The overall goal of this procedure is to isolate fibroblasts using FACS, thereby foregoing in vitro culture, which has been shown to cause a phenotypic change in these cells. This method can help answer key questions in developmental biology and wound healing, such as how to identify fibroblast subtypes involved in scarring and fibrosis. The main advantage of this technique is that in vitro experiments are avoided as adherence to plastic, male to the phenotype of the fibroblast.
Not only can this method provide insights into dermal fibroblasts, but it can also be applied to other systems, such as peritoneal fibroblasts and abdominal adhesions. Begin by shaving and depilating the dorsal skin of the mouse of interest. Then, submerge the denuded mouse in 70%ethanol and place the animal on a clean, sterile surface to dry.
Next, starting at the base of the tail, use forceps to tent the skin and make a transverse cut with dissecting scissors. Then, dissect along the suprafascial plane to harvest the dorsal tissue. When a 60 by 100 millimeter piece of skin has been removed, use the blunt edge of a scalpel to carefully remove any subcutaneous fat.
Then, rinse the harvested skin in betadine, followed by five PBS washes on ice. Using razorblades and dissecting scissors, mince the pieces of dermis in a sterile dish until the samples are uniformly approximately two to three millimeters in size. Then, transfer tissue fragments from up to five mice into the appropriate number of individual 50 milliliter conical tubes containing 20 milliliters of collagenase 4 in dmem.
Agitate the samples vigorously at 37 degrees Celsius. After one hour, transfer the tubes to a sterile hood and pass the tissue flurries through a needless 10 milliliter syringe three to five times. Return the samples to the shaker for another 30 minutes, followed by another three to five pipettes with a 10 milliliter syringe.
Then, filter the samples through a 100 micron strainer into a new 50 milliliter tube and wash the mesh with 20 milliliters of dmem, supplemented with FBS to bring the total volume up to 40 milliliters. Next, spin down the cells and use a sterile glass pipette to aspirate the upper fat layer. When the contaminating adipose sites have been removed, use a new glass pipette to remove the rest of the supernatant and resuspend the pellets in 20 milliliters of dmem plus FBS.
Now, filter the cells through a 75 micron strainer and rinse the mesh with 10 milliliters of dmem plus FBS. Then, spin down the cells again and remove the fat and supernatant as just demonstrated. Resuspend the pellet in 20 milliliters of FACS buffer, setting aside a five milliliter aliquot for the unstained control.
Then, spin down the remaining sample, discard the supernatant and place the pellet on ice. To isolate the fibroblasts by FACS, resuspend the pellets in 500 microliters of lineage antibody incubation mix on ice. After 20 minutes, gently mix five milliliters of FACS buffer, supplemented with DNase with the cells and spin them down.
Wash the pellet in a second five milliliters of DNase. Then, resuspend the cells in 500 microliters of FACS buffer and DNase, reserving a 50 microliter aliquot of cells for the viability dye control. Finally, add viability dye to the remaining sample and sort the cells according to the diagram.
Using a lineage negative depletion approach rather than a positive selection approach avoids pre-selecting for particular subpopulations. Pull transcriptome microarray analysis reveals that cultured fibroblasts isolated by the live harvest and tissue explant methodologies have a high degree of similarity at a transcriptome wide level with a Pearson product-moment correlation coefficient of 0.92. By comparison, cultured fibroblasts differed significantly from a live harvested uncultured fibroblast, establishing the importance of analyzing live harvested fibroblasts over cultured fibroblasts.
After it's development, this technique paved the way for researchers in the field of developmental biology and wound healing to confirm fibroblasts heterogeneity and identify subpopulations responsible for scarring and other forms of fibroses.
