A Large-format Polyacrylamide Gel with Controllable Matrix Mechanics for Mammalian Cell Culture and Conditioned Media Production

Materials and reagents

1. 15 mL conical tubes (CELLTREAT, catalog number: 667016B)

2. Cleanroom wipes (Fisher Scientific, Contec, catalog number: 19-130-5932)

3. Plastic spoons and clear plastic cups (recommend Amazon or any major retailer)

4. Feather scalpel blades #11 (Electron Microscopy Sciences, catalog number: 72044-11)

5. Sylgard 184 silicone elastomer kit (Krayden, catalog number: DC4019862)

6. 145 mm glass Petri dishes (VWR, catalog number: 25354-127)

7. 145 mm plastic Petri dishes (VWR, catalog number: 82050-600)

8. 0.2 μm PES bottle top filter (VWR, catalog number: 10040-436)

9. Autoclavable sterilization pouches (VWR, Cardinal Health, catalog number: 11213-033)

10. Sodium hydroxide (0.1 M NaOH)

11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888)

12. 3-aminopropyltriethoxysilane (APTES) (Fisher Scientific, ACROS organics, catalog number: AC430941000); store at 4 °C, avoid contact with air

13. 25% glutaraldehyde (GA) (VWR, Electron Microscopy Sciences, catalog number: 100504-788); store at 4 °C. We have found that this GA can be stored for up to three years without any changes to the end result

14. 40% acrylamide (Sigma-Aldrich, catalog number: A4058); store at 4 °C

15. 2% bis-acrylamide (Sigma-Aldrich, catalog number: M1533); store at 4 °C

16. Ammonium persulfate powder (APS) (VWR, catalog number: 97064-594), store at room temperature in a desiccator, strong oxidizer

17. Tetramethylethylenediamine (TEMED) (VWR, catalog number: 97064-684); flammable

18. 125 mm clear acrylic disc (127 mm/5 inch acrylic discs may be found on Amazon, or acrylic sheets may be purchased from McMaster-Carr and laser cut into any size or shape)

19. Sulfo-SANPAH (Covachem, catalog number: 13414-100); store at 4 °C

20. HEPES (Sigma-Aldrich, catalog number: H3375-500G)

21. Protein of interest (e.g., collagen, Corning, catalog number: 354236); store at 4 °C

22. Hank’s buffered salt solution (HBSS) (ScienCell, catalog number: 0313)

23. Milli-Q water (or equivalent grade)

24. Isopropanol (70% solution)

25. Ethanol (70% solution)

26. Dimethyl sulfoxide (DMSO) (Sigma-Aldrich, catalog number: 276855)

27. Ammonium persulfate, 10% w/v solution (see Recipes)

28. Sulfo-SANPAH, 100 mg/mL solution (see Recipes)

29. HEPES-buffered saline (HBS) (see Recipes)

Equipment

1. Vacuum desiccator

2. Balance/weighing scale

3. Glass beaker (VWR, catalog number: 13912-284)

4. Filter forceps (VWR, catalog number: 89259-954)

5. Scalpel handle #3 (Fine Science Tools, catalog number: 10003-12)

6. Pipet aid and serological pipets (10, 25, 50 mL)

7. Set of micropipettes and tips (P10, P200, P1000)

8. Oven, set at 65 °C

9. Chemical hood

10. Biosafety cabinet with vacuum aspirator

11. Autoclave

12. UV lamp (e.g., 100-Watt 365 nm UVP lamp)

13. CO₂humidified incubator, set at 37 °C

14. -20 °C freezer

15. 4 °C refrigerator

Procedure

1. Creation of polydimethylsiloxane (PDMS) ring molds

   1. Using a balance, weigh Sylgard 184 elastomer base and Sylgard 184 curing agent into a plastic cup at a 9:1 ratio (e.g., 27 g of elastomer base and 3 g of curing agent).

      Note: Uncured Sylgard 184 elastomer base is highly viscous.

   2. Mix the PDMS pre-polymer thoroughly with a plastic spoon and pour into plastic 145 mm Petri dishes, making sure the surface is evenly coated. The amount of PDMS pre-polymer can be increased or decreased, but 1–1.5 mm final thickness within the Petri dish is recommended.

   3. Degas Petri dishes in a vacuum chamber to remove air bubbles from pre-polymer mixture.

   4. Cure the PDMS pre-polymer overnight in an oven set at 65 °C.

      Note: Make sure the oven surface is leveled for a uniform PDMS thickness. As little as ~1° slope will adversely impact downstream processes.

   5. With gloved hands, remove the cured PDMS discs from the Petri dishes and cut into rings. Using a scalpel, cut out a ring with an outer diameter of 145 mm and an inner diameter of 120 mm to serve as the gasket.

      Suggestion: Trace a circle with a diameter of 120 mm on the PDMS disc using a pen or non-solvent resistant marker. The acrylic cover must overlap the PDMS to get a good seal during polymerization.

   6. Clean PDMS rings with 70% isopropanol followed by 70% ethanol. Dry completely.

   7. Seal PDMS rings inside an autoclavable pouch and use a 30 min sterilization cycle with a minimum temperature of 120 °C (see section F for reusability).

