Name: fabricating a kidney cortex extracellular matrix-derived hydrogel
Uploaded: 2018-10-13T14:30:00
Description: Watch this Scientific Journal Video about Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel at JoVE.com

The authors would like to acknowledge the Lynn and Mike Garvey Imaging Laboratory at the Institute for Stem Cell and Regenerative Medicine and LifeCenter NorthWest. They would also like to acknowledge the financial support of National Institutes of Health grants, UH2/UH3 TR000504 (to J.H.) and DP2DK102258 (to Y.Z.), NIH T32 training grant DK0007467 (to R.J.N.), and an unrestricted gift from the Northwest Kidney Centers to the Kidney Research Institute.

Human kidneys were isolated by LifeCenter Northwest following ethical guidelines set by the Association of Organ Procurement Organizations. This protocol follows animal care and cell culture guidelines outlined by the University of Washington.
1. Preparation of Human Kidney Tissue
<ol>
	<li>Preparation of decellularization solution
	<ol>
		<li>Sterilize a 5000 mL beaker and a 70 x 10 mm stir bar.</li>
		<li>Mix 1:1000 (weight:volume) sodium dodecyl sulfate (SDS) in autoclaved deionized water...

Matrices provide important mechanical and chemical cues that govern cell behavior. Synthetic hydrogels are able to support complex 3-dimensional patterning but fail to provide the diverse extracellular cues found in physiological matrix microenvironments. Hydrogels derived from native ECM are ideal materials for both in vivo and in vitro studies. Previous studies have used decellularized ECM hydrogels to coat synthetic biomaterials to prevent host immunological responses33,<...

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. The complex molecular composition regulates both structural and functional properties of tissue. Structural proteins provide cells with spatial awareness and allow for adhesion and migration1. Bound ligands interact with cell surface receptors to control cell behavior2. Kidney ECM contains a plethora of molecules whose composition and structure varies depending on anatomical location, developmental stage, and disease state3,4. Recapitulating the complexity of ECM is a key aspect in studying kidney-derived cells in vitro.
Previous attempts at replicating ECM microenvironments have focused on decellularizing whole tissue to create scaffolds capable of recellularization. Decellularization has been performed with chemical detergents such as sodium dodecyl sulfate (SDS) or non-ionic detergents, and it utilizes either whole organ perfusion or immersion and agitation methods5,6,7,8,9,10,11,12,13. The scaffolds presented here preserve the structural and biochemical cues found in native tissue ECM; furthermore, recellularization with donor-specific cells has clinical relevance in reconstructive surgery14,15,16,17,18,19. However, these scaffolds lack structural flexibility and are therefore incompatible with many current devices used for in vitro studies. To overcome this limitation, many groups have further processed decellularized ECM into hydrogels20,21,22,23,24. These hydrogels are compatible with injection molding and bioink and circumvent micrometer scale spatial constraints that decellularized scaffolds place on cells. Furthermore, molecular composition and ratios found in native ECM are preserved3,25. Here we demonstrate a method to fabricate a hydrogel derived from kidney cortex ECM (kECM).
The purpose of this protocol is to produce a hydrogel that replicates the microenvironment of the kidney cortical region. Kidney cortex tissue is decellularized in a 1% SDS solution under constant agitation to remove cellular matter. SDS is commonly used to decellularize tissue because of its ability to quickly remove immunological cellular material6,7,9,26. The kECM is then subject to mechanical homogenization and lyophilization5,6,9,11,26. Solubilization in a strong acid with pepsin results in a final hydrogel stock solution20,27. Native kECM proteins that are important for structural support and signal transduction are preserved3,25. The hydrogel can also be gelled to within one order of magnitude of native human kidney cortex28,29,30. This matrix provides a physiological environment that has been used to maintain the quiescence of kidney-specific cells compared to hydrogels from other matrix proteins. Furthermore, matrix composition can be manipulated, for example, through the addition of collagen-I, to model disease environments for the study of renal fibrosis and other kidney diseases31,32.

