Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Obtaining stromal vascular fractions, or SVFs, from lipoaspirates requires digestion with collagenase, which increases the risk of biological contamination. A better way to obtain SVF without collagenase should be developed. These techniques enable us to obtain the SVF in its native extracellular matrix through a simple mechanical process.

Demonstrating the procedure will be Xihang Chen, a grad student from our lab. After obtaining the fat samples, transfer the lipoaspirates into one 50 milliliter tube, and allow the harvested fat to equilibrate for 10 minutes. Next, use a wide tip pipette to pull the fat from the top layer of the tube into a new 50 milliliter tube and spin down the fat by centrifugation.

The upper layer in the tube is the Coleman fat fraction. Transfer the upper 2/3 of the Coleman fat into a new 15 milliliter tube. This portion is considered the low density fat.

Transfer the lower third of the Coleman fat into a second 15 milliliter tube. This portion is considered the high density fat. To produce an extracellular matrix stromal vascular fraction gel from the low density fat, use two 20 milliliter syringes connected by a female to female luer lock connector to intershift 10 milliliters of the low density fat.

This is the key step to destroy adipocytes. The speed and the time that the intersyringe shifting is performed contributes to the quality of the final product. After the last intershift session, collect the fat by centrifugation and transfer the top oil layer to a 15 milliliter tube.

Then transfer the sticky middle extracellular matrix stromal vascular fraction gel layer into a new 15 milliliter conical tube. To produce an extracellular matrix stromal vascular fraction gel from the high density fat, add two milliliters of the low density oil fraction to five milliliters of the high density fat sample. Intershift the mixed fat as demonstrated until a flocculate is observed within the emulsion.

Collect the flocculate by centrifugation and discard the top oil layer. Then collect the sticky middle layer and mix this fraction with the low density extracellular matrix stromal vascular fraction gel sample. After processing the Coleman fat to extracellular matrix stromal vascular fraction gel, the volume of discarded oil takes up 80%of the final volume, and only 20%of the adipose tissue preserved under the oil layer is regarded as extracellular matrix stromal vascular fraction gel.

Extracellular matrix stromal vascular fraction gel has a smooth liquid-like texture that enables it to pass through a 27 gauge fine needle. However, Coleman fat is comprised of an integral adipose structure with large fibers that can only pass through an 18 gauge cannula. On day three after the transplantation, large numbers of small sized pre-adipocytes with multiple intracellular lipid droplets, extensive well-vascularized connective tissue, and infiltrated inflammatory cells appear.

Beginning on day 15, the number of inflammatory cells starts to reduce gradually, and the adipocytes begin to mature. By day 90, most of the vascularized connective tissue in the grafts has been replaced by mature adipocytes. Extracellular matrix stromal vascular fraction gel grafts also contain a few perilipin positive adipocytes three days after the transplantation, with small sized pre-adipocytes, with multiple intracellular lipid droplets beginning to appear on day 15.

Each field of the day 90 extracellular matrix stromal vascular fraction gel graft sections exhibit numerous perilipin positive adipocytes and newly formed blood vessels. Flow cytometry can be performed to determine the cellular population in the product. And scanning electron microscopy can be performed to visualize the microstructure of the product.

This technique provides a simple method for surgeons and researchers to obtain a product with high concentration of SVF cells with native extracellular matrix fibers from adipose tissue.