Human Stromal Cell Aggregates Concentrate Adipose Tissue Constitutive Cell Population by In Vitro DNA Quantification Analysis.

BACKGROUND: Regenerative cell strategies rely on stromal cell implants to attain an observable clinical outcome. However, the effective cell dose to ensure a therapeutic response remains unknown. To achieve a higher cell dose, the authors hypothesized that reducing the volume occupied by mature adipocytes in lipoaspirate will concentrate the stromal vascular fraction present in the original tissue. METHODS: Human standardized lipoaspirate (n = 6) was centrifuged (1200 g for 3 minutes) and the water phase was discarded. Mechanical disaggregation was achieved by shearing tissue through 2.4- and 1.2-mm Luer-to-Luer transfers. After a second centrifugation (800 g for 10 minutes), stromal cell aggregates were separated from the supernatant oil phase. Lipoaspirate percentage composition was determined by its constituent weights. Cell content was measured by total DNA quantification, and partial cell viability was determined by image cytometry. Tissue sections were evaluated histologically (hematoxylin and eosin and Masson trichrome stains). RESULTS: Stromal cell aggregates reduced the standardized lipoaspirate mass to 28.6 ± 4.2 percent. Accordingly, the cell density increased by 222.6 ± 63.3 percent (from 9.9 ± 1.4 million cells/g to 31.3 ± 6.6 million cells/g; p < 0.05). Cell viability was unaffected in stromal cell aggregates (71.3 ± 2.5 percent) compared to standardized lipoaspirate (72.2 ± 2.3 percent), and histologic analysis revealed high-density areas enriched with stromal cells (622.9 ± 145.6 percent) and extracellular matrix (871.2 ± 80.3 percent). CONCLUSION: Stromal cell aggregates represent a biological agent that triplicates the cell density versus unprocessed lipoaspirate, low on oil and water fluids, and enriched extracellular matrix components.

Nanofat Cell Aggregates: A Nearly Constitutive Stromal Cell Inoculum for Regenerative Site-Specific Therapies.

BACKGROUND: Recent technology developed by Tulip Medical Products allows clinicians to mechanically disaggregate fat tissue into small fat particles known as nanofat. The present study aimed to evaluate the cell yield obtained from nanofat generation in comparison to traditional methods involving enzymatic dissociation (stromal vascular fraction). METHODS: Nanofat preparations were characterized by cell content and viability, based on DNA quantification and image cytometry, respectively. DNA analysis was also used to determine the cell content in unprocessed dry lipoaspirate and native adipose tissue (excised adipose tissue). To evaluate cell yield, the authors compared the number of cells recovered from 1 g of lipoaspirate between stromal vascular fraction and nanofat preparations, and subsequently determined the final cell inoculum obtained following their respective protocols. RESULTS: The data showed that nanofat samples presented a cell burden of 7.3 million cells/g, close to 80 percent of unprocessed dry lipoaspirate, and 70 percent of native excised adipose tissue. Moreover, cell viability was not altered by mechanical disaggregation in nanofat samples compared to unprocessed dry lipoaspirate. Nanofat samples exhibited a cell yield of 6.63 million cells/g lipoaspirate, whereas stromal vascular fraction preparations resulted in only 0.68 million cells/g lipoaspirate. The final cell inoculum obtained from stromal vascular fraction isolation was 120 million cells and it required 200 to 250 cc of raw lipoaspirate as starting material, whereas nanofat preparation resulted in 125 million cells with only 20 cc of raw lipoaspirate. CONCLUSION: Mechanical disaggregation offers a better cell inoculum than conventional enzymatic dissociation methods by using 10 times less fat tissue as starting material and delivering a higher cell yield.