      
      

2. Glass dish surface activation

   Note: This section should be performed in a chemical fume hood due to hazardous chemical use.

   1. Add enough 0.1 M NaOH to cover the bottom of the 145 mm glass Petri dishes and let sit for 30 min.

   2. Remove excess NaOH and let dishes dry completely.

   3. In a glass beaker, mix APTES into isopropanol to make a 2% solution.

   4. Pour APTES solution into dishes until surfaces are fully covered (20–30 mL/dish) and let sit for 10 min.

      Note: APTES is reactive to oxygen. The container from the listed manufacturer has a regenerative rubber seal where the contents are meant to be removed via syringe needle and volumetrically replaced with an inert gas (e.g., N₂). APTES loses its reactivity when exposed to molecular oxygen and will therefore become less effective over time. This step extends the shelf life of APTES.

      CAUTION: APTES is corrosive; chemical-resistant gloves, lab coat, and goggles should be used.

   5. Following the reaction, remove the APTES solution and add ~30 mL of Milli-Q water to each dish; swirl dishes to mix, let sit for 5 min, and then remove water.

      1. Repeat this step three more times for a total of four exchanges.

      2. For each exchange, dispose in appropriate hazardous waste stream.

   6. Add 20–30 mL of 0.5% glutaraldehyde diluted with Milli-Q water to each of the glass dishes and let sit for 30 min.

   7. Dispose of the glutaraldehyde solution in an appropriate hazardous waste stream and allow dishes to air dry. Proceed to polymerization step (section C) within 24 h of activation for optimal gel attachment.

      
      

3. Polyacrylamide gel polymerization

   Note: Parts of this section are recommended to be performed in a biosafety cabinet (BSC) and/or laminar flow hood to minimize contamination. Wipe down all materials with 70% ethanol before placing them in the BSC.

   1. Prepare pre-polymer solutions of acrylamide.

      1. Many polyacrylamide gelrecipesare published in the literature that report the ratios of bis-acrylamide and acrylamide to produce the desired elastic modulus (Aratyn-Schaus et al., 2010;Tse and Engler, 2010; Syed et al., 2015; Charrier et al., 2020).

      2. Using any published reference table, add 40% acrylamide, 2% bis-acrylamide, and Milli-Q water in the desired ratios in a 15 mL conical tube for a total volume of 15 mL of solution per 145 mm glass dish. Optional: Pre-polymer solutions can be made up in larger volume batches, wrapped in parafilm, and stored at 4 °C for 3–4 days. Fresh solutions should be used when possible because even minor evaporation of solution over time can cause noticeable shifts in the storage modulus upon polymerization.

      3. Degas solutions in a vacuum chamber for at least 30 min to remove dissolved oxygen.

   2. In a BSC, use forceps to remove sterile PDMS rings from the autoclave pouch and place them inside the activated glass Petri dishes. Press down firmly on the PDMS ring with the forceps to ensure good contact with the glass.

   3. Spray down acrylic discs with ethanol and dry with cleanroom wipes or compressed air and set inside BSC.

   4. Bring degassed pre-polymer solutions inside BSC. Initiate the reaction by adding ammonium persulfate (1:100 dilution) and TEMED (1:1,000 dilution).

   5. Gently invert tube 2–3 times and pour ~15 mL into the glass Petri dish.

   6. Place the acrylic cover on top of the PDMS ring to create a seal.

      1. Like mounting a coverslip to a slide, grab one side of the acrylic cover with forceps and set onto the PDMS ring at an angle.

      2. Lower the acrylic cover—watch as it makes contacts with the solution and check for any bubbles in the process.

      3. Pipette any excess solution from around the ring, place solution back into the conical tube, and cap the tube. Use the extra solution in the conical tube to gauge when the polyacrylamide has gelled, as it will indicate when the dish is ready.

   7. After the polyacrylamide has fully polymerized (~15 min at room temperature), remove the acrylic cover.

      Aspirate unpolymerized solution at the PDMS–acrylic interface.

      Note: Unpolymerized solution is expected. The overlap between the PDMS ring and the acrylic cover is designed to prevent uneven gelation at the edges and facilitate removal of the acrylic cover.

      1. Using a P1000 micropipette, add 100% isopropanol into the PDMS–acrylic interface where the unpolymerized solution sat until it penetrates the polyacrylamide–acrylic interface. Gently lift up on the acrylic cover to help the isopropanol enter the gel-cover interface while pipetting.

      2. Once the isopropanol has completely infiltrated the polyacrylamide–acrylic interface, the acrylic cover should pop off very easily.

   8. Wash polyacrylamide gel three times with HBS.

      Note: Do not put vacuum aspirator on or directly above the gel as it may get caught in the vacuum. Instead, place the aspirator on the PDMS ring and tip the dish so the fluid falls into the vacuum aspirator.

   9. Polyacrylamide gel dishes can be stored at 4 °C in HBS, sealed with parafilm or plastic wrap.

      
      

4. Matrix protein-polyacrylamide conjugation

   Note: This section is recommended to be performed in a BSC with aseptic technique whenever possible, to minimize contamination. The extracellular matrix (ECM) protein of choice (fibronectin, collagen, laminin, etc.) depends on the cell line used and will need to be tested for suitability.

