This protocol describes a platform technique to isolate microvascular endothelial cells from skeletal muscles. These cells can be used, for example, to gain further insights into blood muscle barrier functions. The main advantage of this technique over existing methods is that it's possible to isolate primary microvascular endothelial cells with high purity.
This technique can help to answer key questions in the field of immune-mediated muscle diseases such as the interaction between muscle and endothelial cells in health and disease. First, prepare all needed solutions and six-well plates as described in the protocol. To begin the practical procedure, obtain a pair of surgical sharp/blunt scissors, a pair of surgical sharp/sharp scissors, and straight and curved forceps.
Disinfect all surgical instruments with 70%ethanol. Position a euthanized adult male mouse on its back and moisten the legs with 70%ethanol. Using the sharp/blunt surgical scissors, sever each whole leg by cutting at the hip joint.
Place the extremities in a closed cell culture dish. Transfer the dish to a sterile laminar flow hood. Use sharp scissors and curved forceps to cut the skin open from the hip to the tip of the toe.
Use the straight forceps to hold the toe or foot pad while using the curved forceps to peel the skin off from the toe to the hip. To isolate the musculus quadriceps femoris, cut the tendon from the knee, and sever the muscle along the femur to the hip. Sever the Achilles tendon to isolate the musculus triceps surae.
Next, cut along the tibia to the popliteal fossa and remove the muscle. To remove tendons which cannot be dissociated, hold the muscles with the curved forceps and cut off each appropriate tendon. Add 2, 445 microliters of prepared digestion solution to a fresh, labeled cell culture dish and determine the weight.
Transfer all of the muscle pieces to this dish. Then, determine the weight. The difference of both measured values provides the dry weight of the muscle tissue which must not exceed one gram.
Using the surgical sharp scissors, cut the whole muscle tissue into small pieces. Incubate the tissue suspension at 37 degrees Celsius with 5%carbon dioxide for 1.5 hours, making sure to mix the suspension carefully with a one-milliliter insulin syringe every 20 minutes for approximately five minutes. After the incubation, transfer the suspension to a 70-micrometer nylon strainer placed on a 50-milliliter labeled tube and collect the flow through.
Wash the cell strainer with eight milliliters of DMEM and collect the flow through. If the pieces are plugging the strainer, resuspend the dissociated solution. Discard the cell strainer and centrifuge the suspension at 300 times gravity and at 20 degrees Celsius for 10 minutes.
Carefully remove the supernatant and resuspend the cell pellet in one milliliter of an ammonium chloride potassium lysing buffer for the lysis of red blood cells. Incubate at room temperature for 30 seconds. Then, add nine milliliters of DMEM containing 10%FCS to stop the reaction.
Transfer the cell suspension to a 15-milliliter tube. Then, use a Neubauer cell counting chamber to determine the cell numbers. To begin depleting the CD45-positive cells, centrifuge the suspension at 300 times gravity and at four degrees Celsius for 10 minutes.
Remove the supernatant completely and resuspend the cell pellet in 90 microliters of MCS buffer. Add 10 microliters of CD45 microbeads and mix the suspension. Incubate in a refrigerator at four to eight degrees Celsius for 15 minutes.
After this, add one milliliter of MCS buffer. Centrifuge at 300 times gravity and at four degrees Celsius for 10 minutes. Remove the supernatant completely and resuspend the cells in 500 microliters of MCS buffer.
Position a large magnetic column in the magnetic field of the separator. Rinse the reservoir of the column with three milliliters of MCS buffer. Then, place a 15-milliliter conical tube below the column to collect the flow through.
Apply the whole cell suspension to the column and let it flow through completely. Wash the column three times by adding three milliliters of MCS buffer into the reservoir for each washing step and waiting until the column's reservoir is empty before beginning the next washing step. Collect the unlabeled cells passing through the column for use in further separation steps.
To begin accumulating CD31-positive cells, use a Neubauer cell counting chamber to determine the cell numbers. Afterwards, centrifuge the unlabeled cells at 300 times gravity and at four degrees Celsius for 10 minutes. Remove the supernatant completely and resuspend the pellet in 90 microliters of MCS buffer.
