The overall goal of this procedure is to observe the effect of specific treatments of interest on cell migration in a three dimension wound associated fibrin scaffold. This method can help answer key questions in the wound healing field, about how alterations in the fiber network structure influence cell migration and to fibrin clots at wound sites, the main advantage of this technique is that it provides a simple and reproducible method for analyzing cell migration within fibrin clots in 3D. Generally, individuals new to this method will struggle because transferring the spheroids can be difficult.
Begin by warming a vial of frozen neo-natal human dermal fibroblast in a 37 degree celsius water bath. When the cells are just thawed, collect them by centrifugation and re-suspend the pellet in fresh neo-natal human dermal fibroblast medium at 1 x 10 to the 6th cells per milliliter concentration. Then seed the cells in a T75 culture flask for a 72 hour incubation at 37 degree celsius and 5%carbon dioxide.
When the culture reaches 80%confluency, detach the cells with five milliliters of trypsin EDTA. After five minutes at 37 degree celsius, neutralize the trypsin with five milliliters of warmed medium and mix 40 microliters of cells with trypan blue at a 1:1 ratio. Reserve a 10 microliter aliquot of cells for counting.
Collect the cells by centrifugation and re-suspend the pellet at a 1.25 x 10 to the 5th cells per milliliter concentration. Next, plate the bottom of a 10 centimeter petri dish with five milliliters of sterile PBS. Then invert the petri dish lid and add multiple 20 microliter drops of the cell suspension onto the inner surface of the lid.
When all of the drops have been placed, re-invert the lid onto the dish, taking care not to dislodge the hanging drops and gently place the dish into a 37 degree celsius incubator for 72 hours. At the end of the incubation, add 100 microliters of clot solution per spheroid per well, to a 48 well plate and gently shake and tap the plate to ensure that the fibrin clot mixture, coats the entire well. Place the plate in the incubator for one hour.
When the clot layer has polymerized, confirm the presence of spheroids in the hanging drop culture, under a light microscope and transfer the 48 well plate and the hanging drop culture dish into a cell culture hood. Carefully invert the petri dish lid to access the hanging drop cultures and use a one milliliter syringe equipped with a 21 gauge needle, to transfer one drop into each fibrin coated well. I take extra care when aspirating and plating the spheroids, as the most difficult and critical step in the process is transferring the spheroids without destroying them.
Examine the plate under the microscope to confirm that the spheroids have been properly seeded into the appropriate wells. Gently layer 100 microliters of freshly prepared clot solution onto each spheroid containing drop and re-examine the spheroids under the microscope, to confirm that they are still intact and in the center of each well. Then return the 48 well plate to the incubator to allow the second layer of fibrin to polymerize.
After one hour, treat each well with 500 microliters of the appropriate medium for the corresponding experimental spheroid group and return the plate to the 37 degree celsius incubator. Obtain images of the spheroids by bright field microscopy every 24 to 72 hours, using a z stack to view successive cross sections of the 3D cell culture. Then, in the appropriate image analysis software, trace the cell border of each spheroid at each successive time point and use the measure function to access the percent increase in area for each spheroid.
Following their culture, in the pro or anti-migratory agent of interest, the initial areas of the spheroids can be determined by bright field microscopic imaging and used as a reference for determining the degree of subsequent cell outgrowth from the spheroid bodies at each successive time point. In this representative experiment, the spheroids that were exposed to a pro-migratory agent, exhibited a significantly increased degree of migration away from the original spheroid body compared to the spheroids exposed to a migration inhibitory or control agent. Conversely, the spheroids exposed to an anti-migratory agent exhibited a significantly decreased degree of migration away from the original spheroid body, compared to the control treated spheroids.
Once mastered, this technique can be completed in two and a half hours, excluding the cell culture time, if it is performed properly. After watching this video, you should have a good understanding of how to culture and embed cell spheroids for three dimensional in vitro cell migration assays. Following this procedure, other methods like histological and immunohistochemistry, can be performed to answer additional questions about how the fibrin network structure within the clot or the actin alignment in the spheroid cells correspond to increased or decreased cell migration.
Don't forget that working with cell cultures and needles can be extremely hazardous and that precautions such as, wearing the proper protective equipment and working in a sterile culture hood, should always be taken while performing this procedure.