   1. Prepare working solutions of the ECM protein of interest (e.g., collagen at 100 μg/mL) by diluting in HBS.

   2. Remove the storage buffer from the polyacrylamide dishes.

   3. Dilute sulfo-SANPAH stock solution to 0.5 mg/mL in Milli-Q water and pipette directly on the surface of polyacrylamide gels (~5 mL). Sulfo-SANPAH hydrolyzes rapidly in water, so this step needs to be done promptly.

   4. Place the dishes under the UV light for up to 10 min to activate the surface.

      Note: Remove the lids to the dishes before placing under UV light.

      1. Sulfo-SANPAH should turn burgundy (dark orange) when it has reacted with the polyacrylamide.

      2. Activation time may change based on UV lamp wavelength, power, and distance of dish from light source. For the lamp listed above, a distance of three inches from the light source for 10 min is sufficient.

   5. Wash the polyacrylamide dishes three times with HBS to remove excess sulfo-SANPAH.

   6. Add the ECM protein solution directly on top of the polyacrylamide and place the dishes in a cell culture incubator at 37 °C overnight.

      1. Roughly 10 mL of solution is sufficient for a uniform conjugation.

      2. We found that an overnight incubation worked well, but less time (1–2 h) may be sufficient.

   7. After incubating with the protein solution, wash once with HBS and replace with fresh HBS.

   8. Soak the gels in HBS overnight (or 2–3 h) at 37 °C.

      1. Repeat this step with one more exchange of fresh HBS to remove excess unconjugated sulfo-SANPAH and/or ECM protein if necessary.

      2. The gel should turn to a very light transparent orange.

      3. Collagen can be visualized under a light microscope equipped with a 10× objective, which can be used to check for any non-uniformity.

   9. Dishes can be stored at 4 °C in HBS sealed with parafilm or plastic wrap for weeks.

      
      

5. Cell culture

   Note: This section should be performed in a BSC with aseptic technique whenever possible, to minimize contamination.

   1. Remove HBS and incubate dishes with fresh cell culture media for 1 h at 37 °C prior to seeding cells.

   2. Plate cells by dripping the cell suspension over the entire surface and allow to adhere for ~1 h.

   3. Add 25–30 mL of media once cells have adhered to the surface.

   4. When cells are ready to be passaged, remove the cell media, add warm HBSS or serum-free media to the dishes, and let sit for 15–30 min in a cell culture incubator.

      Note: This will wash the cells in preparation for trypsinization.

      1. If cell media contains a high concentration of serum (e.g., 10% fetal bovine serum) or polyacrylamide gels are softer (< 10 kPa), more than one exchange may be necessary to remove excess protein within the gels.

      2. Serum proteins must be adequately removed to prevent inhibition of the trypsin.

      3. If phenol is present in cell media, the polyacrylamide gels should change from a pink color to almost clear, indicating the cells are ready to be trypsinized.

   5. Remove HBSS or serum-free media and proceed with routine passaging.

      
      

6. Cleaning and reusing of the glass dish

   1. With gloved hands, remove PDMS ring, clean with warm, soapy water, and sterilize in the autoclave. PDMS rings can be placed inside a beaker of isopropanol and/or detergent solution before sterilization to remove absorbed material.

   2. Remove polyacrylamide from the dish. Polyacrylamide can be removed by gloved hand or with forceps.

   3. Bleach glass dish for at least 15 min, rinse with copious water, and clean with warm soapy water.

   4. Sterilize dishes in autoclave. Dishes are ready to be re-used (start at section B: Glass dish surface activation).

Data analysis

Cell viability and cell debris measurements were performed in technical and experimental triplicates using an Agilent NovoCyte flow cytometer. An ordinary one-way ANOVA was performed on cell viability data and Brown-Forsythe and Welch’s ANOVA were performed on cell debris data.

Recipes

1. Ammonium persulfate, 10% w/v solution

   Dissolve in deionized water at a concentration of 10% w/v. Ammonium persulfate should crackle as it dissolves in water.

   Aliquot and store at -20 °C for up to six months.

   
   

2. Sulfo-SANPAH, 100 mg/mL solution

   Dissolve sulfo-SANPAH in DMSO at 100 mg/mL.

   Aliquot in 25 μL volumes in microcentrifuge tubes and freeze at -80 °C for up to one year.

   
   

3. HEPES buffered saline (HBS)

   Dissolve 50 mM HEPES and 0.9% w/v NaCl in Milli-Q water.

   Bring to pH 8.0 with NaOH.

   Sterile filter solution using 0.2 μm PES bottle top filter.

Acknowledgments

This work was funded by the National Institutes of Health (R01GM26643, R01HL133163, R01HL145147, U19AI158930). The protocol was adapted from Millar-Haskell et al. (2022). The authors would like to acknowledge John Sperduto and Bryan Ferrick for their assistance in acquiring materials.

Competing interests

The authors declare no competing interests.

Ethics

No animal or human subjects were used in this work.

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