Human Adipose Tissue Micro-fragmentation for Cell Phenotyping and Secretome Characterization

Using this system, we can collect the sub-millimeter clusters of human adipose tissue, in which the whole microenvironment of regenerative cells is intact. The whole microvascular system is also intact, and so we can use these sort of micro organs for in-vivo transplantation for in-vitro culture and for cell isolation for further characterization. The main rationale for the use of the system in the research laboratory is that the process is totally enzyme-free, and so it allows the rapid and efficient processing of human adipose tissue while maintaining a micro-architecture and it will be the microvasculature of the tissue totally intact.

And so we can directly study a cell therapy product which has proven already very useful in the clinic. This closed system technology is an easy to use technique for harvesting, processing and re-injection refined fat tissue intra-operatively, and can be successfully applied in cosmetics orthopedics, proctology and also gynecology. This method allows further study of the impact of fat in tissue regeneration.

The procedure will be demonstrated by Bianca Vezzani, a postdoctoral fellow and by Mario Gomez-Salazar, who is a PhD student. Working under aseptic conditions within 16 hours of harvesting, place the abdominoplasty sample on top of a surgical cloth with the skin facing upwards. Use a disposable tissue infiltration cannula to inject 50 to 100 milliliters of 37 degrees Celsius 0.9%sodium chloride solution into the subcutaneous adipose tissue.

Connect a 10 milliliter Luer lock syringe to a disposable liposuction cannula and grasping the sample at the cutaneous region near an edge of the sample, carefully introduce the cannula into the adipose tissue. Once the cannula is inside the tissue, extract the plunger and move the cannula radially within the adipose tissue sample. It is important to handle the adipose tissue firmly during the lipo-aspiration.

If not enough fat is collected, consider performing an additional saline injection. When the syringe is full of lipo-aspirate, carefully remove the cannula from the tissue and replace the full syringe with a new empty syringe until a total of 60 milliliters of aspirate has been harvested. For micro-fragmentation of the lipo-aspirate, remove the device from the package and verify that the main unit is connected to the waste bag.

Place the waste collection bag on the ground, and make sure that the valves are secured to the process unit caps. Pierce the bag connection port to connect the terminal spike of the input line into the saline bag and position the saline bag higher than the processing unit. Verify that all five of the clamps connected to the tubes of the circuits are open, place the processing unit in a vertical position, allow the saline solution to fill the processing unit, and confirm that the flow reaches the waste bag.

Shake the processing unit to remove any air bubbles and place the unit in a vertical position with the blue cap facing upwards. Close the clamp next to the input line and connect a syringe of lipo-aspirate to the self-occluding valve of the blue input cap to begin injecting the lipo-aspirate. Keeping the processing unit vertical, slowly push down on the syringe plunger until all of the lipo-aspirate has been transferred to the unit.

When all of the lipo-aspirate has been injected, open the input clamp to restore the saline flow and vigorously shake the processing unit for at least two minutes, regularly checking the saline solution flow into the waste bag. When the saline solution in the processing unit turns transparent, place the unit with the gray cap upwards and close the clamps located on the drain near both the blue and the gray caps. The processed tissue will float at the top.

Next, fill a 10 milliliter Luer lock syringe with saline solution, and connect the syringe to the loading valve of the blue cap. Connect an empty 10 milliliter Luer lock syringe with the plunger completely inserted to the valve of the gray cap. Firmly inject the saline into the processing unit from the blue cap, checking that the syringe connected to the gray valve is consequently being filled with the processed tissue.

When all of the saline has been injected, carefully remove both syringes and repeat the saline infusion until all of the processed tissue has been collected. At the end of the processing, remove all of the syringes, close all of the clamps, detach the saline bag, and dispose of the device according to local protocols. When all of the processed tissue has been collected, transfer the micro-fragmented adipose tissue into a sterile container, and add an equal volume of digestion medium to the container.

Place the sealed container in a 37 degrees Celsius shaking water bath, set to 120 rotations per minute for 45 minutes, stopping the digestion with an equal volume of blocking solution at the end of the incubation. Sequentially filter the sample, first through a 100 micrometer cell strainer followed by filtration through a 70 micrometer cell strainer and centrifuge the filtered suspension for five minutes at 200 times g. We suspend the pellet in approximately 5 milliliters of erythrocyte lysis buffer for 15 minutes at room temperature.

Stop the lysis with an equal volume of blocking solution and filter the solution through a 40 micrometer cell strainer. Then collect the micro-fragmented adipose tissue cells by centrifugation and we suspend the pellet in fresh blocking solution with floure-mixing for counting. After dissociation and antibody staining, micro-fragmented fat cells can be taken to the flow cytometer for cell analysis and/or sorting.

Mechanical dissociation of manual lipo-aspirates results in the production of micro-fragmented adipose tissue which consists of an aggregate of adipocytes enveloping a microvascular network. Immunofluorescence analysis of gelatin embedded in cryofixed micro-fragmented adipose tissue highlights the structure, showing the vascular network marked by the endothelial cell marker Ulex Europaeus Agglutinin 1 receptor and mainly consisting of small capillary-like vessels. Notably, pericytes expressing neuron-glial antigen 2 or platelet-derived growth factor receptor beta are normally distributed, ensheathing endothelial cells.

By excluding CD31 positive endothelial and CD45 positive hematopoietic cells, the presence of CD146 positive CD34 negative pericytes and CD146 negative and CD34 positive adventitial cells can be identified within the MAT. Manual lipo-aspiration is a critical part of the procedure and must be performed under sterile conditions no more than 16 hours after the abdominoplasty. Micro-fragmented adipose tissue can be further processed by immunohistochemical staining, in vitro culture or secretome analysis to obtain a deeper insight into adipose tissue biology.

This technique allows the study of diverse tissues in their natural, native, three-dimensional arrangement as a sort of small organoids, which are small enough to be transplanted in mice or to be maintained in long-term culture. And we use this system principally to approach experimentally the different organ-specific microenvironments. Our adipose tissue donors are not known as carriers of transmissible infections.

However in the absence of any further screening, all human tissues should be considered as potentially contaminated and handled accordingly.