The Generation of Tissue-Specific ECM Hydrogels From Melanoma and Associated Organs to Study Cancer Biology

Materials and reagents

Biological materials

1. YUMM 1.7 murine melanoma cells (ATCC, CRL-3362)

2. B16-F10 murine melanoma cells (ATCC, CRL-6475)

3. C57BL/6 mice (Harlan Laboratories Israel)

Reagents

1. Double-distilled water (DDW)

2. 1× phosphate-buffered saline (PBS) (Sigma-Aldrich, catalog number: L0615-500ML)

3. 10× PBS (Sigma-Aldrich, catalog number: D1408-500ML)

4. Triton X-100 (Sigma-Aldrich, catalog number: T8787)

5. Ammonium hydroxide solution 25% (Biolab, catalog number: 125050100)

6. DNase I powder 3,520 U/mg (Worthington, catalog number: LS002139)

7. Ethanol, absolute >99.8% (Gadot Group, catalog number: 64-17-5)

8. Hydrogen peroxide (H₂O₂), 33% (Panreac, catalog number: 131077.1211)

9. Acetic acid glacial 99.7% (BioLab, catalog number: 107052100)

10. Tris base (Sigma-Aldrich, catalog number: T1503)

11. Ethylenediaminetetraacetic acid (EDTA) (Sigma-Aldrich, catalog number: EDS-100G)

12. Trypsin 0.25%–EDTA (0.038%) in HBSS (IMBH, catalog number: L0931-500ML)

13. Pepsin from porcine gastric mucosa powder (Sigma-Aldrich, catalog number: P7012-1G)

14. Sodium hydroxide pearls (NaOH) (Biolab, catalog number: 1908059100)

15. Hydrochloric acid 32% (HCl) (BioLab, catalog number: 846050100)

Solutions

1. Triton X-100/ammonium hydroxide solution (see Recipes)

2. Triton X-100/0.26% EDTA/0.69% Tris (see Recipes)

3. 0.4% peracetic acid/4% ethanol solution (see Recipes)

4. Pepsin solution (see Recipes)

5. DNase I solution 50 U/mL (see Recipes)

6. 70% ethanol (see Recipes)

7. 3% H₂O₂ (see Recipes)

Recipes

1. Triton X-100/ammonium hydroxide solution

2. Triton X-100/0.26% EDTA/0.69% Tris

3. 0.4% peracetic acid/4% ethanol solution

4. Pepsin solution

Note: This recipe is calculated based on preparing solutions for processing 50 mg of dry ECM powder. Volumes and reagent quantities should be scaled accordingly if processing larger or smaller tissue amounts. Ensure precise weighing of the ECM powder prior to preparation and adjust all components proportionally to maintain the specified final concentrations.

5. DNase I solution 50 U/mL

6. 70% ethanol

7. 3% H₂O₂

Note: All recipes should be made fresh.

Laboratory supplies

1. Eppendorf tubes 1.7 mL (Corning Inc., catalog number: 28324032)

2. Scintillation vials (Sigma-Aldrich, catalog number: 986546)

3. Depilatory cream

4. 27G needle (Bactlab, catalog number: BD300635)

5. Scissors (Sigma-Aldrich, catalog number: 41122408)

6. Butterfly needle (Bar Naor, catalog number: BNSCLPV23g)

7. 20 mL syringe (BD Emerald, catalog number: 307736)

8. 200 μL pipette tips (TH Geyer, catalog number: 7695884)

9. 50 mL conical tubes (Greiner Bio-One, catalog number: 227261)

Equipment

1. Lyophilizer (Labogene, model: CoolSafe 110-4, catalog number: 7001000115)

2. Shaker-incubator (Benchmark, catalog number: H2010)

3. Horizontal shaker (ELMI, catalog number: S-3.02)

4. Cellular centrifuge (Heraeus, catalog number: PICO17)

5. Intelli-Mixer (ELMI, catalog number: RM-2L)

6. Handheld tissue homogenizer (VISOSCI, catalog number: B0CG4KPQW5)

7. 37 °C dry incubator (Heracell, catalog number: 150i)

8. -80 °C freezer

9. Refrigerator (2–8 °C)

Procedure

Note: The following protocol is divided into two parts. Part I is the decellularization process for tumor, lung, and skin tissues. Part II is the freeze-drying and gelation processes. The overall protocol is illustrated in the graphical abstract. It is important to note that the procedure does not require aseptic conditions or prior filtration of solutions and reagents, as the final pre-gel solution can be irradiated with UV light for 15 min before use in downstream assays.

