Stromal Vascular Fraction Isolation Research Articles

Evaluation of Three Devices for the Isolation of the Stromal Vascular Fraction from Adipose Tissue and for ASC Culture: A Comparative Study.

Adipose-derived stem/stromal cells (ASCs) reside in the stromal vascular fraction (SVF) of adipose tissue (AT) and can be easily isolated. However, extraction of the SVF from lipoaspirate is a critical step in generating ASC, and semiautomated devices have been developed to enhance the efficacy and reproducibility of the outcomes and to decrease manipulation and contamination. In this study, we compared the reference method used in our lab for SVF isolation from lipoaspirate, with three medical devices: GID SVF-1™, Puregraft™, and Stem.pras®. Cell yield and their viability were evaluated as well as their phenotype with flow cytometry. Further on, we determined their proliferative potential using population doublings (PD), PD time (PDT), and clonogenicity assay (CFU-F). Finally, we checked their genetic stability using RT-qPCR for TERT mRNA assay and karyotyping as well as their multilineage potential including adipogenic, chondrogenic, and osteogenic differentiation. Our results demonstrate that all the devices allow the production of SVF cells with consistent yield and viability, in less time than the reference method. Expanded cells from the four methods showed no significant differences in terms of phenotype, proliferation capabilities, differentiation abilities, and genetic stability.

Shift toward Mechanical Isolation of Adipose-derived Stromal Vascular Fraction: Review of Upcoming Techniques.

Background:Standard isolation of adipose stromal vascular fraction (SVF) requires the use of collagenase and is considered more than “minimally manipulated” by current good manufacturing practice requirements. Alternatively, nonenzymatic isolation methods have surfaced using physical forces to separate cells from the adipose matrix. The purpose of this study was to review the literature on the use of mechanical isolation protocols and compare the results. The implication for use as a standard procedure in practice is discussed.Methods:A systematic review of the literature was performed on mechanical isolation of SVF with a search of six terms on PubMed and Medline databases. One thousand sixty-six articles were subject to evaluation by predetermined inclusion and exclusion criteria.Results:Two level 2 evidence articles and 7 in vitro studies were selected. SVF was isolated using automated closed systems or by subjecting the lipoaspirate to centrifugation only or by shaking or vortexing followed by centrifugation. Six articles reported isolation in laboratory settings and three inside the operating room. Stromal vascular cells expressed CD34, and CD44, CD73, CD90, and CD105, and differentiated along adipogenic and osteogenic lineages. When compared with enzymatic methods, mechanical isolation required less time but yielded fewer cells. Both case–control studies reported improved volume retention with cell-supplemented fat grafts for breast reconstruction.Conclusions:Mechanical isolation methods are alternatives to circumvent safety issues posed by enzymatic protocols. However, randomized comparative studies with long-term clinical outcomes using mechanically isolated stromal vascular cells are needed to identify their ideal clinical applications.

Microcirculatory Response In Vivo on Local Intraarterial Infusion of Autogenic Adipose-derived Stem Cells or Stromal Vascular Fraction.

Fat tissue was surgically harvested from the flanks of male Sprague-Dawley rats (n = 12) and processed for SVF isolation. Some SVF samples were cultured for 24 hours for ASC purification. The autogenic SVF (1 × 105) cells (n = 6) or purified ASC (1 × 105) cells (n = 6) cells were infused into the microcirculation of cremaster muscle at a speed of 0.05 mL/min through the cannulation of femoral artery. As this is a vascular pedicle isolated preparation, the infused SVF or ASC cells went nowhere but the cremaster muscle. The video image of the microcirculation was monitored in real time during infusion. Arteriole diameter was measured as A1 (100-160 µm), A2 (40-80 µm), and A3/A4 (10-30 µm). Capillary perfusion was quantified in 18 capillary fields of each muscle. There was a significant increase in the diameter of terminal arterioles (P = 0.049) and the capillary density (P = 0.02) after ASC intraarterial infusion. However, a significant cell aggregation, embolisms, and arterial obstruction were observed in the microcirculation in every case during SVF infusion. Intraarterial infusion is an appropriate route for the delivery of autogenic ASCs, but not of SVF. SVF-induced microembolisms were the reason for narrowing or blocking the lumen of terminal arterioles, resulting in no flow in the corresponding capillaries.

