To begin, in a cell culture hood, add the harvested lipoaspirate into a 50 milliliter centrifuge tube. Transfer the lipoaspirate into a sterile 20 milliliter Luer lock syringe. Then attach a 1.4 millimeter connector to the syringe.
Now attach a second 20 milliliter Luer lock syringe to the contralateral side of the connector. Push the adipose tissue from one syringe to the other about 30 times. Then transfer the emulsified fat into a fresh 50 milliliter centrifuge tube.
Centrifugate the fat at 500 G for 10 minutes. Then discard the oily top layer. Collect the central purified layer containing the separated stromal vascular layer and transfer it into a fresh 50 milliliter centrifuge tube.
Now fill the centrifuge tube with supplemented DMEM up to the 40 milliliter mark. Centrifuge the tube again at 500 G for five minutes. Collect the resulting mSVF layer and transfer it into a new 50 milliliter centrifuge tube.
Into a sterile 1.5 milliliter tube, pipette 100 microliters of the isolated stromal vascular fraction. To this, add 10 microliters of thrombin, then pipette 10 microliters of calcium chloride. Finally, add 70 microliters of tranexamic acid to the mix.
With a fresh pipette tip, add 10 microliters of fibrinogen to the tube shortly before application. After the polymerization of the mSVF hydrogel mixture, pipette 200 microliters of the hydrogel into a 12-well plate for analytical purposes. Now add 100 microliters of the fibrin hydrogel into one well as a negative control.
Incubate the 12-well plate at 37 degrees Celsius under 5%carbon dioxide for 30 minutes. Then add one milliliter of pre-warmed culture medium to each well. Place the 12-well plate back into the incubator again for four hours.
Add one milliliter resazurin to each well before incubating again for 24 hours. Pipette the resazurin samples into a 96-well plate. The next day, use a cell imaging multi-mode microplate reader to measure the first fluorescence intensity.
For histological analysis on days one, three, and seven, incubate the fibrin hydrogel in 0.5 milliliters of 4%paraformaldehyde. Now add 1%PBS to the plate and store it at four degrees Celsius. The resazurin assay was performed to quantify the in vitro cell viabilities of the vascular fraction and the hydrogel.
The vascular fraction showed reduced viability on day three, whereas the mSVF-fibrin hydrogel combination remained close to baseline. By day seven, the viability of the mSVF dropped, while the mSVF-fibrin hydrogel combination increased. Histological analysis showed no reduction in the number of cell nuclei on days three and seven.
The fibrin hydrogel displayed minimal degradation with evenly distributed visible cell clusters.
