The Combination of Mechanically Isolated Stromal Vascular Fraction and Fibrin Hydrogel: A Processing Protocol

Chronic wounds and large area skin defects pose a major clinical burden with limited therapeutic options. We aim to investigate the use of a readily available autologous cell source, fat tissue, as a possible treatment opportunity. Stromal vascular fraction is isolated from fat tissue obtained either by excision or liposuction.

Mechanically isolated stromal vascular fraction is used as an alternative to enzymatically processed SVF in order to bypass the problems faced by extra corporeal laboratory processing. So typical problems include long processing time, risk of contamination, and incurred costs. We believe that the diverse regenerative capabilities of MSVF cells mentioned, and the advantageous combination with a fibrin hydrogel can provide an innovative approach to enhance wound healing processes.

Overall, this approach allows for an efficient topical delivery of viable MSVF cells. In future, we will address how to translate our acquired knowledge into a clinical setting, specifically the effect of each cell type represented in SVF in an in vitro pathological condition. To begin in a cell culture hood, add the harvested lipo-aspirate into a 50 milliliter centrifuge tube.

Transfer the lipo-aspirate into a sterile 20 milliliter lure lock syringe. Then attach a 1.4 millimeter connector to the syringe. Now, attach a second 20 milliliter lure 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 500xg 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 500xg 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 1 milliliter of pre-warmed culture medium to each well, place the 12 well plate back into the incubator again for four hours. Add 1 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 multimode 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 4 degrees Celsius.

The resazurin assay was performed to quantify the in vitro cell variabilities 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.