Tumescent Liposuction without Lidocaine

Our previous study demonstrated that lidocaine has a negative impact on adipose-derived stem cell (ASC) survival. Currently for large-volume liposuction, patients often undergo general anesthesia; therefore, lidocaine subcutaneous anesthesia is nonessential. We hypothesized that removing lidocaine from tumescent might improve stromal vascular fraction (SVF) and ASC survival from the standard tumescent with lidocaine. Ropivacaine is also a commonly used local anesthetic. The effect of ropivacaine on ASC survival was examined. Adults who underwent liposuction on bilateral body areas were included (n = 10). Under general anesthesia, liposuction on 1 area was conducted under standard tumescent with lidocaine. On the contralateral side, liposuction was conducted under the modified tumescent without lidocaine. Five milliliters of lipoaspirate were processed for the isolation of SVF. The adherent ASCs were counted after 24 hours of SVF culture. Apoptosis and necrosis of SVF cells were examined by Annexin/propidium iodide staining and analyzed by flow cytometry. Average percentage of live SVF cells was 68.0% ± 4.0% (28.5% ± 3.8% of apoptosis and 3.4% ± 1.0% of necrosis) in lidocaine group compared with 86.7% ± 3.7% (11.5% ± 3.1% of apoptosis and 1.8% ± 0.7% of necrosis) in no-lidocaine group (P = 0.002). Average number of viable ASC was also significantly lower (367,000 ± 107) in lidocaine group compared with that (500,000 ± 152) in no-lidocaine group (P = 0.04). No significant difference was found between lidocaine and ropivacaine on ASC cytotoxicity. Removing lidocaine from tumescent significantly reduced SVF and ASC apoptosis in the lipoaspirate. We recommend tumescent liposuction without lidocaine, particularly if patient's lipoaspirate will be used for fat grafting.

A rapid sonication based method for preparation of stromal vascular fraction and mesenchymal stem cells from fat tissue.

Introduction: Much attention has been paid to the idea of cell therapy using stem cells from different sources of the body. Fat-derived stem cells that are called adipose derived stem cells (ADSCs) from stromal vascular fraction (SVF) are the subject of many studies in several cell therapy clinical trials. Despite production of some GMP-grade enzymes to isolate SVF for clinical trials, there are critical conditions like inconsistency in lot-to-lot enzyme activity, endotoxin residues, other protease activities and cleavage of some cell surface markers which significantly narrow the options. So we decided to develop a new method via sonication cavitation to homogenize fat tissue and disrupt partially adipose cells to obtain SVF and finally ADSCs at a minimum of time and expenses. Methods: The fat tissue was chopped in a sterile condition by a blender mixer and then sonicated for 2 s before centrifugation. The next steps were performed as the regular methods of SVF harvesting, and then it was characterized using flow cytometry. Results: Analysis of the surface markers of the cells revealed similar sets of surface antigens. The cells showed slightly high expression of CD34, CD73 and CD105. The differentiation capacity of these cells indicates that multipotent properties of the cells are not compromised after sonication. But we had the less osteogenic potential of cells when compared with the enzymatic method. Conclusion: The current protocol based on the sonication-mediated cavitation is a rapid, safe and cost-effective method, which is proposed for isolation of SVF and of course ADSCs cultures in a large scale for the clinical trials or therapeutic purposes.

Roll, Spin, Wash, or Filter? Processing of Lipoaspirate for Autologous Fat Grafting: An Updated, Evidence-Based Review of the Literature.

