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 in the beaker. Leave the solution on a stir plate at approximately 200 rpm for 24 h or until the SDS is completely dissolved. 
		Note: Typically, 2500 mL of 1% SDS solution is sufficient to decellularize a single human kidney.</li>
		<li>Transfer the solution to a 500 mL sterile vacuum filter and filter it into sterilized sealable containers.</li>
	</ol>
	</li>
	<li>Processing of kidney tissue
	<ol>
		<li>Wash and autoclave a pair of forceps, two hemostat clamps, a pair of general service grade scissors, two scalpel blade handles, a 1000 mL beaker covered with aluminum foil, and a 36 x 9 mm stir bar.</li>
		<li>Line a tissue culture hood with underpad. Place the beaker, a sterile tissue culture dish (150 x 25 mm), and the whole kidney organ into the hood. Fill the beaker with 500 mL of 1% SDS solution. 
		Note: Human kidneys were received on ice from LifeCenter NorthWest.</li>
		<li>Place the kidney in the sterile tissue culture dish (Figure 1A). Remove all perirenal fat by lightly shaving around the renal capsule with a scalpel (Figure 1B).</li>
		<li>Make a shallow 8-10 cm incision with the scalpel, just deep enough to break open the renal capsule without damaging the underlying cortex tissue, across the superior end of the kidney. Remove the renal capsule by peeling it away from the cortex tissue with two hemostat clamps (Figure 1C).</li>
		<li>Bisect the kidney along the coronal plane by using the scalpel along the lateral side of the kidney (Figure 1D). Isolate cortex tissue from both halves by carving out the medullar region with the scalpel (Figure 1E) and dice the cortex tissue into 0.5 cm3 pieces (Figure 1F). Remove any large visible vessels.</li>
	</ol>
	</li>
	<li>Isolation of extracellular matrix
	<ol>
		<li>In a tissue culture hood, fill a 1000 mL beaker with 500 mL of 1% SDS solution. Place the diced cortex tissue and stir bar into the beaker containing SDS solution. Cover the beaker with autoclaved aluminum foil and place it on a stir plate at approximately 400 rpm outside of the tissue culture hood.</li>
		<li>After the cortex tissue has been on the stir plate for 24 h, bring the beaker into a tissue culture hood and add a 40 &#181;m sterile cell strainer made with nylon mesh. Fill a separate 1000 mL beaker with 200 mL of bleach and place it in the tissue culture hood.</li>
		<li>Pipette out the SDS solution through the cell strainer into the beaker containing bleach. Pipette out all SDS solution until only decellularized tissue and the cell strainer remain in the beaker. 
		Note: The cell strainer should prevent any tissue from being removed during solution aspiration.</li>
		<li>Leave the cell strainer in the beaker and fill with 500 mL of fresh SDS solution. Cover the beaker with the same aluminum foil and place onto a stir plate at the same speed as before.</li>
		<li>Repeat steps 1.3.1-1.3.3 every 24 hours with fresh SDS solution for a total of five days.</li>
		<li>Rinse decellularized tissue with autoclaved DI water every 24 h for 3 days total, following the technique outlined in steps 1.3.1-1.3.3.</li>
		<li>Rinse decellularized tissue with cell culture grade water every 24 h for 2 days total, following the technique outlined in steps 1.3.1-1.3.3.</li>
		<li>Repeat steps 1.3.1-1.3.2. Transfer the decellularized tissue (referred to as kECM from this point on) into a 30 mL self-standing conical tube and fill it with cell culture grade water until all the tissue is submerged.</li>
	</ol>
	</li>
</ol>
2. Fabrication of Hydrogel Stock Solution
<ol>
	<li>Mechanical processing of decellularized tissue
	<ol>
		<li>In a tissue culture hood, mechanically homogenize the kECM within the conical tube with a tissue homogenizer for 2 min. 
		Note: Homogenized kECM should resemble an opaque solution with no visible pieces of ECM.</li>
		<li>Submerge the conical tube containing the kECM in liquid nitrogen until boiling surrounding the tube no longer persists. Store the kECM at -4 &#730;C overnight.</li>
	</ol>
	</li>
