), for 20 min to stain live cells.
After staining, hydrogels were washed thrice and images were taken using Zeiss fluorescence microscope (Carl Zeiss, Jena, Germany).
3.2.5. THP-1 cell culture and polarization in 2D
Human acute monocytic leukemia cell line (THP-1) was purchased through ATCC (Manassas, VA). Cells were cultured in RPMI media supplemented with 10% FBS and 1% antibiotics. Cells were cultured in T175 flasks at optimal density and expanded in a humidified incubator (37 ºC, 5% CO2). For macrophage polarization study, 5 × 104 cells per well were seeded in Corning transwells plates (Fisher Scientific, USA). THP-1 cells were induced to macrophage (M0) using 100 ng/mL (162 nM) of phorbol 12- myristate 13-acetate (PMA) (Sigma-Aldrich, USA) in basal RPMI media for 16 h.
After PMA treatment, macrophage differentiation (M0) was confirmed by their adherence to well plates.
Figure 3.2. Study design for in vitro macrophage polarization and plasticity using three different approaches. General scheme showing monocyte (THP-1) to macrophage (M0) conversion using PMA. Further, macrophage polarization and plasticity studies were conducted using pro-inflammatory IFN-γ and anti-inflammatory IL-4 cytokines (A). In the first approach, M0 cells were cultured in TCP wells and cytokine-loaded hydrogels were placed in transwell inserts. H-IFN-γ and H-IL-4 represent IFN-γ and IL-4 encapsulated in hydrogels, respectively (B). In the second approach, M0 cells were encapsulated in hydrogels (placed in transwell inserts); polarizing cytokines were provided through cell culture media. H-M0, H-M1 and H-M2 represent hydrogel- encapsulated M0, polarized M1 and polarized M2, respectively at different stages of polarization (C). In the third approach, M0 cells and individual cytokines were co-
encapsulated in silk hydrogels. H-IFN-γ-M0, H-IFN-γ-M1, H-IL-4-M0 and H-IL-4-M2 represent M0 encapsulated with IFN-γ, polarized M1 encapsulated with IFN-γ, M0 encapsulated with IL-4, and polarized M2 encapsulated with IL-4, respectively (D).
After PMA treatment, the medium was switched to polarization conditions through cytokine-releasing hydrogels (8% SP, 8% SH and 8% SS) placed in the transwell inserts (Figure 3.2B). Macrophages (M0) were incubated with different silk hydrogels loaded with M1 (IFN-γ) and M2 (IL-4) polarizing cytokines to evaluate macrophage polarization potential of hydrogels on a time-dependent basis for 60 h and 120 h. Cell morphology was analyzed to assess macrophage polarization. At pre-specified time points (60 h and 120 h), cells were lysed and preserved for gene expression analysis for M1 and M2 markers.
3.2.6. Polarization in hydrogel-encapsulated M0 macrophages
Cell-encapsulating hydrogels were prepared as described in section 3.2.4. Briefly, THP-1 cells were induced to M0 using 100 ng/mL (162 nM) PMA in basal RPMI media for 16 h and cells were collected from tissue culture flasks using cell scrapper (Corning, USA). Macrophage polarization was conducted with M0 cells encapsulated within the hydrogels. The hydrogel volume (100 μL), final silk concentration (5% w/v) and THP-1 cell density (2 × 106/mL) were held constant for 3D polarization studies similar to hMSCs encapsulation. Cell-encapsulated hydrogels were added to transwell inserts and wells were flooded with media after gelation (Figure 3.2C). All cell culture studies were performed in an incubator (37 ºC, 5% CO2). Polarization media was used as RPMI supplemented with 10% FBS and 20 ng/mL IFN-γ (for M1) and 20 ng/mL IL- 4 (for M2) (Figure 3.2C). Imaging of hydrogel-encapsulated M0 cells was performed with 4 μM calcein AM (prepared in incomplete media) after 5 days of culture.
Hydrogels were washed twice with PBS and incubated in staining solution for 20 min.
Post incubation, hydrogels were washed thrice with PBS and images were taken under fluorescence microscope.
