In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

We created an individual collagen-rich model to mimic the environment that cells experience in vivo using the macromolecular crowding technique. Skin is a crowded environment, unlike the liquid environment that we already use to grow cells in vitro. Compared to the traditional monolayer cell culture system, this model recreates the crowded molecular environment that cells experience in vivo, especially in scar tissues where abundant extracellular matrix displaces liquid water.

Besides hypertrophic scars, this macromolecular crowding technique can be used for studies where the extracellular matrix is abundant, such as pulmonary fibrosis. Grow scar-derived and normal fibroblasts according to manuscript directions. Then seed them into 24 well plates at 50, 000 cells per well in one milliliter of medium.

Incubate the plate overnight at 37 degrees Celsius and 5%carbon dioxide. Prepare macromolecular crowding or MMC media by mixing Ficoll 70, Ficoll 400 and ascorbic acid into 10%FCS DMEM. Incubate the media in a water bath to disperse the crowders into the solution.

Then sterilize it with a 0.2 micrometer filter. Aspirate the spent media from the fibroblasts and replace it with freshly made MMC media. Incubate the cells at 37 degrees Celsius for six days, changing the MMC media every three days.

Prepare a Sirius Red solution by dissolving 0.2 grams of Direct Red 80 powder in 200 milliliters of distilled deionized water with one milliliter of acetic acid. Aspirate the MMC media from the cells and add 300 microliters of Sirius Red solution to each well. Then return the cells to the incubator for 90 minutes.

After the incubation, gently rinse the Sirius Red solution with tap water and allow the plate to air dry overnight. On the next day, extract the Sirius Red by adding 200 microliters of 0.1 molar sodium hydroxide into each well and shaking the plate for five to 10 minutes. Transfer 100 microliters of the extracted stain into a transparent 96 well plate and measure the absorbance at 620 nanometers with a micro plate reader.

Wash each well with 200 microliters of PBS and fix the cells with methanol at four degrees Celsius for 10 minutes. Then add 3%BSA to the wells and incubate the plate for 30 minutes at room temperature. Aspirate the blocking solution and add 200 microliters of anti-collagen one antibody to each well.

Incubate the plate for 90 minutes. Then aspirate the antibody And wash the wells three times with PBS for five minutes per wash. Add 200 microliters of Goat Anti-Rabbit-FITC Secondary Antibody and DAPI to each well.

Cover the plate with aluminum foil and incubate it for 30 minutes at room temperature. Discard the secondary antibody and DAPI and repeat the washes with PBS. Then visualize the fluorescence directly under a microscope.

After washing the cells twice with PBS, add 40 microliters of lysis buffer into each well and scrape the cell layer with a pipette tip. Then transfer the protein lysate into micro centrifuge tubes to measure the protein concentration. Load 10 micrograms of protein into each well of a four to 12%Bis-Tris protein gel and perform SDS-PAGE at 200 volts for 30 minutes.

When finished, transfer the protein to a nitrocellulose membrane by running a western blot at 90 volts for 90 minutes, taking care to avoid bubble formation between the gel and the membrane. Block the membrane with 10 milliliters of blocking buffer. Then incubate it with primary antibodies at four degrees Celsius overnight.

On the next day, wash the membrane five times with 0.1 percent TBS Tween 20 for five minutes per wash. Then incubate the membrane with a species-appropriate secondary antibody for one hour at room temperature. Repeat the washes and visualize fluorescence with an imaging system.

After purifying the total RNA with a commercial RNA extraction kit, measure RNA concentration using a micro volume spectrophotometer. Then use a cDNA synthesis kit to perform a first strand DNA synthesis. Mix the cDNA with 10 microliters of SYBR Green Supermix and transfer the samples to a custom 96 well plate pre-coated with oligonucleotide primers.

Adjust the total volume to 20 microliters per well with water and run RT-PCR according to manuscript directions. Cell density of hypertrophic scar-derived human skin fibroblasts significantly increased after culturing with Ficoll compared to using PVP or the control. Macromolecular crowding with Ficoll at 9%fractional volume occupancy significantly enhanced the deposition of collagen, including collagen one, compared to other formulations.

This was further demonstrated with quantitative analysis. Scar-derived fibroblasts and normal dermal fibroblasts cultivated in MMC environments were found to regulate the expression of ECM species in addition to collagen. Notably, an elevated expression of matrix metalloproteinases or MMPs was found to accumulate in hypertrophic scar tissues compared to native tissues.

It was observed that the expression of MMP2, nine and 13 were significantly up-regulated in both cell cultures cultivated in macromolecular crowding conditions. MMPs play an important role during wound healing and scar formation, regulating ECM assembly and remodeling. Synthesis of interleukin six and vascular endothelial growth factor was undetectable in a western blot.

But RT-PCR analysis revealed that the expression of interleukin six was significantly up-regulated, while the expression of vascular endothelial growth factor was down-regulated in fibroblasts cultivated in MMC conditions. When attempting this protocol, it is very important to choose dermal fibroblasts that have very few CO pathogens. We also have to keep in mind that ascorbic acid is an essential ingredient in the MMC medium.

We are particularly interested in further detecting the collagen deposition and alignment in this MMC model. We plan to use advanced microscopic techniques to compare this in vitro model with native scar tissues in vivo. We encourage others to use this macromolecular crowding technique to improve the in vitro authenticity of the ancillary modal tissues.

Of course, macromolecular crowding can also be used to recalculate models of disease and pathology in vitro.