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Fybrix Protocols

Fybrix hydrogel preparation:

timing: approx. 24 hrs

  1. Weigh the desired amount of dry polymer, dissolve in a pre-cooled (4 °C) buffer and leave overnight at 4 °C until a clear solution is obtained. For cell culture application, we recommend using PBS or appropriate cell culture media as dissolving buffer.
  2. Place the samples in the fridge during the dissolution process. Occasionally, pipette the solution up and down or gently shake the vial by hand if the polymer has not been dissolved overnight and leave for another 1–2 hours. Repeat this until fully dissolved. Note that PIC dissolution becomes increasingly slow at concentrations > 6 mg mL–1.
  3. Once dissolved, use the PIC solution freshly, or aliquot the solution and store at –20 °C.

Tip: Choose the aliquot volume wisely; we recommend using the volume necessary for a single experiment to avoid freeze-thaw cycles.

3D Cell seeding in Fybrix gels:

Generic protocol for starting 3D cell culture (timing: approx. 1 hr)

  1. Prepare a Fybrix solution with double the desired concentration by mixing the stock solution with pre-cooled buffer.
  2. Pre-warm the cell culture plate and medium at 37°C. Transfer the Fybrix solution to the flow hood on ice to prevent early gelation.
  3. Count the cell numbers, and prepare a cell suspension at double the desired concentration and place it on ice. Keep the cell suspension on ice!
  4. On ice, mix the Fybrix solution and the cell suspension at a 1:1 ratio (v/v) by gently pipetting up and down. Avoid bubble formation.
  5. Seed the cell/gel mixture into a cell culture plate and place it in the incubator to allow gel formation (roughly 5 min, larger gel volumes require more time).
  6. Add the desired amount of pre-warmed (37 °C) cell culture medium to the wells.
  7. To collect or replace the medium, remove the plate from the incubator and place it on a warm plate to prevent the culture from cooling.
  8. Gently pipette the supernatant conditioned media from 3D culture and, when desired, collect it in a separate container. To prevent gel damage, do not pipet too harshly, tilt the plate while aspirating the media and remove only ~80% of the medium instead of the full 100%.
  9. When desired for analysis, store the conditioned medium at –80 °C for downstream (e.g., secretome, MMP, exosome, etc) analysis. 
  10. Add the desired amount of fresh pre-warmed (37 °C) cell culture medium to the wells and return the culture to the incubator.
2D Cell seeding on top of Fybrix gels:

Generic protocol for starting 2D cell culture (timing: approx. 1 hr)

  1. Pre-warm the cell culture plate and medium at 37 °C. Transfer the Fybrix solution to the flow hood on ice to prevent early gelation.
  2. Place the desired volume of Fybrix solution in the culture plate and leave it in the incubator for at least 5 min to allow gel formation. Larger volumes need longer gelation times.
  3. Trypsinize the cells, count them, and resuspend them in fresh culture media.
  4. Seed proper volume of warm (37 °C) cell suspension with a desired cell density on top of the gelled Fybrix.
  5. Culture the cells at 37 °C and change the cell culture media when necessary but always in a gentle fashion. Note: To prevent gel damage, do not pipet too harshly, tilt the plate while aspirating the media and remove only ~80% of the medium instead of the full 100%.
In situ cell culture analysis:

To analyze cell-matrix constructs, follow option A for immunostaining or option B for proliferation assays. Note that the protocol below is for the cell and organoids in 3D condition. Protocols for 2D cell cultures, follow those on TCP, but a warm gel and gentle pipetting is needed for the gel to maintain its integrity.

Immunostaining:

Use a 37 °C cell heating plate to keep the temperature, and gently pipetting during washing and adding buffers to avoid mechanical disintegration the samples.

  1. Wash cell culture samples with warm (37 °C) PBS.
  2. Fix the cells using 3–4% paraformaldehyde in PBS for 1 h at 37 °C and wash the samples with warm (37 °C) PBS.
  3. Permeabilize cells using prewarmed Triton X-100 (0.1 % in PBS) for 30 mins at 37 °C and wash the samples with prewarmed PBS.
  4. Block the samples with BSA (1% in PBS) overnight in an incubator. Do not wash!
  5. Add a primary antibody in 1% BSA/PBS (concentrations recommended by the supplier for 3D cultures) overnight in an incubator. When concentrations are only given for 2D cultures, we suggest a higher concentration of the primary antibody than the 2D staining protocols. For instance, if a certain primary antibody is used at 1:500 for 2D, then 1:200 is recommended for 3D. We recommend case-by-case optimization of the exact concentration and timing, especially for higher FYBRIX concentrations.
  6. Wash with warm PBS (37 °C); then add warm PBS (37 °C) on top, incubate for 1 h, and aspirate.
  7. Add the secondary antibody in 1% BSA/PBS (concentration recommended by the supplier) for 4 h at 37 °C (with aluminum foil to protect from light).
  8. Optional: nuclear counterstaining with DAPI (>2 μg/mL) can be carried out simultaneously with the secondary antibody. Wash with warm PBS (37 °C); then add warm PBS (37 °C) on top, incubate for 1h, and aspirate.
Proliferation Assays:
  1. Aspirate the medium from the samples.
  2. Add the cell proliferation reagent of interest and follow the manufacturer’s instructions; Table below provides details on typical dilution and incubation times used in our lab.
AssayIncubation timeNotes
Cell counting Kit-8
(CCK-8, Sigma, USA)		2hBe cautious not to disturb gels when collecting supernatant working solution
WST-1 (Roche, Germany)1hBe cautious not to disturb gels when collecting supernatant working solution
CellTiter-Glo (Promega, USA)2 min (mixing) + 10 minLiquify hydrogel before adding reagent (after step  #16-B-i) and add equal volume of CellTitel-Glo
Live-Dead assay (Invitrogen, USA)1 hAfter incubation, wash 3× with PBS before imaging
Cell extraction from Fybrix gels:

timing: approx. 24 hrs

  1. Weigh the desired amount of dry polymer, dissolve in a pre-cooled (4 °C) buffer and leave overnight at 4 °C until a clear solution is obtained. For cell culture application, we recommend using PBS or appropriate cell culture media as dissolving buffer.
  2. Place the samples in the fridge during the dissolution process. Occasionally, pipette the solution up and down or gently shake the vial by hand if the polymer has not been dissolved overnight and leave for another 1–2 hours. Repeat this until fully dissolved. Note that PIC dissolution becomes increasingly slow at concentrations > 6 mg mL–1.
  3. Once dissolved, use the PIC solution freshly, or aliquot the solution and store at –20 °C.

Tip: Choose the aliquot volume wisely; we recommend using the volume necessary for a single experiment to avoid freeze-thaw cycles.

Cell extraction