Name: measuring global cellular matrix metalloproteinase and metabolic activity in 3d hydrogels
Uploaded: 2019-01-22T15:00:00
Description: Watch this Scientific Journal Video about Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels at JoVE.com

The authors would like to acknowledge Ohio Cancer Research (OCR), OH, USA for funding this work as well as King Saud University (KSU), Riyadh, KSA for sponsoring the first author.&#160;Molecular weight of the fluorescent peptide sensor was measured using matrix assisted, laser desorption-ionization, time-of-flight (MALDI-TOF) mass spectrometry with assistance from the Campus Chemical Instrument Center Mass Spectrometry and Proteomics Facility at The Ohio State University.

1. Hydrogel components preparation
<ol>
	<li>Synthesize the fluorescent protease-degradable peptides as described elsewhere8, utilizing fluorescein as the fluorescent molecule and dabcyl as the quencher. Dissolve the peptide in DMSO to a concentration of 10 mM and store in a -80 &#176;C freezer in small (~30 &#181;L) aliquots to avoid repeated freeze-thaw cycles. 
	NOTE: These peptides can also be purchased commercially. This protocol requires a C-terminal cysteine in the peptide sequence t...

<ol>
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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension+-+how+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension - how 3D culture microenvironments alter cellular cues.</a> Journal of Cell Science. 125 (13), 3015-3024 (2012).</li><li>Duval, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Physiological+Events+in+2D+vs.+3D+Cell+Culture.">Modeling Physiological Events in 2D vs. 3D Cell Culture.</a> Physiology. 32 (4), 266-277 (2017).</li><li>Hoarau-V&#233;chot, J., Rafii, A., Touboul, C., Pasquier, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halfway+between+2D+and+Animal+Models:+Are+3D+Cultures+the+Ideal+Tool+to+Study+Cancer-Microenvironment+Interactions.">Halfway between 2D and Animal Models: Are 3D Cultures the Ideal Tool to Study Cancer-Microenvironment Interactions.</a> International Journal of Molecular Sciences. 19 (1), (2018).</li><li>Lee, S. -. 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M., Schroeder, M. E., Payne, S. Z., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+High-Throughput+Cell+Encapsulation+Platform+to+Study+Changes+in+Cell-Matrix+Interactions.">Three-Dimensional High-Throughput Cell Encapsulation Platform to Study Changes in Cell-Matrix Interactions.</a> ACS Applied Materials &amp; Interfaces. 8 (34), 21914-21922 (2016).</li><li>Sridhar, B. V., Doyle, N. R., Randolph, M. A., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Covalently+tethered+TGF-&#946;1+with+encapsulated+chondrocytes+in+a+PEG+hydrogel+system+enhances+extracellular+matrix+production.">Covalently tethered TGF-&#946;1 with encapsulated chondrocytes in a PEG hydrogel system enhances extracellular matrix production.</a> Journal of Biomedical Materials Research. Part A. 102 (12), 4464-4472 (2014).</li><li>Sridhar, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+cellularly+degradable+PEG+hydrogel+to+promote+articular+cartilage+extracellular+matrix+deposition.">Development of a cellularly degradable PEG hydrogel to promote articular cartilage extracellular matrix deposition.</a> Advanced Healthcare Materials. 4 (5), 702-713 (2015).</li><li>Fairbanks, B. D., Schwartz, M. P., Bowman, C. N., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoinitiated+polymerization+of+PEG-diacrylate+with+lithium+phenyl-2,4,6-trimethylbenzoylphosphinate:+polymerization+rate+and+cytocompatibility.">Photoinitiated polymerization of PEG-diacrylate with lithium phenyl-2,4,6-trimethylbenzoylphosphinate: polymerization rate and cytocompatibility.</a> Biomaterials. 30 (35), 6702-6707 (2009).</li><li>Mucha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Type-1+Matrix+Metalloprotease+and+Stromelysin-3+Cleave+More+Efficiently+Synthetic+Substrates+Containing+Unusual+Amino+Acids+in+Their+P1'+Positions.">Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1' Positions.</a> Journal of Biological Chemistry. 273 (5), 2763-2768 (1998).</li><li>Chai, S. C., Goktug, A. N., Chen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+Validation+in+High+Throughput+Screening+-+from+Concept+to+Application.">Assay Validation in High Throughput Screening - from Concept to Application.</a> Drug Discovery and Development. , (2015).</li><li>Iversen, P. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HTS+Assay+Validation.">HTS Assay Validation.</a> Assay Guidance Manual. , (2004).</li><li>Leight, J. L., Tokuda, E. Y., Jones, C. E., Lin, A. J., Anseth, K. 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3D in vitro cell culture recapitulates many important aspects of the in vivo environment. However, 3D culture also makes assessing cell function and signaling challenging, as many biological assays require cellular retrieval and large numbers of cells. Therefore, the development of a simple 3D culture system that enables measurement of cellular function without further sample processing would greatly increase the utility of 3D culture systems. The 3D system described here can be adapted for a variety of different applica...

