Authors are grateful to C. Luzuriaga for the support in the porosimetry measurements. Authors also thanks to Ministerio de Econom&#237;a y Competitividad of Spain for financial support (Project MAT2014-59116-C2-2-R) and PIUNA (ref. 2018-15). The authors also would like to acknowledge Dr. Amir Maldonado from Departamento de F&#237;sica-UNISON for support and helpful comments and Dr. SE Burruel-Ibarra from DIPM-UNISON for SEM images and Rubio Pharma y Asociados S. A. de C. V. for financial support. ME Mart&#237;nez-Barbosa would like to thank CONACyT (M&#233;xico) projects No. 104931 and No. 256753, besides the financial support from Red Tem&#225;tica de Nanociencias y Nanotecnolog&#237;a del programa de Redes Tem&#225;ticas del CONACyT. And, also project USO316001081. MD Figueroa-Pizano would like to acknowledge CONACyT for financial support (scholarship 373321).

1. Preparation of chitosan-PVA hydrogels
<ol>
	<li>Prepare 2% (w/w) chitosan and 10% (w/w) PVA solutions. Dissolve 0.2 g of chitosan in 10 mL of 0.1 M CH3COOH solution (previously filtered) at room temperature and maintain continuous mechanical stirring overnight. Dissolve 1 g of PVA in 10 mL of distilled water and stir at 80 &#176;C for 1 h.</li>
	<li>Mix both solutions 1:1 using a magnetic stirrer until they are homogeneous at room temperature, and pour the mixtures on Petri dishes. Leave the samples for 2 h at atmospheric pressure to degas.</li>
	<li>Freeze the hydrogels at -4 &#176;C, -20 &#176;C or -80 &#176;C for 20 h and 4 cycles (samples CP4-4, CP4-20 and CP4-80, respectively). Freeze another hydrogel at -80 &#176;C for 20 h using 5 or 6 freezing cycles (samples CP5-80 and CP6-80). After the third freezing cycle, wash the hydrogels with deionized water. At the end, freeze-dry the hydrogels at -46 &#176;C for 48 h and store for further characterization (methodology adapted from2).</li>
</ol>
2. FT-IR characterization
<ol>
	<li>Place a little piece (1 mm x 2 mm) of hydrogel in the FT-IR spectrometer in ATR mode. Take the FT-IR spectra from 4000 to 600 cm-1 (2 cm-1 of resolution and average of 32 scans).</li>
</ol>
3. Swelling assays
<ol>
	<li>Cut out discs (13 mm in diameter and 10 mm in height) from the hydrogel and weigh them. Incubate the discs in 50 mL of deionized water with shaking at 25 &#176;C. Repeat three times.</li>
	<li>Every 30 min remove the sample from the medium, blotter to eliminate the excess of water, and weigh. Calculate the swelling degree using the equation 1 and calculate the equilibrium state of swelling, <img alt="Equation 1" src="/files/ftp_upload/59636/59636eq1.jpg" />, at 24 h using the equation 2. 
	<img alt="Equation 2" src="/files/ftp_upload/59636/59636eq2.jpg" />) 
	Where <img alt="Equation 3" src="/files/ftp_upload/59636/59636eq3.jpg" /> is the weight of the dry hydrogel and <img alt="Equation 4" src="/files/ftp_upload/59636/59636eq4.jpg" /> is the weight of the wet hydrogel. 
	<img alt="Equation 5" src="/files/ftp_upload/59636/59636eq5.jpg" /></li>
</ol>
4. Electronic Microscopy
<ol>
	<li>Cover a little piece of hydrogel with a thin gold layer (30 s and 10 mA) in a sputter coater.</li>
	<li>Put the sample in a scanning electron microscope (SEM). Analyze the samples under vacuum at 20 kV and take the images with a 500x and 1500x magnification.</li>
</ol>
5. Porosimetry
<ol>
	<li>Place discs 15 mm in diameter weighing around 0.26 g into the penetrometer (a solid penetrometer, having a bulk volume of 0.3660 mL and 5.7831 mL of stem volume). Analyze the porosity and pore size by Mercury Intrusion Porosimetry (MIP).</li>