<table>
	<tbody>
		<tr>
			<td>Preparation of Kidney Tissue</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5000 mL Beaker</td>
			<td>Sigma-Aldrich</td>
			<td>Z740589</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>436143</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile H2O</td>
			<td></td>
			<td></td>
			<td>Autoclaved DI H2O</td>
		</tr>
		<tr>
			<td>Stir Bar (70 x 10 mm)</td>
			<td>Fisher Science</td>
			<td>14-512-128</td>
			<td></td>
		</tr>
		<tr>
			<td>500 mL Vacuum Filter</td>
			<td>VWR</td>
			<td>97066-202</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Plate</td>
			<td>Sigma-Aldrich</td>
			<td>CLS6795420D</td>
			<td></td>
		</tr>
		<tr>
			<td>1000 mL Beaker</td>
			<td>Sigma-Aldrich</td>
			<td>CLS10031L</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Sigma-Aldrich</td>
			<td>F4642</td>
			<td>Any similar forceps may be used</td>
		</tr>
		<tr>
			<td>Scissor-Handle Hemostat Clamp</td>
			<td>Sigma-Aldrich</td>
			<td>Z168866</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting Scissors</td>
			<td>Sigma-Aldrich</td>
			<td>Z265977</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle, No. 4</td>
			<td>VWR</td>
			<td>25859-000</td>
			<td>Any similar scalpel handle may be used</td>
		</tr>
		<tr>
			<td>Scalpel Blade, No. 20</td>
			<td>VWR</td>
			<td>25860-020</td>
			<td>Any similar scalpel blade may be used</td>
		</tr>
		<tr>
			<td>Stir Bar (38.1 x 9.5 mm)</td>
			<td>Fisher Science</td>
			<td>14-513-52</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbent Underpad</td>
			<td>VWR</td>
			<td>82020-845</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri Dish (150 x 25 mm)</td>
			<td>Corning</td>
			<td>430597</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable Biohazard Bag</td>
			<td>VWR</td>
			<td>14220-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Cell Strainer (40 um)</td>
			<td>Fisher Science</td>
			<td>22-363-547</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Grade Water</td>
			<td>HyClone</td>
			<td>SH30529.03</td>
			<td></td>
		</tr>
		<tr>
			<td>30 mL Freestanding Tube</td>
			<td>VWR</td>
			<td>89012-778</td>
			<td></td>
		</tr>
		<tr>
			<td>Fabrication of ECM Gel</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Homogenizer Machine</td>
			<td>Polytron</td>
			<td>PCU-20110</td>
			<td></td>
		</tr>
		<tr>
			<td>Freeze Dryer</td>
			<td>Labconco</td>
			<td>7670520</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Glass Scintillation Vials and Cap</td>
			<td>Sigma-Aldrich</td>
			<td>V7130</td>
			<td></td>
		</tr>
		<tr>
			<td>Stir Bar (15.9 x 8 mm)</td>
			<td>Fisher Science</td>
			<td>14-513-62</td>
			<td></td>
		</tr>
		<tr>
			<td>Pepsin from Porcine Gastric Mucosa</td>
			<td>Sigma-Aldrich</td>
			<td>P7012</td>
			<td></td>
		</tr>
		<tr>
			<td>0.01 N HCl</td>
			<td>Sigma-Aldrich</td>
			<td>320331</td>
			<td>Dilute to 0.01 N HCl with cell culuture water</td>
		</tr>
		<tr>
			<td>Kidney ECM Gelation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 N NaOH (Sterile)</td>
			<td>Sigma-Aldrich</td>
			<td>415413</td>
			<td>Dilute to 1 N in cell culture grade water</td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M4530</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Conical Tube</td>
			<td>ThermoFisher</td>
			<td>339651</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Media</td>
			<td>ThermoFisher</td>
			<td>11330.032</td>
			<td>Dulbecco&#39;s Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Gibco</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic 100X</td>
			<td>Life Technologies</td>
			<td>15240-062</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin, Transferrin, Selenium, Sodium Pyruvate Solution (ITS-A) 100X</td>
			<td>Life Technologies</td>
			<td>51300-044</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>Sigma-Aldrich</td>
			<td>Z192325</td>
			<td></td>
		</tr>
		<tr>
			<td>Microspatula</td>
			<td>Sigma-Aldrich</td>
			<td>Z193208</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The kECM hydrogel provides a matrix for kidney cell culture with similar chemical composition as the native kidney microenvironment. To fabricate the hydrogel, kidney cortex tissue is mechanically isolated from a whole kidney organ and diced (Figure 1). Decellularization with a chemical detergent (Figure 2A.1-A.3) followed by rinses with water to remove detergent particles (Figure 2A...

Watch this Scientific Journal Video about Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel at JoVE.com

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

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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