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

Summary

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.

Abstract

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.

Introduction

Three-dimensional (3D) culture systems often more closely recapitulate in vivo cellular responses than traditional two-dimensional (2D) culture systems, see several excellent publications^1,2,3. 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 environments^4,5,6,7. 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 MMPs^9,10. MMPs play critical roles in physiological processes such as wound healing and in diseases such as arthritis, atherosclerosis, Alzheimer'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 phenomena^13,14. 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-β (TGF-β)^15,16. More recently, PEG hydrogels have been functionalized with sensor peptides that enable measurement of cell function as well^5,8. 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.

Protocol

1. Hydrogel components preparation

1. 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 °C freezer in small (~30 µL) 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.
2. 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 °C freezer in (~300 µL) aliquots.
   NOTE: PEG functionalized with norbornene can also be purchased commercially.
3. 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 °C freezer in (~300 µL) aliquots.
   NOTE: As an alternative, Irgacure (2-Hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone) can be used as the photo-initiator. LAP and Irgacure can be purchased commercially.
4. Dissolve the MMP-degradable peptide crosslinker (KCGPQG↓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 °C freezer in (~300 µL and ~30 µL) aliquots, respectively.

2. Assay media preparation

1. 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.
   1. Inactivate 100 mL of FBS by heating for 30 min at 55 °C.
      NOTE: 100 mL of FBS was utilized here for aliquoting and storage at -20 °C for future use. Smaller volumes can be used as needed.
   2. 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 °C.
   3. Centrifuge at 1,962 x g for 20 min at 4 °C. Then transfer the supernatant to another vessel.
   4. Repeat step 2.1.2 but at 37 °C followed by step 2.1.3. Sterilize the supernatant using a 0.45 µm filter and then a 0.2 µm filter.
2. Prepare assay media using media supplemented with 1% charcoal stripped FBS, 2 mM L-glutamine, 10 U/mL penicillin, 10 µg/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).
3. Dilute bacterial collagenase enzyme type I at 10 and 1,000 µg/mL in the assay media as a positive control.

3. Hydrogel preparation and cell encapsulation

1. Prepare the hydrogel precursor solution.
   1. 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 µL 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.
   2. 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.
2. Encapsulate cells in hydrogels.
   1. 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 °C and 5% CO₂ for 3 min. Count cells with a hemocytometer to determine total cell number.
   2. 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.
   3. Count the cells again to ensure an accurate cell concentration.
   4. 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.
   5. Dispense 10 µL 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.
   6. 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.
   7. Add 150 µL of assay media to all wells except for the positive control conditions without encapsulated cells. To the positive controls, add 150 µL of collagenase enzyme solution.
   8. Add 150 µL of PBS to the outer wells of the plate to reduce evaporation during incubation.

4. MMP and metabolic activity measurement

1. 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.
2. Incubate plate in a humidified incubator at 37 °C and 5% CO₂ for 18 h.
3. Add metabolic activity reagent (resazurin) at 1:10 (v/v) for each well.
4. Incubate plate in a humidified incubator at 37 °C and 5% CO₂ for an additional 6 h.
   NOTE: This incubation time may vary depending on cell type.
5. 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.

Results

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)↓WARKDDK(AdOO)C (↓ 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 molecul...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1X Phosphate Buffered Saline	Fisher Scientific	10-010-049
Activated charcoal	Sigma-Aldrich	C3345
Black round bottom 96-well plate	Brand-Tech	89093-600
Cell adhesion peptide (CRGDS)	GenScript USA Inc.	custom made
Collagenase enzyme type I	Life Technologies	17100-017
Dextran	Sigma-Aldrich	D4876
DMEM High Glucose Media	Invitrogen	11965118
Fetal bovine serum (FBS)	Seradigm	1500-500
Fluorescence microplate reader	BioTek	Cytation 3
Hemocytometer	Hausser Scientific Co.	3200
L-glutamine	Life Technologies	25030-081
MMP-degradable peptide crosslinker (KCGPQG↓IWGQCK)	GenScript USA Inc.	custom made
NaOH	Fisher Scientific	S318500
Penicillin /streptomycin	Life Technologies	15140-122
Resazurin (Alamar Blue)	Life Technologies	DAL1100
UV light	UVP	95-0006-02