Three-dimensional (3D) culture systems often more closely recapitulate in vivo cellular responses than traditional two-dimensional (2D) culture systems, see several excellent publications1,2,3. However, utilizing 3D culture systems to measure cell function has been challenging due to the difficulty of cellular retrieval and further sample processing. This difficulty limits the measurement of many cellular functions in 3D culture systems. To overcome this difficulty, new techniques are needed that enable easy measurement of cell function within 3D environments. One way to address this need is the development of materials that not only support 3D cell culture but also incorporate sensors to measure cell function. For example, several hydrogel systems have incorporated fluorogenic protease cleavable moieties to enable visualization of protease activity within 3D environments4,5,6,7. While these systems were originally utilized for microscopic imaging, these systems can also be adapted for use in a global matrix metalloproteinase (MMP) activity assay using a standard plate reader, enabling facile measurement of a cell function in a 3D environment8.
MMPs, a superfamily of zinc proteases, play critical roles in normal tissue homeostasis and in many diseases. MMPs degrade and remodel extracellular matrix (ECM), cleave cell surface receptors and cytokines and activate other MMPs9,10. MMPs play critical roles in physiological processes such as wound healing and in diseases such as arthritis, atherosclerosis, Alzheimer&#39;s, and cancer (see reviews in 9,10,11). In cancer, high MMP expression levels are strongly correlated with cancer metastasis and poor prognosis12. Furthermore, MMPs contribute to tumor progression by promoting cancer cell invasion and migration, cellular processes that are inherently 3D phenomena13,14. Therefore, there is much interest in the ability to measure MMP activity in 3D culture in many contexts, including fundamental biological studies and drug screening assays.
PEG hydrogels are widely used for 3D cell culture due to their high water content, resistance to protein adsorption, and tunable nature. PEG hydrogels have been functionalized with a number of moieties to direct cell function, such as with ECM mimetic peptides like RGD, RLD, and IKVAV to facilitate cell adhesion, or direct tethering of growth factors such as transforming growth factor-&#946; (TGF-&#946;)15,16. More recently, PEG hydrogels have been functionalized with sensor peptides that enable measurement of cell function as well5,8. Specifically, the use of a PEG hydrogel system functionalized with a fluorogenic MMP-degradable peptide enabled measurement of cellular MMP activity in 3D cultures with a standard plate reader and required no further processing. These systems are also compatible with other measurements of cellular function, including metabolic activity. Here, a protocol is described for the measurement of MMP activity of cells cultured in a 3D PEG hydrogel functionalized with a fluorogenic MMP-degradable peptide, and results presented demonstrating the initial optimization experiments needed for use of this assay. Human melanoma cells (A375) were encapsulated in the fluorogenic hydrogels over a range of seeding densities to determine the appropriate seeding densities that are within the working range of the assay. After 24 h of encapsulation, MMP and metabolic activity were measured utilizing a standard microplate reader. Next, MMP activity was normalized to metabolic activity to determine the seeding densities within the linear range of the assay. Finally, intra-plate coefficient of variation percentages (% CV) were calculated between triplicates to reflect the reproducibility of the obtained results. This method enables simple and fast 3D cell culture, and easy measurement of protease activity with minimal sample processing.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">1X Phosphate Buffered Saline</td>
			<td style="width:268px;">Fisher Scientific</td>
			<td style="width:119px;">10-010-049</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Activated charcoal&#160;</td>
			<td style="width:268px;">Sigma-Aldrich</td>
			<td style="width:119px;">C3345</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Black round bottom 96-well plate</td>
			<td style="width:268px;">Brand-Tech</td>
			<td style="width:119px;">89093-600</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Cell adhesion peptide (CRGDS)</td>
			<td style="width:268px;">GenScript USA Inc.</td>
			<td style="width:119px;">custom made</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Collagenase enzyme type I&#160;</td>
			<td style="width:268px;">Life Technologies</td>
			<td style="width:119px;">17100-017</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Dextran</td>
			<td style="width:268px;">Sigma-Aldrich</td>
			<td style="width:119px;">D4876</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">DMEM High Glucose Media</td>
			<td style="width:268px;">Invitrogen</td>
			<td style="width:119px;">11965118</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fetal bovine serum (FBS)&#160;</td>
			<td style="width:268px;">Seradigm</td>
			<td style="width:119px;">1500-500</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Fluorescence microplate reader&#160;</td>
			<td style="width:268px;">BioTek</td>
			<td style="width:119px;">Cytation 3</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Hemocytometer</td>
			<td style="width:268px;">Hausser Scientific Co.</td>
			<td style="width:119px;">3200</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">L-glutamine</td>
			<td style="width:268px;">Life Technologies</td>
			<td style="width:119px;">25030-081</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:272px;">MMP-degradable peptide crosslinker (KCGPQG&#8595;IWGQCK)</td>
			<td style="width:268px;">GenScript USA Inc.</td>
			<td style="width:119px;">custom made</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">NaOH</td>
			<td style="width:268px;">Fisher Scientific</td>
			<td style="width:119px;">S318500</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Penicillin /streptomycin</td>
			<td style="width:268px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:272px;">Resazurin (Alamar Blue)</td>
			<td style="width:268px;">Life Technologies</td>
			<td style="width:119px;">DAL1100</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:272px;">UV light&#160;</td>
			<td style="width:268px;">UVP</td>
			<td style="width:119px;">95-0006-02</td>
			<td style="width:64px;"></td>
		</tr>
	</tbody>
</table>

measuring global cellular matrix metalloproteinase and metabolic activity in 3d hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

The current assay was adapted from a previously developed and characterized 3D hydrogel culture system functionalized with a fluorogenic MMP cleavable sensor8. The fluorogenic MMP sensor used here consists of a peptide sequence, GPLAC(pMeOBzl)&#8595;WARKDDK(AdOO)C (&#8595; indicates the cleavage site) that was previously optimized for cleavage by MMP-14 and MMP-1119. The peptide is labeled with a fluorescent molecule (fluorescein) and a quen...

Watch this Scientific Journal Video about Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels at JoVE.com

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

3D culture makes it more difficult to measure cellular function, using standard biological assays. This protocol enables measurement of cellular and somatic activity without further sample processing, using a fluorescent sensor and a standard microplate reader. This protocol enables a number of new applications, including high throughput drug screening.Additionally, the fluorescent sensor can be exchanged with a different sensor to measure other proteases or cell functions. It is important to carefully plan out the experiment in advance, complete all calculations for the hydrogel preparation, and setup all equipments and reagents to minimize the time cells are in suspension. To begin, prepare the assay media with 1%charcoal stripped Fetal Bovine Serum, two mM L-glutamine, 10 units per mL of penicillin, and 10 micrograms per mL of streptomycin.Do not use phenol red media, which has more fluorescence interference. To make positive controls, add bacterial collagenase enzyme type one to the assay media at the concentrations of 10 and 1000 micrograms per mL. Next, prepare the hydrogel precursor solution by adding the reagents to a 1.5 mL tube.Make sure to vortex after addition of each component. Then divide the solution into multiple 1.5 mL tubes for different conditions. To encapsulate cells in hydrogels, first prepare a single cell suspension by washing a 10 cm dish of A375 melanoma cells with 10 mL of PBS.Then, add 0.05%trypsin to the dish, to trypsinize cells. Incubate the dish at 37 degrees Celsius and 5%carbon dioxide, for three minutes. After incubation, count the cells with a hemocytometer.Next, centrifuge the cell solution at 314 times G for three minutes. Aspirate the culture media, and resuspend cells in PBS buffer at approximately three times the final encapsulated density. Count the cells again to ensure an accurate cell concentration.Then, add suspended cells in PBS to each tube of the hydrogel precursor solution according to the required seeding density, and mix by pipetting up and down. For controlled conditions, only add PBS and vortex. Next, pipette 10 microliters of the hydrogel precursor solution into the middle of each well of a sterile, black, round-bottomed 96 well plate.Visually inspect the placement of the hydrogels within the wells. Non-centered hydrogels can be repositioned using a pipette tip before polymerization. To polymerize the hydrogel precursor solution, expose the plate to UV light at 4 milliwatts per square centimeter, for three minutes.Next, add 150 microliters of the assay media to all wells containing encapsulated cells, and 150 microliters of the collagenase enzyme solution to the wells of positive controls. Add 150 microliters of PBS buffer to the outer wells of the plate to reduce evaporation during incubation. Use a microplate reader to measure the fluorescent intensity of the plate at zero hour, immediately post encapsulation.Select the opaque 96 well plate protocol with 494 nanometer excitation, and 521 nanometer emission wavelengths. Next, incubate the plate at 37 degrees Celsius in 5%carbon dioxide, for 18 hours. Then add metabolic activity reagents at a one to 10 volume to volume ratio per well for all conditions and controls.Incubate the plate in 37 degrees Celsius, 5%carbon dioxide, for six hours. Finally, measure the fluorescent intensity of the plate at 24 hours, post encapsulation. Select the opaque 96 well plate protocol, with 494 nanometer excitation, and 521 nanometer emission wavelengths, for MMP activity.To measure the metabolic activity, select 560 nanometer excitation, and 590 nanometer emission wavelengths. In this study, upon exposure of the fluorogenic sensor to the appropriate protease, the quencher and fluorophore are separated, and fluorescence increases. Fluorescence measurements of the incubated hydrogels with bacterial collagenase enzyme type one, show that the lowest detected signal was produced by negative controls, with no collagenase.While the highest detected signal was produced by 1000 microgram per mL of collagenase or above, when the signal begins to plateau. The calculated working range was from approximately 0.16 to 474 micrograms per mL of collagenase. Fluorescence readings for A375 melanoma cell line, encapsulated in a range of densities right after encapsulation, were low across seeding densities, and similar to control gels without cells as expected.24 hours after encapsulation, MMP activity was directly proportional to the seeding density, and seeding densities at or greater than 1 million cells per mL, fall within the limits of the working range. Metabolic activity measurements of the A375 cell line, were also directly proportional to the cell seeding density. By normalizing MMP activity to metabolic activity, there was no significant difference in MMP activity per cell at seeding densities greater than 2 million cells per mL.Proper pipetting techniques is important. This include pre-wetting tips to achieve accurate volumes of viscous solutions. Also, centering hydrogel precursor solution within the well, is critical to achieve accurate measurements by the plate reader.To identify specific MMPs that contribute to MMP activity measured here, expression assays such as western blot or PCR could be used. The use of live cells requires proper biosafety procedure, aseptic technique, and a biological safety cabinet. The UV light is hazardous, use a shield to protect the user, and do not look directly into the light.

measuring-global-cellular-matrix-metalloproteinase-metabolic-activity

Research

JoVE Journal

Bioengineering

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Planar Gradient Diffusion System to Investigate Chemotaxis in a 3D Collagen Matrix

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to their surrounding area, leading to cell movement and migration. In order to understand the mechanisms that can alter and/or affect cell migration, one can study these forces. In theory, understanding the fundamental mechanisms and forces underlying cell migration holds the promise of effective approaches for treating diseases and promoting cellular transplantation. Unfortunately, modern chemotaxis chambers that have been developed are usually restricted to two dimensions (2D) and have complex diffusion gradients that make the experiment difficult to interpret. To this end, we have developed, and describe in this paper, a direct-viewing chamber for chemotaxis studies, which allows one to overcome modern chemotaxis chamber obstacles able to measure cell forces and specific concentration within the chamber in a 3D environment to study cell 3D migration. More compelling, this approach allows one to successfully model diffusion through 3D collagen matrices and calculate the coefficient of diffusion of a chemoattractant through multiple different concentrations of collagen, while keeping the system simple and user friendly for traction force microscopy (TFM) and digital volume correlation (DVC) analysis.

Cell migration is an important part of human development and life. In order to understand the mechanisms that can alter cell migration, we present a planar gradient diffusion system to investigate chemotaxis in a 3D collagen matrix, which allows one to overcome modern diffusion chamber limitations of existing assays.

The importance of cell migration can be seen through the development of human life. When cells migrate, they generate forces and transfer these forces to ...

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 ...

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 ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...

A Human 3D Extracellular Matrix-Adipocyte Culture Model for Studying Matrix-Cell Metabolic Crosstalk

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of cellular function. The ECM plays a particularly important role in adipose tissue function in obesity, and alterations in adipose tissue ECM deposition and composition are associated with metabolic disease in mice and humans. Tractable in vitro models that permit dissection of the roles of the ECM and cells in contributing to global tissue phenotype are sparse. We describe a novel 3D in vitro model of human ECM-adipocyte culture that permits study of the specific roles of the ECM and adipocytes in regulating adipose tissue metabolic phenotype. Human adipose tissue is decellularized to isolate ECM, which is subsequently repopulated with preadipocytes that are then differentiated within the ECM into mature adipocytes. This method creates ECM-adipocyte constructs that are metabolically active and retain characteristics of the tissues and patients from which they are derived. We have used this system to demonstrate disease-specific ECM-adipocyte crosstalk in human adipose tissue. This culture model provides a tool for dissecting the roles of the ECM and adipocytes in contributing to global adipose tissue metabolic phenotype and permits study of the role of the ECM in regulating adipose tissue homeostasis.

We describe a 3D human extracellular matrix-adipocyte in vitro culture system that permits dissection of the roles of the matrix and adipocytes in contributing to adipose tissue metabolic phenotype.

The extracellular matrix (ECM) plays a central role in regulating tissue homeostasis, engaging in crosstalk with cells and regulating multiple aspects of ...

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 ...

Measuring 3D In-vivo Shoulder Kinematics using Biplanar Videoradiography

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, multiple ligaments, and approximately 20 muscles. Unfortunately, shoulder pathologies (e.g., rotator cuff tears, joint dislocations, arthritis) are common, resulting in substantial pain, disability, and decreased quality of life. The specific etiology for many of these pathologic conditions is not fully understood, but it is generally accepted that shoulder pathology is often associated with altered joint motion. Unfortunately, measuring shoulder motion with the necessary level of accuracy to investigate motion-based hypotheses is not trivial. However, radiographic-based motion measurement techniques have provided the advancement necessary to investigate motion-based hypotheses and provide a mechanistic understanding of shoulder function. Thus, the purpose of this article is to describe the approaches for measuring shoulder motion using a custom biplanar videoradiography system. The specific objectives of this article are to describe the protocols to acquire biplanar videoradiographic images of the shoulder complex, acquire CT scans, develop 3D bone models, locate anatomical landmarks, track the position and orientation of the humerus, scapula, and torso from the biplanar radiographic images, and calculate the kinematic outcome measures. In addition, the article will describe special considerations unique to the shoulder when measuring joint kinematics using this approach.

Biplane videoradiography can quantify shoulder kinematics with a high degree of accuracy. The protocol described herein was specifically designed to track the scapula, humerus, and the ribs during planar humeral elevation, and outlines the procedures for data collection, processing, and analysis. Unique considerations for data collection are also described.

The shoulder is one of the human body's most complex joint systems, with motion occurring through the coordinated actions of four individual joints, ...

Cellular Affinity of Particle-Stabilized Emulsion to Boost Antigen Internalization

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug delivery and immune response. The present study stemmed from the observation that the effects of charge, size, and shape of solid particles on cell affinity are usually considered, but we seldom realize the essential role of softness, dynamic restructuring phenomenon, and complex interface interaction in cellular affinity. Here, we developed poly-lactic-co-glycolic acid (PLGA) nanoparticle-stabilized Pickering emulsion (PNPE) that overcame the shortcomings of rigid forms and simulated the flexibility and fluidity of pathogens. A method was set up to test the affinity of PNPE to cell surfaces and elaborate on the subsequent internalization by immune cells. The affinity of PNPE to bio-mimetic extracellular vesicles (bEVs)-the replacement for bone marrow dendritic cells (BMDCs)-was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D), which allowed real-time monitoring of cell-emulsion adhesion. Subsequently, the PNPE was used to deliver the antigen (ovalbumin, OVA) and the uptake of the antigens by BMDCs was observed using confocal laser scanning microscope (CLSM). Representative results showed that the PNPE immediately decreased frequency (&#916;F) when it encountered the bEVs, indicating rapid adhesion and high affinity of the PNPE to the BMDCs. PNPE showed significantly stronger binding to the cell membrane than PLGA microparticles (PMPs) and AddaVax adjuvant (denoted as surfactant-stabilized nano-emulsion [SSE]). Furthermore, owing to the enhanced cellular affinity to the immunocytes through dynamic curvature changes and lateral diffusions, antigen uptake was subsequently boosted compared with PMPs and SSE. This protocol provides insights for designing novel formulations with high cell affinity and efficient antigen internalization, providing a platform for the development of efficient vaccines.

To rationally design efficient adjuvants, we developed poly-lactic-co-glycolic acid nanoparticle-stabilized Pickering emulsion (PNPE). The PNPE possessed unique softness and a hydrophobic interface for potent cellular contact and offered high-content antigen loading, improving the cellular affinity of the delivery system to antigen-presenting cells and inducing efficient internalization of antigens.

The cellular affinity of micro-/nanoparticles is the precondition for cellular recognition, cellular uptake, and activation, which are essential for drug ...

Microengineering 3D Collagen Hydrogels with Long-Range Fiber Alignment

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark of cardiac and musculoskeletal tissues. To study cell response to aligned microenvironments in vitro, several protocols have been developed to generate COL1 matrices with defined fiber alignment, including magnetic, mechanical, cell-based, and microfluidic methods. Of these, microfluidic approaches offer advanced capabilities such as accurate control over fluid flows and the cellular microenvironment. However, the microfluidic approaches to generate aligned COL1 matrices for advanced in vitro culture platforms have been limited to thin "mats" (&lt;40 &#181;m in thickness) of COL1 fibers that extend over distances less than 500 &#181;m and are not conducive to 3D cell culture applications. Here, we present a protocol to fabricate 3D COL1 matrices (130-250 &#181;m in thickness) with millimeter-scale regions of defined fiber alignment in a microfluidic device. This platform provides advanced cell culture capabilities to model structured tissue microenvironments by providing direct access to the micro-engineered matrix for cell culture.

This protocol demonstrates the use of a microfluidic channel with changing geometry along the fluid flow direction to generate extensional strain (stretching) to align fibers in a 3D collagen hydrogel (&lt;250 &#181;m in thickness). The resulting alignment extends across several millimeters and is influenced by the extensional strain rate.

Aligned collagen I (COL1) fibers guide tumor cell motility, influence endothelial cell morphology, control stem cell differentiation, and are a hallmark ...