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

Introduction:

Fat auto graft transplantation method for different reconstructive plastic surgeries has been fueled recently by encouraging reports from Japan, United Kingdom, United States and others. Hundreds of performed procedures around the world didn’t confirm major concern of inducing malignant transformation upon fat transplantation. Based on these report American Association of Plastic Surgeons has removed restrictions on this kind of treatments. However, the efficacy of the method remains to be improved due to low survival of fat tissue upon transplantation of large auto grafts, particularly in breast reconstructive and augmentation surgeries.

We assume that adding of purified autologous adipose-derived mesenchymal stem cells (ADMSC) could improve survival of fat transplant. 

Vascular Endothelium Gross Factor (VEGF) persists in human body, including fat tissue, and participates in induction of and endothelium formation and vascular development. Nevertheless, we suggest that external supplement of VEGF could increase neo-vascular development thereby improving support of fat transplant. 

Previous reports on this subject are controversial and do not provide scientific evidences on effect of MSCs and VEGF on fat viability.

We are aiming by this study to investigate the effect of human autologous ADMSCs and VEGF supplement on human adipose tissue viability in vitro. 

Study design:

Model: 3gr fat in 5ml DMEM, supplemented 7.5% FBS and Antibiotics 1X

Calculation of VEGF concentration

Final work concentration is 50ng/ml.  In order to prep 10ml of working medium we need 500ng, which is 0.5ug.

When reconstituted original 20ug of VEGF in 1ml of distil water, we achieve concentration of 20ug/ml. Adding 9ml of DMEM (suppl. 7.5% FBS) bring to conc. 20ug/10ml or 2ug/ml.  0.25ml of this solution contains 0.5ug of VEGF, which required for prep of 10ml of working solution.

We aliquot 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 concentration of 30.000 cells per 50ul of medium for each 1ml of fat.

The imitation of proposed clinical concentration was calculated as follow:

We assumed that for 400ml of transplanted fat 20ml of MSCs suspension could be mixed in. Therefor for each 1ml of fat 50ul of cell suspension should be added.

We suggest based on our results that 12 millions of purified MSCs could be prepared for clinical application within 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 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 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 same amount of MScs as in the first group;

And Group #4: control – 3gr of fat in 5ml of medium without any of above supplements.

In current study we used donor MSCs obtained in advance from fat tissue following 

clinical liposuction. Informed consent has been signed and procedure approved by IRB.

Fat digestion and MSCs extraction was performed as described elsewhere.

All samples were cultured in 12cm2 flasks in incubator at 370C and 5.5% CO2. 3ml of appropriate medium were exchange on fresh one every three days.

On day 14 and day 21 the free oil was measured as indicator for degenerated lipocytes.

On day 21 the remaining fat was fixed and embedded in paraffin blocks toward histological analysis of markers, specific for endothelial cells.

Results

Our preliminary results indicated significant development of neo-vascular network in the fat graft following MSCs enrichment. However, VEGF alone did not provide measurable increase in endothelial expression. The total volume of remaining fat graft in group 1 and 3 was almost twice more than in control. Even though the volume of the survived fat graft in group 2 exceeded the control, the observed fat quality and appearance was incomparably worse than in MSCs containing groups 1 and 3.

Conclusion

Further investigation is required toward scientific significance of the results.

Nevertheless, preliminary results confirm feasibility of adipose-derived mesenchymal stem cells for improvement of fat graft survival.

We are aiming to expand the study forward application of non-purified cells, adipose-derived stromal vascular fraction, in term of neo-vascularization of grafted tissues and organs.