The use of autologous adipose tissue harvested through liposuction techniques for soft-tissue augmentation has become commonplace among cosmetic and reconstructive surgeons alike. Despite its longstanding use in the plastic surgery community, substantial controversy remains regarding the optimal method of processing harvested lipoaspirate before grafting. This evidence-based review builds on prior examinations of the literature to evaluate both established and novel methods for lipoaspirate processing. A comprehensive, systematic review of the literature was conducted using Ovid MEDLINE in January of 2015 to identify all relevant publications subsequent to the most recent review on this topic. Randomized controlled trials, clinical trials, and comparative studies comparing at least two of the following techniques were included: decanting, cotton gauze (Telfa) rolling, centrifugation, washing, filtration, and stromal vascular fraction isolation. Nine articles comparing various methods of processing human fat for autologous grafting were selected based on inclusion and exclusion criteria. Five compared established processing techniques (i.e., decanting, cotton gauze rolling, centrifugation, and washing) and four publications evaluated newer proprietary technologies, including washing, filtration, and/or methods to isolate stromal vascular fraction. The authors failed to find compelling evidence to advocate a single technique as the superior method for processing lipoaspirate in preparation for autologous fat grafting. A paucity of high-quality data continues to limit the clinician's ability to determine the optimal method for purifying harvested adipose tissue. Novel automated technologies hold promise, particularly for large-volume fat grafting; however, extensive additional research is required to understand their true utility and efficiency in clinical settings.

Breast Reconstruction with Enhanced Stromal Vascular Fraction Fat Grafting: What Is the Best Method?

Actually, there are 2 main methods to obtain stromal vascular fraction (SVF): enzymatic digestion and mechanical filtration; however, the available systems report heterogeneous and sometimes not univocal results. The aim of this study is to evaluate different procedures for SVF isolation and compare their clinical efficacy in the treatment of soft-tissue defects in plastic and reconstructive surgery. The authors evaluated Celution and Medikhan, enzymatic systems, and Fatstem and Mystem system, mechanical separation systems. Fifty patients affected by breast soft-tissue defects were treated in the Plastic and Reconstructive Surgery Department of Tor Vergata University of Rome. Four groups of 10 patients were managed with enhanced SVF fat grafts using cells obtained by Celution (Cytori Therapeutics, Inc., San Diego, Calif.), Medikhan (Medi-Khan Inc., West Hollywood, Calif.), Fatstem (Fatstem CORIOS Soc. Coop, San Giuliano Milanese, Italy), and Mystem (Mystem evo Bi-Medica, Treviolo, Italy) systems. A control group of 10 patients was treated with only centrifuged fat according to Coleman's technique. In enhanced SVF-treated patients treated with cells obtained by Celution system, we observed a 63% ± 6.2% maintenance of contour restoring after 1 year, compared with 39% ± 4.4% of control group. In patients treated with SVF obtained by Medikhan system, we observed a 39% ± 3.5% maintenance, whereas enhanced SVF with Fatstem and Mystem systems gave a 52% ± 4.6% and 43% ± 3.8% maintenance of contour restoring, respectively. SVF cell counting indicated that Celution and Fatstem were the most efficient systems to obtain SVF cells. Celution and Fatstem were the 2 best automatic systems to obtain SVF and to improve maintenance of fat volume and prevent the reabsorption.

Development of a System and Method for Automated Isolation of Stromal Vascular Fraction from Adipose Tissue Lipoaspirate.

Autologous fat grafting for soft tissue reconstruction is challenged by unpredictable long-term graft survival. Fat derived stromal vascular fraction (SVF) is gaining popularity in tissue reconstruction as SVF-enriched fat grafts demonstrate improved engraftment. SVF also has potential in regenerative medicine for remodeling of ischemic tissues by promoting angiogenesis. Since SVF cells do not require culture expansion, attempts are being made to develop automated devices to isolate SVF at the point of care. We report development of a closed, automated system to process up to 500 mL lipoaspirate using cell size-dependent filtration technology. The yield of SVF obtained by automated tissue digestion and filtration (1.17 ± 0.5 × 105 cells/gram) was equivalent to that obtained by manual isolation (1.15 ± 0.3 × 105; p = 0.8), and the viability of the cells isolated by both methods was greater than 90%. Cell composition included CD34+CD31− adipose stromal cells, CD34+CD31+ endothelial progenitor cells, and CD34−CD31+ endothelial cells, and their relative percentages were equivalent to SVF isolated by the manual method. CFU-F capacity and expression of angiogenic factors were also comparable with the manual method, establishing proof-of-concept for fully automated SVF isolation, suitable for use in reconstructive surgeries and regenerative medicine applications.