3.2.7. Polarization in hydrogel co-encapsulated macrophages and cytokines
Cell-encapsulated hydrogels were prepared as described in section 3.2.4. Macrophage polarization was done with M0 cells encapsulated in hydrogels with cytokines (IFN-γ/
IL-4). Cytokines were added during the cell encapsulation step. After gelation, cytokine-loaded hydrogels were incubated for 1 h in cell culture media to remove any
surface bound cytokine. These polarization studies were performed for five days with RPMI basal media changed every other day (Figure 3.2D). Hydrogel volume (100 μL), final silk concentration (5% w/v), cell concentration (2 × 106 per mL) and cytokine loading (200 ng/gel, IFN-γ /IL-4) were kept constant for 3D polarization studies. M0 cells entrapped in respective hydrogels were used as control for each time point. For cell imaging, M0 and IL-4 encapsulating hydrogels were incubated for 30 min in staining solution (1% (w/v) BSA, 0.5% (v/v) Triton X-100 in PBS) containing 0.1%
(v/v) Hoechst 33342 (Sigma-Aldrich, USA) and AdipoRed (AdipoRed Assay Reagent, Lonza, USA). Cytoskeleton was stained using Fluorescein Phalloidin (ThermoFisher scientific) for F-actin and counterstained with Hoechst 33342 for nucleus. After staining, hydrogels were washed thrice and images were taken using KEYENCE all-in- one fluorescence microscope BZ-X700 (USA) and Z-stacking was performed using BZ-X Analyzer software.
3.2.8. RNA extraction and gene expression by real-time RT-qPCR
Analysis of gene expression specific to M1 and M2 was performed by quantifying the mRNA levels of respective genes. Cells were lysed and RNA was isolated and purified using RNeasy Plus Mini Kit (Qiagen, CA) according to manufacturer’s protocol for on- column treatment. From hydrogels, total RNA was extracted by thoroughly pipetting the hydrogel to disrupt the gel network. Hydrogels were treated with Trizol (Invitrogen, USA) for 15 min. The treated hydrogels were centrifuged (12000g, 10 min, 4 ºC) and the supernatant was incubated in a new tube with chloroform. After centrifugation (12000g, 15 min, 4 ºC), the upper aqueous layer was transferred to RNeasy Plus Mini Kit and extracted as above. The RNA concentration and quality was measured by NanoDrop 1000 Spectrophotometer (Thermo Scientific). For reverse transcription, 1 µg of RNA was converted to cDNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA). RT-qPCR was performed using Power SybrGreen (Thermo Fisher Scientific) using the Stratagene Mx3000P QPCR system (Stratagene, CA). All assays were carried out in duplicate in 96-well format plates.
Primers (Table 3.1) were used at 1 µM concentration and run with cycles of 15 s at 95 ºC and 45 s at 60 ºC for 40 cycles. To quantify relative gene expression changes, the ΔΔCT method was used with Glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) as a housekeeping gene (Livak and Schmittgen, 2001). Changes in target gene expression in M1 and M2 macrophages were expressed as fold change from control groups.
Table 3.1. Primers for RT-qPCR
Gene Forward sequence Reverse sequence Phenotype
GAPDH ACAACTTTGGTATCGTGGAAGG GCCATCACGCCACAGTTTC N/A CD68 CTTCTCTCATTCCCCTATGGACA GAAGGACACATTGTACTCCACC M0 CCR7 TGAGGTCACGGACGATTACAT GTAGGCCCACGAAACAAATGAT M1 CD206 CTACAAGGGATCGGGTTTATGGA TTGGCATTGCCTAGTAGCGTA M2 IL1B ATGATGGCTTATTACAGTGGCAA GTCGGAGATTCGTAGCTGGA M1 CD209 AATGGCTGGAACGACGACAAA CAGGAGGCTGCGGACTTTTT M2
3.2.9. Statistical analysis
All experiments were performed in triplicate and results represented as mean ± standard deviation (S.D.). One-way analysis of variance (ANOVA) was performed to calculate statistical significance between different groups followed by Tukey’s test. All statistical analysis was performed using OriginPro 8 (Originlab Corporation, USA). Differences between groups were considered statistically significant when p ≤ 0.05.