Immunostaining of Cells Encapsulated in a 3D Hydrogel System

1. Cell Encapsulation in 3D Elastin-like Protein (ELP) Hydrogels

1. Preparation of silicone molds
   1. Use a biopsy punch with the desired diameter to create holes in a 0.5 mm thick silicone sheet and cut out a square around each hole. Repeat for the number of molds desired.
      ​NOTE: The diameter and thickness of the molds can be adjusted for the particular application and cell culture conditions. In practice, for cell cultures of 50 x 10⁶ cells/mL, 0.5 mm thick molds with 4 mm and 5 mm diameters are recommended for sufficient DNA/RNA and protein extraction, respectively. For immunostaining, a 2 mm biopsy punch can be used to instead create three adjacent holes per square mold so that three replicates can be stained per well.
   2. Remove the plastic wrap on each side of the individual mold with tweezers.
      NOTE: Avoid contact with the exposed silicone surface as contamination can decrease future plasma bonding efficiency.
   3. Using tweezers, arrange the same number of bare silicone molds and glass coverslips (No. 1, 12 mm diameter) in alternating rows on the inverted lid of a 48-well plate. Once completed, cover the lid of the 48-well plate to avoid contamination.
   4. Oxygen plasma treat the entire 48-well plate lid (i.e., molds and glass slides). Immediately after, use forceps to invert the silicone mold onto the adjacent glass coverslip. Press firmly on the mold to ensure bonding. Let the molds incubate at room temperature for 1 h.
      NOTE: The duration of oxygen plasma treatment will vary depending on the instrument used. Typical conditions for our instrument are an operating gas pressure window between 0.3-4 mbar, oxygen gas flow at 20 cm³/min, and sample exposure to plasma for 10-20 s.
   5. Sterilize the molds by autoclaving. Store the molds at room temperature in a sterile environment until use.
      NOTE: The molds can be stored at this stage indefinitely.
2. Preparation of elastin-like protein stock solution​
   1. Remove lyophilized ELP from 4 °C storage. Warm the protein to room temperature before opening the tube to ensure no condensation builds on the protein over repeated use.
   2. Dissolve ELP in Dulbecco's phosphate-buffered saline (DPBS) at 4 °C with constant agitation (i.e., spinning) overnight.
      NOTE: ELP stock solution concentration will be further diluted with the addition of crosslinker solution at a user-defined volumetric ratio. For example, for a 3% (w/v) final ELP concentration, prepare a 3.75% (w/v) ELP stock solution that will be diluted at a 4:1 volumetric ratio of ELP solution:crosslinker solution. Adjust the concentration as necessary for desired application.
   3. Sterile filter ELP stock solution using a 0.22 µm syringe filter. Store ELP on ice when not in use.
3. In the biosafety cabinet, transfer the sterile molds using sterile tweezers to a 24-well tissue-culture plate.
4. Preparation of tetrakis(hydroxymethyl)phosphonium chloride (THPC) stock solution​
   1. Dilute THPC in DPBS just prior to use. Adjust the concentration of THPC solution according to the final ELP concentration and desired crosslinking ratio.
      NOTE: For the protocol, a final ELP concentration of 3% (w/v) will be made by mixing the ELP stock solution with the THPC solution at a volumetric ratio of 4:1. Adjust as necessary.
   2. Add 2.6 µL of THPC solution (80% in water) to 997.4 µL of DPBS.
      NOTE: This concentration of THPC corresponds to a 1:1 stoichiometric ratio of hydroxy methyl groups on THPC and primary amines on the ELP protein when mixing solutions at the described 4:1 volumetric ratio for a final 3% (w/v) ELP hydrogel. The THPC solution is viscous and droplets of solution may stick to the side of the pipette tip. For accurate concentrations, avoid such droplets when diluting into DPBS. Purge the THPC stock container with nitrogen gas to prevent oxidation of the phosphine and inactivation of the crosslinker.
   3. Vortex the solution to mix and keep on ice.
   4. Sterile filter the THPC stock solution using a 0.22 µm syringe filter. Dilute the THPC stock solution further with sterile DPBS to achieve lower stoichiometric crosslinking ratios (e.g., 0.5:1 or 0.75:1).
      NOTE: THPC is oxygen-sensitive, and the diluted solution should be used within a few hours after preparation.
5. Dissociate the cells by incubation with Trypsin-EDTA into a single-cell suspension, pellet the cells, and count the cells re-suspended in medium using a hemocytometer.
   NOTE: Exact protocols for this step will depend heavily on desired cell type and application. For the neural progenitor cells used throughout this protocol, a 0.025% Trypsin-EDTA incubation at room temperature for 1.5 min was conducted. The cells were pelleted at 200 x g for 2 min. For increased cell viability, cells should be suspended in normal medium conditions. Typical cell densities used for cell encapsulation range from 1 – 50 x 10⁶ cells/mL of final hydrogel volume. Adjust as necessary.
6. Aliquot the desired number of cells into a sterile 1.5 mL centrifuge tube.
7. Centrifuge the cells at ~200 x g for 3 min. Carefully, aspirate the supernatant and keep the cell pellet on ice.
   NOTE: It is critical to completely aspirate all the supernatant to mitigate the further dilution of the ELP and THPC crosslinker. Specific centrifugation speeds will vary with cell type.
8. Re-suspend the cell pellet in the ELP stock solution such that the volume is 80% of the final volume (assuming a 4:1 ratio of ELP solution:THPC solution). Pipette mix 20-25 times to produce a homogenous mixture of the cells and ELP.
   NOTE: Avoid gripping the bottom of the tube to mitigate temperature increase and subsequent phase transition of ELP. Each 2 mm mold with three replicates requires 7.5 µL of final volume (i.e., 6 µL of ELP stock solution and 1.5 µL of THPC stock solution) divided equally into the 3 holes (i.e., 2.5 µL final volume per hole). For the 4 and 5 mm molds, a final volume of 7.5 µL and 15.5 µL is required, respectively.
9. Add THPC stock solution to the cell/ELP suspension for the remaining 20% final volume. Pipette mix 20-25 times to produce a homogenous mixture.
10. Immediately pipette the corresponding final volumes of cell/ELP/THPC mixture into each mold by a circular motion. Repeat for all molds.
11. Incubate the samples at room temperature for 15 min, followed by an additional incubation at 37 °C for 15 min.
    NOTE: The first incubation period will help facilitate initial crosslinking of the hydrogel before increasing the temperature to that above the ELP's lower critical solution temperature (LCST) and inducing its thermal phase segregation.
12. Slowly add 750 µL of warm cell culture medium to each well of the 24-well plate, avoiding disrupting the gels.
13. Incubate the hydrogels at 37 °C for 7 days.
    NOTE: Full medium changes are recommended every 1-2 days depending on cell type. To limit the stress on the gel, use a glass Pasteur pipette affixed with a 200 µL pipette tip when aspirating medium from the well.