	<li>Lyophilization of frozen decellularized tissue
	<ol>
		<li>Slightly loosen the conical tube cap to allow for gas exchange and place the tube into a lyophilization machine. Lyophilize the kECM for three days or until it resembles a fine white powder. Store at -4 &#730;C.</li>
	</ol>
	</li>
	<li>Chemical digestion and solubilization of gel
	<ol>
		<li>Autoclave a 20 mL scintillation vial and cap, a 15.9 x 7.9 mm stir bar, and one pair of fine-tip forceps.</li>
		<li>Weigh the lyophilized kECM and calculate the volume of HCl and mass of pepsin needed to solubilize the kECM to a 3% (30 mg/mL) solution using the following equations, where mpepsin is the mass of pepsin, mtissue is the mass of lyophilized tissue, and VHCl is the volume of 0.01 N HCl: 
		<img alt="Equation 1" src="/files/ftp_upload/58314/58314eq1.jpg" /> 
		<img alt="Equation 2" src="/files/ftp_upload/58314/58314eq2.jpg" /></li>
		<li>In a tissue culture hood, add porcine gastric pepsin, 0.01 N HCl, and the stir bar to the scintillation vial, and leave it on a stir plate at approximately 500 rpm until all the pepsin has dissolved. Transfer the lyophilized kECM to the scintillation vial and leave the solution on a stir plate at approximately 500 rpm for three days.</li>
	</ol>
	</li>
</ol>
3. Hydrogel Gelation
<ol>
	<li>Kidney ECM hydrogel preparation
	<ol>
		<li>Gel the hydrogel by mixing the kECM hydrogel stock solution with 1 N NaOH, 10x Media Supplement (M199), and cell culture media. Keep all the solutions on ice. 
		Note: Final gel concentrations of 7.5 mg/mL were used for cell culture. 1 mL of kECM gel was sufficient for cell culture experiments presented.</li>
		<li>Determine the volume of workable kECM gel produced and volume of stock kECM hydrogel needed by using the following equation, where Vfinal is the volume of gel created, Vstock kECM is the volume of stock kECM hydrogel needed, Cstock kECM is the concentration of the stock kECM hydrogel, and Cfinal is the concentration of the final gel: 
		<img alt="Equation 3" src="/files/ftp_upload/58314/58314eq3v2.jpg" /></li>
		<li>Determine the volume of neutralizing reagents needed by using the following equations, where VNaOH is the volume of 1 N NaOH, V10X is the volume of M199 10X media supplement, and V1X is the volume of cell culture media: 
		<img alt="Equation 4" src="/files/ftp_upload/58314/58314eq4.jpg" /> 
		<img alt="Equation 5" src="/files/ftp_upload/58314/58314eq5.jpg" /> 
		<img alt="Equation 6" src="/files/ftp_upload/58314/58314eq6.jpg" /></li>
		<li>In a tissue culture hood, pipette the neutralizing reagents (NaOH, M199, and cell culture media) into a sterile 30 mL self-standing conical tube. Mix the neutralizing reagent solution with a microspatula.</li>
		<li>Use a sterile 1 mL syringe to transfer the appropriate volume of stock kECM hydrogel to the neutralizing reagent solution. Use a microspatula to gently mix the solution until a homogeneous in color hydrogel solution is obtained. 
		Note: Avoid introducing air bubbles by stirring slowly and gently.</li>
		<li>To incorporate cells into the kECM hydrogel, subtract 10 &#181;L of cell culture media (V1X) from the neutralizing solution volume calculations in step 3.1.1.3.
		<ol>
			<li>Suspend cells into 10 &#181;L of cell culture media. Determine the number of cells to be suspended by using the following equation, where #cells implies the number of cells to suspend and Vfinal is the volume of gel created: 
			<img alt="Equation 7" src="/files/ftp_upload/58314/58314eq7.jpg" /> 
			Note: 300,000 cells/mL is the concentration of cells used in the kECM gel.</li>
			<li>Pipette the 10 &#181;L of cell suspended solution into the final kECM gel after the kECM stock solution has been mixed with neutralizing reagent solution. Stir the solution with a microspatula until the cells are evenly distributed.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Use a 1 mL syringe to fill a desired cell culture device with the kECM hydrogel.</li>
	<li>Allow the gel to set at 37 &#730;C for 1 h before transferring or plating cells.</li>
</ol>