	<li>Conduct the experiment in the hysteresis mode (intrusion-extrusion). Measure the total intrusion volume (mL/g), total pore area (m2/g), pore diameter (&#181;m), porosity (%), permeability (mDarcy) and tortuosity. Repeat twice.</li>
</ol>
6. Drug loading and release
<ol>
	<li>Before loading, prepare 4 L of 15 mg/L diflunisal solution and stir overnight. Confirm the concentration of the solution by UV-Vis spectroscopy (initial concentration). Indeed, swell 400 mg of freeze-dried samples of hydrogel in 6 mL of distilled water for 24 h.</li>
	<li>For loading, fill a flask with 50 mL of diflunisal solution and maintain at 25 &#176;C with constant stirring. Submerge each swelled hydrogel in the flask.
	<ol>
		<li>Take aliquots of remaining diflunisal solution (2 mL) at different times in order to determine the plateau region of the curve, for example: 3, 6, 24, 27, 30 and 48 h. After 24 h replace the solution with a fresh one.</li>
	</ol>
	</li>
	<li>Measure the absorbance at 252 nm of each aliquot, and determine the concentration of diflunisal present in the solution, using a calibration curve of diflunisal. Calculate the amount of diflunisal retained in the hydrogel at 24 and 48 h, as the difference of initial and final concentrations, taking into account the total volume (56 mL).
	<ol>
		<li>Determine the encapsulation efficiency (EE) using the equation 3. 
		<img alt="Equation 6" src="/files/ftp_upload/59636/59636eq6.jpg" /></li>
		<li>Freeze the loaded hydrogels at -80 &#176;C and lyophilize them at -50 &#176;C.</li>
	</ol>
	</li>
	<li>For drug release, submerge 300 mg of freeze-dried diflunisal loaded hydrogels in 50 mL of phosphate buffer (pH 7.4) at 25 &#176;C. Maintain constant stirring. Withdraw aliquots of 2 mL at different times and replace with fresh medium to keep a constant volume.
	<ol>
		<li>Determinate the diflunisal released spectrophotometrically at 252 nm, according to a calibration curve.</li>
	</ol>
	</li>
	<li>Deduce the predominant drug release mechanism in the hydrogels adjusting the drug release data corresponding to the first 60%, to the Korsmeyer-Peppas model (Equation 4), to obtain the kinetic (k) and the diffusion (n) constants. The n values indicate the mechanism of drug release24,25. Then, n values close to 0.5 are related to Fickian diffusion, meanwhile values of 0.5-1.0 for anomalous transport, where are involved diffusion and relaxation chains, and finally, values of 1.0 are related to case II transport. 
	<img alt="Equation 7" src="/files/ftp_upload/59636/59636eq7.jpg" />
	<ol>
		<li>To confirm the results, use the Higuchi, First order, and Zero order mathematical models (Equations 5 to 7) and select the better fit.</li>
		<li><img alt="Equation 8" src="/files/ftp_upload/59636/59636eq8.jpg" /> 
		<img alt="Equation 9" src="/files/ftp_upload/59636/59636eq9.jpg" /> 
		<img alt="Equation 10" src="/files/ftp_upload/59636/59636eq10.jpg" /> 
		where t represents the release time, Mt the amount of drug delivered at a given time, and M&#8734; the total amount of drug delivered at the end of the process.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

The freeze-thawing method is a suitable process to prepare biocompatible hydrogels focused in biomedical, pharmaceutical or cosmetical applications34,35,36. The most important advantage of this method, compared with other well-known methods to prepare hydrogels, is that crosslinking agent use is avoided, which could cause an inflammatory response or adverse effects in the human body34. This is a versatile...

