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.
