Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

1. Dissolution of peptide

1. In a tissue culture hood, add 800 μL of sterile water to a 15 mL tube using a P1000 pipette.
2. Using a fine balance, weigh the peptide powder into a non-static weighing-boat. Use a mass (in mg) of 1.25x the desired final peptide gel concentration (in mg/mL, Table 1).
   NOTE: The method described here will produce a volume of approximately 1.25 mL of peptide gel per tube at the point of final gelation. Multiple tubes may be prepared at a time, or alternatively the volume per tube can be increased when the user is experienced with the method presented.

Table 1: Peptide mass and suggested initial NaOH addition for typical final gel concentrations. The ranges of peptide concentrations listed may be extended in either direction, however, it is more likely that lower peptide concentrations may not form stable gels, whereas at high concentrations the resulting gel may be too dense to allow sufficient nutrient exchange and cell viability. The appropriate concentration will require optimization for different cell types and peptide batches.

3. Add the weighed peptide into the 15 mL tube. Flick the weighing boat to ensure no powder is left behind.
   NOTE: The peptide powder can be very static, take care to minimize the loss of powder. It is not necessary to maintain sterile conditions during steps 1.2 and 1.3 (or for any pH measurements) as sterilization occurs during the incubation at 80 °C later in the process.
4. Vortex for 3 min, then centrifuge at 200 x g for 3 min.
5. Incubate the peptide solution in an oven set at 80 °C for a minimum of 2 h. If undissolved peptide is present after incubation, repeat step 1.4.
   NOTE: As uniform heating is essential, a heatblock is not suitable for this step. However, we have found that hybridization ovens work well as does a water bath as long as the peptide solution is fully submerged.

2. Formation of gel precursors

1. Prepare a sterile 0.5 M sodium hydroxide (NaOH) solution using sterile water.
   NOTE: Freshly diluted NaOH solution is recommended for most consistent results.
2. In a tissue culture hood, add 0.5 M NaOH to the center of the dissolved peptide and mix by slowly stirring with the pipette tip (Table 1).
3. Vortex the tube for 10 s and centrifuge at 200 x g for 10 s to remove bubbles.
4. If the gel precursor is cloudy, repeat steps 2.2 and 2.3, adding 5 µL increments of NaOH.
   NOTE: pH measurement can help determine whether sufficient NaOH has been added: the optimal pH is between 9–10.5 and it is preferable not exceed this as using acids to bring the pH back down can result in gel inhomogeneity. The precise volume of NaOH required may vary with peptide source.
5. Once the gel precursor is optically clear and self-supporting (or only just flowing) on inversion of the 15 mL tube, add 100 μL of sterile 10x PBS in a tissue culture hood. Vortex for 10 s and centrifuge at 200 x g for 10 s.
   NOTE: If the gel precursor is cloudy, repeat step 2.4 until it becomes clear and semi-solid. If the gel precursor is liquid (above pH ~10.5), it will not form a stable gel and should be discarded.
6. Incubate overnight in an oven at 80 °C.
7. Visually check the gel precursor to ensure it is fully liquid at 80 °C.
   1. If the gel precursor is not fully liquid at 80 °C, it has not been sufficiently neutralized, add NaOH following step 2.4.
   2. If the gel precursor is liquid but air bubbles or small precipitates are present, flick the tube sharply to disperse them. If air bubbles or precipitates persist after flicking the tube, vortex the gel precursor and centrifuge at 200 x g for 10 s each.
8. After following either of steps 2.7.1 or 2.7.2, incubate precursor gels for a further 2 h at 80 °C before proceeding to final gelation.
9. Keep the gel precursors at 80 °C until needed (maximum 48 h).
   NOTE: The protocol can be paused here, store gel precursors at 4 °C for up to 4 weeks. If restarting from this pause point, incubate gel precursors at 80 °C for at least 2 h, and restart from step 2.7. It is strongly recommended to prepare gel precursors well in advance of them being needed for the final gelation, to ensure there is sufficient time for the above modifications if required.

3. Preparation of matrix components for seeding

NOTE: An example calculation for steps 3–5 is shown in Figure 1. Step 3 and step 4 may be omitted to produce a matrix-free and/or a cell-free gel respectively.