Recently, hydrogels have attracted great interest in the biomedical field because they are three-dimensional networks with high water content and are soft and flexible, so they can mimic natural tissues easily1. Also, they do not dissolve in aqueous medium at physiological temperature and pH but present a large swelling2. Hydrogels can act as tissue engineering scaffolds, hygiene products, contact lenses, and wound dressings; because they can trap and release active compounds and drugs, they are used as drug delivery systems3. Depending on their application, hydrogels can be made from natural or synthetic polymers, or a combination of both, in order to obtain the best characteristics4.
The properties of hydrogels are a consequence of many physical and chemical factors. At the physical level, their structure and morphology depend on their porosity, pore size and pore distribution5. At the chemical and molecular level, the polymer type, the hydrophilic group content in the polymer chain, the crosslinking point type, and the cross-linking density are the factors that determine the swelling capacity and the mechanical properties6,7.
According to the type of crosslinking agent used to form the network, the hydrogels are classified as chemical hydrogels or physical hydrogels. Chemical hydrogels are joined by covalent interactions between their chains, which are formed through UV and gamma irradiation or using a crosslinking agent7,8. Chemical hydrogels usually are strong and resistant but, generally, the crosslinking agent is toxic to the cells and its removal is difficult, so its application is limited. On the other hand, physical hydrogels form by the connection of the polymer chains through non-covalent interactions, avoiding the use of crosslinking agents4,9. The main non-covalent interactions in the network are hydrophobic interactions, electrostatic forces, complementary and hydrogen bounds7.
Poly(vinyl alcohol) (PVA, Figure 1a) is a synthetic and water-soluble polymer with excellent mechanical performance and biocompatibility that can from crosslink agent-free hydrogels through the freeze-thawing method10,11. This polymer has the capacity to form concentrated zones of hydrogen bonds between -OH groups of their chains (crystalline zones) when they are freezing12. These crystalline zones act as crosslinking points in the network, and they are promoted by two events: the approaching of the polymer chains when the crystal water expands and the PVA conformational changes from isotactic to syndiotactic PVA during freeze13. Because of the freeze-drying, the water crystals are sublimated, leaving void spaces that are the pores in the hydrogel14. To obtain hydrogels with better properties, PVA can been easily combined with other polymers.
In that sense, chitosan constitutes an option as it is the only biopolymer from natural sources with positive charges. It is obtained by the deacetylation of chitin and it is composed of random combinations of &#946;-1,4 linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit)15,16 (Figure 1b). Chitosan is biodegradable by human enzymes and it is biocompatible. Also, by its cationic nature, it can interact with the negative charge of the cell surface, and this property has been associated with its antimicrobial activity17. This polymer is easy to process; however, their mechanical properties are not sufficient and some materials have been added to form complexes with better characteristics.
Considering specific characteristics of chitosan and PVA, the successful manufacture of hydrogels has been reached by the freeze-thawing method2,18 to avoid the use of toxic crosslinking agents. In chitosan-PVA hydrogels, the crystalline zones of PVA are also formed, and chitosan chains are interpenetrated and form simple hydrogen bonds with -NH2 groups and -OH groups in PVA. The final chitosan-PVA hydrogel is mechanically stable, with high rates of swelling and low toxicity, and with antibacterial effect18. However, depending on the freezing conditions used in the preparation (temperature, time and number of cycles), the final characteristics may change. Some studies report that increasing the number of freezing cycles decreases the swelling degree and increases the tensile strength19,20. In order to strengthen the network, other agents such as gamma and UV radiation and chemical crosslinkers have been used additionally after the freeze-thawed preparation21,22,23. Hydrogels with a higher chitosan proportion have a more porous network and high swelling capacity but less strength and thermal stability. In this context, it is important to consider the preparation conditions to obtain suitable hydrogels for their target application.
The purpose of this work is to present in detail how the freezing conditions (temperature of freezing and number of cycles) affect the final characteristics of CS-PVA hydrogels. FT-IR spectra, morphological and porosity characteristics and swelling capacity were evaluated, as well as drug loading and release capacity. In the release studies, diflunisal (Figure 1c) was used as model drug, due to its size suitable to the hydrogel structure.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Chitosan medium molecular weight</td>
			<td>Sigma-Aldrich</td>
			<td>448877</td>
			<td>Mw determined by capillary viscometry (637,000 Da) and deacetylation degree of 70%</td>
		</tr>
		<tr>
			<td>Diflunisal (2&#39;-4&#39;-difluoro-4-hydroxy-3-biphenyl-carboxylicacid)</td>
			<td>Merck</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>1005706</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly(vinyl alcohol)</td>
			<td>Sigma-Aldrich</td>
			<td>341584</td>
			<td>Mw 89,000-98,000, 99+% hydrolyzed</td>
		</tr>
		<tr>
			<td>Equipment:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cressington Sputter Coater 108 auto</td>
			<td>TED PELLA INC</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryodos Lyophilizator</td>
			<td>Telstar</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon tubes</td>
			<td>Thermo Fisher Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FT-IR spectroscopy</td>
			<td>Nicolet iS50</td>
			<td>in ATR mode</td>
			<td></td>
		</tr>
		<tr>
			<td>Lyophilizator</td>
			<td>LABCONCO</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Micromeritics Autopore IV 9500</td>
			<td>Micromeritics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning electron microscope</td>
			<td>Pemtron SS-300LV</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV-visible spectrophotometer</td>
			<td>Agilent 8453</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a freeze-thawing method to prepare chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents and diflunisal release studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these systems are limited by their characteristics (e.g., porosity, flexibility, swelling capacity, drug loading and drug release capacity), which depend on the freezing conditions and the kind and ratio of polymers. This protocol describes how to prepare hydrogels from chitosan and poly(vinyl alcohol) at 50/50 w/w % of polymer composition and varying the freezing temperature (-4 &#176;C, -20 &#176;C, -80 &#176;C) and freeze-thawing cycles (4, 5, 6 freezing cycles). FT-IR spectra, SEM micrograph and porosimetry data of hydrogels were obtained. Also, the swelling capacity and drug loading and release of diflunisal were assessed. Results from SEM micrographs and porosimetry show that the pore size decreases, while the porosity increases at lower temperatures. The swelling percentage was higher at the minor freezing temperature. The release of diflunisal from the hydrogels has been studied. All the networks maintain the drug release for 30 h and it has been observed that a simple diffusion mechanism regulates the diflunisal release according to Korsmeyer-Peppas and Higuchi models.

