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

Published: April 30, 2023

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.

Offenlegungen

The authors have nothing to disclose.

Materials

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

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Diesen Artikel zitieren
Extracellular Matrix-derived Foam Fabrication via Lyophilization: A Procedure to Generate Biological Scaffolds from ECM of Decellularized Tissues. J. Vis. Exp. (Pending Publication), e20485, doi: (2023).

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