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>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 ...

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

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JoVE Journal

Bioengineering

10.3K 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

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively for tissue regeneration, a number of variables must be taken into account. These variables include: the total volume and surface area of the implantation site, the mechanical properties of the tissue and the tissue microenvironment, which includes the amount of vascularization and the components of the extracellular matrix. Therefore, the materials being used to deliver these cells must be biocompatible with a defined chemical composition while maintaining a mechanical strength that mimics the host tissue. These materials must also be permeable to oxygen and nutrients to provide a favorable microenvironment for cells to attach and proliferate. Chitosan, a cationic polysaccharide with excellent biocompatibility, can be easily chemically modified and has a high affinity to bind with in vivo macromolecules4-5. Chitosan mimics the glycosaminoglycan portion of the extracellular matrix, enabling it to function as a substrate for cell adhesion, migration and proliferation. In this study we utilize chitosan in the form of microspheres to deliver adipose-derived stem cells (ASC) into a collagen based three-dimensional scaffold6. An ideal cell-to-microsphere ratio was determined with respect to incubation time and cell density to achieve maximum number of cells that could be loaded. Once ASC are seeded onto the chitosan microspheres (CSM), they are embedded in a collagen scaffold and can be maintained in culture for extended periods. In summary, this study provides a method to precisely deliver stem cells within a three dimensional biomaterial scaffold.

A major hurdle in current stem cell therapies is determining the most effective method to deliver these cells to host tissues. Here, we describe a chitosan-based delivery method that is efficient and simple in approach, while allowing adipose-derived stem cells to maintain their multipotency.

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Engineering a Bilayered Hydrogel to Control ASC Differentiation

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in vivo. An area of research that holds promise in regenerative medicine is the combinatorial use of novel biomaterials and stem cells. A fundamental strategy in the field of tissue engineering is the use of three-dimensional scaffold (e.g., decellularized extracellular matrix, hydrogels, micro/nano particles) for directing cell function. This technology has evolved from the discovery that cells need a substrate upon which they can adhere, proliferate, and express their differentiated cellular phenotype and function 2-3. More recently, it has also been determined that cells not only use these substrates for adherence, but also interact and take cues from the matrix substrate (e.g., extracellular matrix, ECM)4. Therefore, the cells and scaffolds have a reciprocal connection that serves to control tissue development, organization, and ultimate function. Adipose-derived stem cells (ASCs) are mesenchymal, non-hematopoetic stem cells present in adipose tissue that can exhibit multi-lineage differentiation and serve as a readily available source of cells (i.e. pre-vascular endothelia and pericytes). Our hypothesis is that adipose-derived stem cells can be directed toward differing phenotypes simultaneously by simply co-culturing them in bilayered matrices1. Our laboratory is focused on dermal wound healing. To this end, we created a single composite matrix from the natural biomaterials, fibrin, collagen, and chitosan that can mimic the characteristics and functions of a dermal-specific wound healing ECM environment.

This protocol focuses on utilizing the inherent ability of stem cells to take cue from their surrounding extracellular matrix and be induced to differentiate into multiple phenotypes. This methods manuscript extends our description and characterization of a model utilizing a bilayered hydrogel, composed of PEG-fibrin and collagen, to simultaneously co-differentiate adipose-derived stem cells1.

Natural polymers over the years have gained more importance because of their host biocompatibility and ability to interact with cells in vitro and in ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

An Injectable and Drug-loaded Supramolecular Hydrogel for Local Catheter Injection into the Pig Heart

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the limited access to donor hearts. An example of a treatment to recover the function of the heart consists of the local delivery of drugs and bioactives from a hydrogel. In this paper a method is introduced to formulate and inject a drug-loaded hydrogel non-invasively and side-specific into the pig heart using a long, flexible catheter. The use of 3-D electromechanical mapping and injection via a catheter allows side-specific treatment of the myocardium. To provide a hydrogel compatible with this catheter, a supramolecular hydrogel is used because of the convenient switching from a gel to a solution state using environmental triggers. At basic pH this ureido-pyrimidinone modified poly(ethylene glycol) acts as a Newtonian fluid which can be easily injected, but at physiological pH the solution rapidly switches into a gel. These mild switching conditions allow for the incorporation of bioactive drugs and bioactive species, such as growth factors and exosomes as we present here in both in vitro and in vivo experiments. The in vitro experiments give an on forehand indication of the gel stability and drug release, which allows for tuning of the gel and release properties before the subsequent application in vivo. This combination allows for the optimal tuning of the gel to the used bioactive compounds and species, and the injection system.

