Katz, N., Koukharenko, V. and Geldner, P2,3.
Laboratory of Stem cells and Bioengineering, Jointechlabs, Inc., Skokie, IL
North Michigan Surgical Center, Chicago, IL
Geldner Center, Chicago, IL
Fat auto graft transplantation method for different reconstructive plastic surgeries has been fueled recently by encouraging reports from Japan, the United Kingdom, the United States, and others. Hundreds of performed procedures around the world didn’t confirm the major concern of inducing malignant transformation upon fat transplantation. Based on these reports American Association of Plastic Surgeons has removed restrictions on this kind of treatment. However, the efficacy of the method remains to be improved due to the low survival of fat tissue upon transplantation of large auto grafts, particularly in breast reconstructive and augmentation surgeries.
We assume that adding purified autologous adipose-derived mesenchymal stem cells (ADMSC) could improve the survival of fat transplants.
Vascular Endothelium Gross Factor (VEGF) persists in the human body, including fat tissue, and participates in the induction of endothelium formation and vascular development. Nevertheless, we suggest that external supplements of VEGF could increase neo-vascular development thereby improving support for fat transplant.
Previous reports on this subject are controversial and do not provide scientific evidence on the effect of MSCs and VEGF on fat viability.
We are aiming by this study to investigate the effect of human autologous ADMSCs and VEGF supplements on human adipose tissue viability in vitro.
Model: 3gr fat in 5ml DMEM, supplemented 7.5% FBS and Antibiotics 1X
Calculation of VEGF concentration
The final work concentration is 50ng/ml. In order to prep 10ml of the working medium, we need 500ng, which is 0.5ug.
When reconstituting the original 20ug of VEGF in 1ml of distilled water, we achieve a concentration of 20ug/ml. Adding 9ml of DMEM (suppl. 7.5% FBS) brings to conc. 20ug/10ml or 2ug/ml. 0.25ml of this solution contains 0.5ug of VEGF, which is required for the prep of 10ml of the working solution.
We aliquot the final 10ml of diluted VEGF into 0.25ml aliquots and freeze in liquid nitrogen vapor at -1700C.
For each 10ml of required medium with 50ng/ml of VEGF we have to thaw 1 vial of 0.25ml aliquot and add to 9.75ml of work medium.
Calculation of MSCs concentration
We applied a concentration of 30.000 cells per 50ul of medium for each 1ml of fat.
The imitation of the proposed clinical concentration was calculated as follows:
We assumed that for 400ml of transplanted fat 20ml of MSCs suspension could be mixed in. Therefore for each 1ml of fat 50ul of cell suspension should be added.
We suggest based on our results that 12 million purified MSCs could be prepared for clinical application within a short time of lab culture (data not presented yet). Assuming that 12 mills of cells would be applied in 20ml of solution in case of clinical application, we calculated and prepared for the presented experiment 30.000 of MSCs per 50ul of medium for each 1ml of fat.
Our experiment contained 4 study groups:
Group #1: 3gr of fat in 5ml of work medium (see above) supplemented with 50ng/ml
VEGF and 90.000 MSCs, suspended in 150ul of the same medium;
Group #2: 3gr of fat in 5ml of medium supplemented with 50ng/ml VEGF without MSCs supplement;
Group #3: 3gr of fat in 5ml of medium without VEGF supplementation but with the same amount of MScs as in the first group;
And Group #4: control – 3gr of fat in 5ml of medium without any of the above supplements.
In the current study, we used donor MSCs obtained in advance from fat tissue following
clinical liposuction. Informed consent has been signed and the procedure approved by IRB.
Fat digestion and MSCs extraction were performed as described elsewhere.
All samples were cultured in 12cm2 flasks in an incubator at 370C and 5.5% CO2. 3ml of the appropriate medium was exchanged for a fresh one every three days.
On day 14 and day 21 the free oil was measured as an indicator for degenerated lipocytes.
On day 21 the remaining fat was fixed and embedded in paraffin blocks for histological analysis of markers, specific for endothelial cells.
Our preliminary results indicated significant development of neovascular network in the fat graft following MSCs enrichment. However, VEGF alone did not provide a measurable increase in endothelial expression. The total volume of remaining fat graft in groups 1 and 3 was almost twice more than in the control. Even though the volume of the survived fat graft in group 2 exceeded the control, the observed fat quality and appearance were incomparably worse than in MSCs containing groups 1 and 3.
Further investigation is required toward the scientific significance of the results.
Nevertheless, preliminary results confirm the feasibility of adipose-derived mesenchymal stem cells for the improvement of fat graft survival.
We are aiming to expand the study forward application of non-purified cells, adipose-derived stromal vascular fraction, in terms of neo-vascularization of grafted tissues and organs.