Extracellular Matrix-derived Foam Fabrication via Lyophilization: A Procedure to Generate Biological Scaffolds from ECM of Decellularized Tissues

1. ECM-derived Foam Fabrication

1. Prepare the homogenous ECM suspension by dispersing ECM fine powder in buffer solution and incubate at 37 °C with continuous agitation at 120 rpm until the suspension is warm.
2. Dilute the ECM suspension in 0.2 M acetic acidic to the desired concentration.
   NOTE: Depending on the ECM source, stable foams may be prepared in the range of 10–100 mg/mL, with 15–50 mg/mL as the recommended range for DAT, DDT, and DLV. Typically, foams fabricated at higher concentrations will be slightly less porous but more stable in culture and easier to handle.
3. Using a 3 mL syringe with an 18G needle to prevent bubble formation in the ECM suspension, fill the desired mold with the ECM suspension. To fabricate the DAT, DDT, and DLV foams, dispense 400 μL of 35 mg/mL ECM suspension into a 48-well cell culture-treated plate.
   NOTE: The shape of the selected mold and the volume of ECM suspension will determine the geometry of the resultant foam.
4. Cover and freeze the molds overnight by placing them in a -20 °C or -80 °C freezer.
   NOTE: The freezing temperature may affect the porosity of the foams. Larger pores are expected when the samples are frozen at -20 °C as compared to -80 °C due to the formation of larger ice crystals. To fabricate foams with a uniform porous structure, ensure that the mold is not in contact with a conductive surface to prevent directional cooling.
5. Place the molds containing the frozen samples into a lyophilizer flask. Connect the lyophilizer flask to the laboratory freeze dryer system and dry for 24 h.
   NOTE: It is important that the sample remains frozen prior to lyophilization.
6. Store the lyophilized foams in a desiccator until required.