Tissue Dissociation Enzymes for Adipose Stromal Vascular Fraction Cell Isolation: A Review

With the increasing clinical translation of adipose stromal cell based technologies, it is important to thoroughly report on all aspects of these technologies. In this article, we review the commonly used enzymes for adipose tissue dissociation and stromal vascular fraction isolation. The enzymes reviewed include collagenase, trypsin, clostripain, and the neutral proteases dispase and thermolysin. Enzyme characteristics, the advantages of enzymatic isolation, and other relevant practical and regulatory issues are discussed.

Safety of stromal vascular fraction cells applications in chronic pain

Autologous stromal vascular fraction (SVF) can be enzymatically released from lipoaspirate obtained under local anesthesia. SVF is known to have regenerative, anti-inflammatory, pain mitigating, and immune-modulatory properties. Our translational research network has been studying the safety and efficacy of SVF since 2012. Almost 100 related physician teams around the world are applying the same institutional review board–approved methods of SVF production, which use a surgically closed SVF isolation system. During the same outpatient surgical procedure, procured SVF is administered according to strict investigative protocols to mitigate diseases associated with chronic pain including arthritis, autoimmune disease, neurodegenerative disease, and various inflammatory conditions. The shared research collaborative online database contains safety and efficacy data on more than 3500 patients. Our processed SVF contains valuable anti-inflammatory cytokine growth factors in addition to both adult mesenchymal and hematopoietic stem cells targeting damaged, or inflamed tissue. SVF administration may potentially play a large role in the outpatient treatment of pain. In this article, we describe our protocol for the production and administration of SVF, and its safety and efficacy in the treatment of pain associated with chronic conditions.

Lidocaine-induced ASC apoptosis (tumescent vs. local anesthesia).

The purpose for the present study was to determine which anesthetic method, local anesthesia versus tumescent, is superior for liposuction in terms of adipose-derived stem cell (ASC) survival in lipoaspirate; which component, lidocaine versus lidocaine with epinephrine, in anesthetic solutions could affect ASC survival; and which mechanism, necrosis versus apoptosis, is involved in lidocaine-induced ASC death. Human lipoaspirates were harvested using standard liposuction technique. Individuals scheduled for liposuction on bilateral body areas gave consent and were included in the study. On one area, liposuction was conducted under local anesthesia with lidocaine/epinephrine. On the contralateral area, liposuction was accomplished with tumescent wetting solution containing lidocaine/epinephrine. Lipoaspirates were processed for the isolation of stromal vascular fraction (SVF). ASC survival was determined by the number of adherent ASCs after 24 h of SVF culture. Lidocaine dose-response (with or without epinephrine) on cultured ASCs was examined. Lidocaine-induced ASC apoptosis and necrosis was determined by Annexin V-FITC/Propidium Iodide (PI) assay and analyzed by flow cytometry. All of the participants were female adults. The average age was 45 ± 4.0 years (±SEM) and the average BMI was 28 ± 1.0 (±SEM). Lipoaspirate samples (n = 14) treated by local anesthesia (n = 7/group) or tumescent anesthesia (n = 7/group) were investigated. Liposuction sites were located in the hip or thigh. The average number of adherent ASCs was 1,057 ± 146 k in the local anesthesia group, which was significantly lower than the 1,571 ± 111 k found in the tumescent group (P = 0.01). ASC survival was significantly lower in the lidocaine group and in a dose-dependent manner as compared to the correspondent PBS controls (P < 0.05 or P < 0.01). ASC survival was significantly lower in both the lidocaine and lidocaine with epinephrine groups when compared to PBS controls. Annexin/PI assay showed that ASC apoptosis (but not necrosis) in the lidocaine group was significantly higher than that in the corresponding PBS control (P = 0.026). Tumescent anesthesia is the superior method for liposuction with respect to ASC preservation compared to local anesthesia. Lidocaine could cause significant ASC apoptosis.

