This protocol allows us to isolate both quiescent and injury-induced activated fibroblast populations with significant purity. The main advantage of this technique is its flexibility for improvisation. For flow, as well as bead-based isolation, one can incorporate antibodies against contaminating cells to exclude them from the isolated target cells.
This technique is primarily used for cardiac fibroblast isolation. However, it can be utilized to isolate fibroblasts from other tissues. Demonstrating this procedure will be Meiling Melzer, an undergraduate intern, and David Beier, a research assistant from my laboratory.
To prepare a six-well plate for storing six hearts during dissection, dispense two milliliters of cold KHB into each well, and place the plate on ice. Spray the body with 70%ethanol. Orient it so the ventral side is facing the experimenter.
Pin the appendages to prevent interference. Without piercing the liver, cut open the abdominal skin and muscle. Cut vertically toward the sternum, carefully opening the thorax without piercing the heart.
Next, continue to cut through the ribcage to expose the heart. Using forceps, gently lift the heart out of the chest, cutting away any lung or excess tissue attached to the outside of the heart. Separate the left ventricle.
For each dissection, place the ventricle in a separate well of the six-well plate. First, use forceps to repeatedly squeeze and agitate the ventricle in KHB to remove excess blood. Transfer the ventricle to a clean, sterile, 10-centimeter-square plate.
Next, using a single-edged blade, quickly mince the ventricle into small pieces. Add one milliliter of collagenase digestion cocktail, and continue mincing. When the pieces are small enough to transfer with a one-milliliter micropipette, transfer them to a 50-milliliter conical tube.
Wash the plate twice with two milliliters of cocktail, and transfer the wash to the tube. Incubate the tube for 30 minutes, and resuspend the cells as described in the manuscript using a five-milliliter pipette. After the initial resuspension, incubate the cells for 15 minutes, and resuspend again using a 10-milliliter pipette.
Then, place a 40-micrometer cell strainer on top of a new, 50-milliliter conical tube, and prime the strainer by wetting it with one to two milliliters of KHB. Add 25 milliliters of KHB to the digestion suspension, and resuspend. Filter the suspension through the strainer, replacing the strainer as needed.
Centrifuge the filtrate at 400 times g and four degrees Celsius for 10 minutes. Remove the supernatant, and resuspend the pellet in 1x RBC lysis buffer using five milliliters of buffer per heart. After incubating the suspension for two minutes at room temperature, centrifuge the suspension at 400 times g for 10 minutes at room temperature.
Remove the supernatant, and then resuspend the pellet in one milliliter of KHB. Then, add nine milliliters of KHB to the suspension. Filter the suspension through a primed, 40-micrometer cell strainer into a new, 50-milliliter conical tube.
Centrifuge the conical tube at 400 times g for 10 minutes at room temperature. Finally, remove the supernatant, and resuspend the pellet in one milliliter of fibroblast media or PBS. After the cell number has been determined, fibroblasts can be isolated from the suspension by three different methods described in the manuscript.
To begin isolation by differential plating, prepare a six-well plate by adding two milliliters of fibroblast media to each well. Swirl the plate to cover the well bottoms. If the mouse heart cells are suspended in PBS, centrifuge and resuspend them in one milliliter of fibroblast media per heart.
Add one milliliter of cell suspension, the equivalent of one heart, to each well. Swirl the plate to evenly distribute cells. Add an additional one to two milliliters of fibroblast media per well for a total volume of up to four milliliters per well.
Incubate at 37 degrees Celsius for four hours. Because the fibroblasts will selectively adhere to the wells, they can be isolated by removing and discarding the media. Wash the remaining attached cells with two milliliters of PBS, and then add two to four milliliters of sterile fibroblast media per well.
Incubate at 37 degrees until confluent, changing the media every two to four days. To begin the magnetic labeling and separation of CD45-positive hematopoietic cells, centrifuge a suspension of mouse heart cells at 500 times g for five minutes. Remove the supernatant, and resuspend the cell pellet in one milliliter of equilibration buffer.
Count the cells using a hemocytometer. After centrifuging the cells again, resuspend the cell pellet in equilibration buffer. Add CD45-positive magnetic beads, and mix well.
Incubate for at least 15 minutes at four degrees Celsius. Wash the cells by adding equilibration buffer and centrifuging at 500 times g for 10 minutes at four degrees Celsius. Remove the supernatant, and count the cells using a hemocytometer.
Resuspend the pellet in equilibration buffer. Pass the suspension through a 40-micrometer filter to prevent cell aggregations from clogging the separation column. Next, place a separation column in the magnetic field of a suitable separator, and equilibrate the column with at least three milliliters of PBS.
Pour the cell suspension through the column, and collect the flow-through, which will contain the unlabeled cells, in a 15-milliliter conical tube. Wash the column three times with three milliliters of equilibration buffer, and combine the washes with the flow-through. Remove the column from the separator, and place the column on a 15-milliliter conical tube.
To elute the labeled CD45-positive cells, pipette five milliliters of equilibration buffer into the column, and plunge the column firmly. Centrifuge the tube of eluent and the tube of flow-through at 500 times g. Count the cells using a hemocytometer.
To complete the isolation, follow the protocols in the manuscript for the CD31-positive endothelial cells and the MEFSK4-positive fibroblasts, which are similar to the protocol for the CD45-positive cells. Fluorescence-activated cell sorting was performed on single cells isolated from alpha-SMA-GFP mouse hearts following myocardial infarction. A representative gating scheme demonstrates GFP-positive cells co-expressing CD31, CD45, or AN2.
In the injured mouse hearts, a small percentage of endothelial cells and hematopoietic cells expressed GFP. However, GFP-positive/CD31-negative/CD45-negative cells did not express AN2, a pericyte marker. GFP-positive cells expressed alpha-SMA, collagen one alpha one, periostin, and vimentin.
Uninjured fibroblasts isolated by selective adhesion expressed vimentin but did not demonstrate expression of alpha-SMA, periostin, or collagen one alpha one. Both uninjured and activated fibroblasts expressed the MEFSK4 antigen. Both uninjured fibroblasts, isolated by selective adhesion, and myofibroblasts, isolated and sorted from alpha-SMA-GFP mice, demonstrated an ability to contract collagen.
It is important to mince the tissue thoroughly before incubation. Before bead-based separation, it is important to pass the suspension through a 40-micrometer filter to prevent cell aggregations. The cells purified by this method could be further purified using magnetic beads associated with pericyte-specific antibodies to ensure a high level of purity.
With the availability of new bead-conjugated antibodies, as well as reporter transgenic mice, one can utilize these techniques to isolate and study distinct cellular populations, not just fibroblasts.