Hydrogels preparation 
Chitosan-PVA hydrogels were obtained at -4 &#176;C, -20 &#176;C and -80 &#176;C with 4 freezing cycles and at -80 &#176;C with 5 and 6 freezing cycles by the previously reported freeze-thawing method2. All hydrogels were homogeneous, semi-transparent, flexible and resistant against manipulation.
FT-IR characterization 
The FT-IR spectra ar...

Watch this Scientific Journal Video about A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies at JoVE.com

A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

A Freeze-Thawing Method to Prepare Chitosan-Poly(vinyl alcohol) Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Chitosan-poly(vinyl alcohol) hydrogels can be produced by the freeze-thawing method without using toxic crosslinking agents. The applications of these ...

a-freeze-thawing-method-to-prepare-chitosan-poly-vinyl-alcohol

Research

JoVE Journal

Bioengineering

17.2K Views.  University of Sonora. The freezing-thawing method is used to produce chitosan-poly(vinyl alcohol) hydrogels without crosslinking agents. For this method, it is important to consider the freezing conditions (temperature, number of cycles) and polymer ratio, which can affect the properties and applications of the obtained hydrogels.

Video: A Freeze-Thawing Method to Prepare Chitosan-Polyvinyl alcohol Hydrogels Without Crosslinking Agents and Diflunisal Release Studies

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

A Novel Three-dimensional Flow Chamber Device to Study Chemokine-directed Extravasation of Cells Circulating under Physiological Flow Conditions

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell homing and tumor metastasis. The three-dimensional flow chamber device (hereafter the 3D device) is a novel in vitro technology that recreates physiological shear stress and allows each step of the cell extravasation cascade to be quantified. The 3D device consists of an upper compartment in which the cells of interest circulate under shear stress, and a lower compartment of static wells that contain the chemoattractants of interest. The two compartments are separated by porous inserts coated with a monolayer of endothelial cells (EC). An optional second insert with microenvironmental cells of interest can be placed immediately beneath the EC layer. A gas exchange unit allows the optimal CO2 tension to be maintained and provides an access point to add or withdraw cells or compounds during the experiment. The test cells circulate in the upper compartment at the desired shear stress (flow rate) controlled by a peristaltic pump. At the end of the experiment, the circulating and migrated cells are collected for further analyses. The 3D device can be used to examine cell rolling on and adhesion to EC under shear stress, transmigration in response to chemokine gradients, resistance to shear stress, cluster formation, and cell survival. In addition, the optional second insert allows the effects of crosstalk between EC and microenvironmental cells to be examined. The translational applications of the 3D device include testing of drug candidates that target cell migration and predicting the in vivo behavior of cells after intravenous injection. Thus, the novel 3D device is a versatile and inexpensive tool to study the molecular mechanisms that mediate cellular extravasation.