Age influence on stromal vascular fraction cell yield obtained from human lipoaspirates

Background aimsThe adipose stromal vascular fraction (SVF) is a heterogeneous population of mononuclear cells that includes approximately 1–10% mesenchymal stromal cells. These SVF cells can be freshly obtained from human lipo-aspirates and represent and ideal candidate for regenerative medicine applications. In the present study, we analyzed the SVF yield as a function of the patient's age. MethodsAdipose tissue samples from 52 informed subjects (all women) were processed by means of an innovative point-of-care technology for SVF isolation (GID platform). After enzymatic dissociation of adipose tissue and SVF pellet resuspension, we measured the concentration of mononucleated cells as well as other cell quality analyses on the cell suspension obtained. We then calculated the cell yield as total nucleated cells per milliliter of dry adipose processed. ResultsThe mean SVF yield obtained was 7.19 × 105 ± 2.11 × 105 total nucleated cells per milliliter of adipose tissue. Our results show that there is a clear statistically significant decline in SVF cell yield with increasing age. ConclusionsBecause all samples were obtained from the same donor area and the isolation technique used was the same in all cases, we conclude that the SVF cell yield in women is affected by patient age. Specific age-related factors should be analyzed in the future to explain these results.

Expansion of Semi-Automatic Processed Human Adipose-Derived Stem Cells in Medium Supplemented with Autologous Serum and Antioxidants

Objective: Excised fat tissue (EFT) is an excellent source of adipose-derived stem cells (ADSCs) for autologous cell therapy. However, the current technique for excising fat tissues with conventional surgical scissors and using culture medium containing fetal bovine serum (FBS) may not comply with Good Tissue Practice (GTP) guidelines. We thus developed a semi-automated process complying with the abovementioned guideline to manufacture clinicalgrade ADSCs from small volumes of EFT. Methods: A semi-automated process was developed by using a disposable disperser tube operated on a Tube Disperser Workstation to obtain stromal vascular fraction (SVF) from EFT. A culture medium supplemented with human autologous serum (HAS) and mesenchymal stem cell culture adjuvant (MCA) consisted of basic fibroblast growth factor (bFGF/FGF-2) and antioxidants, was formulated to expand ADSCs from SVF in vitro. Results: The semi-automated process can markedly enhance cell yields and reduce the operation time for mincing tissues from an hour down to a few minutes. The growth of ADSCs was slower in the 10% HAS medium than that in medium supplemented with FBS, but their growth in the 10% HAS medium containing MCA (10% HAS+MCA) was superior to both media. The 10% HAS+MCA medium also promoted adipogenesis, osteogenesis, chondrogenesis and increased the expression of CD44, CD73 and the stemness genes in the ADSCs. Conclusion: Our novel semi-automated process provides an efficient isolation of SVF, and the 10% HAS+MCA medium accelerates the growth of ADSCs from small amounts of EFT without losing their differentiation ability.

Selected Abstracts from the Voice of Europe Session of the 5th Annual Congress of the EASAPS