Part I. Decellularization

Decellularization is the essential first step in producing intact, tissue-specific ECM for hydrogel preparation. Its goal is to efficiently remove cellular content without disrupting the native structural and biochemical composition of the matrix. Because tissues differ significantly in cellularity, density, and ECM architecture, we employ modified protocols for tumors, lungs, and skin to ensure consistent removal of cells while preserving ECM integrity for downstream freeze-drying and gelation.

A. Decellularization of tumors and lungs

1. Euthanize mice according to institutional animal care and use committee (IACUC) guidelines.

2. Cardiac perfusion: Insert a butterfly needle into the left ventricle of the mouse heart and make a small cut on the right ventricle.

3. Connect a 20 mL syringe to the tubing distal edge and flow DDW through it using low-to-medium pressure.

4. Remove the lungs or tumor and soak in 50 mL of DDW within a 50 mL tube. Rotate for 2 h at 4 °C using an Intelli-Mixer device at 30 rpm.

Note: Tumors were generated by injecting YUMM 1.7 or B16-F10 melanoma cells intradermally into the dorsal flank of C57BL/6 mice (typically, 5 × 10⁵ cells in 100 μL of HBSS per mouse). Tumors were allowed to grow until they reached approximately 1,500 mm³.

5. After 2 h, replace the DDW.

6. Rotate further for at least 48 h at 4 °C and 30 rpm. Replace the DDW once every 24 h.

Note: Proceed to the next step only when the lungs or tumors no longer appear reddish.

7. Exchange the DDW with 50 mL of the Triton X-100/ammonium hydroxide solution. Rotate on Intelli-mixer for an additional 48 h at 4 °C and 30 rpm.

Note: Triton X-100 solubilizes cellular membranes, while ammonium hydroxide helps remove nuclear material and loosens tissue architecture.

8. Monitor the clearness of the lungs or tumors. If the organs are not clear yet, replace the Triton X-100/ammonium hydroxide solution with a fresh solution and continue to rotate for an additional 24 h.

9. Wash with DDW for 15 min × 3 times on a horizontal shaker at room temperature (RT) and 300 rpm.

10. Fat removal (for tumor only, performed at RT): Change to 50 mL of 70% ethanol and rotate for an additional 12 h, followed by 15 min in 50 mL of 3% H₂O₂, and an additional 2 h in 50 mL of 0.4% peracetic acid solution/4% ethanol. Follow with three washes with DDW for 15 min, all performed on a horizontal shaker at RT and 300 rpm.

Note: 70% ethanol removes lipids and helps dehydrate tissues, H₂O₂ oxidizes and bleaches residual pigments, and peracetic acid acts as a potent disinfectant and removes residual organic material while preserving ECM structure.

11. Insert tissues in 5 mL of DNase I solution and rotate at 150 rpm on a shaker-incubator at 37 °C for 1 h.

Note: DNase I digests residual nucleic acids, ensuring complete removal of DNA before downstream processing.

12. Wash with DDW for 2 h on an Intelli-mixer device at 4 °C and 30 rpm.

13. After 2 h, replace the DDW with fresh DDW and wash for another 24 h at 4 °C and 30 rpm.

14. Transfer decellularized tissue sections into a 1.7 mL Eppendorf and add 200 μL of PBS 1×.

15. Using scissors, cut the sections into small pieces.

16. Centrifuge at 11,000× g for 2 min, then gently discard as much fluid as possible.

17. Store at -80 °C.

Note: Issues can be stored for up to 1 year at this point.

B. Decellularization of skin

1. Euthanize according to institutional animal care and use committee (IACUC) guidelines.

2. Insert a butterfly needle into the left ventricle of the mouse heart and make a small cut on the right ventricle.

3. Connect a 20 mL syringe to the tubing distal edge and flow DDW through it at low-medium pressure.

Note: Relevance of perfusion: Although the skin is collected externally, cardiac perfusion is performed to remove blood from dermal and subdermal vessels. This reduces residual hemoglobin and cellular components, improves decellularization efficiency, and enhances the clarity and consistency of the resulting ECM for hydrogel preparation.