Add 10 microliters of CD31 microbeads and mix the whole suspension. Incubate in a refrigerator at four to eight degrees Celsius for 15 minutes. After this, add one milliliter of MCS buffer and centrifuge at 300 times gravity and at four degrees Celsius for 10 minutes.
Remove the supernatant and resuspend the cells in 500 microliters of MCS buffer. Position the medium magnetic column in the magnetic field of the separator. Rinse the column with 500 microliters of MCS buffer.
Next, place a 15-milliliter conical tube below the column to collect the flow through. Apply the whole cell suspension on to the column and let it completely flow through. Wash the column three times by adding 500 microliters of MCS buffer into the reservoir for each washing step, and wait until the column's reservoir is empty before beginning the next washing step.
Unlabeled cells passing the column represent the CD45-negative, CD31-negative fraction. Store them for further quality controls. Then, remove the column from the separator and place it on a suitable collection tube.
Pipette two milliliters of MCS buffer onto the column. Immediately push the plunger into the column to flush the magnetically-labeled cells. This CD45-negative, CD31-positive fraction represents the enriched primary murine ECs.
Centrifuge the cell suspension at 350 times gravity and at 20 degrees Celsius for five minutes. Remove the supernatant completely and resuspend the cells in one milliliter of endothelial cell medium. Transfer the cells to a single well of a coated six-well culture plate containing one milliliter of endothelial cell medium.
For cultivation, incubate the cells at 37 degrees Celsius with 5%carbon dioxide in a sterile incubator, making sure to refresh the medium every two to three days. When the cells are at 80 to 90%confluence, rinse each well containing cells twice with two milliliters of PBS in two consecutive washing steps. Then, add 800 microliters of trypsin EDTA solution to each well and incubate at 37 degrees Celsius with 5%carbon dioxide in a sterile incubator for three to five minutes.
After this, add 1, 200 microliters of DMEM containing at least 10%FCS to stop the enzymatic activity. Centrifuge at 350 times gravity and at 20 degrees Celsius for five minutes. Then, accumulate the CD31 cells as previously described to increase the purity.
Use a bright field or standard phase contract microscope with 20%magnification and 0.35 lens numerical aperture to observe the cell confluence. In this study, primary murine skeletal muscle microvascular endothelial cells are isolated. One day after isolation, primary murine MMECs and residual other cells form conglomerates and adhere to the bottom of culture dishes.
From day seven onward, flat and elongated cells can be observed, however, contamination of other, mostly spheroid cells is still visible. Thus, another cycle of CD31-positive selection via MCS is required. Hereafter, primary murine MMECs proliferate to a density of approximately 80 to 90%Upon confluence, they typically form a non-overlapping monolayer of longitudinally-aligned cells.
Proliferation stops upon confluence due to contact inhibition. Quality control via flow cytometry shows values for both viability and purity ranging around 70%each for cells immediately after the isolation. Cells cultivated after another CD31-positive selection via MCS show satisfying values for purity as well as for viability, ranging up to 95%each.
Obtained cells are then investigated for the gene expression of the muscle satellite cell marker genes paired box protein 7 and M-cadherin on mRNA level by quantitative PCR. As expected, only the CD45-negative, CD31-negative fraction as well as the differentiated primary murine muscle cells expressed box protein 7 and M-cadherin, whereas CD45-negative, CD31-positive and the primary murine MMECs are negative for these markers. After the second CD31 MCS step, qPCR is used to evaluate the expression of tight junction proteins in confluent primary murine MMECs.
Primary murine MMECs are seen to express high levels of claudin-5, occludin, and zonula occludens-1, whereas pmMC only show a low expression of zonula occludens-1. This technique can be performed in five to six hours. Following this protocol, other methods such as migration assays or low-shear stress experiments can be performed to answer additional questions.
After watching this video, you should be able to isolate primary microvascular endothelial cells from skeletal muscles by mechanic and enzymatic dissociation and magnetic cell sorting.
