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Cytokine-Rich Adipose Tissue Extract Production from Water-Assisted Lipoaspirate: Methodology for Clinical Use

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ABSTRACT

Proper functioning wound healing strategies are sparse. Adequate vascular formation to the injured area, as well as replacement of the volume loss, is fundamental in soft tissue repair. Tissue engineering strategies have been proposed for the treatment of these injury sites. Novel cell-free substance, human adipose tissue extract (ATE), has been previously shown to induce in vitro angiogenesis and adipogenesis and in vivo soft tissue formation. This study reports the translation of ATE preparation from laboratory to the operating room (OR). ATE samples for this study were derived from adipose tissue obtained with the water-jet assisted liposuction technique from 27 healthy patients. The variables studied included incubation time (15, 30, and 45 min), temperature (room temperature vs. 37°C), and filter type to determine the optimal method yielding the most consistent total protein content, as well as consistent and high expression of adipose-derived growth factors and cytokines, including: vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, leptin, and insulin-like growth factor. Following the optimization, samples were produced in the OR and tested for their sterility. No significant differences were observed when comparing extract incubation time points or incubation temperature. Nonetheless, when studying the different filter types used, a syringe filter with PES membrane with larger filter area showed significantly higher protein concentration (p ≤ 0.018). When studying the different growth factor concentrations, ELISA results showed less variation in cytokine concentrations in the OR samples with the optimized protocol. All of the OR samples were tested sterile. The devised protocol is an easy and reproducible OR-ready method for ATE generation. As an attractive source of growth factors, ATE is a promising alternative in the vast field of tissue engineering. Its clinical applications include volume replacement as a complement to fillers and improvement of the permanence of fat grafts and wound healing, among other bioactive functions.

No MeSH data available.


Protein concentrations obtained with four different filters. Comparison of total protein concentration when ATE was produced with 30 min incubation at RT using four distinct filters; PES Filter 1 (7.5 cm2), cellulose acetate Filter 2 (6.2 cm2), cellulose acetate Filter 3 (5.3 cm2), and PES Filter 4 (4.5 cm2). Statistical analysis was performed with One-way ANOVA with Tukey's posttest, p < 0.05* and p < 0.01** and n = 4.
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f3: Protein concentrations obtained with four different filters. Comparison of total protein concentration when ATE was produced with 30 min incubation at RT using four distinct filters; PES Filter 1 (7.5 cm2), cellulose acetate Filter 2 (6.2 cm2), cellulose acetate Filter 3 (5.3 cm2), and PES Filter 4 (4.5 cm2). Statistical analysis was performed with One-way ANOVA with Tukey's posttest, p < 0.05* and p < 0.01** and n = 4.

Mentions: To estimate whether the protein yield varied among different filter membranes, four different 0.2 μm filters were selected. ATE was produced at 30 min incubation at RT and subsequently strained through the filters. Close to 1000 μg/mL protein was obtained with all filters, but with polyethylene sulfone (PES) membrane and largest surface area, showed a significantly higher protein yield (multiplicity adjusted p ≤ 0.018) compared to the other filters (Fig. 3).


Cytokine-Rich Adipose Tissue Extract Production from Water-Assisted Lipoaspirate: Methodology for Clinical Use
Protein concentrations obtained with four different filters. Comparison of total protein concentration when ATE was produced with 30 min incubation at RT using four distinct filters; PES Filter 1 (7.5 cm2), cellulose acetate Filter 2 (6.2 cm2), cellulose acetate Filter 3 (5.3 cm2), and PES Filter 4 (4.5 cm2). Statistical analysis was performed with One-way ANOVA with Tukey's posttest, p < 0.05* and p < 0.01** and n = 4.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035828&req=5

f3: Protein concentrations obtained with four different filters. Comparison of total protein concentration when ATE was produced with 30 min incubation at RT using four distinct filters; PES Filter 1 (7.5 cm2), cellulose acetate Filter 2 (6.2 cm2), cellulose acetate Filter 3 (5.3 cm2), and PES Filter 4 (4.5 cm2). Statistical analysis was performed with One-way ANOVA with Tukey's posttest, p < 0.05* and p < 0.01** and n = 4.
Mentions: To estimate whether the protein yield varied among different filter membranes, four different 0.2 μm filters were selected. ATE was produced at 30 min incubation at RT and subsequently strained through the filters. Close to 1000 μg/mL protein was obtained with all filters, but with polyethylene sulfone (PES) membrane and largest surface area, showed a significantly higher protein yield (multiplicity adjusted p ≤ 0.018) compared to the other filters (Fig. 3).

View Article: PubMed Central - PubMed

ABSTRACT

Proper functioning wound healing strategies are sparse. Adequate vascular formation to the injured area, as well as replacement of the volume loss, is fundamental in soft tissue repair. Tissue engineering strategies have been proposed for the treatment of these injury sites. Novel cell-free substance, human adipose tissue extract (ATE), has been previously shown to induce in vitro angiogenesis and adipogenesis and in vivo soft tissue formation. This study reports the translation of ATE preparation from laboratory to the operating room (OR). ATE samples for this study were derived from adipose tissue obtained with the water-jet assisted liposuction technique from 27 healthy patients. The variables studied included incubation time (15, 30, and 45&thinsp;min), temperature (room temperature vs. 37&deg;C), and filter type to determine the optimal method yielding the most consistent total protein content, as well as consistent and high expression of adipose-derived growth factors and cytokines, including: vascular endothelial growth factor, basic fibroblast growth factor, interleukin-6, adiponectin, leptin, and insulin-like growth factor. Following the optimization, samples were produced in the OR and tested for their sterility. No significant differences were observed when comparing extract incubation time points or incubation temperature. Nonetheless, when studying the different filter types used, a syringe filter with PES membrane with larger filter area showed significantly higher protein concentration (p&thinsp;&le;&thinsp;0.018). When studying the different growth factor concentrations, ELISA results showed less variation in cytokine concentrations in the OR samples with the optimized protocol. All of the OR samples were tested sterile. The devised protocol is an easy and reproducible OR-ready method for ATE generation. As an attractive source of growth factors, ATE is a promising alternative in the vast field of tissue engineering. Its clinical applications include volume replacement as a complement to fillers and improvement of the permanence of fat grafts and wound healing, among other bioactive functions.

No MeSH data available.