Michael Henry, Dublin City University, kindly assisted with the data collection and analysis shown in Figure 5. This work was supported partially by (1) George Mason University, (2) the Italian IstitutoSuperiore di Sanita&#8217; in the framework of the Italy/USA cooperation agreement between the U.S. Department of Health and Human Services, George Mason University, and the Italian Ministry of Public Health, (3) NIH, IMAT program grants 1R21CA137706-01 and 1R33CA173359-01 to LAL, and (4) Ceres Nanosciences, Inc.

Human plasma and urine was collected from healthy volunteer donors, with written informed consent, following George Mason University Institutional Review Board approved protocols. Donors were equally distributed between Caucasian males and females between the ages of 25 and 42. Samples were analyzed individually and were not pooled.
1. Nanoparticle Processing of Serum or Plasma Samples
Potential low abundant biomarkers in plasma are captured, in solution, with hydrogel nanoparticles. The particles are added to the plasma, incubated, separated by centrifugation, washed, and the captured proteins are eluted. The eluted proteins are dried under nitrogen flow for downstream mass spectrometry sequencing and identification.
<ol>
	<li>Dilute 500 &#181;l of serum 1:2 with 50 mM TrisHCl pH 7 (500 &#181;l serum + 500 &#181;l TrisHCl) in a microcentrifuge tube.</li>
	<li>Add 500 &#181;L of poly(NIPAm/AAc) core nanoparticles. Incubate for 15 min at RT.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge equipped with a fixed-angle rotor. Remove and discard the supernatant.</li>
	<li>Add 500 &#181;l sodium thiocyanate (25 mM) to the pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.</li>
	<li>Add 500 &#181;l of MilliQ water to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and discard the supernatant.</li>
	<li>Prepare fresh elution buffer: Add 700 &#181;l acetonitrile to 300 &#181;l ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.</li>
	<li>Add 300 &#181;l of elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.</li>
	<li>Spin the sample at 16,100 x g, 25 &#176;C for 10 min in a centrifuge with a fixed angle rotor. Remove and save the eluate in a clean, labeled microcentrifuge tube.</li>
	<li>Repeat steps 1.9 &#8211; 1.10. Combine the two eluates into one microcentrifuge tube.</li>
	<li>Dry the eluates under nitrogen flow in a nitrogen evaporator manifold at 42.1&#160;&#176;C, with air flow set to 8 (Figure 2F).</li>
	<li>Store the dried eluate at RT for O/N storage, or at -20&#160;&#176;C for long term storage, prior to mass spectrometry, western blotting, or ELISA assays (optional).</li>
</ol>
2. Nanoparticle Processing of Urine Samples
Normal urine contains less than 30 mg/dl protein and less than 1+ blood. However, many diseases/conditions may alter the normal levels of urine protein and blood. To aid in determining the optimal volume of nanoparticles to add to the urine sample, a urinalysis is performed prior to nanoparticle harvesting. Urine biomarkers may exist in extremely low concentrations, which may require optimizing the ratio of nanoparticles to urine volume. This procedure describes nanoparticle harvesting of urine samples for downstream western blot analysis.
<ol>
	<li>Collect urine samples in a clean, dry plastic cup. A 22 ml minimum volume is required. Store urine specimens at -80 &#176;C until ready to analyze.</li>
	<li>Thaw the frozen urine at RT or at 4 &#176;C O/N. Mix briefly on a vortex mixer. Pour at least 22 ml of urine into a 50 ml conical bottom polypropylene tube.</li>
	<li>Spin the urine in a centrifuge with a swing-out rotor at 3,700 x g for 15 min. Decant the urine, without disturbing the pellet, into a clean 50 ml conical bottom polypropylene tube. Discard the pellet.</li>
	<li>Perform urinalysis using a multi-analyte &#8220;urine dipstick&#8221; reagent strip. Note: store the reagent strips in a tightly closed container away from direct sunlight and humidity17, 18.
	<ol>
		<li>Lay the reagent strip, face-up, on a clean, dry paper towel.</li>
		<li>Use a disposable pipette to aspirate 1 ml of the urine prepared in step 2.3.</li>
		<li>Set a timer for 2 min but do not start it. Quickly dispense 1 - 2 drops of urine on each test pad of the reagent strip. Immediately start the timer. Note: Do not submerge the reagent strip directly in the urine container. The dye/chemical indicators in the reagent strip can potentially leach out of the reagent strip into the urine container.</li>
		<li>At the time indicated on the reagent strip container, record the qualitative results for the various analytes on the reagent strip by comparing the color of the individual reagent strips to the corresponding color-coded indicators on the reagent container. Typical normal urine results are: (normal or negative) for blood, protein, leukocytes, nitrite, glucose, ketone, bilirubin, and urobilinogen; Urine pH (5.5 - 7.0); specific gravity (1.001 - 1.020). Note: High protein levels in a urine specimen may compete with the protein of interest for binding sites on the nanoparticles. To maximize protein harvesting with nanoparticles, the nanoparticle volume/urine volume ratio can be adjusted to either limit competition from high abundance proteins, or can be optimized to harvest proteins that exist in very low concentrations. If the urine protein value is 1+ or greater, add 2x volumes of nanoparticles in step 2.5 below. If the analyte of interest is a very low abundant protein, it may be necessary to increase the volume of nanoparticles up to 2 ml&#160;(Figure 3).</li>
	</ol>
	</li>
	<li>Transfer 20 ml of the clarified urine from step 2.3 into a clean 50 ml conical bottom polypropylene tube. Do not disturb any debris/pellet that may be in the bottom of the urine tube. Add 200 &#181;l of nanoparticles to the 20 ml urine sample. Mix briefly on a vortex mixer. Note: If the urine protein value is 1+ or greater, add 400 &#181;l of nanoparticles to 20 ml urine.</li>
	<li>Incubate the urine/nanoparticle mixture for 30 min at RT, without rocking/mixing.</li>
	<li>Spin the urine/nanoparticle suspension in a centrifuge equipped with a swing-out rotor at 3,700 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 &#8211; 7 min.</li>
	<li>Remove and discard the supernatant. Add 500 &#181;l MilliQ water to the nanoparticle pellet.</li>
	<li>Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Transfer the nanoparticle solution to a clean 1.5 ml microcentrifuge tube.</li>
	<li>Spin the nanoparticles in a centrifuge equipped with a fixed-angle rotor at 16,100 x g for 10 min. Note: If a pellet is not visible following centrifugation, spin the samples for an additional 2 &#8211; 7 min.</li>
	<li>Remove and discard the supernatant.</li>
	<li>Repeat steps 2.8 and 2.11 (twice) with 200 &#181;l 18 MilliQ water to wash the nanoparticles.</li>
	<li>Prepare fresh elution buffer: Add 970 &#181;l acetonitrile to 30 &#181;l ammonium hydroxide in a clean tube. Caution: Ammonium hydroxide is corrosive. Use with appropriate ventilation. Note: The elution buffer must be prepared immediately before use. Do not store the elution buffer.</li>
	<li>Add 20 &#956;l elution buffer to the nanoparticle pellet. Resuspend the nanoparticles by vigorously pipetting up and down multiple times. Incubate for 15 min at RT.</li>
	<li>Spin the nanoparticle samples at 16,100 x g for 10 min. Remove and save the supernatant in a clean, 1.5 ml microcentrifuge tube. Do not disrupt the nanoparticle pellet. Discard the pellet in the biohazard trash.</li>
	<li>Place the samples in a rack in a chemical fume hood. Open the caps and incubate at RT for 30 min. Alternatively, place the samples under nitrogen flow at 40 &#176;C until dry (1 - 2 hr).</li>
	<li>Add 15 &#181;l Tris-glycine SDS sample buffer (2x) to the samples. Heat at 100 &#176;C in a dry heat block for 5 min with the caps open or until the volume left in the tube is no more than 20 &#181;l. Note: do not use a boiling water bath. The humidity from the water bath will prevent evaporation of the buffer.</li>
	<li>Place the cap on the tubes. Remove the tubes from the heat block. The sample can be stored at -80 &#176;C or used immediately for downstream western blot analysis.</li>
</ol>

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Benjamin Espina is an employee of Ceres Nanosciences, Inc. that produces reagents used in this Article. Lance Liotta, Alessandra Luchini and Virginia Espina hold patents (US patent 7,935,518 and/or 8,497,137) on the nanoparticle technology used in this Article. As university employees they are entitled to receive royalty from these patents per university policy. Lance Liotta and Alessandra Luchini are shareholders in Ceres Nanosciences and serve on the Scientific Advisory Board.

Clinical Relevance
A serum or plasma sample is thought to contain low-abundance circulating proteins and peptides which can provide a rich source of information regarding the state of the organism as a whole. Despite the promise of serum proteomics, there are three fundamental and serious physiologic barriers thwarting biomarker discovery and translation to clinical benefit10, 11, 16, 25.
1. Important diagnostic biomarkers may exist in ex...

Despite the completion of the human genome sequencing, significant progress has not been made in identifying biomarkers predictive of early stage disease, or that correlate with therapeutic outcome, or prognosis1. One reason for this lack of progress is that many potentially significant biomarkers exist at a concentration below the detection limit of conventional mass spectrometry and other biomarker discovery platforms. Mass spectrometry (MS) and Multiple Reaction Monitoring (MRM) have a detection sensitivity typically greater than 50 ng/ml while the majority of the analytes measured by immunoassays in a clinical laboratory fall in the range between 50 pg/ml and 10 ng/ml. This means that many biomarkers, particularly in the early stage of a disease cannot be detected by conventional MS and MRM2. In addition the presence of high-abundance proteins such as albumin and immunoglobulin in complex biological fluids often mask by billion-fold excess low-abundance, low molecular weight proteins and peptides3, 4. For this reason several sample preparatory steps are required prior to mass spectrometry sequencing and identification. One such preparatory step employs the depletion of high-abundance proteins with commercially available depletion columns5-8. Unfortunately this step leads to the reduction of the yield of candidate biomarkers because they are often non-covalently associated with carrier proteins that are being removed. Another challenge is represented by the stability of candidate biomarkers ex-vivo once the samples are collected. Proteins are subject to degradation by endogenous or exogenous proteases9. Hydrogel nanoparticles can transcend these critical challenges by amplifying the putative biomarker concentration to a level within the range of the assay, while protecting the protein from degradation10-13.
It&#8217;s important to note that LMW proteins in blood are a mixture of small intact proteins as well as fragments of large proteins. Tissue-derived proteins larger than 60 kDa are too large to passively enter the blood stream through the vascular basement membrane, but they can be represented in blood as peptides or protein fragments14. Our goal is to measure novel circulating biomarkers that can be candidates for early detection of disease, patient stratification for therapy, and monitoring the response to therapy. Our nanoparticles are created to selectively exclude high abundance immunoglobulins and albumin, while simultaneously capturing smaller proteins and peptides and concentrating them up to 100-fold depending on the starting volume.
Our group identified a set of small organic dyes which can successfully act as high affinity molecular baits for proteins and peptides. Protein-dye binding is thought to be due to a combination of hydrophobic and electrostatic interactions. The aromatic rings on the dye interleave with proteins via hydrophobic pockets on the protein surface11.
The baits, depending on their chemistry, show a particular affinity for selected classes of analytes. The baits compete with the carrier proteins, such as albumin, for the proteins or peptides. The low-molecular weight proteins/peptides become trapped in the particle. High-molecular weight proteins such as albumin and immunoglobulin are prevented from entering the particle because of the sieving capability due to the restrictive pore of the hydrogel11(Figure 1).
Hydrogel nanoparticles are synthesized by precipitation polymerization initiated by ammonium persulfate11. N-isopropylacrylamide (NIPAm), co-monomers of acrylic acid (AAc) and allylamine (AA) and cross-linker N,N&#8217;-Methylenebisacrylamide (BIS) are allowed to react at 70 &#176;C for 6 hr in dilute conditions11, 13. The high protein binding affinity of poly(N-isopropylacrylamide-co-acrylic acid) (poly(NIPAm-co-AAc) nanoparticlesis achieved by covalently incorporating amino-containing dyes (i.e., sulfonatedanthraquinonetriazine dyes) into the nanoparticles through an amidation reaction performed in aqueous or organic solvents depending on the hydrophilic/hydrophobic characteristics of the dyes11, 13. Nucleophilic substitution of the amine groups in the nanoparticle with the chloride atom of an anthraquinonetriazine dye is utilized to create dye-containing poly(NIPAm-co-Allylamine) (AA) nanoparticles11, 12. A two-step polymerization process is utilized to create hydrogel nanoparticles containing an outer shell of vinylsulfonic acid (VSA)11, 13.
Hydrogel nanoparticles can be applied to various biological fluids, including whole blood, plasma, serum, cerebrospinal fluid, sweat, and urine. In one step, in solution, the nanoparticles perform a rapid (within minutes) sequestration and concentration of low molecular weight analytes10, 11, 13, 15-18. Proteins are subsequently eluted from the nanoparticles and detected using western blotting19-21, mass spectrometry10, 11, 13, 15, 18, 22, 23, immunoassays/ELISA10, 11, 15, 18, or reverse phase protein microarray16, 24 assays. Nanoparticles functionalized with chemical bait, and presenting a core or core shell architecture, capture and concentrate proteins based on the bait/shell physicochemical properties. Different dyes incorporated into the nanoparticles will therefore capture different subsets of proteins with varying efficiency based on the dye affinity, pH of the solution, and the presence/absence of competing high-abundant proteins13. Furthermore, the quantity of nanoparticles in relation to the volume of the solution will affect the protein yield from the nanoparticles. These aspects of hydrogel nanoparticle harvesting are demonstrated using three different nanoparticle baits for harvesting proteins from plasma samples which contain high amounts of protein, and from urine samples which typically do not contain large amounts of protein. In this protocol we demonstrate harvesting and concentrating tumor necrosis factor alpha (TNF&#945;) from plasma samples using poly(NIPAm-co-AAc), Poly(NIPAm/dye), and core- shell nanoparticles (Poly(NIPAm-co-VSA)). Poly(NIPAm/dye) nanoparticles are shown to concentrate Mycobacterium species antigen that was added to human urine samples, to mimic Mycobacterium tuberculosis infected individuals.

<table border="0" cellpadding="0" cellspacing="0" width="957">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">hydrogel nanoparticles</td>
			<td style="width:187px;">Ceres Nanoscience</td>
			<td style="width:119px;">CS003</td>
			<td style="width:336px;">NanoTrap ESP particles</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl, 50mM pH7.0</td>
			<td style="width:187px;">VWR</td>
			<td style="width:119px;">IC816116</td>
			<td>50mM, pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acetonitrile</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH1103-4LP</td>
			<td>available from VWR</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Ammonium Hydroxide NH4OH</td>
			<td style="width:187px;">BDH</td>
			<td style="width:119px;">BDH3014</td>
			<td>available from VWR, assayed at 28-30% NH3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">sodium thiocyanate 25mM</td>
			<td style="width:187px;">Acros Organics</td>
			<td style="width:119px;">419675000</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multi-analyte Urine Reagent Strips</td>
			<td style="width:187px;">Siemens</td>
			<td style="width:119px;">2161</td>
			<td style="width:336px;">for urine samples</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Tris-Glycine SDS Sample Buffer (2X)</td>
			<td style="width:187px;">Life Technologies</td>
			<td style="width:119px;">LC2676</td>
			<td style="width:336px;">use at room temperature to prevent SDS from precipitating</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Dry bath incubator (100 oC) with heating block</td>
			<td style="width:187px;">Barnstead</td>
			<td>11-715-125DQ</td>
			<td>do not substitute a boiling water bath</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nitrogen evaporator manifold</td>
			<td style="width:187px;">Organomation Associates</td>
			<td>Microvap118</td>
			<td>for serum/plasma samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, swing-out rotor</td>
			<td style="width:187px;">Sorvall</td>
			<td style="width:119px;">Legend series</td>
			<td>50ml tube capacity, rcf 3700 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Centrifuge, fixed angle rotor</td>
			<td style="width:187px;">Eppendorf</td>
			<td style="width:119px;">5424</td>
			<td>1.7ml microcentrifuge capacity, rcf 16,000 x g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">50ml conical centrifuge&#160;tubes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">14-432-22</td>
			<td>with screw caps for urine samples</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">1.5ml microcentrifuge tubes</td>
			<td style="width:187px;">Eppendorf</td>
			<td>22363204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Disposable plastic transfer pipettes</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">13-711-7M</td>
			<td>at least 1ml capacity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex mixer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td>50-949-755</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Timer</td>
			<td style="width:187px;">Fisher Scientific</td>
			<td style="width:119px;">S04782</td>
			<td>seconds/minutes</td>
		</tr>
	</tbody>
</table>

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.

Hydrogel Nanoparticle Size and Uniformity
Poly(NIPAm-AAc) particles have been produced with extremely high yield and reproducibility between and within batches. The particles have very good colloidal stability at RT during the time required for capture, storage, and elution of proteins (at least 48 hr), and nanoparticle precipitation has not been observed (Figure 1)11. The colloidal stability may be very important for rapid protein/peptide uptake by...

Watch this Scientific Journal Video about Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins at JoVE.com

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

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 ...

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13.8K Views.  George Mason University. 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.

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

Plasma Lithography Surface Patterning for Creation of Cell Networks

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular phenomena. To address this requirement, we have developed a plasma lithography technique to manipulate the cellular microenvironment by creating a patterned surface with feature sizes ranging from 100 nm to millimeters. The goal of this technique is to be able to study, in a controlled way, the behaviors of individual cells as well as groups of cells and their interactions.
This plasma lithography method is based on selective modification of the surface chemistry on a substrate by means of shielding the contact of low-temperature plasma with a physical mold. This selective shielding leaves a chemical pattern which can guide cell attachment and movement. This pattern, or surface template, can then be used to create networks of cells whose structure can mimic that found in nature and produces a controllable environment for experimental investigations. The technique is well suited to studying biological phenomenon as it produces stable surface patterns on transparent polymeric substrates in a biocompatible manner. The surface patterns last for weeks to months and can thus guide interaction with cells for long time periods which facilitates the study of long-term cellular processes, such as differentiation and adaption. The modification to the surface is primarily chemical in nature and thus does not introduce topographical or physical interference for interpretation of results. It also does not involve any harsh or toxic substances to achieve patterning and is compatible for tissue culture. Furthermore, it can be applied to modify various types of polymeric substrates, which due to the ability to tune their properties are ideal for and are widely used in biological applications. The resolution achievable is also beneficial, as isolation of specific processes such as migration, adhesion, or binding allows for discrete, clear observations at the single to multicell level.
This method has been employed to form diverse networks of different cell types for investigations involving migration, signaling, tissue formation, and the behavior and interactions of neurons arraigned in a network.

A versatile plasma lithography technique has been developed to generate stable surface patterns for guiding cellular attachment. This technique can be applied to create cell networks including those that mimic natural tissues and has been used for studying several, distinct cell types.

Systematic manipulation of a cell microenvironment with micro- and nanoscale resolution is often required for deciphering various cellular and molecular ...

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 ...

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

Rod-based Fabrication of Customizable Soft Robotic Pneumatic Gripper Devices for Delicate Tissue Manipulation

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations at the point of contact. It can be achieved by complementing traditional rigid grippers with soft robotic pneumatic gripper devices. This manuscript describes a rod-based approach that combined both 3D-printing and a modified soft lithography technique to fabricate the soft pneumatic gripper. In brief, the pneumatic featureless mold with chamber component is 3D-printed and the rods were used to create the pneumatic channels that connect to the chamber. This protocol eliminates the risk of channels occluding during the sealing process and the need for external air source or related control circuit. The soft gripper consists of a chamber filled with air, and one or more gripper arms with a pneumatic channel in each arm connected to the chamber. The pneumatic channel is positioned close to the outer wall to create different stiffness in the gripper arm. Upon compression of the chamber which generates pressure on the pneumatic channel, the gripper arm will bend inward to form a close grip posture because the outer wall area is more compliant. The soft gripper can be inserted into a 3D-printed handling tool with two different control modes for chamber compression: manual gripper mode with a movable piston, and robotic gripper mode with a linear actuator. The double-arm gripper with two actuatable arms was able to pick up objects of sizes up to 2 mm and yet generate lower compressive forces as compared to elastomer-coated and non-coated rigid grippers. The feasibility of having other designs, such as single-arm or hook gripper, was also demonstrated, which further highlighted the customizability of the soft gripper device, and it's potential to be used in delicate surgical manipulation to reduce the risk of tissue grip damage.

This protocol describes a rod-based approach, combining 3D-printing and soft lithography techniques for fabricating the soft gripper devices. This approach eliminates the need for an external air source by incorporating a chamber component and reduces the chance of occlusion during the sealing process, particularly for miniaturized pneumatic channels.

Soft compliant gripping is essential in delicate surgical manipulation for minimizing the risk of tissue grip damage caused by high stress concentrations ...

Targeted Plasma Membrane Delivery of a Hydrophobic Cargo Encapsulated in a Liquid Crystal Nanoparticle Carrier

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. Recently, nanoparticles (NPs) have shown significant promise in the development of such delivery systems. Here, a liquid crystal NP (LCNP)-based delivery system has been employed for the controlled delivery of a water-insoluble dye, 3,3&#8242;-dioctadecyloxacarbocyanine perchlorate (DiO), from within the NP core to the hydrophobic region of a plasma membrane bilayer. During the synthesis of the NPs, the dye was efficiently incorporated into the hydrophobic LCNP core, as confirmed by multiple spectroscopic analyses. Conjugation of a PEGylated cholesterol derivative to the NP surface (DiO-LCNP-PEG-Chol) enabled the binding of the dye-loaded NPs to the plasma membrane in HEK 293T/17 cells. Time-resolved laser scanning confocal microscopy and F&#246;rster resonance energy transfer (FRET) imaging confirmed the passive efflux of DiO from the LCNP core and its insertion into the plasma membrane bilayer. Finally, the delivery of DiO as a LCNP-PEG-Chol attenuated the cytotoxicity of DiO; the NP form of DiO exhibited ~30-40% less toxicity compared to DiOfree delivered from bulk solution. This approach demonstrates the utility of the LCNP platform as an efficient modality for the membrane-specific delivery and modulation of hydrophobic molecular cargos.

A liquid crystal nanoparticle (LCNP) nanocarrier is exploited as a vehicle for the controlled delivery of a hydrophobic cargo to the plasma membrane of living cells.

The controlled delivery of drug/imaging agents to cells is critical for the development of therapeutics and for the study of cellular signaling processes. ...

Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors ...

Apparatus for Harvesting Tissue Microcolumns

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns of full-thickness skin tissue. The small size of the microcolumns allows donor sites to heal quickly without causing donor site scarring, while harvesting full-thickness tissue enables the incorporation of all cellular and extracellular components of skin tissue, including those associated with deeper dermal regions and the adnexal skin structures, which have yet to be successfully reproduced using conventional tissue engineering techniques. The microcolumns can be applied directly into skin wounds to augment healing, or they can be used as the autologous cell/tissue source for other tissue engineering approaches. The harvesting needles are made by modifying standard hypodermic needles, and they can be used alone for harvesting small amounts of tissue or coupled with a simple suction-based collection system (also made from commonly available laboratory supplies) for high-volume harvesting to facilitate studies in large animal models.

Here we describe a protocol for producing harvesting needles that can be used to collect full-thickness skin tissue without causing donor site scarring. The needles can be combined with a simple collection system to achieve high-volume harvesting.

This manuscript describes the production process for a laboratory apparatus, made from off-the-shelf components, that can be used to collect microcolumns ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

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

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

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

Photodegradable Hydrogel Interfaces for Bacteria Screening, Selection, and Isolation

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to develop practical technologies that couple microscopic cell screening with cell isolation at high purity for downstream genetic analysis. Here, the use of photodegradable poly(ethylene glycol) hydrogels for screening and isolation of bacteria with unique growth phenotypes from heterogeneous cell populations is described. The method relies on encapsulating or entrapping cells with the hydrogel, followed by culture, microscopic screening, then use of a high-resolution light patterning tool for spatiotemporal control of hydrogel degradation and release of selected cells into a solution for retrieval. Applying different light patterns allows for control over the morphology of the extracted cell, and patterns such as rings or crosses can be used to retrieve cells with minimal direct UV light exposure to mitigate DNA damage to the isolates. Moreover, the light patterning tool delivers an adjustable light dose to achieve various degradation and cell release rates. It allows for degradation at high resolution, enabling cell retrieval with micron-scale spatial precision. Here, the use of this material to screen and retrieve bacteria from both bulk hydrogels and microfabricated lab-on-a-chip devices is demonstrated. The method is inexpensive, simple, and can be used for common and emerging applications in microbiology, including isolation of bacterial strains with rare growth profiles from mutant libraries and isolation of bacterial consortia with emergent phenotypes for genomic characterizations.

The use of photodegradable hydrogels to isolate bacterial cells by utilizing a high-resolution light pattering tool is reported. Essential experimental procedures, results, and advantages of the process are reviewed. The method enables rapid and inexpensive isolation of targeted bacteria showing rare or unique functions from heterogeneous communities or populations.

Biologists have long attempted to understand the relationship between phenotype and genotype. To better understand this connection, it is crucial to ...

Synthesis of Strong Adhesive Hydrogel, Gelatin O-Nitrosobenzaldehyde

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - gelatin o-nitrosobenzaldehyde (gelatin-NB) - which is mainly used for tissue regeneration and has been validated in animal models of corneal injury and inflammatory bowel disease. This is a novel hydrogel formed by modifying biological gelatin with o-nitrosobenzaldehyde (NB). Gelatin-NB was synthesized by activating the carboxyl group of NB-COOH and reacting with gelatin through 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The obtained compound was purified to generate the final product, which can be stably stored for at least 18 months. NB has a strong adhesion to -NH2 on the tissue, which can form many C = N bonds, thus increasing the adhesion of gelatin-NB to the tissue interface. The preparation process comprises steps for the synthesis of the NB-COOH group, modification of the group, synthesis of gelatin-NB, and purification of the compound. The goal is to describe the specific synthesis process of gelatin-NB in detail and to demonstrate the application of gelatin-NB to damage repair. Moreover, the protocol is presented to further strengthen and expand the nature of the material produced by the scientific community for more applicable scenarios.

The protocol presented here shows the synthesis of a strong adhesive hydrogel gelatin o-nitrosobenzaldehyde (gelatin-NB). Gelatin-NB has rapid and efficient tissue adhesion ability, which can form a strong physical barrier to protect wound surfaces, so it is expected to be applied to the field of injury repair biotechnology.

Adhesive materials have become popular biomaterials in the field of biomedical and tissue engineering. In our previous work, we presented a new material - ...