This work was supported by NIH-NIDCR IRO1DE019355 (MJ Yost, PI), and NSF-EPSCoR (EPS-0903795).

1. Determine the Percentage of Solids in the Collagen Batch
<ol>
	<li>Extract the collagen following a previously published procedure13. Thaw a minimum of 20 ml of collagen. Determine the initial percentage of collagen solids in the batch in order to manipulate the collagen concentration in the formed hydrogels.
	<ol>
		<li>Cut three pieces of aluminum foil (about 6 x 6 cm) and shape each one as a pan by using the bottom of a 25 ml beaker. Record the weight of each pan.</li>
		<li>Add a small amount of collagen to each pan and record the total weight of the aluminum pan and collagen. Add 0.5 - 0.8 g of collagen on each aluminum pan. 
		Note: After this step, there are three aluminum foil pans and each one should have a small amount of collagen. Make sure to record the weight of each (total 3) empty aluminum pan and the weight of the pan after the addition of collagen.&#160;</li>
		<li>Calculate the weight of the collagen in each pan using the following formula: 
		Collagen weight = Pan and collagen weight - Pan weight</li>
		<li>Place the three aluminum pans that hold the collagen in the oven at 100 &#176;C for 24 hr.</li>
		<li>After 24 hr, record the weight of each aluminum pan and the dehydrated collagen.</li>
		<li>Calculate the weight of the dehydrated collagen using the following equation: 
		Dehydrated collagen weight = Pan and dehydrated collagen weight - Pan weight</li>
		<li>Calculate the percentage of solid for the three samples to determine the collagen solid concentration using the formula below: 
		<img alt="Equation 2" src="/files/ftp_upload/53578/53578eq2.jpg" /></li>
		<li>Calculate the average collagen solid content of the batch using the percentage of collagen solids for each of the three samples. 
		Note: The collagen that will be used is what is left of the hydrated collagen. None of the dehydrated collagen will be used.</li>
		<li>After determining the percentage of collagen solid (initial collagen concentration) of the batch, continue using the remaining hydrated collagen. Use a calibrated pH meter to adjust the pH of the collagen batch to 3.
		<ol>
			<li>Add small amounts (2-5 &#181;l at a time) of 12 N Hydrochloric Acid (HCl). Keep on ice at all times. Do not add the hydrochloric acid directly to the collagen&#160;&#8212; add the acid to the side of the tube. After adding the acid, use the spatula to push the acid to the collagen and quickly stir the mixture.</li>
		</ol>
		</li>
		<li>Once it reaches a pH of 3, let the collagen sit O/N at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Preparation of the BSA Rubber
<ol>
	<li>Prepare the Bovine Serum Albumin (BSA) solution following the procedure listed below.
	<ol>
		<li>Prepare 2x Phosphate Buffered Saline solution (PBS). Add two PBS tablets to 100 ml of water to make 0.02 M PBS solution.</li>
		<li>Combine BSA with 2x PBS to create a 30% BSA solution using the procedure listed below.
		<ol>
			<li>For example, to make a 30% BSA with 30 ml of 2x PBS, use 12.9 g of BSA. Add 1/3 of the 2x PBS (e.g., 10 ml) to a flask with a stir bar. Add 1/2 of the BSA (e.g., 6.45 g BSA) to the flask and using a spatula, wet the dry solute.</li>
			<li>Repeat this process of adding PBS and then BSA until all the solute and solvent is in the flask. Use the spatula to wet all the solute. It will look like a mixture of some liquid and clumps. Let it sit for approximately 30 min.</li>
			<li>Then, turn the stirrer on a low cycle and make sure that there are no clumps around the stir bar. It should take 90 min to get everything in solution or it can be left stirring O/N at 4 &#176;C. After the solute is all dissolved, place the solution in a 20 ml syringe and capped with a 0.20 &#181;m syringe filter. Press the plunger to expel liquid through filter and collect the sterilized solution in a new tube. Store at 4 &#176;C.</li>
		</ol>
		</li>
		<li>Prepare 3% glutaraldehyde solution by diluting 25% glutaraldehyde solution with sterile filtered 2x PBS. For example, for 10 ml of 3% Glutaraldehyde solution, use 2 ml of 25% glutaraldehyde and 8 ml of 2x PBS. Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
3. Molds Treatment
Note: The prototype described in this paper uses a custom made stainless steel Y mold piece. The mold contains an inflow and two outflow channels of 4 and 3 mm, respectively. First, clean molds, spray them with unsaturated lard, and sterilize them. Prepare the molds following the procedure described below.
<ol>
	<li>Clean stainless steel molds using sonicator at a frequency of 35 kHz. Place the molds in the sonicator and submerge them with water and ice. Keep the molds cold at all times while the sonicator is running. Run the sonicator for 2 periods of 90 min.
	<ol>