The three-dimensional flow chamber device is a novel in vitro technology for the quantitative and step-wise evaluation of the extravasation cascade of cells circulating under conditions of physiological shear stress. The device therefore fills a critical need for basic, preclinical, and clinical studies of cell migration.

Extravasation of circulating cells from the bloodstream plays a central role in many physiological and pathophysiological processes, including stem cell ...

The Synthesis of RGD-functionalized Hydrogels as a Tool for Therapeutic Applications

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and functionalize polymer chains with compounds that are able to improve biocompatibility, mechanical properties, or cell viability allows the design of novel materials to meet new challenges in the biomedical field. With the polymer functionalization strategies, click chemistry is a powerful tool to improve cell-compatibility and drug delivery properties of polymeric devices. Similarly, the fundamental need of biomedicine to use sterile tools to avoid potential adverse-side effects, such as toxicity or contamination of the biological environment, gives rise to increasing interest in the microwave-assisted strategy.
The combination of click chemistry and the microwave-assisted method is suitable to produce biocompatible hydrogels with desired functionalities and improved performances in biomedical applications. This work aims to synthesize RGD-functionalized hydrogels. RGD (arginylglycylaspartic acid) is a tripeptide that can mimic cell adhesion proteins and bind to cell-surface receptors, creating a hospitable microenvironment for cells within the 3D polymeric network of the hydrogels. RGD functionalization occurs through Huisgen 1,3-dipolar cycloaddition. Some PAA carboxyl groups are modified with an alkyne moiety, whereas RGD is functionalized with azido acid as the terminal residue of the peptide sequence. Finally, both products are used in a copper catalyzed click reaction to permanently link the peptide to PAA. This modified polymer is used with carbomer, agarose and polyethylene glycol (PEG) to synthesize a hydrogel matrix. The 3D structure is formed due to an esterification reaction involving carboxyl groups from PAA and carbomer and hydroxyl groups from agarose and PEG through microwave-assisted polycondensation. The efficiency of the gelation mechanism ensures a high degree of RGD functionalization. In addition, the procedure to load therapeutic compounds or biological tools within this functionalized network is very simple and reproducible.

We present a protocol for the synthesis of RGD-functionalized hydrogels as devices for cell and drug delivery. The procedure involves copper catalyzed alkyne-azide cycloaddition (CuAAC) between alkyne-modified polyacrylic acid (PAA) and a RGD-azide derivative. The hydrogels are formed using microwave-assisted polycondensation and their physicochemical properties are investigated.

The use of polymers as biomaterials has provided significant advantages in therapeutic applications. In particular, the possibility to modify and ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

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

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

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

Human Pluripotent Stem Cell Culture on Polyvinyl Alcohol-Co-Itaconic Acid Hydrogels with Varying Stiffness Under Xeno-Free Conditions

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by several researchers. However, most of these investigators have used polyacrylamide hydrogels for stem cell culture in their studies. Therefore, their results are controversial because those results might originate from the specific characteristics of the polyacrylamide and not from the physical cue (stiffness) of the biomaterials. Here, we describe a protocol for preparing hydrogels, which are not based on polyacrylamide, where various stem, cells including human embryonic stem (ES) cells and human induced pluripotent stem (iPS) cells, can be cultured. Hydrogels with varying stiffness were prepared from bioinert polyvinyl alcohol-co-itaconic acid (P-IA), with stiffness controlled by crosslinking degree by changing crosslinking time. The P-IA hydrogels grafted with and without oligopeptides derived from extracellular matrix were investigated as a future platform for stem cell culture and differentiation. The culture and passage of amniotic fluid stem cells, adipose-derived stem cells, human ES cells, and human iPS cells is described in detail here. The oligopeptide P-IA hydrogels showed superior performances, which were induced by their stiffness properties. This protocol reports the synthesis of the biomaterial, their surface manipulation, along with controlling the stiffness properties and finally, their impact on stem cell fate using xeno-free culture conditions. Based on recent studies, such modified substrates can act as future platforms to support and direct the fate of various stem cells line to different linkages; and further, regenerate and restore the functions of the lost organ or tissue.