Supramolecular hydrogelators based on ureido-pyrimidinones allow full control over the macroscopic gel properties and the sol&#8211;gel switching behavior using pH. Here, we present a protocol for formulating and injecting such a supramolecular hydrogelator via a catheter delivery system for local delivery directly in relevant areas in the pig heart.

Regeneration of lost myocardium is an important goal for future therapies because of the increasing occurrence of chronic ischemic heart failure and the ...

Fabricating Superhydrophobic Polymeric Materials for Biomedical Applications

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial applications. Here we describe how electrospinning or electrospraying a polymer mixture containing a biodegradable, biocompatible aliphatic polyester (e.g., polycaprolactone and poly(lactide-co-glycolide)), as the major component, doped with a hydrophobic copolymer composed of the polyester and a stearate-modified poly(glycerol carbonate) affords a superhydrophobic biomaterial. The fabrication techniques of electrospinning or electrospraying provide the enhanced surface roughness and porosity on and within the fibers or the particles, respectively. The use of a low surface energy copolymer dopant that blends with the polyester and can be stably electrospun or electrosprayed affords these superhydrophobic materials. Important parameters such as fiber size, copolymer dopant composition and/or concentration, and their effects on wettability are discussed. This combination of polymer chemistry and process engineering affords a versatile approach to develop application-specific materials using scalable techniques, which are likely generalizable to a wider class of polymers for a variety of applications.

Two- and three-dimensional superhydrophobic polymeric materials are prepared by electrospinning or electrospraying biodegradable polymers blended with a lower surface energy polymer of similar composition.

Superhydrophobic materials, with surfaces possessing permanent or metastable non-wetted states, are of interest for a number of biomedical and industrial ...

Fabricating Cotton Analytical Devices

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and provide information for follow-up treatment. Here, we demonstrate the development of a cotton-based urinalysis (i.e., nitrite, total protein, and urobilinogen assays) analytical device that employs a lateral flow-based format, and is inexpensive, easily fabricated, rapid, and can be used to conduct multiple tests without cross-contamination worries. Cotton is composed of cellulose fibers with natural absorptive properties that can be leveraged for flow-based analysis. The simple but elegant fabrication process of our cotton-based analytical device is described in this study. The arrangement of the cotton structure and test pad takes advantage of the hydrophobicity and absorptive strength of each material. Because of these physical characteristics, colorimetric results can persistently adhere to the test pad. This device enables physicians to receive clinical information in a timely manner and shows great potential as a tool for early intervention.

To investigate simple fabrication approaches for multiple assay needs, we created a fluid-absorbing channel system made of cotton material. This device was used to establish a multiple detection platform, and solve contamination issues that commonly affect lateral flow-based biomedical devices, for clinical urinalysis of nitrite, total protein, and urobilinogen.

A robust, low-cost analytical device should be user-friendly, rapid, and affordable. Such devices should also be able to operate with scarce samples and ...

Bioprinting Cellularized Constructs Using a Tissue-specific Hydrogel Bioink

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as organ replacements for implantation in patients, and also, when created on a smaller size scale as model "organoids" that can be used in in vitro systems for drug and toxicology screening.
Despite development of a wide variety of bioprinting devices, application of bioprinting technology can be limited by the availability of materials that both expedite bioprinting procedures and support cell viability and function by providing tissue-specific cues. Here we describe a versatile hyaluronic acid (HA) and gelatin-based hydrogel system comprised of a multi-crosslinker, 2-stage crosslinking protocol, which can provide tissue specific biochemical signals and mimic the mechanical properties of in vivo tissues. 
Biochemical factors are provided by incorporating tissue-derived extracellular matrix materials, which include potent growth factors. Tissue mechanical properties are controlled combinations of PEG-based crosslinkers with varying molecular weights, geometries (linear or multi-arm), and functional groups to yield extrudable bioinks and final construct shear stiffness values over a wide range (100 Pa to 20 kPa). Using these parameters, hydrogel bioinks were used to bioprint primary liver spheroids in a liver-specific bioink to create in vitro liver constructs with high cell viability and measurable functional albumin and urea output. This methodology provides a general framework that can be adapted for future customization of hydrogels for biofabrication of a wide range of tissue construct types.

We describe a set of protocols that together provide a tissue-mimicking hydrogel bioink with which functional and viable 3-D tissue constructs can be bioprinted for use in in vitro screening applications.

Bioprinting has emerged as a versatile biofabrication approach for creating tissue engineered organ constructs. These constructs have potential use as ...

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 ...