		<li>After each period, use a needle to make sure that there is no material in the Luer lock stainless steel or brass connector. Use soap and water to clean the entire surface of the two sides of the molds. Verify that there is no obstruction in the channels.</li>
	</ol>
	</li>
	<li>Place the molds, screws, and Luer lock connector in an autoclave bag and autoclave it.</li>
	<li>Fill the bottle that attaches to an air sprayer half way with commercially available lard (mixed fatty acid release agent). Replace the cap with a regular cap bottle. Place it in an autoclave bag and autoclave it. 
	Note: Lard is used to facilitate the release of the material that will be reaction injected later on (BSA rubber). Do not place the sprayer bottle lid in the autoclave- it can damage the internal seal.</li>
	<li>Warm up the lard for 45 sec or until its clear and liquid in a microwave. Screw the air sprayer lid to the lard bottle. Connect the lid with the sprayer. Attach the sprayer to the air source at the lab bench. Open the air valve, and open the nozzle of the sprayer until it starts wetting the surface of a paper towel.</li>
	<li>Spray lard perpendicular to the mold surface until the surface is fully covered. After each piece has been sprayed, place them in a Petri dish and seal it. Place the molds at 4 &#176;C for 2 hr.</li>
	<li>Proceed to sterilize the molds by exposing the surface to UV light for 30 min. Place them back at 4 &#176;C until they are ready to be reaction injected.</li>
</ol>
4. Reaction Injection of the BSA Rubber
Note: All the materials and solution should be keep cold until ready to use to prevent premature setting of the BSA rubber in the next steps.
<ol>
	<li>Prepare the dispenser for delivering the BSA rubber to the molds following these steps.
	<ol>
		<li>Sterilize all of the mixing components (two O rings, syringe cap, double syringe, mix tip, and 4:1 dispenser) by exposing them to UV light for 30 min in the polymerase chain reaction (PCR) hood. 
		Note: A PCR hood was used because this procedure involves fixatives. These chemicals cannot be used in the cell/tissue culture hood due to the risk of exposure and toxicity to the cells. Any other hood that contains a UV light will be suitable.&#160;</li>
		<li>Place the tip cap on the solution holder.</li>
		<li>Perform the mixing and injection at a 4:1 ratio of BSA:Glutaraldehyde. Add the 30% BSA to the double syringe chamber that will deliver the highest amount of solution (It will take approximately 4 ml to fill). Make sure to leave enough space to place the O ring in order to prevent overflowing and contamination of the adjacent chamber.</li>
		<li>Add 3% glutaraldehyde solution to the other chamber (it will take about 1 ml to fill). Make sure to leave enough space to place the O ring to prevent overflowing and contamination of the adjacent chamber.</li>
		<li>Place the double syringe on the dispenser. Tilt the assembly vertically so that the syringe cap is on top. Replace the cap with the mixing tip.</li>
		<li>Screw the two stainless steel mold pieces together.</li>
		<li>Place the assembly inside of an autoclave bag.</li>
		<li>To remove any air in the dispenser, hold it in the upright position and quickly squeeze the handle one time to release a small amount of the BSA mixed with the glutaraldehyde. Then quickly attach the stainless steel Y mold&#39;s Luer lock connector to the syringe tip.</li>
		<li>Hold the stainless steel Y mold with the left hand and the BSA-Glutaraldehyde mixture dispenser on the right. Alternate covering each of left and right exhaust of the outflow channels by pressing the exhaust to the sides of the autoclave bag to make sure the inside voids are filled with solution. Then, place the molds horizontally and inject again.</li>
		<li>Detach the molds from dispenser and place in a 25 mm Petri dish.</li>
		<li>Place Parafilm around the Petri dish to prevent dehydration of the rubber.</li>
		<li>Place the mold in the 4 &#176;C fridge O/N.</li>
	</ol>
	</li>
</ol>
5. Adjusting the Collagen Concentration
Note: The collagen should be kept on ice at all times during the process.
<ol>
	<li>Modify the collagen concentration using the percentage of collagen solids.
	<ol>
		<li>Make 10 ml of 1.75% collagen by adjusting the initial collagen concentration with cold water.</li>
		<li>Using a calibrated pH meter, adjust the pH to 3 using 12 N Hydrochloric Acid. Do not add the hydrochloric acid directly to the collagen- add the acid to the side of the tube. After adding the acid, use the spatula to push the acid into the collagen and quickly stir the mixture.</li>
		<li>Weigh 4 g of collagen in a separate conical tube.</li>
		<li>Centrifuge the collagen to remove air at 4 &#176;C and 9,343 x g for 20-30 min.</li>
		<li>UV sterilize the cell culture hood for 30 min, and add 14 &#181;l of laminin to the 4 ml collagen. This will result in a final laminin concentration of 10 &#181;g/&#181;l. Note: Laminin provides structural integrity, adhesion, and promotes various cellular responses.</li>
