The overall goal of this procedure is to isolate microvascular fragments from murine adipose tissue for the generation of vascularization units for tissue engineering. This method can help answer key questions in the field of tissue engineering about the in vivo vascularization of tissue substitues. An advantage of this technique is that it yields fully functional microvascular segments without complex in vitro processing that can be seeded onto scaffolds and implanted into tissue defects.
This method can also be applied to other systems such as in vitro angiogenesis assays to unravel molecular mechanisms underlying microvascular network formation and remodeling. Begin by placing an anesthetized male wild type C57 black six mouse in the supine position under a surgical stereomicroscope and confirm a lack of response to toe pinch. Immobilize the paws with tape and disinfect the abdomen.
Next, use dissecting scissors to separate the abdominal layer from the underlying muscle tissue and make a midline laparotomy. Laterally unfold the flaps of the abdominal wall and bilaterally identify the testes, epididymus and the epididymal fat pads. Then use small preparation scissors and fine forceps to harvest the fat pads, taking care not to collect any epididymal tissue and place the fat pads into a petri dish containing 15 ml of 37 degrees celsius DMEM.
In a laminar flow hood, wash the fat pads in three separate petri dishes containing 15 ml of PBS and place the washed fat into an empty 14 ml polypropylene tube. Use a tube scale to determine the volume of harvested fat. Then use fine scissors to mechanically mince the fat tissue.
When a homogenous tissue suspension is obtained, use a 10 mL measuring pipet to transfer the minced tissue with two volumes of collaginase NB 4G into a 50 mL erlenmeyer flask containing a 25 millimeter stir bar. Place the flask in a cell culture incubator for 10 minutes under vigorous stirring. Then, observe 10 mcL of the digested tissue under a microscope.
The most difficult step in this isolation procedure is identifying the appropriate time to stop the digestion. Digesting the fat pads for too long will result in a single cell suspension without any microvascular fragments. If the digestate contains free adipose tissue derived microvascular fragments next to single cells, neutralize the enzyme with two volumes of PBS supplemented with 20%FCS and transfer the cell vessel suspension into new polypropylene tubes.
Incubate the solution for five minutes at 37 degrees celsius to separate the microvascular fragments from the remaining fat by gravity. Then, use a one mL precision pipet to carefully remove one mL of the main fat supernatent followed by the use of a 100 mcL precision pipet to remove the rest of the supernatent until the suspension appears to be fat free. When all of the fat has been removed, use a 10 mL measuring pipet to filter the cell vessel suspension through a 50 micron strainer over a 50 mL conical tube and transfer the filtered suspension into a new 14 mL polypropylene tube for each individual microvascular fragment isolate.
Collect the microvascular fragment pellets by centrifugation and remove all but the last one mL of supernatent from each tube. Resuspend the pellets in the remaining supernatents. Then, transfer each suspension into individual 1.5 mL conical microcentrifuge tubes and pellet the microvascular fragments again for resuspension in the appropriate final volume of PBS and 20%FCS for the planned downstream analysis.
Here, the length distribution of freshly isolated microvascular fragments from six, seven to twelve month old wild type C57 black six mice is shown, with a mean average length of approximately 42 micrometers observed. As this representative cell vessel suspension smear demonstrates, large, medium and small microvascular fragments as well as single cells are observed when the fat tissue has been sufficiently digested. Higher magnification reveals that the microvascular fragments exhibit a mature microvessel morphology with hierarchical microvessel segments.
Characterization of the microvascular fragments by flow cytometry indicates that they contain CD31 positive endothelial cells, alpha smooth muscle acton positive paravascular cells and CD117 positive mesenchymal stem cells, marker expressing cells. After seeding on a dermal skin substitute, larger microvascular fragments are mainly localized on the implant's surface with some capillary vessel segments detected within the implant cores. Immunohystochemical staining of the seeded microvascular fragments further reveals physiological microvessel configurations with arteriolar, venular and capillary like fragments.
Once mastered, this technique can be completed in two hours if performed properly. After watching this video, you should have a good understanding of how to isolate microvascular fragments from murine adipose tissue as vascularization units for tissue engineering.