The authors have nothing to disclose.

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>&lt;strong&gt;Preparation of Kidney Tissue&lt;/strong&gt;</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 H&lt;sub&gt;2&lt;/sub&gt;O</td><td></td><td></td><td>Autoclaved DI H&lt;sub&gt;2&lt;/sub&gt;O</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>&lt;strong&gt;Fabrication of ECM Gel&lt;/strong&gt;</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>&lt;strong&gt;Kidney ECM Gelation&lt;/strong&gt;</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&#039;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 ...

fabricating-a-kidney-cortex-extracellular-matrix-derived-hydrogel

Research

JoVE Journal

Bioengineering

10.4K Views.  University of Washington. 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.

Video: Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Printing Thermoresponsive Reverse Molds for the Creation of Patterned Two-component Hydrogels for 3D Cell Culture

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact bioprinting1-4 (extrusion, dip pen and soft lithography), contactless bioprinting5-7 (laser forward transfer, ink-jet deposition) and laser based techniques such as two photon photopolymerization8. It can be used for many applications such as tissue engineering9-13, biosensor microfabrication14-16 and as a tool to answer basic biological questions such as influences of co-culturing of different cell types17. Unlike common photolithographic or soft-lithographic methods, extrusion bioprinting has the advantage that it does not require a separate mask or stamp. Using CAD software, the design of the structure can quickly be changed and adjusted according to the requirements of the operator. This makes bioprinting more flexible than lithography-based approaches.
Here we demonstrate the printing of a sacrificial mold to create a multi-material 3D structure using an array of pillars within a hydrogel as an example. These pillars could represent hollow structures for a vascular network or the tubes within a nerve guide conduit. The material chosen for the sacrificial mold was poloxamer 407, a thermoresponsive polymer with excellent printing properties which is liquid at 4 &deg;C and a solid above its gelation temperature ~20 &deg;C for 24.5% w/v solutions18. This property allows the poloxamer-based sacrificial mold to be eluted on demand and has advantages over the slow dissolution of a solid material especially for narrow geometries. Poloxamer was printed on microscope glass slides to create the sacrificial mold. Agarose was pipetted into the mold and cooled until gelation. After elution of the poloxamer in ice cold water, the voids in the agarose mold were filled with alginate methacrylate spiked with FITC labeled fibrinogen. The filled voids were then cross-linked with UV and the construct was imaged with an epi-fluorescence microscope.

A bioprinter was used to create patterned hydrogels based on a sacrificial mold. The poloxamer mold was backfilled with a second hydrogel and then eluted, leaving voids which were filled with a third hydrogel. This method uses fast elution and good printability of poloxamer to generate complex architectures from biopolymers.

Bioprinting is an emerging technology that has its origins in the rapid prototyping industry. The different printing processes can be divided into contact ...

Immunology and Infection

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. Several commercial extracellular matrix products are available as hydrogels or membranes, but these do not possess tissue-specific biological activity. Because perfusion decellularization is usually not possible with human heart tissue, we developed a 3-step immersion decellularization process. Human myocardial slices procured during surgery are first treated with detergent-free hyperosmolar lysis buffer, followed by incubation with the ionic detergent, sodium dodecyl sulfate, and the process is completed by exploiting the intrinsic DNase activity of fetal bovine serum. This technique results in cell-free sheets of cardiac extracellular matrix with largely preserved fibrous tissue architecture and biopolymer composition, which were shown to provide specific environmental cues to cardiac cell populations and pluripotent stem cells. Cardiac extracellular matrix sheets can then be further processed into a microparticle powder without further chemical modification, or, via short-term pepsin digestion, into a self-assembling cardiac extracellular matrix hydrogel with preserved bioactivity.

Processing of Human Cardiac Tissue Toward Extracellular Matrix Self-assembling Hydrogel for In Vitro and In Vivo Applications

This protocol describes the complete decellularization of human myocardium while preserving its extracellular matrix components. Further processing of the extracellular matrix results in the production of microparticles and a cytoprotective self-assembling hydrogel.

Acellular extracellular matrix preparations are useful for studying cell-matrix interactions and facilitate regenerative cell therapy applications. ...

Developmental Biology

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Construction of Modular Hydrogel Sheets for Micropatterned Macro-scaled 3D Cellular Architecture

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue geometry. The resulting 3D hydrogel-based cellular constructs have been introduced as an alternative to animal experiments for advanced biological studies, pharmacological assays and organ transplant applications. Although hydrogel-based particles and fibers can be easily fabricated, it is difficult to manipulate them for tissue reconstruction. In this video, we describe a fabrication method for micropatterned alginate hydrogel sheets, together with their assembly to form a macro-scale 3D cell culture system with a controlled cellular microenvironment. Using a mist form of the calcium gelling agent, thin hydrogel sheets are easily generated with a thickness in the range of 100 - 200 &#181;m, and with precise micropatterns. Cells can then be cultured with the geometric guidance of the hydrogel sheets in freestanding conditions. Furthermore, the hydrogel sheets can be readily manipulated using a micropipette with an end-cut tip, and can be assembled into multi-layered structures by stacking them using a patterned polydimethylsiloxane (PDMS) frame. These modular hydrogel sheets, which can be fabricated using a facile process, have potential applications of in vitro drug assays and biological studies, including functional studies of micro- and macrostructure and tissue reconstruction.

