We want to understand the role of the subcutaneous adipose tissue microvasculature in people with and without cardiometabolic disease. To do this, we've been isolating subcutaneous adipose tissue microvascular endothelial cells from patients undergoing cardiac implantable electronic devices. This is to study the endothelial cell phenotype and cell-cell crosstalk with adipocytes.
Historically, we have used immortalized human cell lines or animal models to study cardiometabolic disease in vitro. However, these are not representative of diseases in human subjects. Our technique overcomes this by allowing us to understand the cellular basis of cardiometabolic disease using tissue from real-world patients.
Our group is currently investigating the mechanism of dysregulated subcutaneous adipose tissue endothelial cell adipocyte crosstalk in people with diabetes, in particular, the impact of diabetic endothelium on adipocyte function and how this can be targeted to improve cardiometabolic health. To begin, obtain 250 milligrams of SAT above the pectoralis major muscle during CIED implantation in magnetic activated cell sorting tissue storage solution. Prepare one milligram per milliliter of collagenase/dispase in 10 milliliters of cold Hanks'Balanced Salt solution.
Under a laminar flow cabinet, mince tissues into one cubic millimeter pieces in 500 microliters of collagenase/dispase solution. Add 4.5 milliliters of the collagenase/dispase solution to the mix and transfer the triturated tissue to a clean 50 milliliter centrifuge tube. Wash the Petri dish lid with five milliliters of collagenase/dispase solution and add it to the tube.
Incubate the tube for 30 minutes at 37 degrees Celsius in a tube rotator set to 20 revolutions per minute. To halt the digestion, pour 10 milliliters of complete endothelial cell growth media into the tube. After triturating the digested mix with a 14 gauge Venflon cannula, strain it through a 70 micrometer cell sieve and rinse it with 10 milliliters of 0.5%PBS-BSA buffer.
Centrifuge the filtrate at 300 G for 10 minutes at room temperature and discard the supernatant. To remove the dead cells, add 200 microliters of dead cell removal beads to the cell pellet, taken in a microcentrifuge tube and incubate. Then attach a liquid separation column with a 30 micrometer filter to the separator magnet and place a 15 milliliter centrifuge tube underneath.
After washing the column with dead cell removal binding buffer, add the sample/bead suspension. Let it pass through under gravity and collect the eluate. Next, spin the collected live cell eluate and resuspend the resulting pellet in 400 microliters of 0.5%PBS-BSA.
Incubate the suspended cells with 20 microliters of anti-CD 31 coated magnetic beads at four degrees Celsius for 20 minutes. After centrifuging, resuspend the cell pellet in 500 microliters of 0.5%PBS-BSA. Attach a column with a 30 micrometer filter to the separator magnet and place a 15 milliliter centrifuge tube underneath.
Add the cell microbead suspension to the column and let it pass through under gravity. After washing the column, place it in a fresh 15 milliliter centrifuge tube and add one milliliter of 0.5%PBS-BSA to the column. Push the column plunger to collect the CD31 positive fraction.
Centrifuge the sample as demonstrated earlier and resuspend the cell palette in one milliliter of complete microvascular endothelial cell growth media. Dispense the suspension into two wells of a 2%gelatin coated 24 well plate. Culture the cells at 37 degrees Celsius with 5%carbon dioxide for two to four weeks until cells become nearly 80%confluent.
To begin, thaw the passage one human white subcutaneous preadipocytes and add the cells into a T75 flask containing 12 to 15 milliliters of warm preadipocyte growth media two. After the cells attain 90%confluency, wash them with PBS and incubate them with one milliliter of 0.25%trypsin/EDTA solution for two minutes. Using a light microscope, confirm the cell detachment and add 23 milliliters of preadipocyte growth media two to the cells.
Dispense one milliliter of cell suspension into each well of a 24 well co-culture companion plate and incubate the plate to attain cell confluency. Then replace the media with one milliliter of preadipocyte differentiation medium and incubate at 37 degrees Celsius with 5%carbon dioxide for 10 days. On day six of differentiation, seed five times 10 to the power of four human SAT-MVECs in 500 microliters of complete MVEC growth media on a transwell insert.
Place the insert in a well containing 500 microliters of complete MVEC growth media and incubate at 37 degrees Celsius with 5%carbon dioxide. On day 10, when the adipocytes are fully differentiated, remove half the media from each well and transfer the transwell inserts containing confluence SAT MVECs to each well. Incubate the plate for 24 hours to obtain a co-culture.
As the human subcutaneous white preadipocytes become more differentiated, the development of lipid vacuoles can be noticed.