4. Gently shave the desired skin section and apply depilatory cream for 1–2 min.

5. Collect the skin section by separating it gently from the underlying tissues.

6. Separate the subcutaneous layer manually using a scalpel.

7. Cut the skin into 1 cm² rectangles, put in a 1.7 mL Eppendorf tube, and store at -80 °C until use.

Note: Tissues can be stored for up to 1 year at this point.

8. Place 1–2 skin sections into one 50 mL tube and incubate sequentially in 50 mL of each of the following solutions on a horizontal shaker (300 rpm) at RT (unless otherwise specified):

a. 0.25% Trypsin-EDTA (0.038%) in HBSS: 6 h at 37 °C and 300 rpm.

b. DDW: 15 min × 3 washes.

c. 70% Ethanol: 10–12 h (once).

d. 3% H₂O₂: 15 min (once).

e. DDW: 15 min × 2 washes.

f. 1% Triton X-100/0.26% EDTA/0.69% Tris: 6 h (once) followed by overnight incubation.

g. DDW: 15 min × 3 washes.

h. 0.4% peracetic acid/4% ethanol: 2 h (once).

i. PBS: 15 min × 2 washes.

j. DDW: 15 min (once).

k. DNase I solution (50 U/mL): 1 h at 37 °C and 300 rpm.

l. PBS: 15 min × 2 washes.

m. DDW: 15 min × 3 washes, followed by overnight incubation.

9. Transfer the decellularized tissue sections into a 1.7 mL Eppendorf tube and add 200 μL of PBS.

10. Using scissors, cut the section into small pieces.

11. Centrifuge at 11,000× g for 2 min, then gently discard the fluid as much as possible.

12. Store at -80 °C.

Note: Tissues can be stored for up to 1 year at this point.

Note: An example of a successful decellularization process in lungs is provided in Figure 1, where the lungs no longer appear reddish. In addition, the decellularization process can be evaluated using hematoxylin and eosin (H&E) staining to determine the lack of nuclei and cells in the decellularized tissue (Figure 2). Moreover, additional validation for the decellularization process can be done by analyzing the content of DNA in the decellularized tissue (Figure 3).

Figure 1. Gross morphology of murine lungs before and after decellularization. (A) Mouse lung tissue prior to decellularization, showing a characteristic red coloration of intact cellular and vascular components. (B) Lungs following completion of the decellularization protocol, demonstrating loss of cellular material and blood components, resulting in a pale, translucent tissue. This comparison visually illustrates the effectiveness of the decellularization process described in the protocol.

Figure 2. Histological evaluation of native and decellularized tissues. Representative hematoxylin and eosin (H&E) staining images of murine tissues: skin (A–B), lung (C–D), B16-F10 (E–F), and YUMM 1.7 (G–H) melanoma tumors, before (top row) and after decellularization (bottom row). n ≥ 3 samples/group, imaged at 20× magnification, scale bar, 100 μm. Decellularized matrices retained overall extracellular structure while showing effective removal of cellular material. In B16-F10 tumors (F), residual pigment is evident, representing melanin spillover from melanoma cells, which persists despite decellularization.


Figure 3. DNA content of native vs. decellularized tissues. DNA concentration (ng/μL) of murine skin (A), lung (B), and B16-F10 tumors (C) before (Control) and after (Decell) the decellularization process (n = 3 samples/group, mean ± SD). Genomic DNA was isolated using the NucleoSpin Tissue kit (Macherey-Nagel) according to the manufacturer’s protocol and quantified using NanoDrop. All tissues show a significant reduction in DNA following the decellularization process, confirming effective removal of cellular material. The dashed line represents the threshold of the effective decellularization process, as previously published [16,17]. *p < 0.05, using an unpaired two-tailed t-test.

Part II. Freeze-drying and gelation

The purpose of this step is to process the decellularized tissue into a soluble form that can subsequently self-assemble into hydrogels. The freeze-dry procedure removes water content while preserving ECM proteins, ensuring long-term storage and accurate downstream quantification. Mechanical fragmentation and pepsin digestion are then used to solubilize the ECM into small protein fragments. After pH neutralization and adjustment to the desired concentration, the solution is incubated at 37 °C, enabling ECM proteins to reassemble and form hydrogels.

