ECM Suspension Preparation: A Technique to Obtain Homogenous Suspension from Tissue-derived Extracellular Matrix

1. Decellularized Tissue Processing

1. Decellularization and lyophilization
   1. Decellularize tissue(s) of interest following an established protocol.
      NOTE: Scaffolds in the current study have been prepared following published decellularization protocols for human decellularized adipose tissue (DAT), porcine decellularized dermal tissue (DDT), and porcine decellularized left ventricle (DLV). Commercially available, insoluble collagen can also be used to fabricate foams and microcarriers, such as collagen sourced from bovine tendon, which has been used successfully over a concentration range of 10–50 mg/mL. Other insoluble collagen sources may be utilized but may require optimization to identify the concentration range that will yield stable scaffolds.
   2. Transfer the hydrated decellularized tissue or purified collagen into a 50 mL centrifuge tube with forceps and add sufficient double distilled water (ddH₂O) to submerge the tissue, to a maximum total volume of 35 mL.
   3. Freeze the sample overnight at -80 °C, with the centrifuge tube positioned horizontally during freezing to maximize the surface area for sublimation during subsequent lyophilization.
   4. Remove the cap from the centrifuge tube and transfer the frozen sample into a lyophilization jar.
   5. Lyophilize the sample using a laboratory freeze dryer for 48–72 h or until fully dried.
      NOTE: Drying time may vary for different lyophilizers and decellularized tissues.
   6. Finely mince the lyophilized ECM into small pieces (~1–2 mm³) using sharp surgical scissors.
   7. Store the minced ECM in a desiccator until ready for further processing.
      NOTE: It is recommended that the minced ECM be used immediately to prevent moisture absorption from the environment.
2. Cryomilling
   1. Fill a 25 mL stainless steel milling chamber for a laboratory ball mill system with minced lyophilized ECM and add two 10 mm stainless steel milling balls.
   2. Close and completely submerge the loaded milling chamber in liquid nitrogen for 3 min.
   3. Mill the frozen sample for 3 min at 30 Hz (1,800 rpm) into a fine powder.
      NOTE: The number of times these steps should be repeated may vary between ECM sources. For example, DAT typically requires 3 milling cycles to generate a fine powder.
3. ECM suspension preparation
   1. Weigh out 250 mg of either minced (for foams) or cryomilled (for foams or microcarriers) ECM and transfer it to a 15 mL centrifuge tube. Refer to step 1.1.6 and section 1.2 for preparation of minced ECM and cryomilled ECM, respectively.
      NOTE: This protocol is designed to prepare a 50 mg/mL ECM suspension, which is recommended for human DAT, porcine DDT, and porcine DLV. Depending on the specific ECM source, uniform suspensions may be generated at higher or lower starting concentrations.
   2. Add 5 mL of 0.22 M NaH₂PO₄ (pH 5.4) buffer to the centrifuge tube and mark the liquid level on the tube.
   3. Prepare an α-amylase stock solution by adding 7.5 mg of α-amylase to 1 mL 0.22 M NaH₂PO₄ (pH 5.4) buffer. Add 100 μL of the α-amylase stock solution (0.75 mg of α-amylase: 0.3% w/w of dry tissue) to the sample. Add 0.22 M NaH₂PO₄ to obtain a final volume of 10 mL.
   4. Agitate the suspension continuously at 300 rpm for 72 h at room temperature.
   5. Centrifuge the suspension at 1,500 x g for 10 min. Carefully collect and discard the supernatant without disturbing the digested ECM pellet. Resuspend the pelleted material in 10 mL of 5% NaCl diluted in ddH₂O.
   6. Repeat step 1.3.5 for a total of two rinses with 5% NaCl in ddH₂O.
   7. Discard the supernatant and resuspend the pelleted material in 10 mL of ddH₂O.
   8. Agitate the suspension continuously at 300 rpm for 10 min at RT.
   9. Centrifuge the suspension at 1,500 x g for 10 min. Carefully collect and discard the supernatant without disturbing the digested ECM pellet.
   10. Add 0.2 M acetic acid to the 5 mL mark made in step 1.3.2.
   11. Agitate the suspension continuously at 120 rpm O/N at 37 °C.
   12. Homogenize the ECM suspension at room temperature in 10-second intervals using a hand-held homogenizer equipped with a saw-tooth 10 mm wide probe until no visible fragments remain. Place the suspension in a beaker of cold water between the homogenization intervals to prevent overheating.
       NOTE: Depending on the ECM source, further dilution in 0.2 M acetic acid may be required to obtain a homogeneous suspension. Excessive cooling of the ECM suspension may result in an increase in viscosity that can interfere with efficient homogenization. If an increase in viscosity is noted, the sample can be rewarmed to 37 °C and subjected to further processing.
   13. Store at 4 °C for up to one month prior to foam or microcarrier fabrication.