A protocol is presented to prepare polyvinyl alcohol-co-itaconic acid hydrogels with varying stiffness, which were grafted with and without oligopeptides, to investigate the effect of the stiffness of biomaterials on the differentiation and proliferation of stem cells. The stiffness of the hydrogels was controlled by the crosslinking time.

The effect of physical cues, such as the stiffness of biomaterials on the proliferation and differentiation of stem cells, has been investigated by ...

Production of Elastin-like Protein Hydrogels for Encapsulation and Immunostaining of Cells in 3D

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue culture systems lack a three-dimensional (3D) matrix, resulting in a significant disconnect between results collected in vitro and in vivo. To address this limitation, researchers have engineered 3D hydrogel tissue culture platforms that can mimic the biochemical and biophysical properties of the in vivo cell microenvironment. This research has motivated the need to develop material platforms that support 3D cell encapsulation and downstream biochemical assays. Recombinant protein engineering offers a unique toolset for 3D hydrogel material design and development by allowing for the specific control of protein sequence and therefore, by extension, the potential mechanical and biochemical properties of the resultant matrix. Here, we present a protocol for the expression of recombinantly-derived elastin-like protein (ELP), which can be used to form hydrogels with independently tunable mechanical properties and cell-adhesive ligand concentration. We further present a methodology for cell encapsulation within ELP hydrogels and subsequent immunofluorescent staining of embedded cells for downstream analysis and quantification.

Recombinant protein-engineered hydrogels are advantageous for 3D cell culture as they allow for complete tunability of the polymer backbone and therefore, the cell microenvironment. Here, we describe the process of recombinant elastin-like protein purification and its application in 3D hydrogel cell encapsulation.

Two-dimensional (2D) tissue culture techniques have been essential for our understanding of fundamental cell biology. However, traditional 2D tissue ...

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Controlled Strain of 3D Hydrogels under Live Microscopy Imaging

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized in vitro stretching methods. Various available systems use 2D substrate-based stretchers, while the accessibility of 3D techniques to strain soft hydrogels, is more restricted. Here, we describe a method that allows external stretching of soft hydrogels from their circumference, using an elastic silicone strip as the sample carrier. The stretching system utilized in this protocol is constructed from 3D-printed parts and low-cost electronics, making it simple and easy to replicate in other labs. The experimental process begins with polymerizing thick (&#62;100 &#956;m) soft fibrin hydrogels (Elastic Modulus of ~100 Pa) in a cut-out at the center of a silicone strip. Silicone-gel constructs are then attached to the printed-stretching device and placed on the confocal microscope stage. Under live microscopy the stretching device is activated, and the gels are imaged at various stretch magnitudes. Image processing is then used to quantify the resulting gel deformations, demonstrating relatively homogenous strains and fiber alignment throughout the gel&#8217;s 3D thickness (Z-axis). Advantages of this method include the ability to strain extremely soft hydrogels in 3D while executing in situ microscopy, and the freedom to manipulate the geometry and size of the sample according to the user&#8217;s needs. Additionally, with proper adaptation, this method can be used to stretch other types of hydrogels (e.g., collagen, polyacrylamide or polyethylene glycol) and can allow for analysis of cells and tissue response to external forces under more biomimetic 3D conditions.

The presented method involves uniaxial stretching of 3D soft hydrogels embedded in silicone rubber while allowing live confocal microscopy. Characterization of the external and internal hydrogel strains as well as fiber alignment are demonstrated. The device and protocol developed can assess the response of cells to various strain regimes.

External forces are an important factor in tissue formation, development, and maintenance. The effects of these forces are often studied using specialized ...