We describe the fabrication of micropatterned hydrogel sheets using a simple process, which can be assembled and manipulated in a freestanding form. Using these modular hydrogel sheets, a simple macro-scaled 3D cell culture system can be generated with a controlled cellular microenvironment.

Hydrogels can be patterned at the micro-scale using microfluidic or micropatterning technologies to provide an in vivo-like three-dimensional (3D) tissue ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies of endothelial-epithelial bilayers have traditionally relied on permeable support models that enable bilayer culture, but permeable supports are limited in their ability to replicate the diversity of human basement membranes. In contrast, hydrogel models that require chemical synthesis are highly tunable and allow for modifications of both the material stiffness and the biochemical composition via incorporation of biomimetic peptides or proteins. However, traditional hydrogel models are limited in functionality because they lack pores for cell-cell contacts and functional in vitro migration studies. Additionally, due to the thickness of traditional hydrogels, incorporation of pores that span the entire thickness of hydrogels has been challenging. In the present study, we use poly-(ethylene-glycol) (PEG) hydrogels and a novel zinc oxide templating method to address the previous shortcomings of biomimetic hydrogels. As a result, we present an ultrathin, basement membrane-like hydrogel that permits the culture of confluent cellular bilayers on a customizable scaffold with variable pore architectures, mechanical properties, and biochemical composition.

Current bilayer culture models do not allow for functional in vitro studies that mimic in vivo microenvironments. Using polyethylene glycol and a zinc oxide templating method, this protocol describes the development of an ultrathin biomimetic basement membrane with tunable stiffness, porosity, and biochemical composition that closely mimics in vivo extracellular matrices.

The basement membrane is a critical component of cellular bilayers that can vary in stiffness, composition, architecture, and porosity. In vitro studies ...

Renal Cancer and Healthy Tissue Co-Culture Using Microphysiological Systems

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating intricate morphological features in three-dimensional environments with dynamic interactions lacking in conventional models. We implemented a renal cell carcinoma (RCC) co-culture model to recreate the cross-talk between healthy and malignant renal tissue. 
This model is based on the referenced multi-organ platform and consists of co-culturing a reconstructed renal proximal tubule with RCC spheroids. Custom-designed 3D-printed chambers were used to culture human renal epithelial proximal tubule cells (RPTEC) and facilitate their self-assembly into a renal tubule contained in a collagen type I matrix. Caki-1 RCC cells were embedded in an agar collagen matrix, subsequently forming cancer spheroids. Both collagen and agar/collagen gels were optimized to maintain their integrity during cyclic perfusion and withstand shear stress during a minimum culture period of 7 days. 
The gels also enable adequate nutrient supply and cell secretions. Moreover, the agar/collagen gels limit the overproliferation of RCC cells, ensuring relatively homogeneous spheroid size. The MPS chip microfluidic circuits comprise two independent culture chambers with the size of a standard 96-microplate well. The renal tubule and RCC gels populate separate chambers and share the same culture media, which is recirculated approximately twice per minute. Under these conditions, we observed upregulation of immune factor expression and secretion in the renal tubules (interleukin-8 and tumor necrosis factor-alpha). The renal tubules also shift their metabolic activity towards glycolysis under the influence of RCC. This novel approach demonstrates that a co-culture-based MPS can amplify the complexity of RCC in vitro and be employed to study the impact of cancer on non-tumor cells.

Here, we present protocols that detail instructions for implementing 3D cell cultures using collagen and collagen-agarose matrixes in a microphysiological system. These protocols support renal proximal tubule and renal cell carcinoma spheroids co-culture, simulating in vivo conditions and enabling advanced investigation of kidney cancer cell interactions.

Microphysiological systems (MPS) have enabled the introduction of more complex and relevant physiological elements into in vitro models, recreating ...

Cancer Research

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Summary

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Ultrathin Porated Elastic Hydrogels As a Biomimetic Basement Membrane for Dual Cell Culture

Microfluidic Co-culture of Renal Healthy and Tumor Epithelium to Model Kidney Cancer Progression

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels