Name: measuring global cellular matrix metalloproteinase and metabolic activity in 3d hydrogels
Uploaded: 2019-01-22T15:00:00.000+00:00
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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 to enable covalent incorporation into the hydrogel polymer network.</li>
	<li>Prepare 8 arm 40 kDa poly(ethylene glycol) amine (PEG)-norbornene (NB) as described17. Verify end group functionalization of greater than 90% using 1H NMR. Dissolve PEG-NB in sterile phosphate buffer saline (PBS) at 25% w/v and store in -80 &#176;C freezer in (~300 &#181;L) aliquots. 
	NOTE: PEG functionalized with norbornene can also be purchased commercially.</li>
	<li>Synthesize the photo-initiator lithium phenyl 2,4,6 trimethylbenzoylphosphinate (LAP) as described elsewhere18. Dissolve LAP in sterile water to a concentration of 68 mM and store in a -80 &#176;C freezer in (~300 &#181;L) aliquots. 
	NOTE: As an alternative, Irgacure (2-Hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone) can be used as the photo-initiator. LAP and Irgacure can be purchased commercially.</li>
	<li>Dissolve the MMP-degradable peptide crosslinker (KCGPQG&#8595;IWGQCK) and the cell adhesion peptide (CRGDS) in sterile water to a concentration of 200 mM and 100 mM respectively, and store them in a -80 &#176;C freezer in (~300 &#181;L and ~30 &#181;L) aliquots, respectively.</li>
</ol>
2. Assay media preparation
<ol>
	<li>Prepare the heat-inactivated, charcoal stripped fetal bovine serum (FBS) for the MMP assay: 
	NOTE: Proteases in FBS can produce a high background signal with the MMP assay; therefore, it is recommended to heat-inactivate and charcoal strip the FBS for the assay media.
	<ol>
		<li>Inactivate 100 mL of FBS by heating for 30 min at 55 &#176;C. 
		NOTE: 100 mL of FBS was utilized here for aliquoting and storage at -20 &#176;C for future use. Smaller volumes can be used as needed.</li>
		<li>Add 0.25% w/v of activated charcoal and 0.025% w/v of dextran to a small amount of FBS (approximately 5 mL) and stir until a slurry is formed. Then, add the rest of the FBS and stir for 30 min at 55 &#176;C.</li>
		<li>Centrifuge at 1,962 x g for 20 min at 4 &#176;C. Then transfer the supernatant to another vessel.</li>
		<li>Repeat step 2.1.2 but at 37 &#176;C followed by step 2.1.3. Sterilize the supernatant using a 0.45 &#181;m filter and then a 0.2 &#181;m filter.</li>
	</ol>
	</li>
	<li>Prepare assay media using media supplemented with 1% charcoal stripped FBS, 2 mM L-glutamine, 10 U/mL penicillin, 10 &#181;g/mL streptomycin. 
	NOTE: Media without phenol red is recommended because it has less fluorescence interference. Other additions to the assay media such as insulin, growth factors, etc. may be added as long as the absorbance and fluorescence spectrum peaks do not overlap with the sensor (494 nm/521 nm).</li>
	<li>Dilute bacterial collagenase enzyme type I at 10 and 1,000 &#181;g/mL in the assay media as a positive control.</li>
</ol>
3. Hydrogel preparation and cell encapsulation
<ol>
	<li>Prepare the hydrogel precursor solution.
	<ol>
		<li>Add the reagents to a 1.5 mL tube in the following order, vortexing after addition of each component: 20 mM 8 arm 40 kDa PEG-NB, 12.75 mM crosslinker MMP-degradable peptide, 17.8 mM NaOH, 1 mM CRGDS, 2 mM LAP, and 0.25 mM fluorogenic MMP-degradable peptide. 
		NOTE: Table 1 shows hydrogel precursor solution contents, stock concentrations, working concentrations, volume calculation formulas and the required volumes needed to make 120 &#181;L of hydrogel precursor solution, which is sufficient to conduct an experiment with 10 hydrogels. To account for loss of the hydrogel solution due to pipetting, increase the total volume by 20%. The commercial peptides are often supplied in an acidic hydrogen chloride solution; therefore, NaOH is added to achieve a final pH of 7. pH of the final solution should be confirmed by the user.</li>
		<li>Divide the hydrogel precursor solution into multiple 1.5 mL tubes, one tube per condition being tested. 
		NOTE: Several control conditions in which hydrogels are prepared without the addition of cells are suggested. For a negative control, to account for non-specific degradation of the fluorogenic sensor, one hydrogel condition can be incubated with the vehicle control or the experimental media alone if there are no treatment conditions. For a positive control and for calibration between experiments, hydrogels can be incubated with a protease known to cleave the fluorogenic sensor. For example, two concentrations of bacterial collagenase were used here.</li>
	</ol>
	</li>
	<li>Encapsulate cells in hydrogels.
	<ol>
		<li>Prepare a single cell suspension as appropriate for the cell type being used. For example, wash a 10 cm dish of A375 melanoma cells with 10 mL of PBS. Trypsinize cells using 0.05% trypsin and incubate at 37 &#176;C and 5% CO2 for 3 min. Count cells with a hemocytometer to determine total cell number.</li>
		<li>Centrifuge the cell solution at 314 x g for 3 min, aspirate culture media, then re-suspend cells in PBS at approximately three times the highest required seeding density for the experiment. For example, a cell suspension with a density of 21 x 106 cells was used here to achieve a final encapsulated density of 7 x 106 cells/mL.</li>
		<li>Count the cells again to ensure an accurate cell concentration.</li>
		<li>Add suspended cells and PBS to each tube of hydrogel precursor solution according to the required seeding density (0.25, 0.5, 1, 2, 3, 4, 5, 6, and 7 x 106 cells/mL in this example). Add PBS to conditions with no cells in lieu of suspended cells. 
		NOTE: All conditions should have the same final hydrogel precursor solution volume to ensure the ratio between the hydrogel components and PBS is constant. Do not vortex tubes that have cells in them, pipette up and down vigorously without creating bubbles in order to mix the precursor solution.</li>
		<li>Dispense 10 &#181;L of the hydrogel precursor solution into a sterile black round bottom 96-well plate, ensuring that the tip is centered in the middle of the each well while dispensing.</li>
		<li>Polymerize hydrogel precursor solution by exposing the plate to ultra violet (UV) light at 4 mW/cm2 for 3 min. 
		NOTE: The UV lamp (UVL-56 Handheld UV Lamp, UVP, Upland, CA) produces UV-A light at a long UV wavelength (365 nm), which does not affect cellular viability.</li>
		<li>Add 150 &#181;L of assay media to all wells except for the positive control conditions without encapsulated cells. To the positive controls, add 150 &#181;L of collagenase enzyme solution.</li>
		<li>Add 150 &#181;L of PBS to the outer wells of the plate to reduce evaporation during incubation.</li>
	</ol>
	</li>
</ol>
4. MMP and metabolic activity measurement
<ol>
	<li>Measure fluorescence immediately post-encapsulation to establish a baseline fluorescence measurement and ensure uniformity in hydrogel polymerization. Read the plate using a fluorescence microplate reader utilizing an opaque 96-well plate protocol with an area scan setting at 494 nm/521 nm (excitation/emission). This will be the 0 h read.</li>
	<li>Incubate plate in a humidified incubator at 37 &#176;C and 5% CO2 for 18 h.</li>
	<li>Add metabolic activity reagent (resazurin) at 1:10 (v/v) for each well.</li>
	<li>Incubate plate in a humidified incubator at 37 &#176;C and 5% CO2 for an additional 6 h. 
	NOTE: This incubation time may vary depending on cell type.</li>
	<li>Measure fluorescence at 24 h post-encapsulation. Read the plate using a fluorescence microplate reader utilizing an opaque 96-well plate protocol with an area scan setting at 494 nm/521 nm (excitation/emission) for MMP activity and 560 nm/590 nm (excitation/emission) for metabolic activity.</li>
</ol>

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The authors have nothing to disclose.

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 applications. The sequence of the fluorogenic sensor can be changed to detect other proteases. For instance, the fluorogenic cleavable sensor sequence (GPQG&#8595;IWGQK) which can be cleaved by MMP-1, -2, -3, -7, -8, and -9, was previously utilized to detect melanoma MMP activity in response to chemotherapeutics22. The use of the PEG hydrogel system also enables precise independent tuning of the cellular microenvironment, including the mechanical properties, the degradability, and the matrix adhesion moieties within the hydrogel. The adhesion molecule RGD used in this work is a fibronectin-derived sequence and enables integrin-mediated adhesion. Other adhesion molecules such as (RLD) and (IKVAV) that are derived from fibrinogen and laminin respectively could be utilized to activate other types of integrin receptors. For example, it was demonstrated that altering the adhesion molecules changed the elongation of aortic valvular interstitial cells (VIC), which may have an effect on aortic valve stenosis15. The hydrogel crosslinker sequence can control the degradability of the hydrogel and encapsulated cellular behavior within the hydrogel. For example, it was demonstrated that fibroblasts had increased proliferation and cell spreading when cultured in faster degrading hydrogels, which may expedite the healing process in vivo23. Mechanical properties of the microenvironment can also regulate cell function, and hydrogel stiffness can be modified by altering the number of arms in the PEG macromer, PEG molecular weight and the ratio between PEG and crosslinker.
Because MMP activity varies by cell type, for each new cell type it is important to conduct an encapsulation density optimization experiment as demonstrated here. After 24 h of encapsulation, MMP and metabolic activity were directly proportional to cell seeding density. However, at longer times the fluorescent signal can plateau, therefore the timing of the assay may have to be optimized by the user8. The specific protease sensor can also affect the working range and timing of the assay. The working range of the assay can be determined by conducting a signal range experiment with no cells and a proteolytic enzyme over a wide concentration. Inclusion of several no cell hydrogel conditions incubated with enzyme controls enables establishment of the low (background noise), medium and high levels of signal within each assay (0, 10 and 1,000 &#181;g/mL of collagenase here). This gives an indication of where the signals produced by cells fall within the working range of the assay. This assay is also compatible with other fluorescent sensors that do not have overlapping excitation and emission spectra, such as the metabolic activity assay used here as an internal control to calculate MMP activity on per cell basis, as previously reported8. MMP activity normalized to metabolic activity demonstrated the validity of this approach for seeding densities at or above 2 x 106 cells/mL, in which there was no significant change across seeding densities. This normalization is crucial to identify the appropriate seeding densities that are within the working range of the MMP activity curve and within the linear portion of the normalization curve.
While the assay can be adapted for several purposes, there are several limitations and critical aspects the user should consider, specifically regarding interference with the fluorescent signal and preparation of the hydrogels. First, care must be taken with the choice of culture media and additional treatments, as these can have an overlapping absorbance spectrum or opaqueness that may interfere with the detection of fluorescence or quench the signal. It is recommended to use culture media that does not contain phenol red. Also, some drug treatments are fluorescent (e.g., doxorubicin), or have absorbance spectrum that overlaps with the excitation/emission spectrum of the fluorophore (e.g., curcuminoids). A second aspect that can affect the performance of the assay is the hydrogel preparation. Because of the high viscosity of the hydrogel precursor solution, care needs to be taken to ensure thorough mixing of the hydrogel solution and careful pipetting practices to prevent unequal fluorophore content or hydrogel solution volume in each well. Pre-wetting of the pipette tips and using low-retention tips can help reduce variability. Another important aspect of hydrogel preparation is the hydrogel shape and location in wells. The hydrogel should be centered within the well and of a uniform shape to enable accurate fluorescence measurements with less variability15. Here, the use of round bottom plates aids in centering the pipette tip in each well and the production of semi-spherical hydrogels consistently across all wells.
By adapting the 3D protease-degradable hydrogel system, real-time protease and metabolic activity readouts can be detected in a 3D microenvironment with minimal sample processing. In addition, the use of the synthetic PEG hydrogel reduces the batch-to-batch variability observed with naturally derived ECM hydrogels. Furthermore, utilizing PEG hydrogels enables fine-tuning of chemical and mechanical cues of hydrogels for an improved control of the microenvironment. Moreover, the PEG hydrogel polymerization described here is a photo-initiated process, making it quick and more amenable to high throughput methodologies than classic natural ECM hydrogels (i.e., collagen or Matrigel), which are slower and temperature sensitive. This quick, user-controlled polymerization allows the scaling-up of the system to be further automated using robotic liquid handlers, as demonstrated by others15.

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.

<table><tbody><tr><td>1X Phosphate Buffered Saline</td><td>Fisher Scientific</td><td>10-010-049</td><td></td></tr><tr><td>Activated charcoal&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C3345</td><td></td></tr><tr><td>Black round bottom 96-well plate</td><td>Brand-Tech</td><td>89093-600</td><td></td></tr><tr><td>Cell adhesion peptide (CRGDS)</td><td>GenScript USA Inc.</td><td>custom made</td><td></td></tr><tr><td>Collagenase enzyme type I&amp;nbsp;</td><td>Life Technologies</td><td>17100-017</td><td></td></tr><tr><td>Dextran</td><td>Sigma-Aldrich</td><td>D4876</td><td></td></tr><tr><td>DMEM High Glucose Media</td><td>Invitrogen</td><td>11965118</td><td></td></tr><tr><td>Fetal bovine serum (FBS)&amp;nbsp;</td><td>Seradigm</td><td>1500-500</td><td></td></tr><tr><td>Fluorescence microplate reader&amp;nbsp;</td><td>BioTek</td><td>Cytation 3</td><td></td></tr><tr><td>Hemocytometer</td><td>Hausser Scientific Co.</td><td>3200</td><td></td></tr><tr><td>L-glutamine</td><td>Life Technologies</td><td>25030-081</td><td></td></tr><tr><td>MMP-degradable peptide crosslinker (KCGPQG&amp;darr;IWGQCK)</td><td>GenScript USA Inc.</td><td>custom made</td><td></td></tr><tr><td>NaOH</td><td>Fisher Scientific</td><td>S318500</td><td></td></tr><tr><td>Penicillin /streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Resazurin (Alamar Blue)</td><td>Life Technologies</td><td>DAL1100</td><td></td></tr><tr><td>UV light&amp;nbsp;</td><td>UVP</td><td>95-0006-02</td><td></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 quencher molecule (dabcyl) on either side of the cleavage site (Figure 1). Upon exposure of the fluorogenic sensor to the appropriate protease, the fluorophore and quencher are separated, and fluorescence increases. Here, the assay was miniaturized from a 24-well plate to a 96-well plate format, eliminating several steps in the encapsulation process and reducing the time needed to perform an experiment by 50%. Further, it reduced the volume of reagents consumed by 80%, as hydrogel volumes were reduced from 50 &#181;L to 10 &#181;L per well. Moreover, by using a 96-well plate, 20 conditions in triplicates could be tested instead of the 12 conditions in duplicates per 24-well plate. A schematic of the cell encapsulation process is illustrated in Figure 2. Label 1 and 2 correspond to steps 3.1 and 3.2 in the protocol, respectively. Labels 3 to 5 correspond to steps 3.2.5 to 3.2.7 in the protocol. Labels 6 to 9 correspond to steps 4.2 to 4.5 in the protocol.
To establish the detection limits and signal range of the assay, the hydrogels functionalized with the fluorogenic MMP-degradable peptide were incubated with a range of concentrations (0 to 2,000 &#181;g/mL) of bacterial collagenase enzyme type I. After 24 h of incubation at 37 &#176;C, a plate reader was used to measure the fluorescence (Figure 3). From these measurements, the dynamic (lowest and highest detected signals) and working range (3 standard deviations above the lowest detected signal and 3 standard deviations below the highest detected signal) were determined. After 24 h of incubation, it was observed that the lowest detected signal was produced by negative controls (background noise) at 0 &#181;g/mL collagenase, while the highest detected signal was produced by 1,000 &#181;g/mL collagenase or above, where the signal begins to plateau (Figure 3). From the dynamic range, the working range was calculated to be between &#8776;0.16 &#181;g/mL and &#8776;474 &#181;g/mL of collagenase, a wide signal range across three&#160;orders of magnitude.
For cell culture assays, the appropriate cell density that results in fluorescence readings within the working range of the assay must be determined for each cell type. Here, representative data is presented for the melanoma cell line A375, encapsulated in a range of densities from 0.25 to 7 x 106 cells/mL. Fluorescence intensity was acquired utilizing a standard microplate reader at two different time points: 1) directly after encapsulation (0 h) and 2) after 24 h of encapsulation. At 0 h, fluorescence readings were low across seeding densities (Figure 4A), as expected. After 24 h of culture (Figure 4B), MMP activity was directly proportional to the seeding density, in which more cells resulted in more cleavage of the fluorogenic MMP cleavable sensor and higher fluorescence intensity. As internal controls, hydrogels containing no cells were incubated with 0, 10, and 1,000 &#181;g/mL of collagenase type I enzyme to indicate the low, medium and high levels of the signal respectively (as determined by the collagenase signal range characterization in Figure 3) and represented by dashed lines in Figure 4B. Further, the working range limits were calculated from the 0 and 1,000 &#181;g/mL signals and represented by dotted lines in Figure 4B. Seeding densities at or greater than 1 x 106 cells/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 (Figure 4C). Previously, MMP activity has been normalized to metabolic activity as an internal control to determine MMP activity on a per cell basis8. Normalizing MMP activity to metabolic activity across seeding densities resulted in no significant difference in MMP activity at seeding densities greater than 2 x 106 cells/mL (Figure 4D). To determine variability between triplicates in each plate (intra-plate variability), the coefficient of variation percentage (% CV) was calculated for both MMP and metabolic activity and is summarized in Table 2. % CV below 20% indicates that the intra-plate variability within triplicates is acceptable for the system to produce consistent results20,21.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59123/59123fig1.jpg" /> 
Figure 1: The fluorogenic MMP-degradable peptide design. The fluorogenic MMP-degradable peptide consists of a backbone peptide GPLAC(pMeOBzl)&#8595;WARKDDK(AdOO)C (&#8595; indicates the cleavage site) that determines the specificity of the sensor. The peptide is labeled with a quencher (dabcyl) and a fluorophore (fluorescein), which is unquenched (fluoresces) when the backbone peptide is cleaved by MMP. A thiol group is conjugated to the backbone peptide to enable covalent reaction with norbornene functional groups in the PEG molecule, coupling the sensor to the hydrogel. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59123/59123fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59123/59123fig2.jpg" /> 
Figure 2: Assay schematic. The hydrogel precursor solution components are mixed with cells suspended in PBS. The precursor solution is then pipetted into black, round bottom, 96-well plates and polymerized by exposure to UV light for 3 min. Assay media is added, and plates are incubated for 18 h (37 &#176;C, 5% CO2). The metabolic activity reagent resazurin is then added, and plates are incubated for an additional 6 h. MMP and metabolic activity are measured using a fluorescent microplate reader with a well scan protocol at the indicated excitation/emission wavelengths. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59123/59123fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59123/59123fig3.jpg" /> 
Figure 3:&#160;Dynamic and working range of the assay. Hydrogels functionalized with fluorogenic MMP-degradable peptide were incubated with a range of concentrations of collagenase enzyme type I for 24 h at 37 &#176;C and fluorescence intensity was measured. Dotted lines represent the dynamic and working range. n = 3, mean &#177; standard deviation (SD). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59123/59123fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59123/59123fig4.jpg" /> 
Figure 4:&#160;Effect of seeding density of melanoma cell line A375 on MMP activity. (A) Initial measurement (0 h) of MMP activity for A375 cell line encapsulated over a range of seeding densities. n = 3 mean &#177; SD. (B) Measurement of MMP activity at 24 h. 0, 10 and 1000 &#181;g/mL of collagenase are represented by dashed lines. Dotted lines represent the working range (WR) calculated from collagenase controls. n = 3, mean &#177; SD. (C) Measurement of metabolic activity for A375 cell line encapsulated for 24 h over a range of seeding densities and incubated with resazurin for 6 h. n = 3, mean &#177; SD. (D) A375 MMP activity normalized to metabolic activity. n&#160;= 3, mean &#177; SD. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/59123/59123fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Table 1" src="/files/ftp_upload/59123/59123table1.jpg" /> 
Table 1:&#160;Hydrogel precursor solution preparation.
<img alt="Table 2" src="/files/ftp_upload/59123/59123table2.jpg" /> 
Table 2:&#160;Intra-plate % CV of the assay with A375 cell line.

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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) ...

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

Nanyang Technological University

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JoVE Journal

Bioengineering

7.4K Views.  The Ohio State University. 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.

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

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position- and time-dependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(&#969;). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment.

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and ...

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to tumor and cancer metastasis. Despite intense research efforts, the basic biochemical and biophysical principles of cell migration are still not fully understood, especially in the physiologically relevant three-dimensional (3D) microenvironments. Here, we describe an in vitro assay designed to allow quantitative examination of 3D cell migration behaviors. The method exploits the cell&#8217;s mechanosensing ability and propensity to migrate into previously unoccupied extracellular matrix (ECM). We use the invasion of highly invasive breast cancer cells, MDA-MB-231, in collagen gels as a model system. The spread of cell population and the migration dynamics of individual cells over weeks of culture can be monitored using live-cell imaging and analyzed to extract spatiotemporally-resolved data. Furthermore, the method is easily adaptable for diverse extracellular matrices, thus offering a simple yet powerful way to investigate the role of biophysical factors in the microenvironment on cell migration.

The mechanical properties and microstructure of the extracellular matrix strongly affect 3D migration of cells. An in vitro method to study the spatiotemporal cell migration behavior in biophysically variable environments, at both population and individual cell levels, is described.

The ability of cells to migrate is crucial in a wide variety of cell functions throughout life&#160;from embryonic development and wound healing&#160;to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous microscale cryogels (microcryogels), which can be loaded with a variety of cell types to form 3D microtissues. Herein, we present the protocol to fabricate versatile 3D microtissues and their applications in regenerative therapy and drug screening. Size and shape-controllable microcryogels can be fabricated on an array chip, which can be harvested off-chip as individual cell-loaded carriers for injectable regenerative therapy or be further assembled on-chip into 3D microtissue arrays for high-throughput drug screening. Due to the high elastic nature of these microscale cryogels, the 3D microtissues exhibit great injectability for minimally invasive cell therapy by protecting cells from mechanical shear force during injection. This ensures enhanced cell survival and therapeutic effect in the mouse limb ischemia model. Meanwhile, assembly of 3D microtissue arrays in a standard 384-multi-well format facilitates the use of common laboratory facilities and equipment, enabling high-throughput drug screening on this versatile 3D cell culture platform.

This protocol describes the fabrication of elastic 3D macroporous microcryogels by integrating microfabrication with cryogelation technology. Upon loading with cells, 3D microtissues are generated, which can be readily injected in vivo to facilitate regenerative therapy or assembled into arrays for in vitro high-throughput drug screening.

To upgrade traditional 2D cell culture to 3D cell culture, we have integrated microfabrication with cryogelation technology to produce macroporous ...

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Biology

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 ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

The present protocol describes an experimental platform to assess the effects of mechanical and biochemical cues on chemotherapeutic responses of patient-derived glioblastoma cells in 3D matrix-mimetic cultures using a custom-made UV illumination device facilitating high-throughput photocrosslinking of hydrogels with tunable mechanical features.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes ...

Phototunable Bioprinted Hydrogels to Probe Cellular Responses to ECM Stiffening

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture platforms and dynamically triggering changes, such as increasing microenvironmental stiffness, enables researchers to model changes in the extracellular matrix (ECM) that occur during fibrotic disease progression. Herein, a method is presented for 3D bioprinting a phototunable hydrogel biomaterial capable of two sequential polymerization reactions within a gelatin support bath. The technique of Freeform Reversible Embedding of Suspended Hydrogels (FRESH) bioprinting was adapted by adjusting the pH of the support bath to facilitate a Michael addition reaction. First, the bioink containing poly(ethylene glycol)-alpha methacrylate (PEG&#945;MA) was reacted off-stoichiometry with a cell-degradable crosslinker to form soft hydrogels. These soft hydrogels were later exposed to photoinitator and light to induce the homopolymerization of unreacted groups and stiffen the hydrogel. This protocol covers hydrogel synthesis, 3D bioprinting, photostiffening, and endpoint characterizations to assess fibroblast activation within 3D structures. The method presented here enables researchers to 3D bioprint a variety of materials that undergo pH-catalyzed polymerization reactions and could be implemented to engineer various models of tissue homeostasis, disease, and repair.

Author Spotlight: Understanding Chronic Lung Diseases Using 3D Printed Phototunable Hydrogels 

This article describes how to 3D bioprint phototunable hydrogels to study extracellular matrix stiffening and fibroblast activation.

Phototunable hydrogels can transform spatially and temporally in response to light exposure. Incorporating these types of biomaterials in cell-culture ...

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and tunable 3D hydrogel, suitable for culturing cells and tissue in a setting that matches their native environment. This is particularly important for researchers investigating the initiation, growth, and treatment of cancer where the interaction between cells and their local extracellular matrix is a fundamental part of the model. Moving to 3D culture can be challenging and is often associated with a lack of reproducibility due to high batch-to-batch variation in animal-derived 3D culture matrices. Similarly, handling issues can limit the usefulness of synthetic hydrogels. In response to this need, we have optimized a simple self-assembling peptide gel, to enable the culture of relevant cell line models of cancer and disease, as well as patient-derived tissue/cells. The gel itself is free from matrix components, apart from those added during encapsulation or deposited into the gel by the encapsulated cells. The mechanical properties of the hydrogels can also be altered independent of matrix addition. It, therefore, acts as a &#8216;blank slate&#8217; allowing researchers to build a 3D culture environment that reflects the target tissue of interest and to dissect the influences of mechanical forces and/or biochemical control of cell behavior independently.

We present a method for creating a 3D cell culture environment, which can be used to investigate the importance of cell/matrix interactions in cancer progression. Using a simple self-assembling octapeptide, the matrix surrounding encapsulated cells can be controlled, with independent regulation of mechanical and biochemical cues.

There is a growing awareness that cells grown in 3D better model in vivo behavior than those grown in 2D. In this protocol, we describe a simple and ...

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

Summary

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Chapters in this video

Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Concentric Gel System to Study the Biophysical Role of Matrix Microenvironment on 3D Cell Migration

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

3D Microtissues for Injectable Regenerative Therapy and High-throughput Drug Screening

Detection of Functional Matrix Metalloproteinases by Zymography

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

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models

3D Bioprinting Phototunable Hydrogels to Study Fibroblast Activation

Preparation of a User-Defined Peptide Gel for Controlled 3D Culture Models of Cancer and Disease