In practice, decellularized tissues are rapidly transferred from the freezer to the lyophilizer to prevent thawing. After lyophilization, samples are mechanically crushed (e.g., with a handheld tissue grinder) and digested in pepsin solution. The digested ECM is neutralized with NaOH and diluted to ~8 mg/mL protein. For fat-rich samples, especially dermal/subdermal tumors, a further clarification step is included.

1. Punch Eppendorf tube cups with a 27G needle (7–10 holes). Cut out the punched cup and place it on the tissue-containing Eppendorf tubes.

2. Quickly apply the samples to a lyophilizer at -110 °C for 24 h (tumors and lungs) or 48 h (skin). When the lyophilization ends, quickly remove the punched cups and close the Eppendorf tube with the original cups.

3. Put the tubes at -80 °C.

Note: The samples can be kept in a -80 °C freezer for several months.

4. Before crushing, weigh the tissues and record the weight.

5. Grind the tissue using a handheld tissue grinder by adding 200 μL of pepsin solution to the Eppendorf tube containing the tissue sample. Grind until the solution is homogeneous. If needed, add more pepsin solution and grind until it becomes homogeneous. Mark how much pepsin solution is being used during the grinding process.

6. Transfer the ground tissue into a scintillation vial.

Critical: After the tissue is transferred to the scintillation vial, add pepsin solution to achieve a final ratio of 10 mg of lyophilized decellularized tissue powder per 1 mL of pepsin solution (if this ratio has already been achieved during the grinding process, no additional pepsin is required).

7. Incubate for 24 h (skin) or 48 h (lungs and tumors) on a horizontal shaker at 150 rpm at RT.

Critical: After 24 h, titrate the pepsin-digested solution to pH 7.2–7.4 using 0.1 M NaOH. Add NaOH gradually in small increments to avoid rapid pH shifts and carefully monitor the pH to prevent exceeding pH 7.5. Record the volume of NaOH added.

8. Add sterile 10× PBS equivalent to one-ninth of the final solution volume.

9. Add sterile 1× PBS to reach the target ECM concentration (typically ~8–10 mg/mL protein).

10. Optional clarification step: If visible debris is present, transfer the digested solution to a new 1.7 mL Eppendorf tube and centrifuge at 11,000× g for 2 min. Carefully collect the supernatant to enhance gel clarity.

11. The pre-gel can be stored at 4 °C for up to 1 week before use.

12. Transfer the pre-gel solution to a required tissue culture plate, transwell insert, or other platform according to the intended downstream application. Incubate at 37 °C for 1 h to allow self-assembly and hydrogel formation.

a. Recommended hydrogel volumes and thicknesses:

i. 96-well plates: 30–60 μL per well → ~0.5–1 mm gel thickness.

ii. 24-well plates: 100–200 μL per well → ~1.5–2 mm thickness.

iii. Transwell inserts: 50–100 μL per insert.

iv. In vivo injections (mice): 100–300 μL per injection site.

Notes:

1. Gelation behavior: Gels ≥ 8 mg/mL are formed consistently and readily at 37 °C. Concentrations below ~3–4 mg/mL may result in incomplete polymerization.

2. The hydrogel end-product can be stained or used for various applications, such as Sirius Red staining (Figure 4).

Figure 4. Collagen assessment in extracellular matrix (ECM) hydrogels by Sirius Red staining. Sirius Red staining of hydrogels derived from skin (A–B) and lung (C–D) decellularized murine tissues. Images were taken at brightfield (top row) and corresponding polarized light highlighting collagen birefringence (bottom row). Collagen fibers are preserved in both preparations, confirming retention of structural ECM components after decellularization and hydrogel formation. Under polarized light, skin-derived hydrogels display thicker, more organized collagen bundles, whereas lung-derived hydrogels exhibit finer, loosely arranged fibers, reflecting site-specific ECM architecture. Scale bar, 100 μm.