1. Calculate the total combined volume of cells, matrix and media required for seeding, allowing for 250 µL to be added to each 1 mL precursor gel. Multiply this volume by 1.1 to allow for pipetting error. This is the seeding volume, V_S.
2. For each matrix component, calculate the volume of stock solution to be added to the seeding volume using Equation 1:
   (1)
   NOTE: V_S corresponds to the total volume of seeding medium required for seeding all gels (step 3.1). Ensure that the sum of all matrix component volumes does not exceed V_S.
3. Thaw all matrix components thoroughly (refer to manufacturer’s instructions) and store on ice until needed.
   NOTE: Some matrix components will be very prone to gelation. Refer to manufacturer’s instructions to avoid this occurring.
4. If an acidic collagen stock solution requiring neutralization is to be added (refer to manufacturer’s instructions), prepare the neutralization solution as follows:
   1. Calculate the volume of collagen stock solution, V_C, required using Equation 1.
   2. Determine a suitable volume for the neutralization solution, V_N. This should be chosen such that the combined volume of all matrix additions does not exceed the seeding volume, V_S.
      NOTE: Generally, a sensible value for V_N = 2 x V_c, but this may be adjusted according to the number and volume of other matrix components to be added.
   3. Calculate the volume of 1 M NaOH required for neutralization using Equation 2.
      (2)
   4. Calculate the volume of 10x PBS required in the neutralization solution using Equation 3.
      (3)
   5. Combine the calculated volumes of 1 M NaOH and 10x PBS, then make up to V_N with sterile water. Mix well, and store on ice until needed.
      NOTE: Do not add the acidic collagen at this point. Premature mixing of the collagen with the neutralization solution will lead to the initiation of collagen fibrillogenesis, which can cause inconsistency in the properties of the final peptide gel.

4. Preparation of cells for seeding

1. If not already completed, calculate the seeding volume, V_S according to step 3.1.
2. Calculate the cell density required in this cell suspension, by taking the desired cell density in the final peptide gel and multiplying by 5.
   NOTE: The cell suspension is 5x the desired end concentration to account for dilution upon mixing with the gel precursor. Cell densities should be optimized for each new cell line under consideration. Depending on the cell type, densities between 1 x 10⁴ and 1 x 10⁶ cells/mL in the final peptide gel may be appropriate.
3. Calculate the total number of cells required for seeding (multiply the cell density by the seeding volume V_S calculated in step 4.1.).
4. Using standard culture/passage methods for the cells in use, prepare a cell pellet containing the required quantity of cells as calculated in step 4.3.

5. Final gelation/cell encapsulation

1. When ready to begin final gelation, transfer the gel precursors from the 80 °C oven into a 37 °C water bath.
   NOTE: The gel precursors should be self-supporting at 37 °C. If they are liquid, it is unlikely that complete gelation will occur, and the gel precursors should be discarded.
2. Prepare a 96 well plate, or alternatively a 24 well plate (or similar) with cell culture inserts.
   NOTE: Ensure there is a gap between the base of the insert and the well plate when plating peptide gels in 24 well inserts. This ensures the gel is in contact with media.
3. If matrix is to be added, combine all matrix components and neutralization solutions (step 3). Make up to V_S (step 3.1) using cell culture medium and mix thoroughly. If cells are to be added, resuspend the cell pellet prepared in step 4 using the seeding volume V_S.
   NOTE: If no matrix components have been prepared in step 3, use the cell culture medium for the seeding volume. This is generally the standard culture medium for the cell type in use, although this may need to be validated if a mixture of cell types is being used.
4. Using a P1000 pipette, add 250 μL of the cell/matrix mix gently on top of the gel precursor.
   NOTE: Matrix components, particularly basement membrane extract, may begin to polymerize once added to the gel precursor. It is, therefore, important to move to the next step as quickly as possible.
5. Gently mix by the combined action of pipetting and stirring. The gel is shear thinning so will become easier to mix with gentle pipetting/stirring. When thoroughly mixed, add 100 μL to each well of a 96 well plate, or 200 μL to each cell culture insert.
   NOTE: When mixing, reverse pipetting using a P1000 set at 200 μL can be beneficial to avoid introducing air bubbles. The gel may initially be difficult to mix, but on continued mixing should become easier – this indicates that the mixing is efficient.
6. Incubate for 10 min at 37 °C in 5% CO₂ and a humidified atmosphere.
   NOTE: This step is not necessary if the peptide gel contains no matrix additions.
7. Add 200 μL of media to each well of the 96 well plate, or 1 mL to the outside of the cell culture insert with a few drops on the top of the gel. Incubate at 37 °C in 5% CO₂ and a humidified atmosphere.
8. Change media twice within the next hour, and again after several hours (or the following day).
   NOTE: Take care here as the gels will be unstable for several hours.
9. Change media every 2–3 days (or following standard culture protocol for the cells in use).

6. Indirect co-culture

NOTE: This method is only applicable where the peptide gels are seeded into 24 well plate inserts, or similar formats in which the gel can be supported above a cell monolayer. Indirect co-culture can be introduced in this case by preparing a 2D feeder layer of cells on the bottom of the well plate.