2. Immunocytochemistry of Cells in 3D ELP Hydrogels

1. Prepare the fixation solution by mixing 10 mL 16% (w/v) paraformaldehyde (PFA) in 30 of mL DPBS. Warm the solution to 37 °C.
2. Aspirate the medium from the 24-well plate and gently wash with 1 mL of DPBS.
3. Add 750 µL of the fixation solution to each well and incubate at 37 °C for 30 min. Do not use the tissue culture incubator to avoid contamination of other cultures with PFA vapor.
4. Carefully aspirate the fixation solution from each well, discarding the PFA to an appropriate hazardous waste container.
   CAUTION: Exposure to PFA can cause skin and eye irritation. Wear gloves/safety glasses and work in a chemical fume hood.
5. Add 1 mL of DPBS to each sample. Immediately aspirate the DPBS and discard into PFA waste container.
6. Wash the samples twice with 1 mL of DPBS for 10 min each.
   NOTE: Samples can be stored in DPBS at 4 °C for up to a week after sealing the plate with Parafilm.
7. Permeabilize each sample with 750 µL of permeabilization solution (100 mL of DPBS and 0.25 mL of Triton X-100; PBST) for 1 h at room temperature on a rocker at 15 rpm.
8. Aspirate the PBST and add 750 µL of blocking solution (95 mL of PBS, 5 g of bovine serum albumin (BSA), 5 mL of serum, and 0.5 mL of Triton X-100) to each sample. Incubate at room temperature for 3 h on a rocker at 15 rpm.
   NOTE: The blocking solution should contain serum from the host species in which the secondary antibodies were raised (e.g., goat, donkey) and sterile-filtered through a 0.22 µm filter prior to use.
9. Prepare the antibody dilution solution (97 mL of DPBS, 2.5 g of BSA, 2.5 mL of serum (from same host species from which EPL is prepared), and 0.5 mL of Triton X-100). Dilute the primary antibody using the antibody dilution solution and add 500 µL of the solution to each sample. Seal the plate with Parafilm and incubate overnight at 4 °C on a rocker.
   NOTE: Nestin and Sox2 primary antibodies were diluted 1:400 in antibody dilution solution from the manufacturers' original concentration.
10. Aspirate the antibody solution from each sample and wash the samples with PBST for 60 min at room temperature on a rocker at 15 rpm. Repeat the wash step 3 times.
11. Dilute the secondary antibodies and 5 mg/mL stock DAPI (1:2,000) using the antibody dilution solution and add 500 µL of the solution to each sample. Cover the 24-well plate with aluminum foil and incubate overnight at 4 °C on a rocker.
    NOTE: As the secondary antibodies are light sensitive, the samples must be protected from photobleaching for all subsequent steps. Goat anti-mouse and goat anti-rabbit secondary antibodies were diluted 1:500 in antibody dilution solution from the manufacturers' original concentration.
12. Aspirate the antibody solution from each sample and wash the samples with PBST for 30 min at room temperature on the rocker. Repeat the wash step 3 times.
13. Position a drop of hard-set mounting medium onto the surface of a glass slide. Using forceps, remove excess solution from the mold by lightly blotting the edge of the mold on a paper towel. Carefully place the mold upside down onto the mounting medium and avoid introducing bubbles.
14. Allow the mounting medium to harden for 48 h at room temperature. Store the samples at room temperature or 4 °C. Allow the mounting medium to fully set for 48 h before imaging as refractive index of the medium will change over the course of hardening. Seal the samples to the glass cover slide with clear nail polish to mitigate contamination or sample movement.
15. Image the samples using a confocal microscope.