S EXPERIMENTAL/SPECIAL TOPICS Selected Abstracts from the Voice of Europe Session of the 5th Annual Congress of the EASAPS (Editorial Coordinator Cristino Suarez Lopez de Vergara) Kemal Findikcioglu • Radu Ionescu • Nicolae Antohi • Tiberiu Bratu • Dana Jianu • Maria Filipescu • Doru Chirita • Jesus Benito-Ruiz • Jose Carlos Parreira • Paul Harris • Reza Nassab • James Southwell-Keely • Rajiv Grover • Barry Jones • Cristino Suarez Lopez de Vergara Published online: 16 April 2013 Springer Science+Business Media New York and International Society of Aesthetic Plastic Surgery 2013 The Hinderer Award Winner 2012 The Impact of Breast Reduction Surgery on the Vertebral Column Kemal Findikcioglu (Gazi Universitesi Tip Fakultesi Plastik Rekonstruktif ve Estetik Cerrahi A.D. Turkey. email: kemaldoctor@yahoo.com) Background: Although many studies have shown that breast reduction surgery is effective in reducing neck, back and lumbar pain; most of these studies are subjective evaluations that usually provide data through pain scales. This study was undertaken to objectively evaluate the radiologic effects of breast reduction on the vertebral column. Methods: Thirty breast reduction patients were included in the study. A lateral thoracolumbar radiograph was taken preoperatively and three months postoperatively for each patient. The thoracic kyphosis, lumbar lordosis and sacral inclination angles were measured for each radiograph. The impact of breast reduction surgery on posture was evaluated according to the comparison of these angles preoperatively and postoperatively. Effect of the age, BMI and the total amount of removed tissue was also taken into account and the relationship between these parameters and their effects on the change in preoperative and postoperative angle measurements were evaluated. Results: There was a significant decrease in all three angles after breast reduction surgery. A significant correlation was determined between BMI and the total amount of removed tissue on the change in angle measurements while a definite relationship was not observed between the angles and the patient’s age. Conclusion: This study has shown the objective impact that breast reduction surgery has on the vertebral column. Even though the symtomatic relief of breast reduction surgery on the musculoskeletal system is widely accepted, the objective assessment of this relief will be beneficial in persuading health insurance companies and those who think of this surgery as a purely aesthetic procedure. Junior Section—Young Investigator Winner Stromal Vascular Fraction Isolation Manual Protocol: A Cheap and Easy Way of Controlling the Production of Adipose-Derived Stem Cells Authors: Radu Ionescu MD, Nicolae Antohi MD, Tiberiu Bratu MD FAT!!! and the discovery of adipose-derived stem cells put the plastic surgery in the front line of new frontiers of science, the place where we, with our ability for public relation and safeness of good practicing in lipotransfer, we develop a powerful marketing tool, maybe too powerful, performing this type of surgery with a certain lack of knowledge and scientific data, fact that can make the ADRC research to be estompated by a increased number of questions concerning the safety and efficacy of Adipose-Derived Stem Cells treatments. The main objective of this work is to standard the protocol of: Fat harvesting, SVF preparation, and Lipotransfer. Fat harvesting have to be performed using low aspiration pressure 0.35 bar, cannula of maximum 3 mm, tumescent or super wet, buffer solution for infiltration—with low dosage of xyline at 37 Celsius (see photo 1). Preparation of SVF is performed in the operation room on using GMP devices and drugs, in approx. 60 min, on a standard protocol of 9 steps (see photo 2). Collagenase is Serva NB6 a GMP approved, and K. Findikcioglu R. Ionescu N. Antohi T. Bratu D. Jianu M. Filipescu D. Chirita J. Benito-Ruiz J. C. Parreira P. Harris R. Nassab J. Southwell-Keely R. Grover B. Jones C. S. L. de Vergara (&) Santa Cruz de Tenerife, Avenida de la Asuncion, 30 2nd, 38007 Tenerife, Spain e-mail: cristinosuarez@gmail.com 123 Aesth Plast Surg (2013) 37:602–604 DOI 10.1007/s00266-013-0106-4

Analysis of Stromal Vascular Fraction from Lipoaspirates: Our Institute's Experiences

Today, adipose tissue derived mesenchymal stem cells (ADSC) have gained a great interest in various medical fields due to the characteristics of its self-renewal and multilineage differentiation capacity. The stromal vascular fraction (SVF) of adipose tissue is known to contain mesenchymal stem cells and it is obtained by processing the lipoaspirate which is usually collected from tumescent liposuction. In this study, we reviewed the records of patient epidemiology and results of SVF isolation. 30 patients (8 males and 22 females) had been underwent tumescent liposuction between April 2012 and January 2013, and the collected lipoaspirates were processed to isolate SVF in GMP facility in CHA Bundang Medical Center. The average stem cell count per 1 cc of lipoaspirate was 52,252 ± 26,704 and cell count including red blood cells per 1cc of lipoaspirate was 970,607 ± 873,436. The stem cell viability was proven to be 84 ± 4%. Bacteria were not detected in all the SVF samples. Compared to previous reports concerning the yield of SVF, our results coincide well with the results of previous studies. Because there were no domestic report about the yield and viability of SVF, this report may provide a reference value of the Korean SVF for the clinicians who want to use SVF as a therapeutic purpose.

