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

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)

This video describes the method for synthesizing pure ECM-derived foams without the need for chemical crosslinking. These ECM-derived foams find applications as advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Analytical balance	Sartorius	CPA225D
Centrifuge tubes (15 mL)	Sarstedt	62.554.205
Centrifuge tubes (50 mL)	Sarstedt	62.547.205
Collagen from bovine achilles tendon (insoluble)	Sigma	C9879	Or similar insoluble collagen source; Can be used as an alternative to decellularized tissues to fabricate the foams and microcarriers
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 (ddH_{2} O)			From Barnstead GenPure xCAD Water Purification System
D-PBS (Phosphate-buffered Saline)	Wisent	311425125
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 (ref # 28, 8, 32)
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
18 G needle	VWR	C ABD305185	For dispensing ECM suspension into moulds
Orbital incubator shaker	SciLogex	832010089999	Temperature controlled (37 °C)
Serological pipettes (10 mL)	Sarstedt	86.1254.001
Serological pipettes (25 mL)	Sarstedt	86.1685.001
Sodium chloride	BioShop	7647-14-5
Sodium phosphate monobasic	BioShop	10049-21-5