		<li>Turn the PCR UV light on for 20-30 min prior to using the hood for sterilization.</li>
		<li>Place the Luer lock cap, to attach to a 20 ml syringe, in ethanol for a 2 hr. Then, allow it to dry and place it in UV light.</li>
		<li>Gamma irradiate the collagen for 8.6 min to reach 1,200 cGy. 
		Note: The time will depend on the decay of the Cesium source. Adjust the time to reach the same dosage.</li>
	</ol>
	</li>
</ol>
6.Casting Collagen on BSA Rubber
<ol>
	<li>To polymerize the collagen, use an 8:1:1 ratio (collagen:HEPES:MEM). The following procedure is based on an initial 4 g of acidified collagen (from Step 5.1.8).
	<ol>
		<li>Make a 0.2 N HEPES solution in water, and adjust the pH to 9 by adding small amounts (1-5 &#181;l) of 1-5 M sodium hydroxide (NaOH) solution. Using a calibrated pH meter, monitor the pH of the solution after each addition. Store at 4 &#176;C or keep on ice.</li>
		<li>Turn on the UV light of the tissue culture hood for 20-30 min to sterilize the hood.</li>
		<li>Autoclave forceps, spatula, and scalpel for sterilization.</li>
		<li>Mix 1.5 ml of 0.2 N HEPES (pH 9) and 1.5 ml of 10x MEM using the tissue culture hood. Make sure to keep it on ice and vortex it for 5 sec prior to use. Store at 4 &#176;C or keep on ice.</li>
		<li>To sterilize the PCR hood, turn on the UV light for 30 min.</li>
		<li>Place a 12 well plate and a 20 ml syringe in -20 &#176;C for 10 min, or until ready to continue. Note: Keep all materials cold until ready to use. The increase in temperature induces premature collagen fibrillogenesis.</li>
		<li>Spray all tubes and well plates that are going to be in the hood with 70% ethanol and let them dry for sterilization. Place a 20 ml syringe on ice to cool for later use.</li>
		<li>In the PCR hood, open the stainless steel molds to release the BSA rubber and using a scalpel, cut the exhaust channels of the BSA rubber mold.</li>
		<li>Under the PCR hood, open the sterile collagen tubes and add 1 ml of the HEPES-MEM solution (make sure that before extracting the HEPES-MEM solution, that it is well mixed and there are no solid deposits).</li>
		<li>Using the sterile spatula, thoroughly mix the collagen and buffer solution.</li>
		<li>Close and vortex it quickly to ensure a well-mixed hydrogel.</li>
		<li>Transfer to a cold 20 ml syringe.</li>
		<li>With one hand, hold the BSA Rubber inside of the well and, using the other, dispense half of the collagen hydrogel solution onto the bottom of the well.</li>
		<li>Using the sterile tweezers, ensure that the rubber inflow and outflow ends are touching the sides of the well.</li>
		<li>Pour collagen solution on top of the rubber until is completely covered.</li>
		<li>Ensure that the BSA rubber is suspended within the collagen and that there are no bubbles, especially near the ends of the rubber.</li>
		<li>Place the cover and wrap Parafilm around the circumference of the well.</li>
		<li>Put in the incubator for 1 hr at 37 &#176;C. Keep the PCR UV light on.</li>
	</ol>
	</li>
	<li>After the polymerization of the collagen, UV crosslink the hydrogel via the following procedure. 
	Note: The crosslinking of the collagen will be done using a UV crosslinker apparatus in which the amount of energy can be controlled.
	<ol>
		<li>Turn on the UV crosslinker and use the energy setting to irradiate the empty chamber with 630,000 &#181;J/cm2.</li>
		<li>Remove the gels from the incubator.</li>
		<li>Spray hands with ethanol, and, inside the chamber, remove the lid as quickly as possible.</li>
		<li>Close the chamber and UV crosslink the hydrogels by selecting the energy setting and irradiating 630,000 &#181;J/cm2.</li>
		<li>After the crosslinking cycle, turn off the UV light on the PCR hood</li>
		<li>Spray hands with ethanol and open the chamber, quickly placing the lid back onto the well plate. Move the well plate to the PCR hood.</li>
		<li>Using the sterile spatula, gently loosen and remove the gel from the well. Flip the gels under the hood to crosslink the bottom of the hydrogel. Repeat step 6.2.3 and 6.2.4.</li>
	</ol>
	</li>
</ol>
7. Enzyme Digestion of the BSA Rubber
<ol>
	<li>In order to have a hollow collagen scaffold, remove BSA Rubber in a way that does not affect the dimensions embedded in the hydrogel. The procedure is described below.
	<ol>
		<li>Turn on the UV light for 20-30 min to sterilize the tissue culture hood.</li>
		<li>Make 0.25% trypsin solution pH 7.8. For example, for 15 ml of water, add 0.0376 g of trypsin in a 50 ml conical tube. Adjust the pH to 7.8 by adding small amounts (2-5 &#181;l) of 1 M NaCl. Place the solution in a 20 ml syringe and capped with a 0.20 &#181;m syringe filter. Press the plunger to expel liquid through filter and collect the sterile solution in a new tube.</li>
		<li>Turn on the water bath and set the temperature to 30 &#176;C.</li>
		<li>After 30 min, turn off the UV light. Spray ethanol on all the tubes and materials that will be used in the hood.</li>
		<li>Transfer the collagen hydrogel under the hood and place in separate conical tube.</li>
		<li>Add around 3-5 ml (just enough to cover the gels) of 0.25% trypsin solution with a pH of 7.8 to each tube.</li>
		<li>Seal the tubes with Parafilm and vortex lightly for approximately 1 min.</li>
		<li>Place in the 30 &#176;C water bath for 15-24 hr. While in the water bath, lightly vortex the gels frequently until the BSA rubber has been digested or removed from the hydrogel. 
		Note: In order to determine if the BSA rubber has been removed, either the rubber is floating in the trypsin solution or there are broken-down pieces. There must be no visual dark areas within the hydrogel.</li>
	</ol>
	</li>
	<li>To ensure that all the BSA rubber and trypsin has been removed from the hydrogels, rinse it as described below.
	<ol>
		<li>Turn on the PCR hood UV light for 20-30 min.</li>
		<li>Prepare Mosconas solution. Combine potassium chloride (KCl, 28.6 mM), (NaHCO3, 11.9 mM), glucose (9.4 mM) and (NaH2PO4, 0.08 mM) in water. Adjust pH to 7.4 with 1 M NaOH or 12 M HCl solution. Place the solution in a 20 ml syringe and use a syringe filter of 0.20 &#181;m to sterilize the solution.</li>
		<li>Spray everything with ethanol prior placing them on the hood and allow the ethanol to dry.</li>
		<li>Open the tube and transfer 5-10 ml of sterile Mosconas solution to a new 50 ml conical tube.</li>
		<li>Transfer the collagen hydrogel to the conical tube that contains the enzyme solution. Make sure that the hydrogel is completely covered with the solution. Leave the tube in the shaker in the fridge at 4 &#176;C for 30 min.</li>
		<li>Aspirate the Mosconas solution and repeat step 7.2.5 twice.</li>
		<li>Store at 4 &#176;C.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Biofabrication is a highly multidisciplinary field in which biology and engineering principles merge to generate complex materials that mimic native tissue. In order to achieve this, there is a need to develop techniques that use the information gathered from in vivo tissue and translate it into an in vitro scaffold. In this way, a platform can be engineered that closely resembles the architectural, functional, and mechanical properties of the in vivo tissue. The optimal scaffolding material should possess certain properties, such as being biocompatible, mimic the mechanical properties of the tissue of interest, be capable of controlled degradation, able to support cell viability, and capable of allowing tissue remodeling2,3.
A multitude of fabrication techniques have been developed to generate viable, three-dimensional constructs. These technologies fall into two major categories: conventional and advanced. The conventional techniques include the use of synthetic and natural traditional materials to make porous structures. Some examples are solvent-casting, freeze drying, and melt molding. Disadvantages of these techniques include poor control of porosity within the structure (pore size and pore interconnectivity) and difficulty making internal channels within the scaffolds. Advanced techniques include stereolithography, molding, 3D printing, and electrospinning, among others1. These techniques have drawbacks such as the lack of long-range microarchitecture channels, material selection to provide optimal mechanical strength while being capable of dispensing through small-diameter nozzles, optimization dependent on the material, require extensive post-processing that might be toxic, and restriction in the design of inner architectures in an in vitro construct. A major drawback in 3D printing is the availability of biomaterials that are adequate cell carriers, while also having the mechanical properties required to maintain a defined architectural organization12. The technology presented here incorporates both conventional and advanced fabrication techniques. The best of both worlds is derived from the convergence of computer-aided manufacturing to create, or import, the desired architecture with the development of an enzyme-labile rubber. The stainless steel molds were created using a milling machine, but their fabrication isn&#39;t limited to this technique. Using a 3D Printer (e.g., Makerbot), the same molds were fabricated from polylactic acid (PLA). The negative molds were milled using architectural directives designed by the CAD program, which provides solid molds that create an easy and reliable transfer of features to any material. This technology is significant in that it allows not only the control of the external tissue composition, but also of the highly complex internal architecture. The work presented here focuses primarily on the characterization of the BSA rubber, which can be mixed homogeneously, is easily digested in a reasonable amount of time, was resistant to alterations in its structure, and is able to mimic minute features, while holding its stability during the casting process. The limitation of this technique was tested and the sacrificial material&#39;s resolution can maintain dimensions as small as 300 &#181;m in diameter. However, Figure 7 show that the channel is surrounded by a thin layer of mold release agent. Using a microscope, this additional area is clearly distinguished and can be removed to have the desired dimension. This biofabrication technique allows the replication of a wide variety of internal structures that range from macro- to micro-scale.
Serum albumin is the most abundant protein in the circulatory system. Since the proteins are polyelectrolytes, solubility was determined by electrostatic interactions15-17. It has been shown that at low salt concentrations, there is a salting-in effect on the protein facilitating its solubility18. Glutaraldehyde is a crosslinking agent that causes changes in the properties of albumin. The color change seen in Figure 1 is attributed to the formation of the aldimine linkages19-21. The glutaraldehyde reacts predominantly with the BSA through the amino groups of lysine to form the intermolecular covalent bonds14. Data indicates that the increase of salts improved the elasticity of the rubber (from 1x PBS to 2x PBS). The higher glutaraldehyde concentration produces stiffer gels that set earlier compared to low concentration ones. However, they are more difficult to dispense, mix homogenously, and they form brittle gels. To deliver the appropriate amount of BSA rubber into the molds, every part of the assembly must be kept cold because higher temperatures accelerate the glutaraldehyde and BSA reaction. The ideal rubber (30% BSA 3% glutaraldehyde in 2x PBS or 1x PBS) behaves as a viscoelastic material that can withstand loading without permanently deforming. This becomes very important when handling and casting material around the BSA rubber structure.
Trypsin is a serine protease that hydrolyzes proteins. Trypsin is a widely used enzyme that has high cleavage specificity. It cleaves the peptide chains mainly at the carboxyl side of the amino acids lysine and arginine22. BSA is readily soluble at low concentrations in water, and the trypsin readily digested the BSA rubber leaving the collagen intact and relatively untouched. The material will not have any contact with cells. The construct was tested for the presence of free glutaraldehyde and there were no traces of this fixative. This demonstrates the efficacy of the BSA rubber as a sacrificial material for biofabrication. In this protocol, collagen was used as the scaffold material, but any other material that is resistant to trypsin digestion could be used.
Future work will be focused on reconstituting the vascular components within the hydrogels by seeding the scaffold with endothelial and fibroblast cells. One of the challenges is to create a uniform distribution of cells throughout the inside of the channels that mimic the native 3D distribution. To address this issue, a strategy is being developed that allows the sealing or plugging of the channels and the injection of the cell suspension. The material tested is pluronic F127, which is a thermoreversible gel, liquid at 4 &#176;C and a solid above 30 &#176;C23,24. A high concentration of pluronic was successful in creating the necessary temporal seal. After the cell suspension is within the channels of the scaffold, the entity is rotated for a specific amount of time until the cells adhere to all sides of the 3D structure. The inside channel will have adequate media for maintaining cell survival. Pluronic maintains its gel form and is readily soluble in an aqueous environment. Once the cells adhere, the hydrogel will be flooded with media, and can be cultured in stationary or flow conditions, depending on the purpose of the study. This methodology will be further assessed and will become the follow up to this publication. The biofabrication technique described herein is currently being used to develop a scaffold replica of a human renal artery. The same approach could be done with other types of tissues, such as cardiac, to expand the applicability of this technique to a wide range of clinical applications.
The biofabrication technique developed here is a step forward in the generation of in vitro scaffolds that can recapitulate intrinsic geometrical features quickly and reliably. A natural material, such as collagen, was selected because it offers optimal chemical and physical cues to cells over synthetic materials. These natural materials can be used for therapeutic research, as in vitro models of development, malformation, and disease tissue, as well as for replacement of damaged tissue.

In the tissue engineering field the ability to fabricate tissue scaffolds is vital. A suitable tissue scaffold has a 3D structure, is composed of biocompatible materials, and mimics in vivo tissue architecture to facilitate cell and tissue growth and remodeling. This scaffold must allow the transport of nutrients and the removal of wastes1-4. One of the main obstacles in the production of these scaffolds is the ability to recapitulate specific geometrical features into a biocompatible material. Several biofabrication techniques have been reported to control the geometrical features of these scaffolds, examples are electrospinning5-8, solvent-casting9, stereolithography10, and 3D-printing11, among others. These techniques fall short in providing a relatively easy transfer of controllable internal and external architectural features, are expensive, are limited by their resolution and printability (e.g., nozzle gauge, material restriction), or require post-fabrication techniques which demands a long period of time to produce viable scaffolds12.
In many commercial fabrication systems, the creation of internal voids, channels, and features is achieved using sand or other suitable removable or sacrificial materials. The metal or plastic part is formed around the sand mold, and once it is solidified, the sand is removed. In much the same manner, the next generation of biomaterials needs the biosand equivalent. Therefore, the BSA rubber was developed as a substitute for biosand. The BSA rubber is a newly formulated material that consists of bovine serum albumin crosslinked with glutaraldehyde. The ultimate goal is to recreate specific architectural features into a biodegradable collagen scaffold. The characteristics of the sacrificial biorubber that maintains dimensional fidelity with the mold of the original tissue are described.
Several combinations of BSA and glutaraldehyde concentrations were tested using a variety of solvents. This material was created by the reaction between BSA and glutaraldehyde. BSA rubber can be reaction injected into the intricate geometries of the tissue molds. Crosslinked BSA is trypsin labile and readily digested by the enzyme at mild pH and temperature conditions. Conversely, intact type I collagen is very resistant to trypsin digestion. These features were capitalized to selectively remove the BSA rubber leaving the collagen behind. The present work consisted of determining the ideal parameters needed to obtain a labile mold that can deliver specific architectural features to a biocompatible scaffold. The specific features that were evaluated included mixability, enzyme digestion, load bearing, and ability to be reaction injected into a negative mold. The combination of 30% BSA and 3% glutaraldehyde fulfills these requirements. This protocol provides the necessary guidelines to create these three-dimensional scaffolds. The prototype consists of a collagen scaffold that represents a branched architecture with one inflow and two outflow channel with diameters of 4- and 3-mm, respectively. This technique has the potential to mimic macro- and micro-environments of the tissue of interest. This technology provides a viable technique to deliver a specific geometrical instructive to a biodegradable material in a relatively easy and timely matter with high fidelity, which can be tuned to mimic the in vivo tissue elasticity and other characteristics of the tissue of interest.

<table border="0" cellpadding="0" cellspacing="0" width="770">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:281px;">Collagen type I</td>
			<td style="width:156px;"></td>
			<td style="width:116px;"></td>
			<td style="width:217px;">Collagen extracted from calf hide</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Hydrocloric Acid (HCl)</td>
			<td>Sigma-Aldrich</td>
			<td>7647-01-0</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Phosphate Buffer Solution (PBS Tablets)</td>
			<td>MP Biomedical</td>
			<td>U5378</td>
			<td>1 tablet per 100 mL makes 1XPBS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Albumium from bovine serum (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Glutaraldehyde</td>
			<td>Sigma -Aldrich</td>
			<td>G5882</td>
			<td>Toxic</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lard</td>
			<td>Fields</td>
			<td>3090</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Stainless Steel Molds</td>
			<td></td>
			<td></td>
			<td>Milled using Microlution Machine</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Air Brush Kit</td>
			<td>Central Pneumatic</td>
			<td>47791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mixing Tip for double syringe</td>
			<td>Medmix</td>
			<td>ML2.5-16-LLM</td>
			<td>Mixer, DN2,5X16, 4:1 brown, med</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Small O ring for double syringe</td>
			<td>Medmix</td>
			<td>PPB-X05-04-02SM</td>
			<td>Piston B, 5mL, 4:1, PE natural</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe cap&#160;</td>
			<td>Medmix</td>
			<td>VLX002-SM</td>
			<td>Cap, 4:1/10:1, PE brown, med</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Big O ring for double syringe</td>
			<td>Medmix</td>
			<td>PPA-X05-04-02SM</td>
			<td>Piston A, 5 mL, 4:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe&#160;</td>
			<td>Medmix</td>
			<td>SDL X05-04-50M</td>
			<td>Double syringe, 5 mL, 4:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Double Syringe Dispenser</td>
			<td>Medmix</td>
			<td>DL05-0400M</td>
			<td>Dispenser, 5 mL, 4:1, med , plain</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Laminim</td>
			<td></td>
			<td></td>
			<td>3.6 mg/mL- extracted USC lab</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">20 mL Syringe Luer Lock Tip</td>
			<td>BD</td>
			<td>302830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Luer Lock Caps</td>
			<td>Fisher</td>
			<td>JGTCBLLX</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HEPES</td>
			<td>Sigma -Aldrich</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gibco Minimum Essential Media 10X (MEM)</td>
			<td>&#160;Life Technologies</td>
			<td>1143-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trypsin</td>
			<td>Life Technologies</td>
			<td>27250-018</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">UV Crosslinker&#160;</td>
			<td>Spectroline UV</td>
			<td>XLE1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium Cloride (NaCl)</td>
			<td>Fisher</td>
			<td>S271-10</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Potassium chloride (KCl)</td>
			<td>Sigma -Aldrich</td>
			<td>P5405-250</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Bicarbonate (NaHCO3)</td>
			<td>Fisher</td>
			<td>BP328-500</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glucose</td>
			<td>Sigma -Aldrich</td>
			<td>G-8270</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Sodium Phosphate didasic (NaH2PO4)</td>
			<td>Sigma-Aldrich</td>
			<td>S-7907</td>
			<td>To prepare Mosconas</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sterile Filter for syringes</td>
			<td>Corning</td>
			<td>431224</td>
			<td></td>
		</tr>
	</tbody>
</table>

