Name: Extracellular Matrix-derived Foam Fabrication via Lyophilization: A Procedure to Generate Biological Scaffolds from ECM of Decellularized Tissues
Uploaded: 2023-04-30T13:41:35
Description: Source: Kornmuller, A. et al. Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms. ...

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1. ECM-derived Foam Fabrication
<ol>
	<li>Prepare the homogenous ECM suspension by dispersing ECM fine powder in buffer solution and incubate at 37 &#176;C with continuous agitation at 120 rpm until the suspension is warm.</li>
	<li>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&#8211;100 mg/mL, with 15&#8211;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.</li>
	<li>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 &#956;L 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.</li>
	<li>Cover and freeze the molds overnight by placing them in a -20 &#176;C or -80 &#176;C freezer. 
	NOTE: The freezing temperature may affect the porosity of the foams. Larger pores are expected when the samples are frozen at -20 &#176;C as compared to -80 &#176;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.</li>
	<li>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.</li>
	<li>Store the lyophilized foams in a desiccator until required.</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Analytical balance</td>
			<td>Sartorius</td>
			<td>CPA225D</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL)</td>
			<td>Sarstedt</td>
			<td>62.554.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>Sarstedt</td>
			<td>62.547.205</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen from bovine achilles 
			tendon (insoluble)</td>
			<td>Sigma</td>
			<td>C9879</td>
			<td>Or similar insoluble collagen 
			source; Can be used as an 
			alternative to decellularized 
			tissues to fabricate the foams and 
			microcarriers</td>
		</tr>
		<tr>
			<td>Dessicator</td>
			<td>Fisher Scientific</td>
			<td>8624426</td>
			<td>For lyophilized ECM and 
			bioscaffold storage</td>
		</tr>
		<tr>
			<td>Dewar flask</td>
			<td>Fisher Scientific</td>
			<td>10-196-6</td>
			<td>Low form; volume range of 250 -500 mL</td>
		</tr>
		<tr>
			<td>Double distilled water (ddH_{2} O)</td>
			<td></td>
			<td></td>
			<td>From Barnstead GenPure xCAD 
			Water Purification System</td>
		</tr>
		<tr>
			<td>D-PBS (Phosphate-buffered 
			Saline)</td>
			<td>Wisent</td>
			<td>311425125</td>
			<td></td>
		</tr>
		<tr>
			<td>ECM (Extracellular Matrix)</td>
			<td></td>
			<td></td>
			<td>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)</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>VWR</td>
			<td>37-501-32</td>
			<td>For transferring the foams</td>
		</tr>
		<tr>
			<td>Freezer (-20 &#176;C)</td>
			<td>VWR</td>
			<td>97043-346</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer ( -80 &#176;C)</td>
			<td>Thermo Scientific</td>
			<td>EXF40086A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vials</td>
			<td>Fisher Scientific</td>
			<td>03-339-26D</td>
			<td>To store lyophilized cryomilled 
			ECM</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td>For electrospraying</td>
		</tr>
		<tr>
			<td>Lyophilizer</td>
			<td>Labconco</td>
			<td>7750021</td>
			<td>FreeZone4.5</td>
		</tr>
		<tr>
			<td>18 G needle</td>
			<td>VWR</td>
			<td>C ABD305185</td>
			<td>For dispensing ECM suspension 
			into moulds</td>
		</tr>
		<tr>
			<td>Orbital incubator shaker</td>
			<td>SciLogex</td>
			<td>832010089999</td>
			<td>Temperature controlled (37 &#176;C)</td>
		</tr>
		<tr>
			<td>Serological pipettes (10 mL)</td>
			<td>Sarstedt</td>
			<td>86.1254.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipettes (25 mL)</td>
			<td>Sarstedt</td>
			<td>86.1685.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>BioShop</td>
			<td>7647-14-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic</td>
			<td>BioShop</td>
			<td>10049-21-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

extracellular matrix-derived foam fabrication via lyophilization: a procedure to generate biological scaffolds from ecm of decellularized tissues

Source: Kornmuller, A. et al. <a href="https://www.jove.com/video/55436" target="_blank">Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms.</a> 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.

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

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

ECM-derived foams are highly porous scaffolds obtained using the extracellular matrix - a non-cellular component of the tissue microenvironment.
To fabricate an ECM-derived foam, begin by taking a homogenous suspension of ultra-fine ECM powder in a tube. Incubate the suspension at a warm temperature with constant agitation to prevent ECM clumping.
Next, using a syringe, transfer the liquified ECM suspension to the desired mold. Refrigerate the mold overnight for the ECM to solidify. The ECM solidifies to form a three-dimensional network structure.
Finally, place the solid ECM-containing mold into a lyophilizer flask. Connect the flask to a freeze dryer system. Inside the lyophilizer, the sample is subjected to an ultracold temperature and low pressure. This results in the formation of ice crystals in the interstitial spaces of the ECM network.
In due course, the pressure and the temperature of the lyophilizer rise gradually. These conditions facilitate sublimation - a process in which the solid directly converts to its gaseous form rather than undergoing melting.
The gaseous vapors evaporate, leaving behind empty spaces inside the ECM network, thereby generating a porous ECM-derived scaffold.

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1.1K Views. 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. 

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

Studying Normal Tissue Radiation Effects using Extracellular Matrix Hydrogels

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue and its role in local recurrence at the primary tumor site are unknown. Here we present a method for the decellularization, lyophilization, and fabrication of ECM hydrogels derived from murine mammary fat pads. Results are presented on the effectiveness of the decellularization process, and rheological parameters were assessed. GFP- and luciferase-labeled breast cancer cells encapsulated in the hydrogels demonstrated an increase in proliferation in irradiated hydrogels. Finally, phalloidin conjugate staining was employed to visualize cytoskeleton organization of encapsulated tumor cells. Our goal is to present a method for fabricating hydrogels for in vitro study that mimic the in vivo breast tissue environment and its response to radiation in order to study tumor cell behavior.

This protocol presents a method for decellularization and subsequent hydrogel formation of murine mammary fat pads following ex vivo irradiation.

Radiation is a therapy for patients with triple negative breast cancer. The effect of radiation on the extracellular matrix (ECM) of healthy breast tissue ...

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Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed from JBNTs and FN through self-assembly in an aqueous solution. JBNTs measure 200-300 &#181;m in length with inner hydrophobic hollow channels and outer hydrophilic surfaces. JBNTs are positively charged and FNs are negatively charged. Therefore, when injected into a neutral aqueous solution, they are bonded together via noncovalent bonding to form the NM bundles. The self-assembly process is completed within a few seconds without any chemical initiators, heat source, or UV light. When the pH of the NM solution is lower than the isoelectric point of FNs (pI 5.5-6.0), the NM bundles will self-release due to the presence of positively charged FN.
NM is known to mimic the extracellular matrix (ECM) morphologically and hence, can be used as an injectable scaffold, which provides an excellent platform to enhance hMSC adhesion. Cell density analysis and fluorescence imaging experiments indicated that the NMs significantly increased the anchorage of hMSCs compared to the negative control.

The goal of this protocol is to show the assembly of a biomimetic nanomatrix (NM) with Janus base nanotubes (JBNTs) and fibronectin (FN). When co-cultured with human mesenchymal stem cells (hMSCs), the NMs exhibit excellent bioactivity in encouraging hMSCs adhesion.

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

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