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

Abstract

Source: Kornmuller, A. et al. Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms. J. Vis. Exp. (2017)

In this video, we present the protocol to obtain finely ground extracellular matrix powder from lyophilized decellularized tissue by cryomilling. The powdered ECM retains its biochemical composition and can be used for microcarrier synthesis.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alligator clip leads	VWR	470149-728
Aluminium foil	Fisher Scientific	01-213-101
Analytical balance	Sartorius	CPA225D
Cryomilling system	Retsch	20.745.0001	MM 400
Dessicator	Fisher Scientific	8624426	For lyophilized ECM and bioscaffold storage
Dewar flask	Fisher Scientific	10-196-6	Low form; volume range of 250 - 500 mL
Double distilled water (ddH2O)			From Barnstead GenPure xCAD Water Purification System
ECM (Extracellular Matrix)			Isolated from human adipose tissue, porcine dermis or porcine myocardium, as described in Flynn et al. 2010, Reing et al. 2010, and Wainwright et al. 2010
Forceps	VWR	37-501-32	For transferring the foams
Freezer (-20 °C)	VWR	97043-346
Freezer ( -80 °C)	Thermo Scientific	EXF40086A
Glass vials	Fisher Scientific	03-339-26D	To store lyophilized cryomilled ECM
Liquid nitrogen			For electrospraying
Lyophilizer	Labconco	7750021	FreeZone4.5
Milling chamber	Retsch	02.462.0213	25 mL volume recommended
Milling balls	VWR	16003-606	10 mm diameter, stainless steel recommended
Penicillin-streptomycin	Life Technologies	15140-122	Used for proliferation media
Pipet-Aid XP	Mandel Scientific	DRU-4-000-101
Scoopula	VWR	89259-968	For collecting microcarriers
Surgical scissors	VWR	82027-590