Selection of Donor Site for Fat Grafting and Cell Isolation

Autologous fat grafting has rapidly become an important treatment for soft tissue defects in cosmetic and reconstructive surgery. However, consensus is lacking on the ideal donor site for harvesting and isolating stromal vascular fraction (SVF) cells to improve survival of fat grafts. We aimed to determine the best donor site for tissue harvesting and isolation of SVF cells for fat graft survival. Adipose tissue samples were harvested from six women who underwent an aesthetic procedure. The samples were harvested by needle aspiration of five commonly used donor sites: flank, upper and lower abdomen, and lateral and inner thigh. The adipose tissue was injected subcutaneously into nude mice and grafts were harvested at 12weeks. We evaluated graft volume, weight, and histologic parameters of the grafts: integrity, cysts/vacuoles, inflammation, fibrosis, and neovascularization. SVF cells isolated from donor sites were counted and assayed by flow cytometry. At 12weeks post-transplantation, weight, volume, and histologic parameters did not differ among the grafts from the five tissue donor sites. Also, SVF and levels of cell surface markers did not differ by donor site. This study revealed no ideal tissue donor site for fat grafting and SVF isolation. Choosing a site should be based on ease and safety of access and the preference and request of the patient. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Abstract 92: A Novel Role for Dipeptidyl Peptidase-4 and Adenosine Deaminase in Regulating Macrophage and Dendritic Cell Function: Implications for Treatment of Type 2 Diabetes

Introduction: In addition to its powerful effects on glycemic control via enzymatic degradation of incretins, Dipeptidyl peptidase-4 (DDP4) also exerts non-metabolic functions. Despite the ubiquitous use of DPP4 inhibitors in the treatment of Type 2 diabetes (T2D), their effects on immune cell function are poorly understood. Hypothesis: In this study, we investigated the hypothesis that DPP4 may play a critical role in modulating T cell proliferation through its expression on dendritic cells (DCs) and further characterized its role in adipose inflammation in patients with morbid obesity. Methods: Monocytes were isolated from peripheral blood of healthy donors. DCs were induced from monocytes by rhGM-CSF and rhIL-4. DPP4 expression and T-cell proliferation were detected by flow cytometry. DPP4 knockdown was accomplished by transient transfection with DPP4-specific siRNA. Human visceral adipose tissues were used for stromal vascular fraction isolation. Results: 7.5±3.1% (n=10) of peripheral monocytes expressed DPP4. DPP4 dramatically increased when monocyte was differentiated to DC/Macrophage or activated by LPS. Interestingly, DPP4 expression was much higher on the DC/Macrophage in adipose tissue than that of peripheral blood. Furthermore, an increase of DPP4 on adipose tissue macrophages in obese patients was observed (13.2±2.4 vs. 7.9±1.6 for obese vs. lean, n=5). And there was a correlation between DPP4 expression and insulin level (y = 0.9094x-3.6088, R 2 =0.6943). Cell surface DPP4 was able to bind adenosine deaminase (ADA). The binding enhanced the capability of DCs to stimulate T-cell proliferation by relieving the suppressive effect of adenosine on T-cell proliferation. Knockdown of DPP4 on DCs by siRNA attenuated their ability to stimulate T-cell proliferation without influence on the secretion of pro-inflammatory cytokines by DCs. Conclusions: DPP4 on DCs mediates binding of ADA and may re-create a microenvironment suitable for T-cell proliferation. This interaction in DCs between ADA and DPP4 may promote adipose T-cell inflammation and play facilatatory role in T2D. Pro-inflammatory effects of DPP4 and the elucidation of its enzymatic v.s. non-enzymatic contributions may be of fundamental importance in the understanding of T2D.

Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis

The heterogeneous stromal vascular fraction (SVF), containing adipose-derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell-processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side-effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell-based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice-level cell processing area.