1. Prepare a 24 well plate for seeding. This should be a separate plate to the peptide gels but should be of the same brand to ensure compatibility with the inserts used (see step 5.2).
2. Calculate the cell density needed for seeding the indirect co-culture, according to the cell type under consideration.
   NOTE: The cell seeding density should be optimized for each new cell line under consideration. Cells should be plated to give approximately 30–50% confluence. As an example, the human mammary fibroblast cell line HMFU19 is typically plated at a density of 1-5 x 10⁴ cells/well.
3. Using standard culture/passage methods for the cells in use, prepare a cell suspension suitable for seeding at 1 mL/well, using the typical growth medium for these cells.
4. Seed the cell suspension into the well plate, 1 mL per well.
5. Incubate at 37 °C in 5% CO₂ and a humidified atmosphere to attach for several hours, or overnight. Then remove the medium from the wells.
6. Using sterile forceps, transfer the 24 well plate inserts containing the peptide gels into the new wells containing the pre-seeded cells in 2D. Add 1 mL of medium dropwise to the exterior of the insert and a few drops on the surface of the gel.
   NOTE: Typically, the medium used at this point is the one suitable for the cells encapsulated in the gel, but the suitability of this medium for the cells in 2D may need verification or optimization for the particular experiment under consideration.
7. Regularly prepare fresh feeder layers to avoid over-confluence, and transfer peptide gels to these new wells following the same method as above.
   NOTE: Typically, cells for indirect co-culture are prepared at the same time as the peptide gel seeding. Peptide gels can then be transferred to co-culture at the point of media change after a few hours or overnight incubation, see step 5.8.

7. Bulk oscillatory rheology of peptide gels

NOTE: As standard, rheological characterization is carried out 24 h after gel seeding, which should take place in 24 well plate inserts.

1. Set up and calibrate the rheometer according to manufacturer’s instructions. Use a parallel plate geometry with plate diameter as close as possible to the diameter of the cell culture insert.
   NOTE: Tests may be carried out at 37 °C if desired, replicating the environment during culture.
2. Remove the first peptide gel sample to be tested from the cell culture insert by inverting the insert and cutting out the plastic membrane using a scalpel.
   NOTE: Ensure the plate containing the peptide gels is outside the cell culture incubator for as short a time as possible prior to testing. Media with a bicarbonate buffering system relies on the presence of CO₂ to maintain pH. Gels that have been outside the incubator too long will drift in pH, which can affect rheological assessment. It can be beneficial to maintain the gels with 10 mM HEPES added to the media to prevent this effect.
3. Carefully transfer the gel to the rheometer plate. Then, using a scalpel, trim the height of the gel to approximately 1 mm to minimize gel deformation when loaded under the rheometer plate.
   NOTE: Be careful not to touch or damage the rheometer plate when using the scalpel.
4. Set the parallel plate spacing to 1 mm. Trim any excess gel that is not covered by the rheometer plates.
5. Run the desired test settings on the rheometer, according to manufacturer’s instructions.
   NOTE: For each new sample condition, it is advised to run an amplitude sweep from 0.1–100% strain to ensure that all tests are carried out at a strain level within the sample’s linear viscoelastic region.

8. Live/dead staining of encapsulated cells

1. Remove the medium from the wells, and wash peptide gels twice with 1x PBS, using the same technique as for a media change (step 5.7).
2. Remove peptide gels that have been cultured in 24 well plate inserts following step 7.2. Keep gels in 1x PBS in the original well plate until ready to stain.
   NOTE: Take care as the gels can be fragile at this point, especially after extended culture.
3. Prepare a live/dead staining solution, allowing for 500 μL stain per 24 well plate insert, or 50 μL per well of a 96 well plate. A typical stain is 4 μM ethidium homodimer and 2 μM calcein AM in 1x PBS. Protect the resulting solution from light.
   NOTE: Concentrations of live/dead reagents may require further optimization depending on the cell type used and reagent supplier.
4. Carefully remove PBS from each gel and replace with a few drops of the staining solution, making sure each gel is well-covered.
5. Incubate gels in the staining solution in the dark for 10–15 min, then visualize using a confocal/fluorescent microscope.
   NOTE: For higher quality images, it can be beneficial to transfer the gels to glass-bottom dishes of coverslip thickness.

9. Fixing peptide gels for end-point imaging

1. Wash peptide gels following step 8.1.
2. Add 4% paraformaldehyde (PFA) in 1x PBS: 100 μL for each well of a 96 well plate and 1 mL for each gel in a 24 well plate insert (a few drops should be added on top of the gel inside the insert).
   CAUTION: Paraformaldehyde (PFA) is highly toxic and is readily absorbed through the skin. It is extremely destructive to the skin, eyes, mucous membranes, and upper respiratory tract. PFA should be handled in a fume hood and users should wear protective clothing and gloves. Alternative chemical fixatives may be used in the same way according to the end application.
3. Incubate peptide gels in PFA fixative for 1 h at room temperature.
4. Remove PFA fixative and wash peptide gels twice with 1x PBS.
   NOTE: The protocol can be paused here, store fixed peptide gels at 4 °C in 1x PBS for up to 4 weeks, making sure the plate is well sealed with paraffin film.