Validation of protocol

The reproducibility and robustness of this protocol were validated through independent preparations of ECM hydrogels derived from murine skin, lung, and melanoma tumors. Each tissue type was processed in at least three independent biological replicates, following identical decellularization, lyophilization, digestion, and polymerization steps. The following validation steps were carried out: (A) An effective decellularization process was confirmed by histological evaluation (H&E staining) showing complete removal of nuclei and cellular material. Representative microscopy images of the different tissues were obtained from three replicates. (B) DNA quantification in decellularized tissue demonstrated a significant reduction in DNA content after the decellularization process. The experiment was carried out in triplicate (Figure 3). (C) Collagen preservation and fibrillar organization after hydrogel formation were verified using Picrosirius Red staining under polarized light, revealing site-specific ECM architectures that remained consistent between preparations. Representative images from three to four replicates are shown (Figure 4).

General notes and troubleshooting

General notes

1. Tissue variability and protocol limitations: The biochemical composition and stiffness of ECM differ between tissues and even between animals, affecting digestion efficiency, gelation, and downstream cellular behavior. Therefore, results obtained using ECM from a specific site or species may not be directly comparable across models. This protocol is optimized for murine tissues and may require adjustments (e.g., detergent strength or digestion time) for other organs or species.

2. Compatibility with downstream assays: The resulting hydrogel is compatible with spheroid embedding, migration assays, immunostaining, and protein extraction.

3. Compatibility with in vivo and histological workflows: The hydrogels can be injected intradermally or subcutaneously, forming stable, retrievable depots that remain identifiable and compatible with standard histopathological staining (Figure 5).

4. Applicability to additional tumor models: This protocol is compatible with a wide range of murine solid tumors. We successfully applied the decellularization and hydrogel-generation workflow to LLC (Lewis Lung Carcinoma), 4T1 mammary carcinoma, and PyMT mammary tumors, achieving effective decellularization with only minor adjustments in detergent exposure time based on tumor cellularity and lipid content. This indicates the flexibility and robustness of the protocol for use beyond melanoma-derived tissues.

Figure 5. Host cell infiltration into intradermally injected extracellular matrix (ECM) hydrogel. Representative hematoxylin and eosin (H&E) staining images of murine skin, 7 days after intradermal injection of 300 μL of B16-F10 tumor-derived hydrogel. The central region corresponds to the hydrogel (black arrows), surrounded by normal skin tissue (red arrows). Infiltrating host cells are visible within the hydrogel, indicating cellular migration into the implanted matrix. (A) 10× magnification; scale bar, 1 mm. (B) Zoom magnification based on OlyVIA software; scale bar, 200 μm.

Troubleshooting

Problem 1: Incomplete decellularization.

Possible causes: Insufficient duration of detergent incubation, inadequate perfusion leading to retained blood, tissue pieces cut too large, limited detergent penetration, low agitation speed, or insufficient solution volume.

Solutions: Extend immersion in Triton X-100/ammonium hydroxide solution by 24–48 h and replace with fresh solution, ensure proper perfusion before tissue collection, and cut tissues into small pieces (2–3 mm).

Problem 2: Inhomogeneous hydrogel formation.

Possible causes: Incomplete solubilization of the ECM or poor mixing procedure during neutralization.

Solution: Centrifuge at 11,000× g for 2 min.

Problem 3: Batch-to-batch variation in hydrogel stiffness.

Possible causes: Variable tissue composition, incomplete lyophilization, or inconsistent ECM dry weight normalization.

Solutions: Standardize tissue weight-to-volume ratios and record all intermediate weights; normalize digestion to 10 mg of ECM powder per milliliter of pepsin solution.

Problem 4: Hydrogel does not polymerize at 37 °C.

Possible cause: Over-digestion or low ECM concentration (<3 mg/mL).

Solution: Reduce digestion time or prepare a more concentrated ECM solution. Verify if the pH is within the range of 7.2–7.6.

Acknowledgments

This protocol was adapted from previous work carried out on human and swine [12–14, 17].

The following figures were created using BioRender: Graphical overview, https://app.biorender.com/illustrations/6862806011ac182ce6b5b171?slideId=869c84f1-4568-4975-a770-520931227036.

Competing interests

The authors declare that they have no competing interests.

Ethical considerations

All animal procedures were conducted in accordance with institutional guidelines and approved by the Animal Ethics Committee of the Technion, Israel Institute of Technology (protocol number IL-074–05-2025).

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