three-dimensional biomimetic technology: novel biorubber creates defined micro- and macro-scale architectures in collagen hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

Watch this Scientific Journal Video about Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels at JoVE.com

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

three-dimensional-biomimetic-technology-novel-biorubber-creates

Nanyang Technological University

Research

JoVE Journal

Bioengineering

9.1K Views.  Medical University of South Carolina. An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Video: Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Three-dimensional Optical-resolution Photoacoustic Microscopy

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As the mainstays of in vivo three-dimensional (3-D) optical microscopy, single-/multi-photon fluorescence microscopy and optical coherence tomography (OCT) have demonstrated their extraordinary sensitivities to fluorescence and optical scattering contrasts, respectively. However, the optical absorption contrast of biological tissues, which encodes essential physiological/pathological information, has not yet been assessable. 
The emergence of biomedical photoacoustics has led to a new branch of optical microscopy optical-resolution photoacoustic microscopy (OR-PAM)1, where the optical irradiation is focused to the diffraction limit to achieve cellular1 or even subcellular2 level lateral resolution. As a valuable complement to existing optical microscopy technologies, OR-PAM brings in at least two novelties. First and most importantly, OR-PAM detects optical absorption contrasts with extraordinary sensitivity (i.e., 100%). Combining OR-PAM with fluorescence microscopy3 or with optical-scattering-based OCT4 (or with both) provides comprehensive optical properties of biological tissues. Second, OR-PAM encodes optical absorption into acoustic waves, in contrast to the pure optical processes in fluorescence microscopy and OCT, and provides background-free detection. The acoustic detection in OR-PAM mitigates the impacts of optical scattering on signal degradation and naturally eliminates possible interferences (i.e., crosstalks) between excitation and detection, which is a common problem in fluorescence microscopy due to the overlap between the excitation and fluorescence spectra. 
Unique for optical absorption imaging, OR-PAM has demonstrated broad biomedical applications since its invention, including, but not limited to, neurology5, 6, ophthalmology7, 8, vascular biology9, and dermatology10. In this video, we teach the system configuration and alignment of OR-PAM as well as the experimental procedures for in vivo functional microvascular imaging.

Optical-resolution photoacoustic microscopy (OR-PAM) is an emerging technology capable of imaging optical absorption contrasts in vivo with cellular resolution and sensitivity. Here, we provide a visualized instruction on the experimental protocols of OR-PAM, including system configuration, system alignment, typical in vivo experimental procedures, and functional imaging schemes.

Optical microscopy, providing valuable insights at the cellular and organelle levels, has been widely recognized as an enabling biomedical technology. As ...

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

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 ...