Cryomilling of Decellularized Tissue: A Technique to Obtain Fine Extracellular Matrix Powder

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 DAT, porcine DDT, and porcine 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 (optional for foam fabrication)
   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).
   4. Repeat steps 1.2.2 and 1.2.3 until the ECM is milled 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.
   5. Transfer the powder using a scoopula into a glass vial, seal tightly, and store in a desiccator.