Our simple and practical methodology can be used to obtain abundant ADSCS for studying PVAT adipogenesis in vitro and to the test normal drugs against obesity and related cardiovascular diseases. The advantage of this method is the inherent fluorescence of the NCADSCs of biotin you need to label the cells with flou-chrome conjugated antibodies for FACS, or magnetic activated cell sorting. This method can be used to acquire abundant ADSC derived from neural crest cells from the ectoderm to study PVAT adipogenesis, or lipogenesis in vitro.
Using young mice and having experience with cautery and induced adipogenesis means 3T3 cells is critical to the success of this protocol. Demonstrating the procedure will be Yiding Qi, a grad student, and Lian Xu, a technician from my laboratory. For PAAT dissection, after sterilizing the mouse carcass in 75%ethanol for five minutes, use scissors to snip and separate the skin on the abdomen.
Cut along the ventral midline from pelvis to the neck, and open the abdomen. Move the liver to expose the diaphragm, and cut the diaphragm and the ribs on both sides of the midline. Peel back the ribs to expose the heart and lungs.
And remove the lungs and the thymus. Extract the PAAT along with the aorta and heart into a petri dish. And cut off the aorta at the aortic root to remove the heart.
Then, make a cut between the aortic arch and descending aorta. Carefully separate the adipose tissue surrounding both structures, and the left and right common carotid arteries from the posterior chest wall. It is important to separate the PAAT from the root and as a larger vascular vessels carefully, is the presence of vascular wall cells can affect FACS efficiency.
Place the tissue into a petri dish containing ice cold HBSS buffer. And use sterile forceps to remove as much of the vasculature and fascia as possible. Then, transfer the adipose tissue into a two milliliter Eppendorf tube, containing point five milliliters of ice cold HBSS buffer on ice.
When all of the PAAT has been collected, transfer the harvested adipose tissue into a new two milliliter micro-centrifuge tube containing one milliliter of freshly prepared digestion medium. And use surgical scissors to mince the tissues. Transfer the tissue slurry into a 50 millimeter tube containing nine milliliters of fresh digestion medium.
And use a one milliliter pipette to triturate the tissues 10 times. When a homogenous solution has been obtained, incubate the tube for 30 to 45 minutes at 37 degrees Celsius and 100 revolutions per minute, checking every five to 10 minutes to prevent overdigestion. When a light yellow homogenous adipose tissue solution can be observed upon gentle swirling of the tube, stop the digestion with five milliliters of HDMEM supplemented with 10%fetal bovine serum.
After a thorough mixing, centrifuge the adipose tissue sample. The stromal vascular cell fraction will be visible as a brownish pellet. Re-suspend the pellet in 10 milliliters of culture medium, and filter the cell suspension through a 70 micrometer cell strainer.
Collect the cells by another centrifugation. And gently re-suspend the pellet in five milliliters of erythrocytes lysis buffer. Transfer the suspension to a 15 milliliter tube.
After 10 minutes, stop the reaction with two centrifugations in 10 milliliters of PBS, supplemented with 1%fetal bovine serum. After the second centrifugation, re-suspend the pellet in five milliliters of culture medium on ice. And collect the cells with a final centrifugation.
Then, re-suspend the cells in five milliliters of FACS buffer for counting. To set up an NCADSC culture, after their isolation by FACS according to standard protocols, place the cells at a five times 10 to the third cells per centimeters squared concentration in each well, a twelve well culture plate. Incomplete culture medium at 37 degrees Celsius and 5%carbon dioxide for 20 to 24 hours.
The next day, wash the cells with 37 degrees Celsius PBS, and feed the culture with fresh culture medium. When the culture reaches 80 to 90%confluency, treat the cells with two milliliters of 0.25%trypsin EDTA per well for three to five minutes at 37 degrees Celsius. When the cells have been detached from the bottom of the plate, neutralize the enzymes with two milliliters of cultured medium, and collect the harvested cells by centrifugation.
Re-suspend the pellet in one milliliter of fresh culture medium for counting. And seed the cells in a new 12 well culture plate in a five times 10 to the third cells per centimeters squared concentration. Then, re-suspend the remaining cells in culture medium supplemented with 10%dimethyl sulfoxide for frozen storage in liquid nitrogen.
To induce adipogenic differentiation, when the cells reach an 80 to 90%confluency, treat the cells with brown or white adipogenic induction medium for two days as appropriate. At the end of the incubation, gently wash the cells two times with PBS before adding the appropriate fresh adipogenic induction medium to each well, and returning the cells to the cell culture incubator for an additional three to five days of incubation. Cultured NCADSCs reach an 80 to 90%confluency after seven to eight days of culture.
And demonstrate an expanded fiberglass like morphology. To further confirm their adipogenic potential, oil red staining of the differentiated NCADSCs can be performed to detect mature adipocytes. Typically, mature adipocytes are observed after eight days of white or brown adipogenic induction with over 60%of the NCADSCs exhibiting adipogenic differentiation.
Note that NCADSCs demonstrate a greatly reduced adipogenic potential after passaging. Immunoblotting and quantitative real-time PCR reveal that the expression of adipocyte specific relative proteins and genes significantly increases in adipogenically differentiated NCADSCs after eight days of white adipogenic induction. Similarly, the expression of adipocyte specific genes, and brown adipocyte specific genes, also significantly increases after eight days of brown adipogenic induction.
Since most NCADSC isolation protocol is simple, economical, and doesn't require the use of antibodies, for that, the strong fluid reasons intensity of IFP can improve the FACS nourishment efficiency.