10. Embedding peptide gels for sectioning

NOTE: Embedding peptide gels in 4% agar is a crucial step prior to paraffin-embedding for immunohistochemistry. Alternatively, gels may be embedded in 2% agar and sectioned using a vibratome (typically 500 μm sections give good results). This is an optional step, producing hydrated gel sections that can be beneficial for staining extracellular matrix localization in the gel, using the methods in section 11.

1. Prepare molten 2% or 4% agar solution in 1x PBS (see note above), by boiling in a microwave. Allow to cool for a few minutes before using.
   NOTE: Molten agar presents a heat hazard – handle with care using hand and face protection. Once prepared, agar solution may be stored at 4 °C until needed.
2. Remove peptide gel from the cell culture insert following step 7.2.
3. Using a plastic Pasteur pipette, cover the base of a histological embedding mold with a thin layer of agar solution. Leave to cool at 20 °C for a few seconds.
4. Using a spatula, place the peptide gel into the center of the agar. Then completely cover the peptide gel in agar.
   NOTE: The gel should not sink into the agar. If it does, remove the gel and wait a few more seconds until the agar has solidified more and try again. Try not to let too much solidification take place or there will be a weak join between the two layers.
5. Allow the embedded gel to cool for 1 h at 4 °C before removing from the histological mold.
   NOTE: The protocol can be paused here, store embedded gels at 4 °C in 1x PBS for up to 4 weeks.
6. If immunohistochemistry is to be carried out, put embedded gels into a tissue processor, and proceed using standard lab methods.
   NOTE: Alternatively, embedded gels may be cut into hydrated sections using a vibratome. Hydrated sections should be stored in sealed plates at 4 °C in 1x PBS for up to 4 weeks.

11. Staining cells in gels using immunocytochemistry

1. Remove the 1x PBS covering the peptide gels/gel sections. Remove any gels still in 24 well plate inserts following step 7.2.
2. Cover gels/gel sections in a blocking buffer and incubate for 30 min at 20 °C.
   NOTE: A typical blocking buffer consists of 0.5% bovine serum albumin (BSA) in 1x PBS with 0.1% Triton X-100. Triton X-100 is toxic and causes serious eye damage, skin irritation and is very toxic to aquatic life. Users should wear protective clothing, eye protection and gloves.
3. Prepare primary antibodies in blocking buffer at optimized working concentrations. Allow 200 μL per 24 well plate gel, 100 μL per gel section, and 50 μL per 96 well plate well.
   NOTE: Typically, the antibody concentrations used for 3D staining in gels should be double the concentration used in 2D.
4. Remove blocking buffer and add antibody solution to the gels dropwise.
5. Seal plate with paraffin film and incubate overnight at 4 °C.
6. Remove antibody solution and wash twice with blocking buffer.
7. Add secondary antibody following the same procedures described in steps 11.3 and 11.4.
8. Incubate in the dark overnight at 4 °C, or for 3 hours at 20 °C.
9. Remove antibody solution and wash twice with 1x PBS.
10. Cover samples in 1:1,000 DAPI solution and incubate at 4 °C in the dark for 1 h.
11. Transfer the gel to a glass coverslip and image by fluorescent/confocal microscopy.

12. RNA extraction

NOTE: The volumes used in this method are applicable where the peptide gels are seeded into 24-well plate inserts. Other gel formats can be used, and volumes adjusted accordingly.

1. Remove the medium from the wells, and wash peptide gels twice with 1x PBS, using the same technique as for a media change (step 5.7). 
2. Remove peptide gels that have been cultured in 24 well plate inserts following step 7.2. Place each gel into a separate 15 mL centrifuge tube.
   NOTE: Take care as the gels can be fragile at this point, especially after extended culture. 
3. Using a P1000, add 500 μL Trypsin-EDTA (0.25%) to each tube and pipette up and down to mix, and disrupt the gel.
4. Incubate gels in the Trypsin-EDTA at 37 °C for 3-5 min.
   NOTE: Incubation times may require optimization depending on the cell type used. 
5. Add 5 mL of 1x PBS to dilute the Trypsin-EDTA.
6. Centrifuge at 200 x g for 5 min to pellet cells.
7. Remove the supernatant.
   NOTE: Take care as a gel layer may have formed in between the cell pellet and the supernatant.
8. Resuspend the cell pellet in lysis buffer, according to the manufacturer’s instructions, and proceed following standard protocols for RNA extraction.